key: cord-272437-gvzfl8c3 authors: zhao, jing; zhang, keran; cheng, jinlong; jia, wenfeng; zhao, ye; zhang, guozhong title: replicase 1a gene plays a critical role in pathogenesis of avian coronavirus infectious bronchitis virus date: 2020-08-20 journal: virology doi: 10.1016/j.virol.2020.08.009 sha: doc_id: 272437 cord_uid: gvzfl8c3 avian coronavirus infectious bronchitis virus (ibv) is an important pathogen threatening poultry production worldwide. here, two recombinant ibvs (ryn-1a-ayn and ryn-1b-ayn) were generated in which orf1a or orf1b of the virulent yn genome were replaced by the corresponding regions from the attenuated strain ayn. the pathogenicity and virulence of ribvs were evaluated in ovo and in vivo. the results revealed that mutations in the orf1a gene during passage in embryonated eggs caused the decreased pathogenicity of virulent ibv yn strain, proven by determination of virus replication in eces and cek cells, the observation of clinical signs, gross lesions, microscopic lesions, tracheal ciliary activity and virus distribution in chickens following exposure to ribvs. however, mutations in orf1b had no obvious effect on virus replication in both eces and cek cells, or pathogenicity in chickens. our findings demonstrate that the replicase 1a gene of avian coronavirus ibv is a determinant of pathogenicity. . those of the ryn group at 5 and 7 dpi (p < 0.01 or 0.001) (fig. 4b ). in the lungs, the 264 virus copy numbers in the ryn-1a-ayn infected group were also significantly lower 265 than those of the ryn group at 7 and 10 dpi (p < 0.01 or 0.001) (fig. 4c) . the numbers of viral rna copies in the kidneys of the ryn-1a-ayn infected group were 267 lower than in the ryn group at 10 dpi (p < 0.001) (fig. 4d ). there were no orf1a limited viral replication in chicken tissues. ibv is a highly infectious pathogen in domestic fowl that replicates mainly in the this may indicate that avirulent strain beaudette was not the ideal background in 296 which to study the role of ibv-specific mutations in pathogenicity. we suspect that 297 mutations in the replicase gene may play an important role in attenuation of ibv. in this study, we used an ibv reverse genetics system based on virulent strain fig. 2. growth characteristics of ibv ryn, ryn-1a-ayn or ryn-1b-ayn, and 466 clinical scores and survival rates of chickens inoculated with these ibv strains. ibv ryn, ryn-1a-ayn and ryn-1b-ayn strains. statistical significance was considered as follows: highly significant at p < 0.01 (**) 482 and extremely significant at p < 0.001 (***). is critical for viral virulence infectious bronchitis virus variants: a 425 review of the history, current situation and control measures chapter three -the nonstructural proteins directing 427 coronavirus rna synthesis and processing recombinant live attenuated avian coronavirus 431 vaccines with deletions in the accessory genes 3ab and/or 5ab protect against infectious 432 bronchitis in chickens coronavirus pathogenesis attenuation, safety, and efficacy of a qx-like infectious bronchitis virus serotype 445 vaccine porcine deltacoronavirus: overview of infection dynamics, diagnostic methods, 447 prevalence and genetic evolution attenuated chinese qx-like infectious bronchitis virus strain as a candidate vaccine successful establishment of a reverse 452 genetic system for qx-type infectious bronchitis virus and technical improvement of the 453 rescue procedure s gene and 5a accessory gene 455 are responsible for the attenuation of virulent infectious bronchitis coronavirus coronavirus replicative proteins virus-encoded proteinases and proteolytic 460 processing in the nidovirales key: cord-266230-ia04jc9j authors: rott, m. e.; tremaine, j. h.; rochon, d. m. title: comparison of the 5′ and 3′ termini of tomato ringspot virus rna1 and rna2: evidence for rna recombination date: 1991-11-30 journal: virology doi: 10.1016/0042-6822(91)90801-h sha: doc_id: 266230 cord_uid: ia04jc9j abstract the sequences of the 5′ terminal 1140 and 3′ terminal 1546 nt of tomato ringspot virus (tomrsv) rna1 have been determined. these sequences share a high degree of nucleotide sequence similarity with the previously determined tomrsv rna2 sequence. eighty-eight percent of the 5' terminal 907 nt of tomrsv rna1 and rna2 contain identical nucleotide residues; the first 459 nt are identical at all positions, whereas the next 447 nt are identical at only 75.8% of the nucleotide positions. the region of similarity includes not only the 5' nontranslated leader but also sequence probably encoding polyproteins. the 3′ terminal 1533 nt of tomrsv rna1 and rna2 are identical and are noncoding. the sequences common to rna1 and rna2 account for almost 35% of the total genomic sequence. it is possible that the similar sequences at both ends of tomrsv rna1 and rna2 are a result of recombination between these two genomic rna components. o 1991 academic press. inc. involving the exchange of genetic information between genomic rna molecules of rna viruses, appears to play a number of important functions during the evolution and life cycle of rna viruses (for reviews see 7, 2). the importance of nonhomologous rna recombination in the generation of evolutionary diversity can be seen in the modularity of viralencoded genes. in addition, many rna viruses produce defective-interfering rnas which may be a result of recombination. homologous rna recombination has been observed among the picornaviruses (3), coronaviruses (4), and bromoviruses (5) and is suspected to occur in the tobraviruses (6). it has been suggested that homologous recombination in the picornaviruses and coronaviruses is important for repairing defective genomes. in addition, coronaviruses undergo site-specific recombination to express downstream genes from leader-primed subgenomic transcripts (see 7). tomato ringspot virus (tomrsv) is a member of the nepovirus group (8) . nepoviruses consist of 28-nm spherical particles composed of 60 copies of a single coat protein species and two separately encapsidated genomic rnacomponents. nepoviruses share similarities in genomic structure and translational strategies with the plant coma-and potyviruses as well as the animal picornaviruses (9). previously, we reported that the 3' termini of tomrsv rna1 and rna2 share an ' sequence data from this article have been deposited with the embugenbank data libraries under accession numbers m27935 and m73822. 2 to whom requests for reprints should be addressed. extended region of nucleotide sequence similarity, as determined by restriction enzyme cleavage maps and hybridization analysis (10). we report that extensive nucleotide sequence similarity also exists between the 5'termini of rna1 and rna2. the possibility that these repeated sequences may facilitate replication of tomrsv rna, perhaps through recombination, will be discussed. two cdna clones derived from tomrsv rna1 (see fig. 1 ) were used to determine the 5' and 3' terminal sequences of tomrsv rnal. clone j27, which has been previously described (io), was used to sequence the 3' terminal 1546 nt of tomrsv rna1 and clone 25p6 was used to sequence the 5' terminal 1108 nt. 25p6 was obtained in essentially the same manner as j27 except that random priming was used for firststrand cdna synthesis. subcloning, sequencing, sequence assembly, and analysis were essentially as described previously (1 i). clone 25p6 was found to hybridize to both tomrsv rna1 and rna2 in northern hybridization studies (12) (data not shown). however, the restriction enzyme map of 25p6 matched that of the rna1 -specific clone b54 (10) ( fig. 1 ) but was distinct from that of the rna2-specific clone 035 (see 7 7). to confirm that 25p6 was derived from rnal, the region 5' to the hindill site of b54 was partially sequenced in one direction and was found to be identical to the corresponding region obtained from 25p6 (data not shown). the 5' terminal sequence not encoded by 25p6 was determined by dideoxynucleotide sequence analysis using tomrsv rna as a template and a specific oligonucleo"pi3 tide primer (s'gccticgatggcaacc 3') complementary to nucleotide positions 115-l 30 as described previously (i i). the sequencing gel obtained from this experiment terminated with two strong stop points in all four lanes which likely correspond to the first and second nucleotides of tomrsv rna1 (each denoted n in fig. za) . nucleotide sequence comparison of the 5'termini of tomrsv rna1 and rna2 revealed that within the first 907 nt, 88.8% of the nucleotide positions are identical. the first 459 nt of tomrsv rna1 and rna2 are identical. this region includes the 5' noncoding regions of rna1 and rna2 as well as two potential in-frame translation initiation sites at augtb and aug,,, . beginning from the first potential in-frame initiation site at aug,b, the n-terminal regions of the tomrsv rna1 and rna2 polyproteins are identical for the first 132 amino acids, and of the next 145 amino acid residues 75.3% of the positions are identical (fig. 3a ). it is perhaps significant that the second in-frame initiation site at aug,, occurs shortly after the point where the homology between the rna1 and the rna2 polyproteins becomes less than perfect. as described in a previous paper (1 i), aug,,, is in a better kozak context for the initiation of translation than aug,, (13). since it is unknown whether aug,, and/or aug,,, act as initiation sites for translation, it cannot be said whether it is the conservation of amino acid or nucleotide sequence between aug,, and aug,,, on rna1 and rna2 which is most significant. the deduced amino acid sequence of the regions beginning shortly after aug,,, on rna1 and rna2 could be aligned with the deduced amino acid sequences encoded by the 5' terminal region of rna1 of the nepoviruses tomato blackring (tbrv) and grapevine chrome mosaic (gcmv) (14, 15) ( fig. 3b ). the fact that these regions of similarity are present only at the n-termini of the tbrv and gcmv rnal-encoded polyproteins but are present at the n-termini of both tomrsv rna1 -and rna2encoded pofyproteins suggests that a large portion of coding and noncoding sequences at the 5' terminus of tomrsv rna1 have been duplicated and are now present at the 5' terrnini of both rna1 and rna2. the function of this coding region in rna1 is unknown, however, it has been suggested that it may have a role in proteofytic prmessing of the viral-encoded polyprotein (75). it is interesting that in vitro translation studies of cherry feafroll virus (clrv) rna2 (16) another nepovirus with a large rna2 component ( 17) resulted in proteolytic processing of the rna2 polyprotein in the absence of rnalencoded protease. it is possible that a subgroup of nepoviruses with large rna2 components, which would include tomrsv and clrv (181, may encode another protease on rna2 which is involved in proteolytic processing. however, it is possibtethirt proteotytic processing of the clrv rna2 polyprotein may not be due to a specific viral-encoded protease. the 3' noncoding regions of tomfz@v rna1 and rna2 are almost identical for 1533 nt (excluding the 3 poly(a) tail sequence) with only three nucleotide differences at positions 703, 720, and 770 as shown in fig. 2 b. these sequences are preceded by 13 and 17 nt of noncoding sequence which are unique to rna1 and rna2, respectively (uaaauccucwuug and tj&jg-uuggcuuccugaa, underlined nuclsotides indicate stop codons for the large orfs of rna1 and rna2, respectively). when we first reportedthesequence similarity at the 3'termini of tomrsv rpval and rna2 ( ici), we proposed that extensive 3' terminel identity between rna1 and rna2 may be characteristic for other nepoviruses with large rna2 components. this has been confirmed for the nepovirus clrv (19) ** ****** * * **** ******** * ** ******** *********** * x** aaltapwllcsyksgvssppppmtqrqqfaaikrrlvqkgqq~irelirarkaakyalbfaarkka aaitapwllrpck-geapppppltqrqqfaalkkrlavkgqqiirehirarkaakyaaiakakka ** ** ** ************ ****** *******x******r****** ** x**** aavaaqkaraeaprlaaqkaaiakilrdrqlvslpppppps~rl~~a~sksaslqrl~fh aalflavkaaqeaprlaaqkiskilrdrdrdvaalpppppps~rl~eablaskaeslrrlkafk **** ** *** ** ranrvrpvlnnsfpspp tfsrvrpalntsfpppp shows 3' noncoding sequence identity between rna1 in summary, the total amount of duplicated seand rna2 for a length which is similar to that found in quences between tomrsv rna1 and rna2 as well as tomrsv. however, very little sequence similarity is de-within rna2 (see 11) accounts for almost 35% of the tectabje between the 3' noncoding regions of tomrsv total genomic sequence. the extensive amount of nuand clrv (19). the 3' noncoding regions of the two cleotide sequence identity at the 5' and 3' termini of rna components for several other nepoviruses are tomrsv rna1 and rna2 may be required for recognialso identical but much shorter (less than 300 nt) (74, tion by a highly selective replicsee. it is slso possible 15). extensive sequence similarity at the 3'termini has that rna recombination is responsible for maintaining also been reported for members of the tobravirus nucleotide sequence identity at the 5' and 3'termini of group (20, 21) and includes both potential coding se-rna1 and rna2. rna recombination has been postuquences and noncoding sequences. lated to explain the duplication of 820 nt at the 3'terfig. 2. nucleotide sequence and deduced amino acid sequence of the 5'(a) and 3'(b) regions of tomrsv rna1 (a) the first two nuolaotides which could not be determined from the sequencing gel are each represented by an n. the second in-frame aug which is a potential initiation site for translation is underlined. nucleotides are numbered on the left beginning at the first n. amino acids are numbered on the right snd begin at the first m. numbering in (b) begins at the termination codon (wa) for the rnal-encoded long orf as determined from clone j.27. three nucleotide substitutions in the 3' noncoding region of rna2 (i 7) compared to rna1 are shown below the sequence of rna1 mini of rna1 and rna2 of the tobravirus tobacco rattle virus (trv) strain plb (6). however, after several passages of a pseudorecombinant consisting of rna1 from trv strain tcm and rna2 from strain plb, which differ at their 3'termini in 39 of 820 nt, a recombinant rna molecule was not detected. not only was rna recombination in this system not detected experimentally, but the viability of a pseudorecombinant consisting of heterologous 3' termini in rna1 and rna2 suggests that in this system precise 3'terminal sequences in rna1 and rna2 are not essential for replication. it has been suggested that the high frequency of homologous recombination in the animal picornaviruses is important for removing deleterious mutations introduced by the poor fidelity of the rna replicase (22) . since it has been suggested that the picornavirus replicative machinery may not function efficiently in trans, nondefective genes, located on different rna molecules which possess errors in genes involved in replication, can only be utilized after genetic recombination with an rna molecule which encodes functional replicative genes (23, 24) . it is possible that, in tomrsv, replication begins in cis with rna1 and that vans replication of rna2 occurs only following disassociation and reassociation of the initial negative-strand transcript with the corresponding region in rna2. a similar mechanism involving recombination could account for the sequence conservation observed between the 5' termini of rna1 and rna2. such a mechanism has recently been proposed for leader-primed generation of subgenomic rnas in coronaviruses (see 7). the size of the duplicated sequences in tomrsv may be the minimum required to facilitate efficient replication through rna recombination between rna1 and rna2. alternatively, the entire length may not be required for recombination but may serve other important functions in addition to a postulated role in recombination. we are planning further experiments to determine the biological significance of the repeated sequences between tomrsv rna1 and rna2 and whether they are in fact involved in rna recombination during replication. the technical assistance of l. lee is gratefully acknowledged. this work was partially supported by an nserc operating grant awarded to j. h. tremaine. /n "the molecular biology of the positive strand rna viruses cold spring harbor symp. quart biol descriptions of plant viruses nematode vectors of plant viruses v///rh hf key: cord-258379-v3lceirh authors: liu, d. x.; inglis, s. c. title: association of the infectious bronchitis virus 3c protein with the virion envelope date: 1991-12-31 journal: virology doi: 10.1016/0042-6822(91)90572-s sha: doc_id: 258379 cord_uid: v3lceirh abstract a highly purified radiolabeled preparation of the coronavirus infectious bronchitis virus (ibv) was analyzed, by immunoprecipitation with monospecific antisera, for the presence of a series of small virus proteins recently identified as the products of i bv mrnas 3 and 5. one of these, 3c, a 12.4k protein encoded by the third open reading frame of the tricistronic mrna3 was clearly detectable and was found to cofractionate with virion envelope proteins on detergent disruption of virus particles. these results, together with the hydrophobic nature of 3c and its previously demonstrated association with the membranes of infected cells, suggest strongly that 3c represents a new virion envelope protein, which may have counterparts in other coronaviruses. infectious bronchitis virus (ibv) is the prototype virus of the coronaviridae, a family of viruses containing a large single-stranded rna genome of positive sense. the morphology of the virion is characterised by a "corona" of widely spaced bulbous projections which consist of the virus spike glycoprotein (s) (27) . this s protein is composed of two glycopolypeptides, gp90 and gp84, which are produced by post-translational cleavage of a precursor, gp155 (7, 8, 25) . in addition to the spike protein, only two other major virion structural proteins are currently recognized. these are the nucleocapsid protein (n), which is a 5 1 k phosphoprotein found in close association with the genomic rna (26) and the membrane protein (m), an integral envelope protein which appears as a heterogeneous collection of molecules differing in their extent of glycosylation (~23, gp28, gp31, and gp36) (8, 24) . boursnell and colleagues (4) have deduced the complete sequence of ibv genomic rna through cloning of cdna. their results indicated that the ibv genome is 27.6 kb in length with at least ten open reading frames (orfs), of which only three were known certainly to encode virus proteins (the s, n, and m genes). the seven unassigned orfs were clustered in three groups on the genome-two very large orfs at the 5' end of the rna (1 a and 1 b), three small ones between the s and n genes (3a, 3b, and 3c), and two small ones between the m and the n genes (5a and 5b). recently through expression of nucleotide sequences from these unassigned orfs as bacterial fusion proteins, and production of monospecific antisera against the bacterial products, we have been able to establish posi-' to whom reprint requests should be addressed. tively the coding function of several of these orfs. it now appears that the 3a, 3b, and 3c orfs encode polypeptides in virus-infected cells (17, 23) and that the 5a and 5b orfs also represent functional genes (18) . it was therefore of interest to examine whether any of these newly identified proteins are associated with the virus particle. although their function is as yet unknown, certain of the polypeptides, particularly the 12.4k 3c and 7.5k 5a polypeptides, possess hydrophobic sequences characteristic of integral membrane proteins (2, 3) and we have shown previously that 3c can be found in association with ibv-infected cell membranes (23) . we were therefore particularly interested in the possibility that these proteins might represent previously unidentified virion envelope proteins. we report here that the 3c protein is indeed virion-associated and that it cofractionates with other virion envelope proteins on detergent disruption of virus particles. confluent monolayers of primary chicken kidney cells were prepared from 2-to 3-week-old birds as previously described (23 mm diameter) of ibv-infected cells was layered onto two tubes each containing 10 ml of 20% (w/v) tne-buffered sucrose solution (tne buffer: 50 mm tris-hci, ph 7.4, 100 mm naci, 1 rnm edta) and centrifuged at 75,000 g for 3 hr at 4'. the resulting virus pellets from the two tubes were resuspended in 10 ml of tne buffer, layered onto a 20-ml 20-55% linear sucrose gradient prepared with tne buffer, and centrifuged at 75,000 g for 18 hr at 4". the gradient was fractionated (22 samples) and the radioactivity present in each fraction determined directly by liquid scintillation. three adjacent fractions which contained most (70%) of the radioactivity were pooled, diluted three times in tne buffer, and applied to a second density gradient consisting of 20 ml of 1 o-50% renografin (meglumine diatrizoate, mw 809.1, sigma) prepared with tne buffer. this gradient was centrifuged and fractionated as before. aliquots of 13 fractions starting from the bottom of the gradient were analyzed by sds-polyacrylamide gel electrophoresis (page) (fig. 1 a) . prominent radiolabeled bands corresponding to the known structural proteins of ibv (6, 24) were observed in fractions 7-l 0 (lanes 1 o-l 4). the sedimentation rate of these proteins corresponded with that reported originally for ibv virions by stern et al. (24) whose basic protocol was used for the purification carried out here. we also observed that a polypeptide of approximately 12k, with an electrophoretic mobility similar to that of in vitrotranslated 3c (23) cofractionated exactly on the gradient with the virus major structural proteins, suggesting that it was associated with virus particles. polypeptides corresponding to in vitro-translated 5a, 5b, 3a, and 3b (17, 18) however, were not detectable. a radiolabeled protein with an apparent molecular weight of 44k, appearing in fractions l-6 (lanes 4-9), probably represents cellular actin as previously reported (19) . to confirm further the association of the putative 12k polypeptide with virions, fractions 7, 8, and 9 (lanes 10, 11, and 12) which contained most of the radioactive virions, were diluted threefold with tne buffer, pooled, and fractionated on a second 20-55% linear sucrose gradient under the same conditions as before. twenty-two fractions were collected, of which 16 (starting from the bottom of the gradient) were analyzed by sds-page ( fig. 1 b) . once again the 12k polypeptide was clearly present and cofractionated exactly with the major virion structural proteins, appearing mostly in fractions 5-9 (lanes 6-l 0). there was considerably less evidence of contaminating cellular proteins in this gradient, and the relative proportion of the 12k protein to the other virion proteins appeared similar to that observed at the previous stage in purification, strongly supporting the idea of a specific association between the 12k protein and virus particles. a single band migrating at the position expected of one of the cleaved portions of the spike protein, gp90, was observed in the upper part of the gradient, which may represent the degraded product of the spikes; morphological examination of unlabeled equivalent preparations at this stage by electron microscopy after negative staining showed that a quarter of the virions had lost their spike structures (data not shown). in order to establish the identity of the 12k polypeptide as the product of the 3c gene, and to examine the possibility that ibv virions might contain other new virus polypeptides in amounts too small to detect directly, we next carried out immunoprecipitation experiments using monospecific antisera directed against the predicted products of the 3a, 3b, 3c, 5a, and 5b orfs (17, 18, 23) . for this purpose, fractions 5, 6, and 7 from the second sucrose gradient (lanes 6, 7, and 8, fig. 1 b) were pooled, diluted three times in tne buffer, and centrifuged at 65,000 g for 3 hr at 4' to pellet the virus. the virus pellet was then resuspended in ripa buffer (50 mll/l tris-hci, ph 7.5, 150 mm naci, 1% np-40, 0.1% sds, 10% sodium deoxycholate) and aliquots were immunoprecipitated with a series of specific and control antisera as described (5) . the result of this experiment (fig. 2a) showed clearly that the 12k polypeptide was immunoprecipitated by anti-3c antiserum, but not by any of the other antisera. further evidence for the specificity of the association between 3c and purified virions comes from analysis of the relative amount of the 12k protein in virus-infected cells before virion assembly (fig. 2b) ; in this case the 3c protein is completely undetectable except by immunoprecipitation. thus the likelihood of the 3c protein appearing in virions by chance from contaminating cellular material which has survived three cycles of gradient purification is remote. these experiments indicate therefore that 3c is indeed a virion-associated polypeptide. no evidence was obtained, however, for the presence of the other small virus polypeptides. in order to assess the approximate amount of the 3c polypeptide present in the purified virion preparation, the autoradiograph shown in fig. 2a , lane "total v", was analyzed by scanning densitometry (fig. 2c) , and the relative peak areas for the virion proteins np, m, s, and 3c were measured. using these data, and taking account of the methionine content of these proteins (3c has only one aside from its initiator, and the s, np, and m proteins have 17, 4, and 4, respectively), we calculated the relative molar proportion of the four proteins to be approximately 1 .o: 1 1 .o: 10.1 :1.7 (s:np:m:3c). these relative values for the major structural proteins are broadly similar to those reported previously for ibv (6, 7) except that the proportion of envelope proteins to np is rather lower. this calculation also assumes that 2. (a) lmmunoprecipitation of radiolabeled virion proteins with specific antisera. virions were recovered from fractions 5, 6, and 7 of the sucrose density gradient shown in fig. 1 b as described in the text and immunoprecipitated with different antisera as indicated above each track. the monospecific test antisera were prepared as described elsewhere (17, 18, 23) and control anti-whole ibv serum was a gift from dr. d. cavanagh (afrc institute for animal health, houghton laboratory, uk). plbt3-radiolabeled 3a, b, and c polypeptide markers were prepared by in vitro transcription and translation as described for fig. 1 a. total 'v'unprecipitated virions. polypeptides were separated on a 22% polyacryamide gel and detected by fluorography. lanes containing anti-3a, b, and c and anti-5a and b antisera were exposed for 10 days, and other lanes for 2 days. (b) synthesis of 3c protein in ibv-infected cells. ck cells were mock-infected or infected with ibv and labeled with [?slmethionine from 6 to 7.5 hr p.i. as described before (23) . cells were harvested immediately after labeling and analyzed on a 22% sds-polyacrylamide gel the initiator methionine is not removed from any of the proteins; if one were to assume that cleavage does occur, the calculated relative proportion of 3c would increase. figures are not available for the number of molecules of each structural protein present in ibv virions, but using those reported for human coronavirus oc43 as a guide (np-726; m-726; s-88) (12) , we suggest that each virion may contain in excess of 100 molecules of 3c, as many or perhaps even more than the number of spike proteins. this relatively large proportion of 3c further supports the idea that the 3c protein is a genuine virion structural protein which may play an important role in virion assembly or infectivity. since the product of the 3c orf is known to be associated with the membranes of infected cells (23) it seemed likely that it could be located in thevirion envelope. to test this possibility, a sample of purified radiolabeled virions purified by sucrose and then renografin density gradient sedimentations as described above was pelleted bycentrifugation and disrupted according to the procedure of sturman et al. (27) by treatment with 1 ml of tne buffer containing 0.25% np-40. the ribonucleocapsid (rnp) and envelope components of the virus were then separated by centrifugation, at 180,000 g for 18 hr at 4', through a lo-ml lo-55% linear sucrose gradient containing 0.1% np-40, layered on top of a l-ml cushion of 75% renografin also containing 0.1% np-40. eighteen fractions were collected from the gradient (of which the first two correspond to the renografin cushion), and samples were analyzed by sds-page (fig. 3) . it can be seen clearly that the major virion envelope proteins sl , s2, and m cofractionate with each other, appearing maximally in fractions 15-l 8, but are well separated from the nucleocapsid protein (fractions l-l 0), indicating successful separation of the virion components. it is also evident that the 12k virion-associated protein cofractionates with the spike and membrane proteins rather than the nucleocapsids, suggesting strongly that it is indeed associated with the virion envelope. lmmunoprecipitation of the pooled rnp and envelope fractions confirmed that the 12k protein present in the envelope fraction was indeed 3c and further indicated the absence of the protein in the rnp fraction (data not shown). the function of the 3c protein is not yet clear. however, we are intrigued by an overall similarity in organization between 3c and the influenza avirus m2 protein (13, 74) . the ibv 3c protein is of a similar size and, like m2, has a stretch of hydrophobic amino acids near its n-terminus which could span the membrane. by analogy with m2, which is known to be an integral membrane protein present in small amounts in the virion (75, 31) this would leave the n-terminal 1 1 amino acids exposed on the external surface of the membrane with an internal c-terminal tail of 76 residues. recently it has been proposed that the m2 protein forms an ion channel (28) which selves to allow both uncoating of incoming virions and proper assembly of progeny virus, and this hypothesis is consistent with the observation that the m2 protein is the target for the action of the anti-viral agent amantadine (10) . m2 appears to be present in the membrane as a homotetramer composed of two disulfide-linked dimers held together by noncovalent interactions, which provides a structural basis for its proposed function in proton translocation (28) . comparison of the predicted amino acid sequence of 3c for five strains of ibv (17) indicates that the hydrophobic character of the proposed transmembrane region near the n-terminus of the protein is preserved, although the actual amino acid sequences show slight differences. there are also two conserved cysteine residues 45 and 46 amino acids from the nterminus of the predicted sequence of the 3c products in all five strains examined, which might serve the same function as cysl7 and cysl9 of the m2 protein. it will be of considerable interest to explore this possible parallel further. are 3c-like proteins a general feature of coronaviruses? on the basis of currently available information, this seems likely. messenger rna 4 of mouse hepatitis virus (mhv) is known to encode a 15k polypeptide, the predicted amino acid sequence of which contains a highly hydrophobic region from residues 8 to 41 (9, 22) , although the protein has not so far been found in virions. wesley and woods (29) have identified a 17k polypeptide in transmissible gastroenteritis virus (tgev)-infected cells, which could be a minor viral structural protein. intriguingly, sequence data deduced from the genomic rna of several species of coronaviruses have indicated that the orf immediately upstream of the membrane protein gene, in the equivalent position to that of 3c, has the potential to encode a hydrophobic protein with similar characteristics. for example the second orf (5b) of mhv mrna 5 can encode a 10.2k protein with a hydrophobic amino terminal region (16, directly or after immunoprecipitation with anti-3c antiserum. plbt3-radiolabeled 3a, b, and c polypeptide markers were prepared by in vitro transcription and translation as described for fig. 1 22 ) and the corresponding genetic location on the tgev genome has the potential to encode a 1 ok polypeptide with striking similarity to 3c (30) . furthermore, the bovine coronavirus genome at this point contains an orf which could encode a 9.5k hydrophobic protein (1). comparison of the predicted amino acid sequences of these four proteins (fig. 4 ) reveals some interesting common features. first, the amino acids flanking the proposed transmembrane region of the four proteins are negatively charged at the n-terminus and positively charged at the c-terminus. this arrangement could allow them to adopt the orientation of class iii integral membrane proteins (i i), i.e., with the n-terminus on the outside. it has also been observed that the predicted molecular weight of the 3c protein of ibv, the 5b protein of mhv, and the 9.5k protein of bcv is consistent with their migration rate in sds-page (i, 16, 17, 22) suggesting that these proteins contain uncleaved signal peptides; this is again consistent with the possibility that they are, like the m2 protein of influenza a virus, class iii integral membrane proteins (11, 20) . a further common feature is the occurrence of a cysteine residue 34 or 35 nt downstream of the charged amino acid at the n-terminal end of the proposed transmembrane region, which may play a role, as described above for the influenza m2 protein, in forming disulfide linkages. thus it may be that 3c-like proteins are conserved across the coronaviridae and may play a similar role in viral replication. proc. nat/. acad. sci. usa ce// 40 key: cord-269193-a647hwu9 authors: lin, debby a.; roychoudhury, sonali; palese, peter; clay, william c.; fuller, frederick j. title: evolutionary relatedness of the predicted gene product of rna segment 2 of the tick-borne dhori virus and the pb1 polymerase gene of influenza viruses date: 1991-05-31 journal: virology doi: 10.1016/0042-6822(91)90641-n sha: doc_id: 269193 cord_uid: a647hwu9 abstract the complete nucleotide sequence of the second largest rna segment of dhori/india/1313/61 virus was determined and the deduced amino acid sequence was compared with the polymerase (p) proteins of influenza a, b, and c viruses. rna segment 2 (2224 nucleotides) of dhori virus contains a single long open reading frame that can encode a 716-amino amid polypeptide (81.3 kda). the predicted polypeptide shares between 27 and 31% sequence identities with the pb1 polypeptides of influenza a, b, and c viruses. among the regions most highly conserved are the sequences around the asp-asp motif common to many rna polymerases. in spite of the high level of sequence identity between the dhori rna segment 2 gene product and the influenza a, b, and c virus pb1 proteins the amino acid composition of the dhori protein indicates an acidic charge feature at ph 7.0 in contrast to the basic nature of the pb1 proteins of the influenza viruses. we suggest that the dhori pb1-like protein be designated the pα protein of this virus. members of the dhori virus serogroup are as yet unclassified tick transmitted viruses that share structural and genetic properties with the orthomyxoviridae (clerx et al., 1983) . dhori viruses have been isolated from a variety of tick and vertebrate species (anderson and casals, 1973; filipe and casals, 1979; karabat-sos, 1985; williams et a/., 1973) . the genome of the virus consists of seven unique segments of singlestranded rna with a total size of approximately 1 1.9 kb. the viral rnas have been shown to encode information in the negative-sense (clerx et al., 1983; fuller et al., 1987; freedman-faulstich and fuller, 1990) and it was previously shown that the dhori nucleoprotein (encoded by rna segment 5) shares conserved amino acid sequences with the influenza a, b, and c virus nucleoproteins (fuller et al., 1987) . the sequence of rna segment 4 encodes the single envelope protein of dhori virus (freedman-faulstich and fuller, 1990) . the envelope protein does not share sequence identity with any orthomyxovirus envelope protein. we have also determined the nucleotide sequence of the dhori segment 6 rna and this segment most likely encodes the viral matrix protein (unpublished data). the 3'and 5' nontranslated ends of rna segments 4, 5, and 6 are very similar to the conserved ends of the genes of the recognized members of the orthomyxoviridae (clerx et ' to whom correspondence and requests for reprints should be addressed. a/., fuller et a/., 1987; freedman-faulstich and fuller, 1990) and segment 3 of thogoto virus (staunton et al., 1989) . a second group of tick-transmitted viruses, the thogoto viruses, possess a segmented genome with six or seven segments of single stranded rna (clerx et al., 1983; staunton et al., 1989) . thogoto virus has been shown to replicate in rhipicephalus appendiculatus ticks and to be transmitted to laboratory animals (davies et a/., 1986) . the sequence of the third largest segment of thogoto/siar/l26/72 virus was recently determined (staunton et al., 1989) unique in having a net negative charge unlike the net positive charge of the corresponding a, b, and c virus proteins. despite these differences the data reported here establish that the influenza viruses and dhori virus share a common evolutionary ancestor. dhori/lndia/l313/61 virus was grown in green monkey kidney (gmk-vero) cells as previously described (clerx et a/., 1983) . virus purification and rna extraction have also been described previously (clerx et a/., 1983) . dhori virus-infected cellular rna was isolated from vero cells at 48 hr postinfection, phenol extracted, ethanol precipitated, and purified by oligo(dt)-cellulose chromatography as previously described (collins eta/., 1982; fuller er a/., 1983). cloning of virus-specific dna and identification of clones the dhori/lndia/l3 13/6 1 virus strain was used to infect gmk-vero cells. infected cell mrna was reverse transcribed with an oligo(dt),,m,s primer. second-strand cdna synthesis was performed by the method of gubler and hoffman (1983) . the blunt-ended double-stranded cdnas were ligated to pbr322 which had been cut by pvull and dephosphorylated. clones bearing dhori virus specific inserts were identified by colony hybridization using radiolabeled cdna which had been obtained using viral rna as template and the oligonucleotide 5' agcaa(a/t)aacaagcagt 3' as primer. this oligonucleotide is complementary to sequences common to the 3' ends of the seven viral rna segments (clerx et a/., 1983) . virus-specific clones were organized by cross-hybridization into several pools. viral rna was extracted from cesium chloride gradient (12-420/o (w/w)) purified dhori virus and resolved on a 1.5% agarose gel containing 10 mm methylmercuric hydroxide. the viral rnas were transferred from the agarose gel to biotrans nylon membranes (icn inc., irvine, ca) and hybridized with nick-translated plasmid dna. both strands of insert dna of pd50-40 were directly sequenced using specific synthetic oligonucleotide primers and the modified t7 dna polymerase sequenase (united states biochemical corp.). all oligonucleotides were synthesized using an applied biosystems 380a synthesizer (applied biosystems, foster city, ca). for plus-strand as well as for minus-sense sequencing specific primers were synthesized to permit the reading of new sequences over a distance of 150-200 nucleotides. in order to obtain sequences located at the 3' end terminus of the rna segment 2, polymerase chain reactions were performed using purified viral rna and the following oligonucleotides, yagcaa(a/t)aacaagcagt 3' and 5'atctctgtg-gaagccac 3'. the sequence located at the 5' end terminus of the rna segment 2 was also obtained by polymerase chain reaction performed on purified viral rna and the following oligonucleotides, 5'agtagaq a)atcaaagca 3' and 5'acctcttttgttgaag 3'. the resulting dsdnas were cloned into ml 3mpl8rf and directly sequenced using universal primer. an analysis of the dhori virus genome was attempted by dna cloning of the poly(a)-containing rna of virus-infected gmk-vero cells. virus-specific cdna clones were obtained and by cross-hybridization were grouped into seven pools. nick-translated dnas of plasmids pd21-39, pd50-40, pd43-22, pd68-30, pd22-39, pd26-16, and pd12a-35 hybridized to rnas 1, 2, 3, 4, 5, 6, and 7, respectively in a northern blot analysis of dhori virus rna (fig. 1) 35, contains a small insert of segment 1 as well as an belonging to the group that hybridized to rna seginsert for segment 7 (unpublished results). plasmid ment 2. pd50-40 was selected for further study since it had the the insert of pd50-40 was then sequenced using largest insert among a total of nine different clones specific synthetic oligonucleotide primers and the mod(yamashita et a/., 1989) . 'alignment score (above diagonal) determined by the program pcompare (intelligenetics) using the unitaty matrix, a bias of 0, a gap penalty of 3, and 100 random runs. a score of 5 or greater is considered significant (dayhoff, 1983) . ' percent amino acid identity (below diagonal) based on an alignment of the two sequences by the method of needleman and wunsch (1970) . ified t7 dna polymerase-sequenase. sequences of the 3'-and 5'-terminal regions of the rna segment 2 absent from the insert in pd50-40 were determined by polymerase chain reaction amplification, cloning of the amplified dnas into m 13mpl8rf, and subsequent sequencing of the inserts. the complete sequence of segment 2 of dhori/lndia/l313/61 virus is shown in the plus sense in fig. 2 . the rna is 2224 nucleotides in length and the cdna insert of pd50-40 only lacks 25 nucleotides at the 5' end (in the plus-sense orientation). the rna encodes a single long open reading frame that could be initiated by an aug codon at nucleotides 25 through 27. no other reading frame is open in the antigenome sense for longer than 64 amino acids and the longest open reading frame in the genome sense is 109 amino acids. we do not know if these smaller reading frames can be expressed but we have not detected any subgenomic messenger rnas derived from rna segment 2 (unpublished data). the aug codon at nucleotides 25 through 27 is in a strong context for protein synthesis initiation (g at -3, a at +4; kozak, 1986) . a 716amino acid polypeptide is predicted from the largest open reading frame and it is terminated with a single uag codon at nucleotides 2173 through 2 175. a search of the nbrf (national biomedical research foundation) protein database using the fastp program (lipman and pearson, 1985) indicated that the predicted gene product of dhori segment 2 was most closely related to the pbl proteins of the influenza virus group. the pbl polymerase proteins are the most highly conserved among the proteins of the influenza a, b, and c viruses (yamashita et a/,, 1989 ) and they are most likely required for nucleotide addition during viral rna synthesis (braam et al., 1983) . a pairwise comparison of the amino acid sequence identities of the dhori virus protein and the influenza a, b, and c virus pbl proteins are shown in table 1 . the optimized alignments were made by the method of needleman and wunsch (1970) and indicate that the dhori pbllike protein shares sequence identities of 30.7, 26.9, and 29.8% with the pbl proteins of influenza aiprf8/ 34, influenza b/ann arbor/i/66, and influenza c/jj/50 virus, respectively (table 1 ). in addition, we compared the sequence of the dhori pbl-like protein to the pbl proteins of influenza a, b, and c viruses using the pcompare (intelligenetics) program. this program determines the probability that the similarity between two sequences could occur by chance. the score is expressed as the number of standard deviation units by which the maximum score for the real sequences exceeds the average maximum score for 100 randomizations of the sequence. an alignment score greater than 5 (probability of similarity occurring by chance < 3 x 10p7) implies an evolutionary relationship between proteins (dayhoff et al., 1983) . pairwise comparisons (see table 1 above diagonal) between dhori and influenza pbl proteins yield alignment scores of 25-26 which clearly indicate an evolutionary relationship between the dhori virus pbl-like protein and the influenza pbl proteins. a similar comparison between the dhori segment 2 gene product and the influenza a/pr/ 8/34 pb2 protein and pa protein gave alignment scores of 0.25 and 0.015, respectively. comparative analysis of the deduced amino acid sequences revealed highly conserved regions in the pbl proteins of influenza a, b, and c viruses (kawaoka et al., 1989; yamashita et al., 1989) . although the functional domains of the pbl proteins of influenza viruses have not been determined, the conserved regions may be important for basic functions of the pbl proteins, such as initiation and chain elongation (braam et al., 1983) . it has also been observed that there are four highly conserved motifs among rna-dependent rna polymerases (plus-or minus-strand virus polymerases) as described by poch et a/. (1989) and the dhori pa protein shares all of these motifs (fig. 3) . a comparison of the dhori virus protein with the pbl proteins shown in fig. 3 also reveals regions of highly conserved sequences. a 15-residue sequence motif (a double aspartic acid core flanked by hydrophobic residues) is found in many dna and rna polymerases (argos, 1988 (yamashita et al., 1990) with the pcu protein of dhori/lndia/l313/61 virus. the alignment was obtained by a compilation of the pairwise comparisons by the method of needleman and wunsch (1970) . an asterisk (*) denotes identity with the amino acid of the dhori pa protein at that position and a dash (-) indicates a gap introduced in the sequence for optimal pairing. the four domains designated by the lines above and below the sequence represent those highly conserved regions defined in rna polymerases by poch et a/. (1989) . b, and c shares this motif (w/fwd/tglqssddfa/vli/ fv/a), at residues 435/438 to 449/452 (domain 3, fig. 3 ). in the dhori virus protein, we found a similar sequence at residues 419 to 433 (tgdhvessddfihff). all rules of this asp-asp polymerase motif are fulfilled except for position 7 which contains a serine instead of the traditional gly, met, cys, val, or leu residue (ar-gos, 1988). thus it is likely that the dhori virus protein also possesses a polymerase function similar to that of the pbl proteins of influenza viruses. the influenza virus pbl protein is transported to the nucleus independently of other viral proteins (smith et al., 1987; akkina et al., 1987) . nath and nayak (1990) have recently shown that the nuclear localization sig-nal of influenza virus (a/wsn/33) pbl protein was located within residues 180-252 of pbl the study suggested that two discontinuous regions of the pb 1 both containing a stretch of basic amino acids were required for its nuclear localization. homologous sequences, however, were not found in the dhori virus protein. a comparison of some of the physical properties of the dhori and influenza pbl proteins indicates that the dhori protein is the smallest in this group (716 amino acids; m, 81,292) and unlike the influenza pbl proteins which have a strong positive charge at neutral ph (+23-a/pr/8/34; +12-b/aa/i/66; +14-c/u/50) the dhori protein is slightly acidic with a charge of -10 (peptidesort; devereux et al., 1984) . since the nomenclature of the influenza virus polymerase proteins is based on their charge characteristics a different name for the related protein of dhori virus may be appropriate and we thus suggest the designation pa protein. this nomenclature is proposed for the designation of the dhori polymerase proteins [pa-(pbl), pp-(pb2), and pr-(pa)] since the charge characteristics do not appear to be constant across virus groups. for example, the pa-like polymerase protein of influenza c/jj/50 virus (yamashita eta/., 1989 ) encodes a weakly basic protein designated p3 (segment 3 gene product). the third largest rna segment of thogoto virus encodes a pa-like protein with only a very weak negative charge (-2.5 at ph 7.0; staunton et al., 1989) . genes of other viruses also share sequence identity with influenza virus segments. it has been previously demonstrated that some members of the coronavirus group and influenza c virus have a hemagglutinin-esterase (he) glycoprotein that recognizes the same nacetyl-9-0-acetylneuraminic acid receptors on cells (vlasak et a/., 1988) . the influenza c virus he glycoprotein shares an approximately 30% amino acid sequence identity with a coronavirus he glycoprotein present in bovine coronavirus (kienzle et al., 1990 ) and mouse hepatitis virus (luytjes et a/., 1988) . this example of significant identity between a plus-strand rna and a minus-strand rna virus supports the concept of a cassette model of evolution for the he gene and is most likely an example of a recombinational event. in contrast, we feel that the relationship of the dhori virus group to the orthomyxovirus group is most likely an example of divergent evolution in light of the similarities in genome structure and common replication strategies and of the observation that several dhori virus gene products share significant identity with orthomyxovirus genes. intracellular localization of the viral polymerase proteins in cells infected with influenza virus and cells expressing pbl protein from cloned cdna. 1. v;ro dhori virus a sequence motif in many polymerases molecular model of a eukaryotic transcription complex: function and movements of influenza p proteins during capped rna primed transcription tick-borne viruses structurally similar to orthomyxoviruses experimental studies on the transmission cycle of thogoto virus, a candidate orthomyxovirus establishing homologies in protein sequences sequence comparison of wildtype and cold-adapted b/ann arbor/l/66 influenza virus genes a comprehensive set of sequence analysis programs for the vax antibodies to congo-crimean haemorrhagic fever, dhori, thogoto and bhanja viruses in southern portugal isolation of dhori virus from hyalemma marginatum ticks in portugal nucleotide sequence of the tick-borne complete nucleotide sequence of the tick-borne, orthomyxo-like dhori/lndia/l313/61 virus nucleoprotein gene a simple and very efficient method for generating cdna libraries international catalogue of arboviruses including certain other viruses of vertebrates avian-to-human transmission of the pbl gene of influenza a viruses in the 1957 and 1968 pandemics structure and orientation of expressed bovine coronavirus hemagglutinin-esterase protein point mutations define a sequence flanking the aug initiation codon that modulates translation by eukaryotic ribosomes rapid and sensitive protein similarity searches sequence of mouse hepatitis virus a59 mrna 2: indications for rna recombination between coronaviruses and influenza c virus function of two discrete regions is required for nuclear localization of polymerase basic protein 1 of a/wsn/33 influenza virus (hl nl) a general method applicable to the search for similarities in the amino acid sequence of two proteins identification of four conserved motifs among the rna-dependent polymerase encoding elements synthesis and cellular location of the ten influenza polypeptides individually expressed by recombinant vaccinia viruses sequence analyses of thogoto viral rna segment 3: evidence for a distant relationship between an arbovirus and members of the orthomyxoviridae human and bovine coronaviruses recognize sialic acid containing receptors similar to those of influenza c viruses isolation of wanowrie, thogoto, and dhori viruses from hyalomma ticks infecting camels in egypt nucleotide sequence of human influenza a/pri8/34 segment 2 comparison of the three large polymerase proteins of influenza a, b, and c viruses this work was supported by national institutes of health grants al-l 8998 (p-p.), al-l 1823 (p.p.) and al-20939 (f.f.). this work was in partial fulfillment of a westinghouse project (d.l.). key: cord-260108-osg8q89i authors: park, yon mi; kim, jeong-hoon; gu, se hun; lee, sook young; lee, min-goo; kang, yoon kyoo; kang, sung-ho; kim, hak jun; song, jin-won title: full genome analysis of a novel adenovirus from the south polar skua (catharacta maccormicki) in antarctica date: 2012-01-05 journal: virology doi: 10.1016/j.virol.2011.10.008 sha: doc_id: 260108 cord_uid: osg8q89i adenoviruses have been identified in humans and a wide range of vertebrate animals, but not previously from the polar region. here, we report the entire 26,340-bp genome of a novel adenovirus, detected by pcr, in tissues of six of nine south polar skuas (catharacta maccormicki), collected in lake king sejong, king george island, antarctica, from 2007 to 2009. the dna polymerase, penton base, hexon and fiber genes of the south polar skua adenovirus (spsadv) exhibited 68.3%, 75.4%, 74.9% and 48.0% nucleotide sequence similarity with their counterparts in turkey hemorrhagic enteritis virus. phylogenetic analysis based on the entire genome revealed that spsadv belonged to the genus siadenovirus, family adenoviridae. this is the first evidence of a novel adenovirus, spsadv, from a large polar seabird (family stercorariidae) in antarctica. adenoviruses have linear, non-segmented, double-stranded dna genomes, which range between 26 and 43 kb and are generally characteristic of each genus (davison et al., 2003; klempa et al., 2009; kovács and benkö, 2011; mase et al., 2009) . the family adenoviridae is comprised of five genera: mastadenovirus, aviadenovirus, atadenovirus, siadenovirus and ichtadenovirus (houng et al., 2006; kovács and benkö, 2011; kovács et al., 2010; lehmkuhl and hobbs, 2008; wellehan et al., 2004) , which infect a wide range of vertebrate species (davison et al., 2003; morrison et al., 1997) . mastadenovirus has been identified in mammals, including human, sea lion, canine, bovine, porcine, murine and bat (aggarwal and mittal, 2000; goldstein et al., 2011; klempa et al., 2009; kovács et al., 2004; li et al., 2010; morrison et al., 1997; rusvai et al., 2000) . aviadenovirus contains falcon and other fowl adenoviruses (davison et al., 2000) . atadenovirus has been found in snake, marsupial and ruminants (dan et al., 1998; farkas et al., 2008; thomson et al., 2002) . siadenovirus has been detected in frog, raptor and turkey (beach et al., 2009; davison and harrach, 2002; davison et al., 2000; kovács and benkö, 2009) . a new genus, ichtadenovirus, has been identified recently in fish (benkö et al., 2005) . typically, adenovirus infection in most species is characterized by enteritis and respiratory disease (beach et al., 2009; russell, 2009; rux and burnett, 2004; schrenzel et al., 2005) . however, other clinical manifestations have been observed. for example, turkey hemorrhagic enteritis virus (thev) causes inclusion body hepatitis, depression, splenomegaly, immunosuppression and death (beach et al., 2009; jucker et al., 1996; pitcovski et al., 1998) ; falcon adenovirus also causes hepatitis ; and agamid adenovirus infection can be subclinical or lethal (wellehan et al., 2004 ). an understanding of virus diversity in wildlife provides epidemiological and ecological information about potential pathogens and may lead to the identification of newly emerging microbial threats. a previous study reported that some antarctic avifauna is infected with various viruses, which may have been spread by antarctic birds (austin and webster, 1993; stannard et al., 1998) . the south polar skua (catharacta maccormicki, previously known as stercorarius maccormicki), which migrates for their breeding season (yogui and sericano, 2009) initially, the dna polymerase (pol) gene of a previously unknown adenovirus was amplified by pcr from the kidney of a south polar skua (sps t03). using newly designed primers based on the obtained sequence, the full genome of the novel adenovirus (spsadv) was extended from the left-end inverted terminal repeat (itr) region to the right-end itr region. the entire viral dna genome was 26,340 bp and encoded 24 adenoviral genes. the complete nucleotide sequence of spsadv was deposited in genbank (accession number hm585353). the g + c content was found to be 34.2%. the pol, penton and hexon genes of spsadv from four other skuas (sps t01, sps t02, sps t06 and sps t09) were also sequenced (table 2) , while from sps t08 only the penton gene was sequenced from the liver, suggesting lower virus concentration. nucleotide sequences of the pol, penton and/or hexon genes of spsadv identified in the six skuas were identical, with no evidence of polymorphism. since separate sterile instruments were used in collecting tissues from each animal under bsl-2 containment, it is unlikely that the identical sequences were due to cross contamination. the positions and coding directions of the 24 genes and open reading frames (orf) are shown in the schematic genome map (fig. 2) . the locations, as well as lengths of each gene (nucleotide and amino acid) and their g + c content, are indicated in table 3 . the itr regions were located on left and right ends. iva2, polymerase, protein precursor (ptp), dna binding protein (dbp), u exon and orf8 were transcribed leftward, and sialidase, orf4, 52k, piiia, iii (penton), pvii, px, pvi, hexon, protease, 100k, 22k, 33k, pviii, e3 region, fiber and orf7 were transcribed rightward. the length of the itr region differed depending on the adenovirus species. for example, the itr of thev (ay849321) was 40 bp, whereas that of hadv-1 (ac000017) was 103 bp. by contrast, the itr of spsadv was 31 bp, whereas the lengths of the 5′ and 3′ terminal ends were the same as those of other species. the sialidase of spsadv, located immediately downstream of the itr region, was composed of orf1, orf2 and orf3. in spsadv, orf4, located immediately next to the sialidase, was identified as hydrophobic, as in radv-1, thev and fradv-1. iva2, a delayed early gene located downstream of the polymerase gene, was slightly shorter than that in the avirulent turkey enteritis virus (1104 bp) and its g + c content of 29.69% was lower than that of other genes. the e2 region, containing the genes for dna polymerase, ptp and dbp, consisted of two cleavage sites. the penton base, encoding a major capsid protein of adenovirus, was located between the piiia and pvii. and the hexon gene, encoding a capsid protein with a penton base and a fiber knob, had a g + c content of 34.36%. the protease gene encoded one of the most conserved proteins among all adenovirus genes (russell, 2009; weber, 2007) . the length of the e3 gene was 891 bp, and the 1389-nucleotide fiber gene encoded a 462-amino acid capsid protein, which was located between the u exon and orf 7 (22,600-23,988) and transcribed in the rightward direction. orf7 and orf8 were genus specific, existing only in siadenovirus. the viral genome and phylogenetic analysis showed that spsadv belonged to genus siadenovirus in the family adenoviridae ( fig. 3) . at the nucleotide level, the spsadv pol, penton base and hexon genes exhibited somewhat higher sequence similarity of 73.8%, 79.2% and 77.5% with radv-1 than with thev (68.3%, 75.4% and 74.9%) ( table 4 ). the fiber gene of spsadv revealed only 57.2% and 48.0% nucleotide sequence similarity with that of radv-1 and thev, respectively. compared with other genera, the pol, penton base and hexon genes of spsadv shared b61% nucleotide sequence similarity with that of mastadenovirus, atadenovirus and aviadenovirus. the nucleotide and amino acid sequences of the pol, penton base, hexon and fiber genes showed nearly equi-distant differences between spsadv and other siadenoviruses. in fig. 4 , trees were based on the polymerase and hexon genes. only siadenovirus and aviadenovirus sequences were compared. these trees, which also included siadenoviruses from great tit, psittacine and sulawesi tortoise, showed that spsadv was most closely related to radv-1. only a limited number of viruses have hitherto been discovered among animals in the polar region. infectious bursal disease virus (ibdv) and poxvirus were detected in penguins (gauthier-clere et al., 2002; stannard et al., 1998) and serum antibodies to influenza a viruses and paramyxoviruses were reported in skua and adelie table 1 oligonucleotide primers for full genome amplification of south polar skua adenovirus. primer nucleotide sequence (5′-3′) p o l a r i t y (austin and webster, 1993) . in this study, viruses were targeted for discovery in antarctic birds. although no evidence of influenzavirus and coronavirus was found, a novel adenovirus was detected by pcr in the south polar skua, a predatory seabird species whose migratory route includes antarctica. based on genetic and phylogenetic analyses, the newly identified viral sequences from six south polar skuas could be classified as a novel siadenovirus. other members of the genus siadenovirus include thev (beach et al., 2009) , radv-1 (kovács and benkö, 2011) and great tit adenovirus (gtadv) (kovács et al., 2010) , all from avian hosts, as well as frog adenovirus 1 (fradv-1) (davison et al., 2000) , originating from an amphibian host. at first, we assumed that spsadv would belong to the aviadenovirus genus because the south polar skua is an antarctic bird. however, phylogenetic analysis revealed that spsadv was similar to radv-1 and thev (pitcovski et al., 1998) . comparison between spsadv and its closest relatives (radv-1 and thev) showed 21-43% and 25-52% nucleotide dissimilarity at the pol, penton base, hexon and fiber genes, and 15-56% and 25-71% amino acid difference, respectively. also, the nucleotide sequences of the pol, penton base, hexon and fiber genes of spsadv, compared with fradv-1, showed 34-53% dissimilarity. interestingly, although birds serve as host species of aviadenoviruses (jiang et al., 1999; oaks et al., 2005; schrenzel et al., 2005) , aviadenovirus encodes more distinct proteins than siadenovirus . the g + c content of the spsadv (34.2%) is similar with that of the other three siadenoviruses (radv-1: 38.5%, tadv-3: 34.9%, fradv-1: 37.9%). the pvii gene of spsadv also shows significantly higher g + c content (46.9%). the g + c content does not vary across the genome in a systematic fashion, and this may suggest that a recombination event between disparate viruses did not occur. designation of a novel siadenovirus species is predicated on more than 10% sequence dissimilarity at the nucleotide and amino acid levels and a previously unrecognized host species (benkö et al., , 2005 . based on these criteria, we conclude that spsadv represents a novel adenovirus species in the genus siadenovirus. recently, the entire genome of radv-1 was obtained by pcr without virus isolation (kovács and benkö, 2011) . thus, apart from thev, radv-1 and fradv-1, this is only the fourth complete viral genome sequence in the genus siadenovirus. partial siadenovirus genomes have also been reported from the great tit (kovács et al., 2010) , budgerigar (katoh et al., 2009) , psittacine and sulawesi tortoise (rivera et al., 2009) . siadenovirus from different avian, reptilian and amphibian host species share the same genome organization (kovács and benkö, 2009) , suggesting that the evolutionary history may have involved host-switching events (davison et al., 2003; kovács and benkö, 2009) . other than the genetic and phylogenetic features, an important consideration is the clinical signs of adenovirus infection. it is well known that thev infection is occasionally lethal and is characterized by depression, diarrhea, splenomegaly (beach et al., 2009) . because of the close phylogenetic relationship between thev and spsadv, we speculate that certain disease manifestations may be shared. that six of nine dead south polar skuas had evidence of spsadv genomic sequences, as determined by pcr in one or more organs (heart, lung, liver, kidney, intestine and/or trachea), suggests systemic or disseminated infection, presumably with viremia and clinically significant disease outcome. spsadv infection in the south polar skua may indicate acquisition and spread of infection as a result of stress from migration. future studies are warranted to ascertain the biology, epizootiology and pathogenic potential of this newfound polar-region siadenovirus. frozen carcasses of nine south polar skuas (sps t01-t09), without readily discernable signs of disease, were collected in lake king sejong near king sejong station (latitude 62°13′ s and longitude 58°47′ w) in antarctica (fig. 1) , when ambient temperatures ranged from −5.6°c to 2.1°c. tissue samples from various organs (heart, trachea, lung, esophagus, intestine, liver, kidney) were obtained using separate sterile instruments from each bird and stored at −70°c until used. autopsy was conducted in a bsl2 laboratory. total dna was extracted from blood and tissue samples using the high pure pcr template preparation kit (roche, indianapolis, in), according to the manufacturer's instructions. first and nested pcr were performed in a 50-μl reaction volume containing 1 μl of 10 mm dntp, 2 μl (10 pm) of each primer, 1 unit of super-therm taq polymerase (jmr holdings, london, uk) and 2.5 μl (400 ng) of template. primers used for pcr amplification and sequencing are provided in table 1 . initially, adenovirus sequences available in genbank were aligned using clustal w, megalign program. regions exhibiting high homology were then selected for designing oligonucleotide primers. for amplification of the full genome, specific primers were designed based on newly acquired sequences. initial denaturation at 95°c for 5 min was followed by six cycles each of denaturation at 94°c for 30 s, annealing at 37°c for 30 s and elongation at 72°c for 1 min, then 32 cycles of denaturation at 94°c for 30 s, annealing at 42°c for 30 s and elongation at 72°c for 1 min and finally 72°c for 5 min in a peltier ptc-200 thermal cycler (mj research, inc., watertown, ma). elongation time was altered between 1 and 3 min depending on the expected product size. pcr products were separated by electrophoresis in 1% agarose gels containing ethidium bromide. the right and left ends of the genome were determined by race pcr kit (takara, shiga, japan) and amplified by pcr using enzyme-tagged primer in table 1 (kovács and benkö, 2011) . amplicons were purified by using a pcr purification kit (qiagen, chatsworth, ca) and were sequenced with the big-dye® terminator kit version 3.1 (applied biosystems, foster city, ca) and abi 3730 automated sequencer (applied biosystems) after cloning into the pstblue-i vector (novagen, san diego, ca). the identity of the sequences was searched by blast (altschul et al., 1990) . sequences were edited with editseq programs in the lasergene 6 (dnastar) (www.dnastar.com) and aligned using clustal w (thompson et al., 1994) . phylogenetic trees were constructed, using maximum-likelihood (ml) and neighbor-joining (nj) algorithms implemented, rooted at the midpoint, in paup (swofford, 2003) based on the full length of amino acid sequences of polymerase, penton base and hexon. the nj trees using the partial nucleotide sequences of polymerase and hexon were performed for the analysis of partially characterized siadenoviruses including viruses from parrots, great tit and sulawest tortoises. an initial ml tree estimation was performed by modeltest 3.7 (posada and crandall, 1998) . topologies were evaluated by bootstrap analysis of 1000 nj and 100 ml replicates. the genetic distances were computed by the paup program. sequence analysis of porcine adenovirus type 3 e1 region, pix and piva2 genes, and two novel open reading frames basic local alignment search tool evidence of ortho-and paramyxoviruses in fauna from antarctica comparison of 12 turkey hemorrhagic enteritis virus isolates allows prediction of genetic factors affecting virulence family adenoviridae adenoviridae analysis of the hexon gene sequence of bovine adenovirus type 4 provides further support for a new adenovirus genus (atadenovirus) the springer index of viruses dna sequence of frog adenovirus genetic content and evolution of adenoviruses completion of the genome analysis of snake adenovirus type 1, a representative of the reptilian lineage within the novel genus atadenovirus serological survey of the king penguin, aptenodytes patagonicus, in crozet archipelago for antibodies to infectious bursal disease, influenza a and newcastle disease viruses isolation of a novel adenovirus from california sea lions zalophus californianus emergence of a new human adenovirus type 4 (ad4) genotype: identification of a novel inverted terminal repeated (itr) sequence from majority of ad4 isolates from us military recruits application of the polymerase chain reaction to detect fowl adenoviruses characterization of the haemorrhagic enteritis virus genome and the sequence of the putative penton base and core protein genes a novel budgerigar-adenovirus belonging to group ii avian adenovirus of siadenovirus a novel cardiotropic murine adenovirus representing a distinct species of mastadenoviruses confirmation of a novel siadenovirus species detected in raptors: partial sequence and phylogenetic analysis complete sequence of raptor adenovirus 1 confirms the characteristic genome organization of siadenoviruses analysis of the first complete genome sequence of an old world monkey adenovirus reveals a lineage distinct from the six human adenovirus species recognition and partial genome characterization by non-specific dna amplification and pcr of a new siadenovirus species in a sample originating from parus major, a great tit serologic and hexon phylogenetic analysis of ruminant adenoviruses host range, prevalence, and genetic diversity of adenoviruses in bats identification of group i -iii avian adenovirus by pcr coupled with direct sequencing of the hexon gene complete dna sequence of canine adenovirus type 1 isolation and epidemiology of falcon adenovirus the complete dna sequence and genome organization of the avian adenovirus, hemorrhagic enteritis virus modeltest: testing the model of dna substitution systemic adenovirus infection in sulawesi tortoises (indotestudo forsteni) caused by a novel siadenovirus adenoviruses: update on structure and function identification and sequence analysis of the core protein genes of bovine adenovirus 2 adenovirus structure. hum characterization of a new species of adenovirus in falcons evidence for incomplete replication of a penguin poxvirus in cells of mammalian origin paup*: phylogenetic analysis using parsimony (*and other methods), version 4 sinauer associates clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice molecular confirmation of an adenovirus in brushtail possums (trichosurus vulpecula) synthesis and assay of recombinant adenovirus protease detection and analysis of six lizard adenoviruses by consensus primer pcr provides further evidence of a reptilian origin for the atadenoviruses siadenovirus infection in two psittacine bird species levels and pattern of polybrominated diphenyl ethers in eggs of antarctic seabirds: endemic versus migratory species we thank dr. hae ji kang for phylogenetic analysis and dr. richard yanagihara for editorial assistance. this research was supported in part by a core research support for senior researchers, national research foundation of korea (2010-002-7564), kopri (grant number pe11030), and institute of biomedical science & food safety, korea university. key: cord-262245-eb7g9p1x authors: monica, nicola la; banner, lisa r.; morris, vincent l.; lai, michael m.c. title: localization of extensive deletions in the structural genes of two neurotropic variants of murine coronavirus jhm date: 1991-06-30 journal: virology doi: 10.1016/0042-6822(91)90635-o sha: doc_id: 262245 cord_uid: eb7g9p1x abstract the intracellular rna of two neurotropic variants of the jhm strain of mouse hepatitis virus (mhv) independently isolated from the brain and spinal cord of an infected wistar furth rat were compared with that of the parental virus. the mrnas corresponding to the genes encoding the peplomer (s) and the hemagglutinin-esterase (he) proteins of the variant viruses were found to be smaller in size. the possible sequence changes were studied by oligonucleotide fingerprinting and direct rna sequencing. both variants have a large deletion of 246 amino acids in the carboxy-terminal end of the he protein. however, this truncated protein was not detected in the infected cells, suggesting either a translational regulation or rapid degradation of the truncated protein in these cells. the variant virus isolated from the spinal cord has a second deletion of 147 amino acids in the amino-terminal half of the s protein. this deletion site corresponds to a hypervariable region where deletions have been frequently noted among mhv variants with different biological properties. these findings suggest that the changes in pathogenic properties of the two neural isolates are associated with drastic alterations of the viral structural glycoproteins. mouse hepatitis virus (mhv), a member of the coronaviridae, contains a nonsegmented positive-stranded rna genome of 31 kb (1) (2) (3) . the rna genome is enclosed in a helical nucleocapsid structure formed by association with nucleocapsid proteins (n). the virions are enveloped and contain three virus-specific glycoproteins of 180 (s), 65 (he), and 23 kda (m) (4, 5) . the s (spike) glycoprotein forms the projecting peplomers of the virus, contains neutralizing epitopes, binds target cells, and is required for viral infectivity (4) . m is a transmembrane protein (8) the function of which is not yet clear. the he glycoprotein contains an esterase activity (7, 8) and shares sequence and functional homology with the influenza c hemagglutinin (9) . its expression appears to be optional in murine coronaviruses. in bovine coronavirus (bcv), the he protein has been found to contribute to viral infectivity (10) . the structural proteins of mhv are translated from virus-specific subgenomic mrnas in the infected cells. these mrnas have a 3'-coterminal nested-set structure (i 1, 12) and only their 5'-end terminal unique regions, which include more than one open reading frame in some mrnas, are utilized for translation (13, 14) . the s protein is translated from mrna 3, he from mrna 2-1, which is synthesized only by certain strains of mhv (9, and m from mrna 6. the 5'-end of the genomic rna of mhv contains a leader sequence of ' to whom correspondence and reprint requests should be addressed. approximately 70 nucleotides (15, 18) , which is also present at the 5'-end of every subgenomic rnas. at the 3'-end of the leader rna, there are several repeats of a pentanucleotide sequence ucuaa, whose repeat number varies among different mhv strains (17). the number of ucuaa repeats appears to correlate with the efficiency of transcription of specific mhv genes. for instance, the jhm(3) strain of mhv, which contains three ucuaa repeats at the 3'-end of the leader rna, makes only a small quantity of he protein. in contrast, the jhm(2) strain with two ucuaa repeats makes a large quantity of this structural glycoprotein (5, 17) . among many strains of mhv, jhm is of particular interest because of its capacity to cause neurological diseases in murine species (18) (19) (20) . lntracerebral (ic) or intranasal inoculation of susceptible mice and rats with the jhm virus often results in acute, subacute, and chronic infections of the central nervous system (cns). the symptoms of these infections depend on the age of the animal, and range from encephalitis, associated with lesions in the gray matter of the cns, to demyelination, characterized by hindleg paralysis or paresis (18, 21, 22) . in view of the interesting pathogenic properties of the jhm virus, extensive studies have been undertaken to understand the genetic basis for mhv-induced neurological diseases. studies on monoclonal antibody-resistant mhv variants and on variants isolated from persistent infections have shown that mutations in the peplomer protein s are associated with marked variations in disease patterns of these virus isolates, suggesting that this structural gly-883 0042.6822/91 $3.00 copyright 0 1991 by academic press, inc. all rlghrs of reproduction i" any form reserved coprotein plays an important role in the establishment of coronavirus infection and pathogenesis (23, 24) . the m protein may also play a role in the development of viral disease since several monoclonal antibodies against this protein can alter the pathogenicity of the virus (25). the role of he protein in mhv pathogenesis has not been studied. recently, two jhm variant viruses at1 if have been isolated from the brain and the spinal cord, respectively, of a single wistar furth rat with a jhm-induced demyelinating disease. the virus isolated from the spinal cord was named at 11 f cord, while the other, at 11 f brain, was independently isolated from the brain of the same rat (26). the at1 if cord variant differs from the parental jhm virus in its pathogenic properties in that it induces a chronic demyelinating disease in wistar furth rats when inoculated intracerebrally, independently of their age. also, unlike the parental jhm virus, the at1 1 f cord variant forms massive syncytia and replicates extensively in the murine oligodendroglioma cell line g26-24. in contrast, the at1 if brain isolate produced an acute encephalitis when inoculated into 2and lo-day-old wistar furth rats. both the brain and the spinal cord isolates displayed a different pattern of virus-specific mrnas from the parental jhm virus in the infected cells (26). however, the brain isolate did not replicate as well as the at1 if cord variant in the g26-24 cell line and did not form syncytia (26). the parental jhm virus and the at1 if cord virus were plaque-purified three times and passaged less than ten times in l-2 cells. both the plaque-purified and unpurified stocks of the at1 1 f brain variant were used in this study. the unpurified at1 if brain variant used had been passaged in g26-24 and l-2 cells less than ten times, and had the same passage history as the one used previously for its biological characterization (26). to obtain a better understanding of the genetic changes that may contribute to the altered biological and pathogenic properties of the at1 1 f brain and cord isolates, we have begun to characterize the genomic structure of these viruses. in this paper, we have identified large deletions within the genes encoding the s and he proteins of these variant viruses. to examine the virus-specific rna patterns of these variants and the parental jhm strain, virus-infected dbt cells treated with actinomycin d (2.5 pg/ml) were labeled with [32p]orthophosphate between 5 and 9 hr p.i. at the end of this period, rna was extracted from the infected cells, denatured, and analyzed by gel electrophoresis according to the published method (27). because some mrnas of the parental jhm virus were synthesized only in a very small quantity, intracellular rna from another well-characterized mhv, jhm (2), was included for comparison. jhm (2) (2). the parental jhm and the neural isolates at1 if brain and cord. the viruses were grown in mouse astrocytoma cell line dbt (34) and labeled with [3zp]orthophosphate between 5 and 9 hr p.i. in the presence of actinomycin d (2.5 rg/ml). rna was then extracted from the infected cells, denatured with glyoxal and dmso, and analyzed by gel electrophoresis on a 1% agarose gel as described (27). this photograph is a composite of different time exposures of the same gel. a jhm strain maintained in laboratories in japan (17, 29). as shown in fig. 1 , all the virus strains examined synthesized seven to eight mrna species in varying quantity. the size of several of these mrnas appeared different; most notably, the mrna 3 of the at1 lf cord isolate was smaller than those of the parental jhm strain and of the brain isolate but similar to that of jhm (2), which has a deletion of 423 nucleotides in the s gene (28). in addition, the sizes of mrna 2 and mrna 2-1 of both neural isolates were smaller than those of the corresponding mrna synthesized by the jhm(2) strain; the parental jhm virus synthesizes only negligible quantity of mrna 2-l (see below). as expected from the nested-set structure of mhv mrnas, the mrna 2 of the parental jhm strain is larger than that of jhm(2) because of the presence of the deletion in gene 3 of jhm(2) (28). the amounts of mrna 2 in both neural isolates were low; however, a longer exposure clearly showed that mrna 2 of both neural isolates was smaller than that of the parental jhm strain (data not shown). this result suggests that there are internal deletions in genes 2-1, 3, and possibly also gene 2 of the neural jhm variants. however, these size differences could also be due to aberrant transcriptional initiation. furthermore, because mhv mrnas have a nested-set structure, the genetic alterations affecting the size of mrna 2 could be the result of deletions or insertions in the coding sequences of mrnas 2-1 or 3. to characterize the structure of mrna 3 of the at1 if cord isolate, we first determined by tl -oligonucleotide fingerprinting analysis whether the coding region and leader sequence of mrna 3 of this variant virus contained any missing or altered sequence. the oligonucleotide map showed that the cord isolate lacked oligonucleotide spots numbered 4, 9, and 19 , which have previously been mapped contiguously to the middle portion of this gene (27) (data not shown). this result suggests that the smaller size of mrna 3 of at1 if cord isolate is most likely caused by a deletion in the coding sequence of gene 3. the site of the deletion was determined by direct sequencing (28) of the purified genomic rna of both the parental jhm virus and the variant viruses. after identifying the approximate site of the deletion by preliminary sequencing, the region from nucleotides 842 to 1830 of gene 3 was completely sequenced. figure 2 shows that, when compared with the sequence of the parental jhm, gene 3 of the variant virus has a deletion of 441 bases extending from nucleotide 1324 to 1765. the reading frame downstream of the deletion remains unaltered. the rest of the gene 3 sequence is identical with that of the parental virus. therefore, the smaller size of mrna 3 of the at1 1 f cord variant is the consequence of a deletion within the coding sequence of gene 3. the loss of 441 nucleotides from base 1324 to 1765 of the gene 3 sequence results in a smaller peplomer protein lacking an internal 147 amino acids. we next examined the size variation of mrna 2-l. when compared with jhm(2), which makes a large quantity of mrna 2-l (5) both the brain and spinal cord isolates appeared to have a smaller mrna 2-l (fig. 1) . however, because of the nested-set structure of mhv mrnas, it could not be determined with certainty whether the coding region of gene 2-l had any deletion, since the size comparison of mrna 2-l was complicated by the size differences in mrna 3 between these viruses. we therefore performed pcr amplification of the gene 2-l of the parental jhm and the two variant viruses, using two specific primers corresponding to both ends of the gene. the results showed that the gene 2-l of the parental jhm has exactly the same size as that of the published jhm-2 ( is homologous to nucleotides 781-801 of gene 2 located 36 nucleotides upstream from the intergenic site of gene 2-l (5). after an initial denaturation at 94" for 2 min, 20 units of amv reverse transcriptase (seigakaku) were added and the samples were incubated at 45" for 30 min. the cdna products were then amplified by addition of 2.5 units of taql dna polymerase (perkin-elmer-cetus, norwalk, ct). each cycle of amplification consisted of a 1.5.min denaturation at 94", followed by 2-min annealing at 58", and 3-min extension at 72". after 35 cycles, the final product was extended for 10 min. 20-~1 aliquots of the pcr reaction were electrophoresed on a 1.4% agarose gel. trast, the pcr-amplified gene 2-1 of both variants are smaller than that of the parental jhm, suggesting that the gene 2-l of these two variants has a deletion. the sites of the deletion were determined by dna sequencing of the pcr products and also direct rna sequencing of the genomic rnas. the results showed that both variants contain a deletion of 739 nucleotides extending from nucleotide 555 to 1294 of gene 2-1, which translates into a deletion of 246 amino acids in the gene 2-l of both viruses (fig. 4) . furthermore, because the reading frame was changed after the deletion, the orf of this gene in both variant viruses has a capacity to encode a protein of only 182 amino acids. this truncated protein product would be expected to lack the carboxy-terminal transmembrane domain. the finding that the parental jhm did not synthesize an appreciable amount of mrna 2-1, while both variants do (fig. l) , is reminiscent of our previous finding with jhm (2) , in which there is a strong correlation between the amount of mrna 2-l and the number of ucuaa repeats in the leader rna (5) . therefore, the leader sequences of these viruses were determined. the results showed that the parental virus has three ucuaa repeats while the variants have two (data not shown). this result is in agreement with the previous findings concerning the transcriptional regulation of this gene (5, 17) . to determine the size of the proteins encoded by the structural genes of these variants, we examined the intracellular virus-specific proteins. infected cells were pulse labeled with 35s-translabel (amersham) for 15 min, and immunoprecipitated with various antibodies. figure 5a shows that the s protein of the at1 lf cord variant is smaller than that of the jhm parental virus, but similar in size to that of the jhm(2), which has a deletion of 153 amino acids (28). this result is consistent with the presence of a deletion in the s gene of the at1 if cord virus. figure 5a also shows that the jhm parental virus produced a small amount of he protein, which has the same size as that produced by the jhm(2)virus, indicating that the mrna 2-l of the parental jhm virus does not contain a deletion. in contrast, neither of the two neural variants produced this protein. the reason for the failure to detect the he protein in the variant viruses is not yet clear. this could be due to rapid degradation of the truncated proteins or the failure to initiate translation. we have also examined whether a truncated protein immunoprecipitable with anti-he antibodies could be detected in purified virions or infected cell media since the truncated protein is expected to lack a c-terminal transmembrane domain; no protein was detected (data not shown). all of the viruses examined synthesized a 30-kda polypeptide, which was precipitated by an antiserum prepared against the trpe-mhv fusion protein containing 1 18 amino acids of the predicted gene 2 product (fig. 5b) . thus, all of these viruses have an intact gene 2; the differing size of the mrna 2 in these viruses is due to deletions in the downstream genes. these results showed that the neural variants have undergone multiple changes from the parental virus. both of them have a large deletion of 739 nucleotides in the he gene, and the cord variant had an additional deletion of 441 nucleotides in the s gene. in addition, both of them have two ucuaa repeats at the 3'-end of the leader rna, resulting in the high expression of mrna 2-1, in contrast to the parental virus, which has three ucuaa repeats and, thus, a small amount of mrna 2-l. however, the truncated gene product (he) of mrna 2-l was not detected in cells infected by either of the variant viruses, while the truncated s protein was expressed by the at1 if cord variant (fig. 5) . it is possible that these changes have contributed to the changes of biological and pathogenic properties of these viruses. variation in the size of the s glycoprotein has been observed during both in viva and in vitro passages of mhv. the previously published jhm sequence (30) has been shown to lack 423 nucleotides in the same region of the s gene as the at1 1 f cord variant, although the exact deletion sites are slightly different (28, 31) . a59 virus and several other mhv isolates are also missing from 156 to 474 nucleotides in the same area (28). thus, this region appears to be prone to deletions fig. 5. lmmunoprecipitation of mhv-specific intracellular proteins. virus-infected cells were radiolabeled with 35s-translabel at 9 hr p.i. for 15 min. cell lysates were prepared as previously described (7) have reported similar deletions in the s protein of several jhm variants which had been selected for resistance to neutralization with monoclonal antibodies (23). these viruses also display altered pathogenicity in mice similar to that of the at1 if cord isolate. also, taguchi et al. have reported the isolation, from both the brain of lewis rats and primary rat neuronal cultures, of neurotropic jhm variants, which have a larger mrna 3 (32, 33). thus, the variation of the sequence in this region of the s gene may be related to the alteration of the pathogenicity of these viruses. the deletion observed in the he protein of mhv has not previously been described. we have reported the increased synthesis of the he glycoprotein after in vitro passages, which correlated with the decrease in the number of ucuaa repeats at the 3'-end of the leader (5). here we observed a similar variation of the ucuaa repeat number during the passage of the virus in the cns of rats. however, the he protein was not synthesized as a result of the deletions. it is unlikely that the observed deletions and increased mrna expression in the neural variants originated from the passaging of these viruses in tissue culture after isolation. in fact, no change in the biological and physical properties of the viruses has been observed during passage of these neural isolates in l-2 and g26-24 cells. furthermore, we have not detected any changes in the rna pattern and protein profile of the parental jhm virus after passaging under our culture conditions. it should be noted that the at1 1 f brain isolate initially used in this and a previous study (26) was not cloned because of technical reasons. however, the he sequence reported here was obtained by direct sequencing of genomic rna, suggesting that the major population of this variant virus is characterized by a deletion in the he glycoprotein. we have now confirmed this deletion with several plaque-purified at1 if brain isolates (data not shown). furthermore, the fact that both the at1 if brain and cord variants have an identical he sequence suggests that this he deletion was selected early during jhm infection of the rat (26). the biological significance of this deletion in gene 2-l is more difficult to assess since the gene in both variants probably is not expressed, and these viruses are characterized by strikingly different biological and pathogenic properties. however, the variation in the expression of he glycoprotein has been associated with the changes in the pathogenic properties of the virus. the jhm(2) virus, which expresses a larger amount of the he protein, is more neurovirulent than, but does not replicate as well in glial cells as the jhm (3) virus, which synthesizes a small amount of he (yokomori eta/., unpublished data). thus, the amount of this structural glycoprotein may in some way influence the viral pathogenicity, depending on the genetic background of the virus. whether the truncated mrna 2-l of both the at1 if brain and cord variants is actually translated in viva is not clear at this time. the sequence analysis of the truncated 2-l gene suggests that the mrna 2-l should be functional. however, the failure to detect such a protein in infected cell lysates may be due to a short half-life of the protein product. alternatively, there is a possibility that the translation of this gene may be regulated by additional factors in the virus-infected cells. proc. nat/. acad. sci. usa we thank lee tan for helpful suggestions during the course of this project. we also thank kyoko yokomori for helping in the preparation of virus stocks, and daphne shimoda for editorial assistance. key: cord-258232-br4z3na6 authors: maeda, junko; maeda, akihiko; makino, shinji title: release of coronavirus e protein in membrane vesicles from virus-infected cells and e protein-expressing cells date: 1999-10-25 journal: virology doi: 10.1006/viro.1999.9955 sha: doc_id: 258232 cord_uid: br4z3na6 abstract coronavirus e protein is a small viral envelope protein that plays an essential role in coronavirus assembly; coexpression of coronavirus m and e proteins results in the production of virus-like particles. the present study demonstrated that mouse hepatitis virus (mhv) e protein was released as an integral membrane protein in lipid vesicles from e-protein-expressing mammalian cells, in the absence of other mhv proteins. furthermore, our data indicated that the e-protein-containing vesicles, which had a slightly lighter buoyant density than that of mhv, were released from mhv-infected cells. these data implied that e protein alone can drive the production and release of coronavirus envelope in the absence of m protein. coronavirus is an enveloped virus containing a large positive-stranded rna genome. the coronavirus envelope typically contains three virus-specific proteins: s, m, and e. the s protein forms 180-to 90-kda peplomers, binds receptors on coronavirus-susceptible cells, and induces cell fusion. the m protein, a transmembrane protein, is the most abundant glycoprotein in infected cells as well as in the virus particle. the m protein has three domains; these include a short n-terminal ectodomain, a triple-spanning transmembrane domain, and a c-terminal endodomain. the e protein, previously referred to as the small membrane protein or sm protein, is an acylated protein and is present only in minute amounts in infected cells as well as in the virus envelope. the structure inside the viral envelope is a helical nucleocapsid that consists of viral rna and n protein. each coronavirus-specific protein is translated from each of six to eight species of virus-specific mrnas that have a 3ј-coterminal nested set structure (1) . e protein is crucial to coronavirus assembly. coexpression of coronavirus m protein and e protein results in the production of virus-like particles (vlps), while expression of m protein alone does not produce vlps (2) (3) (4) . complementation studies using two defective interfering (di) rnas of a prototypic coronavirus, mouse hepatitis virus (mhv), showed that e and m proteins are both required for the production of mhv particles containing the viral nucleocapsid (5) . e protein also plays a role in coronavirus morphogenesis, because mhv mutants encoding mutated e protein are morphologically aberrant compared to wild-type mhv (6) . even though the importance of e protein in coronavirus assembly is known, more detailed characterization of its function has been limited because of its very low abundance in the virus and in infected cells. in infected cells e protein has been shown to be an integral membrane protein (3), yet its presence as an integral membrane protein in virus particles has not been demonstrated. the orientation of e protein in the membrane is also unknown. in the present study we demonstrated that e-proteincontaining membrane vesicles were released from eprotein-expressing cells. furthermore, our data indicated that the e-protein-containing membrane vesicles were released from mhv-infected cells. these data were unexpected, because m protein is also considered essential for coronavirus envelope formation. the data presented here suggest the possibility that e protein alone can drive the production and release of the coronavirus envelope in the absence of m protein. we used a sindbis virus expression vector, psinrep5 (7) (invitrogen, san diego, ca), to express mhv e protein and m protein at high levels in mammalian cells. this vector contains a promoter for subgenomic transcription and the sindbis virus nonstructural protein genes 1-4 that are required for replication of vector rnas. we constructed one sindbis vector that expressed mhv m protein (psinm) and another that expressed mhv e protein (psine) by respectively inserting gene 6 (encoding the m protein) from the jhm strain of mhv (mhv-jhm) and gene 5b (encoding the e protein) from the a59 strain of mhv (mhv-a59) downstream of the promoter for subgenomic transcription. sindbis vector expressing mhv-jhm e protein was also constructed. however, expression of e protein from this vector was significantly lower than that from psine (data not shown). for this reason, psine was used for subsequent studies. rna transcripts from these plasmids were synthesized in vitro and each rna was mixed with the sindbis helper transcript, dh(26s), which contains the genes for sindbis virus structural proteins (8) . when dh(26s) rna transcripts are cotransfected with the recombinant rna from psin-rep5, expression of the structural proteins in trans from dh(26s) rna transcripts allows the packaging of recombinant rna into virions (pseudovirions) (7) . because dh(26s) lacks a sindbis virus packaging signal, dh(26s) rna is not packaged into pseudovirions. a mixture of psinm rna transcripts and dh(26s) and another of psine rna transcripts and dh(26s) were independently transfected into bhk cells using electroporation (7) . as a control, dh(26s) and rna transcripts of psinrep/lacz (invitrogen) encoding the lacz gene were cotransfected. culture fluid was collected 30 h after transfection and then inoculated into dbt cells. cells were metabolically labeled with 50-100 ci of tran 35 slabel (icn) from 6.5 to 7 h postinoculation for labeling of m protein and from 5 to 7 h postinoculation for labeling of e protein. the cell lysates were prepared with lysis buffer [150 mm nacl, 50 mm tris-hcl (ph 7.5), 1% triton x-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 1 mm phenylmethylsulfonyl fluoride]. radioimmunoprecipitation analysis of cell extracts from the cells that were inoculated with the culture fluid from cells cotansfected with psinm transcripts and dh(26s) and from cells cotransfected with psine transcripts and dh(26s), respectively, showed excellent expression of m and e proteins (fig. 1) . mhv m protein, n protein, and an unknown protein (shown by an asterisk in fig. 1) were also coimmunoprecipitated by anti-e protein peptide-2 antibody (9) under these experimental conditions. anti-e protein antibody did not immunoprecipitate expressed mhv m and n proteins (unpublished data), eliminating the possibility that anti-e protein antibody cross-reacts with m and n proteins. to determine whether e protein is released from the e-protein-expressing cells, sine pseudovirion-infected dbt cells were radiolabeled with tran 35 s-label from 5 to 9 h p.i. as controls, dbt cells were infected with sinm pseudovirions alone or coinfected with sinm pseudovirions and sine pseudovirions. culture fluid was harvested at 9 h p.i. and briefly centrifuged to remove cell debris. culture media were applied onto a discontinuous sucrose gradient consisting of 20 and 60% sucrose in nte buffer [100 mm nacl, 10 mm tris-hcl (ph 7.5), and 1 mm edta]. after centrifugation at 26,000 rpm for 15 h at 4°c in a beckman sw28 rotor, the interface between 20 and 60% sucrose was collected and diluted threefold with nte buffer. the sample was further purified by the 20-60% continuous sucrose gradient centrifugation at 26,000 rpm for 15 h at 4°c in a beckman sw28 rotor. ten fractions were collected from the bottom of the gradient and an equal amount of 2ϫ lysis buffer was added to each fraction. m and e proteins in each of the sucrose fractions were immunoprecipitated with a mixture of anti-m protein monoclonal antibody j.2.7 (10) and anti-e protein peptide-2 antibody (9) . no m protein signal was detected in the culture fluid from sinm pseudovirioninfected dbt cells (data not shown). from the cells coexpressing m and e proteins, both m and e proteins were detected in three sucrose fractions ( fig. 2a) . these results were expected; expression of m protein alone does not produce vlps, while coexpression of m and e proteins produce vlps (2) (3) (4) . characterization of culture fluids from coexpressing cells showed that the distributions of m and e proteins in the gradient were not identical; the major m protein signal was in fractions 6 and 7, while the major e protein signal was in fractions 7 and 8; m protein signal in fractions 6 and 7 was about 100 times higher than that in fraction 8 and the e protein signal in fractions 7 and 8 was about 15 times higher than that in fraction 6. to our surprise, e protein was released into the culture supernatant from e-proteinexpressing cells (fig. 2b) . the released e protein sedimented to a sucrose density of about 1.13 g/cm 3 , which was slightly lighter than the major signal of m protein (density 1.16-1.14 g/cm 3 ) released from coinfected cells; the relative intensity of e protein signal in fractions 7-9 was 2, 4.3, and 1, respectively. the presence of e protein in culture fluid was not due to the release of an intracellular form of e protein as a result of cellular disruption, since no cytopathic effects were detected in sine pseudovirion-infected dbt cells at 9 h p.i. in coexpressing cells, we saw a slightly different distribution of e and m proteins in the sucrose gradient ( fig. 2a) , indicating that vlps, containing m and e proteins and free e protein, not associated with m protein, were released into the culture media. to know whether the release of e protein from e-protein-expressing cells was an artifact of the expression system, release of e protein from mhv-infected cells was examined. mhv-a59-infected cells were radiolabeled with tran 35 s-label from 5 to 9 h p.i. the culture fluid was collected and mhv was purified on a 20-60% continuous sucrose gradient under the same conditions as described above. mhv structural proteins in each fraction were immunoprecipitated with a mixture of anti-mhv serum, anti-m protein monoclonal antibody, and anti-e protein antibody (fig. 3a) . mhv s protein, n protein, and m protein appeared in a single radioactive peak that corresponded to a sucrose density of 1.18 g/cm 3 , which is the buoyant density of mhv particles (11) . e protein had two radioactive peaks, one corresponded to the mhv buoyant density of 1.18g/cm 3 and the other peak had a density of about 1.13 g/cm 3 , which was similar to the density of released e protein from e-protein-expressing cells. these data suggested that e protein that was not associated with mhv particles was released from mhvinfected cells. a different centrifugation condition was used to further confirm the release of e protein from mhv-infected cells. 35 s-labeled culture fluid from dbt cells, which were infected with mhv-a59 or sine pseudovirions, was collected 9 h p.i., as described above, and then applied onto a discontinuous sucrose gradient consisting of 20 and 60% sucrose in nte buffer. after centrifugation at 26,000 rpm for 15 h at 4°c in a beckman sw28 rotor, the interface between 20 and 60% sucrose was collected and diluted threefold with nte buffer. the sample was layered over a 10-30% continuous sucrose gradient, with a 60% sucrose cushion at the bottom of the gradient. the gradients were centrifuged at 30,000 rpm for 1 h at 4°c in a beckman sw41 rotor. twelve fractions were collected from the bottom of the gradient and mhv structural proteins in each fraction were immunoprecipitated with a mixture of anti-mhv serum, anti-m protein monoclonal antibody, and anti-e protein antibody. densitometric analysis of n protein in the sample from mhv-infected cells showed a single radioactive peak in fraction 4 with a sucrose density of 1.15 g/cm 3 (fig. 3b) . mhv m and s proteins also had a single radioactive peak in fraction 4 (data not shown), demonstrating that fraction 4 represented the major mhv peak. radioactive signal of e protein, which was broader than that of n protein, had two peaks. one of the peak signals was in the heavier fraction 4; this signal most probably represented e protein in mhv particles. the other peak signal of e protein had a lighter density of about 1.11 g/cm 3 . under the same centrifugation condition, released e protein from the e-protein-expressing cells had a single radioactive peak in fractions 6 and 7; the densities of sucrose in fractions 6 and 7 were 1.12 and 1.11 g/cm 3 , respectively (fig. 3c ). e protein released from the e-protein-expressing cells and the e protein signal from mhv-infected cells, corresponding to the lighter density, had very similar sucrose densities. essentially the same result was obtained in three independent experiments. these results strongly indicated that e protein that was not associated with mhv particles was released from mhv-infected cells. to establish the buoyant density of released e protein, culture fluids from e-protein-expressing cells were applied onto a discontinuous sucrose gradient and centrifuged as described above. the interface between 20 and 60% sucrose was collected, diluted, and separated again on a 10-30% continuous sucrose gradient at 30,000 rpm in a beckman sw40 rotor at 4°c for 1, 3, 6, or 12 h. radioimmunoprecipitation of e protein from the sucrose fractions showed that released e protein sedimented to sucrose densities of 1.10-1.13 g/cm 3 after 1 h centrifugation. released e protein sedimented to a sucrose density of about 1.13 g/cm 3 after 3 h centrifugation, and longer centrifugation did not affect the sedimentation profile (data not shown); these data demonstrated that the buoyant density of e protein was about 1.13g/cm 3 . the released e protein had a narrow distribution in the sucrose gradients under all the described centrifugation conditions (data not shown). we conducted flotation analysis to determine whether the e protein was released, from the e-protein-expressing cells, as a membrane protein in vesicles or as a secreted protein. the e protein released from sine pseudovirion-infected cells was partially purified by dis-continuous sucrose gradient centrifugation. sucrose crystals were added to the partially purified e protein to a final concentration of 67% sucrose, and the sample was placed at the bottom of a centrifuge tube. subsequently, 5 ml of 65% sucrose and 3 ml of 10% sucrose solutions were overlaid onto the 67% sucrose solution. the gradients were centrifuged at 35,000 rpm for 18 h at 4°c, and fractions were collected from the top of the gradient. radioimmunoprecipitation of e protein by anti-e protein antibody determined that most of the e protein settled at the interface between 65 and 10% were radiolabeled with tran 35 s-label from 5 to 9 h p.i. culture medium was then applied onto a discontinuous sucrose gradient consisting of 20 and 60% sucrose and centrifuged at 26,000 rpm for 15 h at 4°c in a beckman sw28 rotor. the interface between 20 and 60% sucrose was collected, diluted, and layered over a 10-30% continuous sucrose gradient with a 60% sucrose cushion at the bottom of the gradient. the samples were centrifuged at 30,000 rpm for 1 h at 4°c in a beckman sw41 rotor. twelve fractions were collected and mhv structural proteins in each fraction were immunoprecipitated with a mixture of anti-mhv serum, anti-m protein monoclonal antibody, and anti-e protein antibody. portions of gels showing n and e proteins (b) and e protein (c) are shown. relative intensities of n protein (triangles) and e protein (open boxes) are also shown. sucrose, which indicated that the e protein had migrated from the 67% sucrose solution to the interface during centrifugation (fig. 4a) . when np-40 was added to the 67% sucrose solution at a final concentration of 1% and the same flotation analysis was performed, the majority of e protein was detected in the 65-67% sucrose fractions (fig. 4a) . in flotation analysis, membrane-associated proteins localize at the interface of 65 and 10% sucrose and soluble proteins remain in the heavier sucrose loading zone of the gradient (12) . therefore, these data demonstrated that released e protein was associ-ated with lipid membrane vesicles and that np-40 treatment disrupted this association. next we used a sodium carbonate treatment method to determine whether the e protein was released as an integral membrane protein. culture fluids from e-proteinexpressing cells were incubated in 100 mm na 2 co 3 at ph 11 for 30 min on ice. as a control, the culture fluid was incubated in a buffer containing no na 2 co 3 at ph 7.0. sodium carbonate treatment disrupts membrane vesicles, releasing the peripheral membrane proteins from these vesicles, while integral membrane proteins are not (3, 13) . after incubation, the samples were centrifuged at 50,000 rpm for 1 h at 4°c in a beckman sw50 rotor. e protein was present in the pellet after both alkali treatment and the neutral ph treatment (fig. 4b) , leading us to conclude that e protein was released as an integral membrane protein in lipid vesicles. the release of e-protein-containing membrane vesicles from mhv-infected cells and e-protein-expressing cells further emphasized the pivotal role of e protein in coronavirus assembly. other examples, where expression of a single viral protein results in production and release of lipid vesicles, exist: the gag protein of rous sarcoma virus (rsv) and the m protein of vesicular stomatitis virus (vsv) both have the ability to bud from cells independently of other viral proteins (14, 15) . recent studies indicate that a proline-rich motif found within the vsv m protein and the rsv gag protein interacts with ww domains of cellular proteins (16, 17, 18) and this interaction may be important for virus budding (18, 19) . the ww domain is a highly structured, modular domain of 38-40 amino acids that facilitates protein-protein interactions. the ww domain is present in a wide variety of cellular proteins with diverse functions and consists of a hydrophobic pocket that is flanked by two perfectly conserved tryptophan (w) residues (16) . involvement of the ww domain of some host protein in budding of mhv e-protein-containing vesicles seems less likely, as mhv e protein lacks the proline-rich motif found in rhabdoviruses and rsv. nevertheless, some host protein may possibly interact with e protein and this interaction may drive the budding of coronavirus. vsv and rsv bud from the cytoplasmic membrane, while coronavirus buds from an internal compartment between the endoplasmic reticulum and the golgi apparatus; cellular protein(s) that localizes at the budding site may affect coronavirus budding. although e protein is a major player in coronavirus envelope formation and budding, e protein alone may be insufficient for nucleocapsid incorporation into the virus particles. earlier we established a complementary di the sucrose fraction containing partially purified e-protein-containing vesicles was adjusted to 67% sucrose in nte (nte) or 67% sucrose in nte plus 1% np-40 (np-40) and overlaid with 65 and 10% sucrose. after centrifugation fractions were collected and e protein was immunoprecipitated using anti-e protein antibody. fraction 11, fractions 6-10, and fractions 1-4 represent 67, 65, and 10% sucrose, respectively. fraction 5 represents the interface between 65 and 10% sucrose. the 14 c-labeled 14-kda size marker is shown on the left of each gel. (b) analysis of e-protein-containing vesicles after sodium carbonate treatment. culture fluid from e-protein-expressing cells was incubated in 100 mm na 2 co 3 at ph 11 (lane 3) or ph 7 (lane 2) for 30 min on ice. after incubation, the samples were centrifuged at 50,000 rpm for 1 h at 4°c in a beckman sw50 rotor. pelleted protein was immunoprecipitated with anti-e protein antibody. lane 1, the 14 c-labeled 14-kda size marker. system, in which mhv particles are released after coinfection of two di rnas; one is a self-replicating di rna encoding viral polymerase function and n protein, and the other subgenomically expressing m and e proteins (5) . release of mhv particles containing nucleocapsid requires synthesis of both e and m proteins in the complementary di system (5) . presumably, in the complementary di system e-protein-containing vesicles were released from the cells, in which self-replicating di rna and another di rna encoding only e protein were replicated. we know that no mhv nucleocapsid was detected in the supernatant (5), suggesting that e-proteincontaining vesicles may not include nucleocapsid. furthermore, sucrose gradient centrifugation of mhv particles showed two e protein radioactive peaks, whereas n protein had only one peak corresponding to the mhv buoyant density (fig. 3) , indicating that membrane vesicles containing only e protein do not include nucleocapsid. another envelope protein, s protein, is not involved in the incorporation of the nucleocapsid, because mhv particles containing nucleocapsid are produced in the absence of s protein (5) . most probably, m protein functions to incorporate nucleocapsid into virus particles. likely, m protein interacts with the nucleocapsid and e protein at the coronavirus budding site, and these intermolecular interactions facilitate the envelopment of the nucleocapsid. data demonstrating that purified m protein and viral rna interact in vitro (20) and that m protein and nucleocapsid interact in mhv-infected cells (narayanan, maeda, maeda, and makino, unpublished data) support this model. this model is also consistent with the present data that m, n, and e proteins were coimmunoprecipitated by anti-e protein antibody in mhv-infected cells (fig. 1b) . vlps that are made of m and e proteins are morphologically similar to coronavirus particles (3, 4) , whereas the morphology of e-protein-containing vesicles is not known. poor production of e-protein-containing vesicles in e-protein-expressing cells and a lack of anti-e protein monoclonal antibodies which are suitable for immunogold-labeling studies are the main obstacles in determining the shape of e-protein-containing vesicles. characterization of e-protein-containing vesicles is important. if e-protein-containing vesicles and mhv particles have similar sizes and morphologies, then e protein alone may determine the size and shape of coronavirus particles. if e-protein-containing vesicles are significantly different from mhv particles in size and shape, then perhaps m protein is important for the determination of the size and morphology of coronavirus particles. e-protein-containing vesicles were released from mhv-infected cells (fig. 3) , demonstrating that the production of e-protein-containing vesicles was not an artifact of the expression system. probably e-protein-containing vesicles are also produced in coronavirus-infected humans and animals. do e-protein-containing vesicles have any biological functions? recently baudoux et al. (4) showed that vlps that are made of coronavirus transmissible gastroenteritis virus (tgev) m and e proteins can induce ␣-interferon in leukocytes. their study suggests that m protein in vlp is important for ␣-interferon induction. further studies will show whether mhv e-protein-containing vesicles have any biological functions. the molecular biology of coronaviruses the production of recombinant infectious di-particles of a murine coronavirus in the absence of helper virus nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes coronavirus pseudoparticles formed with recombinant m and e proteins induce alpha interferon synthesis by leukocytes assembled coronavirus from complementation of two defective interfering rnas analysis of constructed e gene mutants of mouse hepatitis virus confirms a pivotal role for e protein in coronavirus assembly sindbis virus expression vectors: packaging of rna replicons by using defective helper rnas molecular basis of sindbis virus neurovirulence in mice mouse hepatitis virus gene 5b protein is a new virion envelope protein antigenic relationships of murine coronaviruses: analysis using monoclonal antibodies to jhm (mhv-4) virus physico-chemical properties of mouse hepatitis virus (mhv-2) grown on dbt cell culture characterization of the membrane association of the influenza virus matrix protein in living cells isolation of intracellular membrane by means of sodium carbonate treatment: application to endoplasmic reticulum viral liposomes released from insect cells infected with recombinant baculovirus expressing the matrix protein of vesicular stomatitis virus an assembly domain of the rous sarcoma virus gag protein required late in budding ww domains and retrovirus budding fine mapping and characterization of the rous sarcoma virus pr76gag late assembly domain a prolinerich motif within the matrix protein of vesicular stomatitis virus and rabies virus interacts with ww domains of cellular proteins: implications for viral budding late domain function identified in the vesicular stomatitis virus m protein by use of rhabdovirus-retrovirus chimeras isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid we thank john fleming for anti-m protein monoclonal antibody and julian leibowitz for anti-e protein antibody. we also thank krishna narayanan for proofreading the manuscript. this work was supported by public health service grant ai29984 from national institutes of health (to s.m.) and partially by a grant from the research fellowships of the japanese society for the promotion of science for young scientists (to a.m.). key: cord-269419-68kja6bg authors: hu, weibin; chen, aizhong; miao, yi; xia, shengli; ling, zhiyang; xu, ke; wang, tongyan; xu, ying; cui, jun; wu, hongqiang; hu, guiyu; tian, lin; wang, lingling; shu, yuelong; ma, xiaowei; xu, bianli; zhang, jin; lin, xiaojun; bian, chao; sun, bing title: fully human broadly neutralizing monoclonal antibodies against influenza a viruses generated from the memory b cells of a 2009 pandemic h1n1 influenza vaccine recipient date: 2013-01-20 journal: virology doi: 10.1016/j.virol.2012.09.034 sha: doc_id: 269419 cord_uid: 68kja6bg whether the 2009 pandemic h1n1 influenza vaccine can induce heterosubtypic cross-protective anti-hemagglutinin (ha) neutralizing antibodies is an important issue. we obtained a panel of fully human monoclonal antibodies from the memory b cells of a 2009 pandemic h1n1 influenza vaccine recipient. most of the monoclonal antibodies targeted the ha protein but not the ha1 fragment. among the analyzed antibodies, seven mabs exhibited neutralizing activity against several influenza a viruses of different subtypes. the conserved linear epitope targeted by the neutralizing mabs (fieggwtgmvdgwygyhh) is part of the fusion peptide on ha2. our work suggests that a heterosubtypic neutralizing antibody response primarily targeting the ha stem region exists in recipients of the 2009 pandemic h1n1 influenza vaccine. the ha stem region contains various conserved neutralizing epitopes with the fusion peptide as an important one. this work may aid in the design of a universal influenza a virus vaccine. because of their highly flexible genomes, influenza a viruses cause annual epidemics and sometimes pandemics around the world. for nearly 100 years, influenza a viruses have been a global threat to humans (palese, 2004) . based on the antigenicity of the hemagglutinin (ha) protein, influenza a viruses are classified into two groups and at least 16 different subtypes (h1-h16). the ha protein is the functional protein that mediates the entry of influenza viruses into susceptible host cells and thus contains various epitopes that are recognized by neutralizing antibodies (skehel and wiley, 2000) . however, heterosubtypic neutralizing or protective antibody responses are rarely observed in the general population, largely because of the high mutation rate of the ha protein, especially in the globular head (ha1) region, which is the primary target of the humoral immune response. consequently, when a new reassortant influenza virus emerges that the human immune system has not previously encountered, a pandemic may occur. the 2009 swine-origin h1n1 influenza is an example of such a pandemic. the 2009 pandemic h1n1 influenza virus contains gene segments that are in both the american and the eurasia swine genetic linkages (garten et al., 2009) . nucleotide sequence alignment has shown that the ha sequence of the 2009 pandemic h1n1 influenza virus is divergent from the sequences of the seasonal h1 influenza viruses that have previously been contents lists available at sciverse sciencedirect journal homepage: www.elsevier.com/locate/yviro virology circulating in humans. the antigenicity of the ha in this strain is also highly distinct from that of the previously circulating h1 influenza viruses (garten et al., 2009; hancock et al., 2009) . people, especially young people, generally lacked protection against this new virus (hancock et al., 2009) , and the 2009 pandemic h1n1 influenza vaccines have been proven effective in inducing neutralizing antibody responses against the pandemic influenza virus (liang et al., 2010; zhu et al., 2009) . it is important to determine whether cross-reactive neutralizing antibodies against both seasonal and pandemic influenza viruses are present in individuals who were infected with or vaccinated against 2009 pandemic h1n1 influenza. recently, wrammert et al. discovered that plasmablasts from 2009 pandemic h1n1 influenza patients produced cross-subtype neutralizing antibodies that targeted both the ha stalk and the head domain (wrammert et al., 2011) . we examined whether such antibodies existed in individuals vaccinated against pandemic influenza. in this study, we used the full-length ha protein from the 2009 pandemic h1n1 influenza virus to raise fully human neutralizing mabs. we obtained 19 monoclonal antibodies from the memory b cells of a 2009 pandemic h1n1 influenza vaccine recipient and confirmed that all 19 of the monoclonal antibodies recognized the lysates of both the pandemic virus and the recently circulating seasonal h1n1 influenza virus. seven of the human monoclonal antibodies were further found to have apparent neutralizing effects against different subtypes of influenza a viruses, including viruses belonging to both group 1 and group 2 and the pandemic influenza virus. interestingly, we found that most of the monoclonal antibodies, including the seven neutralizing mabs, bound to the ha stem region (ha2), which is relatively conserved among different influenza a virus strains. these findings indicate that a broad cross-subtype neutralizing antibody response targeting the ha stem region exists in individuals vaccinated against 2009 pandemic h1n1 influenza and that these broadly reactive memory b cells may be important for protecting humans from infection with different influenza a viruses. a functional analysis revealed that the ha2 region contained several (at least four) conserved neutralizing epitopes that could be recognized by the raised mabs. further experiments showed that one of them was a linear epitope (fieggwtgmvdgwygyhh), which was in the region of the fusion peptide on ha2. these results may be helpful in the design of universal influenza vaccines. a 27-year-old healthy female adult volunteer who had been vaccinated with a 2009 pandemic h1n1 influenza split-virion vaccine for one month was enrolled in this study. we used flow cytometry to separate pandemic h1n1 ha-specific memory b cells with three surface markers: cd19, igg, and ha-specific bcr. a baculovirus-expressed ha protein was used for cell sorting. as shown in fig. 1 , the memory b cells accounted for approximately 0.6% of the total peripheral blood small lymphocytes, and less than 1% of the selected memory b cells were ha-specific . the antibody variable genes of these memory b cells were identified with single-cell rt-pcr and nested pcr wrammert et al., 2008) . nineteen human monoclonal antibodies (constructed using the igg1 and k framework) were obtained that bound to the ha protein ( fig. 2a) . these mabs used different v, d, and j gene segments in their heavy and light chain variable genes; the v, d, and j usage is presented in detail in table 1 . the presence of somatic mutations in the 19 mabs revealed that most of the b cells producing these mabs were from the germinal center. notably, five of the mabs used the vh1-69 gene; the use of this gene is considered to be a non-exclusive characteristic of heterosubtypic binding to the ha stem region, with a neutralizing effect (ekiert et al., 2009; sui et al., 2009 ). these five mabs used different dh, vl, and jl genes, indicating that they are from different b cell clones. further experiments showed that after the ha protein was denatured with heat, four mabs (1e1, 3d2, 3e2, and 5a2) fully maintained their binding abilities to the antigen, one mab (3e1) completely lost its binding ability to the antigen, and the rest of the mabs partially retained their binding abilities to the ha protein ( fig. 2a) . all of the mabs bound to the 2008-09 seasonal h1 influenza (a/brisbane/59/2007) virus lysate and the 2009 pandemic h1n1 influenza virus lysate, but their binding avidities were different (fig. 2b ). an insect cell-expressed ha1 domain protein from the 2009 pandemic h1n1 influenza virus was also used to analyze the binding activity between ha1 and the mabs. as shown in fig. 2a , only one mab, 3d2, exhibited binding activity to the ha1 domain, whereas all of the other mabs did not bind to ha1. these results suggest that the other mabs may bind to the ha2 domain. these results indicate that memory b cells from people vaccinated with the 2009 pandemic h1n1 influenza vaccine can secrete antibodies that primarily target the ha2 region, and these antibodies are cross-reactive with the 2008-09 seasonal h1 influenza virus. to investigate the neutralizing effect of all of the fully human mabs, a microneutralization assay was adopted to screen the mab neutralizing activities. the experiment demonstrated that seven mabs (1c4, 1e1, 1f2, 1f4, 1g1, 3c4, and 3e1) showed relatively higher neutralizing activities against the 2009 pandemic h1n1 influenza virus. the ic 50 of the seven neutralizing mabs against the pandemic influenza virus sc09 were about 1 mg/ml or less (table 2) , while the ic 50 of the other mabs against sc09 were more than 200 mg/ml (data not shown). interestingly, all seven of the mabs also showed neutralizing activities against several other influenza virus strains, including subtypes of h3 (h3n2), h5 (pr8-h5), h7 (pr8-h7), and h9 (h9n2) ( fig. 3 and table 2 ). sequence analysis revealed that three mabs (1f2, 1f4, and 1g1) used the vh3-23 and vl3-15 genes and one mab, 3c4, used the vh1-69 gene (table 1) . next, we analyzed the neutralizing mechanism of the broad neutralizing mabs. a cell-cell fusion experiment was adopted. the results showed that all the seven broad neutralizing mabs exhibited apparent inhibition activities against syncytia formation of hek293t cells transfected with the ha expressing plasmids ( fig. 4) , which probably indicated that these mabs took effect in the virus-cell membrane fusion phase and hindered the function of ha2. the broad neutralizing effect probably resulted from the relatively conservation of ha2. to map the epitopes recognized by the tested mabs, a competitive elisa was used. as fig. 5 shows, 1f2, 1f4, and 1g1, which used the same vh and vl genes, bound to ha at the same site. it is consistent with the sequence analysis results (table 1) . 1c4 and 3c4 were derived from different variable genes but recognized the same epitope. as shown in fig. 2a , the neutralizing epitope recognized by 1e1 was linear (epit-3), whereas the epitope recognized by 3e1 was completely conformational (epit-4). the other two neutralizing epitopes (recognized by 1f2/1f4/1g1 and 1c4/3c4, respectively) were partially conformational epitopes (epit-1 and epit-2). these results revealed that the ha2 region contained several (at least four) conserved neutralizing epitopes that co-exist in different strains of the influenza virus. to further confirm whether the selected neutralizing mabs are capable of binding to ha2, a series of synthetic peptides (from g345 to p504 of ha) that cover the entire ha2 region with ten-amino-acid overlaps were used to detect the binding sites of the seven mabs (table 3) . as shown in fig. 6 , 1f2, 1f4, and 1e1 could specifically recognize peptide no. 2 (fieggwtgmvdgwygyhh) in the elisa assay, which is in the location of the fusion peptide that is believed to be very conserved among different strains of influenza a viruses (skehel and wiley, 2000) . sequence alignment shows that some amino acid residuals in the linear epitope are very conserved among the tested viruses of different subtypes (fig. 7) . the conserved amino acid residuals may play critical roles in recognition of the epitope by the cross-subtype neutralizing mabs. based on this observation, it is concluded that 1f2, 1f4, and 1e1 recognize a linear epitope in the fusion peptide region. the other mabs (1g1, 1c4, 3c4, and 3e1) showed no significant reaction with the peptides, suggesting that they target conformational epitopes. we will analyze the complex of the ha protein together with the mabs and use structural biology methods to reveal the characteristics of the epitopes recognized by the mabs (1g1, 1c4, 3c4, and 3e1). in this study, we demonstrated that memory b cells that secrete broadly neutralizing heterosubtypic antibodies that target ha2 were present in a recipient of the 2009 pandemic h1n1 influenza vaccine. corti et al. reported that cross-subtype neutralizing mabs from immortalized b cells were raised in individuals vaccinated against seasonal influenza, and they confirmed that almost all of the heterosubtypic mabs recognized epitopes within the ha stem region (corti et al., 2010) . our results are in line with the observation of corti et al. (2010) and wrammert et al. (2011) . they show that broadly reactive cross-subtype neutralizing antibody responses are induced in influenza virus infected patients and in individuals who accept vaccination against the pandemic or seasonal influenza viruses. the influenza ha stem region is crucial for recognition by broadly neutralizing cross-subtype antibodies, and this region can be a promising target for cross-subtype influenza vaccines. it contains a linear b-cell neutralizing epitope and several conformational ones, as shown by this study. importantly, the fusion peptide is a critical target for the binding of broad neutralizing mabs. we believe that studies on these broad neutralizing monoclonal antibodies would be beneficial for the development of a broadly effective heterosubtypic influenza vaccine. we generated seven neutralizing monoclonal antibodies in this study. it is interesting to note that partial v gene differences can influence the neutralizing activity without changing the binding activity; for example, the 3a6, 3b6, and 3c4 mabs show similar antigen-binding but different neutralizing activities. the reason may be that the neutralizing mabs target the postadhesion phase and interfering with ha2 function during the virus-cell membrane fusion process. the binding activity alone may be not sufficient to prevent viral entry into susceptible cells. additionally, 1c4 and 3c4 used completely different vh genes, but they recognized the same site in the ha2 region. our work also indicates that the broadly reactive human neutralizing monoclonal antibodies obtained in this study may be potentially used for the treatment of severe cases of influenza a virus infection (hung et al., 2011) . several neutralizing monoclonal antibodies targeting different epitopes can be used in a cocktail that covers as many influenza subtypes as possible. so far, many neutralizing mabs with the target of the ha stem region have been generated and carefully studied. these antibodies are incompletely enumerated in table 4 . the information obtained is useful for designing broadly protective influenza vaccines. nevertheless, the antigenic character of the ha stem region is complicated. including the fusion peptide, there are a number of linear or conformational neutralizing epitopes within this region. therefore, further research is needed to obtain a 3-11n01 6-25n01 4n01 9/121 3-15n01 2n01 8/109 1d2 1-69n09 4-17n01 4n02 7/121 3-15n01 2n01 4/108 1e1 a 3-23n04 1-1n01 4n02 7/121 3-11n01 5n01 0/108 1f2 a 3-23n04 5-5n01 5n02 6/121 3-15n01 5n01 2/108 1f4 a 3-23n04 2-8n02 3n02 6/121 3-15n01 4n01 0/108 1g1 a 3-23n04 3-16n01 4n02 9/121 3-15n01 4n01 1/108 1g6 3-23n04 6-19n01 5n02 7/123 2d-40n01 4n01 3/108 3a4 3-33n01 5-24n01 3n02 6/127 3-15n01 3n01 3/108 3a6 1-69n09 3-22n01 4n02 5/125 3-11n01 1n01 2/108 3b6 1-69n09 5-24n01 4n02 5/121 3-11n01 1n01 6/108 3c4 a 1-69n09 deeper and more comprehensive understanding of the interaction between the neutralizing antibodies and the ha stem region. minimizing the interference of non-neutralizing antibodies will aid in the successful development of a heterosubtypic broadly protective influenza vaccine. single-cell rt-pcr is a rapid method for the production of fully human neutralizing monoclonal antibodies. the cd19, igg, and haspecific bcr markers used in this work are suitable for selecting antigen-specific memory b cells, and this screening strategy significantly increased the efficiency of obtaining high-affinity antigen-specific human monoclonal antibodies. the platform established in this study can be applied to the development of fully human mabs that target other infectious agents, such as hiv, hcv, and hbv. this study was approved by the biomedical research ethics committee of the shanghai institutes for biological sciences, cas (er-sibs-211101). a 27-year-old healthy female adult who had received the pandemic influenza vaccine was recruited for this study. the influenza virus strains used in this study were as follows: a/sichuan/1/2009 (h1n1) (referred to as sc09), a/jiangxi-donghu/ 312/2006 (h3n2) (referred to as h3n2), and a/guangzhou/333/99 (h9n2) (referred to as h9n2) were kindly provided by prof. yuelong shu from the chinese center for disease control and prevention, 5 . a competitive elisa to detect the binding epitopes of the seven neutralizing mabs. the percentage of competition between two mabs in the elisa binding assay is shown. the percentage of competition was calculated as the reduction in the absorbance when mab binding competition occurred. shadowed grids: complete competition (percentage of competition 490%). epit-1 to epit-4: four neutralizing epitopes. the vertical row: non-conjugated mabs. the horizontal row: biotin-conjugated mabs. for which the four basic amino acid residues at the ha cleavage site were removed and has been confirmed to be non-pathogenic in mammalian (ferrets) and avian (chickens) hosts ). the h1 subtype virus lysates used in this study were from the 2009 pandemic h1n1 split-virus vaccine (ha from strain a/california/7/2009 (h1n1)) and the 2008-09 trivalent seasonal influenza vaccine (ha from strain a/brisbane/59/2007 (h1n1)). both of the vaccines were produced by hualan biological bacterin company, china. the ha gene was synthesized by generay china with the sequence from the influenza strain a/california/4/2009 (h1n1). the full-length ha and the ha1 sub-domain (18-344 aa) were expressed using the bac-to-bac baculovirus expression system (invitrogen) according to the manufacturer's protocol. ha was purified according to a previously described method (wang et al., 2006) . ha1 (c-terminal fusion with a human igg fc tag) was purified using a protein g sepharose 4 fast flow column (ge) according to the manufacturer's instructions. venous blood was collected from the volunteer in a tube containing 0.4% sodium citrate as an anticoagulant. lymphocytes and monocytes were prepared using the density separation medium lympholytes-h (cedarlane) according to the manufacturer's instructions. single-cell sorting was performed using an aria ii (bd) cell sorter. a fitc-labeled mouse anti-human cd19 antibody and an apclabeled mouse anti-human igg antibody were purchased from bd. a streptavidin-cy3 conjugate was purchased from sigma-aldrich . the ha protein was labeled with biotin using the ez-link sulfo-nhs-lc-biotin biotinylation reagent (thermo scientific) according to the manufacturer's instructions. the ha-specific memory b cells were gated and isolated as cd19 ã¾ /igg ã¾ /ha-specific bcr ã¾ cells. the flow cytometry data were analyzed with flowjo software. the generation of human monoclonal antibodies was performed using single-cell rt-pcr as previously described wrammert et al., 2008) . briefly, ha-specific memory b cells were sorted into 96-well pcr plates containing an rnase inhibitor (promega) at one cell per well. the antibody variable genes (vh and vk) were amplified from each cell with rt-pcr and nested pcr using panels of primers as previously described . restriction sites were incorporated with pcr with specific primers designed according to the characteristics of the v and j gene families to which the amplified genes belonged. the vh and vk genes were cloned into igg and igk expression vectors, respectively. the igg and igk expression plasmids were then co-transfected into hek293t cells using lipofectamine transfection reagent (invitrogen) according to the manufacturer's instructions. the supernatants were harvested, and the human monoclonal antibodies were purified using an rmp protein a sepharose fast flow column (ge) according to the manufacturer's instructions. ninety-six microwell plates (nunc) were coated with viral lysate or recombinant protein at a concentration of 10 mg/ml in 0.1 m sodium carbonate-bicarbonate buffer (ph 9.6) at 41 c overnight. phosphate-buffered saline (pbs, ph 7.4) containing 10% bovine serum albumin and 0.1% tween-20 was used as the blocking and dilution buffer. after blocking at 37 1c for two hours, the plates were washed, and human monoclonal antibodies were added. the plates were then incubated at 37 1c for another two hours. next, a horseradish peroxidase-conjugated goat antihuman igg (fc-specific) antibody (sigma-aldrich) was added, and the plates were incubated at 37 1c for one hour. tetramethylbenzidine (sigma-aldrich) was used as the substrate, and the optical density was measured at 450 nm (od 450 nm) with a microwell plate autoreader (thermo scientific). as for the peptide elisa, 19 overlapped peptides (table 3) were designed according to the protein sequences of ha2 (g345 to p504) of the pandemic h1n1 influenza virus strain: a/california/4/ 2009 (h1n1). the peptides (with n-terminal biotinylation and 90% purity) were synthesized by shanghai science peptide, china. streptavidin was coated onto the 96-microwell plate (nunc) at 20 mg/ml in 0.1 m sodium carbonate-bicarbonate buffer (ph 9.6) at 4 1c overnight. after washing of unbound streptavidin and blocking at 37 1c for two hours, the overlapping peptides were added at 2 mg/ml and incubated at 37 1c for another one hour. thereafter, procedures were the same as mentioned above. the ha protein was used to coat 96-microwell plates (nunc). human monoclonal antibodies were labeled with biotin using the ez-link sulfo-nhs-lc-biotin biotinylation reagent (thermo scientific) according to the manufacturer's instructions. after blocking at 37 1c for two hours, excess amounts of human monoclonal antibodies not conjugated to biotin were added to the plates, and the plates were incubated at 37 1c for two hours. the biotinlabeled monoclonal antibodies were then added to the plates, and the plates were incubated at 37 1c for another two hours. next, the streptavidin-horseradish peroxidase conjugate (bd) was added, and the plates were incubated at 37 1c for one hour. tetramethylbenzidine (sigma-aldrich) was used as the substrate, and the optical density was measured at 450 nm (od 450 nm) with a microwell plate autoreader (thermo scientific). the percentage of competition was calculated as the reduction in the absorbance when mab binding competition occurred. the experiment was performed generally as previously described (godley et al., 1992; goto and kawaoka, 1998; prabhu et al., 2009) . briefly, hek293t cells (atcc) were transfected with pbudce4.1 (invitrogen) plasmids expressing the pandemic h1n1 ha0 protein with the sequence from a/california/4/2009. the cells were cultured in 6-well plates and transfected with plasmids using lipofectamine transfection reagent (invitrogen). 24 h after transfection, the cells were treated with (tosylsulfonyl phenylalanyl chloromethyl ketone)-trypsin (2.5 mg/ml) at 37 1c for 10 min in order to cleave ha0 into ha1 and ha2. after that, the cells were incubated at 37 1c for 30 min with the mabs of 100 mg/ml. afterwards, the cells were washed with pbs, and the ph was decreased to 5.0 at 37 1c for 10 min. next, the cells were incubated with the mabs at the concentration of 100 mg/ml in complete medium for 2 h. the cells were then fixed with 4% (wt/vol) paraformaldehyde and stained with 1% (wt/vol) toluidine blue in pbs. results were observed in optical microscope and photographed. the microneutralization assay was performed as previously described (rowe et al., 1999) . briefly, antibodies were serially diluted two-fold in 50 ml and then mixed with influenza virus (100 tcid 50 in 50 ml per well). positive-control wells (virus only) and negative-control wells (without virus) were included on each plate. after a two-hour incubation at 37 1c in a 5% co 2 humidified atmosphere, 100 ml of trypsin-edta -suspended mdck cells (1.5 ã� 10 5 /ml) was added to each well. the plates were incubated for 18 h at 37 1c in a 5% co 2 humidified atmosphere. the cell monolayers were washed with pbs and fixed in cold 80% acetone for 10 min. the presence of viral protein was detected with an elisa as previously described (rowe et al., 1999; walls et al., 1986 ) with a polyclonal antibody against the influenza a np protein (prepared in our lab). the neutralizing end point was assessed as previously described (rowe et al., 1999) . the inhibition ratio (%) was calculated as [od (pos. control) ã� od (sample)]/ [od (pos. control)ã� od (neg. control)] ã� 100%. monoclonal antibodies isolated from human b cells neutralize a broad range of h1 subtype influenza a viruses including swine-origin influenza virus (s-oiv) heterosubtypic neutralizing antibodies are produced by individuals immunized with a seasonal influenza vaccine a human monoclonal antibody with neutralizing activity against highly divergent influenza subtypes a neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza a hemagglutinins a non-vh1-69 heterosubtypic neutralizing human monoclonal antibody protects mice against h1n1 and h5n1 viruses development of a new candidate h5n1 avian influenza virus for pre-pandemic vaccine production antibody recognition of a highly conserved influenza virus epitope a highly conserved neutralizing epitope on group 2 influenza a viruses introduction of intersubunit disulfide bonds in the membrane-distal region of the influenza hemagglutinin abolishes membrane fusion activity a novel mechanism for the acquisition of virulence by a human influenza a virus cross-reactive antibody responses to the 2009 pandemic h1n1 influenza virus convalescent plasma treatment reduced mortality in patients with severe pandemic influenza a (h1n1) 2009 virus infection a common neutralizing epitope conserved between the hemagglutinins of influenza a virus h1 and h2 strains influenza: old and new threats monoclonal antibodies against the fusion peptide of hemagglutinin protect mice from lethal influenza a virus h5n1 infection detection of antibody to avian influenza a (h5n1) virus in human serum by using a combination of serologic assays receptor binding and membrane fusion in virus entry: the influenza hemagglutinin rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen structural and functional bases for broad-spectrum neutralization of avian and human influenza a viruses a cross-reactive neutralizing monoclonal antibody protects mice from h5n1 and pandemic (h1n1) 2009 virus infection characterization and evaluation of monoclonal antibodies developed for typing influenza a and influenza b viruses expression and purification of an influenza hemagglutinin-one step closer to a recombinant protein-based influenza vaccine rapid cloning of high-affinity human monoclonal antibodies against influenza virus broadly crossreactive antibodies dominate the human b cell response against 2009 pandemic h1n1 influenza virus infection a novel influenza a (h1n1) vaccine in various age groups we thank dr. patrick c. wilson from the university of chicago for kindly providing the igg and igk expression vectors. this work was supported by grants from the national 863 project ( key: cord-256703-eaj63c2k authors: matsuoka, yumiko; ihara, takeshi; bishop, david h.l.; compans, richard w. title: intracellular accumulation of punta toro virus glycoproteins expressed from cloned cdna date: 1988-11-30 journal: virology doi: 10.1016/0042-6822(88)90075-x sha: doc_id: 256703 cord_uid: eaj63c2k abstract the punta toro virus (ptv) middle size (m) rna encodes two glycoproteins, g1 and g2, and possibly a nonstructural protein, nsm. a partial cdna clone of the m segment which contains g1 and g2 glycoprotein coding sequences but lacks most of the nsm sequences was inserted into the genome of vaccinia virus under the control of an early vaccinia promoter. cells infected with the recombinant virus were found to synthesize two polypeptides with molecular weights of 65,000 (g1) and 55,000 (g2) that reacted specifically with antibody against ptv. studies using indirect immunofluorescence microscopy revealed that these proteins accumulated intracellularly in the perinuclear region. the results of endoglycosidase h digestion of these glycoproteins suggested that both g1 and g2 glycoproteins were transported from the rer to the golgi complex. these proteins were not chased out from the golgi region during a 6-hr incubation in the presence of cycloheximide. surface immune precipitation and 125i-protein a binding assays also demonstrated that the majority of the g1 and g2 glycoproteins are retained intracellularly. these results indicate that the ptv glycoproteins contain the necessary information for retention in the golgi apparatus. the family bunyaviridae comprises a large and heterogeneous group of arthropod-borne viruses with certain structural features in common (bishop and shope, 1979; bishop et a/., 1980) . the viruses are enveloped, and contain two surface glycoproteins, gl and g2, an internal nucleocapsid-associated protein n, and a large internal protein (l) believed to be a transcriptase component. the nucleocapsid consists of three singlestranded rna genome segments, l, m, and s. on the basis of genetic recombination and sequence analysis, it has been concluded that the s rna encodes the nucleocapsid protein n and a nonstructural protein ns,; the m rna encodes two glycoproteins gl and g2 and in some genera a nonstructural protein, ns,; and the l rna probably contains the information for the viral transcriptase (bishop et al., 1982; bouloy et a/., 1984; cabradilla et al., 1983; collett et a/., 1985; eshita and bishop, 1984; fuller et al., 1983; lhara et a/., 1985; lees et al., 1986; ronnholm and pettersson, 1987; schmaljohn et a/., 1987 , 1982, 1984) . ' to whom requests for reprints should be addressed. it has also been demonstrated by using temperaturesensitive mutants that the intracellular accumulation of glycoproteins occurs in the absence of virus maturation (gahmberg er a/., 1986). lntracytoplasmic virus budding has also been described for coronaviruses (dubois-dalcq eta/., 1982; holmes et al., 1981; tooze et a/., 1984; tooze and tooze, 1985) , flaviviruses (lear-y and blair, 1980) , toroviruses (fagerland et a/., 1986; weiss and horzinek, 1986) , and rotaviruses (altenburg et al., 1980; petrie et al., 1984) . it has been reported that the el glycoprotein of the coronaviruses, when expressed from cloned dna, accumulated intracellularly (machamer and rose, 1987; rottier and rose, 1987) . furthermore, evidence has recently been reported which indicates that one of three hydrophobic membrane-spanning domains of this protein is responsible for retention in the golgi complex (machamer and rose, 1987) . punta toro virus (ptv) is one of some 36 arthropodborne viruses assigned to the phlebotomus fever serogroup (phlebovirus genus, bunyaviridae, bishop et a/., 1980) and has structural and morphological features similar to other members of the family bunyaviridae. the s rna of ptv has an unusual ambisense coding strategy. one protein (n) is coded in a subgenomic, viral-complementary s mrna species and a second protein (nss) is coded by a viral sense mrna (ihara et a/., 1984) . the m rna segment possesses a single open reading frame in the viral-complementary sequence that is presumed to code for a polyprotein precursor. in addition to the gi and g2 glycoprotein coding sequences, a sequence capable of coding a polypeptide of approximately 30 kda was found preceding the gi coding sequences, although the putative nsm protein product corresponding to this sequence has not been identified either in virions or in infected cells. the estimated sizes of n&, gl , and g2 from the predicted amino acid sequence are 30,510, 60,897, and 55,005 da, respectively (ihara et a/., 1985) . we have constructed vaccinia virus recombinants expressing the gl and g2 glycoproteins of ptv, in order to study the possible existence of signals for intracellular retention in bunyavirus glycoproteins. we report here the expression of the two glycoproteins of ptv, and their localization in infected cells in the absence of other viral components. cv-1, vero, and helat4+cells(maddoneta/., 1986) were maintained in dulbecco's modified eagle's medium (dmem) supplemented with 10% newborn bovine serum. human tk-143 cells maintained in the above medium supplemented with 25 pg/ml of 5bromo-2-deoxyuridine (5-budr). vaccinia virus stocks were prepared and titrated by plaque assay in cv-1 cells. ptv stocks were prepared and titrated in vero cells. of a vaccinia recombination plasmid containing the gl and g2 glycoprotein genes all dna manipulations were carried out as described by maniatis et a/. (1982) . four cdna clones, 5-79, 1 o-99, 6-37, and 4-148, which were previously used for sequence analysis, had been cloned into the pstl site of pbr322 (ihara et a/., 1985) . by a series of restriction digestions and ligations with these clones, a full-length ptv m cdna clone was constructed and inserted into pgem-3 (promega biotech, madison, wi), designated as pgem-m. it was found, however, by analysis of protein synthesis in an in vitro transcription and translation system using pgem-m and by subsequent sequence analysis that pgem-m contained deletion mutations in the middle of the gl glycoprotein coding region. it was also found that the sequences preceding the gl coding sequences seem to have some inhibitory effects on growth of bacterial colonies. because of these problems, most of the 5'sequence preceding the gl coding region was removed by digestion with ba13 1 exonuclease. in order to repair the mutations, the ncol-xbal restriction fragment which contains the mutated region was replaced by the corresponding fragment from clone 5-79, one of the original clones used for sequence analysis (fig. 1) . after transformation and colony selection, two clones were obtained, one of which contained an m fragment beginning with nucleotide residue 702 that had an atg at nucleotide residue 716, whereas the other contained a fragment beginning with residue 765 and lacked an initiation codon for synthesis of g 1 and g2 glycoproteins. the clone containing an atg codon in the region preceding the g! coding sequences was designated as pgem-g(c) since it possesses a c residue at the -3 position upstream from the translation initiation site. the clone which did not have an atg sequence was further modified to introduce a translation initiation codon. after digestion with ecori and treatment with klenow dna polymerase, c/al linkers were added at the ends of the dna in order to provide an atg sequence. this segment was then digested with c/al and treated with klenow polymerase following digestion with either pstl or sali in order to remove plasmid sequences. these segments were inserted into either pgem-3 digested with sal1 and treated with klenow polymerase following pstl digestion or pgem-3 digested with xbal and treated with klenow polymerase following sali digestion. as a result, pgem-g(a) which contains an a residue at the -3 position from the initiation codon, and pgem-g(g) with a g residue at the -3 position, were obtained (fig. 1 ). the ptv glycoprotein gene was excised by barnhi from the three clones, pgem-g(c), pgem-g(a), and pgem-g(g), treated with klenow polymerase, and inserted into the smal site of the psc1 1 vaccinia recombination plasmid (chakrabarti et a/., 1985) . of a vaccinia recombination plasmid containing the influenza ha gene the cdna clone of the wsn-ha gene in pbr322 was kindly provided by dr. debi nayak. the ha gene was excised from pbr322 and recloned into the pstl site of puc13 (stephens et al., 1986) . the plasmid was liniarized by bamhl digestion, treated with ba/31 exonuclease to remove dg and dc homopolymeric tails at the 5' end, treated with klenow dna polymerase, and then digested with hindill. the ha gene fragment was separated from the pucl3 vector, inserted into hindill-hincll-digested pgem-3, excised byxbal-hindill digestion, treated with klenow dna polymerase, and inserted into the smal site of pscl1. , 1983) . to select for recombinants, tk-143 cells were infected with 50-l 00 pfu of tk-vaccinia virus in the presence of budr (25 pg/ml). at 48 hr after infection, the monolayers were overlayed with 19/o low-melting-point agarose containing 300 pg/ml of 5-bromo-4-chloro-3-indolyl-p-d-galactopyranoside (x-gal). at 4-6 hr, blue plaques were picked and further purified by two additional rounds of plaque purification. hyperimmune mouse ascitic fluids specific for ptv and monoclonal antibodies against gl and g2 glycoproteins were kindly provided by drs. j. f. smith and d. pifat (usamriid, for petrick, md). rabbit antiserum against a/wsn/33 influenza virus was prepared as described elsewhere (roth and compans, 1980) . cv-1 cells or hela t4+ cells were infected with vaccinia virus recombinants or ptv at an m.o.i. of 5 to 10. at 16 hr p.i., cells were washed with phosphate-buffered saline (pbs) and incubated in methionine-free medium for 2 hr. at 18 hr p.i., cells were labeled with [35s]methionine (100 &i/ml) in methionine-free medium for 1 hr. for pulse-chase experiments, cells were pulsed for 10 min and then chased in eagle's medium containing 10 mlvl methionine for an appropriate period. cells were then washed three times in ice-cold pbs and lysed with 0.3 ml of lysis buffer (50 mm tris-hci (ph 7.5), 0.15 m naci, 1 o/o triton x-l 00, 0.1 oh sds, 20 mm edta). nuclei were removed by centrifugation at 13,000 g for 5 min at 4". the cell lysates were incubated with 5 /-cl of mouse ascites against ptv for 4 hr at 4" with constant mixing. for immunoprecipitation of cell-surface antigens, antibody was added before cells were lysed and incubated for 30 min on ice. after washing to remove unbound antibody, radiolabeled polypeptides were immunoprecipitated with protein a-sepharose cl-4b (pharmacia, inc., piscataway, nj). the precipitates were washed three times with cold lysis buffer, resuspended in sample buffer, boiled for 5 min, and analyzed on 10% sds-polyacrylamide gels (laemmli, 1970) . hela t4+ during the labeling period, pmsf and tm were maintained in medium of treated cells. half of each sample was further treated by endo h at 37" for 16 hr (lanes b, d, f, h, and j). all samples were then analyzed on 10% sds-page. laboratories, inc., san mateo, ca) in some experiments. the cells were washed with pbs mounted, and observed with a nikon optiphot microscope equipped with a modified b2 cube. h digestion lmmunoprecipitated samples were boilded for 5 min in sds-page sample buffer and the protein a-sepharose was removed. the supernatants were diluted 15fold with 0.1 m sodium acetate, ph 6.0, and incubated for 16 hr at 37" in the presence of 8 mu/ml of endoglycosidase h (endo h) (boehringer-mannheim biochemicals, indianapolis, in) and 1 mm of pmsf. samples were then precipitated with looh tca for 1 hr at o", washed with cold ethanol/ether (1 :l), and resuspended for sds-page analysis. to quantitate antigen expressed on cell surface, surface radioimmunoassays were performed by a method described by stephens er al. (1986) . hela t4+ cells on glass coverslips were infected with vaccinia virus or the vaccinia virus recombinant at an m.o.i. of 10. at appropriate times after infection, cells were washed three times with pbs/l % bovine serum albumin (bsa), overlaid with 10 ~1 of antibody diluted 1: 10 in pbs/l % bsa, and incubated for 30 min at 4". cells were then washed three times with pbs/l% bsa and incubated with 200,000 cpm of '251-protein a (amersham, arlington heights, il) prepared in pbs/is/o bsa for 30 min at room temperature. the label was removed, the cells were washed three times with pbs/l% bsa, and the bound radioactivity was determined. nonspecific binding of the '251-protein a to cells infected with wild-type vaccinia virus was determined for each antisera and subtracted from each value. to compare the levels of plv glycoprotein synthesis, monolayers of cv-1 cells were infected with wildtype vaccinia virus, ptv, or the vaccinia recombinants described above which contain the ptv glycoprotein gene with the different sequences in the translation initiation region. at 18 hr p.i., cells were labeled for 1 hr with [35s]methionine, harvested, and immunoprecipitated. two polypeptides, which have electrophoretic mobility similar to those of the gl and g2 glycoproteins synthesized in ptv-infected cells, were precipitated from cells infected with vaccinia recombinants using antibody specific for ptv ( fig. 2a) . the polypeptide corresponding to the g2 glycoprotein occasionally appeared as a doublet. the appearance of the lower band was not consistently observed, and it may be a differently glycosylated form of the g2 glycoprotein. a similar observation of the synthesis of a heterogeneous hantaan (htn) gl glycoprotein was reported by pensiero et a/. (1988) . the level of protein synthesis was much higher for the recombinants which contain g or a at the -3 translation initiation position than the one which contained c. therefore, one of the recombinants which contains g at the -3 position was chosen as a prototype (designated vv-g) and used for further experiments. the effect of tunicamycin (tm) on the synthesis of the proteins was also examined as shown in fig. 2b . at 16 hr p.i., cells infected with ptv or recombinants were exposed to medium containing tm for 2 hr. cells were then labeled with [35s]methionine for 1 hr, lysed, and immunoprecipitated. the gl glycoproteins synthesized from the vv-g recombinant, in eitherthe presence or absence of tm, showed slightly slower mobility on the gel then those synthesized in ptv-infected cells. the g2 glycoprotein appeared as multiple bands in the presence of tm, which may indicate a partial inhibition of glycosylation. the amount of gl and g2 glycoproteins detected in the presence of tm was significantly lower than that in the absence of tm. in contrast, under the same condition, the glycosylation of influenza ha glycoprotein was completely inhibited and similar amounts of both glycosylated and unglycosylated forms of ha were detected. when a higher concentration of tm (1.5 pg/ml) was used, neither the gl nor g2 glycoprotein was detected (data not shown). therefore, the unglycosylated forms of gl and g2 glycoproteins appear to be relatively unstable and quickly degraded even in the presence of pmsf. the intracellular localization of the expressed glycoproteins was examined in hela t4+ cells because this cell line shows minimal cytopathic effect with vaccinia virus infection when compared to most other cell lines, and is therefore well suited for studies of protein localization (r. owens and r. compans, submitted for publication). hela t4+ cells were infected with the vv-g recombinant, and at 18 hr p.i., cells were fixed and examined using indirect immunofluorescence microscopy. the proteins expressed from vv-g were localized in a perinuclear region which coincided with the region stained by wheat germ agglutinin, a marker for the golgi complex (figs. 3a and b) . recombinant vv-g-infected cells were also treated with cycloheximide to determine whether gl and g2 glycoproteins are chased out from the golgi region. as shown in fig. 3 , both glycoproteins were retained in the golgi region after a 6-hr chase with cycloheximide. the accumulation of these proteins in a localized region of the cytoplasm was much more apparent in cycloheximide-treated cells (d, e, and f) compared to the cells before treatment (c). in order to further investigate the intracellular location of the glycoproteins, the processing of their oligosaccharide side chains was studied by treatment with endo h. after pulse labeling of vv-g-infected cells and various chase periods, proteins were immunoprecipitated and digested with endo h (fig. 4) . the gl synthesized from the vv-g recombinant acquired endo h resistance within a 2-hr chase period, suggesting that it contains complex-type oligosaccharide side chains. the g2 glycoprotein, on the other hand, appears to contain both high mannose-type and complex-or intermediate-type of sugar moieties. these results indicate that the gl and g2 glycoproteins are transported from the rer to the golgi complex where the maturation of oligosaccharides takes place. no significant amount of surface expression of viral glycoproteins was observed on cells infected with ptv or vv-g when examined by surface immunofluorescence (figs. 5e and f). in contrast, the vaccinia recom-binant expressing the influenza ha glycoprotein is shown as a control which exhibits typical surface protein expression (fig. 5h) . the lack of significant surface expresison of the ptv glycoproteins was also examined by two biochemical approaches. at 18 hr p.i., cells infected with the w-g recombinant were pulsed for 10 min with [35s]methionine, chased with medium containing excess cold methionine for 30, 60, 120, and 180 min, and the presence of proteins on cell surfaces was analyzed by surface immune precipitation. as shown in fig. 6 , lanes fj, only faint bands were seen in the surface immunoprecipitate. no increase in the intensity of gl or g2 proteins was seen during the chase, indicating that the proteins are not being transported to the cell surface during this time period. it was also confirmed that the ascitic fluids specific for ptv possess virus neutralizing activity, and hence have the ability to react with extracellular forms of plv glycoproteins (data not shown). in order to determine whether the plv glycoproteins are secreted, supernatants of w-g-infected cell cultures were examined. at 16 hr p.i., infected cells were labeled with [35s]methioninefor 6 hr, and proteins present in culture media were immunoprecipitated and analyzed by sds-page. no detectable amount of the gl or g2 glycoproteins was found in the medium (data not shown). cell-surface expression was also quantitated by 1251protein a binding assays at different time points of infection. the vaccinia recombinant expressing ha was again used as a control for surface expression. as shown in fig. 7 , ha was detectable on the cell surface at 8 hr p.i. and the amount increased until 16 hr p.i. in contrast, ptv glycoproteins were barely detectable until 16 hr p.i., and the level of maximum '251-protein a binding was significantly lower than that observed with ha. throughout the experiment, no significant cytopathic effect was observed. the percentage of live cells in recombinant-infected cultures determined by trypan blue staining was the same as in uninfected cells. we have constructed vaccinia virus recombinants containing a partial cdna clone of the m genome segment of ptv, which encodes the gl and g2 glycoproteins. although a complete m segment clone was initially obtained, because of a mutation in the gl glycoprotein gene and apparent inhibitory effects of the sequence preceding the gl glycoprotein gene on bacterial growth, which interfered with attempts to repair mutations, most of the 5'sequences preceding the gl coding sequences had to be eliminated to obtain glycoprotein expression. this recombinant virus, w-g, efficiently produced two polypeptides that were recognized by antibody against ptv. it has been suggested that the two glycoproteins of bunyaviruses, including ptv, are derived from a large precursor protein following proteolytic cleavage (collett et a/., 1985; eshita and bishop; 1984 , lhara el a/., 1985 . although the precursor molecule for the glycoproteins has never been identified in virus-infected cells, it was produced in an in vitro translation system using mrna isolated from uukuniemi virus-infected cells (ulmanen et al., 1981) . the vv-g recombinant produced two glycoproteins similar in size to gl and g2 from ptv-infected cells, suggesting that the cleavage between gl and g2 occurs correctly. it was observed, however, by using tunicamycin (tm) that the unglycosylated form of g 1 -synthesized from recombinants had a slightly higher molecular weight than that from ptv-infected cells. since only one predicted glyat 18 hr p.i., hela t4+ cells infected with w-g were labeled with [35s]methionine for 10 min (lanes a and f) and chased for 30 min (lanes b and g), 60 min (lanes c and h), 120 min (lanes d and i). or 180 min (lanes e and j) in medium containing 10 mm unlabeled methionine. cells were lysed and reacted with anti-ptvascites fluid (lanes, a, b, c, d, and e) or treated with anti-ptv ascites fluid and then lysed (lanes f, g, h, i, and j). following immunoprecipitation, radiolabeled polypeptides were analyzed by sds-page. cosylation site was found in the gl glycoprotein sequence including the extra short sequences at the 5' end in w-g (ihara et a/., 1985) it is likely that the decrease in the size of gl glycoproteins in the presence of tm reflects the complete inhibition of glycosylation. also, the apparent molecular weight of the unglycosylated form of gl glycoprotein in ptv -infected cells corresponded to the size predicted from sequence analysis (60,000 da). the observed difference in size (2-3 kda) between unglycosylated gl of ptv and of w-g may, therefore, correspond to the extra 24 amino acids preceding the gl glycoprotein, which are derived from the putative ns~ coding region. in this case, cleavage between gl and the partial nsm sequence may not take place. although there seems to be a consensus sequence after alanine for the potential cleavage sites in the putative precursor protein for ptv as well as rift valley fever virus (rvfv) (ihara et a/., 1985; collett et a/., 1985) the present results indicate that an additional factor such as secondary structure of the protein may also be necessary for proper cleavage. uncleaved precursor proteins were not detected in either ptv-or recombinant-infected cells in the presence of protease inhibitors and/or tm. therefore, glycosylation is apparently not essential for cleavage. the ptv glycoproteins seem to contain the necessary information for intracellular localization as well. the majority of proteins produced from the vv-g, recombinant remain intracellularly thorughout the infection period. the intracellular location of these glycoproteins was further examined by digestion of proteins with endo h. it is generally considered that the time required for the acquisition of endo h resistance corresponds to the transport time from the rer to the golgi complex (strous and lodish, 1980) . the finding of endo h resistant forms of gl and g2 glycoproteins indicates that these glycoproteins are transported from the rer to the golgi complex. furthermore, after 6 hr treatment with cycloheximide, both glycoproteins were still localized in the golgi region by immunofluorescence, indicating that these proteins are retained in the golgi complex. at late times postinfection, small amounts of proteins were detected on the cell surface by 1251-protein a binding assays but not by immunofluorescence. since these glycoproteins were retained in the golgi region for 6 hr in the presence of cycloheximide, any transport from the golgi complex to the cell surface must be extremely slow if it takes place. cell-surface expression of small amounts of glycoproteins has also been reported for other bunyaviruses (kuismanen et a/., 1982; madoff and lenard, 1982; gahmberg et a/., 1986) . however, these proteins may be present on cell-associated virions (smith and pifat, 1982 schmaljohn eta/., 1987). unlike ptv or rvfv, the htn m genome segment lacks the 5' sequences which may encode another protein. instead it possesses 18 amino acids following the first atg of the m segment, and preceding the gl glycoprotein, which may simply act as a signal peptide. these results, as well as the present observations, indicate that the glycoproteins of bunyavirus are synthesized, processed, and localized properly without a requirement for any other viral components, such as viral nucleocapsid or virus-coded nonstructural proteins. the precise location in the glycoproteins of the signals for intracellular retention remains to be determined. a recent study of the el glycoprotein of a coronavirus (machamer and rose, 1987) indicated a role of hydrophobic sequences in protein retention. deletion of one of the hydrophobic membrane spanning domains of these glycoproteins, which are normally retained at intracellular membranes, resulted in their secretion or transport to the surface. it will be of interest to carry out similar studies to determine the precise retention signal for ptv glycoproteins. ultrastructural study of rotavirus replication in cultured cells the complete sequence and coding content of snowshoe hare bunyavirus small (s) viral rna species. nucleic acids res. 10,3703-37 13. bishop, d. h. l., and shope, r. e. (1979) . bunyaviridae.in "comprehensive virology" (h. fraenkel-conrat and r. r. wagner, eds.), vol. 14, pp. l-l 56. plenum, new york. bouloy. m., vialat. p., girard, m., and pardigon. n. (1984) . a transcript from the s segment of the germiston bunyavirus is uncapped and codes for the nucleoprotein and a nonstructural protein. j. viral. 49, 717-723. cabradilla, c. d., holloway, b. p., and obijeski, j. f. (1983) . molecular maddon, p. j., dalgleish, a. g., mcdougal, j. s., clapham, p. r., weiss, r. a., and axel, r. (1986) . the t4 gene encodes the aids in the immune system and the brain. cell47, 333-348. madoff, d. h., and lenard, j. (1982) . a membrane glycoprotein that accumulates intracellularly: cellular processing of the large glycoprotein of lacrosse virus. ce//28,82 l-829. maniatis, t., fritsch, e. f.. and sumbrook, j. (1982) . schmaljohn, c. s., schmauohn, a. l., and dalrymple, j. m. (1987) . hantaan virus m rna: coding strategy, nucleotide sequence, and gene order. virology 157,3 1-39. smith, g. l., and moss, b. (1983) . infectious poxvirus have capacity for at least 25,000 base pairs of foreign dna. gene 2521-28. smith, j. f., and pifat, d. y. (1982) key: cord-260177-xu0elmak authors: collins, arlene r.; knobler, robert l.; powell, harry; buchmeier, michael j. title: monoclonal antibodies to murine hepatitis virus-4 (strain jhm) define the viral glycoprotein responsible for attachment and cell-cell fusion date: 1982-06-30 journal: virology doi: 10.1016/0042-6822(82)90095-2 sha: doc_id: 260177 cord_uid: xu0elmak abstract hybridoma cell lines producing monoclonal antibodies to the jhm strain of mouse hepatitis virus-4 (mhv-4) were established. by indirect immunofluorescence and immune precipitation, monoclonal antibodies of three viral polypeptide specificities were characterized. monoclonal antibodies to nucleocapsid reacted in the cytoplasm of infected cells and precipitated the 60,000d nucleocapsid polypeptide (vp-4) of mhv-4. other monoclonal antibodies reacted both in the cytoplasm and on the surface of infected cells and were found to precipitate the 170,000d viral glycoprotein (gp-1). a third set of monoclonal antibodies reacted both in the cytoplasm and on the surface of infected cells and precipitated the 25,000d viral glycoprotein (gp-5) and its precursor vp-6 (23,000d). antigp-1 alone had direct neutralizing activity for mhv-4 virus, while in the presence of complement both anti-gp-1 and anti-gp-5 neutralized virus. only anti-gp-1 had the ability to inhibit the spread of infection due to fusion in l241 cells. thus, the viral glycoprotein gp-1 likely contains both the attachment and fusion activities of mhv-4. at least four murine coronaviruses are associated with hepatitis and/or encephalitis in mice (gledhill and andrewes, 1951; nelson, 1952; dick et al., 1956; manaker et al., 1961; bailey et al., 1949) , and these viruses are collectively referred to as murine hepatitis viruses (mhv). strains of mhv exhibit distinct organ tropisms, being predominantly either hepatotropic or encephalitogenic, and may cause persistent infections in mouse colonies. mhv-4 (jhm strain) is an encephalitogenic strain which causes fatal acute encephalomyelitis following intracerebral inoculation in its natural host (lampert et al., 1973; weiner, 1973 destruction of oligodendrocytes (haspel et al., 1978) . in viva studies with jhmv and its mutants may provide valuable insights into the pathogenesis of human demyelinating diseases; however, a careful analysis of the pathogenesis of primary demyelination has been hampered by the lack of sensitive and specific probes for viral products as well as a firm understanding of coronavirus structure, replication, and mechanism(s) of persistence. serologic evidence of infection and detection of virus antigens in target organs has been difficult to demonstrate due to the poor quality of serum antibodies elicited in mice. antigens responsible for cross-reactivity among murine coronaviruses have not been well defined. the importance of making accurate comparisons is underscored by a recent report of serologic cross-reactivity between murine coronaviruses and two human coronavirus isolates from brains of multiple sclerosis patients (burks et al., 1980) . of mhv-4 and other murine coronaviruses has not been well defined. the virion contains positivestranded rna and infected cells reportedly contain seven major and two minor mrna species, the largest of which is the same size as the genome . the mode of synthesis and processing of these mrnas has not been elaborated. six polypeptides, four of which are glycosylated, have been reported in infected sac cells by siddell et al. (1981) . of these six polypeptides, the two largest are the glycopeptides gp-l(1'70,oood) and gp-2 (98,oood) which appear to represent dimerit and monomeric forms, respectively, of the same glycopeptide as shown by tryptic peptide mapping. a 60,oood nonglycosylated polypeptide, vp-4, is associated with the viral nucleocapsid. the two smallest polypeptides, oood) and vp-6 (23,oood), found in the infected cells, are identical to those found in the virion envelope (wege et al., 1979) . pulse-chase experiments have shown that vp-6 is processed to gp-5 by the addition of polysaccharide. a minor glycopeptide gp-3 (65,-oood) appeared in gels when dithiothreitol was used as reducing agent, and its dimerit form (125,oood) was found in gels when /3-mercaptoethanol was used (wege et al., 1979) . sturman et al. (1980) have recently proposed a structural model for the membrane proteins of the mouse hepatitis virion based on experimental data obtained with the a-59 strain. in this proposed model, the largest virion glycopeptides gp-1 (180,oood) and gp-2 (98,oood) are associated with the virion peplomers and have been designated e-2 protein. 000d) and vp-6 (23,000d) are deeply embedded in and may transsect the viral envelope, interacting internally with the nucleocapsid protein. in the proposed model, these polypeptides form a structure designated the e-l protein which plays an essential role in virus maturation intracellularly (holmes et al., 1981) . the viral glycoproteins bearing receptor sites for virus adsorption and cell-cell fusion during mhv infection have not been defined. in this report we describe the generation and characterization of monoclonal anti-bodies to mhv-4 and their use in initial studies to define biological activities associated with the virion glycopeptides. v+~.g and cezz culture. the jhm strain of mhv-4 was provided by l. weiner (university of southern california). it was plaque purified in nctc-1469 cells and then propagated and plaque assayed in l-241 cells obtained from l. sturman ( production of nacmochaz antibadies to jhm'v. balb/c mice 4-6 weeks of age were primed by ip inoculation of 103 to lo4 plaque-forming units (pfu) of mhv-4 grown in l-241 cells. after 3 weeks, boosting doses consisting of 0.1 ml of a 10% (v/v) extract of virus-infected cells were inoculated ip for 3 consecutive days prior to fusion. fusion with p3 x 63 ag8 clone 6531 nonsecretor plasmacytoma cells was done essentially by the method of kohler and milstein (1975) as we have described elsewhere . briefly, lo* spleen cells were fused with lo7 plasmacytoma cells using peg 1006 at 50% concentration. cells were diluted in hat medium and plated out in eight 96-well plates. wells were observed for 3-4 weeks for hybrid colonies and these colonies were screened for production of antibody to mhv-4. antibody producing cultures were immediately subcloned by limiting dilution and rechecked. screening of culture fluids for antiviral antibody was usually done by indirect immunofluorescence on both acetone-fixed and paraformaldehyde-fixed cells. as controls, uninfected l-241 cells and culture supernatant from parental myeloma cells were included in each assay. fluorescein isothiocyanate-conjugated goat anti-mouse igg was prepared in this laboratory . clones of each culture producing the highest titers of antibody in vitro were selected for ascites production in pristane (2,6,10,14,-tetramethylpentadecane)-primed balb/c mice. spetifiicities of monoclonal immunoglub ulins. virus-specific antibody concentrations in cell culture and ascites fluids were estimated by indirect immunofluorescence endpoint titration on acetone-fixed target cells. igg concentrations were estimated by radial immunodiffusion using sheep anti-mouse igg. determination of immunoglobulin isotype was determined by agar gel diffusion using subclass-specific antisera (meloy laboratories, springfield, va.). immune precipitation. mhv-4 polypeptide specificities of monoclonal antibodies were determined by immune precipitation of [35s]methionine-labeled viral proteins from infected l-241 cells. previously described methods (schauffhausen et al., 1978; were adapted as follows. radiolabeled cytosol extracts of infected and control l-241 cells were prepared by solubilizing 0.5 to 1.0 x 10' infected cells in 2.5 to 5 ml of lysis buffer (20 mm tris-hcl, ph 9.0; 137 mm nacl; 1 mm cacl,; 0.5 mm mgcib; 1% aprotinin; 1% nonidet p 40; and 10% (v/v) glycerol), then centrifuging at 10,000 g for 30 min to remove nuclear debris. a volume of 200 ~1 of cytosol was mixed with 100 ~1 of hybridoma culture fluid or 5 ~1 of ascites fluid and incubated at 37" for 1 hr. heat-killed, formalin-fixed staphylococcus aureus bacteria (sa) in sac buffer (0.1 mphosphatebuffered saline, ph 7.2; 0.5% nonidet p-40; 2 mm methionine; 0.22% sodium azide; 1 mg/ml ovalbumin) were added to each mixture in volumes of 100 ~1 of a 10% (v/ v) suspension, and incubation was continued for 30 min at 22". pellets were collected by centrifugation at 2500g for 10 min and washed three times in tris-hcl (0.1 m, ph 9) containing 0.5 m licl. the final pellet was resuspended in 100 ~1 of 2% sds, 1% mercaptoethanol sample preparation buffer and heated at 100" for 2 min. bacteria were removed by centrifugation and samples were analyzed by sds-page on 10.5% gels. controls included precipitation of polypeptides from uninfected cytosol and inability to precipitate mhv-4 proteins by monoclonal antibodies directed against an unrelated virus (lcmv). virus neutralization. virus neutralizing capacity of monoclonal antibodies was quantitated by a semimicro plaque reduction assay. ascites fluids were first centrifuged at 10,000 g heat inactivated at 56" for 30 min, then preadsorbed with l-241 cells (1 x lo6 cells per 200 ~1 of ascites) for 30 min at 4". virus was diluted to contain 50-100 pfu in 100 ~1 and mixed with 100 ~1 of diluted antibody. virus-antibody mixtures were incubated for 1 hr at 4", then plated onto l-241 cell monolayers in 24-well plates (flow laboratories, mc-lean, va.). the mixtures were adsorbed for 1 hr and then removed and replaced with culture medium. agar overlay was not necessary due to the short (~24 hr) incubation period and cell-associated nature of jhmv. neutralization endpoint titers were expressed as the reciprocal of the highest antibody dilution which gave a 50% reduction in plaque number (prd 50%). controls included ascites fluids containing neutralizing monoclonal antibodies to an unrelated virus. inhibition of spread of virus in cell cdture. the growth of mhv-4 in l-241 cells is characterized by rapid formation of large syncytia and resultant intercellular virus spread. effect of virus-specific hybridoma antibodies on spread of virus infection in cell cultures was determined by incubation of infected monolayers with monoclonal antibodies in the culture medium. l-241 cell monolayers in 24-well linbro plates were infected at 50-100 pfu per well and incubated at 37" for 1 hr. inoculum was removed and culture medium containing serially diluted monoclonal antibody prepared as described above was added at the concentrations indicated and incubation continued for 22-24 hr at which time plates were fixed and stained with 0.1% crystal violet. the inhibition titer of monoclonal antibody was expressed as the highest dilution which inhibited the number of visible plaques and syncytia by 50%. inhibitory effect of virus-specific monoclonal antibody on syncytium formation was also visualized microscopically by indirect immunofluorescence staining. after 6,9, and 12 hr of incubation in the presence of monoclonal antibody, coverslip cultures were washed three times in pbs and fixed in acetone. fluorescent staining for detection of syncytia was performed using monoclonal antibody to mhv-4 nucleocapsid protein (4b-6.2). surface labeling of mcmoclunal antibody bound to in&ected cells. cell surface binding of monoclonal antibodies was assessed both by indirect immunofluorescence and by electron microscopic examination of immunoferritin-labeled infected cells. l-241 cells were infected for 1 hr at 37" with mhv-4 in suspension at an m.o.i of 1.0, washed, then incubated at 37" for 6 hr. cells were then transferred to an ice bath, washed in cold medium, and viable cells enumerated by trypan blue exclusion. aliquots of 1 x lo6 viable cells were mixed with 100 ~1 of monoclonal ascites fluid at 1:loo dilution for 30 min at 4". cells were then washed three times with cold medium and reacted for 1 hr on ice with 50 ~1 of rabbit anti-mouse igg conjugated to ferritin (cappel laboratory, dorrington, pa.). after incubation, cells for electron microscopy were washed four times with cold medium, once with pbs, and fixed with 2.5% glutaraldehyde in 0.125 m cacodylate buffer and examined as described elsewhere (knobler et al., 1981) . for surface immunofluorescence cells were reacted with monoclonal antibody as described above, then with sheep anti-mouse igg coupled to biotin. after 30 min at 4" cells were washed then reacted with fitc coupled avidin (e-y laboratories, san mateo, calif.) for 5 min. cells were then washed and examined using a zeiss fluorescence microscope equipped for incident illumination. antibodies to mhv-4 fifty-two cell lines making monoclonal antibodies to mhv-4 were established from 243 cultures. initial screening by immunofluorescence segregated these into those which reacted with antigens at the surface of infected cells, and those which reacted with antigens expressed only in the cytoplasm. to determine the polypeptide specificity of these antibodies, we immunoprecipitated radiolabeled viral polypeptides from cytosol extracts of infected cells. under the conditions which we employed, the monoclonal antibodies fell into three groups: those which precipitated gp-1, those reacting with vp-4, and those reacting with gp-5 and vp-6. figure 1 illustrates these results for selected antibodies. correlating these data with immunofluorescence observations, we found that antibodies which stained cell surface viral antigens at 6, 12, or 24 hr reacted with either gp-1 or gp-5, vp-6 whereas antibodies which stained antigens only in the cytoplasm immunoprecipitated vp-4. under the conditions employed with [35s]methionine label, we observed little gp-2 (98,oood) surface glycopeptide; however, preliminary experiments using glucosamine label (data not shown) suggest that anti-gp-1 antibodies also react with gp-2. forty-four of the fifty-two monoclonal antibodies were analyzed for their immunoglobulin isotype using monospecific typing reagents. of these, eight were igg-1, 12 were igg-za, two were igg-zb, six were igg-3, three were iga, four were igm and nine were undetermined. the replication of murine coronaviruses is characterized by synthesis of viral rna and proteins in the cytoplasm, envelopment of the nucleocapsid, and maturation by budding into intracellular vesicles. cell fusion and resultant syncytium formation are not essential for maturation (robb and bond, 1979) ; however, cell-cell fusion between infected cells and uninfected cells may contribute to intercellular spread of infection. viral antigens at the cell surface can also serve as targets for immune attack. presence of viral giycoproteins gp-1 and gp-5 on the surfaces of infected cells was examined using two monoclonal antibodies, 5b-19.2 (anti-gp-1) and 541-5.2 (anti-gp-5). l-241 cells were examined 6 hr after infection with mhv-4 by indirect immunofluorescence and by immunoferritin labeling for surface antigen. six hours was selected for detailed study because this preceded both release of mature virus into the supernatant medium and cell death. both anti-gp-1 (fig. 2b ) and anti-gp-5 (fig. 2c ) stained infected cells at 6 hr; however, anti-vp-4 did not ( fig. 2a) . the binding of anti-gp-1 was visualized by immune electron microscopy using ferritin-conjugated rabbit anti-mouse igg (fig. 3) . antigen at the cell surface was observed in the absence of virions. the mhv-4 antigen responsible for adsorption to target cells has not been identified. in order to address this point, we selected four monoclonal antibodies demonstrated by immunoprecipitation to react with the gp-1 polypeptide, three which precipitated gp-5, and as controls, two which precipitated vp-4. globulin class, igg concentration, and immunofluorescence endpoint titer were also determined. the ability of these monoclonal antibodies to neutralize mhv infectivity is illustrated in fig. 4 . neutralization was mediated only by those monoclonal antibodies which precipitated gp-1. one anti-gp-1 (5b-93.9) which did not neutralize was of the iga class. monoclonal antibodies to the gp-5 glycoprotein and to np showed no significant neutralizing activity in the absence of complement; however, we found that the addition of guinea pig complement to the reaction mixture substantially increased the neutralizing activity of the gp-5 antibodies. in one such experiment with antibody 5a-5.2 (anti-g5) the titer of 10,000 pfu of jhmv was reduced by only 14% without complement, but by 98% with the addition of l/40 fresh guinea pig complement. in contrast, antibody 4b-11.6 (anti-gp-1) neutralized 99 and 97% of the infectivity without and with complement, respectively. neutralizing antibody titers did not always correlate with igg concentration, and this may reflect differences in avidity. other controls consisting of monoclonal antibodies to jhmv vp-4 and neutralizing antibodies to an unrelated virus (lcmv) showed no significant neutralizing activity against mhv-4. an interesting biological property of mhv-4 is its ability to spread infection to adjacent uninfected cells by fusion. this property is especially evident in certain cells such as l-241, primary macrophage, primary brain, and embryonic fibroblast cultures from susceptible mouse strains such as balb/c. other cell lines such as the 17-cl-l line of mouse fibroblasts do not demonstrate extensive fusion after infection with collins, unpublished observation) . the viral glycopeptide associated with fusion activity has not been identified. monoclonal antibodies with specificity for gp-1 and gp-5 were tested for ability to inhibit spread of infection in l-241 cells. inhibition of spread was quantitated by incubating previously infected cells in medium containing monoclonal antibodies and scoring the number of visible plaques and foci of syncytia 22-24 hr after infection. only incubation with monoclonal antibodies to gp-1 resulted in inhibition in plaque and syncytium number (fig. 5) , indicating involvement of that polypeptide in cell to cell spread of infection by fusion. titers observed were unrelated to the neutralizing antibody titer but did correlate with indirect immunofluorescence antibody titers. for example, considering the data summarized in table 1 , the relative titers of three different anti-gp-1 antibodies (5b-170.3, 5b-19.2, and 4b-11.6) in neutralization assays were less than, equal to, and greater than their respective titers in inhibition of spread of infection. measurement of antibody activity by inhibition of spread of infection was approximately lo-fold less sensitive than indirect immunofluorescence. the inhibitory effects of monoclonal antibody to gp-1 upon development of syncytia was also observed morphologically by indirect immunofluorescence on mhv-4-infected l-241 cells. monoclonal antibodies at a final dilution of l/40 were added to the medium of l-241 cultures on coverslips one hour after infection with mhv. as early as 9 hr after infection, a marked inhibition both in the numbers and size of syncytia was observed in cultures incubated with monoclonal antibody to gp-1. inhibition of development of syncytia was even more evident in such cultures 12 hr after infection (fig. 6) . monoclonal antibodies to gp-5, vp-4, and an unrelated virus (lcmv, not shown) had no effect on the development of syncytia. discussion we have prepared a library of monoclonal antibodies to mhv-4 and have identified their polypeptide specificity toward the viral proteins gp-1, vp-4, gp-5, and vp-6. these antibodies have been used in and functional assays to strated that despite this intracellular mode probe mhv infection. of maturation both viral gp-1. and gp-5 coronaviruses mature by budding into are expressed at the surfaces of infected intracytoplasmic vesicles (reviewed in cells. surface expression occurred by 6 hr, robb and bond, 1979 shown by immunoelectron microscopy to occur in areas free of morphologically distinguishable virions. this observation has several implications with respect to hostvirus interactions. first, expression of virus-specific surface components during eclipse may render the infected cell susceptible to specific antibody-dependent or cellular host defense mechanisms. thus the infected cell can potentially be eliminated prior to release of new virus. conversely, spread of mhv infection in culture and presumably in v&o is largely cell to cell, and is facilitated by the ability of viral polypeptides to mediate cell fusion resulting in syncytia formation. presence of these viral polypeptides on the cell sur-face suggests that cell fusion may begin prior to virus release. we have observed cell fusion in culture as early as 6 hr after infection, coincident with the appearance of gp-1 and gp-5 at the cell surface. sturman and co-workers (1980) have proposed that gp-5 and vp-6 are structurally associated with a transmembrane protein complex which they have termed e-l. this protein is thought to be involved in virus maturation along the endoplasmic reticulum. the ability of anti-gp-5 to bind to the infected cell surface and to the intact virion indicates that this putative transmembrane polypeptide of the coronaviruses is expressed at the cell surface independent of virus maturation and differs to had no effect at a l/20 or l/80 dilution. in this regard from the membrane (m) proteins of the orthomyxoviruses and rhabdoviruses which apparently are not exposed to the external environment (gerhard et al., 1980; compans and klenk, 1979) . we investigated the biological roles of the gp-1 and gp-5 polypeptides in initiation and spread of infection using monoclonal antibodies. neutralization tests employing several anti-gp-1, anti-gp-5, and anti-vp-4 monoclonals demonstrated that only anti-gp-1 possessed strong virusneutralizing activity. antibodies to gp-5 did not neutralize virus infectivity unless complement was added to the r,eaction. thus the virion polypeptide gp-1 probably contains the site of attachment of virion to host cell. one anti-gp-1 (5b-93.9) did not neutralize infectivity despite a binding titer of l/16,000. it is likely that this antibody recognizes an epitope on gp-1 that is unrelated to the attachment site. blocking experiments utilizing 5b-93.9 to attempt to competitively inhibit binding to known neutralizing antibodies are currently under way to clarify this question. despite their failure to neutralize, antibodies to gp-5 clearly bind to virions. the addition of fresh guinea pig complement to anti-gp-5 antibody + virus reaction mixtures resulted in virus neutralization comparable to that seen with anti-gp-1 antibody and this may reflect a lytic effect of complement on the virus. since both complement and antibody-dependent cellmediated cytotoxocity (adcc) mechanisms are operative in the whole animal, the ability of these antibodies to modify the course of disease in viva must be considered. virus-specific membrane antigens clearly also play a role in the intercellular spread of infection through the mechanism of cell fusion. fusion activity has been described for many enveloped viruses. among the paramyxoviruses this activity is the function of a distinct fusion glycoprotein (f,) (homma and ohuchi, 1973; scheid and choppin, 1974) . in the orthomyxoviruses a hydrophobic region of the hemagglutinin peplomer has fusion activity (lazarowitz and choppin, 1975; klenk et al., 1975) . when we examined our collection of monoclonal antibodies for the ability to inhibit spread of infection, only anti-gp-1 was effective, suggesting that the virion peplomer contains the active site for cell-cell co % fusion. the mechanism by which anti-gp-1 inhibits spread is not clear. anti-gp-1 may directly inhibit the molecular events of membrane fusion or may bind to a site which sterically hinders membrane penetration. although our experiments suggest that gp-5 is not directly involved in cell fusion the possibility exists that gp-5 participates in events occurring after initiation of fusion by gp-1. alternatively, expression of gp-5 at the surface of the infected cell may be merely a fortuitous occurrence. recent data (holmes et al., 1981) suggest that the gp-5, vp-6 complex e-l plays an essential role in the intracellular maturation of mhv. thus surface expression of this polypeptide may reflect a breakdown in the normal pathways of glycoprotein expression in the infected cell. clearly, monoclonal reagents will be valuable tools to probe the precise mechanisms of infection, cell-cell spread, and cell fusion by coronaviruses. a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin monoclonal antibodies to lymphocytic choriomeningitis and pichinde viruses: generation, characterization and crossreactivity with other arenaviruses protein structure of lymphocytic choriomeningitis virus: evidence for a cell-associated precursor of the virion glycopeptides monoclonal antibodies to lymphocytic choriomeningitis and pichinde viruses: generation, characterization, and crossreactivity with other arenaviruses two coronaviruses isolated from central nervous system tissue of two multiple sclerosis patients viral membranes a virus related to that causing hepatitis in mice (mhv) monoclonal antibody hybridomas: a new dimension in biological analyses a hepatitis virus of mice. &-it temperature-sensitive mutuants of mouse hepatitis virus produce a high incidence of demyelination tunicamycin resistant glycosylation of a coronavirus glycoprotein: demonstration of a novel type of viral glycoprotein trypsin action on the growth of sendai virus in tissue culture cells. iii. structural difference of sendai viruses grown in eggs and in tissue culture cells activation of influenza a viruses by trypsin treatment selective localization of wild type and mutant mouse hepatitis virus (jhm strain) antigens in cns tissue by fluorescence, light and electron microscopy continuous cultures of fused cells secreting antibody of pre-defined specificity mechanism of demyelination in jhm virus encephalomyelitis enhancement of infectivity of influenza a and b viruses by proteolytic cleavage of the hemagglutinin polypeptide a hepatitis virus complicating studies with mouse leukemia acute hepatitis associated with mouse leukemia 1. pathological features and transmission of the disease tumor antigen(s) in cells productively infected by wild type polyoma virus and mutant ng-18 identification of the biological activities of paramyxovius glycoproteins. activation of cell fusion, hemolysis and infectivity by proteolytic cleavage of an inactive precursor protein of sendai virus coronavirus jhm: intracellular protein synthesis isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid coronavirus jhm: characterization of intracellular viral rna structural polypeptides of the murine coronavirus jhm pathogenesis of demyelination induced by a mouse hepatitis virus (jhm) virus this is publication no. 2603 from scripps clinic and research foundation, la jolla, california. we thank michael b. a. oldstone for helpful discussion; ricarda defries, hannah lewicki, and linda tunison for excellent technical assistance, and susan edwards for manuscript preparation.the work described here was supported by nih grants ns 12428, ns14068, and ai 16102, and was performed during the tenure of an american heart association established investigatorship granted to m.j.b. r.l.k. is the recipient of a national multiple sclerosis society postdoctoral fellowship award. key: cord-252615-ajyv95pu authors: lu, yanfang; hou, hongyan; wang, feng; qiao, long; wang, xiong; yu, jing; liu, weiyong; sun, ziyong title: atp1b3: a virus-induced host factor against ev71 replication by up-regulating the production of type-i interferons date: 2016-05-27 journal: virology doi: 10.1016/j.virol.2016.05.013 sha: doc_id: 252615 cord_uid: ajyv95pu enterovirus 71 (ev71) infection can cause severe diseases, and is becoming increasingly common in children. in the current study, we carried out yeast two-hybrid assays to screen human proteins that could interact with 3a protein of ev71. human β3 subunit of na(+)/k(+)-atpase (atp1b3) protein was demonstrated to interact with the 3a protein of ev71. although 3a protein had no effect on the expression of atp1b3, ev71 infection resulted in elevated expression of atp1b3 in rd cell line, both on messenger rna (mrna) and protein levels. interestingly, knockdown of atp1b3 could significantly increase the replication of ev71, whereas overexpression of atp1b3 significantly suppressed the replication of ev71 in rd cells. furthermore, we demonstrated that the expression of atp1b3 could induce the production of type-i interferons. our study demonstrated that atp1b3 inhibit ev71 replication by enhancing the production of type-i interferons, which could act as a potential therapeutic target in ev71 infection. enterovirus 71 (ev71), a common pathogenic agent of handfoot-and-mouth disease (hfmd), can cause severe complications including herpangina, aseptic meningitis, encephalitis, cardiorespiratory failure, poliomyelitis-like syndrome or even fatal disease (chan et al., 2000; mcminn et al., 2001) . ev71 predominantly affects children under 5 years old, and causes severe diseases with high morbidity and mortality (sato et al., 2006) . since it was first isolated in 1969, ev71 outbreaks have occurred frequently in western pacific region countries, including china (lee et al., 2010; samuda et al., 1987) , japan (fujimoto et al., 2002) , malaysia (chan et al., 2000) , and singapore (singh et al., 2002) . the world health organization has estimated the occurrence of one million new hfmd cases in china alone between (world health organization, 2015 . to date, no effective vaccines and antiviral drugs have been available to prevent or treat ev71 infection (wu et al., 2010; yee and poh, 2015) . therefore, it is necessary to identify the host factors involved in the replication of ev71 to control the development and complications of hfmd. ev71 is a non-enveloped virus with a single-stranded, positivesense genomic rna of approximately 7.4 kb nucleotides. protein 3a, a nonstructural viral protein of 87 amino acids in length, is required for enterovirus rna replication (teoule et al., 2013) . 3a proteins play important role in enterovirus replication through the formation (with the 3cd protein) of replication organelles via remodeling of the internal cell membrane. analyses of enterovirus 3a proteins have led to the identification of several cellular partners of this protein. the 3a proteins of pv and of the related coxsackievirus b3 (cv-b3) can interact with the cellular golgi brefeldin a-resistant guanine nucleotide exchange factor 1 (gbf1). by interacting with gbf1, 3a inhibits the cellular secretory pathway. the membrane-anchored 3a protein modulates gbf1/arf1 activity, resulting in the preferential recruitment of pi4kiiiβ to sites of viral rna replication. pi4kiiiβ recruitment leads to the enrichment of virus-induced membranous organelles in pi4p, which has been shown to facilitate viral rna replication. (belov et al., 2005; belov and van kuppeveld, 2012; dorobantu et al., 2015; greninger, 2015; hsu et al., 2010; van der schaar et al., 2013) . however, the function of 3a in the rna replication of ev71 has been rarely reported. so, it is necessary to detect the mechanism of protein 3a and further explore the host factors involved in ev71 infection. thus, to identify the host factors that could interact with 3a protein of ev71 may be a potential target for the therapy of hfmd. the na þ /k þ -atpase is a transporter for na þ and k þ ions across the plasma membranes and widely distributed in prokaryotic and eukaryotic cells. the 43-kda atp1b3, a β-subunit of atpase, is localized to the q22-4 23 region of chromosome (chr) 3 (besirli et al., 1998; malik et al., 1998) . previous studies have demonstrated that atp1b3 can up-regulate lymphocyte activity and promote the production of ifn-γ, il-2, il-4, and il-10 (chiampanichayakul et al., 2002) (chruewkamlow et al., 2015) . however, whether atp1b3 can mediate the innate immune responses during ev71 infection is largely unknown. moreover, type i interferons (ifns), namely ifns-α/β, which lead to the induction of antiviral pathways within hours can be induced in virus-infected cells (wu and chen, 2014) . type i ifns have clinically been used for treatment of many viruses and tumors. thus, identifying the host factors that can induce the production of type i ifns is extremely important in controlling viral infection. in this study we screened human atp1b3 protein that could interact with ev71 3a protein using yeast two-hybrid system. we evaluated the effect of atp1b3 on the inhibition of ev71 replication. furthermore, we found that atp1b3 can induce the production of type-i ifns during ev71 infection. overall, our work demonstrated that atp1b3 might act as a potential therapeutic target in ev71 infection. full-length sequence of ev71 3a was inserted into the pgbkt7 vector for expression as a fusion protein with the gal4 dnabinding domain (gal4-bd). this plasmid was used to transform the ah109 yeast strain (clontech, palo alto, ca), and gal4-bd fused 3a was used as bait in a mating strategy for the screening of human homogenization cdna library. the homogenization cdna library was inserted into the pgadt7 vector (clontech, palo alto, ca) for expression as fusions with the gal4 activation domain (gal4-ad) and was maintained in the y187 strain of yeast (clontech, palo alto, ca). transformed ah109 and y187 yeast cells were mixed together for mating. positive clones were selected on synthetic dropout medium lacking 4 nutrients (leu/trp/ade/his). the blue colonies were kept, and the positive results were confirmed by repeating assays. cdna plasmids isolated from positive colonies were introduced into escherichia coli dh5α and sequenced. the sequences were analyzed with the blast program in ncbi. confocal microscopy experiments were described previously (choe and kirkegaard, 2004) . in brief, the transfected cells were fixed with 3% paraformaldehyde (pfa), washed with pbs and permeabilized with 0.2% triton x-100/pbs. anti-flag and anti-ha antibodies were used as the primary antibodies (1:1000 dilution), alexa fluor 488-conjugated donkey anti-mouse igg and alexa fluor 647-conjugated donkey anti-rabbit igg were used as secondary antibodies (1:500 dilution; molecular probes, abcam). dapi dyes (beyotime institute of technology, china) was used for cell nucleus stains. the cells were examined and images were captured using 100x objectives with a confocal microscope (leica sp8). the images were refined and figures were generated using adobe photoshop software (adobe systems, san jose, ca). the subgenotype c4 strain of ev71 virus (accession no: jx986738) was isolated from a throat swab with severe clinical symptoms at the wuhan medical treatment center (also wuhan infectious diseases hospital) . to prepare virus stocks, viruses were propagated on 90% confluent monolayer cells in dmem with 2% fbs as described previously (yi et al., 2011) . hek 293t and rd cells were grown in dulbecco's minimal essential medium (dmem; lonza) supplemented with 10% fetal bovine serum, 100 μg/ml penicillin and 100 μg/ml streptomycin. all cells were cultured at 37°c in a humidified atmosphere and 5% co 2 . pcmv-flag-2b, pcmv-ha and pcmv-c1 vectors were obtained from clontech (usa). the atp1b3 small interfering rna (sirna) used in this study was designed at website http://sidirect2.rnai.jp/. the atp1b3 sir-na oligonucleotide hairpins were generated with the dna oligomers 5′-uuuuggaaccucaucguugag-3′, and 5′-caacgaugagguuccaaaaua-3′. a nonspecific sirna 5′-uucuccgaacgugucacgutt-3′ and 5′-acgugacacguuc-ggagaatt-3′ was used as negative control. all sirnas were introduced into rd cells by the standard lipofection method using turbofect transfection reagent (theromo scientific) following the manufacturer's protocol. all sirnas were purchased from gema. total rna was extracted from rd cells and the supernatants by using trizol reagent (takara). total rna was reverse transcribed into cdna using moloney murine leukemia virus (mmlv) reverse transcriptase (promega) to detect transcript levels. sybr green pcr master mix was used to conduct real-time qpcr to quantify the expression levels of each target gene. the real-time pcr was performed using a lightcycler s 480 instrument (roche, switzerland). the pcr was set up under the following thermal cycling conditions: 95°c for 10 min, followed by 45 cycles of 95°c for 15 s and 60°c for 1 min. the expression levels were calculated using the comparative method for relative quantification after normalization to gapdh gene expression. primers used in rt-pcr were listed in table 1 (liu et al., 2012; liu et al., 2014) . western blot and co-immunoprecipitation analyses were performed as previously described (li et al., 2013) . briefly, after cell treatment, cells were lysed using lysis buffer (20 mm tris, ph 7.5, 150 mm nacl, 0.5% (vol/vol) nonidet-p40, 1 mm edta, 30 mm naf, 10% proteinase inhibitor mixture). lysates were mixed and precipitated with antibodies or igg and protein g-agarose beads (roche, basel, switzerland) overnight at 4°c. beads were washed five times with lysis buffer containing 0.5 m nacl before the bound proteins were eluted by boiling for 10 min in sds sample lysis buffer. protein samples were separated on polyacrylamide gels and transferred electrically to polyvinylidene fluoride membranes (millipore, billerica, usa). immunoblots were visualized with an enhance chemiluminescent detection kit (pierce, rockford, usa) by amersham imager 600 instrument (ge healthcare, fairfield, usa). after overpression at1b3, rd cells were infected with ev71 at an moi of 1. to neutralize interferon's activity, 2 μg of anti-human ifn-α1 (ab11408; abcam) and anti-human ifn-β (ab6979; abcam) were added to cell culture 3 h before ev71 infection. twenty-four hours after ev71 infection, the viral contents were quantitated by rt-pcr using ev71 specific primers. statistical data are expressed as means 7standard deviations (sd). statistical analyses were performed using prism 5 software (graphpad, san diego, ca). all other data were analyzed using student's t test. values of p o0.05 were considered as statistically significant. yeast two-hybrid system found several positive clones containing the coding sequence of human atp1b3 (genebank: bc011835.2), showing 100% nucleotide sequence identity. data analysis in ncbi indicated that the 3a protein of ev71 could possibly interact with the n-terminal of the atp1b3 protein. to further confirm the interaction between atp1b3 and 3a protein, we performed immunofluorescent co-localization analysis by laserscanning confocal microscopy. results of confocal microscopic analysis of the subcellular localization revealed that the 3a fusion protein showed the same location with the atp1b3 fusion protein on the membrane of 293t cells (fig. 1a) . to further confirm the interaction between ev71 3a and atp1b3, we first proformed exogenous co-ip test. the rd cell line was transiently transfected with a flag-tagged 3a expression vector and ha-tagged atp1b3 expression vector. following anti-flag or anti-ha immunoprecipitation, the interaction between flag-3a and ha-atp1b3 was assessed by immunoblotting the precipitates for the presence of atp1b3 or 3a. atp1b3 or 3a protein was detected in the coimmunoprecipitation reaction from cell lysates that expressed both 3a and atp1b3. to validate the interaction between the endogenous atp1b3 and 3a in the context of ev71 infection, we performed immunoprecipitation experiment in rd cells infected with ev71 or mock infection using anti-3a or anti-atp1b3. in both cases, 3a was revealed to interact with atp1b3 ( fig. 1b and c) . in order to better understand the function of atp1b3 in ev71 infection, we detected the expression levels of atp1b3 after transfection plasmid dna encoding enhanced green fluorescent protein (egfp) with and without 3a into rd cells (ev71-susceptible cells). the fluorescence of egfp was used to detect the transfection efficiency and expression levels of 3a protein ( fig. 2a) . we observed that the mrna expression levels of atp1b3 had no significant difference after the transfection of various doses of plasmid egfp-3a (fig. 2b) . the results showed that ev71 3a protein in rd cells cannot promote the expression of endogenous atp1b3. next we assessed the expression of atp1b3 during ev71 infection. the rd cells were infected with ev71 at a moi of 1 for 0, 6, 12, 24, 48 and 72 h respectively. during the infection of ev71, we found that the mrna expression levels of atp1b3 were elevated continuously in rd cells and had a significant difference compared with that in the uninfected cells. consistent with the results of mrna levels, western blot revealed that protein levels of atp1b3 were also significantly elevated after ev71 infection (fig. 3a) . on the other hand, we infected the rd cells at different mois of ev71 for 72 h and detected atp1b3 expression. we observed that both the mrna and protein levels of atp1b3 in rd cells were positively correlated with the infection doses of ev71 (fig. 3b) . thus, atp1b3 expression levels were elevated along with the increasing of infection time and doses of ev71. since ev71 infection could induce the production of atp1b3, we investigated whether atp1b3 affected the replication cycle of ev71. atp1b3 sirna and its control sirna were transfected into rd cells to knockdown atp1b3 expression and negative control, respectively (fig. 4a) . the presence of ev71 in the cell cultures was determined by quantitative real-time pcr using ev71 specific primers. the expression of ev71 3c protein was analyzed by western blot to represent the replication of ev71 in rd cells. our results showed that knockdown atp1b3 could increase both the mrna and 3c protein levels of ev71 (fig. 4b) . furthermore, pcmv-atp1b3 vector was transfected into rd cells and induced about a three-fold rise of atp1b3 expression compared with the negative table 1 primer sequences of mrna analysis by real-time pcr. nucleotide sequence (5′ to 3′) control group (fig. 4c ). we observed that overexpression of atp1b3 could inhibit the expression of both the mrna and 3c protein levels of ev71 (fig. 4d) . our results showed that atp1b3 could inhibit the replication of ev71. the type i ifns act as the key effectors in the innate immune response against rna virus infection (kuo et al., 2013; randall and goodbourn, 2008) . to explore the mechanism of the atp1b3mediated anti-ev71 response, we examined the relationship between atp1b3 expression levels and type i ifns production. the levels of ifns-α/β mrna in rd cells were detected by quantitative real-time pcr. the intracellular levels of ifns-α/β mrna in rd cells were significantly decreased after knockdown of atp1b3 expression using atp1b3 sirna (fig. 5a) . on the contrary, ifns-α/ β mrna levels were significantly increased when atp1b3 was overexpressed compared with control vector (fig. 5b ). this showed that atp1b3 promoted the production of type i ifns. since ev71 replication was reduced in atp1b3 overpression cells, it would be intriguing to determine if such reduction is associated with the elevated type i interferon expression. we infected atp1b3 overpression cells or vector controls with ev71 at an moi of 1, cultured cells with anti-human interferon-α1/β antibodies or isotype control igg, and examined the viral replication using rt-pcr assay. we found that atp1b3 overpression-induced reduction of viral growth was increased by treatment with antiinterferon-α1/β antibodies but not by its isotype control igg (fig. 5c ), indicating that neutralization of type i interferon in the cell culture by anti-interferon-α1/β antibodies restored ev71 growth in atp1b3 overpression cells. in recent years, ev71 epidemics have occurred in various provinces in china and caused an increasing proportion of severe complications and deaths in children. according to chinese center for disease control and prevention, from january 2015 to december 2015, 2.01 million cases of hfmd had been reported in mainland china, of which 124 were dead (national health and family planning commision of people's republic of china, 2016). more than 80% of the pathogens isolated from patients died from hfmd were identified as ev71. lots of research on developing antiviral drugs and vaccines were largely unsuccessful. intravenous immunoglobulin, ribavirin and pleconaril have been demonstrated the antiviral activity in vitro and in vivo, but these drugs cannot inhibit the cytopathic effect induced by ev71 (yip et al., 2013) . previous studies have shown that 3a protein of fig. 2a upper left) , 0.5 μg egfp-3a ( fig. 2a upper right) , 1 μg egfp-3a ( fig. 2a lower left) , 1.5 μg egfp-3a ( fig. 2a lower right) were transfected into rd cells respectively. in the photographs, green fluorescent represents different 3a fusion protein expression in rd cells. (b) no significant difference in atp1b3 expression level was observed after transfection of different does egfp-3a vector in rd cells. (p40.05). enterovirus plays a critical role in viral rna replication and is a potential target for antiviral therapy (deng et al., 2014; gao et al., 2015; sadeghipour et al., 2012) . itraconazole and enviroxime can target viral protein 3a and/or 3ab and suppress replication of ev71 in vitro (arita et al., 2010; gao et al., 2015) . so we conducted a yeast two-hybrid screening procedure to screen human proteins that could interact with ev71 3a protein. moreover, we confirmed human protein atp1b3 is the target of ev71 3a protein. thus it is important to explore the function of human atp1b3 during ev71 infection. previous studies have shown that some subunits of na þ /k þ -atpase can interact with various viral proteins and affect the replication of viruses. it has been demonstrated that atp1b1 subunit interacts with hcmv ul136 protein, m2 proteins of influenza a and b viruses and inhibit the virus replication (cui et al., 2011; mi et al., 2010) . atp1a1 has also been reported to inhibit entry of coronavirus into host cells by regulating the src signaling pathway (burkard et al., 2015) . atp1b3 is expressed separately from the α subunit, and plays a role in regulation of the immune response (chruewkamlow et al., 2015) . a recent published study suggested that atp1b3 can modulate the restriction of hiv-1 production and nf-κb activation in a bst-2 dependent manner (nishitsuji et al., 2015) . all of these researches suggest that atpase subunits play an important role in innate immunity against virus infection. however, the function of atp1b3 in ev71 infection has been largely unknown. in this manuscript, we found ev71 infection can induce increases in atp1b3 levels. further studies demonstrated that atp1b3 can inhibit ev71 replication along with the induction of ifn-α/ifn-β. therefore atp1b3 play an important role in antiviral immune response. type i ifns serve as key factors in the innate and adaptive immune responses against virus infection. ifns have been demonstrated effective in many viruses such as influenza a virus (stifter et al., 2016) , hepatitis c virus (melian and plosker, 2001) , coxsackievirus type a16 infection (yang et al., 2015) . some studies have demonstrated that type i ifns represent an essential innate defense mechanism for (b) after overpression atp1b3, ifn-α rna and ifn-β rna levels were both upregulated about 4.5 folds. ifn-α rna and ifn-β rna levels were determined by real-time pcr with specific primers. experiments were performed three times with similar results. all graphs represent means 7 sd. (*p o 0.05,**po 0.01). (c)to neutralize interferons, anti-human ifn-α and anti-human ifn-β monoclonal antibodies or isotype control igg were added to cell cultures 3 h before ev71 infection. twenty-four hours after ev71 infection, the cell culture samples were freeze-thawed three times. the viral contents were quantitated by rt-pcr using ev71 specific primers. data are presented as means 7 sd, n¼ 3. (* po 0.05). controlling ev71 infection both in vitro and in vivo (hung et al., 2011; liu et al., 2005; liu et al., 2012) . previous studies have shown that ev71 infection can induce ifn-β production which is dependent on the infectious dose, but the interferon is not cell type specific (lu et al., 2012) . it has also demonstrated that ev71 infection promoted the expression of son of sevenless and increased the secretion of il-1α, il-2, il-6, il-12, tnf-α, ifn-β and ifn-γ . in our current study, we found that the levels of type i ifns (ifn-α/ifn-β) were negatively associated with ev71 replication. previous studies have shown that ifn-mediated resistance to viral infection in vitro is dependent on the inhibition of viral replication (samuel, 2001) . in our manuscript we found overpression atp1b3 inhibited ev71 growth, and this inhibition could be effectively abolished by type i ifns antibodies in the cell culture. our data suggests that atp1b3 inhibits the replication of ev71by inducing the production of type i ifns. to our knowledge, this is the first time to illustrate the function of atp1b3 in innate immunity and the life cycle of ev71 at molecular level. atp1b3 can act as a potential new therapeutic target for ev71 infection and the related disorders. this finding will broaden our understanding of host defense mechanism and the pathogenesis of ev71 infection. a bifunctional anti-enterovirus compound that inhibits replication and the early stage of enterovirus 71 infection ( þ)rna viruses rewire cellular pathways to build replication organelles poliovirus proteins induce membrane association of gtpase adp-ribosylation factor the atp1b3 gene for na,k-atpase beta 3 subunit maps to mouse chromosome 9, and a related gene, atp1b3-rs, maps to mouse chromosome 3 atp1a1-mediated src signaling inhibits coronavirus entry into host cells deaths of children during an outbreak of hand, foot, and mouth disease in sarawak, malaysia: clinical and pathological characteristics of 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interferon subtypes human enterovirus 71 epidemics: what's next? emerg. health threat this work was supported by research grants from the national mega project on major infectious disease prevention (2012zx10004-207). key: cord-265895-ck7eto16 authors: baric, ralph s.; shieh, chien-kou; stohlman, stephen a.; lai, michael m.c. title: analysis of intracellular small rnas of mouse hepatitis virus: evidence for discontinuous transcription date: 1987-02-28 journal: virology doi: 10.1016/0042-6822(87)90414-4 sha: doc_id: 265895 cord_uid: ck7eto16 abstract we have previously shown the presence of multiple small leader-containing rna species in mouse hepatitis virus (mhv)-infected cells. in this paper, we have analyzed the origin, structure, and mechanism of synthesis of these small rnas. using cdna probes specific for leader rna and genes a, d, and f, we demonstrate that subsets of these small rnas were derived from the various viral genes. these subsets have discrete and reproducible sizes, varying with the gene from which they are derived. the size of each subset correlates with regions of secondary structure, whose free energy ranges from −1.6 to −77.1 kcal/mol, in each of the mrnas examined. in addition, identical subsets were detected on the replicative intermediate (ri) rna, suggesting that they represent functional transcriptional intermediates. the biological significance of these small rnas is further supported by the detection of leader-containing rnas of 47, 50, and 57 nucleotides in length, which correspond to the crossover sites in two mhv recombinant viruses. these data, coupled with the high frequency of rna recombination during mhv infection, suggest that the viral polymerase may pause in or around regions of secondary structure, thereby generating pools of free leader-containing rna intermediates which can reassociate with the template, acting as primers for the synthesis of full-length or recombinant rnas. these data suggest that mhv transcription uses a discontinuous and nonprocessive mechanism in which rna polymerase allows the partial rna products to be dissociated from the template temporarily during the process of transcription. we have previously shown the presence of multiple small leader-containing rna species in mouse hepatitis virus (mhv)-infected cells. in this paper, we have analyzed the origin, structure, and mechanism of synthesis of these small rnas. using cdna probes specific for leader rna and genes a, d, and f, we demonstrate that subsets of these small rnas were derived from the various viral genes. these subsets have discrete and reproducible sizes, varying with the gene from which they are derived. the size of each subset correlates with regions of secondary structure, whose free energy ranges from -1.6 to -77. mouse hepatitis virus (mhv), a member of coronaviridae, contains a linear single-stranded and positivesense rna of 5.4 x 1 o6 da (lai and stohlman, 1978) . the genomic rna is enclosed in a helical nucleocapsid structure constructed from multiple copies of nucleocapsid protein (n) (sturman, 1977) . virions are enveloped and contain two virus-specific glycoproteins of 180 kda (e2) and 23 kda (el) (sturman et al., 1980) . upon entry into the host cell, the genomic rna is translated into an early polymerase which directs the synthesis of a full-length negative-sense viral rna (brayton et a/., 1982; lai et al., 1982) . in turn, the negative-sense rna is transcribed by a late or altered early polymerase into the genomic rna and six subgenomic mrnas (bray-ton eta/., 1984) . these mrnas range from 0.6 to 5.4 x lo6 da in length and are arranged in the form of a nested set from the 3'-end of the genome (lai et a/., 1981) . the 5'-ends of each mrna and the genomic rna appear to contain an identical leader sequence of about 72 nucleotides, which are encoded only at the y-end of the genome (lai eta/., 1983 (lai eta/., , 1984 spaan et al., 1983) . the uv transcriptional mapping studies suggest that the mrnas are not derived from cleavage of a large precursor rna (jacobs era/., 1981) nor is there any evidence indicating that nuclear factors are required for mhv replication (brayton et a/., 1981; wilhelmsen et al., 1981) . these data suggest that conventional eukaryotic rna splicing is not involved in mhv transcription. analysis of mhv replicative intermediate and replicative form rnas suggests that a free leader rna may be involved in subgenomic mrna synthesis (baric eta/,, 1983 ). in addition, discrete small leader-related rnas of various sizes have been detected in mhv-infected cells (baric et a/., 1985) . we have also isolated a ts mutant of mhv which synthesizes only small leader rnas, but not mrnas at the nonpermissive temperature (baric et a/., 1985) . these data suggest that the mhv mrnas are synthesized discontinuously and may utilize a single or multiple free leader rnas as primers for the transcription of different subgenomic mrnas. additional proof that free transacting leader rna(s) function in mhv transcription came from the demonstration of reassortment of leader rnas between two different strains of mhv (makino et a/., 1986b) . however, the exact leader rna species which function in mhv transcription has not been identified. rna recombination occurs at very high frequencies during mixed infection with two heterologous strains of mhv (lai et a/., 1985; makino et a/., 1986a) . these data, coupled with the presence of discrete large leader-containing rnas which range from 84 to 1000 nucleotides in length in mhv-infected cells (baric et a/., 1985) suggest that discontinuous rna intermediates may be dissociated and reassert between viral rna templates to generate recombinant viruses by a copy-choice mechanism (makino eta/., 1986a). therefore, the larger leader-containing rnas present in mhvinfected cells may represent functional intermediates of rna transcription and recombination. in this paper, we have analyzed the origin, structure, and probable mechanism of synthesis of these rnas. the data suggest that mhv rna transcription may pause at sites corresponding to hairpin loops in the rna template, and they support a mechanism of discontinuous rna transcription in which rna intermediates can be dissociated and reassociated with the rna template intermittently during the course of transcription. the a59 strain of mhv was propagated in either dbt or l2 cells at 37" in dulbecco's modified mem medium supplemented with 10% fetal calf serum containing 100 pg/ml penicillin and 50 pg/ml streptomycin. infection was performed at a m.o.i. of 1 to 5, as previously described (baric er a/., 1983). preparation of mhv intracellular rna rna was extracted from infected cells by the phenol/ chloroform method between 5 and 7 hr postinfection (70% cpe) (baric et al., 1983) . following ethanol precipitation, the rna was washed once in 70% ethanol to remove excess salts and analyzed by electrophoresis on polyacrylamide gels as previously described (baric et al., 1985) . intracellular rna was extracted from infected cells as described above and applied to 15-30% sucrose gradients made in nte buffer (0.1 ili naci, 0.01 m tris-hci, ph 7.4, and 0.001 1\/1 edta) containing 0.1% sds. rna was sedimented at 45,000 rpm in an sw55ti rotor for 2 hr at 18" and 0.2-ml fractions were collected. in duplicate gradients, 28, 18, and 4 s rna were included as size markers. to isolate mhv ri rnas, the 18-40 s rna fractions were pooled and precipitated with ethanol. it has previously been shown that the ri rnas are contained within this size fraction (bark et al., 1983 (bark et al., , 1985 sawicki and sawicki, 1986) . the 4-10 s rna fractions were collected as the free small rnas. the cdna clones of mhv genomic rna used in this study are summarized in fig. 1 . clones f82 and c96 represent overlapping regions in the 5'-end of the genomic rna (shieh et a/., 1987). f82 is a 3.8-kb clone representing the very 5'end of the genome including the leader rna sequences, while clone c96 represents internal sequences in gene a. two other clones representing the internal sequences of mhv genome were also used: clone d63 contains entire sequences of genes d and e and part of genes c and f (shieh, unpublished), and clone phn42 spans the entire gene f (kindly provided by dr. heiner niemann, giessen, germany). an internal xbal fragment (0.4 kb), designated d63x, of clone d63, was used as the probe for gene d. this fragment includes nucleotides 47-375 in the mrna 4 coding sequence as reported (skinner and siddell, 1985) . the construction of subclones derived from phn 42 followed the procedures described by maniatis er al. (1982) . subclone el-500 was derived by blunt-end ligating a 509 nucleotide ddel fragment, located between nucleotides 90 and 609 in the gene f sequence (armstrong et al., 1984) into plasmid pt7-2 at the smal site (pt7-2 was purchased from amersham). subclone el-300 was derived from a 246-nucleotide fragment, located between nucleotides 534 and 780 in the el rna (armstrong et a/., 1984) and was inserted into plasmid pt7-2 at the /-/indllllsmal site. el-l was excised from phn 42 by an ecoriiscal restriction digestion. it contains the first 84 nucleotides of the mrna 6, including the 72-nucleotide leader rna sequence. this fragment was used directly for nick translation without subcloning. nick translations of cdna clones (0.5-l .o pg) were performed in 50 mlll tris-hci (ph 7.8) 5 mm mgc12, 5 mlll dlt, 50 pg/ml bovine serum albumin (bsa), 10 mm each of datp, dgtp, and l'tp, 100 j&i [a-32p]dctp (3200 ci/mmol), 5ob glycerol, 1 fig/ml dnase i (worthington), and 2 u of dna polymerase i (boehringer mannheim biochem) for 2 hr at 15". unincorporated nucleotides were removed by several precipitations with 80% ethanol. hybridizations were performed at 42" for 36 hr in 50% formamide, 10 mm na phosphate (ph 6.5) 5x ssc (1 x ssc = 0.15 a# naci, 0.015 1\/1 na citrate, ph 7.0) 1 ox denhardt's solution (1 x denhardt's solution: 0.02% each of ficoll, polyvinylpyrrolidone, and bsa) and 125 pg/ml salmon sperm dna (baric et a/., 1985) . approximately 40-60 x lo6 cpm/pg of dna were hybridized to each blot. after hybridization, the filters were washed, dried, and exposed to xar films at -80" in the presence of an intensifying screen. equivalent amounts of intracellular rna (25 or 50 rg) were separated by electrophoresis on 8% polyacrylamide gels in 0.1 m tris-borate buffer (ph 8.4) containing 1 mm edta and 6 m urea. for analysis of small rnas ranging between 30 and 150 nucleotides in length, the samples were electrophoresed at 1000 v (25-30 ma) until the bromphenol blue dye marker had migrated approximately 20 cm from the origin. to analyze larger rnas ranging between 100 and 300 nucleotides in length, electrophoresis was at 1000 v until the xylene cyanol dye marker had migrated 35-40 cm from the origin. following electrophoresis, urea was removed by twice washing the gel in ice-cold tae buffer (0.04 m tris-hci, ph 7.4, 0.02 m na acetate, and 0.001 m edta) for 15 min each, and the rna was electroblotted to zeta-probe paper (bio-rad) in cold tae. electroblotting was performed at 4" in a circulating chamber at 390 ma for 20-24 hr and an additional 2 hr at 1 a to ensure the transfer of larger rnas above 500 nucleotides in length (baric et a/., 1985; stellwag and dahlberg, 1980) . following electrotransfer, the paper was gently washed in tae and baked at 80" for 2 hr. prehybridization was performed for 36 hr at 42" in 50% formamide, 10 mm na phosphate (ph 6.5) 5x ssc, 1 ox denhardt's solution and 125 pg/ml salmon sperm dna. precise sizes of the small rnas were determined by electrophoresis of ml 3 mp 7 sequencing t-ladders in lanes adjacent to intracellular rna preparations. since electrotransfer oftentimes decreases the resolution of individual bands, complete sets of ml 3 sequencing a, t, c, g ladders were also electrophoresed with each set of rna preparations, fixed, dried, and separately exposed to xar-5 film. the ml 3-t ladders electrotransferred with rna preparations were aligned with the a, t, c, g ladders to determine the exact sizes of small rnas. computing stability of mhv rna secondary structure predictions for regions of secondary structure in mhv rna were made using the zucker rna folding program through bionet (zucker and stiegler, 1981) . stability of individual hairpin loops within the mhv sequence was calculated from the thermodynamics of adding a base pair to a double-stranded helix at 25" as reported by tinoco et a/. (1973) and modified by salser (1977) . more specifically, the energies for stacking of internal gu pairs were computed as follows: gu next to gc, -1.3 kcal/mol; gu next to au or gu, -0.3 kcal/mol. in addition, -1 .o kcai/mol was subtracted from loop structures containing u nucleotides (tinoco et al., 1973) . to determine the structure and origin of the small rnas present in mhv-infected cells, we constructed several cdna probes specific for different regions of the mhv genome. we chose three genes for this study, namely, genes a, d, and f. gene a probably encodes an rna polymerase, while genes d and f encode a nonstructural protein p14 and the el protein, respectively. since gene a represents the 5'-most region of the genome, the small rnas originating from this gene represent products of rna replication. the other two genes represent two internal genes which have been well characterized (armstrong eta/,, 1984; skinner and siddell, 1985) . the map positions of the probes used in this study are depicted in fig. 1 . the probe specific for leader region was derived from a gene f-specific clone (phn42) which contains the entire leader sequence. the fragment (el-l) containing the first 84 nucleotides of mhv mrna6 was used as the leader-specific probe in subsequent experiments (see fig. 1 b) . the clones el -500 and el -300 were used to serve as the probes for the 5'-and 3'-ends of the gene f, respectively (fig. 1 b) . the el -300 clone would not detect any leader-containing rnas smaller than 534 nucleotides. clones specific for genes a and d were derived from cdna clones as described under materials and methods. to determine the structure and origin of the small rnas present in mhv-infected cells, we first used a leader-specific cdna probe to identify small rnas terminated within or around the leader rna sequence. these small rnas should represent rna species derived from the 5'-end of genomic or subgenomic mrnas. these rna species have been detected previously (baric et a/., 1985) but their size and structure have not been precisely determined. intracellular rna was extracted from mhv-infected cells and separated by electrophoresis on polyacrylamide gels. following transfer to zeta-probe paper, the blot was probed with a leader-specific cdna probe (el-l) (fig. 1 b) . as shown in fig. 2 , several distinct small rnas ranging from 47 to 84 nucleotides were detected, similar to our previous a cbb\~~ d63pt p fig. 1. the structure of the mhv cdna probes. clone f82 contains the complete 5'-end of the genome and overlaps with clone c96. these clones were used to detect small rnas originating during rna replication (a). clones d63x, el -500, and el -300 were used to detect small rnas originating during transcription of mrna 4 (gene d) and mrna 6 (gene f) (b). probe e 1 -l was used to detect small leader rnas containing the leader sequence. the exact sizes of these clones are described under materials and methods. abbreviations used for cdna fragments: b, bamhi; d, ddel; e, ecori; h, hindlll; p. pstl; s, seal; x, xbal. report (baric et a/., 1985) . the sizes of these rna spe-larger rna species were also reproducibly detected cies were precisely determined from the accompanying by the leader-specific probe, consistent with previously t-ladder of ml 3 sequences. their sizes were repro-published results (baric et a/., 1985); however, under ducible, as evident from the rna patterns of two prep-the conditions used, they were not well resolved. these arations derived from different cell lines, although the leader-containing rnas range from 110 to more than relative amounts of the various rna species varied. 1000 nucleotides in length. to determine the structure the four smallest rna species of 47, 50, 57, and 65 of these larger leader-containing rna species, the rna nucleotides correspond in size to the lengths between blots were hybridized with cdna clone f82, reprethe 5'-end of the leader rna and a region of two weak senting 3.8 kb of the sequences at the 5'-end of the overlapping hairpin loops around nucleotides 42-56 genomic rna (shieh et al., 1987) (fig. 1 a) . several disand 52-72 ( fig. 3; table 1 ) (shieh et a/., 1987) . thus, tinct rna species ranging from 136 to larger than 281 these rna species are likely to represent rna tran-nucleotides were resolved (fig. 4a) . it is notable that scriptional products terminating within the leader se-the two independently isolated preparations of intraquences. the larger leader-containing rnas of 74 and cellular rna gave identical rna bands. when the clone 77 nucleotides, and occasionally an rna of 84 nu-c96, representing the internal region of gene a (apcleotides, also correspond to the region of a hairpin proximately 3.8 kb from the 5'-end) (fig. 1 a) , was used, loop at nucleotides 80-l 43 from the 5'-end of the ge-no distinct small rna bands were detected (fig. 4b ). nome and a postulated transcriptional termination sigthe hybridization with c96 was generally weaker than nal (uuuauaaa) for the mhv leader rna synthesis that with f82, since the c96 probe would not detect (shieh et a/., 1987) (fig. 3) ""g%h c u g'-2 (fig. 2) . the free energy of these hairpin loops is shown in table 1 . data suggest that these rnas represent the transcriptional intermediates derived from the 5'-end of the genomic rna, and that transcription from the 5'-end of the genome terminates or pauses at distinct sites, the length of these rnas correspond to the lengths between the 5'-end and the sites of potential hairpins (table 1; see discussion). the leader-containing rnas larger than 1 10 nucleotides in length detected by the leader-specific cdna probe were more heterogeneous (fig. 2) (baric et al., 1985) suggesting that multiple rna species in this size range were present. however, the probe representing the 5'-end sequences of gene a detected discrete species (fig. 4a) , suggesting that the larger leader-containing rnas may also represent rna species derived from other regions of the genome during subgenomic rna synthesis. therefore, cdna probes specific for the 5'-coding sequences of genes d and f were tested. the probe representing the 5'-end of the gene f (el-500) detected rna species of 118, 123, 127, 143/l 45, 150, 158, 164, 169 , and two groups of poorly resolved rnas ranging between 241-260 and 270-281 nucleotides in length (fig. 5a ). these rna species were different from those detected with the gene a probe (fig. 4a ). in addition, they were not detected with a probe (el-300) representing the 3'-end of the gene between nucleotides 534 and 780 (fig. 5c ). the gene d-specific cdna probe (d63x) (fig. 1 a) also detected a different set of small rna species of 117, 135, 160, 168, 205, and 218 nucleotides in length (fig. 58) . smaller amounts of rna of 173 and 199 nucleotides in length were also detected. these data suggest that transcription of subgenomic mrnas also terminates or pauses at many sites. computer models of rna folding suggest the presence of hairpin loops in regions of nucleotides 105-122, 114-141, 123-167, 193-226 , and 242-285 within the sequence of mrna6 (gene f) (fig. 6b) and in the regions of nucleotides 106-l 31, 105-l 62, and 168-204 and 21 l-235 within mrna4 (gene d) (fig. 6a) . the predicted stability of these hairpin structures is shown in table 1 . these data show that most of the small rnas detected in mhv-infected cells have termination sites within the hairpin loop structures, suggesting a possible correlation between the generation of small rnas and the presence of secondary structure in the template or product rnas. if the small rna species detected in mhv-infected cells represent specific products released from the template rna during transcriptional pausing, rather than degradation products of the mrnas, then the small rna species would be expected to be present in the ri structure which is involved in rna transcription. conversely, if rna transcription proceeds in a continuous manner, the rna species on the ri structure should be heterogeneous, without predominant rna species. to distinguish between these two possibilities, 18-40 s ri rna (baric et a/., 1983; sawicki and sawicki, 1986) was isolated and analyzed for the presence of small leader-containing rna using two probes, f82 and el -500, representing genes a and f, respectively. as shown in fig. 7 , the small rna species present on the ri rna are the same as those present in the whole cell lysates. furthermore, the 4 s rna fractions also contain an identical set of small rnas, indicating that some of the small rnas were dissociated from the ri rna. these results suggest that the small rna species are true transcriptional intermediates. previous findings in our laboratory demonstrate that rna recombination could occur at a very high frequency during a mixed infection with two strains of mhv (makino et a/., 1986a). this high frequency is reminiscent of rna reassortment described for viruses with segmented rnas (fields, 1981) and suggests that mhv replication might involve the generation of free and discontinuous rna intermediates. these intermediates could participate in rna recombination by a copy-choice mechanism. the detection of multiple discrete species of leader-containing rnas, which range from 47 to more than 1000 nucleotides in length, in mhv-infected cells further supports this model (baric et al,, 1985) . in this report, we examined the origin, structure, and mechanism of synthesis of these rnas. the data suggest that these rna species are likely to represent transcriptional pausing products of mhv rna synthesis. the pausing sites correspond, in general, to the potential hairpin loops present in the template or product rnas. this transcriptional pausing mech-anism is reminiscent of the transcriptional pausing of q/3 phage rna, in which rna polymerase pauses in regions of hairpin loop structures (mills et a/., 1978) . pausing also occurs during dna-directed dna synthesis (huang and hearst, 1981; sherman and gefter, 1976) , dna-directed rna synthesis (maizels, 1973; rosenberg et al., 1978) and in reverse transcriptase reactions (efstratiadis et al., 1975; haseltine et al., 1976) . similar to the mechanism of qj3 rna transcription, the majority of mhv small rnas terminate at the 3'-side of hairpin loop structures in the product or template strands, which have free energies ranging from -1.6 to -77.1 kcal/mol ( fig. 6 and table 1 ). the stabilities of these hairpin loops are comparable to those of q/3 (-9.8 to -23.8 kcal) and trna (-4.5 to -15.0 kcal) hairpin loops, whose existence in the rna has been demonstrated by various physical and biochem-ical methods (auron et al., 1982; gehrke et al., 1983; kramer and mills, 1981) . whether the pausing rna intermediates of qp or other pausing transcriptional model systems are dissociated from the rna template is not known; however, the data presented in this paper clearly show that at least some of the mhv rna intermediates are separated from the template rna strand. it has previously been shown that the nascent rna chains of g&i and ms2 phages are probably bound to the template strand only by short duplex regions (weissman, 1974) . it is also possible that the mhv rna polymerase is a nonprocessive enzyme, thus releasing some of the rna intermediates after pausing. currently, there is no direct evidence to prove that these free small rnas actually rebind to the rna template and participate in continuing transcription; however, the high frequency of rna recombination (makino et a/., 1986a) suggests that such rejoining does occur. the recent isolation of several mhv recombinants with multiple crossovers further strengthened this probability (keck et a/., 1987) . most interestingly, the crossover sites in the two recently isolated recombinants, a-l and a-5, have been mapped within a region of the leader sequences (nucleotides 35-60 from the 5'-end) (keck eta/., 1987) , where three small rna species were detected (fig. 2) . the isolation of these recombinants strongly suggests the functional roles of the pausing rna intermediates. it is noteworthy that, during the transduction of the proto-oncogene c-fps by a retrovirus, right-handed recombination occurred near or in stable hairpin loop structures (huang et al., 1986) . these data, coupled with the fact that reverse transcriptase "pauses" in regions of secondary structure (haseltine et al., 1976) suggest a common mechanism of recombination for retroviruses and coronaviruses, involving paused transcriptional intermediates. in addition, it has previously been shown that short oligonucleotides can act as primers for rna synthesis in vitro (minkley and pribnow, 1973; niyogi and stevens, 1965) . thus, it seems likely that the free rnas in mhvinfected cells could also be used for chain elongation. recently, our laboratory has shown that the mhv leader rna can act in vans to participate in rna transcription (makino et al., 1986b) . the four leader-containing rna species of 65-84 nucleotides in length are the most likely candidates for the primers of subgenomic mrna transcription, since they fall within the size range of the leader sequences present in the mhv subgenomic mrnas (lai et a/., 1984; spaan et a/., 1983) . it is not clear, however, whether multiple leader rnas are involved in priming mrna transcription. according to our present model of leader-primed transcription, these leader rnas would have to be cleaved before serving as primer (shieh et a/., 1987) . these rnas are likely generated due to the hairpin loop structures in nucleotides 52-72 and 80-l 43, and the intervening au-rich sequences at the 5'-end of the genome (fig. 3) (shieh eta/., 1987) . it should be noted that the pausing or termination sites of these small rnas were determined by assuming that they were generated from correct transcriptional initiation sites. although the extremely small amount of these rnas makes it impossible to resolve this issue definitively, several reasons argue for the interpretation that they were initiated from the same site: first, the leader-primed transcription mechanism of mhv ( baric et al., 1983; shieh et a/., 1987) indicates that the same primer is used for the transcription of various mrnas; second, these rna species are very reproducible in different rna preparations and are present on replicative intermediate rna; third, rna recombinants have been isolated whose recombination sites may be in an area corresponding to the termination sites of these small rnas (keck et a/., 1986) suggesting that these rnas have biological functions; and finally, correct initiation was noted in a similar study with qb phages (mills et a/., 1978) . the biochemical and biological data presented here and elsewhere (baric eta/., 1985; makino eta/., 1986a) are, thus, consistent with a mechanism of mhv transcription which is discontinuous and nonprocessive (fig. 8 ). in this model, the viral polymerase pauses in molecular cloning-a laboratory manual optimal computer folding of large rna sequence using thermodynamics and auxillaty information. nucleic acids res. 9, 133-l 48. key: cord-258327-03vk6enj authors: schultze, beate; wahn, kurt; klenk, hans-dieter; herrler, georg title: isolated he-protein from hemagglutinating encephalomyelitis virus and bovine coronavirus has receptor-destroying and receptor-binding activity date: 1991-01-31 journal: virology doi: 10.1016/0042-6822(91)90026-8 sha: doc_id: 258327 cord_uid: 03vk6enj abstract bovine coronavirus (bcv) and hemagglutinating encephalomyelitis virus (hev) from swine were found to grow to high titers in mdck i cells, a subline of madin darby canine kidney cells. virus grown in these cells was used to isolate and purify the he-protein. this protein has been shown recently to have acetylesterase activity and to function as the receptor-destroying enzyme of bcv. here we show that hev contains this enzyme, too. the glycoproteins were solubilized by treatment of virions with octylglucoside. following centrifugation through a sucrose gradient the surface proteins s and he (hemagglutinin-esterase) were obtained in purified form. after removal of the detergent by dialysis, he formed rosettes as shown by electron microscopy. the purified he protein retained acetylesterase activity and was able to function as a receptor-destroying enzyme rendering red blood cells resistant against agglutination by both coronaviruses. he protein released from the viral membrane failed to agglutinate red blood cells. however, it was found to recognize glycoconjugates containing n-acetyl-9-o-acetylneuraminic acid as indicated by a binding assay with rat serum proteins blotted to nitrocellulose and by its ability to inhibit the hemagglutinating activity of bcv, hev, and influenza c virus. the purified enzyme provides a useful tool for analyzing the cellular receptors for coronaviruses. it has been reported recently that bovine coronavirus (bcv) contains a receptor-destroying enzyme (vlasak et al., 1 988a) . this is the first example of such an enzyme present on a positive-stranded rna virus . up to then, receptor-destroying enzymes have been known as structural components only of negative-stranded rna viruses : influenza viruses and paramyxoviruses (hirst, 1950) . influenza a and b viruses as well as paramyxoviruses inactivate their receptors by means of a neuraminidase which releases terminal sialic acid from glycoconjugates (klenk et al ., 1955) . the receptordestroying enzyme of influenza c virus, on the other hand, cleaves not a glycosidic linkage but rather an ester linkage . it has been identified as an acetylesterase (herrler et at, 1985c) , which is able to release acetate from various synthetic substrates (vlasak et at, 1987 ; schauer et al., 1988) . the receptor-destroying activity, however, is due to the release of the acetyl group from position c-9 of n-acetyl-9-o-acetylneuraminic acid (neu5,9acz ) (herrler et al., 1 985c) . the 9-o-acetyl residue is crucial for the ability of influenza c virus to recognize a glycoprotein as a receptor (herrler et at, 1985c ; rogers et al., 1986) . ' to whom requests for reprints should be addressed . the intriguing observation of a sequence similarity between the glycoprotein hef of influenza c virus and a protein sequence derived from an open reading frame within the genome of mhv-a59 (mouse hepatitis virus) (luytjes et at, 1988) led to the finding that bcv has the same type of receptor-destroying enzyme as influenza c virus (vlasak et al., 1 988a) . the acetylesterase activity of bcv has been shown to be a function of the glycoprotein he (vlasak et al., 1988b) , the amino acid sequence of which has been derived from the nucleotide sequence of the cloned mrna (parker et al., 1989) . interestingly, this protein is present only on some coronaviruses, while others, such as avian infectious bronchitis virus, porcine transmissible gastroenteritis virus, and feline infectious peritonitis virus, are lacking a corresponding protein . the genome of mhv-a59 contains an open reading frame coding for an he protein, which is, however, not expressed in infected cells (luytjes et al., 1988 ; shieh et al., 1989) . other murine coronaviruses contain such a protein (sugiyama and amano, 1980 ; shieh et al., 1989) and the he protein of the strain mhv-jhm has been shown to have acetylesterase activity pfleiderer et all 1990) . we describe the isolation and purification of he from bcv and from porcine hemagglutinating encephalomyelitis virus (hev) . the activities of the purified glycoprotein are reported . schultze et al . strain l-9 of bcv was obtained from dr . rott (giessen, frg) . strain nt-9 of hev was provided by dr . hess (koblenz . frg) . mdck i cells, a subline of madin-darby canine kidney cells, were maintained as described previously (herrler et al., 1988a) . growth and purification of virus bcv and hev were grown in mdck i cells as reported recently (schultze et al., 1990) . virus was harvested from the supernatant 48 hrp .i . after clarification of the medium by low-speed centrifugation (3000 rpm, 10 min), virus was sedimented by ultracentrifugation at 25,000 rpm for 1 hr in a sw 28 rotor . the pellet was resuspended in pbs and layered on a sucrose gradient (5-50% w/w in pbs) . after centrifugation in a sw 55 ti rotor at 35,000 rpm for 40 min, the virus band was collected, diluted with pbs, and sedimented under the same centrifugation conditions . the virus pellet was resuspended in pbs and used for (i) analysis by polyacrylamide gel electrophoresis ; (ii) determination of the esterase activity ; (iii) hemagglutination and hemagglutination-inhibition assays ; and (iv) purification of the viral glycoproteins . purified coronavirus suspended in 800 µl pbs was incubated in the presence of 1 % n-octylglucopyranoside for 7 min at room temperature . after centrifugation for 10 min at 12,000 g, the supernatant was further incubated for 20 min at 4°. following centrifugation for 30 min at 30,000 rpm in a tla 100 .3 rotor, the supernatant was layered onto a sucrose gradient (10-30% w/w, on a cushion of 0 .5 ml 60% sucrose) in pbs containing 1 % octylglucoside . after centrifugation for 18 hr at 42,000 rpm in a sw55 .1 rotor, fractions of 0 .5 ml were collected from the bottom of the tube . the samples were analyzed by sds-polyacrylamide gel electrophoresis and assayed for acetylesterase activity. for hemagglutination and hemagglutination-inhibition assays, as well as for treatment of cell surface receptors, the fractions were dialyzed against pbs/h 2 0 (1 :4) . if the enzyme activity should be preserved for longer periods, the purified acetylesterase was stored at -20° in the presence of 5 mg bovine serum albumin per milliliter. hemagglutination assays were performed as described previously (herrler et al., 1 985a) . the hemag-glutination titer indicates the reciprocal value of the maximum dilution that caused complete agglutination . hemagglutination-inhibition assays were performed as described previously (herrler et al., 1 985b) . the esterase activity of purified virions or proteins was determined by incubation with 200 µg p-nitrophenyl acetate (pnpa) in 1 ml pbs at room temperature . the substrate was dissolved in 1/50 vol ethanol . using a kinetics program, the release of acetate was monitored by determining the optical density at 405 nm for 10 min at intervals of 2 min . the background level of substrate incubated in the absence of esterase was subtracted from the samples . the amount of esterase which cleaves 1 umol of p-nitrophenyl acetate in 1 min at room temperature was defined as 1 unit of enzyme . labeling of esterases with [ 3 h]dfp was performed as described previously (herrler et al., 1 988b) . purified virions or proteins in 50µl of pbs were incubated with 5 µl of 3 h-labeled dfp (3 .5 ci/mmol) on ice . after 30 min the samples were prepared for electrophoresis . analysis of proteins by sds-polyacrylamide gel electrophoresis was performed as described previously (herrler et at, 1 988a) . samples containing 200 µl of a 10% suspension of chicken erythrocytes were incubated with 100 µl of the gradient fraction containing purified he protein (see above) . prior to use the acetylesterase was dialyzed to remove octylglucoside . after incubation for 2 hr at 37°, the red blood cells were washed twice with pbs and suspended in 4 ml of the same buffer. these erythrocytes and control cells, which had been incubated with pbs, were used to determine the ha titer of bcv, hev, and influenza c virus . solid-phase assay for virus binding different dilutions of rat serum (1 :100, 1 :500, and 1 :1000 in pbs) in 2 µl were applied to nitrocellulose and air-dried . excess protein-binding sites were blocked with 10% nonfat dry milk in pbs overnight at 4°. the nitrocellulose strips were washed three times for 5 min with pbs/0 .1 % tween and incubated for 1 hr at 37° with pbs, purified acetylesterase from influenza c virus (170 mu), or 0 .1 n naoh, all subsequent steps were done at 4°. following three washes with pbs/ tween, the nitrocellulose was incubated for 1 hr with bcv, he protein from bcv (untreated or pretreated with dfp), or influenza a virus (wsn) . after being washed with pbs/tween, strips were incubated with rabbit antiserum directed against bcv (dilution 1 :1000) or against influenza a virus (pr8, dilution 1 :200) . the nitrocellulose was again washed three times and then incubated for 1 hr with biotinylated anti-rabbit immunoglobulins from donkey . after being washed, the strips were incubated with streptavidin-biotinylated horseradish peroxidase complex (1 hr) and washed again . bound bcv, he protein, or wsn was detected by incubation of the nitrocellulose with pbs, 4-chloro-1 -naphtol, and h 2 o 2 (500 :100 :1) . for negative staining, samples were applied to piooforrn-coated copper grids, stained with 2% uranylacetate, and examined in a siemens-elmiskop 101 . growth of bcv and hev in mdck i cells mdck i cells are a subline of madin-darby canine kidney cells which differs from other sublines in both functional and morphological characteristics (richardsonetal ., 1981) . strain johannesburg/1 /66 of influenza c virus was found to grow to high titers in this cell line, while another subline-mdck ii-was resistant to infection because of a lack of cell surface receptors (herrler and klenk, 1987 ; szepanski et al ., 1989) . due to the similarity of the erythrocyte receptors for bcv isolation of the acetylesterase of coronaviruses 2 2 3 and influenza c virus, we tried to grow bcv in mdck i cells . strain l-9 of bcv, which had been grown previously in bovine cell cultures (storz and rott, 1981) , was able to replicate in mdck i cells without adaptation . hemagglutination titers of 256 hau/ml were determined in the supernatant 48 hr p .i . the same growth kinetics was observed with strain nt-9 of hev, which had been grown previously in porcine cell cultures (hess and bachmann, 1978) . these titers are equally high as or higher than those reported for bcv and hev in other cell lines . both mdck-grown coronaviruses were found to have acetylesterase activity . comparing virus suspensions with the same ha titer, the ability of both viruses to release acetate from p-nitrophenyl acetate was similar, hev being somewhat more active than bcv (fig . 1 ) . as expected from previous studies with influenza c virus and bcv (muchmore and varki, 1987 ; vlasak et al., 1 988b) , the esterase activity of the mdck-grown coronaviruses was abolished after treatment with dfp . this compound inhibits serine esterases and proteases by covalently attaching to the active-site serine . up to the time of virus harvest no cytopathic changes were detectable in the monolayer of mdck i cells infected with either bcv or hev . virions released into the medium were purified by centrifugation into a sucrose gradient and analyzed by sds-polyacrylamide gel electrophoresis . as shown in fig . 2 for hev, following coomassie staining of the gel, the major bands visible are the known structural polypeptides : the nucleocapsid protein n, the membrane protein m, the surface protein s, and the hemagglutinin-esterase protein he . the latter polypeptide is detected under nonreducing conditions as a disulfide-linked dimer, (he) z . several other coronaviruses require two rounds of gradient centrifugation for a satisfying degree of purification . using the mdck cell system a single gradient centrifugation step is sufficient to obtain both bcv and hev in purified form . the acetylesterase was isolated from coronaviruses bya modification of the procedure used forthe purification of the influenza c glycoprotein (harrier et al ., 1988a) . purified hev or bcv were treated with octylglucoside to solubilize the components of the lipid envelope . the viral polypeptides n and m were sedimented by centrifugation (not shown) . the glycoproteins remaining in the supernatant (s and he) were centrifuged into a sucrose gradient containing detergent . to determine the location of the viral proteins, the gradient fractions were analyzed by sds-polyacrylamide gel electrophoresis . as shown in fig . 3 for hev, the peak of the he protein was detected in fraction 6 . igg was found to have the same sedimentation behavior (not shown) . the s-protein was well separated from he with the majority being present in fraction 3 . an identical separation was obtained with the glycoproteins of bcv (compare figs . 4 and 5) . the clear separation of both coronavirus glycoproteins was also evident when the individual fractions were analyzed for acetylesterase activity using p-nitrophenyl acetate as a substrate (fig . 4) . the peak fraction of the esterase activity of both bcv and hev coincided with the heprotein (fraction 6) ; no enzymatic release of acetate was detectable with fraction 3 . the purification procedure preserved not only the acetylesterase activity of he, but also the sensitivity of this enzyme to treatment with dfp, which binds covalently to serine hydrolases . as shown in fig . 5 , upon incubation with [3 h]dfp, purified he becomes radioactively labeled . figure 5 also fig . 4 . esterase activity of sucrose gradient fractions after centrifugation of the glycoproteins he and s from bcv (circles) and hev (squares) . compare fig . 3 for a polypeptide analysis of the gradient fractions . illustrates why the initial treatment of virus with detergent was performed in two steps with intermittent centrifugation . if the first centrifugation was omitted, part of the m-protein remained in the supernatant which during gradient centrifugation cosedimented with he (compare lanes e and f) . in order to analyze the effect of purified he-protein on cells, it was necessary to remove octylglucoside . the gradient fraction containing he was dialyzed and analyzed by electron microscopy . removal of the detergent resulted in rosette formation of the glycoprotein (shown in fig . 6 for bcv-he) as has been reported for other viral surface glycoproteins . analysis of the purified he-protein for receptorbinding and receptor-destroying activity the purified he protein was analyzed for its ability to function as a receptor-destroying enzyme . as shown in table 1 , incubation of erythrocytes with purified acetylesterase from either bcv or hev rendered the cells resistant to agglutination by both coronaviruses as well as by influenza c virus . on the other hand, agglutination by influenza a virus was not affected . this result indicates that the specificity of the coronavirus esterase as a sialate 9-o-acetylesterase is preserved during purification of the he protein . next we analyzed whether purified he has receptorbinding activity . as shown in table 2 , the gradient fraction containing he was unable to agglutinate chicken red blood cells . however, it was found to have a low hemagglutination-inhibition activity ; i .e ., purified he was able to prevent intact virions (bcv or hev) from agglutinating erythrocytes . this finding suggests that, following purification, he is still able to attach to coronavirus receptors on red blood cells . however, this interaction appears not to be sufficient for agglutination of erythrocytes . the ability of purified he to bind to neu5,9acz containing receptors was further analyzed by a solid-phase binding assay . rat serum is known to be a potent hemagglutination-inhibitor of influenza c virus (styk, 1955) as well as of bcv and hev (schultze et al ., 1990) . the inhibitory activity of rat serum is mainly due to al -macroglobulin (herrler et al., 1985b ; kitame at al., 1985) , which has been shown to contain a substantial amount of neu5,9ac z (herrler at at, 1 985c) . bcv and purified he from this virus were analyzed for their ability to bind to rat serum which had been spotted on a nitrocellulose filter . as shown in fig . 7 (left lane), attachment of bcv to rat serum could be demonstrated . purified he gave a positive result in this assay only at the highest concentration of rat serum tested (compare sections designated bcv and he) . if he was pretreated with dfp, however, to inactivate the esterase activity, binding of he protein to rat serum was as efficient as binding of whole virus (compare sections designated bcv and he-dfp) . to find out whether attachment of bcv and he-protein was mediated by neu5,9ac z , the serum proteins blotted on the nitrocellulose were preincubated with either acetylesterase or sodium hydroxide . both treatments are known to release 9-o-acetyl residues from neus, 9ac 2 . preincubation of the serum proteins at alkaline ph completely abolished binding of both bcv and he protein, while attachment of influenza a virus (strain wsn) could still be demonstrated (fig . 7 , middle lane) . pretreatment of serum proteins with acetylesterase from influenza c virus (hef) drastically reduced binding of bcv, he, or dfp-treated he protein (fig . 7, right lane) . these results indicate that purified he protein is able to recognize neus,9ac 2 as a receptor determinant for attachment to glycoproteins . binding of he is, however, less efficient than binding of whole virions and can be increased by inactivation of the esterase . coronaviruses grow most readily in cells from their natural host, although adaptation to cells from other species is possible (siddell at al., 1982) . in our attempts to set up a cell culture system for two hemagglutinating coronaviruses, bcv and hev, it turned out that both viruses can be grown to high titers in mdck i 7 . binding of bcv, he-protein from bcv, and influenza avirus (strain wsn) to rat serum proteins . after different dilutions (1 :100, 1 :500, and 1 :1000, from top to bottom for each binding assay) of rat serum were blotted to nitrocellulose, the serum proteins were incubated with pbs (left lane), sodium hydroxide (middle lane), or acetylesterase from influenza c virus (hef, right lane) . the samples were then incubated with virus (bcv, wsn) or purified he-protein, which had been either untreated (designated he) or pretreated with dfp (designated he-dfp) . binding was detected by an enzyme-linked immunoassay using rabbit antiserum directed against bcv or influenza virus . cells without prior adaptation . this subline of madin-darby canine kidney cells has been shown recently to be sensitive to infection by strain johannesburg/1/66 of influenza c virus (szepanski et at, 1989) . this virus initiates infection of cultured cells by attaching to neu5,9acz containing receptors, which area major determinant of the cell tropism of influenza c virus (harrier and klenk, 1987) . neu5,9ac2 serves also as a receptor determinant for attachment of bcv and hev to red blood cells (vlasak et al., 1988a ; schultze et at, 1990) . it remains to be shown whether the same type of receptors is also used for infection of cells . another question to be answered is whether the restricted tropism of coronaviruses is determined by the cellular receptors or at a later stage of the infectious cycle . with mhv-a59 evidence has been presented indicating a crucial role of cellular receptors for the cell tropism . this virus strain has been reported to use a 100 to 110-kda protein as a receptor (boyle et al ., 1987) . mdck cells provide a promising system to analyze the initial stage of infection with other coronaviruses such as bcv and hev, which appear to recognize different receptors on the cell surface . this cell line is also known for its polarized organization, which is reflected -among other characteristics-by a polarity of the virus infection . with vesicular stomatitis virus it has been shown that virus entry and release is restricted to the basolateral domain of the plasma membrane . influenza a virus, on the other hand can infect mdck cells from both the apical and the basolateral side, whereas budding of the virus is restricted to the apical portion of the plasma membrane (rodriguez-boulan and sabatini, 1978 ; fuller et at, 1984) . coronaviruses are known to bud into intracellular vesicles (tooze at at, 1984) . it will be interesting to analyze how viruses with this type of maturation are released by polarized cells . the amount and the purity of both bcv and hev grown in mdck i cells were sufficient to attempt the isolation of the viral glycoproteins . using the protocol described it was possible to get a complete separation of the coronavirus proteins he, 5, and m . a similar method using triton x-100 rather than octylglucoside had been used previously to purify the glycoproteins of hev (callebaut and pensaert, 1980) . as triton x-1 00 cannot be removed by dialysis, these authors used butanol precipitation to obtain detergent-free glycoprotein . he prepared in this way was unable to agglutinate red blood cells . at that time the esterase activity as well as the ability of this virus to recognize neu5,9ac2 as a receptor determinant was unknown . using octylglucoside as a detergent we obtained both he and s in purified form . the isolated s-protein was also subject to a biochemical and functional analysis (manuscript in preparation) . the sedimentation behavior of the isolated he protein was similar to that of igg . considering the molecular weight of the monomeric glycoprotein (parker et at, 1989) , a dimeric structure is suggested for the purified he protein . dinners of he are also detected following polyacrylamide gel electrophoresis under nonreducing conditions (see fig . 2 ), indicating that the two monomers are held together by disulfide bonds . we cannot, however, exclude the possibility that the functional protein is embedded in the lipid envelope as a multimeric structure, e .g ., a tetramer, which is dissociated into dimers upon detergent treatment and/or sucrose gradient centrifugation . for the g-protein of vesicular stomatitis virus it has been reported that gradient centrifugation results in the dissociation of the trimeric glycoprotein into monomers (doms et al ., 1987) . of the two functions which have been assigned to the he-protein (hemagglutinin and esterase) the esterase could be detected easily in the purified protein . the acetylesterase activity was evident both in the color test with the synthetic substrate pnpa (fig . 4) and in the ability of he to inactivate the erythrocyte receptors for bcv, hev, and influenza cvirus (table 1 ) . the hemagglutinin function of he could not be demonstrated with the purified glycoprotein . however, he was still able to recognize neu5,9ac z containing receptors as indicated by the binding-assay with rat serum proteins (fig . 7) and bythe low hemagglutination-inhibition activity of the purified glycoprotein (table 2 ) . this interaction was, however, not sufficient for agglutination of erythrocytes . this situation is reminiscent of influenza c virus, the purified glycoprotein (hef) of which also has esterase and hemagglutination-inhibition but no hemagglutinating activity (herrler et al., 1 988a) . the purified hemagglutinin of influenza a viruses, on the other hand, has been shown to form rosettes which are able to agglutinate red blood cells (laver and valentine, 1969) . the failure of purified he protein to cause hemagglutination is not due to a lack of rosette formation (fig . 6) . the receptor-destroying activity of he counteracts the receptor-binding activity . this is evident from the result of the solid phase binding assay . binding of bcv-he was found to be more efficient after inactivation of the esterase by dfp (fig . 7) . the binding assay has not been applied to hev-he, but because of the similarity of both he proteins in other tests (see figs . 1-5 and tables 1 and 2) it is likely that the results obtained with bcv-he are also valid for hev-he . dfp-treated he was still unable to agglutinate red blood cells . there was, however, some interaction between erythrocytes and the purified coronavirus glycoprotein . erythrocytes incubated with dfptreated he did not pour, when the microtiter plate was placed in a semivertical position (not shown), which is in contrast to control cells . the lack of ha activity in the purified he protein might be due to a conformational change during the purification procedure, which affects the receptor-binding activity more than the receptor-destroying activity . another possibility is that the individual glycoproteins have only a low affinity for neu5,9ac z containing receptors . in this case the multivalent attachment of he-proteins present in the viral membrane may increase the binding activity and enable agglutination of erythrocytes . in any case, the purified acetylesterases of bcv and hev are useful analytical tools to study the importance of neu5,9ac z in general, and specifically the cellular receptors for coronaviruses . the technical assistance of birgit doll and heldrun will is gratefully acknowledged . we thank drs . r . rot : and r . g . hess for providing genetic resistance to mouse hepatitis virus correlates with absence of virus-binding activity on target tissues characterization and isolation of structural polypeptides in haemagglutinating encephalomyelitis virus role for adenosine triphosphate in regulating the assembly and transport of vesicular stromatitis virus g protein trimers vesicular stomatitis virus infects and matures only through the basolateral surface of the polarized epithelial cell line neuraminic acid is involved in the binding of influenza c virus to erythrocytes rat a,-macroglobulin inhibits hemagglutination by influenza c virus the receptor-destroying enzyme of influenza c virus is neuraminate-o-acetylesterase the surface receptor is a major determinant of the cell tropism of influenza c virus the glycoprotein of influenza c virus is the hemagglutinin, esterase and fusion factor . l serine 71 of the glycoprotein hef is located at the active site of the acetylesterase of influenza c virus erbrechen and kommern der ferkel : vorkommen and verbreitung in soddeutschland the relationship of a new strain of virus to those of the mumps-ndv-influenza group isolation and characterization of influenza c virus inhibitor in rat serum uber die enzymetische wirkung von influenza-virus morphology of the isolated hemagglutinin and neuraminidase subunits of influenza virus sequence of mouse hepatitis virus a59 mrna2 : indications for rna-recombination between coronaviruses and influenza c virus selective inactivation of influenza c esterase : a probe for detecting 9-o-acetylated sialic acids cloning and in vitro expression of the gene for the e3 haemagglutinin glycoprotein of bovine coronavirus functional analysis of the coronavirus mhv-jhm surface glycoproteins in vaccinia virus recombinants identification of two strains of mdck cells which resemble separate nephron tubule segments asymmetric budding of viruses in epithelial monolayers : a model system for study of epithelial polarity influenza c virus uses 9-0-acetyl-n-acetylneuraminic acid as a high affinity receptor determinant for attachment to cells isolation and characterization of sialate 9(4)-0-acerylesterase from influenza c virus hemagglutinating encephalomyelitis virus attaches to n-acetyl-9-0-acetylneuraminic acid-containing receptors on erythrocytes: comparison with bovine coronavirus and influenza c virus identification of a newtranscriptional initiation site and the corresponding functional gene 2h in the murine coronavirus rna genome the structure and replication of coronaviruses coronaviruses : structure and genome expression reactivity of antibodies in human serum with antigens of an enteropathogenic bovine coronavirus non-specific inhibitors in normal rat serum for the influenza c virus hemagglutination and structural polypeptides of a new coronavirus associated with diarrhea in infant mice analysis of a mutant of influenza c virus with a change in the receptor s pecificity replication of coronavirus mhv-a59 in sac-cells : determination of the first site of budding of progeny virions the influenza c virus glycoprotein (he) exhibits receptor-binding (hemagglutinln) and receptor-destroying (esterase) activities human and bovine coronaviruses recognize sialic acid containing receptors similar to those of influenza c viruses the e3 protein of bovine ooronavirus is a receptor-destroying enzyme with acetylesterase activity biosynthesis, structure, and biological activities of envelope protein gp 65 of murine coronavirus key: cord-259095-mfptcw8t authors: lu, yiqi; denison, mark r. title: determinants of mouse hepatitis virus 3c-like proteinase activity date: 1997-04-14 journal: virology doi: 10.1006/viro.1997.8479 sha: doc_id: 259095 cord_uid: mfptcw8t abstract the coronavirus, mouse hepatitis virus strain a59 (mhv), expresses a chymotrypsin-like cysteine proteinase (3clpro) within the gene 1 polyprotein. the mhv 3clpro is similar to the picornavirus 3c proteinases in the relative location of confirmed catalytic histidine and cysteine residues and in the predicted use of q/(s, a, g) dipeptide cleavage sites. however, less is known concerning the participation of aspartic acid or glutamic acid residues in catalysis by the coronavirus 3c-like proteinases or of the precise coding sequence of 3clpro within the gene 1 polyprotein. in this study, aspartic acid residues in mhv 3clpro were mutated and the mutant proteinases were tested for activity in anin vitro transcleavage assay. mhv 3clpro was not inactivated by substitutions at asp3386(d53) or asp3398(d65), demonstrating that they were not catalytic residues. mhv 3clpro was able to cleave at a glutamine–glycine (qg3607-8) dipeptide within the 3clpro domain upstream from the predicted carboxy-terminal qs3635-6cleavage site of 3clpro. the predicted full-length 3clpro (s3334to q3635) had an apparent mass of 27 kda, identical to the p27 3clpro in cells, whereas the truncated proteinase (s3334to q3607) had an apparent mass of 24 kda. this 28-amino-acid carboxy-terminal truncation of 3clpro rendered it inactive in atranscleavage assay. thus, mhv 3clpro was able to cleave at a site within the putative full-length proteinase, but the entire predicted 3clpro domain was required for activity. these studies suggest that the coronavirus 3cl-proteinases may have a substantially different structure and catalytic mechanism than other 3c-like proteinases. the role of aspartic acid or glutamic acid residues in 3clpro activity is less well understood. mutagenesis of the coronavirus, mouse hepatitis virus, strain a59 asp/glu residues of 3c and 3c-like proteinases suggests (mhv-a59), contains a chymotrypsin-like proteinase that they might not participate as catalytic residues in all within the 750-kda gene 1 polyprotein ( fig. 1) (lu et al., cases. aspartic/glutamic acid residues have been shown 1995). the 3c-like proteinases of the coronaviruses to be essential for proteinase activity of tobacco etch mhv-a59, infectious bronchitis virus (ibv), and the huvirus (tev), poliovirus, and human rhinovirus 14 (hrvman coronavirus 229e (hcv-229e) are encoded in a con-14) (gorbalenya and koonin, 1993) . the positioning of served region of orf 1a (boursnell et al., 1987; gorbahis, cys, and glu residues in the hrv-14 3cpro has been lenya et al., 1989; lee et al., 1991; herold et al., 1993). shown to be very similar to that of cellular trypsin by mhv, hcv-229e, and ibv encode 3clpro molecules with analysis of the hrv crystal structure (matthews et al., apparent masses of 27, 34, and 35 kda, respectively, 1994) . in contrast, analysis of the crystal structure of as determined by sds-page analysis (lu et al., 1995; hepatitis a virus 3cpro indicates that asp84 most likely ziebuhr et al., 1995; tibbles et al., 1996) . the classificadoes not participate directly in catalysis (allaire et al., tion of the coronavirus proteinases as ''3c-like'' is sup-1994) . more directly relevant to mhv, it has been shown ported by mutagenesis studies of predicted catalytic cysthat mutagenesis at glu residues in ibv 3clpro does teine or histidine residues. we have demonstrated that not abolish activity of the expressed proteinase (liu and mutations at his 41 or cys 145 of mhv 3clpro abolish probrown, 1995) . teolytic activity. similar results have been obtained for several 3clpro cleavage sites within the gene 1 3clpro of ibv and hcv-229e, confirming the essential polyprotein recently have been defined for mhv, ibv, nature of these residues and demonstrating that his and and 229e. the experimentally confirmed coronavirus cys residues are in positions similar to those of the picor-3clpro cleavage sites have a leucine, isoleucine, or navirus 3c proteinases (lu et al., 1995; liu and brown, valine at position p2, glutamine at position p1, and 1995; ziebuhr et al., 1995; tibbles et al., 1996) . serine or alanine at position p1 (liu and brown, 1995; lu et al., 1995 lu et al., , 1996 ziebuhr et al., 1995; tibbles et the amino acid sequences of the coronaviruses were obtained from genbank; mhv (x73559) (bonilla et al., 1994) , ibv (m94356) (boursnell et al., 1987) , hcv-229e (x69721) (herold et al., 1993) , and tgev (z34093) (eleouet et al., 1995) . deduced amino acid sequences of coronavirus 3clpro domains were compared using a pam 250 scoring matrix and a word size of 2 (macvector 4.5.3, ibi-kodak) (fig. 2) . numbering of mhv amino acid sequences was from the beginning of orf 1a. numbering of amino acid residues within mhv 3clpro is based the linear schematic of the mhv genome shows the organization of ser 1 (lu et al., 1995) site-directed mutagenesis of pgpro with bovine chymotrypsin (gorbalenya and koonin, 1993) , 3cpro of human rhinovirus 14 (matthews et al., 1994) , and hepatitis a virus asp53 and asp65 were mutagenized by the chame-3cpro (allaire et al., 1994) . black bars reflect the number of amino acid leon double-stranded, site-directed mutagenesis kit per residues in the proteinase. the proteins are aligned at the confirmed the manufacturer's instruction (stratagene). two primers cys or ser catalytic residues to show the carboxy-terminal extent of the protein. catalytic asp residues of hrv-14 and chymotrypsin have were simultaneously annealed to the template. one sebeen confirmed by crystallography. asp53 and asp65 of mhv are prelection primer changed one nonessential unique restricdicted catalytic residues. lqs indicates cleavage sites at amino termition site alwni on the pgpro vector to a new restriction nus (confirmed) and carboxy terminus (putative) of 3clpro. site. the other primer encoded for a specific mutation. after annealing and extension, all new plasmid dna was incubated with the restriction enzyme alwni. the dialanine at p2 and glycine at p1 (lee et al., 1991) . the gested mixtures were then transformed into repair-defiprecise determinants of 3clpro cleavage site seleccient xlmuts cells, and the resultant colonies were isotion remain to be determined. finally, comparison of lated. the mutant plasmids were then purified and the coronavirus 3clpro sequences with those of other digested with alwni again, and the resultant dna digesproteinases suggests that they may differ from other tion was transformed into xl1-blue cells. all specific muviral and cellular proteinases in their size and structations were confirmed by bidirectional sequencing ture. the coronavirus 3clpro domains contain signifi-(sequenase ii, u.s. biochemicals, per the manufacturer's cantly more amino acids downstream from the putative instructions). substrate binding site than other 3c or 3c-like proteinases ( fig. 1 ) (gorbalenya and koonin, 1993) . the role constructs expressing full-length and truncated of this additional region of polypeptide in structure or versions of 3clpro activity of the coronavirus proteinases is not known. in this study, we demonstrate that aspartic acid resi-the ser3334 to gln 3607(pg-s/fq) and ser3334 to dues in mhv 3clpro are not required for catalytic activity. gln3635(pg-s/lq) fragments were obtained by pcr am-in addition, we show that the mhv 3clpro is able to plification of the region between nt 10212 and 11034 and cleave at a gln-gly cleavage site upstream from the prethat between nt 10212 and 11117, respectively (fig. 5 ). dicted carboxy terminal gln-ser cleavage site and within the pg-s/fq left primer with an added on xbai restricthe proteinase itself. the 3clpro extending from the contion site (5-attctagatgtctggtatagtgaagatgg firmed amino-terminal serine to the internal glutamine/ tgtcg-3) and pg-s/fq right primer with an added on glycine cleavage site is inactive in vitro. in contrast, the hindiii restriction site (5-taaaataagctt tcactggaa-''full-length'' protein extending to the predicted glutamine/ tccagaatgcagcct-3) were used to prime dna synserine cleavage site is identical in size to 3clpro exthesis from the pgpro construct. the pcr products were pressed in vitro and in virus-infected cells and is an digested by xbai and hindiii for 2 hr and then run on an active proteinase. thus, it appears that the entire pre-0.8% low-melting-point agarose gel. the product band was excised and ligated into the ecori and hindiii sites dicted coding region is required for 3clpro activity. 3. with a pam 250 scoring matrix. the mhv-a59 his41 and cys145 residues, and the corresponding residues of ibv, tgev, and 229e are shown in boldface letters. the locations of aspartic acid residues (asp53 and asp65) of mhv-a59 are shown by asterisks. other conserved asparagine (n95) and aspartic/glutamic acid residues (d110) are indicated by a dot. residues predicted to be involved in substrate binding (thr135 and his163) are indicated by a diamond. the solid arrowhead indicates the experimentally confirmed amino-terminal cleavage site of the mhv and 229e 3clpro (lu et al., 1995; ziebuhr et al., 1995) . the open arrowhead indicates the predicted carboxyl terminal lq_s/a cleavage sites of the proteinases (gorbalenya et al., 1989; lee et al., 1991; gorbalenya and koonin, 1993) . the location of the fq_g sequence in mhv is indicated as an underlined arrowhead. numbering of mhv his41, cys145, asp53, asp65, asn95, and glu110 residues is based on identifying ser 3334 of the orf 1a polyprotein as ser1 of 3clpro. mhv-a59 amino acid numbers were derived from the submitted nucleotide sequence of bonilla, et al. (1994) . ''iisvkes'' is a seven-amino-acid region present only in the ibv sequence. of pgem-3zf(0) (promega) behind the t7 promoter which in vitro transcription and translation constituted pg-s/fq. pgopt-s/fq was similarly con-recombinant plasmids were transcribed and transstructed using a left primer with an optimal atg (5lated using a coupled in vitro transcription/translation gggcgaattcgccaccatgagtggtatagtgaagat rabbit reticulocyte lysate system (tnt, promega), as pre-ggtgtcg-3). pc-s/fq was constructed by using a left viously described (lu et al., 1995 (lu et al., , 1996 . approximately primer with an added ncoi restriction site (5-tcatcc-0.5 mg of plasmid dna was incubated at 30њ with 12.5 atggcctctggtatagtgaagat g-3) and a right ml tnt lysate, 1 ml tnt reaction buffer, 0.5 ml t7 rna primer with an added ecori restriction site (5-aatttpolymerase, 20 units rnasin, 0.5 ml 1 mm methionine-gaattcactggaatccagaatgcagcct-3). the fragfree amino acid mixture, and 20 mci [ 35 s]methionine in ment was then subcloned into pcite (novagen). pc-s/ a final volume of 25 ml. samples were taken at various lq was similarly constructed by using a left primer with time points and electrophoresed on an sds 5-18% gradian added ncoi restriction site (5-tcatccatggcctctent polyacrylamide gel (sds-page). ggtatagtgaagatg-3) and a right primer with an added ecori restriction site (5-tgtgcg aattcactg-trans cleavage assay tagcttgacaccagcta-3). the fragment was then subcloned into the ncoi and ecori sites of pcite (nova-inactive site-directed mutants of pgpro (pgproh41g or pgproh41q) were translated in the presence of [ 35 s]-gen) behind the t7 promoter. methionine. the parental pgpro construct, plasmids en-asp 3386 (d53) was conserved as either asp or glu among the four viruses. glu 3443 (e110) of mhv was conserved coding the predicted full-length 3clpro (pc-s/lq), and plasmids encoding the truncated forms of 3clpro (pg-as glu or asp, and asn 3428 (n95) of mhv was identical in all four coronavirus sequences; however, the location s/fq, pgopt-s/fq, and pc-s/fq) were transcribed and translated in the presence of nonradiolabeled l-methio-of these residues relative to the essential his and cys residues makes them less appealing as potential cata-nine. after 40 min, transcription and translation were terminated by the addition of rnase (10 mg/ml) and cyclo-lytic residues. comparison of the coronavirus 3clpro amino acid se-heximide (5 mg/ml) for 5 min. following termination of transcription and translation, labeled mutant and unla-quences with chymotrypsin confirmed the additional amino acids between the putative substrate binding resi-beled 3clpro reaction lysates were mixed 1:1 and incubated for an additional 135 min. the reaction mixtures due his 3496 (h163) and the probable carboxy-terminal qs 3635-6 cleavage site of 3clpro. the comparison of the were checked for residual expression and processing from the pgpro construct by the addition of [ 35 s]-four coronavirus 3clpro sequences revealed two potential cleavage sites present only in mhv, qs 3554-5 , and methionine to an aliquot of the unlabeled reaction mixture after treatment with rnase and cycloheximide and qg 3607-8 . overall, the comparison of the coronavirus sequences indicated that there was variation among the incubation for an additional 135 min. all products were analyzed by electrophoresis by sds gradient page, fol-proteinases in the location of potential catalytic residues and cleavage sites. lowed by fluorography. mutagenesis of aspartic acid residues in vitro transcription and translation were performed based on the analysis of the protein alignments, we in a total volume of 200 ml with 8.0 mg pgpro dna in the chose asp53 and asp65 residues for mutagenesis studpresence of 160 mci [ 35 s]methionine (dupont nen), 400 ies. asp65 has been considered the most likely candi-mci [ 3 h]valine (amersham), or 160 mci [ 3 h]leucine (amerdate for a third residue to be involved in catalysis. asp53 sham) for 120 min at 30њ. the products were separated was in a less favorable position relative to the his, but on 5-18% gradient polyacrylamide gels, transferred to a was conserved among the coronaviruses and provided polyvinylidene difluoride (pvdf) membrane at 50 v at 4њ a good control. in addition, studies of other viruses have for 6 hr in transfer buffer containing 25 mm tris-base, demonstrated that deviation from predictions of active 192 mm glycine, and 10% (v/v) methanol. after transfer, residues is not uncommon. the construct used for these the pvdf membrane was air dried and exposed to x-ray studies (pgpro) encoded amino acids 3239-3687 of film. radiolabeled proteins were identified by autoradiog-mhv gene 1, including 3clpro (3334-3635) and portions raphy, and the corresponding bands were excised from of the flanking domains (fig. 3a) . we have previously the pvdf membrane and subjected to amino-terminal shown that translation of pgpro in vitro results in a presequencing on an abi 470 sequencer. the amino acid cursor polypeptide from which active 3clpro is autoprofraction from each cycle was quantitated in a beckman teolytically cleaved and that 3clpro has an apparent scintillation counter. mass of 27-29 kda (p27) following sds-page (lu et al., 1995) . liberation of p27 3clpro was therefore used results as a marker of proteolytic activity of proteins expressed from different constructs in vitro. the wild-type proteinase construct (pgpro) and mutant proteinase domains proteinase constructs were transcribed and translated in a rabbit reticulocyte lysate (fig. 3b) . the proteinase predictions of catalytic residues of the coronavirus 3clike proteinases have not strongly predicted aspartic or expressed from pgpro was able to process p27 3clpro (fig. 3b, lane 1) , whereas the proteinase with the his41 glutamic acid residues. comparison of the deduced amino acid sequences of 3clpro from the coronaviruses to gln mutation (h41q) did not cleave p27 (fig. 3b , lane 2). mutation of asp65 to pro or ala (d65p and d65a) mhv-a59, ibv, hcv-229e, and tgev revealed no completely conserved asp or glu residues at positions analo-resulted in a proteinase with activity comparable to that expressed from wild-type pgpro (fig. 3b, lanes 3 and 4) . gous to catalytic asp or glu residues of other 3c or 3clike proteinases (fig. 2) . there was relative conservation substitution of asp53 by glu (d53e) did not affect 3clpro activity (fig. 3b, lane 5) , whereas the substitution of of asp, glu, or asn among the coronaviruses at the residue analogous to asp 3398 (d65) of mhv. it has been asp53 by pro (d53p) impaired processing of p27 approximately 70% relative to pgpro (fig. 3b, lane 6) . the d53p shown that the analogous residues within the ibv 3clpro at asp 2841 or asp 2843 (d62 or d64) are not required for change might be expected to cause a change in the proteinase structure with a concomitant alteration of ac-proteolytic activity (liu and brown, 1995). the mhv pgpro in the presence of leupeptin blocked cleavage of p27 and also completely blocked processing of the small polypeptide fragment (fig. 4, lanes 4-5) . the small cleavage fragment indicated by the arrow was consistently seen when pgpro or proteolytically active mutants were translated, but not when proteolytically inactive mutants were expressed (fig. 4b) . the cleavage fragment was detected following translation of pgpro, h127q, h127m, and c142r (fig. 4b, lanes 1, 2, 3, and 4, respectively) , all of which also processed p27. in contrast, no small fragment was seen after translation of c145g or h41q (fig. 4b, lanes 5 and 6) , both of which are inactive in p27 processing. together these results indicated that this cleavage fragment was processed by products expressed from the proteinase constructs in vitro, rather than by proteinases in the reticulocyte lysate. the smallest proteolytic fragment was used for amino terminus radiosequencing since it was the most discrete and abundant. the pgpro construct was transcribed and translated in the presence of [ 3 h]leucine, the peaks of radioactivity were consistent with leucine and asp65 were expressed in a combined transcription and translation at residues 5 and 10, cysteine at residue 8, and valine lysate as previously described (lu et al., 1995) . samples were taken at residue 9. the only cleavage site within the pgpro at 120 min for analysis by 5-18% sds gradient page. the wild-type pgpro construct and the his41 to gln mutant (h41q) were used as expression product that could result in a product with controls. d65a refers to an ala substitution at asp53; other constructs this pattern was gln-gly 3607-8 . are similarly labeled. mass markers are to the right of the gel and the the qg 3607-8 was not conserved in any of the other location of p27 is shown to the left of the gel. processing of p27 coronaviruses and previously had not been predicted as by 3clpro expressed from pgpro was considered as 100%, and the a cleavage site for 3clpro. radiosequencing with three percentage of proteinase activity of each expressed protein is shown beneath the lane markers. different amino acids confirmed specific cleavage between glutamine 3607 and glycine 3608 by the in vitro translated proteinase. we could not define the presumed cartivity; however, the d53p substitution diminished but did boxy-terminal fragment containing the predicted qs 3635-6 not abolish activity, indicating that d53 was not an indiscleavage site, possibly due to the compression of propensable residue. it was interesting that the d65p teins in this region (5.8 kda) of the gel by the nonlabeled change did not diminish proteolytic activity, suggesting globin protein from the lysate. additionally, the protein that even a major change at this location was inconsefrom the construct may have been targeted for rapid degquential for liberation of p27 3clpro in the in vitro system. radation. we also did not detect any prominent alterna-deletion of asp53 (d53del) resulted in complete loss of tive form of p27 3clpro. since we do know the order of proteinase activity (lane 7). this was not surprising since cleavages or pattern of precursors expressed from pgpro such a change might be expected to significantly alter it has not been possible to determine when the fq/g the structure of the proteinase. these experiments demsite is cleaved. direct comparison of these cleavage sites onstrated that neither asp53 nor asp65 was directly inwill require constructs expressing single cleavage sites volved in catalysis with his41 or cys145. to determine specificity. identification of a 3clpro cleavage site truncation of 3clpro and trans cleavage activity during in vitro translation of pgpro several proteins in vitro with apparent masses of less than 14.3 kda were seen along with p27 (fig. 4) . the pulse-label expression (fig. since 3clpro was able to cleave upstream of the predicted qs 3635-6 cleavage site, we determined whether 4a) showed that the smallest of these polypeptides appeared concurrently with p27 3clpro but then decreased ser 3334 to gln 3607 was the entire coding region for the active p27 3clpro protein detected in virus-infected cells over a 4-hr period (fig. 4, lanes 1-3) . translation of and during in vitro translation of pgpro (fig. 5) . the amino proteins by incubating the nonradiolabeled translation products of these constructs with radiolabeled sub-acid sequence extending from ser 3334 to gln 3635 would predict a protein of with a calculated mass of 33 kda, strate expressed from the inactive proteinase mutant pgproh41g (his to gly) (fig. 5b, lane 6) . the ''full-whereas cleavage at gln 3607 would predict a protein of 30 kda in mass, somewhat closer in size to the apparent length'' 27-kda 3clpro expressed from the ser 3334 -gln 3635 construct was able to cleave the pgproh41g mass of p27 (fig. 5a ). we constructed a panel of plasmids containing cdnas encoding amino acids from expressed protein in trans (fig. 5b, lane 8) , whereas the 24-kda truncated 3clpro expressed from the s 3334 -gln 3607 or s 3334 -gln 3635 (fig. 5a) . the cdnas were expressed in a variety of plasmids, using either the first ser 3334 -gln 3607 constructs did not process the mutant protein (fig. 5b , lanes 9, 10, and 11). this result demon-natural aug (pg-s/fq) or an optimized aug before ser 3334 (pgopt-s/fq). we also used vectors containing strated that a 28-amino-acid carboxy terminal truncation of 3clpro abolished proteolytic activity. emcv ires elements to ensure that translation initiated before the ser 3334 (pc-s/fq and pc-s/lq). the constructs were used to direct translation in vitro discussion and the proteins either were radiolabled or were translated in nonlabeled medium and used in a trans cleavage mhv 3clpro is postulated to mediate the majority of assay of the inactive mutant pgproh41g (fig. 5b ). transcleavages in the gene 1 polyprotein during virus replicalation of pc-s/lq, encoding ser 3334 to gln 3635 , resulted in tion. we have shown that aspartic acid residues of mhv a single 27-kda protein, the same migration pattern as 3clpro in locations analogous to essential asp/glu resi-p27 3clpro detected after expression of pgpro (fig. 5b , dues of other 3c and 3c-like proteinases are not neceslanes 1 and 2). in contrast, translation of three different sary for processing of substrate by 3clpro in vitro. reconstructs encoding the truncated 3clpro domain from sults similar to ours have been reported for infectious ser 3334 to gln 3607 resulted in a single 24-kda protein (fig. bronchitis virus (ibv) (liu and brown, 1995) . our study 5b, lanes 3, 4, and 5). these data indicated that proteins demonstrates that conservation of asp/glu in this region expressed from the 3clpro domain differed in their calof the coronavirus 3c-like proteinases is not due to an culated and apparent masses by 6 to 10 kda. the results indispensable catalytic role. the mechanism of the mhv also supported the conclusion that the active 3clpro in 3clpro may be more similar to that of hepatitis a virus, mhv-infected cells and from in vitro translation products in which the asp is on an external motif and not directly incorporated ser 3334 to gln 3635 . involved in the catalytic unit (allaire et al., 1994) . it is we assessed the in vitro cleavage activity of the possible that this variation in the use of a third residue may have coevolved with the specificity for cleavage 24-kda ser 3334 -gln 3607 and the 27-kda ser 3334 -gln 3635 sites. there is a precedent in other virus systems for a consensus substrate binding residues, whereas the mhv sequence extends an additional 137 residues to its contribution of asp residues to proteinase specificity even though they may not be involved in catalysis. for carboxy terminus (lee et al., 1991) . our study demonstrates that a small deletion of this part of the proteinase example, poliovirus contains an frd (d85) sequence that was initially thought to be involved in catalytic activity abolishes its ability to cleave new molecules of p27 3clpro in trans, demonstrating that the entire carboxy-but subsequently was found to be in a flanking turn domain and to be involved in autocatalytic cleavage of 3cd terminal region is essential for 3clpro activity. analysis of confirmed and predicted cleavage sites in (hammerle et al., 1992) . despite the lack of use of an asp residue, the mhv 3clpro should still be classified the gene 1 polyproteins of mhv-a59, hcv-229e, ibv, and tgev has revealed a preference for a gln at p1 and leu, as a chymotrypsin-like enzyme because of the localization of histidine and cysteine residues as well as flanking ile, val, or less often phe or met at p2 (boursnell et al., 1987; breedenbeek et al., 1990; lee et al., 1991 ; herold residues considered to be important in protein structure and substrate binding (gorbalenya and koonin, 1993). et al., 1993; bonilla et al., 1994; eleouet et al., 1995) . although the phe-gln-gly (fq/g) 3clpro cleavage site analysis of the full-length 3clpro domain (ser 3334 to gln 3635 ) reveals several possible differences between the we identified within 3clpro has similarities to other predicted 3clpro sites from p4 to p1, it is not present in mhv 3clpro and other viral proteinases in the group of cysteine-containing enzymes (fig. 1) . first, most of these the other sequenced coronaviruses. we have not determined if the fq/g 3608 site can be cleaved in virus-infected enzymes terminate within 30 amino acids following the intracellular and in vitro translated 27-kda proteins contain the 3c-like proteinase activity of the picornaviral 3c cysteine proteinases have a fold similar to chymocoronavirus mhv-a59 identification and characterizatrypsin-like serine proteinases mouse hepatitis tion of a serine-like proteinase of the murine coronavirus mhv-a59 effect of expression of the aphthovirus protease 3c on viral infection and gene expression completion of the sequence of the virology genome of the coronavirus avian infectious bronchitis virus the primary structure and ase reveals a trypsin-like polypeptide fold, rna-binding site, and means for cleaving precursor polyprotein characterization of a human coronavirus (strain 229e) 3c-like proteinase activity eleouet, j. f., rasschaert, d., lambert, p., levy, l., vende, p., and laude, cells. it is possible that the fragment of gene 1 used in h. (1995) . complete sequence (20 kilobases) of the polyprotein-enthese studies allows presentation of this site in a manner g cleavage site is used by the proteinase in cells, it gorbalenya, a., and koonin, e. (1993) . comparative analysis of aminoacid sequences of key enzymes of replication and expression of might represent a pathway for regulation of the proteinin conclusion, we have identified several unique feaase gene polyprotein processing and virus replication. lawson, m. r., and semler, b. l. (1992) . alternate poliovirus nonstructural protein processing cascades generated by primary sites of 3c key: cord-268416-8hw80qx8 authors: grunewald, matthew e.; fehr, anthony r.; athmer, jeremiah; perlman, stanley title: the coronavirus nucleocapsid protein is adp-ribosylated date: 2018-04-01 journal: virology doi: 10.1016/j.virol.2017.11.020 sha: doc_id: 268416 cord_uid: 8hw80qx8 adp-ribosylation is a common post-translational modification, although how it modulates rna virus infection is not well understood. while screening for adp-ribosylated proteins during coronavirus (cov) infection, we detected a ~55 kda adp-ribosylated protein in mouse hepatitis virus (mhv)-infected cells and in virions, which we identified as the viral nucleocapsid (n) protein. the n proteins of porcine epidemic diarrhea virus (pedv), severe acute respiratory syndrome (sars)-cov and middle east respiratory syndrome (mers)-cov were also adp-ribosylated. adp-ribosylation of n protein was also observed in cells exogenously expressing n protein by transduction using venezuelan equine encephalitis virus replicon particles (vrps). however, plasmid-derived n protein was not adp-ribosylated following transient transfection but was adp-ribosylated after mhv infection, indicating that this modification requires virus infection. in conclusion, we have identified a novel post-translation modification of the cov n protein that may play a regulatory role for this important structural protein. adp-ribosylation is the covalent attachment of adp-ribose (adpr) moieties to a protein, resulting in either mono-adpr (mar) or poly-adpr (par). adp-ribosylation is catalyzed by poly-adpr polymerases (parps), also known as adp-ribosyltransferases (artds). the parp family consists of 17 proteins in humans and 16 in mice, which utilize nad as the adpr donor [reviewed in (bock and chang, 2016) ]. removal of adpr from proteins (de-adp-ribosylation) is catalyzed by different cellular proteins including par glycohydrolase (parg) and macrodomain proteins (bernardi et al., 1997; jankevicius et al., 2013; rosenthal et al., 2013; sharifi et al., 2013) . detection of adp-ribosylated proteins on a proteomic level is difficult due to the reactive nature and short half-life of the modification in cells (cervantes-laurean et al., 1997; wielckens et al., 1982) . despite this, studies of individual parps and adp-ribosylated proteins have elucidated several physiological roles for adp-ribosylation, including dna damage and repair, regulation of rna transcription, cellular stress response, inflammation, differentiation, and apoptosis (bock and chang, 2016) . parps are well-established to have both proviral and antiviral properties. parp1 has been shown to facilitate epstein-barr virus replication and latency, simian virus 40 induction of cellular necrosis, and hiv integration (gordon-shaag et al., 2003; ha et al., 2001; lupey-green et al., 2017; tempera et al., 2010) . tiparp has been shown to inhibit interferon (ifn) production by adp-ribosylation of tbk-1, leading to enhanced replication of several viruses (yamada et al., 2016) . finally, adp-ribosylation of the adenovirus core protein has been implicated in aiding viral replication and modulating stability of viral chromatin-like structures (dery et al., 1986) . other data suggest that parps can also be antiviral. many parps are expressed following ifn stimulation and several parps show evidence of rapid evolution, suggesting a microbial "arms race" between parps and cellular pathogens (atasheva et al., 2012; daugherty et al., 2014; macdonald et al., 2007) . for example, parp7, parp10, and parp12 restrict venezuelan equine encephalitis virus (veev) replication and can block cellular translation when overexpressed (atasheva et al., 2012 (atasheva et al., , 2014 . one notable parp, the zinc-finger antiviral protein (zap) or parp13, has been demonstrated to inhibit replication of several different viruses, potentially by binding to viral rna and directing it to be degraded by the rna exosome (bick et al., 2003; gao et al., 2002; guo et al., 2004 guo et al., , 2007 liu et al., 2015; muller et al., 2007; zhu et al., 2011) . parp13 is enzymatically inactive, and thus its antiviral activity is independent of adp-ribosylation. in addition, liu et al. have described a novel mechanism in which an unknown parp adp-ribosylates two subunits of the influenza rna polymerase, allowing subsequent binding to zap and degradation of these subunits by the proteasome (liu et al., 2015) . three different virus families, hepeviridae, togaviridae, and coronaviridae, encode for a viral macrodomain, which has been shown to de-adp-ribosylate proteins in vitro (li et al., 2016; rosenthal et al., 2013) . these macrodomains have been proposed to counter the antiviral effects of adp-ribosylation during infection. our previous work has focused on the coronavirus (covs) macrodomain. covs are large, positive-sense, single-stranded rna viruses which include human pathogens such as the severe acute respiratory syndrome (sars)-cov and middle east respiratory syndrome (mers)-cov as well as important veterinary pathogens such as bovine cov and porcine epidemic disease virus (pedv). all covs encode a macrodomain within non-structural protein 3 (nsp3) that can remove both mar and par from proteins (li et al., 2016) . covs lacking this enzymatic activity generally replicate normally in vitro but are highly attenuated in vivo and elicit an enhanced innate immune response (eriksson et al., 2008; fehr et al., 2015 fehr et al., , 2016 kuri et al., 2011) . to identify potential targets of the cov macrodomain, we analyzed infected cells for changes in adp-ribosylation patterns utilizing antibodies specific for adpr. we focused on cells infected with a murine cov, mouse hepatitis virus (mhv). mhv causes acute and chronic encephalomyelitis, hepatitis and gastroenteritis (bailey et al., 1949) . surprisingly, we found that the cov nucleocapsid (n) protein was adpribosylated in cells during infection with mhv as well as several other covs. 2.1. cell culture, plasmids and reagents delayed brain tumor (dbt) cells, 17cl-1 cells, vero cells, and hela cells expressing the mhv receptor carcinoembryonic antigen-related cell adhesion molecule 1 (ceacam1) (hela-mhvr) were grown in dulbecco's modified eagle medium with 10% fetal bovine serum as previously described (zhou and perlman, 2007) . codon-optimized mhv-a59 n protein was synthesized and cloned directly into pcdna3 (genscript). a tagged construct was synthesized by inserting a 3x-flag sequence to the c terminus of the n protein using overlapping primers and recombination by in-fusion (clontech). control plasmid pcdna3-gfp was described previously (fehr et al., 2016) . recombinant mouse hepatitis virus (mhv) strains a59 (yount et al., 2002) and jhmv (wild-type and n1347a) (fehr et al., 2015) were propagated on 17cl-1 cells, and titers were determined on hela-mhvr cells. sars-cov (ma15) was propagated and titered on vero e6 cells, and mers-cov (emc12) and pedv (isu13-19338e, a gift from dr. kyoung-jin yoon, iowa state university) were propagated on vero-81 cells. for virus infections, 17cl-1, dbt, calu-3, vero e6, or vero 81 cells were infected with virus at the indicated multiplicity of infection (moi) and collected at the indicated hours post-infection (hpi). all work with sars-cov or mers-cov infectious virus was performed in a biosafety level 3 laboratory according to the guidelines set forth by the university of iowa. dbt cells were infected with mhv-a59 at an moi of 0.5 pfu/cell, and supernatant was collected and filtered at 12 hpi. the filtrate was subjected to ultracentrifugation at 27,000 rpm for 4 h over a 30% sucrose cushion as described previously. pellets were resuspended in 100 mm nacl and 10 mm tris-cl (ph 7.2) and treated with or without proteinase k (new england biolabs) in the presence or absence of sds. the reaction was stopped by incubation at 65°c for 10 min. cells were transfected with polyjet in vitro transfection reagent (signagen labs) as per the manufacturer's instructions. 24 h after transfection, cells were either treated with or without 1000 u/ml of ifn-β (pbl) for 24 h or were infected with mhv-a59 at an moi of 1 pfu/cell for 12 h before collection. veev replicon particles (vrps) encoding either gfp or mers nucleocapsid protein were created and titered as previously reported (zhao et al., 2014 . the vrps were transduced into vero 81 cells at indicated mois and collected at 24 h post-transduction. sample buffer containing sds, β-mercaptoethanol, protease/phosphatase inhibitor cocktails (roche), pmsf, parp inhibitor 3-aminobenzamide (3-ab, tocris bioscience), parg inhibitor adenosine 5ʹ-diphosphate (hydroxymethyl)pyrrolidinediol (adp-hpd, calbiochem) and universal nuclease (thermofisher scientific) was used to collect cell lysates. proteins were resolved on an sds polyacrylamide gel and transferred to a polyvinylidene difluoride (pvdf) membrane. following binding with a primary antibody, blots were then visualized by using a peroxidase-conjugated secondary antibody (thermo fisher scientific) detected with a chemiluminescent substrate (thermo fisher scientific) or by using an infrared (ir) dye-conjugated secondary antibody detected with a li-cor odyssey imager (li-cor, lincoln, ne) . ir secondary antibodies of different wavelengths were used to obtain different signals for antibody bound proteins. images of α-adpror α-nstained immunoblots were merged using image studio software. primary antibodies used for immunoblotting and immunoprecipitation included polyclonal (pab) α-mhv rabbit serum (perlman et al., 1987) , monoclonal (mab) α-mhv n (collins et al., 1982) (mab 5b188.2, a kind gift from dr. m. buchmeier, university of california, irvine), pab α-sars-cov n (novus biologicals), pab α-nsp3 (gift from mark denison, vanderbilt university), mab α-pedv n (gift from dr. kyoung-jin yoon, iowa state university), pab α-mers-cov n , mouse mab α-adpr (10 h, millipore sigma), rabbit pab α-adpr (trevigen), chicken pab α-adpr (tulip biolabs inc.), α-flag (sigma), α-gapdh (poly6314, biolegend), and α-actin (ac15; abcam, inc.) antibodies. secondary antibodies used included horseradish peroxidase-conjugated α-rabbit or α-mouse (sigma #a0545/ a0168) antibodies or ir-conjugated α-rabbit, α-mouse, or α-chicken (li-cor, #926-68071/926-32210/925-32218) antibodies. dbt cells infected with mhv-a59 at an moi of 1 pfu/cell were collected at 12 hpi and pelleted by low-speed centrifugation. cell pellets were lysed with immunoprecipitation (ip) buffer (0.5% np-40, 300 mm nacl, 5% glycerol, and 50 mm tris ph 8.0) containing protease/phosphatase inhibitor cocktails, pmsf, parp inhibitor 3-ab, parg inhibitor adp-hpd, and a universal nuclease for 2 h at 4°c. nuclei were pelleted by centrifugation (16,000 g for 15 min at 4°c). one aliquot of cell lysate was saved as the input control and boiled in sds sample buffer described above. protein g magnetic beads were conjugated to α-adpr or α-n antibodies (described above) as per manufacturer's instructions (thermofisher scientific). protein g antibody-conjugated were mixed with cell lysates overnight at 4°c. beads were washed with pbs-tween before elution by boiling in sds sample buffer. to screen for changes in protein adp-ribosylation during cov infection, we infected dbt cells, an astrocytoma cell line, with the a59 strain of mhv. cells were collected throughout the infection, and cell lysates were immunoblotted with a mouse mab antibody to adpr (mab 10 h). the 10 h antibody has been described to bind preferentially to linear 20+-mers of par with no binding activity to dna, rna, or adenosine-monophosphate. however, more recent reports have demonstrated that mab 10 h also binds to auto-marylated proteins (eckei et al., 2017; goenka et al., 2007; kawamitsu et al., 1984; kleine et al., 2008) . while the adp-ribosylation status of most proteins did not change over the course of infection, we noted the appearance of a ~55 kda band at 8 h post-infection (hpi) which increased in abundance up to 12 hpi (fig. 1a) . immunoblotting with two other commercial α-adpr antibodies raised in two other species also detected a similar protein band (fig. 1a) . a negative control antibody (α-ha) did not bind to this protein, suggesting a specific interaction of the 55 kda protein with adpr antibodies. based on to the size and abundance of this protein, we hypothesized that it was the nucleocapsid (n) protein. staining with monoclonal α-n antibody produced a signal that completely overlapped the signal from the adpr antibody (fig. 1b, left) . to confirm that this overlap was consistent in other cell lines and mhv strains, we infected 17cl-1 cells (a fibroblast cell line) with mhv-a59 or dbt cells with mhv-jhm (jhmv), a neurotropic strain of mhv, and immunoblotted with α-mhv serum or α-adpr antibody. the results showed that in all cases the n protein completely overlapped with thẽ 55 kda adp-ribosylated protein, demonstrating that n protein adpribosylation is not mhv strain or cell type specific (fig. 1b, middle and right) . to confirm the identity of this protein as the n protein, we collected mhv-infected dbt cells at 12 hpi and immunoprecipitated proteins with either monoclonal α-adpr or α-n antibodies and immunoblotted with the reciprocal antibodies. as expected, this~55 kda protein could be stained with the α-adpr antibody after immunoprecipitation with α-n and stained with α-n after immunoprecipitation with α-adpr (fig. 1c) , confirming that the n protein is adp-ribosylated. previous reports have demonstrated that the nsp3 macrodomain removes both mono-and poly-adpr from auto-adp-ribosylated substrates in vitro (fehr et al., 2016; li et al., 2016) . because the n protein is known to associate with nsp3, we speculated that the nsp3 macrodomain may de-adp-ribosylate the n protein (hurst et al., 2013) . to test this, we infected dbt cells with mhv-a59 or mhv-jhm encoding either wild-type or a catalytically-deficient (jhmv-n1347a, a59-n1348a) nsp3 macrodomain and immunoblotted for n protein adpribosylation. if the macrodomain was indeed removing the adpr from the n protein, we would expect to see increased n protein adp-ribosylation in cells infected with mutant virus compared to wild-type infected cells. however, we found that the level of the adp-ribosylated n protein was the same in cells infected with either mhv strain (fig. 2) . these findings suggest that the nsp3 macrodomain does not affect n protein adp-ribosylation under these conditions. next, we tested whether n protein adp-ribosylation was conserved in cov genera and lineages other than mhv. we infected vero cells with pedv (⍺-cov), sars-cov (lineage b β-cov), or mers-cov (lineage c β-cov) and then collected the cells and analyzed whether the n proteins from these viruses were adp-ribosylated (fig. 3) . in all cases, adpr antibody staining overlapped with the n protein of each virus, lysates were collected at 12 and 18 hpi, respectively, and immunoblotted with indicated antibodies. (c) immunoprecipitation with α-adpr antibody confirms n protein is adp-ribosylated. dbt cells were mock infected (m) or infected with mhv-a59 at moi of 1 pfu/cell. at 12 hpi, cells were collected and the lysates were subjected to immunoprecipitation with mouse α-adpr or α-n (mab) bound to protein g beads. cell lysates and eluted proteins were analyzed by immunoblotting with indicated antibodies. asterisk indicates cellular adp-ribosylated protein that is not immunoprecipitated with α-n antibody. m.e. grunewald et al. virology 517 (2018) [62] [63] [64] [65] [66] [67] [68] demonstrating that this modification is conserved across multiple genera and lineages of covs. it has previously been shown that the n protein is phosphorylated and that some phosphorylated residues depend on whether the protein is intracellular or virion-associated (white et al., 2007) . specifically, mhv n protein at amino acid s197 is phosphorylated in infected cells, but this modification is absent on n protein in virions (wu et al., 2014) . to determine if n protein adp-ribosylation is maintained within the virion, we purified virions and analyzed whether n protein from the cov virion was adp-ribosylated by immunoblotting. to rule out the possibility of detecting of any residual n protein from non-virion sources, pelleted virions were treated with proteinase k with or without virion lysis by sds. both the n protein as well as the spike (s) protein were detectable with α-mhv serum in both cells and virions (fig. 4) . although the s protein was not completely eliminated upon proteinase k treatment, the abundance of the s protein was reduced, consistent with the protein being exposed on the surface of the virus. a co-sedimented nonspecific band of~70 kda was also degraded by proteinase k treatment. in contrast, n protein, located in the interior of the virion, was protected from proteinase k treatment unless sds was also added to lyse the viral envelope. importantly, immunoblotting with α-adpr antibody demonstrated that the n protein maintained the adp-ribose modification in virions. to determine if n protein expressed in the absence of cov infection could be adp-ribosylated in cell culture, we transduced vero cells with veev replicon particles (vrps) encoding the mers-cov n protein or control gfp at different mois . transduced cells were collected at 24 hpi, and immunoblotting of cell lysates showed that n protein expressed from an alphavirus replicon was adp-ribosylated ( fig. 5a) . because the vrp platform utilizes a virus infection to express exogenous proteins, we hypothesized that the adp-ribosylation of the n protein may require a virus infection. to examine this possibility, we transfected dbt cells with a plasmid encoding codon-optimized mhv-n protein or gfp and then tested whether the n protein was adp-ribosylated. because several parps are ifn-stimulated genes, we also treated cells with or without ifn-β (atasheva et al., 2012; macdonald et al., 2007) . importantly, we were unable to detect any adp-ribosylation of the exogenous transfected n protein (fig. 5b) . in contrast, a positive control of mhv-a59-infected cell lysate, normalized to total n protein, stained with α-adpr antibody. furthermore, ifn-β treatment did not rescue the modification, suggesting that other factors present during infection drove adp-ribosylation of n protein. to confirm that n protein requires an infection to be adp-ribosylated, we added a 3x-flag tag to our plasmid-expressed n (n-flag) protein. we then 4 . the n protein is adp-ribosylated within the mhv-a59 virion. supernatant from dbt cells infected with mhv-a59 at moi of 0.5 was collected at 12 h. whole virions were purified by filtration and ultracentrifugation with a 30% sucrose cushion. virions were then treated with proteinase k to degrade extramembranous protein or proteinase k in the presence of sds to degrade all viral proteins. proteinase k was then inactivated at 65°c, and proteins were blotted with indicated antibodies. the nucleocapsid (n), spike (s), and a nonspecific (ns) proteins are indicated with arrows. m.e. grunewald et al. virology 517 (2018) 62-68 transfected plasmid expressing gfp or n-flag into dbt cells and then mock infected or infected cells with mhv-a59. at 12 hpi, we collected cells and immunoblotted with α-adpr antibody. the n-flag protein was detectable with α-adpr antibody following infection with mhv-a59 but not in mock infected cells (fig. 5c ). this suggests that n protein is only adp-ribosylated within the context of virus infection. the n protein was initially identified as a major structural protein, binding directly to viral rna, providing stability to the bound rna, and self-oligomerizing into the virus nucleocapsid. n protein also binds to the viral membrane (m) protein to facilitate genome loading and viral assembly (narayanan et al., 2000) . it plays a prominent role in transcription and replication of the viral genome (hurst et al., 2010) . in fact, the addition of n protein from an exogenous plasmid is often utilized to initiate cellular infection from recombinant cov cdna (yount et al., 2002) . the n protein provides additional accessory functions, including the ability to promote cell cycle arrest, inhibit host translation, and block the ifn response during infection [reviewed in (mcbride et al., 2014) ]. many of these functions are regulated by posttranslational modifications, most notably phosphorylation by host proteins. for example, sars-cov n protein phosphorylation modulates n protein oligomerization, translation suppression, and localization to stress granules (peng et al., 2008) . furthermore, phosphorylation of the mhv n protein is implicated to function as a cellular switch to control transcription of either genomic or subgenomic rna (wu et al., 2014) . in this paper, we have identified an additional post-translational modification of the n protein, adp-ribosylation, which could also play a role in the regulation of n protein functions. we have found that the n protein of multiple covs was apd-ribosylated in vitro, using multiple antibodies to adpr (figs. 1 and 3) . the 10 h α-adpr antibody primarily used in this study binds preferentially to par but can be used to detect marylated proteins as well (eckei et al., 2017; goenka et al., 2007; kawamitsu et al., 1984; kleine et al., 2008) . because the n protein band stained with either α-adpr or α-n antibodies did not appear as a smear, which is seen with long-chain parylated proteins, it is likely that the n protein is either marylated or parylated with only a few monomers of adpr. this is further supported by the fact that adp-ribosylation did not alter migration of the n protein to a detectable level ( fig. 5b and c) . furthermore, our data indicate that the n protein adp-ribosylation was detectable during infection ( fig. 1, fig. 5a, fig. 5c ) but not following transfection alone (fig. 5b ). this could be due to a number of factors including, but not limited to, virus infection-dependent expression of an adp-ribosylating enzyme or the localization of the n protein to a distinct cellular compartment during infection. n protein in infected cells has been shown to localize both in the cytoplasm as well as the nucleus, and the sars n protein has been shown to localize to stress granules under stress conditions (hiscox et al., 2001; peng et al., 2008) . several parps are known to colocalize with stress granules, which could potential sites of n protein modification (leung et al., 2011) . future experiments are required to parse out the number of adpr monomers that are attached to the n protein, to identify the amino acid residue(s) that is modified, and to determine where this modification occurs. the fact that adp-ribosylation of n protein was conserved across multiple cov lineages (fig. 3) suggests that it is important either for viral replication or pathogenesis or as a host defense mechanism. individual parps have been identified to be either proviral or antiviral [reviewed in (kuny and sullivan, 2016) ]. because the n protein is the major structural protein of the cov nucleocapsid, it is tempting to speculate that adp-ribosylation is providing a regulatory role for the structure of the genome, similar to adp-ribosylation of histones or of the adenovirus core protein (dery et al., 1986; strickfaden et al., 2016) . it is also possible that adp-ribosylation of n protein could regulate one or more of the other functions of the n protein such as inhibition of cellular translation or of ifn expression (kopecky-bromberg et al., 2007) . on the other hand, adp-ribosylation of the n protein may be an antiviral defense mechanism, similar to adp-ribosylation of influenza a virus polymerase subunits (liu et al., 2015) . if this were the case, the virus must have evolved methods to combat this modification, possibly including removal of the adp-ribose moieties from the n protein to mitigate its antiviral effects. however, we found that the nsp3 macrodomain, a likely candidate to catalyze this de-adp-ribosylation, unexpectedly does not de-adp-ribosylate the n protein based on our assays ( fig. 2) (fehr et al., 2016) . however it is possible that a small, localized subset of the very abundant n protein is de-adp-ribosylated, which would not be detectable in our assay. future experiments will focus on identifying the function of n protein adp-ribosylation and the specific targets of the cov macrodomain. here we have showed that the n proteins of multiple covs are adpribosylated, representing a previously undescribed modification of a rna virus structural protein. this modification only occurred during virus infection and was maintained both in the cell and in virions. future experiments will work to identify the amino acids that are adpribosylated and to demonstrate the effect of this novel modification on virus infection. new parp gene with an antialphavirus function 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cdna of mouse hepatitis virus strain a59 rapid generation of a mouse model for middle east respiratory syndrome airway memory cd4(+) t cells mediate protective immunity against emerging respiratory coronaviruses mouse hepatitis virus does not induce beta interferon synthesis and does not inhibit its induction by double-stranded rna zinc-finger antiviral protein inhibits hiv-1 infection by selectively targeting multiply spliced viral mrnas for degradation we thank the members of the perlman, wendy maury, and pat sinn laboratories for valuable discussion, kyoung-jin yoon for pedv virus and antibodies, and rudragouda channappanavar for critical reading of the manuscript.this study was supported by national institute of health grants to s.p. (p01 ai060699 and r01 ns36592). m.g. and j.a. were supported by institutional predoctoral nrsa training grants (t32 ai007511 and t32 ai007533, respectively). a.r.f was supported by institutional (t32-ai007260) and individual (f32-ai113973) nih nrsa grants. none. key: cord-258691-cd83w9o6 authors: whitman, lucia; zhou, haixia; perlman, stanley; lane, thomas e. title: ifn-γ-mediated suppression of coronavirus replication in glial-committed progenitor cells date: 2009-02-01 journal: virology doi: 10.1016/j.virol.2008.10.036 sha: doc_id: 258691 cord_uid: cd83w9o6 the neurotropic jhm strain of mouse hepatitis virus (jhmv) replicates primarily within glial cells following intracranial inoculation of susceptible mice, with relative sparing of neurons. this study demonstrates that glial cells derived from neural progenitor cells are susceptible to jhmv infection and that treatment of infected cells with ifn-γ inhibits viral replication in a dose-dependent manner. although type i ifn production is muted in jhmv-infected glial cultures, ifn-β is produced following ifn-γ-treatment of jhmv-infected cells. also, direct treatment of infected glial cultures with recombinant mouse ifn-α or ifn-β inhibits viral replication. ifn-γ-mediated control of jhmv replication is dampened in glial cultures derived from the neural progenitor cells of type i receptor knock-out mice. these data indicate that jhmv is capable of infecting glial cells generated from neural progenitor cells and that ifn-γ-mediated control of viral replication is dependent, in part, on type i ifn secretion. inoculation of the neurotropic jhmv strain of mouse hepatitis virus (a positive-strand rna virus and a member of the coronaviridae family) into the cns of susceptible strains of mice results in an acute encephalomyelitis. the resulting infection is characterized by widespread viral replication in astrocytes, microglia, and oligodendrocytes with relatively few infected neurons knobler et al., 1981; parra et al., 1999; perlmane et al., 1999) . jhmv infection of the cns induces localized expression of pro-inflammatory factors that precedes and accompanies the activation and recruitment of immune cells into the cns. during the acute disease phase, infiltrating virus-specific cd8 + t cells control viral replication by two different effector mechanisms: ifn-γ secretion controls viral replication in oligodendrocytes, while a perforin-dependent mechanism promotes viral clearance from astrocytes and microglia (lin et al., 1997; parra et al., 1999) . while a robust and effective cell-mediated immune response is generated in response to jhmv infection, virus persists within the cns and is associated with the development of an immune-mediated demyelinating disease similar to the human demyelinating disease ms. during this stage, both t cells and macrophages are important in amplifying disease severity by contributing to myelin damage (cheever et al., 1949; perlman et al., 1999) . stem cells and neural precursors represent attractive sources for the generation of remyelination-competent cells since they can readily amplify and differentiate into oligodendrocyte committed cells (ben-hur et al., 1998; brustle et al., 1999) . stem cell-derived glial precursors have been shown to myelinate following transplantation into the myelin-deficient rat (brustle et al., 1999) , and neural precursor-derived glial-committed progenitors (ben-hur et al., 1998; keirstead et al., 1999) have been shown to remyelinate following transplantation into regions of acute experimental demyelination (keirstead et al., 1999) . more recently, intracerebroventricular or intrathecal implantation of neural precursors into rodents with eae, an autoimmune model of demyelination, resulted in the migration of transplanted cells into white matter and improved clinical outcome (ben-hur et al., 2003; pluchino et al., 2003) . while implantation of myelin-competent cells has shown to be effective in promoting remyelination in animal models of demyelination initiated by either infiltration of autoreactive lymphocytes or injury, there is limited information available with regards to the ability of these cells to enhance demyelination resulting from viral infection. we believe this is an important and clinically relevant question as the etiology of ms remains enigmatic although viruses have long been considered potential triggering agents for initiating disease (gilden, 2005; olson et al., 2005) . therefore, evaluating potential cellreplacement strategies for inducing remyelination in viral models of neurologic disease may yield insight into whether this method of (totoiu et al., 2004) . moreover, remyelination was not associated with dampened t cell infiltration into the cns as has recently been reported following nsc transplantation in mice with eae (aharonowiz et al., 2008; einstein et al., 2006; hardison et al., 2006) . having demonstrated that engraftment of glial cells promotes remyelination following jhmv-induced demyelination, we next were interested in addressing several interrelated issues including i) if glial cells derived from neural precursor cells were susceptible to infection and ii) how infection may be controlled within this population of cells. we believe these are relevant questions within the context of studying animal models of viral-induced demyelination as cells are being transplanted into the cns in which a persistent virus is present. therefore, analyzing the susceptibility of cellular progeny derived from neural precursor cells to viral infection is important in that these cells may represent important viral reservoirs in the face of persistent infection. understanding consequences of infection and how replication may be controlled within these cells will provide insight into understanding host defense mechanisms of implanted cells as well as potential relevance to disease outcome. the relevance of this is further highlighted by the fact that while previous studies have demonstrated that jhmv is able to infect and replicate within glial cells (dubois-dalcq et al., 1982; lavi et al., 1987; rempel et al., 2005) , the fate of neural progenitor cells as well as cells derived from this population to viral infection is not well characterized. in the present study, we demonstrate that primary cultures of glia derived from neural progenitor cells are susceptible to jhmv infection and support viral replication. additionally, while ifn-β production is dampened in response to viral infection, treatment with recombinant mouse ifn-γ inhibits jhmv replication. the ifn-γ-mediated antiviral effect is dampened in experiments using cells derived from type i ifn receptor-deficient mice (ifnar−/−) indicating a role for type i ifn signaling in limiting jhmv replication in glia-committed progenitor cells. therefore, these findings provide, to our knowledge, the first demonstration that glia-committed cells derived from neural precursors are susceptible to jhmv infection as well as identify a potential mechanism responsible for controlling viral replication. the in vitro culture of neural progenitor cells dissected from the striatal region of the brains of day 1 postnatal c57bl/6 mice resulted in the generation of numerous neurospheres (fig. 1a ) (hardison et al., 2006; totoiu et al., 2004) . after the mature neurospheres were plated on an adherent matrix and incubated in growth medium, the majority of the cells exhibited oligodendrocyte morphology characterized by extensive arborization (fig. 1b) . immunocytochemical staining confirmed the morphology results indicating that ∼70% of the cells differentiated into oligodendrocytes (determined by galc staining) (fig. 1c ). the remaining cells had differentiated into either astrocytes (∼25%, gfap-expression) or neurons (b5%, map2 staining) (fig. 1d ). these differentiated neural progenitor cultures were used for the subsequent studies examining jhmv susceptibility to infection. jhmv is able to infect and replicate in differentiated neural progenitor cultures as demonstrated by increasing viral titers measured at 12, 24, and 48 h p.i. (fig. 2a) . immunocytochemistry revealed viral antigen distributed extensively throughout the monolayer (fig. 2b ). in addition, jhmv infection resulted in cytopathic effects by 24 h p.i. characterized by wide-spread syncytia formation (fig. 2c ). these findings indicate that differentiated cells derived from neural progenitors are susceptible to jhmv infection and are capable of supporting replicating virus which results in extensive cytopathology. previous studies by parra et al. (1999) demonstrated that ifn-γ has an important role in controlling jhmv replication within oligodendrocytes of persistently infected mice. therefore, we next determined whether ifn-γ was capable of inhibiting jhmv replication following infection of differentiated neural progenitor cells. as shown in fig. 3a , treatment of jhmv-infected cells with recombinant mouse ifn-γ inhibited viral replication at 12 (57% reduction, p b 0.05), 24 (56% reduction, p b 0.05), and 48 h p.i. (94% reduction, p b 0.05) compared to media-treated controls. moreover, the ifn-γ-mediated inhibition of jhmv-replication was concentration-dependent; titration of ifn-γ resulted in diminished antiviral effects (fig. 3b ). the pretreatment of cultures with ifn-γ (100 u/ml) resulted in a significant (p b 0.05) reduction in viral titers at 24 h p.i. when compared to cultures incubated with ifn-γ following infection (fig. 3c ). immunocytochemistry revealed that ifn-γ treatment of differentiated neural progenitor cultures limited the extensive cytopathic effects (figs. 3d and e) observed in untreated cells (fig. 3f ) as characterized by diminished syncytium formation. together these data indicate that ifn-γ activates differentiated neural progenitors to inhibit jhmv replication, which correlates with muted cytopathology. it is known that ifn-γ is capable of inducing expression of the non-elr chemokines cxcl9 and cxcl10 in numerous cell types including resident cells of the cns such as astrocytes and microglia (bhowmick et al., 2007; majumder et al., 1998; vanguri and farber, 1994) . moreover, in vivo astrocytes have been shown to express cxcl9 and cxcl10 mrna transcripts during the acute response to jhmv infection and in vitro cultured astrocytes are capable of expressing cxcl10 mrna transcripts (lane et al., 1998; liu et al., 2000 liu et al., , 2001 . neither cxcl9 nor cxcl10 are detectable in differentiated neural progenitor cultures in response to jhmv infection at 12 or 24 h p.i. (figs. 4a and b) . in contrast, ifn-γ treatment of infected cultures resulted in measurable levels of both cxcl9 and cxcl10 at 12 and 24 h p.i. (figs. 4a and b) . levels of cxcl10 were dramatically higher (∼ 45,000 pg/ml at 12 h) compared to cxcl9 levels (∼75 pg/ml at 12 h) suggesting differential promoter sensitivities to ifn-γ treatment or altered stability at either rna or protein levels. to assess the importance of ifn-γ-mediated production of cxcl9 and cxcl10 in the inhibition of jhmv replication, neural progenitor cells were isolated from cxcl10−/− mice and mice deficient in the signaling receptor for cxcl9 and cxcl10, cxcr3 (cxcr3−/− mice). the neural progenitor cells from deficient mice were differentiated in vitro, infected with jhmv and treated with ifn-γ. as we observed in wildtype mice, such treatment resulted in a significant reduction in viral titers at 24 h p.i. compared to infected cells incubated with medium alone (figs. 4c and d). therefore, the ifn-γ-mediated anti-viral effect observed occurs independently of either production of cxcl9, and cxcl10 or cxcr3 signaling. type i ifn (ifn α and β) exhibit potent antiviral activity and have recently been shown to be important in controlling jhmv replication in vivo (ireland et al., 2008) . therefore, we next evaluated production of type i ifn from differentiated progenitor cultures following jhmv infection. as shown in fig. 5a , jhmv infection did not result in detectable levels of ifn-α/β at either 24 or 48 h p.i. however, ifn-γ treatment of jhmv-infected cultures resulted in expression of ifn-α/β that was elevated compared to treatment with ifn-γ alone (fig. 5a) . further, direct treatment with either recombinant mouse ifn-α or ifn-β of jhmv-infected cultures resulted in a dramatic reduction in viral replication compared to media treatment (fig. 5b) . ifn-β exhibited ∼ 90% greater reduction in viral replication compared to ifn-α treatment indicating a more potent antiviral activity associated with ifn-β signaling (fig. 5b) . next, progenitor derived glial cultures were generated from type i ifn receptor-deficient mice (ifn-r−/−) mice and treated with ifn-γ following jhmv infection. as shown in fig. 5c , such treatment did result in a reduction in viral replication (p b 0.05) compared to media-treated controls by 48 h p.i. however, viral replication was reduced, on average, by only 53% in ifn-r−/− cells compared to n90% reduction in wildtype cells (figs. 3a and c). therefore, these data indicate that the ifn-γ-mediated antiviral effect is diminished in the absence of type i ifn signaling indicating that one mechanism by which ifn-γ promotes control of jhmv replication within glial-derived progenitors is through induction of type i ifn. the findings put forth in this paper provide, to our knowledge, the first demonstration that jhmv is capable of infecting and replicating within primary cultures of glia derived from neural progenitor cells. these findings are distinct from earlier studies (dubois-dalcq et al., 1982; lavi et al., 1987; rempel et al., 2005) showing that primary neural cultures are susceptible to viral infection, as we have allowed for differentiation of glial cells from neural progenitor cells into defined glia populations. in addition, we have demonstrated that ifnγ treatment of jhmv-infected cultures suppresses jhmv replication and this is dependent, in part, on secretion of ifn-i. the importance of ifn-i in defense following viral infection of the cns has been documented in several animal models. infection of mice in which ifn-i is genetically silenced or signaling blocked results in uncontrolled proliferation of west nile virus (samuel and diamond, 2005) , sindbis virus (burdeinick-kerr et al., 2007; byrnes et al., 2000) , and semliki forest virus (fragkoudis et al., 2007) . the cellular source of ifn-i production is controlled by viral tropism and the model system employed. for example, neurons are a primary source of ifn-i in response to west nile virus infection and these cells also represent a prominent cellular target for viral infection and replication (samuel and diamond, 2005) . similarly, robust cytokine production, including ifn-β, is observed following infection of astrocyte cultures with theiler's murine encephalomyelitis virus (tmev) (palma et al., 2003) . in the case of jhmv infection, emerging evidence highlights the importance of ifn-i in protection of the cns in response to infection. bergmann and colleagues (ireland et al., 2008) recently demonstrated increased mortality correlating with wide-spread jhmv dissemination throughout the parenchyma including expanded cell tropism with neurons infected in mice deficient in ifn-i receptor (ifnr−/−). additional support for an important role for ifn-i in host defense in response to infection with mouse coronaviruses are derived from studies that demonstrate increased disease severity following anti-ifn antibody treatment (lavi and wang, 1995) and enhanced resistance following treatment with recombinant ifn-β (matsuyama et al., 2000; smith et al, 1987) . further support for mouse coronaviruses in initiating ifn-i production following experimental infection of mice are provided by studies from cervantesbarragan et al. (2007) indicating that peripheral infection with a hepatotropic strain of mhv (mhv-a59) results in increased ifn-i production by plasmacytoid dendritic cells. while it is clear that ifn-i is produced in vivo in response to mouse coronavirus infection and participates in effective host defense, the molecular signals regulating expression on a cellular basis are less well characterized. indeed, ifn-β is not produced in response to jhmv infection of fibroblasts but this is not dependent on the absence of intracellular double-stranded rna or deficiencies in ifn-β signaling (roth-cross et al., 2007) . impaired ifn-β production within jhmvinfected cultures correlated with impaired translocation of transcription factors irf-3 and irf-7 into the nucleus of infection cells (versteeg et al., 2007; zhou and perlman, 2007) . in addition, it may be possible that double-stranded rna generated during the course of jhmv infection is not accessible to cellular pattern recognition receptors (ppr) such as rig-i, mda-5, and tlr-3 (zhou and perlman, 2007) . while the molecular mechanisms associated with inhibited ifn-β production have not been completely defined, the mhv nucleocapsid protein has been suggested to be an ifn-i antagonist (ye et al., 2007) . data provided in the current study demonstrate that jhmv infection of differentiated progenitor cells resulted in muted expression of ifn-i and virus was able to replicate in an unrestricted manner. in addition, secretion of cxcl9 and cxcl10 was also impaired following jhmv infection of differentiated progenitor cultures and this is in contrast to previous findings indicating robust chemokine expression following infection of primary cultures of astrocytes. these results support the earlier hypothesis that within certain host cell populations, double-stranded rna generated during the course of jhmv replication may not be accessible to ppr and this impacts secretion of ifn-i and non-elr chemokines cxcl9 and cxcl10. moreover, since the majority of progenitor cells (∼70-80%) differentiate into oligodendrocyte progenitor cells (opc), it is possible that oligodendrocytes are unable to synthesize either cxcl9 or cxcl10 in response to jhmv infection. treatment of progenitor cultures with ifn-γ resulted in reduced jhmv replication and this highlights the importance of this cytokine in host defense following jhmv infection. additionally, these data support and extend studies by stohlman and colleagues (gonzalez et al., 2005 parra et al., 1999) that have demonstrated ifn-γ is critical in controlling jhmv replication in oligodendrocytes in vivo. infection of ifn-γ−/− mice with jhmv highlighted a critical role for this cytokine in controlling viral replication within oligodendrocytes (parra et al., 1999) . additionally, transgenic mice expressing a dominant-negative ifn-γ-receptor specifically on oligodendroglia demonstrated that ifn-γ is required for inhibiting viral replication (gonzalez et al., 2005 . the findings put forth in the present study clearly indicate that ifn-γ suppresses jhmv replication in glial-committed progenitor cells derived from neural precursors. moreover, the ifn-γ-mediated antiviral effect is not dependent on secretion of cxcr3-binding chemokines. although the in vivo mechanism(s) by which ifn-γ evokes an antiviral response have not yet been defined, our data suggest that production of ifn-i by ifn-γtreated glia may contribute to viral control. further support for type i ifn in controlling jhmv replication within oligodendrocyteenriched cultures is provided by the demonstration that treatment of jhmv-infected oligodendrocytes with either recombinant ifn-α or ifn-β resulted in n1 log decrease in viral titers with ifn-β having a much greater anti-viral effect compared to ifn-α in controlling replication. neurotropic viruses are capable of infecting and replicating within opc (dietrich et al., 2004; levine et al., 1998; mock et al., 2006) . for example, human herpesvirus 6 (hhv6) is capable of infecting and replicating within the human oligodendrocytes and suggested to be involved in the pathogenesis of both acute and chronic inflammatory demyelinating diseases (dietrich et al., 2004; mock et al., 2006) . hhv6 infection of human opc cultures results in formation of multinucleated syncytia and elevated expression of galc (dietrich et al., 2004) . opc proliferation was also impaired in hhv6-infected cultures and infected cells and suggests that infection in vivo may have longlasting effects on precursor cell properties. these findings are interesting in that remyelination is relatively slow in jhmv-infected mice yet opc are present within and surrounding areas of on-going demyelination. this suggests that the ability of opc to successfully remyelinate axons is impaired and/or an environment that is conducive for promoting remyelination is not available. having demonstrated that jhmv is capable of infecting and replicating within primary cultures of opc indicates that these cells are susceptible to infection in vivo. therefore, it is interesting to speculate that early infection of neural progenitor cells impacts either generation of opc and/or the ability of opc to successfully remyelinate demyelinated axons at later stages of infection. we are currently addressing these possibilities. the neurotropic strain jhmv (2.2v-1) of mouse hepatitis virus (mhv) was used for all experiments described here (fleming et al., 1986) . wild type mice for progenitor cell isolation, c57bl/6 mice (on the h-2 b background), were purchased from the national cancer institute (frederick, md). additional mouse strains used for progenitor cultures, cxcl10−/−, cxcr3−/−, and ifn-i receptor deficient (ifnar−/−) (c57bl/6 h-2 b background), were bred in the university of california, irvine animal facility. the animal protocols and procedures used for these studies were reviewed and approved by the institutional animal care and use committee of the university of california, irvine. neural progenitor cells were cultured as previously described (totoiu et al., 2004) . in brief, striata from 5 to 6 postnatal day 1 mice were dissected, triturated and dissociated in 0.05% trypsin-edta. the resulting single cell suspension was cultured for 6-7 days in 25 ml serum free media (dmem:f12 supplemented with b27 supplement, 1× insulin-transferrin-selenium-x supplement, 1× penicillin-streptomycin and t3) with 20 ng/ml human recombinant epidermal growth factor (egf; sigma-aldrich) (ben-hur et al., 1998) . media was replaced on days 1, 3, and 5; culture supernatant and floating clusters were removed, centrifuged at 300 × g for 5 min and resuspended in fresh media with egf. after one week, cells had proliferated into numerous free-floating spheres. after one week, cell spheres were transferred to matrigel (bd bioscience, bedford, ma) coated flasks (use thin coat method, 1:30 dilution) at a low density. individual cells spread out from the attached spheres and formed a monolayer with 1 to 2 days. once the monolayer formed, cells were trypsinized, counted and plated into four chamber imaging slides (nalgene-nunc international, rochester, ny), 6-well plates or t25 flask (costar, corning, ny) previously coated with matrigel (bd biosciences). cells were allowed to equilibrate for an additional 1-2 days before viral infection or staining procedures were done. for all experiments shown, jhmv was added to cultures at a multiplicity of infection (moi) of 0.1 pfu/cell. virus was allowed to adsorb for 1 h, cultures were washed with pbs and replaced with 4 ml of fresh medium. recombinant mouse ifn-γ, ifn-α, and ifn-β cytokines were purchased from cell sciences (canton, ma). viral titers in supernatants of infected cultures were determined on dbt astrocytoma cells at defined time points post-infection (p.i.) (hirano et al., 1978; lane et al., 2000) . to assess differentiation potential, cells were grown on matrigel coated imaging slides for a total of 4 days, fixed in 4% paraformalde-hyde (fisher scientific, fair lawn, nj) for 20 min and immunofluorescence staining was performed using standard protocols. imaging chambers were blocked with 10% normal goat serum (ngs) (vector laboratories, burlingame, ca) for 1 h at room temperature. primary antibodies (polyclonal rabbit anti-galc, chemicon, 1:100 dilution in 10% ngs; monoclonal mouse anti-map2, sigma, 1:750 dilution in 10% ngs; polyclonal rabbit anti-gfap, invitrogen, 1:500 dilution in 10% ngs) or blocking solution (negative control, 10% ngs in pbs) were applied to chambers overnight at 4°c on rocker. slides were rinsed three times with pbs and fluorescent-conjugated secondary antibody (alexa 594, goat anti-rabbit or goat anti-mouse igg h + l, 1:400 dilution in 10% ngs; invitrogen) was applied and incubated for 30 min at room temperature. slides were rinsed three times in pbs, and nuclear staining was with hoechst 33342 (1 μg/ml in pbs, molecular probes, eugene, or) for 10 min. cell quantification was conducted using an olympus bx-60 microscope, 200× magnification. the percentage of immunopositive cells was determined by dividing the total number of immunopositive cells by the total number of hoechst-positive cells in five images from each chamber, and averaging the results from three different chambers per marker. each 4-chamber imaging slide had one no-primary control chamber and three stained chambers for each of the markers mentioned above. only immunopositive cells with a hoechst-positive nucleus were counted. distribution of viral antigen in cultures was determined by immunoperoxidase staining, as specified by the manufacturer (vectastain-abc kit and dab peroxidase substrate kit; vector laboratories). imaging chambers were blocked with triton containing blocking buffer (bb), (0.3% triton x-100 and 10% ngs normal goat serum in pbs), for 1 h at room temperature. the anti-jhmv mab j.3.3 (1:20 dilution in bb) specific for the carboxyl terminus of the viral nucleocapsid (n) protein was applied and incubated overnight at 4ϒc. slides were rinsed three times in pbs and secondary antibody (biotinylated goat-anti-mouse igg h + l, vector laboratories, 1:750 dilution in bb) was applied and incubated for 1.5 h at room temperature. slides were rinsed three times in pbs and counterstained with hematoxylin (bergmann et al., 2003; fleming et al., 1983; walsh et al., 2007) . differentiated neural progenitor cells were infected with mhv, and levels of ifn were measured using a bioassay based on inhibition of vsv growth in l929 cells. supernatants were harvested and exposed to uv light to inactivate infectious virus. l929 cells infected with 1000 pfu vsv were treated with dilutions of supernatants or recombinant murine ifn-β (pbl biomedical laboratories, piscataway, nj) at 30 min post-infection (p.i.). titers of vsv were determined on vero cells. ifn levels were calculated based on standard curves generated with recombinant ifn-β. opc culture supernatants were used to measure chemokines cxcl9 and cxcl10. elisas were performed using the duoset mouse cxcl9 and cxcl10 elisa kit (r & d systems, minneapolis, mn), as specified by the manufacturer. statistically significant differences between groups of mice were determined by student's t test and p values of b0.05 were considered significant. neuroprotective effect of transplanted human embryonic stem cell-derived neural precursors in an animal model of multiple sclerosis growth and fate of psa-ncam+ precursors of the postnatal brain transplanted multipotential neural precursor cells migrate into the inflamed white matter in response to experimental autoimmune encephalomyelitis perforin-mediated effector function within the central nervous system requires ifn-gamma-mediated mhc up-regulation induction of ip-10 (cxcl10) in astrocytes following 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variants of murine coronavirus jhm selected with monoclonal antibodies the type i interferon system protects mice from semliki forest virus by preventing widespread virus dissemination in extraneural tissues, but does not mediate the restricted replication of avirulent virus in central nervous system neurons infectious causes of multiple sclerosis expression of a dominant negative ifn-gammareceptor on mouse oligodendrocytes inhibition of interferon-gamma signaling in oligodendroglia delays coronavirus clearance without altering demyelination transplantation of glial-committed progenitor cells into a viral model of multiple sclerosis induces remyelination in the absence of an attenuated inflammatory response utility of mouse cell line dbt for propagation and assay of mouse hepatitis virus type i interferons are essential in controlling neurotropic coronavirus infection irrespective of functional cd8 t cells polysialylated neural cell adhesion molecule-positive cns precursors generate both oligodendrocytes and schwann cells to remyelinate the cns after transplantation selective localization of wild type and mutant mouse hepatitis virus (jhm strain) antigens in cns tissue by fluorescence, light and electron microscopy dynamic regulation of alpha-and beta-chemokine expression in the central nervous system during mouse hepatitis virus-induced demyelinating disease a central role for cd4(+) t cells and rantes in virus-induced central nervous system inflammation and demyelination the protective role of cytotoxic t cells and interferon against coronavirus invasion of the brain coronavirus mouse hepatitis virus (mhv)-a59 causes a persistent, productive infection in primary glial cell cultures reactions of oligodendrocyte precursor cells to alpha herpesvirus infection of the central nervous system mouse hepatitis virus is cleared from the central nervous systems of mice lacking perforin-mediated cytolysis the t cell chemoattractant ifn-inducible protein 10 is essential in host defense against viral-induced neurologic disease expression of mig (monokine induced by interferon-gamma) is important in t lymphocyte recruitment and host defense following viral infection of the central nervous system regulation of human ip-10 gene expression in astrocytoma cells by inflammatory cytokines protective effects of murine recombinant interferon-beta administered by intravenous, intramuscular or subcutaneous route on mouse hepatitis virus infection infection of murine oligodendroglial precursor cells with human herpesvirus 6 (hhv-6) -establishment of a murine in vitro model a virus-induced molecular mimicry model of multiple sclerosis infection with theiler's murine encephalomyelitis virus directly induces proinflammatory cytokines in primary astrocytes via nf-kappab activation: potential role for the initiation of demyelinating disease ifn-gamma is required for viral clearance from central nervous system oligodendroglia coronaviruses: hepatitis, peritonitis, and central nervous system disease injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis viral induction of central nervous system innate immune responses inhibition of the alpha/beta interferon response by mouse hepatitis virus at multiple levels alpha/beta interferon protects against lethal west nile virus infection by restricting cellular tropism and enhancing neuronal survival intranasally administered alpha/beta interferon prevents extension of mouse hepatitis virus, strain jhm, into the brains of balb/cbyj mice remyelination, axonal sparing, and locomotor recovery following transplantation of glial-committed progenitor cells into the mhv model of multiple sclerosis ifn and virus-inducible expression of an immediate early gene, crg-2/ip-10, and a delayed gene, i-a alpha in astrocytes and microglia group 2 coronaviruses prevent immediate early interferon induction by protection of viral rna from host cell recognition expression of cxc chemokine ligand 10 from the mouse hepatitis virus genome results in protection from viral-induced neurological and liver disease mouse hepatitis coronavirus a59 nucleocapsid protein is a type i interferon antagonist mouse hepatitis virus does not induce beta interferon synthesis and does not inhibit its induction by double-stranded rna this work was supported by the national multiple sclerosis society grants 3847 (t.e.l.) and 2864 (s.p.) and the national institutes of health grant ns41249 to t.e.l. key: cord-254558-gvo0gwjf authors: guo, yan xiang; wei, ting; dallmann, klara; kwang, jimmy title: induction of caspase-dependent apoptosis by betanodaviruses ggnnv and demonstration of protein α as an apoptosis inducer date: 2003-03-30 journal: virology doi: 10.1016/s0042-6822(02)00098-3 sha: doc_id: 254558 cord_uid: gvo0gwjf betanodaviruses, members of the nodaviridae family, are the causative agents of viral nervous necrosis in fish and infection by which cause high mortality in larvae and juveniles in a wide range of marine fish species in asia, europe, australia, martinique, and tahit. greasy grouper (epinephelus tauvina) nervous necrosis viruses (ggnnv) were investigated for their apoptotic activity in culture cells. ggnnv infection of sea bass (sb) cells appeared to induce a typical cytopathic effect (cpe), i.e., cytoplasmic vacuolation, thinning, rounding up, detachment of infected cells from the cultured dish, and eventually cell lysis and death. the infected sb cells underwent dna fragmentation and stained positive in terminal deoxynucleotidyl transferase (tdt)-mediated dutp nick-end labeling (tunel) assay, suggesting that ggnnv infection induced apoptosis in sb cells. in addition, ggnnv-infected sb cells showed an increased activity of caspase-8-like proteases (ietdase) and caspase-3-like proteases (ietdase), whereas inhibitor of caspase-8 and caspase-3 reduced ggnnv-induced apoptosis. this suggests that ggnnv may promote apoptosis via the extrinsic pathway in sb cells. protein α, the precursor of ggnnv capsid proteins, was transiently expressed in sb and cos-7 cells. the dna fragmentation and tunel positive signal were apparent in sb and cos-7 cells expressing protein α, suggesting that protein α may serve as an apoptotic inducer in these cells. moreover, expression of protein α resulted in the activation of caspase-3-like proteases in both cells, which could be inhibited by a caspase-3-like protease specific inhibitor devd-cho peptide. these results suggest that fish caspases are important elements in ggnnv-meditated apoptosis. apoptosis or programmed cell death (pcd) is the process whereby an individual cell of multicellular organisms undergoes systematic self-destruction in response to a wide variety of stimuli. during this process, cells display characteristic morphological changes, including chromatin condensation, plasma membrane blebbing, cell shrinkage, and fragmentation into membrane-bound bodies (majno and joris, 1995; vaux et al., 1994; wyllie et al., 1980) . there are two different mechanisms by which a cell commits suicide by apoptosis. one is generated by signals arising within the cells, which involves b cell lymphoma-2 (bcl-2) and subsequently leads to the release of apoptosis protease activating factor-1 (apaf-1) and cytochrome c. then the released cytochrome c and apaf-1 bind to caspase-9 and other molecules to form the apoptosome. the sequential activation of one caspase by another creates an expanding cascade of proteolytic activity, which leads to digestion of structural proteins in the cytoplasm and degradation of chromosomal dna. the other mechanism is triggered by death activators outside the cell, which bind to receptors at the cell surface, such as fas (cd95 antigen) and tumor necrosis factor (tnf) receptors, and transmit a signal to the cytoplasm that leads to activation of caspase-8, which initiates a cascade of caspase activation to cause the formation of membrane-bound apoptotic bodies (adams and cory, 1998; everett and mcfadden, 2001; jagus et al., 1999; lee et al., 1997; li et al., 1997; teodoro and branton, 1997) . in recent years, many viruses in different families have been found to induce apoptosis during their infection cycles. host cells defend themselves from viral infection by apoptosis, but viruses have also developed a range of strategies to fight against the host immune response and apoptosis; they even make use of apoptosis to propagate. for some viruses, inhibition of apoptosis appears to be essential for the maintenance of viral latency. but for many other viruses, the careful induction of apoptosis during lytic infection may represent the basis for cytotoxity and be on important outlet for dissemination of progeny virus (hardwick, 1998; roulston et al., 1999) . the process of apoptosis is controlled by a large number of genes, many of which have been identified from viral genomes (teodoro and branton, 1997) . nodaviruses are small, nonenveloped, spherical viruses with bipartite positive-sense rna genomes, which are capped but not polyadenylated. two genera have been distinguished within the nodaviridae family: the alphanodaviruses that were originally isolated from insects and the betanodaviruses that infect a wide variety of larval and juvenile marine fish (ball et al., 2000) . the fish disease caused by betanodavirus is spreading worldwide, resulting in severe morbidity and mortality and significant economic losses to the aquaculture industry. infected fish showed abnormal swimming behavior, such as erratic swimming in circles, or to the right, lying on their sides or belly up. histopathological changes are characterized by extensive cellular vacuolations and neuronal degeneration in the central nervous system and retina (delsert et al., 1997; grotmol et al., 1999; mori et al., 1992; munday, 1997; nakai et al., 1994) . despite their greater economic impact, the viruses in this genus are less studied. therefore, it is essential to characterize their molecular biology to understand the mechanism of their infection. ggnnv (greasy grouper (epinephelus tauvina) nervous necrosis viruses) of the singapore strain, a member of betanodavirues, contain two genomic rna segments: complete nucleotide sequences of rna1, 3103 nt (genbank accession number af319555), and rna2, 1433 nt (gen-bank accession number af318942), which have been determined in our previous work . rna1 gene encodes a 110 kda protein a, which is an rnadependent rna polymerase and rna2 encodes a 37 kda protein ␣, which is the precursor of capsid proteins ␤ and ␥. two putative orfs of protein b1 (111 aa) and protein b2 (75 aa) were also confirmed. protein b1-encoding region is in the same reading frame as protein a. protein b2-encoding region is in the ϩ1 reading frame with respect to protein a and overlaps the c terminal of protein a almost entirely. the functions of putative 11 kda protein b1 or 8.5 kda protein b2 are still unknown. a sensitive method to diag-nose ggnnv infection with recombinant protein ␣ has been established . in this study, typical cytopathic effect (cpe), i.e., cytoplasmic vacuolation, fusion, rounding up, detachment of infected cells from the cultured dish, and eventually cell lysis and death were observed in ggnnv-infected sb cells. to understand if there is a relationship between ggnnv infection and apoptosis, a time course study of ggnnv infection was performed and ggnnv was demonstrated to be capable of inducing apoptosis in infected sb cells as indicated by the dna ladder and tunel assays. furthermore, transient expression of protein ␣ revealed that protein ␣ trigged apoptosis in transfected sb and cos-7 cells. to further study the apoptotic signaling pathway utilized by ggnnv and protein ␣, their effect on caspase-3-like protease (devdase) activity and caspase-8-like protease (iet-dase) activity was examined. protein a was expressed in e. coli using the pgex-4t-2 vector which includes a glutathione s-transferase (gst) tag and is under the control of the tac promoter. protein ␣ was expressed in e. coli using the pqe-30 vector which contains a 6x his-tag coding sequence and is under the control of the t5 promoter. the recombinant plasmids pgex-4t-2/rna1 and pqe30/rna2 were transformed into e. coli jm 105, induced with 1 mm isopropyl-d-thiogalactopyranoside (iptg) (final concentration) at midexponential growth phase, and incubated for 4 h. the recombinant proteins were efficiently synthesized and demonstrated to have the respective sizes of 136.4 (fig. 1a , lane 2) and 37 kda (fig. 1a , lane 4) on coomassie brilliant blue stained sds-page gel. the recombinant proteins of proteins a and ␣ were then purified and used to immunize guinea pig to obtain antibody against each protein. to confirm the monospecificity of each antibody, ggnnv-infected sb cells were used as viral antigens to perform western-blotting analyses. consistent with the reported sizes of protein a and ␣ , proteins of 110 and 37 kda were detected by the two different antisera, respectively (fig. 1b, lane 1 and 2) , whereas no signals were detected when the control sera (fig. 1b, lane 3) were reacted with extracts from the infected sb cells. in addition, no signals were detected when extraction from mock-infected sb cells (fig. 1b, lane 4) was reacted with the mixture of both sera. these results indicated that the antibodies against proteins a and ␣ were monospecific and could be used as a tool to carry out the following experiments. the typical cytopathic effects (cpe) characterized by cytoplasmic vacuolation, fusion, rounding up, and detachment of infected cells from the cultured dish were first detected in betanodavirus (ggnnv)-infected sb cells at 24 h postinfection (pi) and the extent of cpe gradually increased by 48 h pi (fig. 2 , image c). to investigate if ggnnv kills cells by apoptosis and if there is a correlation between cpe and apoptosis, the membrane permeable dna-binding dye hoechst 33342 was used to stain the nuclei of the infected cells to see any gradual morphological change of the nuclei. as shown in fig. 2 , image d, the nuclei became distorted and condensed in ggnnv-infected cells at 48 h pi, whereas in mock-infected cells (fig. 2 , image b), the nuclei remained uniformly stained, suggesting that apoptosis might be triggered in sb cells during ggnnv infection. cellular genomic dna was extracted from the ggnnv infected sb cells at 24, 36, 48, 60, and 72 h pi. as shown in fig. 3a , dna fragmentation was observed as early as 24 h pi, and the ladder was not evident until 48 h pi; at 60 pi and 72 h pi (fig. 3a, lanes 3, 3-7) , the number of fragments increased remarkably. meanwhile, no ladder was observed in mock-infected cells (fig. 3a, lane 2) . these data demonstrated that extensive internucleosomal dna cleavage occurred late in ggnnv-infected sb cells. the tunel assay was performed to further confirm dna fragmentation in ggnnv-infected sb cells. tunel-positive cells began to appear at 24 h pi (fig. 3b , image b), and the number of positive cells increased gradually. at 48 h pi, the positive signal was strongest (fig. 3b , images c and d). no positive signal was found in mock-infected cells (fig. 3b , image a). in addition, when the cells were treated with devd-cho, a caspase-3 specific inhibitor, the number of tunel positive cells dramatically decreased at 48 h pi (fig. 3b , image e). apoptotic death is known to involve a cascade of proteolytic events accomplished mainly by caspases. caspase-8, the first caspase in the cd95 apoptotic pathway, directly or indirectly initiates the proteolytic activities of downstream effector caspases, such as caspase-3, caspase-6, and caspase-7. to determine if ggnnv induces apoptosis in fish cells through the cd95 apoptotic pathway, infected sb cells were harvested at 24 pi and 48 h pi, and cell lysates were analyzed for ietdase activity, using ietd as the substrate for caspase-8. as shown in fig. 4 , the ietdase activity in lysates from infected sb cells was 6.5 nmol/h at 24 h pi and increased to 10.5 nmol/h at 48 h pi. no significant ietd activity was observed in mock-infected cells. furthermore, ggnnv-mediated activation of caspase-8 was inhibited by ietd-fmk, a caspase-8 specific inhibitor. to evaluate the possible involvement of downstream effector caspases in apoptosis triggered by ggnnv infection in fish cells, infected sb cells were harvested at 24 pi and 48 h pi, and the cell lysates were analyzed for dev-dase activity, using devd as the substrate for caspase-3. as shown in fig. 5 , the devdase activity in lysates from infected sb cells was 3.2 nmol/h at 24 h pi and increased to 15 nmol/h at 48 h pi. there was no significant devdase activity in mock-infected cells. in addition, when infected cells were treated with devd-cho, reduction of devdase activity in ggnnv-infected cells was observed. to determine which protein encoded by betanovirus ggnnv might be responsible for inducing apoptosis, the transient expression of viral proteins a and ␣ in sb and cos-7 cells was carried out. pegfp-rna1 and pegfp-rna2 were transfected independently to sb or cos-7 cells and the expression was confirmed by western-blot assay using antibodies against protein a (fig. 6, lanes 1 and 4) or ␣ (fig. 6, lanes 2 and 5) . pooled guinea pig anti-protein a and ␣ antibodies failed to detect any viral proteins in cells transfected with pegfp-c1 vector (fig. 6, lanes 3 and 6) . at 48 h post-transfection, the cellular genomic dnas were extracted and analyzed on 2% agarose gel electrophoresis. distinct dna ladder was detected in dna samples from sb and cos-7 cells transfected with pegfp-rna2 (fig. 7a , lane 2), but no dna fragmentation could be observed from cells transfected with pegfp-c1 or pegfp-rna1 (fig. 7a, lanes 3 and 4, respectively) . the apoptosis caused by protein ␣ was further demonstrated by tunel labeling. as shown in fig. 7b , a great number of tunel-positive cells characterized by brown nuclei were detected in pegfp-rna2 transfected sb and cos-7 cells at 48 h post-transfection (fig. 7b , images a and c). in contrast, no brown nucleus signals were observed in the cells transfected with pegfp-c1 vector (fig. 7b, images b and d) . to determine whether the activation of caspase-3 like proteases occurred in the expression of protein ␣, both sb and cos-7 cells were transfected with pegfp-rna2 or pegfp-c1, and lysates of transfected cells were assayed for devdase activity at 24 h post-transfection. as shown in fig. 8 , protein ␣ induced an activation of caspase-3-like proteases characterized by 2.8 and 4.2 nmol/h devdase activity in sb cells and cos-7 cells, respectively. there was no significant devdase activity in sb and cos-7 cells expressing egfp. in addition, when protein ␣ transfected cells were treated with devd-cho, reduction of devdase activity in protein ␣-induced apoptosis in both cells was observed. the interaction between viruses and host cells is very complex. host defense mechanism attempts to limit the virus replication by killing infected cells (mounira et al., 1998) . on the other hand, many viruses have evolved mechanisms to cope with cellular antiviral responses. one of these viral strategies is triggering apoptosis during infection. in the case of virus-infected cells, apoptosis seems to represent a very efficient mechanism by which the virus can induce cell death and disseminate progeny while limiting induction of inflammation. the presence of progeny virions in membrane-bound bodies also protects them from contact with neutralizing antibodies. a lot of viruses, such as adenoviridae, papovaviridae, herpesviridae, etc, have been shown to use apoptosis to kill the infected cells at the end of infection to spread the viral progeny to nearby cells (krajcsi and wold, 1998; roulston et al., 1999; teodoro and branton, 1997) . this is the first report showing that ggnnv is capable of inducing apoptosis. this is demonstrated by several methods including analysis of total cellular dna for low molecular weight dna by agarose gel electrophoresis, tunel labeling for detection of dna fragmentation in situ, and hoechst 33342 staining for nuclear morphology. there was general agreement of the results: in the analysis of total cellular dna, dna fragmentation bands were detected at 24 h pi, which peaked at 48 h pi in ggnnvinfected cells. also, the tunel positive signal was much stronger at 48 h pi than at 24 h pi. similarly, ietdase and devdase activity induced by ggnnv infection was much higher at 48 h pi than at 24 h pi. these results suggested that there is a correlation between appearance of apoptosis and cpe that peaked at 48 h pi. caspases are the central players in apoptosis. at least two major pathways for caspase activation have been delineated, including a pathway linked to the tnf family of death receptors ("extrinsic") and a pathway activated by mitochondria ("intrinsic"). the apical proteases in the exfig. 6 . immunoblotting assay showing the expression of proteins a and ␣ in eukaryotic expression system. cell lysates from cos-7 and sb cells transfected with pegfp-rna1 and pegfp-rna2 were electrophoresed in a 12% sds-page, transferred to nitrocellulose membrane, and detected by the monospecific antibodies against proteins a (lanes 1 and 4) and ␣ (lanes 2 and 5), respectively. lysates from cells transfected with pegfp-c1 (lanes 3 and 6) were indicated as negative control. trinsic and intrinsic pathways are caspase-8 and caspase-9, respectively (salvesen and dixit, 1997) . the results of high activity of ietd-pna which can be inhibited by ietd-fmk in ggnnv-infected sb cells suggested that ggnnv triggers apoptosis of sb cells primarily through mechanisms that engage the extrinsic pathway at or above the level of caspase-8. caspase-8 directly or indirectly initiates the proteolytic activities of downstream effector caspases, such as caspase-3. when downstream "effector" caspase-3 is activated by apoptotic stimuli, it activates a caspase-activated dnase (cad), which is present in the cytosol complexed with its inhibitor of cad (icad). caspase-3 cleaves icad and allows cad to translocate to the nucleus and degrade dna (janicke et al., 1998) . in fact, many viruses induce caspase-dependent apoptosis, such as murine coronavirus mouse hepatitis virus (mhv) , langat flavivirus (lgtv) (prikhod'ko et al., 2001) , and poliovirus (agol et al., 1998) . the results that ggnnv infection induced activation of caspase-3-like proteases and ggnnv-induced apoptosis could be inhibited by devd-cho indicate that fish caspases are important mediators of virus-induced cell death. it has been known that many viral proteins are involved in both the induction and suppression of apoptosis. rubella virus capsid protein (duncan et al., 2000) , vp2 of ibdv (fernadez-arias et al., 1997) , and e protein of lgtv (prikhod'ko et al., 2001) have been shown to induce apoptosis alone in cell culture and play an important role in viral pathogenesis. other viruses, including adenoviruses, simian virus 40, and human papillomavirus, etc. have evolved a myriad of proteins to inhibit various steps in the inflammatory and apoptotic response pathways to protect infected cells from early death (roulston et al. 1999) . to localize possible inducers of apoptosis in betanodavirus ggnnv infection, we transiently expressed the viral protein a and protein ␣ in sb and cos-7 cells and found that protein ␣ was capable of inducing apoptosis in both kinds of transfected cells. protein ␣ is the precursor of capsid proteins and is important for viral assembly. here, protein ␣ of ggnnv was demonstrated to be capable of inducing apoptosis by dna fragmentation and tunel staining in transfected sb and cos-7 cells at 48 h post-transfection. in addition, tunel positive signal was dramatically decreased when cells were treated with devd-cho (data not shown). the expression of protein ␣ might trigger the inat 48 h post-transfection, the cells were fixed and analyzed for detection of apoptosis by tunel staining, which detects the dna breakage (brown color). a and c indicate tunel-labeling of cos-7 and sb cells that were transfected with egfp-rna2 at 48 h post-transfection, respectively. b and d indicate cos-7 and sb cells that were transfected with pegfp-c1 at 48 h post-transfection, respectively. magnification x200. fig. 8 . protein ␣ induces activation of caspase-3 proteases in both sb and cos-7 cells. cell lysates from sb or cos-7 cells transfected with plasmids pegfp-rna2 or pegfp-c1 were harvested at 24 h post-transfection and assayed for devdase activity using the caspase-3 colorimetric substrate devd-pna. in addition, cells transfected with pegfp-rna2 were treated with devd-cho. the pegfp-c1 was used as a negative control and to maintain equal plasmid dna concentration for each of the transfections. values shown are means from duplicate experiments. ternal death signal of cells to induce apoptosis, which serves as a host defense mechanism against viral proliferation. further work needs to analyze its proapoptotic domain and subcellular localization in virus-infected cells to find its apoptosis mechanism. in ggnnv-infected sb cells, high activity of caspase-8 and caspase-3 was detected. meanwhile, the apoptosis was decreased by devd-cho treatment demonstrated in tunel staining, which means the apoptosis induced by ggnnv was caspase dependent. similarly, transient expression of protein ␣ from ggnnv in two cell lines; sb and cos-7 cells, could activate caspase-3-like proteases and its activity could be inhibited by devd-cho. moreover, the reduction of tunel positive signal was observed in cells treated by devd-cho. these results indicate that the apoptosis induced by protein ␣ in fish and mammalian cells possibly uses the same caspase-dependent pathway. but the function of apoptosis in the true life cycle of the ggnnv infection needs more investigation. fish nodaviruses (ggnnv) and sea bass (sb) cell line (chong et al., 1987 (chong et al., , 1990 were obtained from ava (agrifood and veterinary authority of singapore). the sb cells were grown in modified eagle's medium (mem; gibco, usa) supplemented with 10% fetal bovine serum (fbs; gibco, usa), 0.34% nacl, 0.12% hepes, and 2 mm glutamine at 23°c. ggnnv was originally isolated in 1992 from brain, head, kidney, and liver of greasy grouper epinephelus tauvina in singapore. the sb cells were infected with the betanodaviruses ggnnv for propagation and inoculated cultures were harvested when 90% of cells in the monolayer showed specific cpe. cos-7 cells were maintained in dulbecco modified eagle's medium (dmem; gibco, usa) supplemented with 10% fbs and grown at 37°c in 5% co 2 . the e. coli strain was grown in luria bertani (lb) broth. the medium was supplemented with 100 g ml ϫ1 ampicillin or 30 g ml ϫ1 kanamycin for selection of transformants and plasmid maintenance. the protein a-encoding region in pcdna3.1/rna1 which contained the full-length cdna of ggnnv rna1 was amplified by pcr using the pair of primers (5ј)at-ggatccatgcgtcgctttgagtttgca, and (3ј)gt-gtcgactacacttg agtgcgacgtcg and constructed into pgex-4t-2 vector (amercham, pharmacia, uppsala, sweden) by digesting both vector and pcr fragment with bamhi/sali to obtain plasmid pgex-4t-2/rna1. the open reading frame of protein ␣ gene was obtained by pcr amplification with primers: (5ј)cggggtaccatg-gtacgcaagggtgataag and (3ј)cccaagctttt-agtttcccgagtcaaccc containing kpni/hindiii restriction enzyme sites and cloned into pqe30 vector (qiagen hilden, germany) to construct plasmid pqe30/rna2. the fusion proteins were expressed and purified by standard techniques as recommended by the manufacturer. to produce specific antibodies against protein a and protein ␣, two female guinea pigs were administered 200 -250 g of the purified proteins emulsified with adjuvant montanide isa 70 (seppic, france). guinea pigs were boosted twice with the same quantities of antigen emulsion every other day for 14 days. ten days after the final booster injection, the animals were bled and the antibody titers and specificity were determined by western blot with ggnnvinfected sb cells. the coding sequence of proteins a and ␣ were amplified by pcr and inserted into pegfp-c1 vector (clontech) under the control of human cytomegalovirus (cmv) promoter to obtain pegfp-rna1 and pegfp-rna2. they were analyzed by sequencing to confirm that no errors were introduced as a result of pcr amplification. the primers used for these two gene amplification were as follows: rna1-5ј: ccgaattctatgcgtcgcttt-gagtttgcac (ecori); rna1-3ј: gtggatcccta-cacttgagtgcgacgtcg (bamhi); rna2-5ј: gc-gaattctatggtacgcaaaggtgagaag (ecori); rna2-3ј: gtgtcgacttagtttcccgagtcaaccct (sali). in vitro expression of the pegfp-rna1 and pegfp-rna2 constructs was performed in transient expression experiments using sb and cos-7 cells, and 60 -80% confluent monolayers of sb or cos-7 cells grown in 25 ϫ 25 cm flask were transfected with a 2 g/flask of plasmid dna (purified by qiagen plasmid midi kits, chatsworth, ca, usa) mixed with lipofectamin plus reagent according to the instructions of the manufacturer (life technologies, gaithersburg, md, usa). western-blot analysis was carried out to determine the expression of protein a and protein ␣. briefly, at 48-h post-transfection, the cells were washed twice with ice-cold pbs, harvested, and separated by 12% sds-page, then transferred to hybond nitrocellulose membranes (bio-rad, richmond, ca, usa). the membranes were blocked with 5% skimmed milk in phosphatebuffered saline with 0.05% tween 20 (pbst) for 1 h, washed with pbst once, and incubated with guinea pig against protein a and protein ␣, separately, at room temperature for 1 h, then washed with pbst three times and incubated with hrp-conjugated anti-guinea pig igg secondary antibody at room temperature for 40 min. after rinsing three times, the specific proteins were visualized by enhanced chemiluminescence (ecl) (pierce, rockford, il, usa). low-molecular-weight nuclear dna was isolated using apoptotic dna-ladder kit (promega, madison, wi, usa) following the manufacturer's instruction. briefly, the infected or transfected cells were harvested and resuspended in binding/lysis buffer (6 m guanidine-hcl, 10 mm urea, 10 mm tris-hcl, and 20% triton x-100, ph 4.4) and incubated for 10 min at rt. after incubation, isopropanol was added and the mixture was vortexed. the mixture was then placed in a filter tube and centrifuged for 1 min at 8000 rpm at rt. the upper reservoir was washed with washing buffer and centrifuged twice. after centrifugation for 1 min at 13,000 rpm, the nucleic acid was dissolved in prewarmed (70°c) elution buffer, centrifuged, and collected. equal amounts of dna were resolved on a 2% agarose gel containing ethidium bromide and the size of the olignucleosomal dna fragment was measured by comparison with a 100 bp dna marker. the terminal deoxynucleotidyltransferase (tdt)-mediated dutp nick-end labeling (tunel) assay was performed with apoptosis detection system fluorescein kit (promega) and deadend colorimetric tunel system kit (promega) according to the protocol of the manufacturer. briefly, ggnnv-infected sb cells grown on 4-well chamber slides (iwaki, japan) were fixed with 4% paraformaldehyde for 25 min at 4°c rt at postinfection 24, 36, and 48 h and permeabilized with 0.2% triton x-100 in pbs for 5 min at rt. after equilibration for 10 min at rt, cells were overlaid with 50 l tdt reaction buffer and incubated at 37°c for 60 min. the reaction was terminated by immersing slides in 2 ϫ ssc for 15 min. after being washed three times with pbs, the slides were examined under confocal microscope lsm510 and photographed. to perform tunel assay in transfected cells, sb or cos-7 cells grown on chamber slides were transfected with 0.4 g of recombinant plasmid as described above. at 48 h post-transfection, cells were fixed with 4% paraformaldehyde for 25 min at 4°c rt and permeabilized with 0.2% triton x-100 in pbs for 5 min at rt. after equilibration, cells were overlaid with tdt reaction buffer and incubated at 37°c for 60 min, then immersed in 2 ϫ ssc for 15 min to terminate reaction. after being washed three times with pbs, the slides were immersed in 0.3% hydrogen peroxide for 5 min to block the endogenous peroxidases and washed three times with pbs. streptavidin-hrp solution was then added to slides and incubated for 30 min at rt. after being washed three times with pbs, the slides were developed by 3,3ј-diaminobenzidine tetrahydrochloride (dab) components until there was a light brown reaction product and then were rinsed several times in deionized water to stop the reaction. colorimetric assay of caspase-8-like (ietdase) and caspase-3-like (devdase) proteolytic activity was performed using an apoalert caspase-8 and caspase-3 colorimetric assay kit (clontech laboratories, palo alto, ca, usa). sb cells were cultured in 25 ϫ 25 cm flasks and infected with betanodavirus ggnnv. at 24 and 48 h postinfection, cells were centrifuged at 2000 rpm for 5 min to harvest. cells (3-5 ϫ 10 6 ) were then lysed in 50 l of lysis buffer on ice for 10 min and centrifuged at 14,000 rpm for 3 min at 4°c and the supernatant was collected. supernatant (50 l) was added to an equal volume of 2x reaction/ dtt buffer supplemented with ietd-pna (200 m) and incubated at 37°c for 2 h in the dark, and then the optical density was measured at 405. in the case of caspase-3-like proteases, supernatant (50 l) was added to an equal volume of 2x reaction/dtt buffer supplemented with devd-pna (50 m) and incubated at 37°c for 1 h. the nanomoles of pna released per hour were calculated from the standard curve. to detect the caspase-3 like proteolytic activity in transfected cells, sb and cos-7 cells were seeded in 25 ϫ 25 cm flasks on the previous day and transfected with 2 g of pegfp-rna2 and pegfp-c1 plasmids. at 24 h posttransfection, cells were harvested at 2000 rpm for 5 min, then caspase-3-like protease (devdase) activity assay was carried out as described above. the bcl-2 protein family: arbiters of cell survival two types of death of poliovirus-infected cells: caspase involvement in the apoptosis but not cytopathic effect induction of apoptosis in murine coronavirus-infected cultured cells and demonstration of e protein as an apoptosis inducer family nodaviridae growth of lymphocystis virus in a sea bass study of three tissue culture viral isolates from marine food fish a fish encephalitis virus that differs from other nodaviruses by its capsid protein processing rubella virus capsid protein induces apoptosis in transfected rk13 cells viruses and apoptosis: meddling with mitochondria the major antigenic protein of infectious bursal disease virus, vp2, is an apoptotic inducer transmission of viral encephalopathy and retinopathy (ver) to yolk-sac larvae of the atlantic halibut hippoglossus hippoglossus: occurrence of nodavirus in various organs and a possible route of infection viral interference with apoptosis detection of nodavirus in barramundi, lates calcarifer (bloch), using recombinant coat protein-based elisa and rt-pcr pkr, apoptosis and cancer caspase-3 is required for dna fragmentation and morphological changes associated with apoptosis inhibition of tumor necrosis factor and interferon triggered responses by dna viruses the apoptosis pathway triggered by the interferon-induced protein kinase pkr requires the third basic domain, initiates upstream of bcl-2, and involves ice-like proteases cytochrome c and datp-dependent formation of apaf-1/caspase-9 complex initiates an apoptotic protease cascade apoptosis, oncosis, and necrosis. an overview of cell death baculovirus regulation of apoptosis properties of a new virus belonging to nodaviridae found in larval striped jack (pseudocaranx dentex) with nervous necrosis resistance to virus infection conferred by the interferon-induced promyelocytic leukemia protein special topic review: nodaviruses as pathogens in hatchery-reared larvae and juveniles of redspotted grouper occurrence of viral nervous necrosis in kelp grouper and tiger puffer infection with langat flavivirus or expression of the envelope protein induces apoptotic cell death viruses and apoptosis caspases: intracellular signaling by proteolysis determination of the complete nucleotide sequences of rna1 and rna2 from greasy grouper (epinephelus tauvina) nervous necrosis virus, singapore strain regulation of apoptosis by viral gene products caspases: enemies within an evolutionary perspective on apoptosis cell death: the significance of apoptosis this work was supported by the national sciences and technology board (nstb), singapore. key: cord-256316-1odgm6hm authors: godet, murielle; l'haridon, rene; vautherot, jean-francois; laude, hubert title: tgev corona virus orf4 encodes a membrane protein that is incorporated into virions date: 1992-06-30 journal: virology doi: 10.1016/0042-6822(92)90521-p sha: doc_id: 256316 cord_uid: 1odgm6hm abstract the coding potential of the open reading frame orf4 (82 amino acids) of transmissible gastroenteritis virus (tgev) has been confirmed by expression using a baculovirus vector. five monoclonal antibodies (mabs) raised against the 10k recombinant product immunoprecipitated a polypeptide of a similar size in tgev-infected cells. immunofluorescence assays performed both on insect and mammalian cells revealed that orf4 was a membrane-associated protein, a finding consistent with the prediction of a membrane-spanning segment in orf4 sequence. two epitopes were localized within the last 21 c-terminal residues of the sequence through peptide scanning and analysis of the reactivity of a truncated orf4 recombinant protein. since the relevant mabs were found to induce a cell surface fluorescence, these data suggest that orf4 may be an integral membrane protein having a cexo-nendo orientation. anti-orf4 mabs were also used to show that orf4 polypeptide may be detected in tgev virion preparations, with an estimated number of 20 molecules incorporated per particle. comparison of amino acid sequence data provided strong evidence that other coronaviruses encode a polypeptide homologous to tgev orf4. our results led us to propose that orf4 represents a novel minor structural polypeptide, tentatively designated sm (small membrane protein). virology 188, 666-675 (1992) the coding potential of the open reading frame orf4 (82 amino acids) of transmissible gastroenteritis virus (tgev) has been confirmed by expression using a baculovirus vector. five monoclonal antibodies (mabs) raised against the 10k recombinant product immunoprecipitated a polypeptide of a similar size in tgev-infected cells. immunofluorescence assays performed both on insect and mammalian cells revealed that orf4 was a membrane-associated protein, a finding consistent with the prediction of a membrane-spanning segment in orf4 sequence. two epitopes were localized within the last 21 c-terminal residues of the sequence through peptide scanning and analysis of the reactivity of a truncated orf4 recombinant protein. since the relevant mabs were found to induce a cell surface fluorescence, these data suggest that orf4 may be an integral membrane protein having a cexo-nendo orientation. anti-orf4 mabs were also used to show that orf4 polypeptide may be detected in tgev virion preparations, with an estimated number of 20 molecules incorporated per particle. comparison of amino acid sequence data provided strong evidence that other coronaviruses encode a polypeptide homologous to tgev orf4. our results led us to propose that orf4 represents a novel minor structural polypeptide, tentatively designated sm (small membrane protein). transmissible gastroenteritis virus (tgev), an important pathogen of swine, is a member of the coronaviridae, a family of enveloped viruses with a large (-30 kb) continuous, positive rna genome. sequencing data have led to the identification of a number of large open reading frames (orfs). orfla and b, which account for the 5' two-thirds of the coronavirus genome, are assumed to encode nonstructural proteins including the viral replicase/transcriptase. the seven to eight remaining orfs are expressed through a set of 3' coterminal, subgenomic size mrnas of which only the unique region is translationally active. these include the orfs coding for the virion structural proteins, i.e., the nucleocapsid (n) and two or three envelope glycoproteins: the spike (s) and the membrane (m) proteins, and the hemagglutinin-esterase (he) present in a coronavirus subset. these orfs are distributed following the consensus gene order 5' pol-(he)-s-m-n 3'. the other orfs are interspersed within the genome and their number and position differ among coronavirus members (reviewed by spaan et a/., 1988; and lai, 1990) . they have been shown to be expressed by functionally mono-, di-, or tricistronic mrnas and were generally assumed to encode nonstructural proteins, the function of which is still unknown or conjectural. ' to whom correspondence and reprint requests should be addressed. in tgev genome, four such orfs have been deduced. two of them are expressed by the same mrna (mrna 3) in two out of the three virus strains sequenced (rasschaert et a/., 1987; kapke et a/., 1988; britton et al., 1989; wesley et al., 1989) . the predicted product of orf3a is 61 to 71 codon long, with a variable c-terminal end. it appears to be dispensable for virus replication since it was found to be absent in a tgev variant strain sp (wesley et a/., 1990) as well as in the closely related porcine respiratory coronavirus (prcv) (rasschaert eta/., 1990) . orf3b (expressed by a separate mrna species numbered 3-l in miller strain) has a constant length (244 residues); however, one clone of purdue-l 15 strain was reported to have an orf3b which is shortened by 79 codons at the 5' end and in several cdna clones by 67 codons at the 3' end (rasschaert et al., 1987) . orf4 was predicted to encode a 82 amino acid long hydrophobic polypeptide (rasschaer-t et a/., 1987; kapke et a/., 1988; britton et a/., 1989; wesley eta/., 1989) . so far, the putative products of the three above-mentioned orfs have not been identified in infected cells. the last orf, orf7, is located downstream of the n gene (kapke and brian, 1986; rasschaert et al., 1987; britton et al., 1988) an unusual feature among coronaviruses. a polypeptide of m, 14k, reacting with antibodies produced against an orf7 synthetic peptide, has been characterized in tgev-infected cells (garwes et al., 1989) . in this study we report the identification of a product of one of these orfs (orf4) in infected cells and its preliminary characterization. in particular, we show evidence that orf4 represents a novel virion-associated polypeptide with a possible counterpart in other coronaviruses. autographa cahfornica nuclear polyhedrosis virus (acnpv) and recombinant baculoviruses were grown and assayed in confluent monolayers of spocfoptera frugiperda (sf9) cells in medium containing 10% (v/v) fetal bovine serum, according to the procedures described by brown and faulkner (1977) . propagation of the high cell passage purdue-l 15 strain of tgev in swine cell lines pd5 or st was done as previously described (laude et al., 1986) . manipulations of plasmid dna were performed according to the procedures described by sambrook et a/. (1989) . restriction enzymes, t4 dna ligase, and calf intestine alkaline phosphatase (cip) were purchased from boehringer-mannheim. the baculovirus transfer plasmid containing the full-length cdna copy of tgev orf4 coding sequence was constructed following the general scheme outlined in fig. 1 . the ndel-sspl fragment (0.7 kbp) derived from plasmid ptg2-15 (rasschaert et al., 1987) was digested with the ddel restriction enzyme. ddel-sspl dna fragment was repaired with the klenow large fragment of dna polymerase i and cloned into the barnhi site of the pvl941 vector (luckow and summers, 1989) . the resulting plasmid, named pvlorf4, contained a 0.33 kbp insert. a second plasmid, pvlorf4a was constructed by inserting an orf4 gene in which the 3' last 63 nt were deleted through pcr mutagenesis @char-f et a/., 1986) on ptg2-15 using the oligonucleotides 5' g aagaagggatccatacctatgac and 5' clta-tagggatcctaagcatg as 5' and 3' amplimers, respectively. these amplimers were designed to introduce an additional stop codon tag at the 3'end of the gene and a barnhi cloning site at each end. the amplification product was digested by bamhl and ligated into the pvl941 cloning site. the orientation and sequence of the orf4 and orf4a inserts relative to the acnpv polyhedrin leader were determined by restriction analysis and partial dna sequencing. in these constructs, the initiation codon of the orf4 and orf4a sequences were positioned at 54 and 7 bp from the bamhl cloning site, respectively. transfer of the tgev orf4 gene into the acnpv genome was accomplished by transfection of sf9 cells using the calcium phosphate precipitation technique as described by summers and smith (1987) . recombinant baculoviruses were screened by dot blot hybridization using an orf4specific [32p]-labeled dna fragment as a probe. four polyhedrin-negative clones were tested for orf4 expression. tgev orf4a gene was introduced into a linear form of acnpv dna (kitts eta/., 1990) . circular acrpg-sc dna was linearized by digestion at the unique bsu361 site. two hundred nanograms of bsu361 or mock digest viral dna was mixed with 1 pg of pvlorf4a dna and transfected into sf9 cells using the lipofectin method (kitts et a/., personal communication) according to the procedure of the manufacturer (gibco-brl). after a 2-day incubation the culture supernatants were harvested and plated. a dozen well-isolated plaques were picked out and screened for orf4a expression using [35s]methionine-labeled cultures. three balb/c mice were injected threefold intraperitoneally at a 1 month interval with 1 x 10' acorf4-infected cells (disrupted in freund complete adjuvant for the first injection). three days before fusion, the mice were boosted both intraperitoneally and subcutaneously with orf4 protein purified from 4 x 10' infected cells by 15% sds-page. splenocytes from one mouse that tested positive by immunoprecipitation assay were fused with sp,o myeloma cells. supernatants of hybrid clones were tested in a comparative immunofluorescence assay (see below) using acorf4or acnpv-infected sf9 monolayers. subcloning of orf4-specific antibody-producing hybridomas and ig isotyping were done as described elsewhere (l' haridon et al., 1991) . iggs purified from ascites fluids by ammonium sulfate precipitation and gel permeation on a sephacryl-s200 column were used in all experiments. screening of hybridoma was performed on sf9 cell monolayers established in 96-well microplates, infected with baculovirus (m.o.i. 10 pfu) and fixed with acetone/ethanol (v/v) at 38 hr p.i. for surface fluorescence analysis, aliquots of cells in suspension were stained with mabs at 100 pglml in grace medium and then with fitc conjugate (each step 1 hr at 4") and spotted onto glass slides. alternatively, spotted cells were fixed with 4% paraformaldehyde and permeabil-ized or not with 0.1% triton x-l 00 before staining (1 hr at 37"). similar experiments were performed on st cell monolayers infected by tgev at a m.o.i. of 0.1 pfu and fixed 15 hr p.i. the procedures for metabolic labeling of insect and mammalian cells were as reported previously (godet et a/., 1991; . monolayers of 4 x 1 o5 sf9 cells or 6 x 1 o6 pd5 cells labeled with [35s]methionine were washed with pbs and lyzed in 4 ml of pbs-triton (tris, 50 mm, ph 8.5, 1 o/o triton x-l 00, 1 o3 kallicrein units of aprotinin per milliliter). resulting cytosols of sf9 cells and pd5 cells were centrifuged 30 min at 10,000 g or 1 hr at 30,000 rpm in a 50 ti rotor (beckman), respectively and stored in aliquots at -70". lmmunoprecipitation assay aliquots of radiolabeled cytosols or virions were adjusted to 0.5 ml with pbs-triton buffer containing the appropriate mab (100 pg/ml) or 3 ~1 of ascites fluid from a feline infectious peritonitis virus-infected cat (used as a source of anti-tgev polyclonal antibodies) and protein a-sepharose beads (50 ~1 of a 50% suspension); after a 2-hr incubation at room temperature with agitation, the immune complexes were extensively washed with pbs-triton, then with 0.5 m nacl + 50 mn/l tris (ph 8). beads were treated for 3 min at 100" in sample buffer. the immunoprecipitated material thus released was analyzed by 15% or 15-20% sds-page. virions in the supernatant of cultures labeled as above were purified following a described procedure (laude et a/., 1986) . the material pelleted by ultracentrifugation at 35,000 rpm in a 45 ti rotor was resuspended in distilled water and was applied to a linear sucrose gradient (16 ml, 20 to 45% sucrose w/v in distilled water). centrifugation was performed in an sw27 rotor for 3 hr at 25,000 rpm and 4". gradients were collected from top to bottom into 500-p.1 fractions. half of each fractions was run at 100,000 rpm for 30 min and the resulting pellets were analyzed by sds-page electrophoresis on a 8-20% gradient gel. the material remaining in the virus-containing fractions was pooled, pelleted as above, and split into three parts; one was analyzed directly by 15% sds-page; the two others were solubilized in pbs-triton, immunoprecipitated by a different mab each, and analyzed as above. in one experiment, virion-associated material was subjected to a second round of gradient purificalaude et al., 1986) prior to gel analysis. peptides were synthesized on polyethylene pins (geysen eta/., 1984) by using a commercially available kit, according to the procedure given by the manufacturer (cambridge research biochemicals). immunoreactivity of the immobilized peptides was assayed by elisa using anti-orf4 mabs (25 pglml) as primary antibody and an anti-mouse igg (h + l) peroxydase conjugate (biosys). insertion of the entire orf4 coding sequence into the genome of acnpv baculovirus was performed by using the transfer plasmid pvl941 (see methods and specific probe and a polyhedron-negative phenotype were selected, amplified, and tested for the expression of orf4. among four selected orflf-expressing clones, one, designated acorf4, was retained for subsequent studies. analysis of [35s]methioninelabeled acorf4-infected cells revealed the presence of a major polypeptide of i'@ 1 ok (fig. 2) in good agreement with the predicted m.w. of orf4 product (9.2k). the time course for its synthesis was found to be from 24 to 48 hr p.i. screening of positive hybridoma clones was achieved by comparative indirect immunofluorescence assay on acorf4-or acnpv-infected cells. among 268 hybridomas tested, 5 positive clones, all producing mabs of iggl isotype, were subcloned and used for the production of ascites fluids. when assayed by immunoprecipitation of acorf4-infected cells extracts, all 5 mabs recognized a single species of m, 1 ok, identical to the target protein (fig. 3a, lane 4 ). an immunoreactive species comigrating with recombinant orf4 protein was detected in tgev-infected mammalian cells as well (lane 2). analysis of the recombinant protein in nonreducing conditions revealed the existence of oligomeric forms, which were not observed with the authentic orf4 protein (fig. 3b) . orf4 product is a membrane-associated protein indirect immunofluorescence assays were performed to determine the subcellular location of orf4 protein. in acetone-fixed cultures of both acorf4-infected sf9 and tgev-infected cells, all anti-orf4 mabs induced a strong fluorescence, which were polarized in a juxtanuclear region consistent with golgi localization (fig. 4a) . in addition, a bright fluorescence was observed on unfixed (fig. 4b) positively stained cells were consistently observed (fig. 4d) , whereasvirtually all the cells showed the presence of orf4 antigen after permeabilization (not shown). the fluorescence observed in nonpermeabilized cells was unlikely due to cell damage since only a few of them were stained positively with a mab directed against an intracellular antigen (tgev n protein; data not shown). these data were interpreted as reflecting a late accumulation of orf4 at the outer membrane of tgev-infected cells. these observations showing that orf4 protein is found in association with cellular membranes are consistent with the prediction of a membrane-spanning domain in its amino acid sequence (fig. 5) . anti-orf4 monoclonal antibodies recognize the c-terminal domain of the protein assuming that orf4 was an integral membrane protein, it was of interest to determine whether exposed epitopes were located within the carboxy or amino domain of the polypeptide chain. the peptide scanning method (geysen et al., 1984) was used since two anti-orf4 mabs were shown to be reactive toward denatured and reduced protein. a set of peptides encompassing all overlapping linear nonapeptides homologous with orf4 sequence was synthesized and tested against each of the five mabs. as shown in fig. 6 , mab v27 strongly recognized a linear epitope centered on residues ayknf (positions 64 to 68; see fig. 5 ). mab s2 gave comparable results, although a lesser reactivity was observed when compared to mab v27. no reactivity was observed with the three remaining mabs, possibly because of a lower avidity. to test the possibility that these three anti-orf4 mabs may also recognize the carboxy-subterminal region of the molecule, a second recombinant baculovirus designated acorf4a, which expressed a truncated form of orf4 lacking the 21 c-terminal amino acids, was constructed via pcr mutagenesis. immunoprecipitation analysis revealed that a polypeptide of slightly reduced size was expressed by acorf4ainfected cells and recognized by polyclonal antibodies but not by any of the anti-orf4 mabs (partial data in fig. 7) . in order to determine whether the protein is incorporated within the virion as a possible envelope protein, labeled tgev particles were purified by centrifugation and analyzed by gel electrophoresis (fig. 8) . five welldefined major bands were visible, which corresponds to the previously recognized virion-associated polypeptides: (i) a 220k band (s protein), (ii) a 47k band (n peptides fig. 6. epitope mapping of anti-orf4 mab v27. a series of nonapeptides (overlapping 1) spanning the length of the entire orf4 sequence was tested for elisa reactivity toward mab v27. n-terminus is on the left. protein), and (iii) three bands corresponding to different species of m protein; 30-36k identified as complex type glycosylated forms, 29k (high mannose form) and 26k (unglycosylated form) (delmas and laude, 1991) . a single additional minor band of m, 1 ok, similar to that of orf4 polypeptide, was detected (fig. 8a) . the fact that very few, if any, other polypeptides copurified renders unlikely the possibility that the 1 ok polypeptide remained nonspecifically associated to the sedimenting virus particles. true association of the 10k band with virions was confirmed by showing that the observed polypeptide pattern remained unchanged after an additional round of purification by isopycnic centrifugation. lmmunoprecipitation with mab v27 of detergent-dissociated material from virus-containing fractions confirmed the identity between the 10k virion-associated and orf4 polypeptides (fig. 8b) . densitometric tracing of autoradiograms at different times of exposure was performed to evaluate the relative amounts of the virion-associated polypeptides. the resulting values were corrected according to the predicted number of met residues in the respective sequences. this led to an estimated molar ratio of 1:20:300 for orf4, s, and m polypeptides. a common feature of coronavirus genomes appears to be the existence, immediately upstream from the membrane protein m gene, of an orf 250 to 330 nucleotide long, which is predicted to encode a hydrophobic polypeptide. furthermore, recent studies on mouse hepatitis virus (mhv), infectious bronchitis virus (ibv), and bovine (bcv) coronaviruses have shown that the products translated from these orfs were associated with the membrane of infected cells (leibowitz et a/., 1988; abraham et a/., 1990; smith et al., 1990) , as evidenced now for tgev orf4. these observations prompted us to reexamine the relevant amino acid sequences for possible similarities that earlier comparative analysis by us and others failed to detect (except for the closely related mhv and bcv viruses). figure 9 shows a tentative alignment of the orf4-like sequences from 5 coronavirus members, which was outlined using the program multalin (corpet, 1988) and refined manually. on the basis of the deduced consensus sequence, in which 37 residues are conserved in at least 3 out of the 5 sequences aligned (87 positions), we conclude that these proteins share significant similarities. as a striking feature, a 19-20 residue-long hydrophobic stretch, followed by a cluster of 2 or 3 cysteines is present 15 to 20 residues from the n terminus the c-terminal part of the polypeptides seems to be more distantly related; in particular, ibv protein extended the consensus sequence by 14 to 28 residues, depending of the strain (see liu et a/., 1991 for ibv orf3c sequence data). in this study we have confirmed the coding potential of the orf encoded by tgev mrna 4 and characterized several properties of its translation product. baculovirus-vectored expression of orf4 resulted in the synthesis of a 1 ok polypeptide. the same species was identified in tgev-infected cells by immunoprecipitation using monoclonal antibodies (mabs) raised against the recombinant polypeptide. examination of its subcellular localization by immunostaining showed that orf4 translation product is a membrane-associated polypeptide. this finding is consistent with the prediction in the second n-terminal quarter of a stretch of uncharged residues with proper-ties of a membrane-spanning domain (fig. 5) . immunofluorescence data also suggested that orf4 may enter the exocytic pathway. in tgev-infected cells, however, orf4 could be detected in association with the cell surface only at a late stage of the infection. assuming that the observed surface fluorescence was related to externally exposed determinants, it was of interest to map the relevant epitopes on the amino acid sequence of the molecule. peptide scanning led to the identification of residues 64-ayknf-68 as the core sequence of the binding site of 2 of the mabs (cavanagh et a/., 1990) . pairwise homologies are marked by dots and bold letters; gaps are indicated by dashes. bottom line, consensus sequence showing residues identical in at least three of the five sequences. sequence data from rasschaert et al., 1987; raabe and siddel, 1989; skinner et al., 1985; woloszyn et al., 1990; and boursnell et al., 1985. the 22 last n-terminal residues of ibv orf3c (beaudette strain) are not shown. studied. furthermore, a recombinant orf4 protein truncated of its last 21 c-terminal amino acids was no longer recognized by any of the 5 mabs. these results support the view that an antigenic, possibly immunodominant site is expressed in the c-subterminal part of orf4 protein. in addition, they led us to speculate that the region of the molecule which is translocated across the membrane would correspond to its carboxy domain. recently, a striking correlation has been reported between the transmembrane orientation of eukaryotic proteins and the disposition of charged residues surrounding the most n-terminal membranespanning sequence. it has been proposed that the difference in the charge of the 15 residues flanking the presumed anchor segment determines its orientation with the more positive portion facing the cytosol (hartmann et a/., 1989) . applying such a rule to tgev orf4 sequence gives charges of -1 and +2 for the n and c flanking segments, respectively, which predicts a nexo-cendo orientation, in contrast to the available experimental evidence. thus further experiments, including the production of antibodies directed to the n-terminal part of the protein, are needed for a definite assignment of the transmembrane orientation of orf4. the possible role of the cysteine cluster immediately downstream the hydrophobic segment was also examined. gel analysis of recombinant orf4 protein under nonreducing conditions revealed the presence of multimeric, predominantly dimeric forms. in contrast, orf4 synthesized in tgev-infected cells could be detected in a monomeric form only, suggesting that the formation of disulfide-bridged species is an artifact, presumably linked to the high level of expression of orf4 in the insect cells (kiefhaber et al., 1991) . the possibility was tested that the cystein residues could serve as an acylation site, as demonstrated for coronavirus s protein (schmidt, 1982) . however, no incorporation of palmitic acid chains could be detected in recombinant orf4 protein (data not shown), as would be expected if the target residues belong to the orf4 ectodomain. an important implication of the above findings was that orf4 could represent a structural polypeptide present in the virus envelope. indeed, purified preparations of labeled tgev virions revealed the presence of a previously unrecognized 1 ok polypeptide which was specifically immunoprecipitated by anti-orf4 mabs. based on the estimated molar ratio and assuming that coronavirions bear 100 (roseto et a/., 1982) to 200 spikes, each composed of 3 s molecules (delmas and laude, 1990) it can be inferred that approximately 15-30 copies of orf4 protein are incorporated into tgev virions (purdue strain). such a small number of molecules in virus particles does not seem to reflect a selective exclusion since s and orf4 accumulated in infected cells at a ratio comparable to that found in virions (data not shown). these results lead us to conclude that orf4 may represent a minor structural polypeptide, which we propose to designate by the tentative acronym sm, standing for "small membrane" protein. several lines of evidence lend support to the view that a gene encoding an sm-like protein is a common feature of the coronavirus genomes: (i) an orf predicting a polypeptide with striking similarities to tgev orf4 was identified in the genome sequence of each of the 5 coronaviruses examined (fig. 9 ) and the fact that tgev sm was recognized by anti-fipv antibodies argues for the presence of a related gene also in feline infectious peritonitis virus genome; (ii) the product expressed from the relevant mhv, bcv, and ibv orfs was reported to have properties of a transmembrane polypeptide (leibowitz et a/., 1988; smith et al., 1990; abraham et a/., 1990) ; and (iii) although expressed through a mono-, di-, or tricistronic mrna (abraham et a/., 1990; budzilowicz and weiss, 1987; liu et al., 1991 ) the assumed sm-encoding genes are all located upstream and adjacent to the m protein gene. therefore, not only the sequences show significant similarities but the gene order 5'. . s sm/m . 3' is conserved, as would be expected for a structural protein. finally, preliminary experiments in this laboratory allowed us to detect orfs-encoded polypeptide in association with bcv particles (n. woloszyn, p. boireau, and j. f. vautherot, unpublished results). small integral membrane proteins have been described in several other enveloped rna viruses, including sindbis and semliki forest togaviruses, influenza a and b viruses, simian virus 5, and respiratory syncitial paramyxoviruses (garoff et a/., 1980; welch and sefton, 1980; lamb et al., 1985; hiebert, 1985; olmsted and collins, 1989) . the influenza virus m2 protein (15k) and the alphavirus 6k protein are both acylated, nexo-cendo transmembrane polypeptides. m2 and 6k have been shown to represent minor structural polypeptides, with an estimated number of 40 + 25 and 24 f 4 molecules per virion, respectively (zebedee gaedigk-nitschko and schlesinger, 1990 ). m2 has been reported to form tetrameric channels within the membrane and to be a target of the antiviral drug amantadine and of the ctl response to influenza virus infection (hay et al., 1985; lamb et al., 1985; surgrue and hay, 1991) . site-directed mutagenesis studies on sindbis and semliki forest viruses have demonstrated that 6k protein is dispensable for virus production but exerts a role late in the assembly, possibly during virus budding (gaedigk-nitschko and schlesinger, 1990; liljestrom et a/., 199 1). the sh protein of sv5 has been reported to be orientated in the mem-brane with its n-terminus domain exposed at the cytoplasmic face, as it might be the case for tgev sm. whether sh is incorporated into virions is still questioned, as well as its potential role (hiebert eta/., 1988) . the apparent conservation of sm gene in the coronavirus genome strongly implies that its product is essential for an efficient replication of the virus. based on its location and its low copy number in particles, we speculate that sm would more likely play a role in modulating assembly and/or release of the virion. thus, eluci-.dating the function of the coronavirus sm protein might contribute to a better understanding of an important aspect of the biology of enveloped viruses. finally, sm may be a potent surface antigen since murine antibodies recognized a domain of the protein possibly exposed on live infected cells. preliminary experiments indicated that anti-sm antibodies are readily detected in the serum from infected swines. therefore, the role of sm protein in humoral and cellular immune response to tgev infection should be worth investigating in the future. we thank j. gelfi for technical assistance, j. levin for revising the manuscript, and m. nezondb for the artwork, part of this work was carried out with the support of the e.e.c program eclair. note addedinproof. during the submission process of this article, a communication by (d. x. liu and s. c. lnglis (1991, virology 185, 91 1-917) reported the association of ibv orf3c protein with the virion envelope. this strengthens the view that sm-like proteins are a general feature of coronavirus. 5 kda encoded between the spike and membrane protein genes of the bovine 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virus. 1. viral nucleotide sequence of the bovine enteric coronavirus becv f15 mrna 5 and mrna 6 unique regions influenza a virus m, protein: monoclonal antibody restriction of virus growth and detection of m, in virions key: cord-008426-ktn8c0zx authors: othman, yasmin; hull, roger title: nucleotide sequence of the bean strain of southern bean mosaic virus() date: 1995-01-10 journal: virology doi: 10.1016/s0042-6822(95)80044-1 sha: doc_id: 8426 cord_uid: ktn8c0zx the genome of the bean strain of southern bean mosaic virus (sbmv-b) comprises 4109 nucleotides and thus is slightlyshorter than those of the two other sequenced sobemoviruses (southern bean mosaic virus, cowpea strain (sbmv-c) and rice yellow mottle virus (rymv)). sbmv-b has an overall sequence similarity with sbmv-c of 55% and with rymv of 45%. three potential open reading frames (orfs) were recognized in sbmv-b which were in similar positions in the genomes of sbmv-c and rymv. however, there was no analog of sbmv-c and rymv orf 3. from a comparison of the predicted sequences of the orfs of these three sobemoviruses and of the noncoding regions, it is suggested that the two sbmv strains differ from one another as much as they do from rymv and that they should be considered as different viruses. southern bean mosaic virus (sbmv) is the type member of the sobemovirus group of small icosahedral positive-strand rna viruses (for reviews see sehgal, 1981 ; tremaine and hamilton, 1983; hull, 1988) . four major strains of sbmv are recognized which are serologically related (grogan and kimble, 1964) . the type strain (bean strain), sbmv-b, infects several phaseolus spp., including many cultivars of phaseo/us vulgaris, but does not systemically infect cowpeas (vigna unguiculata), whereas the cowpea strain (sbmv-c) infects v. unguicu/ata and pisum sativum but fails to systemically infect beans (shepherd and fulton, 1962) . the ghana and mexican or severe bean mosaic strains (sbmv-g and sbmv-m) (lamptey and hamilton, 1974; yerkes and patino, 1960) , which are transmissible to several cultivars of both bean and cowpea, are distinguished bytheir reaction in certain cultivars and also serologically. the sbmv genome consists of a single molecule of positive sense rna (mr 1.4 x 106) which constitutes approximately 21% of the total particle weight. the 5' terminus of sbmv rna has a covalently linked protein (vpg) (ghosh et al., 1979) of molecular weight between 10 (sbmv-c) and 12 kda (sbmv-b) (mang et al., 1982) , which is essential for infectivity (veerisetty and sehgal, 1980) . the 3' terminus of sbmv does not have a pely(a) sequence data from this article have been deposited with the gen-~enk data libraries under accession no. l34672. present address, depaitment of genetics and cellular biology, univereiti malaya, 59100 kuala lumpur, malaysia. 2to whom correspondence and reprint requests should be addressed. tail (mang et al., 1982) but contains a free hydroxyl group (ghosh et al., 1979) . a subgenomic rna of 0.4 x 105 encodes the gene for viral coat protein ghosh eta/., 1981) . the full nucleotide sequence of the cowpea strain, sbmv-c, has been determined (wu et al., 1987) . the genome consists of 4194 nucleotides with four open reading frames designated orfs 1-4 ( fig. 1 ). in addition, limited sequence data are available for the 3' end of sbmv-b (mang eta/., 1982) , which showed little similarity to the sequence of sbmv-c. the recently published nucleotide sequence (4450 nt) of rice yellow mottle virus (rymv), another sobemovirus (ngon a yassi et al, 1994) , shows that it has a similar genome organization to sbmv-c (fig. 1) . the rnas of sbmv-b, sbmv-c, and sbmv-gh have been translated in the rabbit reticulocyte lysate and wheat germ extract in vitro systems ghosh et al., 1981; rutgers et al, 1980; brisco et al., 1985b) to give four major polypeptides= polypeptide p1 of molecular weight 100-105 kda, p2 (60-75 kda), p3 (28-29 kda), and p4 of molecular weight between 14 and 25 kda. wu et al. (1987) related these products to orfs 1 (p4), 2 (p1 and p2), and 4 (p3, coat protein) of the sbmv-c sequence. we have determined the complete sequence of the genomic rna of sbmv-b. detailed comparison of the sbmv-b sequence with that of sbmv-c rna reveals significant differences in the organization and deduced amino acid sequences of the predicted major open reading frames. these comparisons have been extended to a comparison with other plant viruses. our observations suggest that while sbmv-b and sbmv-c are related they should be considered as distinct viruses. virus purification and rna extraction sbmv-b, originally obtained from dr. j. p. fulton (arkansas) and maintained as dried leaves since 1974, was propagated in p. vu/gar/s (cv. the prince) plants and the virus was purified essentially as described by hull (1985) . viral rna was extracted using phenol-chloroform by the method of zimmern (1975) from virus particles dissociated in the presence of 1% sds. first-and second-strand cdna synthesis was performed according to gubler and hoffman (1983) using the cdna cloning system plus kit (amersham) with superscript reverse transcriptase (brl). second-strand products were treated briefly with t4 dna polymerase (sambrook eta/., 1989) to remove any 3' protruding ends. the blunt-ended double-stranded cdna was size fractionated using a sephacryl column (size-sep 400; pharmacia) and products >400 bp in size were cloned into the sinai site of pbluescript ii sk(+) vectors (stratagene) using standard techniques (sambrook et al, 1989) . the resulting set of ovelapping cdna clones were used as templates for sequencing. cloning of the 3' end. clones representing the 3' end of sbmv-b rna used in the confirmation of the 3' terminal sequences were obtained by polyadenylation of the rna using poly(a) polymerase (pharmacia), and oligo(dt)-primed clones were generated using the amersham cloning kit as described above. these cdna's were ligated into the pbluescript vectors at the hincll site. sequencing of sbmv-b. the dideoxnucleotide chain termination procedure (sanger eta/., 1977) was used to sequence the double-stranded dna clones with sequenase (usb corp.). ambiguities in the sequence were re-solved by using pcr cycle sequencing (murray, 1989) or using ditp with sequenase. the sequence of the 5' end of the rna was established by the dideoxynucleotide chain termination method directly on the rna, with primer extensions using terminal transferase (de borde et al., 1986) and oligonucleotide primers complementary to nucleotides 41 to 58 of sbmv-b. general cloning techniques were as in sambrook et al. (1989) . sequence data were assembled and analyzed using the uwgcg programs (devereux etal, 1984) . the oom-pare and dotplot algorithms were used for rna and protein sequence comparisons. alignment of homologous nucleotide or amino acid sequences were obtained using the gap and bestfit algorithms and amino acid alignments were refined using the somap program (parry-smith and atwood, 1990) . ahomol was used for presentation of some alignments. database searches were carried out either through the fasta program (devereux et al., 1984) or through the dapjob program at the university of kent in canterbury (prosrch). nucleotide sequence and organization of the sbmv-b genome the main strategy employed in the determination of the sequence of sbmv-b involved the use of the dideoxychain termination method together with a selection of overlapping cdna clones which spanned the full-length of the genome of sbmv-b, excluding approximately 40 nucleotides at the 5' end. the use of synthetic oligonucleotide primers designed on both strands of the clones as the sequences were elucidated enabled systematic sequencing along the whole length of the clone. all of the genome was sequenced from at least two independent clones and each clone was sequenced on both strands. attempts at cloning the 5' terminal sequences into the bacterial plasmids puc18 or pbluescript proved unsuccessful and all clones obtained lacked at least 40 terminal nucleotides. the sequence of the 5' terminal region of the rna that was not contained within the cloned cdna was therefore analyzed directly on the viral rna using specific oligonucleotide primers and reverse transcriptase and by additional extension using terminal transferase. the reason for the inability to clone the 5' terminal region in pbluescript and related plasmids is unknown but it did prove possible to clone in plasmids in escherichia coil behind the cauliflower mosaic virus 35s promoter. it appeared that this portion of the sbmv-b genome could be toxic to e. coil ceils if behind the t7 or t3 promoters. the 3' terminal sequence was obtained from independent poly(a)-tailed cdna clones and shows identity in all but eight bases when compared to the published 393 nucleotide terminal sequence of sbmv-b (mang et al, 1982) . six of the changes were u-c, c-u, a-u, or u-a substitutions, the 3' nucleotide was a g-u substitution and nucleotide 91 on the mang eta/. (1982) sequence was absent in our sequence. the differences were conserved in all the clones that were sequenced and could represent isolate heterogeneity rather than sequencing artifacts. the complete nucleotide sequence and the deduced amino acid sequence of the predicted translation products of sbmv-b are shown in fig. 2 . the genome contains 4109 nucleotides and thus is slightly shorter than that of sbmv-c. the base composition of sbmv-b rna comprised 23.7% a, 24.5% c, 25.6% g, and 26.0% u residues, which is similar to that reported from direct analysis (tremaine, 1966; ghabrial et al, 1967) , and the rna has an overall sequence identity with sbmv-c of only 55%. the identity of sbmv-b with rymv at the nucleotide level was 45.1%; sequence homology between rymv and sbmv-c was 45.6%. computer analysis of sbmv-b rna and its complementary strand in all six possible reading frames revealed three potential open reading frames (orfs) of >10 kda on the messenger-sense strand of the rna, referred to as orf 1 to orf 3 in order of their proximity to the 5' end of the sequence (fig. 1) ; there were no 0rfs of >10 kda on the complementary strand. thus, the overall genome arrangement of orfs 1, 2, and 3 appears similar to orfs 1, 2, and 4 of sbmv-c ( fig. 1) and rymv. however, there are differences in detail. in sbmv-b and rymv there are intercistronic regions between orfs 1 and 2 whereas these orfs overlap in sbmv-c. in sbmv-b there is also an intercistronic region between orfs 2 and 3 whereas the analogous orfs of sbmv-c and rymv (orfs 2 and 4) overlap. no potential internal coding region within orf 2 similar to the orfs 3 of sbmv-c and rymv could be identified in sbmv-b. an increasing number of viral genes are now known to utilize non-aug codons, which allow translation of multiple related proteins from a single orf (beccera et al, 1985; prats et al, 1989; gordon et al, 1992) . the efficiency of translation is generally much lower than initiation at aug. however, even with these initiation codons no orf could be defined which was similar to sbmv-c orf3. an a + u rich (61%) 5' noncoding region of 92 bases precedes the first orf of sbmv-b. the leader sequence of sbmv-b was considerably longer than that of sbmv-c (46 fit) and of a similar length to that of rymv (79 nt). the alignment of the 5' noncoding region of sbmv-b with those of sbmv-c and rymv (fig. 3) shows a region of close similarity between the 5' 43 nucleotides of sbmv-b and the leader of sbmv-c but less similarity to the leader of rymv; there was no increase in similarity between the leader sequences of sbmv-b and rymv in the part net shown in fig. 3 . there are various suggestions for significant motifs in the leader sequences of sbmv-c and rymv (wu et al., 1987; ngon a yassi et al., 1994) . these include suggested complementarity to the 3' terminus of 18s rrna for both viruses (highlighted in fig. 3 ) and a repeat ofthe sequence acaauug in rymv. the alignment of the three leader sequences does not lend much support to any significance of these motifs. keese et al. (1990) noted similarities between the 5' terminal sequences of various luteoviruses and $bmv-c. this does appear to be reflected in the consensus sequence between the three sobemoviruses ( fig. 3) . the 3' terminus of sbmv-b is nonpolyadenylated and the 129 nucleetides which constitute the noncoding region exhibit no strong secondary structures using the computer folding programs of zuker and steigler (1981) . although the 3' noncoding sequence of sbmv-c was of similar length (136 nt) and conformation (no trna-like structures), very little sequence similarities (34%) were found between the two noncoding regions. the somewhat longer 3' noncoding region of rymv (245 nucleotides) also shows no significant sequence similarity (40%). although the subgenomic mrnas of sobemoviruses have not been mapped in detail they do encode the 3' orf (rutgers eta/., 1980) and most likely resemble many other viruses in being 3' coterminal with the genomic rna. this would mean that they are promoted from a sequence upstream of the 3' orf which in sbmv-b is an intergenic region. no significant sequence similarity could be found between this intergenic region and the ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 3001 guuauaagg~agaucgauguugguuggcgucguggccuaaaacucuguuuaaauacuugucugagggcaagugguucuuugaggacuuagagcgagagcu 833 ::::::::::::::::::::::::::::::::: k i :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: rg. 2. nucleotide sequence of sbmv-b. below the line of sequence is the amino acid sequence of the three orfs given in single letter code, that in lowercase being the possible readthrough product mentioned in the text. ab1 and 2 indicate the atp-binding domains, sp1-4 the serine protease domains, and pi-vlii the polymerase domains as discussed in the text. region immediately upstream of the coat protein orfs of the other two sobemoviruses. coding regions for nonstructural proteins. the first aug in the sbmv-b sequence at nucleotide 93 marks the start of the coding region (orf 1) which terminates at nucleotide 415 and potentially encodes a protein of mr 11,684. this is somewhat smaller than the product(s) of the equivalent orf of sbmv-c (21k) and of rymv (18 and 19.5k), but is in accord with the in vitro translation product (14k) attributed to this orf (mang etal, 1982) . ngon a yassi et al (1994) suggested that the uga stop codon of orf 1 of rymv might be read through to give a product with an extra 16 residues. readthrough of the uag stop codon of sbmv-b would give a product with an extra 79 amino acids (fig. 2) . however, as in vitro translation did not show a product with the expected molecular weight (about 20,500), readthrough is thought unlikely to occur. there is little similarity between the nucleotide or amino acid sequences of this orf from sbmv-b, sbmv-c, or rymv (table 1) . data base searching did not reveal any other proteins similar to the product of this orf and currently no firm function can be attributed to it. however, most viruses, which like sobemoviruses give a full systemic infection of susceptible host, encode a protein which potentiates cell-to-cell movement. thus, one possible (keese et al, 1990) . the regions with suggested complementarity'to the 3' terminus of 18s rrna are highlighted. function of the product of sobemovirus orf 1 is that it is a movement protein. the second orf of sbmv-b extends from nucleotide 505 to nucleotide 3087 and encodes the largest potential protein product of m, 96,481. overall the product of this orf has an intermediate similarity to orf 2 of sbmv-c and rymv (table 1 ) but detailed comparison (fig. 4) shows that there are regions of high homology. these correspond in the main to motifs found in orf 2 of sbmv-c and rymv which are suggestive of the product being a polyprotein containing polymerase and other activities. toward the 5' end are the motifs characteristic of serine proteases (bazan and fletterick, 1990) shown boxed in fig. 2 (sp 1 -sp 4) with the catalytic serine site at residue 284 (underlined). it has been suggested (gorbalenya et al., 1988) that this protease could process the polyprotein to functional products. the c-terminal domain of sbmv-b orf 2 was identified as the putative rna-dependent rna polymerase as it contained the gdd motif (fig. 2, underlined) . this region shows high homology to those of sbmv-c (83%) and rymv (71%) (fig. 4) . the c-terminus also exhibited obvious consensus with the proposed rna-dependent rna polymerases of the luteoviruses beet western yelo lows virus and potato leafroll virus (veldt et al., 1988; mayo et al, 1989) and the eight conserved motifs (p i-p viii), defined by koonin (1991) , are identified in fig. 2 . such similarities have been used to evaluate the taxonomic position of sbmv-c in relation to other positivestrand rna viruses (habili and symons 1989; poch et al, 1989; koonin, 1991; koonin and dolja, 1993) and are supported by the sequence of sbmv-b. biochemical processes such as replication, recombination and repair, translation, and transcription are coupled.to nucleoside triphosphate hydrolysis and viral genomes are thought to have specific domains which have nucleotide binding functions. we could not find any obvious nucleotide-binding domain in the sbmv-b sequence comparison of orf 3 of sbmv-b with orf 4 of sbmv-c and rymv. b% nucleic acid identity, protein identity, protein familial similarity (using amino acid grouping of dayhoff et al (1983) ). â° 100% identity. i i i i i i i i 200 resembling the hydrophobic stretches proposed as ntpbinding proteins in the potyviruses, comoviruses, and picornaviruses (gorbalenya et al., 1988; gorbalenya and koonin, 1989) . wu et al. (1987) proposed a putative atpbinding domain in sbmv-c based on a loose homology to an atp-binding consensus sequence derived mainly from an alignment of picornaviral sequences. while this designation is fairly speculative, this motif is well conserved between sbmv-b and sbmv-c sequences in two regions (as 1 and ab 2) (fig. 2) . on the other hand, possibly significantly, the sequence lacks the consensus motif gks/t, thought to be the general "signature" sequence of the helicase domain in viral rna polymerase. however, the absence of a clear helicase domain in sbmv sequences is consistent with the observation that no such viral-encoded function has been identified in viruses with a compact (<5 kb) genome (gorbalenya and koonin, 1989) . for the majority of vpg-containing viruses, it has been shown that this protein is encoded within a polyprotein sequence of (helicase)-vpg-protease-rdrp (supergroup i type) (dolja and carrington, 1992). wu eta/. (1987) identified a putative vpg sequence at residues 500-519 0fsbmv-c, based on a loose homologyto several known sequences. as noted by gorbalenya et al. (1988) the location of the vpg in this region is inconsistent with the proposed model of the genome organization of orf 2 taking into account the putative identification of the protease domain as described earlier. should the vpg cistron be located at the nhz-terminal end as proposed, the lack of homology in this region between the two strains may indicate that the viruses possess different species of the protein. the dissimilar sizes of the vpg protein between the two strains (mang et al., 1982) may support this assumption. coat protein orf 3, from nucleotide 3195 to nucleotide 3981, encodes a putative protein of mr 28,107 and its allocation as the coat protein cistron is supported by the close similarity of its amino acid content with that published for isolated sbmv-b capsid proteins (tremaine, 1966; ghabrial et al., 1967) . detailed structural information based on crystallographic studies at resolutions of up to 2.9 a is available for the coat protein subunit of sbmv-c (abad-zapatero etal, 1980; rossmann, 1984; silva and rossmann, 1987) . the sbmv particle consists of 180 coat protein subunits arranged in an icosahedral t = 3 geometry (johnson et al., 1976; silva and rossman, 1987) with each protein subunit comprising two domains, the r (random) and asterisks indicate direct similarity and periods familial similarity. the tertiary structure features as determined for sbmv-cp coat protein (see rossmann, 1984) are shown as are the amino acids implicated in calcium binding (ca). the highlighted residues in the sbmv-cp and rymv sequences are those suggested to be similar to the bipartite nuclear targeting motif (dingwall and laskey, 1991) . the s (shell or surface) domain connected by an "arm." the r domain, made up of the n-terminal portion of the polypeptide chain, is rich in arginine, lysine, proline, and glutamine and penetrates into the interior of the particle where it interacts with the rna (abad-zapatero et al, 1980; tremaine eta/., 1981 tremaine eta/., , 1982 hermodson eta/., 1982; kruse eta/., 1982) . the s domain consists of a core made up of a bundle of eight stranded anti-parallel /3 sheets (/~ barrel) together with five ~ helical regions. the interactions between the protein subunits and the rna also involve the basic amino acids on the inner surface of the s domain (hermodson eta/., 1982) . comparison of the amino acid sequences predicted from sbmv-b orf 3 and the corresponding orf 4 of the other sobemoviruses (table 1) shows that it is more ctosely related to sbmv-c than to rymv. when the amino acid sequence of sbmv-b orf 3 is compared as far as tertiary structure considerations are concerned with that of sbmv-c coat protein there are many similarities but also some differences in potentially structurally important residues that maintain the integrity of the capsid. tremaine eta/. (1981) showed that the nh2-terminal polypeptide binds rna, dna, and sodium dextran sulfate. comparison of the 66 residues which compose the nh2-terminal arm of the two strains of sbmv upstream of the first/3-barrel (fig. 5) shows that, while there are differences between the two sequences (89.4% identity), the residues in this region remain primarily basic, a factor thought to be important in the interaction between protein and rna. significantly, the trypsin cleavage site at arg 61 (erickson and rossmann, 1982) is also conserved. rossmann (1984) proposed several possible docking sites for specific rna-protein interaction in sbmv-c, including a polar polypeptide (188 to 196) which forms a large bulge in strand/~g and protrudes into the rna. interestingly there is little conservation between sbmv-b and sbmv-c in this region, which may suggest subtle differences in the strategies of their protein-rna interactions. factors affecting stabilization of the capsid of sbmv represent a common link with other members of the sobemovirus group and have been extensively studied (hull, 1977 (hull, , 1978 sehgal et al., 1979; abdei-meguid et al., 1981; kruse et al, 1982; brisco eta/., 1986; sehgal, 1990) . the function of metal ions, in particular of calcium and magnesium, in relation to the assembly and stabilization of sbmv protein shells has been investigated in both the bean and cowpea strains (hsu et al., 1976; hull, 1977) . the major calcium-binding site that lies in the quasi-threefold axis between the three subunits (a, b, c) of the sbmv-c capdd has been identified at glu 194 (see rossmann, 1984) (fig. 5) . the amino acid sequence of sbmv-b shows the substitution of a lysine residue at the equivalent site which could indicate an alternative mode for subunit interaction. it is also interesting to note that, while other residues interacting with glu-194 in sbmv-c may mediate subunit contacts and are important in ca 2+ binding (silva and rossmann, 1987) , no metal ion has been detected in the refined crystallized virus at this site. the minor calcium-binding sites proposed for sbmv-c at asp 138 and asp 141 and the main chain carbonyls at residues 199 and 259 (rossmann, 1984) (fig. 5) are, on the other hand, conserved in the sbmv-b sequence. the proposed magnesium-binding sites at residues his 132, glu 229, and glu 77 (rossmann, 1984) (fig. 5) are also conserved in both strains. ngon a yassi eta/. (t994) noted that the sequence of the n-terminal regions of rymv and sbmv-cp coat proteins resembles a bipartite nuclear targetting motif (dingwatl and laskey, 1991) . there is full homology be-tween this region of sbmv-c and that of sbmv-b (fig. 5) , which could relate to the occurrence of sbmv-b in the nucleus (de zoeten and gaard, 1969) . the putative genome organization of the sbmv-b ge-n0me (fig. 1 ) raises interesting questions in terms of its overall translational strategies, which are also relevant to the other sobemoviruses. orf 3, the capsid protein cistron, is thought to be expressed via a subgenomic rnrna (ghosh et al, 11981 mang eta/., 1982) . the expression of orf2 is, hdwever, less certain as there is no evidence for a subgenomic mrna associated with it which would suggest that it is a "closed" orf. there are three possibilities for its expression, frameshift, read through of a weak stop codon, or internal initiation. frameshift would be feasible for sbmv-c and, after read through of a weak stop codon (indicated by amino acids jn lowercase letters in fig. 2 ), in sbmv-b. however, rymv orfs 1 and 2 are in the same frame separated byan amber and ochre stop codon (ngon a yassi et al., 1994) , which are unlikely to be both read through at a significant level. the aug codon for orf 1 of each of the three sobemoviruses is the first in the sequence and is in a poor context for initiation of translation (lutcke et al, 1987; kozak, 1989) . in sbmv-b and sbmv-c there is 0nly one other aug in any reading frame before that of 0rf2 and that is also in a poor context; in rymv there are no augs between those of orfs 1 and 2. for all three sobemoviruses the aug of orf 2 is in a favorable context. this raises the possibility is that the expression of 0rf2 may involve a mechanism of internal initiation which would resemble the expression strategy suggested for some of the cistrons of the coronaviruses (reviewed by spann et al., 1988) . despite their differing host specificities sbmv-b and sbmv-o are classified as two strains of the same virus 0nthe basis of morphological and physiochemical similarities and their serological cross-reactivity (shepherd and fulton, 1962) . detailed structural studies, including extensive studies on their virion assembly and disassembly under various physiochemical conditions (brisco et al,, 1985a; shields et al, 1989) again highlighted the close similarity of the two strains. however, as with the earlier immunological evidence, these data related primarilyto the properties of the virion proteins. the availability of the complete nucleotide sequence of sbmv-b raises the interesting question as to whether it and sbmv-c are truly strains of the same virus or whether or not they should be reclassified as unique viruses within the sobemovirus group. various molecular criteria, such as 3' noncoding se-quences (frenkel et al., 1989) or coat protein sequences , have been used to classify viruses either as distinct entities or as strains. on the basis of 3' noncoding sequence sbmv-b and sbmv-c would be recognized as distinct viruses but on coat protein sequence they would probably be considered as strains. one of the main functions of classification is communication and entities distinguished as distinct viruses indicate that the differences are distinct. the molecular differences between sbmv-b and sbmv-c are notable and indicate that there might be significant functional differences especially between the orf 1 products. therefore, we suggest that consideration should be given to separating sbmv-b and sbmv-c as distinct viruses. structure of southern bean mosaic virus at 2.8 angstrom resolution the location of calcium ions in southern bean mosaic virus structural and catalytic models of trypsin-like viral proteases direct mapping of adeno-associated virus capsid protein b and c; a possible auc initiation codon the effect of extraction protocol on the yield, purity and translation products of rna from an 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seed-borne virus of cowpea southern bean mosaic virus rna remains associated with swollen virions during translation in wheat germ cell-free extracts identification and classification of potyvirus on the basis of coat protein sequence data and serology refined structure of southern bean mosaic virus at 2.9a resolution coronavirus= structure and genome expression the amino acid and nucleotide composition of the bean and cowpea strains of southern bean mosaic virus southern bean mosaic virus comparison of highly basic cyanogen bromide peptides from strains of southern bean mosaic virus intermediates of the assembly and disassembly of southern bean mosaic virus proteinase k-sensitive factor essential for the infectivity of southern bean mosaic virus nucleotide equence of beet western yellows virus rna sequence and organization of southern bean mosaic virus genomic rna the severe bean mosaic virus the 5' end group of tobacco mosaic virus rna is m7g~'ppps'gp optimal computer folding of large rna sequences using thermodynamics and auxiliary information key: cord-007373-livz5zuu authors: gayathri, p.; satheshkumar, p.s.; prasad, k.; nair, smita; savithri, h.s.; murthy, m.r.n. title: crystal structure of the serine protease domain of sesbania mosaic virus polyprotein and mutational analysis of residues forming the s1-binding pocket date: 2006-03-15 journal: virology doi: 10.1016/j.virol.2005.11.011 sha: doc_id: 7373 cord_uid: livz5zuu sesbania mosaic virus (semv) polyprotein is processed by its n-terminal serine protease domain. the crystal structure of the protease domain was determined to a resolution of 2.4 å using multiple isomorphous replacement and anomalous scattering. the semv protease domain exhibited the characteristic trypsin fold and was found to be closer to cellular serine proteases than to other viral proteases. the residues of the s1-binding pocket, h298, t279 and n308 were mutated to alanine in the δn70-protease–vpg polyprotein, and the cis-cleavage activity was examined. the h298a and t279a mutants were inactive, while the n308a mutant was partially active, suggesting that the interactions of h298 and t279 with p1-glutamate are crucial for the e–t/s cleavage. a region of exposed aromatic amino acids, probably essential for interaction with vpg, was identified on the protease domain, and this interaction could play a major role in modulating the function of the protease. sesbania mosaic virus (semv) is an ss-rna plant sobemovirus found infecting sesbania grandiflora in india. its genome of 4149 nucleotides codes for four open reading frames (orfs) (lokesh et al., 2001) . orf1 codes for an 18-kda protein predicted to be a movement protein (sivakumaran et al., 1998) . the orf2 codes for a polyprotein, which encompasses more than one functional domain, while orf3 is present as an internal orf in orf2 and is expressed via ribosomal frame shifting mechanism (lokesh et al., 2001) . the orf4 is expressed from a sub-genomic rna and codes for the coat protein of the virus. polyprotein processing is one of the major strategies employed by both animal and plant viruses to generate more than one functional protein from the same polypeptide chain (wellink and van kammen, 1988) . to accomplish cleavage at specific sites, viruses employ one or more proteases with unique cleavage specificities. in an earlier study, we have shown that in semv, the processing is mediated by the nterminal protease domain coded by orf2 (satheshkumar et al., 2004) . it is a serine protease, similar to cellular proteases like trypsin and chymotrypsin (gorbalenya et al., 1988) . the catalytic residues are h181, d216 and s284. the protease cleaves the orf2 polyprotein at three different positions, at e325-t326, e402-t403 and e498-s499 to release four different domains-protease, vpg (viral protein genome linked), p10 and rdrp (rna-dependent rna polymerase) (satheshkumar et al., 2004) . in most other viruses that have vpg at the 5v end of their genome, the domain arrangement is vpg -protease, whereas, in sobemoviruses, it is protease -vpg. recent biochemical studies on the protease -vpg domains of semv have shown that the interaction of vpg with the protease domain modulates the protease activity (satheshkumar et al., 2005) . the serine protease domain lacking the 70 amino acids (dn70-pro) was found to be inactive in trans. however, the presence of vpg at the c-terminus of dn70-pro rendered the polyprotein active in cis and trans. by mutational analysis, it was demonstrated that interaction of w43 in the vpg domain with the protease was responsible for both cis and trans proteolytic activities and the associated conformational changes. it was suggested that the natively unfolded vpg is an activator of the protease and could regulate the polyprotein processing. in the present study, the crystal structure of semv protease domain was determined to a resolution of 2.4 å by multiple isomorphous replacement coupled with anomalous scattering, with a view to identify the residues involved in substrate binding as well as protease -vpg interactions. based on structural alignment of the s1-binding pocket with proteases of glu/gln specificity, h298, t279 and n308 were identified as glutamate-binding residues, and their role has been established by mutational analysis. a region of exposed aromatic residues, probably essential for interaction with the vpg domain, has been identified on the protease domain. this is the first report of the structure of a non-structural protein from the genus sobemovirus and provides the framework for understanding polyprotein processing in the genus. cloning and expression of the full-length protease domain (residues 1 -325 of the polyprotein) in escherichia coli resulted in insoluble aggregates. examination of the amino acid sequence of the protease domain suggested that its amino terminal residues might correspond to a transmembrane region. it was found that the solubility and the activity of the protease were enhanced by the deletion of 70 (dn70-pro) and 92 (dn92-pro) residues. the dn70-pro and dn92-pro domains of the polyprotein were cloned, expressed and purified as described earlier (satheshkumar et al., 2004) . both dn70-pro and dn92-pro domains were crystallized using microbatch method in the presence of 0.2 m tris, ph 8.0, 0.2 m ammonium sulphate, 0.6 m 1,6-hexane diol, 5 mm hmercaptoethanol and 4% glycerol. the protein crystals were shown to belong to the space group p3 1 21. the cell parameters were determined to be a = b = 74.07 å , c = 68.75 å , a = b = 90-, c = 120-. the asymmetric unit of the crystal was compatible with a monomer of the protease with a matthew's coefficient of 2.5 å 3 /da and a solvent content of 52% (matthews, 1968) . a very mosaic diffraction pattern of limited resolution was observed with many crystals. the quality of diffraction of such crystals could be substantially improved by repeated soaking of the crystals in the cryoprotectant solution (crystallization buffer with 20% ethylene glycol) followed by flash freezing in liquid nitrogen. interestingly, the data from the annealed and non-annealed crystals scaled well. hence, both types of datasets were combined and used for structure solution. the use of a similar annealing procedure to improve diffraction has been reported earlier (kriminski et al., 2002) . surprisingly, both the dn70-pro and dn92-pro crystals had identical cell parameters even though dn70-pro was 22 amino acids longer than dn92-pro. on mass spectroscopic analysis, it was observed that both had the same mass of¨20,000 da suggesting an internal cleavage. n-terminal sequencing of the proteins suggested a possible cleavage between the residues a134 and v135 (data not shown) in both the mutants. the ntermini of both dn70-pro and dn92-pro therefore correspond to the residue v135. henceforth, the dn70-pro and dn92-pro were considered indistinguishable, and data from both constructs were combined for phasing. semv protease does not have any significant sequence similarity to any of the cellular or viral serine proteases. hence, molecular replacement method could not be used for phase determination, and data from heavy atom derivatives and anomalous scattering were used to determine the structure. the data collection statistics for the native and derivative datasets and phasing statistics are shown in tables 1a and 1b, respectively. most of the residues in the final map are in good electron density, except for a few residues at the protein surface. the side chains of a few lysines, arginines and aspartates with poor density have been truncated according to the extent of density observed for them. a section of the electron density map is shown in fig. 1 . electron density is absent for the first three residues at the n-terminus and two residues at the c-terminus. hence, the final model includes residues 138 to 323. the electron density for the stretch of residues from 171 to 173 is poor, and these residues are omitted in the model. the loop region comprising of residues 251 to 254 is partially disordered, and there is a break in the electron density at s252. a total of 45 water molecules have been added. the structure has been refined to a resolution of 2.4 å though the data extended to 2.3 å , due to the presence of very few test reflections in the last resolution bin. table 1c lists the refinement statistics. semv protease belongs to the trypsin-like family of serine proteases. the overall fold exhibits the characteristic features of the trypsin fold. fig. 2 shows the overall fold of the protease and a topology diagram with the secondary structure elements labeled according to the convention followed for trypsin. it consists of two h barrels (domains i and ii) connected by a long inter-domain loop. both the domains belong to the all h class of proteins. the active site and the substrate-binding cleft occur in between the two domains and are fairly exposed to the solvent. there are only three helices in semv protease. the barrel formed by the h-strands ai, bi, ci, di, ei and fi constitutes the domain i. the first h-strand ai of domain i begins at l152. the active site residue h181 occurs in the small helix in the segment connecting ci and di strands (fig. 2) . strand ei extends from residues 197 to 210 and consists of two strands eia and eib connected by the stretch of residues from 201 to 205 in an extended but irregular conformation. d216 of the catalytic triad occurs at the end of the loop ei -fi. the loop connecting the two domains is a long stretch of residues extending from 222 to 243. this region contains a small helix, formed by the residues 223 to 231. the domain ii consists of the strands aii, bii, cii, dii, eii, fii and the c-terminal helix. the strand cii forms part of the wall of the s1-binding pocket. there is an intramolecular disulfide bond connecting strands aii and cii. the loop connecting cii and dii forms the oxyanion hole and contains the active site s284. the polypeptide chain ends in the c-terminal helix formed by residues ranging from 312 to 320. the asymmetric unit consists of a monomer of semv protease. however, it was observed that there is an intermolecular disulfide bond between c256 of 2-fold symmetry related molecules. this bond was formed despite the presence of 5 mm h-mercaptoethanol in the crystallization buffer. fig. 3 shows the two molecules connected by the disulfide. this disulfide is likely to be an artifact of crystal packing as gel filtration studies have shown that the protease is a monomer in solution. this is further reflected in the area of the interface formed between the molecules (470.1 å 2 ), which is not very extensive. hence, the protease can be considered as a monomer in the crystal also, consistent with its behavior in solution. similar intermolecular disulfide bonds have been observed in the tobacco etch virus (tev) nia protease structure (phan et al., 2002) and in a few other cases. a comparison of the three-dimensional structure of semv protease domain with all the available entries in the protein data bank was carried out using the dali server (holm and sander, 1993) . the dali server identifies significant similarities in the three-dimensional structures of polypeptides irrespective of the sequence similarities between them. the highest z score (17.1) was observed for the heparin-binding protein (1a7s), a serine protease homolog. the list of the top 20 structures from the dali server output is shown in table 2a . the z scores and rmsd values suggest that the semv protease is closer to the non-viral proteases than to the viral proteases. the structures listed in table 2a and other viral proteases of known three-dimensional structure (table 2b ) were compared to the semv protease by pair wise alignment. the salient features of such a comparison are described below. a helix is present at the n-terminus in all the viral proteases except the capsid-forming proteases of sindbis, semilki forest and venezeulan equine encephalitis viruses (1svp, 1vcp and 1ep5 respectively). this n-terminal helix is not part of the canonical trypsin fold and is absent in the case of semv protease. the n-terminal residues of the semv protease pack against the bii strand of domain ii and have characteristics of h-strand-forming residues. main chain o and n of s141 form hydrogen bonds with the backbone atoms of the bii strand residue s259. this conformation of the n-terminus agrees with those of the cellular and bacterial proteases namely heparinbinding protein (1a7s), bacillus intermedius glutamyl endopeptidase (1p3c), bovine beta trypsin (5ptp) and staphylococcus aureus epidermolytic toxin a (1agj). the rhino and polio virus proteases 1cqq and 1l1n harbor the conserved rna-binding motif kfrdir (mosimann et al., 1997) in the helical segment occurring in the inter-domain loop. although this motif is absent in semv, the residues h225, k229 and k233 constitute a positively charged patch in this region. however, the rna-binding properties of semv protease have not been investigated. another region, which shows considerable variability among the different families of serine proteases, is the loop connecting bii and cii. in many of the viral serine-like cysteine proteases of picornaviruses (1cqq, 1l1n, 1hav and 2hrv), this loop has a long insertion that forms a h-hairpin, which extends to the substratebinding site. it contributes to the peptide binding and shields the active site from the solvent. the peptide substrate binds as a bridging strand between eii and the h-hairpin. semv protease does not have this insertion, and this leads to an exposed active site. this h-hairpin is not present in most of the non-viral proteases. the loop is also not found in the viral serine proteases equine arteritis virus nsp4 protease, hepatitis c virus protease (1mbm and 1jxp, respectively) and the capsid-forming proteases (1ep5, 1vcp and 1svp), suggesting that an exposed active site is a characteristic of the viral serine proteases. the active site residue h181 forms hydrogen bonds with both s284 and d216, the other two residues of the catalytic triad. the active site and the hydrogen bonds involved are shown in fig. 4a . the side chain of d216 is stabilized by two hydrogen bonds-between its oy1 and the main chain n of h181, and its oy2 and ny1 of h181. the n(2 of h181 is at a hydrogen bonding distance from og of s284, although the geometry for the hydrogen bond is not optimal. the electron density for the imidazole ring of the h181 is not very clear, implying conformational flexibility. this is a feature found in the active site histidines of other serine proteases also. in the hrv protease, the active site h40 exists in alternate conformations (petersen et al., 1999) , while in the polio viral 3c protease, the electron density for the side chain is weak, and the mean temperature factor of the atoms is above the average (mosimann et al., 1997) . the oxyanion hole, a highly conserved region of the active site in terms of sequence (gxsg) and structure, is important for the stabilization of the oxyanion intermediate formed during the reaction. in semv protease, the main chain amide nitrogens of g282 and s284 are involved in the oxyanion hole formation. the structure is in conformity with the earlier mutational studies on the active site residues of semv protease (satheshkumar et al., 2004) . viral proteases involved in polyprotein processing are known to have very stringent substrate specificities. mutational analyses have demonstrated that the serine protease domain of semv cleaves at e -t occurring between protease -vpg, and vpg-p10 domains, and also at e -s between p10 and rdrp (satheshkumar et al., 2004) . in order to understand the structural basis of the stringent cleavage specificity of the enzyme, a comparison was carried out between semv protease and the structures of other proteases that display glutamate specificity (table 2c ). it was observed that the histidine that binds to the carbonyl oxygen of glu/gln is conserved in all glu/gln-specific proteases except the s. griseus proteases (2sga and 2sgp) where a positively charged arginine at the base of the substrate-binding pocket stabilizes the glutamate (read et al., 1983; read and james, 1988) . the corresponding residue in semv protease is h298, and it is directed towards the active site. ny1of h298 forms a hydrogen bond with n(2 of h275 (fig. 4a) . the conformation of h275 side chain is, in turn, stabilized by a hydrogen bond from ny1 to the main chain n of the disulfidebonded c277. in the glutamate-specific s. griseus protease (1hpg), the role of the histidine triad, h213, h199 and h228 (1hpg numbering), in stabilizing the charge on glutamic acid has been emphasized (nienaber et al., 1993) . in semv protease, a corresponding histidine triad does not exist, and h275 hydrogen bonded to the conserved h298 is not in the same position as that of h199 in the s. griseus protease. however, the chain of hydrogen bonds is maintained. this can possibly help in the stabilization of the charge of the glutamic acid side chain. the residues present in the proposed glutamate-binding site or the s1 pocket are t279, a280, h298, f301 and n308 (fig. 4a) . out of these residues, only h298 and t279 are conserved across most of the glu/gln-binding proteins. in a few cases, thr is replaced by ser, whose og substitutes for the thr og. his and thr have been implicated to have a major role in glu/ gln binding, and the mutations of these have resulted in inactive enzymes in poliovirus 3c protease (ivanoff et al., 1986) . in the crystal structure of tev complexed with substrate, it has been observed that the carboxyamide group of glutamine forms hydrogen bonds with both his and thr (phan et al., 2002) . in the present structure, electron density observed near the oxyanion hole has been interpreted as a glycerol molecule. fig. 4b shows the glycerol bound near the s1-binding pocket with the 2f o àf c electron density around it contoured at 1.0r level. it is possible that the orientation of the glycerol is related to the mode of glutamate binding. the main chain carboxyl group of glu should be directed towards the amide nitrogens of gly and ser at the oxyanion hole (perona and craik, 1995) . accordingly, the o3 of glycerol forms hydrogen bond with the main chain n of g282 (fig. 4) . at the other end, o1 forms hydrogen bonds with both n(2 of h298 and main chain o of a280. a water molecule, wat28, is present between h298 and the glycerol. this water molecule is close to the aromatic ring of f301 and comes within hydrogen bonding distance from h298 and t279 side chain atoms. however, f301 aromatic ring and the water molecule are not in well-defined density. f301 is in a position equivalent to s170 of tev protease and s137 of equine arteritis virus nsp4 protease. these serine residues are involved in substrate binding at the p1 site (phan et al., 2002; barrette-ng et al., 2002) . further, all glu/glnbinding proteases, except semv protease, have either a ser or gly at this position. the absence of well-defined density for f301 in semv protease suggests that its side chain might undergo substantial displacement on binding of the substrate or on conformational changes induced by the interaction of the protease domain with vpg. tev and equine arteritis virus proteases, in which a serine residue occurs at the position corresponding to f301, are active in trans. mutation of this f301 to serine, the corresponding residue in other glu/glnbinding proteases, might therefore bestow activity in trans to semv protease, even in the absence of vpg domain. an extended c-terminus results in a buried cleft in the case of tev protease. the absence of this extension makes semv substrate-binding site significantly different from that of tev protease. the residues in this region constitute the specificity pocket in tev protease. in semv protease, the cleft is solvent exposed, and the loop 212-215 appears to be closest to the substrate-binding region. the other side of the substratebinding cleft is similar in tev and semv proteases and is lined by the h strand eii. this is a common feature in most of the proteases and mainly involves main chain interactions. as mentioned earlier, the dn70-pro or dn92-pro was not active in trans. hence, in order to confirm the role of the proposed glutamate-binding residues (fig. 4a ) in protease activity, the residues h298, t279 and n308 were mutated to ala in dn70-protease -vpg (dn70pv) fusion protein, and the cis-cleavage of the expressed protein into protease and vpg was monitored as described in the materials and methods section. the results obtained were confirmed by a western blot analysis using anti-protease antibodies (fig. 5d) . expression of dn70pv showed a band of size 27 kda corresponding to the protease domain (fig. 5a, lane 2; fig. 5d , lane 5) confirming that nearly complete cleavage of dn70pv (35 kda) had occurred. the active site mutant dn70pv-s284a (satheshkumar et al., 2004) showed a prominent band corresponding to 35 kda on expression (fig. 5a, lane 6) . the h298a mutant gave a band of size 35 kda corresponding to the dn70pv (fig. 5a, lane 4; fig. 5d, lane 4) , confirming that the mutation had affected the cleavage activity of dn70pv. similarly t279a mutant also did not show any cis-cleavage activity (fig. 5b, lane 1; fig. 5d, lane 3) . however, n308 to a mutation in the dn70pv resulted only in a partial loss of the protease activity (fig. 5b, lane 2; fig. 5d, lane 2) . the mutant dn70pv-n308a partially cleaved the dn70pv giving a 35-kda dn70pv band and a 27-kda dn70-protease band, implying that n308 is not absolutely essential for the substrate binding, but contributes to the proper binding of the glutamate residue and hence to the optimal activity. disulfides are generally implicated in the stability of the three-dimensional structures of serine proteases (wang et al., 1997) . most of the prokaryotic and eukaryotic trypsins have highly conserved disulfide bonds. on the contrary, disulfide bonds have not been observed in any of the viral protease structures reported so far. in the poliovirus and hepatitis 3c virus proteases (1l1n and 1jxp), binding of a metal ion contributes to the structural integrity (de francesco et al., 1996) . in semv protease, c248 forms a disulfide bond with c277. this disulfide bond connects the h strands aii and cii in domain ii (fig. 4a) . it holds the walls of the s1 specificity pocket and could have a role in the maintenance of its rigid conformation. a disulfide bond is responsible for the rigidity of s1-binding pocket in many of the mammalian trypsins. though the disulfide bond in semv protease does not correspond to any of the conserved disulfide bonds in trypsin, it involves the cii strand of domain ii. the same strand in trypsin has the conserved disulfide between the residues 198 and 220 (wang et al., 1997) . in order to assess the role of the unique disulfide bond in semv protease in the maintenance of the s1-binding pocket, c277 was mutated to alanine in dn70pv, and the effect of the mutation on cis-cleavage was examined. as shown in fig. 5c , lane 2, and fig. 5d , lane 1, the 35-kda dn70pv-c277a mutant was completely cleaved in cis to 27-kda band like the wild-type dn70pv (fig. 5a, lane 1; fig. 5d , lane 5). hence, it can be concluded that this disulfide bond is not essential for the maintenance of the rigidity of the s1-binding pocket. an interesting observation in the crystal structure of semv protease is the occurrence of a stretch of aromatic residues exposed to the surface (fig. 6) . these aromatic residues f269, w271, y315 and y319 are not consecutive in sequence but form a stack near the c-terminus of the protein. two of these residues form part of the c-terminal helix. these residues may be of functional significance to the polyprotein. semv residues f269 and w271 are conserved across genomes of sobemoviruses. the presence of exposed aromatic residues is believed to indicate a protein -protein interaction interface. it has been demonstrated that there are extensive interactions between protease and vpg, and the conformational changes that accompany these interactions enhance and regulate protease activity (satheshkumar et al., 2005) . w43 of vpg was shown to mediate aromatic interactions with the protease, which results in a positive peak at 230 nm in the cd spectrum of protease -vpg fusion protein, but the interacting partner in protease was not identified. the presence of exposed aromatic residues in the present structure strongly suggests that this might be the site of protease -vpg interaction. it is probable that w271 of protease interacts with w43 of vpg domain. the positive peak observed at 230 nm in the cd spectrum of protease -vpg fusion protein might be the result of this interaction. further mutational studies are required to delineate the significance of the aromatic stretch of residues in protease activity. the crystal structure of semv protease provides insights on the possibilities of cis (intramolecular)-or trans (intermolecular)-cleavages. the c-terminal helix ends at residue 320, and this is followed by a stretch of five residues. the helix is a stable structure and is closely packed against the rest of the protein. the presence of a c-terminal helix is a characteristic of many serine proteases. the disordered short segment at the cterminus is not long enough to reach the active site suggesting the possibility of intermolecular cleavage between protease and vpg (trans-cleavage). however, it is possible that there could be a major conformational change because of the presence of the natively unfolded vpg at the c-terminus, and this may position the residues for cis-cleavage at the active site. biochemical evidences (data not shown) as well as the present structure suggest that a cleavage occurs between a134 and v135 at the n-terminus. this is an unexpected finding, as this site does not correspond to the canonical site for substrate cleavage. the relative positions of the n-terminus and the active site suggest that an intramolecular cleavage is indeed possible. the n-terminus in the crystal structure is disordered, and clear electron density is observed only from s138. a further extension of approximately four residues is long enough to approach the active site through the cleft below the bii-cii loop of domain ii. this gains additional support by the observation that the n-terminus of semv protease superposes very well with the glutamyl endopeptidase from bacillus intermedius (1p3c), as shown in fig. 7 . in the case of 1p3c, the n-terminus extends to the active site, and this extension is essential for zymogen activation and charge compensation for glutamate specificity (meijers et al., 2004) . therefore, based on the relative positions of the n-and c-termini and the active site in the crystal structure, it can be concluded that the cleavage between protease and vpg could occur in cis or trans, while the n-terminal cleavage could be cis. the non-specific cleavage at ala-val could be due to the conformation of the polypeptide, which positions the a134 -v135 peptide bond optimally for the cleavage. the residues ala and val are small and could be accommodated in the active site without steric hindrance. these observations also suggest that the specificity of the protease depends not only on sequence but also on the conformation of the polypeptide. a non-specific cleavage on the polypeptide chain has been observed at the cterminus of tev protease also (nunn et al., 2005) . there have also been reports in the literature on other viral proteases such as rhinovirus hrv2-2a protease, where the specificity requirements for cis-cleavage are less stringent than for trans-cleavage. the entropy term for substrate binding is favorable in ciscleavage, and hence, the specificity requirements are less stringent (petersen et al., 1999) . in spite of this, most viral proteases are extremely specific, and they function mainly in the processing of the respective polyproteins. their activities are modulated by the presence of vpg domains either at the n-or cterminus. determination of the structure of the protease with the vpg domain could shed light on the molecular mechanism of such regulations. the dn70-protease (dn70-pro) and dn92-protease (dn92-pro) domains of the polyprotein were cloned in prset c vector fig. 7 . superposition of semv protease (blue) and bacillus intermedius glutamyl endopeptidase (pink). the superposition was generated using the output from dali server. at nhei and bamhi sites. the cloning strategy resulted in the addition of 11 amino acids from the vector sequence at the nterminus including the hexa-histidine residues inserted for affinity purification. the proteins were expressed in e. coli bl21 (de3) plyss cells by the addition of 0.3 mm iptg at a culture density 0.6 od at 660 nm and purified using ni-nta affinity chromatography (novagen) as previously described (satheshkumar et al., 2004) . the final protein preparation was in 50 mm tris, ph 8.0, buffer containing 200 mm nacl. both dn70-pro and dn92-pro domains formed small crystals in the presence of 0.2 m tris, ph 8.0, 0.2 m ammonium sulphate, 0.6 m 1,6-hexane diol, 5 mm hmercaptoethanol and 4% glycerol (crystallization buffer). larger crystals could be obtained using the microbatch method when a mixture of silicone and mineral oils in the ratio 2:3 was layered over the crystallization droplets (5 al of 30 mg/ml protein and 3 al of crystallization buffer). the crystals appeared from a layer of precipitate within 2 weeks and grew to a size of approximately 0.3 â 0.3 â 0.2 mm 3 during a period of 1 month. the crystals were transferred to the crystallization buffer containing 20% ethylene glycol as the cryoprotectant for a few seconds and then mounted in a cryo-loop. the crystals were exposed to cu-k a radiation at liquid nitrogen temperature (100 k). x-ray diffraction data were collected using a rotating anode x-ray generator and a mar research image plate detector system. the datasets were processed using denzo, and the resulting intensities were scaled using scalepack (otwinowsky, 1997) . the frames were processed in the space group p321. systematic absences indicated the presence of a 3 1 or 3 2 screw along the c axis. the structure solution was attempted by multiple isomorphous replacement (mir). four isomorphous mercury derivatives were obtained by soaking the crystals in p-chloro mercury benzene sulfonate (pcmbs), mercuric iodide, mercuric chloride and thiomersal (ethyl mercury thio salicylic acid or emts). the native and derivative datasets were scaled using the scaleit program in the ccp4 program suite version 4.2.1 (ccp4, 1994). both isomorphous and anomalous difference patterson maps were calculated for these derivatives using ccp4 program suite and harker sections for the space group p3 1 21 were plotted. initially, one site in the pcmbs derivative was obtained using the isomorphous differences in the program suite solve version 2.03 (terwilliger, 2004) . the rest of the sites in all the derivatives were identified from the difference fourier maps using mlphare in ccp4 program suite (1994) . the fractional coordinates of all the sites, their occupancies and anomalous occupancies were refined in mlphare for all the derivatives to a resolution of 2.8 å . the b-factors of the sites were not refined and were fixed at the values obtained from the wilson plots. the space group was confirmed to be p3 1 21, as this choice resulted in an interpretable map with the correct handedness. the experimental phases obtained from isomorphous and anomalous signals were used in the program resolve (terwilliger, 2004) to perform phase extension to 2.3 å by density modification followed by automated model building. this resulted in the placement of 34 residues with side chains and 123 without side chains into the electron density map. the coordinates of the partial model and the phases from resolve were used as input for arp/warp version 6.1 (morris et al., 2003) for building the rest of the model. 80% of the residues were built by the program, which included residues 140-161, 174-194, 205 -250, 257 -303 and 306-323 . the rest of the residues were built manually in subsequent cycles of refinement using coot (emsley and cowtan, 2004) and refmac 5.2 (murshudov et al., 1997) . the progress of the refinement was monitored by using 5% (459) of the total 9272 independent reflection measurements for the calculation of the free r-factor. the hendrickson -lattman coefficients were used as restraints during the refinement (skubak et al., 2004) . individual b-factors of non-hydrogen atoms were refined. all structural alignments were done using the output from dali server (holm and sander, 1993) . the figures were prepared using molscript (kraulis, 1991) and bobscript (esnouf, 1999) and rendered using raster3d (merritt and murphy, 1994) . the topology diagram for the protease was prepared using topdraw (bond, 2003) . site directed mutagenesis was performed by pcr-based approach (weiner et al., 1994) . the sense and antisense primers were designed with desired changes in the nucleotides (table 3) , and restriction sites were incorporated in the primers to enable easy screening. the pcr was carried out using deep vent polymerase (new england biolabs) according to the manufacturer's instructions. the pcr product was treated with 0.5 al dpni enzyme at 37 -c for 45 min to remove the template dna and transformed into 2 aliquots of dh5a competent cells. the cells were plated after transformation in antibiotic containing plates. the colonies obtained were inoculated separately. plasmid isolation was carried out and digested with the appropriate restriction enzyme. the mutants screened by restriction enzyme digestion were further confirmed for the presence of mutation by dna sequencing. e. coli bl21 (de3) plyss cells were transformed with the recombinant clones, and the proteins were expressed by induction with 0.3 mm iptg for 4 -5 h at 30 -c. the cell pellet was resuspended in buffer a (50 mm tris -hcl, ph 8.0, 300 mm nacl, 0.2% triton x100, 5% glycerol), sonicated and the expression was checked by sds polyacrylamide gel electrophoresis. the cleavage activity was monitored by the appearance of bands of expected size on the sds polyacrylamide gel. the results were confirmed by western blot analysis as described in satheshkumar et al. (2004) . the coordinates and structure factors for semv protease have been submitted to the protein data bank, and the structure has been assigned the accession code 1zyo. structure of arterivirus nsp4. the smallest chymotrypsin-like proteinase with an alpha/beta c-terminal extension and alternate conformations of the oxyanion hole topdraw: a sketchpad for protein structure topology cartoons the ccp4 suite: programs for protein crystallography a zinc binding site in viral serine proteinases coot: model-building tools for molecular graphics further additions to molscript version 1.4, including reading and contouring of electron-density maps sobemovirus genome appears to encode a serine protease related to cysteine proteases of picornaviruses protein structure comparison by alignment of distance matrices expression and site-specific mutagenesis of the poliovirus 3c protease in escherichia coli molscript: a program to produce both detailed and schematic plots of protein structures flash-cooling and annealing of protein crystals complete nucleotide sequence of sesbania mosaic virus: a new virus species of the genus sobemovirus solvent content of protein crystals the crystal structure of glutamyl endopeptidase from bacillus intermedius reveals a structural link between zymogen activation and charge compensation raster3d version 2.0. a program for photorealistic molecular graphics arp/warp and automatic interpretation of protein electron density maps refined x-ray crystallographic structure of the poliovirus 3c gene product refinement of macromolecular structures by the maximum-likelihood method a glutamic acid specific serine protease utilizes a novel histidine triad in substrate binding crystal structure of tobacco etch virus protease shows the protein c terminus bound within the active site processing of x-ray diffraction data collected in oscillation mode structural basis of substrate specificity in the serine proteases the structure of the 2a proteinase from a common cold virus: a proteinase responsible for the shut-off of host-cell protein synthesis structural basis for the substrate specificity of tobacco etch virus protease structure of the complex of streptomyces griseus protease b and the third domain of the turkey ovomucoid inhibitor at 1.8-a resolution refined crystal structure of streptomyces griseus trypsin at 1.7 a resolution polyprotein processing: cis and trans proteolytic activities of sesbania mosaic virus serine protease natively unfolded'' vpg is essential for sesbania mosaic virus serine protease activity identification of viral genes required for cell-to-cell movement of southern bean mosaic virus direct incorporation of experimental phase information in model refinement solve and resolve: automated structure solution, density modification and model building the role of the cys191-cys220 disulfide bond in trypsin: new targets for engineering substrate specificity site-directed mutagenesis of double-stranded dna by the polymerase chain reaction proteases involved in the processing of viral polyproteins. brief review mrnm and hss thank the department of science and technology (dst) and the department of biotechnology (dbt) of the government of india for financial support. diffraction data were collected at the x-ray facility for structural biology at molecular biophysics unit, indian institute of science (iisc), supported by dst and dbt. we thank the staff in the x-ray laboratory and supercomputer education and research centre of iisc for their co-operation during the course of these investigations. pg and pss acknowledge the council for scientific and industrial research, government of india, for the fellowships. key: cord-259500-ndjbrtrv authors: satyanarayana, tatineni; gowda, siddarame; ayllón, maría a; dawson, william o title: frameshift mutations in infectious cdna clones of citrus tristeza virus: a strategy to minimize the toxicity of viral sequences to escherichia coli date: 2003-09-01 journal: virology doi: 10.1016/s0042-6822(03)00387-8 sha: doc_id: 259500 cord_uid: ndjbrtrv the advent of reverse genetics revolutionized the study of positive-stranded rna viruses that were amenable for cloning as cdnas into high-copy-number plasmids of escherichia coli. however, some viruses are inherently refractory to cloning in high-copy-number plasmids due to toxicity of viral sequences to e. coli. we report a strategy that is a compromise between infectivity of the rna transcripts and toxicity to e. coli effected by introducing frameshift mutations into “slippery sequences” near the viral “toxicity sequences” in the viral cdna. citrus tristeza virus (ctv) has cdna sequences that are toxic to e. coli. the original full-length infectious cdna of ctv and a derivative replicon, ctv-δcla, cloned into puc119, resulted in unusually limited e. coli growth. however, upon sequencing of these cdnas, an additional uridinylate (u) was found in a stretch of u’s between nts 3726 and 3731 that resulted in a change to a reading frame with a stop codon at nt 3734. yet, in vitro produced rna transcripts from these clones infected protoplasts, and the resulting progeny virus was repaired. correction of the frameshift mutation in the ctv cdna constructs resulted in increased infectivity of in vitro produced rna transcripts, but also caused a substantial increase of toxicity to e. coli, now requiring 3 days to develop visible colonies. frameshift mutations created in sequences not suspected to facilitate reading frame shifting and silent mutations introduced into oligo(u) regions resulted in complete loss of infectivity, suggesting that the oligo(u) region facilitated the repair of the frameshift mutation. additional frameshift mutations introduced into other oligo(u) regions also resulted in transcripts with reduced infectivity similarly to the original clones with the +1 insertion. however, only the frameshift mutations introduced into oligo(u) regions that were near and before the toxicity region improved growth and stability in e. coli. these data demonstrate that, when hosts are sufficiently susceptible for infection by transcripts of reduced specific infectivity, introduction of frameshift mutations at “slippery sequences” near toxic regions of viral cdnas can be used as an additional strategy to clone recalcitrant viral sequences in high-copy-number plasmids for reverse genetics. reverse genetics, the ability to produce specific mutations followed by examination of phenotype, revolutionized the study of rna viruses. the first successful attempts initiated infection of cells by inoculation with bacterial plasmids containing the viral cdna inserts (taniguchi et al., 1978; racanicllo and baltimore, 1981) . however, production of in vitro rna transcripts that were identical to the virion rna (except for designed mutations and perhaps a few extra nucleotides at either terminus) allowed genetic manipulation using the standard inoculation procedures previously in use for virion rnas (ahlquist et al., 1984) . the current ideal is to clone a full-length viral cdna in a high-copy-number bacterial plasmid behind a promoter for a commercially available rna polymerase. most viruses with relatively small rna genomes have been amenable for such a genetic system. however, some viruses are inherently difficult to clone due to the instability of the viral cdna, often because the viral sequences are toxic to the bacterium (boyer and haenni, 1994; lai, 2000) . the cloning difficulties of some recalcitrant viral genomes have been surmounted by a variety of methods. in vitro ligation of individually cloned cdna fragments followed by in vitro transcription and transfection was used to overcome the cloning difficulties of yellow fever virus, mouse hepatitis virus, transmissible gastroenteritis virus (tgev), and beet necrotic yellow vein virus rna2 (quillet et al., 1989; rice et al., 1989; yount et al., 2000 yount et al., , 2002 . the disadvantages of this approach, though, are relatively low levels of full-length cdna and reduced levels of rna transcripts compared to that of in vitro transcripts from a complete clone in one plasmid. another approach is to reduce the toxicity of viral sequences in high-copy-number plasmids by inserting introns into the viral sequences. recently, cdna clones of tgev, japanese encephalitis flavivirus, pea seedborne mosaic virus, and plum pox virus were stabilized by this method (johansen, 1996; lópez-moya and garcía, 2000; yamshchikov et al., 2001; gonzález et al., 2002) . this strategy, however, requires cytoplasmic viruses to have a nuclear phase and also requires that the viral sequences not have endogenous intron sequences, which has been suggested as a possible problem of large cytoplasmic rna viruses (lai, 2000) . in fact, tobacco mosaic virus (tmv) appears to be an example of such a virus with an endogenous intron, based on the fact that the infectivity of 35s:cdna constructs are several orders of magnitude less than rna transcripts and often are infectious in only certain plant species (w.o. dawson, unpublished data) . moreover, most cells of tobacco plants with transgenes of the infectious cdna of tmv behind the 35s promoter do not become infected from the transgene, although they do become infected via virus movement from other cells (turpen, 1992) . yet another approach is to avoid the use of high-copy-number plasmids. the reticent rna genome of tgev could be propagated in escherichia coli when cloned into a bacterial artificial chromosome, a lowcopy-number plasmid (almazán et al., 2000) . other approaches avoid the use of bacteria. vaccinia virus was used as a vector to clone the coronaviruses, avian infectious bronchitis virus (ibv) and human coronavirus (casais et al., 2001; thiel et al., 2001) . although this approach allowed the cloning of the full-length coronavirus cdna, the viral cdna in the vaccinia virus vector is not very amenable for mutational analysis, requiring multiple cycles of ligation and recombination. here we report an additional strategy to allow cloning of recalcitrant viral cdnas in high-copy-number plasmids in e. coli that can be added to the repertoire of strategies available. citrus tristeza virus (ctv), a member of the closterovirus genus of the closteroviridae family, has a singlestranded positive-sense rna genome of ϳ20.0 kb that is organized into 12 open reading frames (orfs) (fig. 1a ) (pappu et al., 1994; karasev et al., 1995) . orf 1a encodes a polyprotein containing two papain-like protease domains, plus methyltransferase-like (mt) and helicase-like (hel) domains, and a large interdomain region (idr) between the mt and hel domains (fig. 1a) . orf 1b encodes an rna-dependent rna polymerase-like domain that is thought to be translated by a ϩ1 frameshift from orf 1a . the ten 3ј orfs are expressed by 3ј-coterminal sgrnas that encode an assembly gene block of four proteins , a protein that controls asymmetry of ctv rnas (p23) (satyanarayana et al., 2002a) , and five proteins of unknown function. the development of infectious cdna clones of ctv was difficult due to the large size of the genomic rna and its toxicity to e. coli (satyanarayana et al., 1999) . we were able to maintain the plasmids containing the viral cdna in only one strain of e. coli, which after transformation grew very slowly into minute colonies. however, we found that this level of growth was due to a fortuitous frameshift in the ctv cdna that allowed its cloning by reducing the toxicity to e. coli. the corrected cdna exhibited substantially greater toxicity to e. coli, now requiring 3 days to see visible colonies. examination of other frameshift mutations demonstrated that 6 -7 u's functioned as "slippery" frameshift sites, reducing toxicity to e. coli if positioned appropriately near the toxicity region in the viral cdna. thus, purposely introduced frameshift mutations into oligo(u) regions in a toxic viral cdna can provide a compromise between infectivity and toxicity to e. coli. if hosts cells are susceptible enough to allow a reduction in infectivity, frameshift mutations can be inserted in the viral cdna to allow cloning in high-copy-number plasmids of e. coli. the full-length infectious cdna clones of ctv, pctv9, and its derivatives were constructed in puc119 behind the sp6 rna polymerase promoter and grown in e. coli strain jm109 (satyanarayana et al., 1999) . ctv-⌬cla contains the 5ј-nontranslated region (ntr) plus orfs 1a and 1b and the first 105 nts of orf 2 (nts 1-11,005) fused to the 3ј 768 nts of the genomic rna that includes part of the p23 orf and the 3ј-ntr (nts 18,526 -19,293) . the cdna of this small replicon with the ten 3ј genes (7.5 kb) deleted is much easier to manipulate than the full-length virus (satyanarayana et al., 2002b) . the in vitro generated rna transcripts were biologically active as evidenced by their replication in nicotiana benthamiana protoplasts and synthesis of sgrnas (fig. 1 , ctv9 and ctv-⌬cla), and production of viable ctv9 virions that passaged with a specific infectivity similar to that of the parental wild-type virus satyanarayana et al., 1999 satyanarayana et al., , 2002a satyanarayana et al., , 2002b . the rna transcripts of full-length ctv and the replicon replicated similarly in citrus protoplasts (data not shown). the recombinant virus had a phenotype identical to that of the parental wild-type virus in citrus trees . pctv9 and pctv-⌬cla were equally toxic to e. coli, but grew enough on lb agar-amp plates for minute colonies to be picked for propagation after 24 h, compared to 1.8mm-diameter colonies of the vector puc119 after overnight incubation at 37°c (fig. 2) . the cdna clones grew marginally in 2xyt liquid medium, but failed to grow in lb liquid medium. from numerous e. coli strains examined, the original cdna clones of ctv in puc119 were stable only in jm109. to compare the sequence of the full-length infectious cdna clone with that of the consensus sequence from the native quasispecies of parental ctv t36 isolate (genbank accession no. nc_001661), both strands of pctv9 were sequenced completely (genbank accession no. ay170468). although we found numerous nucleotide differences, we ob-served an additional "u" inserted at nt 3732 into a homopolymeric run of 6 u's that resulted in a shift to a reading frame with a stop codon at nt 3734. a similar frameshift mutation was also found in the derivative replicon pctv-⌬cla. however, the sequence of the progeny virion rna of ctv9 from infected protoplasts or citrus plants did not contain the additional nt at position 3732 that occurred in the cdna clones, suggesting that the frameshift mutation present in the original cdna clone was repaired either during the in vitro transcription or during replication in the protoplasts. the ϩ1 insertion found at nt 3732 of the cdna clones was corrected in pctv9 and pctv-⌬cla by exchanging a , and an rna-dependent rna polymerase (rdrp). the orfs (boxes) with their numbers and corresponding translation products are also indicated. hsp70h, hsp70 homolog; cpm, minor coat protein; cp, major coat protein; (2/11), fusion of orfs 2 and 11 with the deletion of internal sequence. the fortuitous frameshift found at nt 3732 (nt in bold) in ctv9 (a) and ctv-⌬cla (c) was corrected in ctv9r (b) and ctv-⌬clar (d). (b) northern blot hybridization analysis of total rna isolated from protoplasts transfected with ctv9 (a), ctv9r (b), ctv-⌬cla (c), and ctv-⌬clar (d) at 3 and 4 days postinoculation (dpi). the position of genomic and subgenomic (sg) rnas corresponding to orfs 2-11 are indicated by arrowheads and arrows, respectively. the blots were hybridized with 3ј 900-nt positive-stranded rna-specific digoxigenin-labeled probe. rt-pcr amplified product between the unique ncoi (nt 3685) and bsu36i (nt 4430) restriction endonuclease sites creating pctv9r and pctv-⌬clar (fig. 1a) . replication of the full-length and replicon transcripts from the original and corrected cdna clones was compared in n. benthamiana protoplasts. the rna transcripts from the repaired clones accumulated genomic and sgrnas about 30 to 40fold more than those from the original cdnas (fig. 1b , compare a and b, and c and d). we then examined the number of protoplasts infected in the presence or absence of a frameshift mutation by introducing green flourescent protein (gfp) orf into ctv using mutants ctv-gfp-p23 (with a ϩ1 insertion at nt 3732) and ctvr-gfp-p23 (no frameshift at nt 3732) (fig. 3a ). the number of protoplasts infected with ctvr-gfp-p23 that exhibited fluorescence under uv light was approximately 18-to 30-fold more than those inoculated with ctv-gfp-p23 ( fig. 3c) , again with the accumulation of approximately 30 to 40-fold more genomic and sgrnas than those of ctv-gfp-p23 (fig. 3b) . the original ctv clones were toxic to e. coli, resulting in substantially reduced colony sizes (fig. 2 ). bacteria transformed with the repaired clones pctv9r or pctv-⌬clar grew much slower than those containing the original clones, producing visible colonies only 3 days after transformation with ligation mixtures. however, e. coli transformed with purified plasmid dnas of pctv9r or pctv-⌬clar produced visible colonies slightly earlier. the yields of plasmid dna from e. coli strain jm109 harboring pctv9 and pctv9r after overnight growth in 2xyt liquid medium were 2.5-3.0 and 1.5-2.0 g of dna per milliliter of culture, respectively, compared to 8 -10 g dna per milliliter of culture for ptmv (dawson et al., 1988) . thus, although the original ctv clones with the frameshift mutation reduced the growth of e. coli and plasmid dna yields, the correction of the ϩ1 insertion in pctv9r further reduced the growth of e. coli to a level below the range of expectations for routine laboratory work. although not quantified here, the degree of difficulty of ligating and isolating intact cdna clones was proportionally increased with the increased toxicity of the repaired constructs. we expected frameshift mutations in ctv to be lethal. we were surprised that cdna clones of ctv with a frameshift mutation would produce transcripts with an appreciable amount of infectivity in protoplasts. we further examined this phenomenon by introducing additional frameshift mutations into ctv-⌬clar. we introduced ϩ1 insertion mutation in other regions of orf 1a to examine whether the frameshift mutations would result in a viable virus similar to that of original cdna clones that contained a ϩ1 insertion at 3732 nt. two mutations were introduced separately at nt 4079 in the idr (gcc aag cuu ugu aag, frameshift nt in bold) and at nt 8390 in the hel domain (agg aag cuu agc ugc, frameshift mutation in bold), creating ⌬clar-m1 and ⌬clar-m2, respectively (fig. 4a, e and h) . the in vitro rna transcripts from these mutants failed to replicate to detectable levels in protoplasts (fig. 4b, e and h) . in contrast to the frameshifts examined above, the ϩ1 insertion found in the original infectious cdna clones that allowed infectivity and greatly reduced the toxicity of the cdna in e. coli was in a run of 6 u's, which is known to be a "slippery sequence" associated with frameshifting in other viral systems (wilson et al., 1988; brierley et al., 1992) . however, frameshifting also has been correlated with downstream structures (jacks et al., 1988; . it is possible that the 6 u's at 3732 had an adventitious accessory structure downstream, and the frameshifting at this position was unusual. to examine fig. 2 . e. coli colonies on lb-agar plates transformed with pctv9 (with a ϩ1 insertion at nt 3732) and pctv9r (without a frameshift at nt 3732) at 1, 2, and 3 days posttransformation. the parental plasmid puc119 and the cdna clone of tmv in puc19 (ptmv) (dawson et al., 1988) were used as controls for comparison of the size of e. coli colonies to evaluate the toxicity of ctv sequences. whether other u-rich regions of ctv would similarly function as slippery-sequence frameshifting signals to recover infectivity and reduce toxicity in e. coli, we created frameshifts in other oligo(u) sites. a run of 6 u's occurs at four different places in orf 1a at nts 1103-1108, 3726 -3731, 4957-4962, and 7078 -7083. mutations to alter the reading frame of orf 1a in each of the u-rich regions were created (fig. 4a) . the plasmids were evaluated for toxicity to e. coli, and the rna transcripts were evaluated for replication in protoplasts. a ϩ1 insertion mutation was introduced at nt 1109 in the l-proi region to obtain ⌬clar-m3 (fig. 4a, c) . a ϩ2 insertion mutation was introduced at nt 3732 in the mt domain of orf 1a to obtain ⌬clar-m4 (fig. 4a, d) . these mutants replicated in protoplasts and produced genomic and sgrnas at reduced levels, 30-to 50-fold lower when compared to that of ctv-⌬clar, but with approximately comparable levels to that of ctv-⌬cla, which contained a ϩ1 insertion at nt 3732 (fig. 4b, b-d) . similarly, a ϩ1 insertion was introduced at the 6 u's at nt 4963 in the 5ј half of idr to obtain ⌬clar-m5, and an adenylate was deleted after the 6 u's at nt 7084 in the 3ј half of idr to create ⌬clar-m6 (fig. 4a, f and g) . mutant ⌬clar m6 replicated at levels comparable with that of the original ctv-⌬cla, while mutant ⌬clar-m5 replicated slightly less. both accumulated 30-to 50-fold less viral rnas than that of the wild-type ctv-⌬clar (fig. 4b, f and g) . these results suggested that the homopolymeric stretch of u's in orf 1a are shift-prone sequences. we examined the requirements of u-rich regions as shift-prone sequences by introducing silent mutations plus a ϩ1 insertion to disrupt the u-run of the original u-rich region between nts 3726 and 3732 (uuu uuu u changed to uuc uuc u), resulting in mutant ctv-⌬cla-m7 (fig. 5a, b) . as a control, similar silent mutations were introduced in ctv-⌬clar between nts 3726 and 3731 without the reading frame mutation (uuu uuu a changed to uuc uuc a), resulting in ctv-⌬clar-m8 (fig. 5a, d) . mutant ctv-⌬cla-m7 failed to replicate to detectable levels in protoplasts, suggesting that the homopolymeric stretch of u's facilitated frameshifting (fig. 5b, b) . as expected, ctv-⌬clar-m8 replicated approximately similarly to that (satyanarayana et al., 2002a) . ctv-gfp-p23 contained a ϩ1 insertion at nt 3732 (nt in bold) (a), and the ϩ1 insertion was corrected in ctvr-gfp-p23 (b). (b) northern blot analysis of accumulation of positive-stranded genomic, and gfp and p23 sgrnas from n. benthamiana protoplasts transfected with ctv-gfp-p23 and ctvr-gfp-p23 at 3 and 4 dpi using a 3ј positive-stranded rna-specific riboprobe. the genomic and sgrnas are indicated with an arrowhead and arrows, respectively. (c) a representative field of protoplasts expressing gfp transfected with ctvr-gfp-p23 or ctv-gfp-p23 at 4 dpi at approximately the same density viewed by a flourescent microscope. of ctv-⌬clar, demonstrating that the silent mutations did not affect the replication (fig. 5b, d) . e. coli transformed with intact, supercoiled plasmids containing the original cdna clones (pctv9 and pctv-⌬cla) with the ϩ1 insertion mutation produced visible colonies after overnight incubation, whereas 2-3 days were required to see visible colonies with plasmids containing the repaired clones ctv9r and ctv-⌬clar (figs. 2 and 6) , suggesting that the frameshift mutation present in the orig-inal cdna clones reduced the toxicity to e. coli. since the original full-length (ctv9) and replicon (ctv-⌬cla) cdna clones were equally toxic to e. coli (the repaired clones equally more toxic) (fig. 6) , the toxic region appeared to be located within orfs 1a or 1b. the frameshift at nt 3732 reduced the toxicity of the original clones, suggesting that the toxicity domain was between nts 3732 and 11011. this location is supported by the observation that, when constructing cdna clones for assembly of the fulllength cdna clone, we were able to clone the sequences between ncoi (nt 3685) and bsteii (nt 7775) only in the reverse orientation in puc119 (pt36-nco-bst; satyanarayana et al., 1999). the schematic diagram of ctv replicon, ctv-⌬clar, containing orf 1a and 1b plus portions of orfs 2 and 11 (a). selected unique restriction sites used to generate mutants in ctv-⌬clar are indicated above the diagram. the ϩ1 insertion found at nt 3732 (nt in bold) in ctv-⌬cla (b) is shown below the ctv-⌬clar. the expanded view of orf 1a is shown below ctv-⌬clar with the location of ϩ1 or ϩ2 insertion, and ϫ1 deletion mutations in orf 1a (c-h). the inserted nucleotides are shown in bold. the nucleotide in bold and underlined was deleted in ⌬clar-m6 (g). the different domains of orf 1a are as outlined in the legend for we examined the effects of the other frameshift mutations on toxicity to e. coli. the purified plasmids containing the frameshift mutations were used to transform e. coli strain jm109, and bacterial growth on lb agar plates was compared with that of e. coli transformed with pctv9, pctv-⌬cla, pctv9r, pctv-⌬clar, puc119, and ptmv (dawson et al., 1988) . mutants with frameshift mutations between nts 3732 and 7084 (p⌬clar-m1, -m4, -m5, -m6, pctv9, and pctv-⌬cla) produced colonies of 0.2-0.9 mm diameter at 1 day posttransformation (dpt) and 0.9 -1.6 mm diameter at 2 dpt, respectively (figs. 2 and 6) , compared to 1.5-1.8 and 2.6 -2.7 mm diameter colonies containing puc119 and ptmv at 1 and 2 dpt, respectively. however, we failed to observe visible colonies under the microscope with the p⌬clar-m3 (with a frameshift mutation at nt 1109), p⌬clar-m2 (with a frameshift at nt 8390), pctv9r, or pctv-⌬clar after overnight incubation at 37°c; however, the colonies grew to 0.2-0.3 mm diameter after 2 days of incubation (fig. 6) . thus, the frameshift mutations in orf 1a around the idr (p⌬clar-m1, -m4, -m5, -m6) minimized the toxicity to e. coli, while the mutations introduced before (p⌬clar-m3) or after (p⌬clar-m2) failed to minimize the toxicity (fig. 6) , suggesting that the toxicity region was located between nts 3732 and 8390. we found that frameshifts within u stretches in ctv were repaired during transcription or replication, resulting in infection of approximately 2-3% as many protoplasts as the repaired transcripts. oligo(u) stretches have been identified as slippery sequences that are involved in frameshifting of other viruses (wilson et al., 1988; brierley et al., 1992; honda et al., 1995; kim et al., 2001) . however, frameshifting also is often associated with downstream structures in the rna (jacks et al., 1988; brierley et al., 1992; chamorro et al., 1992; vickers and ecker, 1992; chen et al., 1995; marczinke et al., 1998; barry and miller, 2002) . in hiv, a run of 6 u's is required for efficient ϫ1 frameshifting to produce the gag-pol proteins with an extended run of u's resulting in slightly increased levels of frameshifting (wilson et al., 1988; honda et al., 1995) . however, parkin et al. (1992) reported that a stem-loop structure 3ј of the hiv ϫ1 shift site was important for wild-type levels of frameshifting in vivo. nevertheless, downstream structures appeared not to be needed in the observed repair of the frameshift mutations after replication in n. benthamiana protoplasts. we did not identify any common structures by examining the sequences with the mfold program (data not shown). also, since we examined all of the 6 u-regions, none of which are thought to naturally be involved in frameshifting, there is no expectation that all would conserve some accessory structure needed for frameshifting. slippery sequences identified in retrovirus frameshifting include aaaaaac (mouse mammary tumor virus); aaauuua (rous sarcoma virus); uuuuuua (hiv-1 and hiv-2); and uuuaaac in coronavirus frameshifting (ibv) (jacks et al., 1987 (jacks et al., , 1988 wilson et al., 1988; brierley et al., 1992) . additionally, numerous other slippery sequences have been identified (brierley et al., 1992; marczinke et al., 1998; kim et al., 2001) and some of them occur throughout the ctv genome. we did not examine whether other potentially slippery sequences effect frameshifting in ctv. however, we examined six oligo(u) sites that allowed frameshifting when a mutation was created to destroy the reading frame. it would be expected that each of these sites also shift to an incorrect reading frame in the absence of mutations. if there are additional slippery sequences scattered throughout the ctv genome that also occasionally shift the reading frame, it is fortunate that sufficient fulllength translation product is produced to allow efficient the schematic diagram of ctv-⌬cla showing the sequence around nt 3732 containing an additional nt "u" (in bold) (a), the codons uuu uuu in ctv-⌬cla encoding phenylalanine were changed to uuc uuc without changing the amino acid sequence (nts in bold and italics) to create ctv-⌬cla-m7 (b). as a control the condons uuu uuu in ctv-⌬clar (corrected clone: without a ϩ1 u insertion) (c) were also changed to uuc uuc (nts in bold and italics) in ctv-⌬clar-m8 (d). the domains of orf 1a are as outlined in the legend for fig. 1a. (b) northern blot analysis of accumulation of positive-stranded rnas from protoplasts transfected with ctv-⌬cla and ctv-⌬clar and their silent mutants at 3 and 4 dpi. the blots were hybridized with a 3ј positive-stranded rnaspecific riboprobe. the genomic and sgrnas are indicated with an arrowhead and an arrow, respectively. replication. yet it is possible that slippery sequences serve as a survival mechanism for large rna viruses. viral rna polymerases are known to be error prone (drake and holland, 1999) without an error repair mechanism, defining viruses as pseudospecies of sequence variants (domingo et al., 1995) . it has been argued that recombination and selection are the viral mechanisms to repair errors. slippery sequences could serve as an additional repair mechanism. if a mutation occurs upstream of a slippery sequence, a frameshift could repair the mistake if the intervening altered amino acids are tolerated. perhaps there is an advantage for ctv to have slippery sequences scattered throughout orf 1a. this could be one of the reasons some ctv isolates have been observed to have a high degree of evolutionary stasis . the frameshifts inserted into the oligo(u) regions of ctv created a compromise between infectivity and toxicity of the cdna in high-copy-number plasmids in e. coli. the number of protoplasts that became infected with rna transcripts from the frameshifted clone was reduced by 30-to 50-fold when compared to that of the corrected clone. however, the toxicity in e. coli was greatly reduced. bacterial colonies were visible after 1 day, even though they were minute, compared to 2-3 days with the repaired clone. the progeny virus from the frameshifted clone was repaired; it did not contain the original frameshift caused by the inserted nucleotide and had a phenotype identical to the parental wild-type virus . thus, the frameshift mutation appeared to have no negative impact on the value of the clones other than the initial reduc-tion in infectivity. developing an infectious, full-length cdna clone of ctv was difficult. it is unlikely that we would have obtained an infectious full-length clone without a fortuitous frameshift mutation in the original full-length cdna clone. these results suggest that purposely inserting frameshifts at slippery sequences of viral cdnas that are toxic to e. coli could be an additional strategy for cloning these viruses in convenient high-copy-number plasmids. the frameshift provides a compromise: a reduction of infectivity for a decrease in toxicity in e. coli. if the host system is susceptible enough to provide infection with a couple of magnitudes of decrease in infectivity, the frameshift should allow growth of an infectious clone in e. coli. if the host cells are even more susceptible, it is possible that two frameshifts could be used. as is evident from this work, all frameshifts in slippery sequences will not minimize the toxicity to e. coli. the frameshift must be positioned to prevent expression of the toxicity domain, similar to the insertion of introns for the same purpose. we examined only stretches of u's, but if the frameshifting occurred during translation, it is likely that other identified slippery sequences would also work. since the repair of the frameshift mutations was due to in vitro transcription or translation in protoplasts, neither of which are virus-specific functions, the purposeful introduction of frameshift mutations into frameshift-prone sequences in front of toxic regions should work for any recalcitrant positive-stranded rna virus. thus, this strategy provides an additional approach in the virologist's reperfig. 6 . effect of insertion (ϩ1 and ϩ2) and deletion (ϫ1) mutations in orf 1a of ctv on e. coli strain jm109 colony size on lb-agar plate. the purified plasmid dnas of pctv9, pctv9r, pctv-⌬cla, and pctv-⌬clar, and frameshift mutants were used to transform e. coli. the location of frameshift mutations introduced in orf 1a are indicated in upper right corner of the graph. the domains of orf 1a are as described in the legend for fig. 1a . the data represent mean and standard deviation of 25-30 colonies at 1 and 2 days posttransformation. nv, colonies not visible. toire for developing reverse-genetic systems for difficult viruses. the assembly of a full-length infectious cdna clone of ctv t36, pctv9, and a replicon, pctv-⌬cla, behind the sp6 rna polymerase promoter in puc119 was reported previously (satyanarayana et al., 1999) . the ϩ1 insertion mutation found at nt 3732 in pctv9 and pctv-⌬cla was corrected by reverse transcription of the region between nts 3559 and 4630 followed by amplification by pcr from the double-stranded rna extracted from ctv t36 isolateinfected bark tissue. the amplified cdna fragment was digested with ncoi (nt 3685) and bsu36i (nt 4430) restriction endonucleases and followed by ligation into similarly digested pctv9 and pctv-⌬cla to obtain pctv9r and pctv-⌬clar, respectively (fig. 1a) . ctv-gfp-p23 was obtained by precisely exchanging the cp orf with that of gfp in ctv-cp/p23 (satyanarayana et al., 2002a) . subsequently, the ϩ1 insertion mutation found at nt 3732 in ctv-gfp-p23 was removed by exchanging a dna fragment between ncoi and bsu36i from pctv9r to obtain ctvr-gfp-p23 (fig. 3a) . the frameshift mutations in orf 1a were introduced by pcr using a set of positive-and negative-sense oligonucleotides that contained the intended mutation, followed by overlap-extension pcr (ho et al., 1989 ) of the region between unique restriction sites in pctv-⌬clar (fig. 4a) . the ϩ2 and ϩ1 insertion mutations at nts 3732 and 4079, respectively, were introduced by amplifying dna fragments that contained the mutations between nts 3559 and 4630 and then ligated between ncoi (nt 3685) and bsu36i (nt 4430) restriction sites into pctv-⌬clar to obtain ⌬clar-m4 and ⌬clar-m1, respectively (fig. 4a ). insertion mutations (ϩ1) were introduced at nt 8390 by amplifying a pcr product between nts 7747 and 19293 using pctv-⌬clar as a template and ligated between bsteii (nt 7775) and noti (nt 19293) restriction sites into pctv-⌬clar to obtain ⌬clar-m2, and at nt 1109 by overlapextension pcr, and the pcr product was ligated between bg/ii (nt 1029) and ncoi (nt 3685) restriction sites into pctv-⌬clar to obtain ⌬clar-m3 (fig. 4a) . the pcr products amplified between nts 3559 and 7803 that contained ϩ1 and ϫ1 mutations at nts 4963 and 7084 were ligated between ncoi and bsteii sites into pctv-⌬clar to obtain ⌬clar-m5 and ⌬clar-m6, respectively (fig. 4a) . silent mutations in homopolymeric run of u's between nts 3726 and 3732 (uuu uuu u changed to uuc uuc u) were introduced by overlap-extension pcr using mutagenized primers between nts 3559 and 4630 and then ligated into pctv-⌬cla between ncoi and bsu36i restriction sites to obtain pctv-⌬cla-m7 that contained a ϩ1 insertion mutation at nt 3732 (fig. 5a) . pctv-⌬clar-m8 contained silent mutations in wild-type pctv-⌬clar between nts 3726 and 3731 (uuu uuu changed to uuc uuc) (fig. 5a) . sp6 rna polymerase-derived transcripts from noti-linearized ctv cdnas were generated in vitro as described by satyanarayana et al. (1999) . the procedures for the isolation of mesophyll protoplasts from n. benthamiana leaves and polyethylene glycol mediated transfections were carried out as described previously (satyanarayana et al., 1999) . the total rna isolated from 3 and 4 day post inoculated (dpi) protoplasts were analyzed by northern blot hybridization using a 3ј positive-stranded rna-specific riboprobe (satyanarayana et al., 1999) . bands from different exposures of northern hybridization blots of total rna from protoplasts at 4 dpi were quantified by scanning and densitometry with the os-scan program (oberlin scientific, oberlin, oh). results represented at least three to four independent protoplasts transfections using two to three independent clones for each construct. competent cells of e. coli strain jm109 prepared as described by hanahan (1995) were transformed and plated on lb agar-amp plates (1.0% tryptone, 0.5% yeast extract, and 1.0% sodium chloride in water, ph 7.0 plus 1.5% bacto-agar containing 100 g/ml ampicillin). the plates were incubated overnight at 37°c followed by 2 days incubation at room temperature. the size of e. coli colonies was measured with a microscope at 1 and 2 dpt. 2xyt liquid medium (1.6% tryptone, 1.0% yeast extract, and 0.5% sodium chloride in water, ph 7.0) was used to grow e. coli harboring recombinant dnas for the plasmid dna isolation (sambrook and russell, 2001) . sequencing of the full-length infectious clone pctv9 and the frameshift mutants to confirm the mutations was performed with an automatic sequencer (applied biosystems, model 373) at the interdisciplinary center for biotechnology research dna sequencing core facility of the university of florida, gainesville, fl. the complete nucleotide sequence of the infectious fulllength cdna clone of ctv t36 isolate, pctv9r, was deposited in the genbank database under the accession no. ay170468. the nucleotide numbering and positions men-tioned in this investigation correspond to the pctv9r sequence. multicomponent rna plant virus infection derived from cloned viral cdna sequences of citrus tristeza virus separated in time and space are essentially identical engineering the largest rna virus genome as an infectious bacterial artificial chromosome a -1 ribosomal frameshift element that requires base pairing across four kilobases suggests a mechanism of regulating ribosome and replicase traffic on a viral rna infectious transcripts and cdna clone of rna viruses mutational analysis of the "slippery-sequence" component of a coronavirus ribosomal frameshifting signal reverse genetics system for the avian coronavirus infectious bronchitis virus an rna pseudoknot and an optimal heptameric shift site are required for highly efficient ribosomal frameshifting on a retroviral messenger rna structural and functional studies of retroviral rna pseudoknots involved in ribosomal frameshifting: nucleotides at the junction of the two stems are important for efficient ribosomal frameshifting modification of the tobacco mosaic virus coat protein gene affects replication, movement, and symptomatology quasispecies: the concept and the word mutation rates among rna viruses stabilization of a full-length infectious cdna clone of transmissible gastroenteritis coronavirus by insertion of an intron characterization of the cis-acting elements controlling subgenomic mrnas of citrus tristeza virus: production of positive-and negative-stranded 3ј-terminal and positive-stranded 5ј-terminal rnas techniques for transformation of e. coli characterization of citrus tristeza virus subgenomic rnas in infected tissue site-directed mutagenesis by overlap extension using polymerase chain reaction rna signals for translation frameshift: influence of stem size and slippery sequence signals for ribosomal frameshifting in the rous sarcoma virus gag-pol region two efficient ribosomal frameshifting events are required for synthesis of mouse mammary tumor virus gag-related polyproteins intron insertion facilitates amplification of cloned virus cdna in escherichia coli while biological activity is reestablished after transcription in vivo complete sequence of the citrus tristeza virus rna genome comparative mutational analysis of cis-acting rna signals for translational frameshifting in hiv-1 and htlv-2 the making of infectious viral rna: no size limit in sight construction of stable and highly infectious intron-containing cdna clone of plum pox potyvirus and its use to infect plants by particle bombardment secondary structure and mutational analysis of the ribosomal frameshift signal of rous sarcoma virus nucleotide sequence and organization of eight 3ј open reading frames of the citrus tristeza closterovirus genome human immunodeficiency virus type 1 gag-pol frameshifting is dependent on downstream mrna secondary structure: demonstration by expression in vivo in vitro synthesis of biologically active beet necrotic yellow vein virus rna cloned poliovirus cdna is infectious in mammalian cells transcription of infectious yellow fever rna from full-length cdna templates produced by in vitro ligation molecular cloning: a laboratory manual an engineered closterovirus rna replicon and analysis of heterologous terminal sequences for replication closterovirus encoded hsp70 homolog and p61 in addition to both coat proteins function in efficient virion assembly amplification of citrus tristeza virus from a cdna clone and infection of citrus trees mutational analysis of the replication signals in the 3ј-nontranslated region of citrus tristeza virus the p23 protein of citrus tristeza virus controls asymmetrical rna accumulation dna -containing hybrid plasmids giving rise to q phage formation in the bacterial host infectious rna transcribed in vitro from a cdna copy of the human coronavirus genome cloned in vaccinia virus ph.d. dissertation. a molecular genetic analysis of host/viral interactions, implications for the use of plant viruses as gene vectors enhancement of ribosomal frameshifting by oligonucleotides targeted to the hiv gag-pol region hiv expression strategies: ribosomal frameshifting is directed by a short sequence in both mammalian and yeast systems a new strategy in design of (ϩ)rna virus infectious clones enabling their stable propagation in e. coli strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model systematic assembly of a full-length infectious cdna clone of mouse hepatitis virus strain a59 we thank john cook, cecile robertson, and judy harber for excellent technical assistance. this research was supported in part by an endowment from the j.r. and addie s. graves family and grants from the florida citrus production research advisory council, the national citrus research council, the u.s.-israel binational agricultural research and development fund, usda/ars cooperative agreement, the usda/nri, and the florida agricultural experiment station, and approved for publication as journal series no. r-09320. key: cord-255453-7e40rj1y authors: oliver, s.l.; asobayire, e.; dastjerdi, a.m.; bridger, j.c. title: genomic characterization of the unclassified bovine enteric virus newbury agent-1 (newbury1) endorses a new genus in the family caliciviridae date: 2006-06-20 journal: virology doi: 10.1016/j.virol.2006.02.027 sha: doc_id: 255453 cord_uid: 7e40rj1y the pathogenic bovine enteric virus, newbury agent-1 (bo//newbury1/1976/uk), first identified in 1976, was characterized as a possible calicivirus by morphology, buoyant density in cscl and the presence of a single capsid protein but genomic sequence could not be obtained. in the present study, the complete genome sequence of newbury1 was determined and classified newbury1 in a new genus of the caliciviridae. the newbury1 genome, of 7454 nucleotides, had two predicted open reading frames (orfs). orf1 encoded the non-structural and contiguous capsid proteins. orf2 encoded a basic protein characteristic of the family caliciviridae. compared to the 4 recognized caliciviridae genera, norovirus, sapovirus, lagovirus and vesivirus, newbury1 had less than 39% amino acid (47% nucleotide) identity in the complete 2c-helicase, 3c-protease, 3d-polymerase and capsid regions but had 89% to 98% amino acid (78% to 92% nucleotide) identity to the recently characterized nb virus in these regions. by phylogenetic analyses, newbury1 and nb viruses formed a distinct clade independent of the 4 recognized genera. however, amino acid identities showed that newbury1 and the nb virus were distinct polymerase types (90% amino acid identity), but their complete capsid proteins were almost identical (98% amino acid identity). analyses of contemporary viruses showed that the two polymerase genotypes, newbury1 and nb, were circulating in uk cattle and antibody to newbury1-like viruses was common in cattle sera. the present study defined the existence of a new genus in the caliciviridae that we propose be named becovirus or nabovirus to distinguish the new clade from bovine noroviruses. the study of the viral causes of diarrhea has revealed a number of previously unrecognized viruses including viruses now classified in the two calicivirus genera norovirus and sapovirus (green et al., 2001) . in the uk before 1984, studies into the viral causes of calf diarrhea identified at least two pathogenic calici-like viruses, newbury agent 1 (newbury1 virus) and newbury agent 2 (newbury2 virus), using electron microscopy and animal cross-protection experiments woode and bridger, 1978) . newbury2 was classified recently by genomic approaches as a member of a third genogroup in the genus norovirus (dastjerdi et al., 1999; oliver et al., 2003) . newbury1 remained unclassified despite numerous failed attempts to amplify any region of its genome using calicivirus-specific oligonucleotides (a. m. dastjerdi and s. oliver personal observations) . in experimental calves, newbury1 was more pathogenic than the newbury2 virus, causing fecal color change and increased fecal output, anorexia and xylose malabsorption . the most severe small intestinal lesions caused by newbury1 infection were in the anterior small intestine where villi were atrophied with complete loss of enterocytes that exposed parts of the lamina propria . virus particles were seen by electron microscopy in feces (woode and virology 350 (2006) 240 -250 www.elsevier.com/locate/yviro bridger, 1978) . viral antigen was found in the cytoplasm of enterocytes in the anterior small intestine up to 3 days postinfection . all of these features were consistent with an enteropathogenic virus. the newbury1 virion had several properties consistent with the caliciviridae: a typical morphology showing cup-like depressions, a particle diameter of 36.6 nm, a buoyant density of 1.34 g/cm 3 in cscl and a single capsid protein with a molecular mass of 49 kda (dastjerdi et al., 2000) . there are currently 4 genera recognized in the family caliciviridae (green et al., 2000) . each one has been named after the disease symptom (vesicular lesions-vesivirus), hosts infected (lagomorphs-lagovirus) or the geographical location where the type species was first isolated (sapporo, japan-sapovirus; norwalk, usa-norovirus). all of these small, non-enveloped, icosohedral viruses contain positive-sense, single-stranded, polyadenylated rna genomes that range from 7.3 to 8.5 kbp in length flanked by 5′ and 3′ untranslated regions (reviewed by green et al., 2001) . the division of the caliciviruses into the 4 distinct genera has been based on differences in their genomic organization and extensive genomic sequence diversity of the polymerase and capsid genes that form 4 clades by phylogenetic analyses (reviewed by green et al., 2000) . the genomes of the lagoviruses and sapoviruses are organized into two open reading frames (orfs) whereas those of the noroviruses, into which newbury2 has been classified, and the vesiviruses are organized into three. orfs 1 and 2 of the noroviruses overlap but those of the vesiviruses are separated by a few nucleotides. in addition, the capsid proteins of the vesiviruses have a leader sequence that is proteolytically cleaved during maturation (matsuura et al., 2000; sosnovtsev et al., 1998) . in all the genera, the 3′ terminal orf, orf2 for the sapoviruses and lagoviruses and orf3 for the noroviruses and vesiviruses, overlap with the capsid gene (reviewed by green et al., 2001) . the proteins encoded by the genome follow the order nh 2 -terminal, 2c-helicase, 3a, 3b, 3c-protease, 3d-polymerase, capsid and a basic protein (3′ terminal orf), which distinguished the caliciviridae from other families of positive-sense, single-stranded rna viruses. the translated proteins of all the genera have at least one conserved amino acid motif for the 2c-helicase (gxxgxgks/ t), 3c-protease (gdcg), 3d-polymerase (glpsg, ygdd) and capsid (ppg) proteins. in the present study, the complete genomic sequence and genome organization of the newbury1 virus were determined and analyzed to characterize newbury1 and verify its relationship with established members of the 4 caliciviridae genera. further newbury1-like viruses were sought in contemporary samples to determine whether a distinct extant calicivirus genus was circulating in uk cattle. the newbury1 genome had a g + c content of 56.2% and was 7454 nucleotides in length. it had a 5′ untranslated region of 75 nucleotides and a 3′ untranslated region of 67 nucleotides flanking 2 orfs (table 1) . orf1 was 6633 nucleotides (2210 amino acids) long, started from nucleotide 76 and was terminated by the ochre stop codon uaa. the short 3′ terminal orf (orf2) was 678 nucleotides (225 amino acids) long. it did not overlap with orf1 but was separated by a single nucleotide so that the gene was in a +2 frame-shift. the non-structural proteins (nh 2 -terminal, 2c-helicase, 3a, 3b, 3c-protease and 3d-polymerse) and the capsid protein were predicted to be encoded by orf1 with a combined molecular mass predicted to be 239 kda. the organization of the orf1 polyprotein was determined by the presence of amino acid motifs (conserved amino acids underlined) for the 2c-helicase (gppghgks), 3cprotease (gycg), 3d-polymerase (glpsg and ygdd) and capsid (ppg) proteins. the observation that the 5′ end of the newbury1 genome had 12 nucleotides conserved within a 22nucleotide region of orf1 (nucleotides 5036 to 5047) predicted a possible internal initiation codon ( 5059 atg 5061 ) for the capsid gene that encoded a 58 kda protein. a second possible initiation codon ( 5326 atg 5328 ) was predicted to produce a 49 kda capsid protein similar to that detected previously for newbury1 by western blot (dastjerdi et al., 2000) . however, it is unknown, at present, if translation occurs from a subgenomic rna. the polyprotein translated from orf1 had 6 protease cleavage sites as predicted from those identified experimentally for the 3 genera lagovirus, norovirus and vesivirus (fig. 1) . the dipeptides at the predicted cleavage sites consisted of e/ qg/a. increased hydrophilicity of the polyprotein, as determined by the method of kyte-doolittle, corresponded with the position of the cleavage dipeptides. the predicted nh 2terminal, 2c-helicase and 3a cleavage products for the newbury1 polyprotein contained both a hydrophobic and a hydrophilic region. the predicted 3b cleavage product was exclusively hydrophilic with the 3c-protease predominantly neutral. whether alternative cleavage sites exist is unknown at present. the capsid protein could be divided into 2 hydrophobic and a hydrophilic region that coincided with the s, p1 and p2 domains based on the crystal structure determined for the capsid protein of the norwalk virus (prasad et al., 1999) . in contrast to viruses in the 4 recognized caliciviridae genera, cysteine, important for disulfide bridge formation, was not present in the capsid protein. consistent with a calicivirus, the translated protein from the 3′ terminal orf, orf2, had a predicted basic isoelectric point (ph11.1). many of the above findings were consistent with viruses classified in the family caliciviridae. newbury1 amplicons from the polymerase gene were detected by rt-pcr in the feces of 2 clinically affected, experimental calves using the primers nbu(f)/na1orf1_07 (data not shown). in the one calf (1424) for which daily fecal samples were available, an amplicon was not produced on the day of inoculation, a weak amplicon was produced the day after inoculation, before clinical signs commenced, and strong amplicons were produced from day 2 to at least day 4 postinoculation when diarrhea, increased temperature and diminished appetite were present. the morphology of newbury1 virions resembled classical calicivirus morphology in contrast to the genogroup iii bovine norovirus newbury2 (fig. 2) . surface depressions were visible on many newbury1 virions. antigenically, newbury1 was distinct from newbury2 using an elisa specific for newbury2 antibody. sera from 3 experimental calves inoculated orally with newbury1 , and shown to have newbury1 antibodies by spiem (dastjerdi et al., 2000) , failed to react in the newbury2 elisa which detected homologous igg titers of log 10 3.0 to 4.0 in newbury2 convalescent calf sera. the relationship of bo/newbury1/1976/uk with members of the caliciviridae although many features of the newbury1 genome organization were consistent with the caliciviridae, the newbury1 genome had low nucleotide (≤47%) and amino acid (≤39%) identities to all representative viruses of the 4 caliciviridae genera by comparison of complete non-structural genes (2chelicase, 3c-protease, 3d-polymerase), complete capsids, capsid subdomains and the 3′ terminal orf genes ( table 2 ). the highest amino acid identity (39%) was seen with the lagovirus polymerase. the lowest amino acid identity (≤9%) was seen with the 3′ terminal orf for the sapoviruses (orf2) and vesiviruses (orf3). in contrast, the entire newbury1 orf1 polyprotein had ≥89% amino acid (≥78% nucleotide) identity compared to the previously characterized nb virus (smiley et al., 2002) . surprisingly, the least variation was seen between the p-domains of the capsid protein of the 2 viruses. newbury1 failed to group phylogenetically with the 4 recognized caliciviridae genera but formed a separate clade with nb virus by upgma, fitch-margoliash, parsimony and maximum likelihood analyses of the nucleotide and translated amino acids of orf1 (excluding the capsid gene), the individual regions that encoded the 2c-helicase, 3c-protease, 3d-polymerase and complete capsid genes (not shown). the nh 2 -terminal, 3a and 3b regions were not used for phylogenetic analyses because of the poor nucleotide and amino acid alignments caused by the extensive diversity in these sequences between the 4 calicivirus genera. both newbury1 and nb viruses consistently grouped more closely with the lagoviruses but the branch lengths were very long, indicative of evolutionary diverse viruses. the distant relationship of newbury1 to the 4 recognized caliciviridae genera was confirmed using additional phylogenetic analyses based on amino acid alignments generated from homology models of structurally conserved regions of the partial polymerase and the capsid s-domain (fig. 3) . homology models are useful to compare sequences with low levels of identity, as observed in the present study. newbury1 and nb were again phylogenetically closer to the lagoviruses in their polymerase sequences ( fig. 3a ) but were more closely related to the noroviruses in their conserved capsid s-domain sequences (fig. 3b) . however, the statistical support at the node between the newbury1 and nb viruses and the noroviruses was weak (72%), indicating that this relationship was uncertain. the divergence of the newbury1 and nb genomes from the other calicivirus genera was confirmed by higher mean values for maximum likelihood distances, a measure of evolutionary divergence for nucleotide or amino acid sequences. newbury1 and nb had twice the nucleotide maximum-likelihood distances when compared to the 4 recognized calicivirus genera for the non-structural genes (x = 1.419; σ = 0.158) and the complete capsid genes (x = 1.377; σ = 0.214) than the means within each of the 4 genera (non-structural genes: vesivirus-x = 0.726; σ = 0.323). the inability to classify newbury1 and nb genomically into any of the 4 recognized genera using all of the above approaches provided strong support for a new genus within the family caliciviridae. simplot analysis of the complete newbury1 genome confirmed a close genomic relationship with the nb virus. the region of orf1 that encoded for the non-structural proteins of the 2 viruses had a mean nucleotide identity of 81%. this increased to 92% for the capsid and orf2 proteins (fig. 4) . the sharp increase in nucleotide identity, from 72% in the polymerase to 97% in the nh 2 -terminal region of the capsid fig. 2 . electron microscopy of newbury1 and newbury2 virions negatively stained with 2% potassium phosphotungstate (ph 6.0). scale bar, 50 nm. was a feature that has been seen for caliciviruses in the genera norovirus and sapovirus. the levels of identities seen in the 2c-helicase (93% amino acid; 81% nucleotide), 3c-protease (89% amino acid; 80% nucleotide) and 3d-polymerase (90% amino acid; 78% nucleotide) suggested that newbury1 and nb were different genotypes in 3 of their non-structural proteins but not in their capsid proteins, which had 98% amino acid (92% nucleotide) between the two viruses ( table 2 ). the lack of capsid genotypes for the newbury1 and nb viruses was evident in their capsid pdomains that were almost identical (99% amino acid; 92% nucleotide). the 3′ terminal orf (orf2), which usually shows high levels of variability between genotypes within the same genus, had a remarkably high amino acid identity of 96% (91% nucleotide). newbury1 was identified in a fecal sample taken in 1976 but serological evidence showed that newbury1 virus or newbury1-like viruses were circulating in uk cattle in 1990 and 1993. fourteen of 18 sera (78%) were positive for newbury1 antibody at 1:10 dilution by spiem, trapping up to 45 particles per field. subsequently, newbury1-like viruses were identified in feces of diarrheic farm calves in 2000 from 2 geographically distant locations in the uk, penrith and starcross. out of 107 samples examined, 7 samples from penrith and 2 from starcross (a mean prevalence of 8.4%) produced amplicons of the expected size for the polymerase (509 bp for primer pair nbu(f)/na1orf1_07) and polymerase-capsid (522 bp for primer pair na1orf1_12/ na1orf1_29) regions of the orf1 gene. the newbury1 and nb polymerase genotypes circulating in uk cattle were determined by amino acid and nucleotide sequence identities of 7 partial polymerase sequences (amino acids 1490 to 1662 [nucleotides 4541 to 5058]) from the penrith and starcross isolates of 2000 (table 3 ). the 5 newbury1-like viruses (penrith142, penrith143, penrith150, penrith151 and starcross117) had 95% amino acid (88 to 89% nucleotide) identity to newbury1 and 88% amino acid (76 to 77% nucleotide) identity to the nb virus. the 2 nb-like viruses (penrithc39 and starcross93) had 88% amino acid (78% nucleotide) identity to newbury1 and 96 to 97% amino acid (89 to 90% nucleotide) identity to nb. in contrast, evidence was not obtained for more than one capsid type. sequence data generated for the nh 2 -terminal of the capsid protein (amino acids 1663 to 1794 [nucleotides 5059 to 5454]) from 8 of the uk isolates showed a close relationship with both newbury1 and nb, having 96 to 99% amino acid (92 to 94% nucleotide) identity. the partial polymerase sequence of the nb-like virus, starcross93, had 19 (11.1%) amino acid substitutions compared with newbury1, of which 15 were identical in position and composition to those of the nb virus (fig. 5) . in contrast to starcross93, the newbury1-like virus penrith150 had just 8 (4.7%) amino acid substitutions in the polymerase when compared with newbury1. the remaining newbury1-like viruses (penrith142, penrith143, penrith151 and starcross117) had similar substitutions in frequency (7 to 8; 4.1 to 4.7%), location and composition to penrith150 when they were compared to newbury1, whereas the nb-like virus penrithc39 had 20 (11.6%) substitutions almost identical in location and composition to starcross93 (not shown). few amino acid substitutions (1 to 4; 0.8 to 3.0%) were seen in the genomic sequence generated for the nh 2 -terminal of the capsid protein. the relationship of uk isolates from penrith and starcross with fig. 4 . nucleotide identity plot between the two bovine enteric caliciviruses newbury1 and nb generated using simplot with a 250-nucleotide window moved along in 20 nucleotide steps. the two horizontal grey lines show the mean nucleotide identity for the non-structural (dotted-81%) and structural (dashed-92%) proteins encoded by orf1 and orf2. orfs 1 and 2 encoded by the newbury1 genome are shown by the two shaded boxes (grey). the numbers in italics show the predicted length of the orf1 and orf2 proteins. the newbury1 and nb viruses was supported by phylogenetic analyses of the partial polymerase and nh 2 -terminal region of the capsid genes and endorsed the existence of 2 polymerase but not capsid genotypes (data not shown). the genomic analyses reported in the present study, plus previously published physicochemical studies, showed that newbury1 virus was a calicivirus in its morphology, virion size, buoyant density, possession of a single major capsid protein, genome organization and the presence of conserved amino acid motifs dastjerdi et al., 2000; hall et al., 1984; woode and bridger, 1978) . the finding that newbury1 and the newbury1-like viruses failed to group phylogenetically with any of the known viruses of the caliciviridae, apart from the recently described bovine enteric calicivirus nb and nblike viruses from the us (han et al., 2004; smiley et al., 2002) , endorsed the existence of a new genus within the caliciviridae as proposed by smiley et al. (2002) . this was in agreement with the calicivirus study group's definition of a new calicivirus genus: "a genus in the caliciviridae will be defined as a genetically distinct clade of viruses" (green et al., 2000) . we propose that the new genus be named either becovirus or nabovirus to distinguish them from the bovine noroviruses that are also associated with bovine enteric disease. becovirus is based on sigla for 'bovine enteric calicivirus' and provides information on the species infected, the disease caused and follows precedents used for the calicivirus genera lagovirus and vesivirus. nabovirus is based on newbury1 and nb, the first two viruses to be identified in the new genus. newbury1 was evidently not a member of the families picornaviridae, astroviridae or hepeviridae (matsui and greenberg, 2001; purcell and emerson, 2001; racaniello, 2001) as there were major differences in the number of orfs and gene order that were consistent with those of the family caliciviridae (green et al., 2001) . the genomic organization was typical of the calicivirus genera lagovirus and sapovirus, not the genera norovirus or vesivirus, as a single orf encoded the non-structural (nh 2 -terminal, 2c-helicase, 3a, 3b, 3cprotease, 3d-polymerase) and single major capsid protein (green et al., 2001) . however, three significant differences in the newbury1 genome organization distinguished it from the 4 recognized calicivirus genera. firstly, the 5′ utr was at least 3 times longer than any other calicivirus. secondly, orf1 and orf2 did not overlap but were separated by a single nucleotide. a similar separation between orfs has been seen for the vesiviruses but between the non-structural and major capsid proteins (glenn et al., 1999; matsuura et al., 2002) . thirdly, the 3c-protease had a gycg motif that was identical to some members of the picornaviridae (carrillo et al., 2005; doherty et al., 1999; skern et al., 1985; wutz et al., 1996) , but not the typical calicivirus gdcg motif. the newbury1 orf2 protein was at least 60 residues longer than those for the lagoviruses, sapoviruses and vesiviruses but was of a similar size to the analogous 3′ terminal norovirus proteins (orf3). all of these genomic features and the low levels of sequence identities, combined with phylogenetic analyses and the maximum likelihood distances, proved that newbury1 was a calicivirus distinct from those of the 4 recognized genera of the caliciviridae. the evidence for classification of the newbury1 and nb viruses in the same genus was their identical genome organization, identical composition and location of predicted orf1 protease cleavage sites and almost identical capsid proteins. the latter was surprising as the two viruses were isolated from diarrheic calves on different continents and 4 years apart (smiley et al., 2002; woode and bridger, 1978) . the (95) the bovine enteric calicivirus isolates in bold had a newbury1-like polymerase. na-genomic sequence for region not available. a the sequence analyzed was 489 nucleotides (162 translated amino acids) at the 3′ end of the polymerase gene. b the sequence analyzed was 396 nucleotides (132 translated amino acids) at the 5′ end of the capsid (nh 2 -terminal region) gene. c smiley et al. (2002) . d han et al. (2004) . predicted molecular mass of 58 kda for the newbury1 and nb capsid proteins contrasted with the 49 kda previously determined for newbury1 by western blot analysis (dastjerdi et al., 2000) . the smaller mass might be due to cleavage of a precursor capsid protein, as described for the genus vesivirus (matsuura et al., 2000; sosnovtsev et al., 1998) , or translation of a subgenomic rna of the newbury1 capsid gene at a predicted initiation codon located at nucleotide 5326 in the newbury1 genome (5327 for the nb genome). the molecular mass of the nb capsid protein has not been reported. as the viruses have not been propagated in cell culture, in vitro molecular studies are required to determine the biologically active orf1 polyprotein protease cleavage sites and translation of the capsid genes for this new calicivirus genus. the present study and one previous study demonstrated that newbury1-like viruses are currently circulating in cattle on two continents (smiley et al., 2003) . for the first time, the present study indicated that two polymerase, but not capsid, genotypes exist. it was unlikely that analysis of the nh 2terminal regions of the newbury-like and nb-like viruses failed to recognize different capsid types as capsid genotypes within the genera norovirus and sapovirus can be distinguished by analysis of the conserved nh 2 -terminal region of the capsid protein (kageyama et al., 2004; okada et al., 2002) . it is uncertain whether both polymerase types exist in the usa as, to date, polymerase sequence data are available only for a single contemporary nb-like virus, bo/cv23-oh/00/us. there are precedents within the genera norovirus and sapovirus for different polymerase genes being associated with similar capsid genes (bull et al., 2005; han et al., 2004; jiang et al., 1999a; katayama et al., 2004; katayama et al., 2002; lochridge and hardy, 2003; oliver et al., 2004) . this is thought to be due to genomic recombination. further studies are required to examine the diversity of the caliciviruses within the new genus. our data has substantially advanced the knowledge about caliciviruses that cause diarrhea in cattle. outbreaks of calf diarrhea cause significant economic losses in the uk (stott and gunn, 1995) . enteropathogens are not identified for approximately 30% of calf diarrhea cases (andrews, 2000; reynolds et al., 1986; snodgrass et al., 1986) , even though the presence of rotaviruses, coronaviruses, cryptosporidia, coccidia, salmonella spp. and escherichia coli are sought. however, caliciviruses are not routinely tested for. in the present study, newbury1-and nb-like viruses were found with similar frequency (8.4%) to the bovine noroviruses (oliver et al., 2003; smiley et al., 2003; van der poel et al., 2003; wise et al., 2004) although this was substantially lower than the frequency of 28% for nb-like viruses found in the usa (smiley et al., 2003) . hence, in the uk, our data indicate that caliciviruses (noroviruses and the caliciviruses in the new genus) are associated with about 17% of calf diarrhea cases, leaving about 13% of cases still undiagnosed. it seems likely that newbury1-and nb-like viruses are responsible for a proportion of diarrhea in farm calves as newbury1 and nb caused enteric disease after oral inoculation of experimental calves and produced small intestinal lesions typical of enteropathogenic viruses hall et al., 1984; smiley et al., 2002) . further studies are required to assess the antigenic diversity of caliciviruses within the proposed new genus, their incidence and prevalence in calf diarrhea outbreaks, their species specificity and whether similar viruses exist in man. fecal samples from experimentally inoculated calves contained either the newbury1 virus (formerly named srv-1 and identified in 1976 in diarrheic feces from a farm calf, woode and bridger, 1978) , the genogroup iii bovine norovirus bo/nv/newbury2/1976/uk or the genogroup iii bovine norovirus bo/nv/dumfries/1994/uk (oliver et al., 2003) . for newbury1, feces were available from two experimentally infected calves (1424 and 2010) (dastjerdi et al., 2000) . fecal samples from 107 diarrheic farm calves collected in 2000 from two geographically distant locations in the uk, penrith (n = 57) and starcross (n = 50), were kindly provided by the veterinary laboratories agency. sera from 18 cattle were collected from two herds; 8 were from mixed sexes of 6-month-old cattle at the institute for animal health, compton, berkshire, uk in 1990 and 10 were from yearling female cattle collected in 1993 from wiltshire, uk, kindly provided by dr. ian thompson, royal veterinary college, uk. amplification and sequence analysis of the bo/newbury1/ 1976/uk genome rna extraction and reverse transcription were performed as described previously (oliver et al., 2003) . reverse transcription was performed at 42°c using 200 iu of moloney murine leukemia virus (h-) reverse transcriptase (promega). the pcr primers used to initially amplify newbury1 were those designed to anneal with viruses in the family caliciviridae bocvpol001 (5′-atcaaggagcacg-tggtggag-3′)/ygdd , 290/289 (jiang et al., 1999b) , noro(f)/noro(r) (smiley et al., 2003) or the nb-like viruses nbu(f)/nb(r) (smiley et al., 2003) . to generate amplicons that spanned the entire newbury1 genome, pcr was performed with hotstar taq (qiagen) using primers partly based on newbury1 sequence and the nb virus (smiley et al., 2002) that overlapped the entire length of the newbury1 genome. an initial denaturation at 95°c for 15 min, then, dependant on the primer pairs, 40 cycles of 95°c for 30 s, 50°c to 55°c for 30 to 60 s and 72°c for 1 to 2 min were performed, with final extension at 72°c for 10 min. amplicons from 0.2 to 1.7-kbp in length were generated and gel purified using a qiaquick gel extraction kit (qiagen). purified amplicons were cloned using a topo-ta pcr2.1 cloning kit (invitrogen ltd.). nucleotide sequencing of 3 to 5 clones for each pcr amplicon from newbury1 was performed by mrcgeneservice, hinxton, uk. the consensus nucleotide sequence of newbury1 was prepared using the staden sequencing package (staden et al., 2000) . orfs were predicted using the ncbi orf finder (http://www.ncbi.nlm. nih.gov/gorf/gorf.html). the gc content of the newbury1 genome was calculated, the molecular masses and isoelectric points of proteins were predicted and hydropathy plots were generated using the bioinformatics software jemboss (carver and bleasby, 2003) . to reduce computation time, 17 viruses from the family caliciviridae (abbreviation, if used; accession number for the complete genome; reference, if available) were used: lagovirus, european brown hare syndrome virus strain gd (ebhsv-gd; nc_002615; le gall et al., 1996) and rabbit hemorrhagic disease virus strain frg (rhdv-frg; m67473; meyers et al., 1991) ; vesivirus, feline calicivirus strain urbana (fcv-urbana; nc_001481; sosnovtsev and green, 1995) , feline calicivirus strain cfi/68 (fcv-cfi/68; u13992), vesicular exanthema of swine virus serotype a48 (vesv-a48; u76874; neill et al., 1998) , san miguel sea lion virus serotype 1 (smsv; u15301; neill and meyer, 1995) , canine calicivirus (ccv; nc_004542); sapovirus, manchester (x86560; dingle et al., 1995) , dresden (nc_006269) and porcine enteric calicivirus (pec; nc_000940; guo et al., 1999) ; norovirus, norwalk (nc_001959; jiang et al., 1993) , southampton (l07418; lambden et al., 1993) , snow mountain virus (smv; ay134748; lochridge and hardy, 2003) , lordsdale (x86557; liu et al., 1995) , newbury2 (af097917; oliver et al., 2003) and jena (aj011099; liu et al., 1999) ; and the unclassified viruses, nebraska virus (nb; nc_004064; smiley et al., 2002) and newbury1 (dq013304). multiple alignments of translated amino acid sequences were prepared for the complete orf1 (excluding the capsid gene), 2c-helicase, 3cprotease, 3d-polymerase and capsid proteins using clustal x version 1.8 (thompson et al., 1997) . multiple alignments of the nucleotide sequences for these proteins, which were generated using tranalign of the bioinformatics software jemboss, were based on the multiple amino acid alignments. in order to retain the maximum number of conserved evolutionary sites, all columns that contained gaps were removed, then identities were calculated using genedoc (www.psc.edu/biomed/genedoc). phylogenetic analyses were performed with multiple nucleotide and amino acid alignments generated for the 2chelicase, 3c-protease, 3d-polymerase and the complete capsid genes. the nh 2 -terminal, 3a-b and orf3 genes were not used due to their extensive diversity between the caliciviruses. phylip (j. felsenstein, department of genetics, university of washington, seattle; phylogeny inference package, version 3.5c) was used for parsimony (dnapars and protpars), upgma, fitch-margoliash, maximum likelihood and bootstrap analyses. treepuzzle 5.2 was used for additional maximum likelihood with quartet puzzling analyses (schmidt et al., 2002) . in addition, phylogenetic analyses were performed using amino acid alignments generated from homology models of the polymerase and the s-domain of the capsid protein. this was done to compensate for the high diversity between newbury1 and the 4 caliciviridae genera; amino acid alignments were generated based on the 3dstructure of the polymerase and the s-domain of the capsid. homology models of the polymerase and s-domain of the capsid were generated using the swiss-model server (http:// swissmodel.expasy.org//swiss-model.html). amino acids sequences for the 17 caliciviruses plus newbury1 were aligned by eye using the homology model alignments for the conserved regions of the polymerase and the s-domain of the capsid protein. phylogenetic trees were prepared by using treeview (page, 1996) and edited for clarity with microsoft word. to determine the evolutionary divergence of the calicivirus genome sequences, maximum likelihood distances were calculated using treepuzzle 5.2. negative stain electron microscopy newbury1 and newbury2 virus particles were partially purified from feces by differential centrifugation and examined by electron microscopy after staining with 2% potassium phosphotungstate ph 6.0 as described previously . the antigenic relationship between newbury1 and new-bury2 was determined by elisa as described previously (s.l. oliver, e. asobayire, a. charpilliene, j. cohen and j.c. bridger, submitted for publication) using sera from bridger et al. (1984) . spiem was used to detect newbury1 antibody as described previously with some modifications (dastjerdi et al., 2000) . all incubations were performed in humidified chambers for 1 h at 37°c. formvar, carbon-coated electron microscope grids were incubated with 10 μg/ml protein-g (sigma, uk), washed 6 times with ultra pure h 2 o, then incubated with 1:10 dilution of test serum. grids were washed again, incubated with 10 μl volume of fecal suspensions containing newbury1 then negatively stained with 2% potassium phosphotungstate ph 6.0 (pta; agar scientific, uk). grids were examined with a jeol 1200ex electron microscope at a magnification of 50,000. serum taken 39 days post-inoculation with newbury1 was used as a positive control and trapped an average of 225.6 ± 23.6 newbury1 particles per field. the newbury1 serum was free from antibodies to bovine astrovirus, the uk strain of bovine rotavirus, bovine enteric coronavirus, bovine viral diarrhea virus, feline calicivirus and the haden strain of bovine parvovirus (j.c. bridger personal observation). a hyperimmune serum to rotavirus uk was used as a negative control and trapped 0.48 ± 0.85 particles per field. test sera that trapped more than 2.2 particles per field (the mean number of newbury1 particles per field trapped by the negative control serum +2 standard deviations) were considered positive for newbury1 antibody. detection by rt-pcr of newbury1-like viruses in the feces of uk diarrheic calves rna extraction and reverse transcription were performed as described previously (oliver et al., 2003) . pcr was performed with hotstar taq (qiagen) using primers nbu(f)/ na1orf1_07 (5′-tgg cag act cac atg ttg gat gc-3′), which amplified a 509 bp region (nucleotides 4519 to 5026 of the newbury1 orf1 gene) of the polymerase gene. a fecal sample from calf 1424 2 days post-inoculation was used as a positive control and h 2 o was used as a negative control for rna extraction and reverse transcription. cdna generated from fecal samples that produced an amplicon of the expected size for the primer pair nbu(f)/na1orf1_07, was used to generate amplicons with primer pair na1orf1_ 12 (5′-ccagatattctctagaagaagc-3′)/na1orf1_29 (5′-ttgacactctccgggtccacg-3′). na1orf1_12/ na1orf1_29 amplified a 522 bp region that spanned the 3′ end of the polymerase and the 5′ end of the capsid genes (nucleotides 4954 to 5474 of the newbury1 orf1 gene). thermocycling conditions were an initial denaturation at 95°c for 15 min followed by 40 cycles of 94°c for 45 s (nbu (f)/na1orf1_07) or 1 min (na1orf1_12/na1orf1_29), 54°c for 45 s and 72°c for 1 min, with a final extension at 72°c for 10 min. cdna generated from newbury1 was used as a positive control and h 2 o as a negative control for pcr. amplicons were cloned, sequenced and compared to newbury1 as described previously. calf enteritis-new information from nadis. uk vet 5 characterization of a calici-like virus (newbury agent) found in association with astrovirus in bovine diarrhea norovirus recombination in orf1/orf2 overlap comparative genomics of foot-and-mouth disease virus the design of jemboss: a graphical user interface to emboss the bovine newbury agent-2 is genetically more closely related to human srsvs than to animal caliciviruses characterisation of the bovine enteric calici-like virus, newbury agent 1 human enteric caliciviridae: the complete genome sequence and expression of virus-like particles from a genetic group ii small round structured virus sequence analysis of a porcine enterovirus serotype 1 isolate: relationships with other picornaviruses nucleotide sequence of uk and australian isolates of feline calicivirus (fcv) and phylogenetic analysis of fcvs polymerase chain reaction detection of small round-structured viruses from two related hospital outbreaks of gastroenteritis using inosine-containing primers taxonomy of the caliciviruses human caliciviruses molecular characterization of a porcine enteric calicivirus genetically related to sapporo-like human caliciviruses lesions of gnotobiotic calves experimentally infected with a calicivirus-like (newbury) agent genetic recombination between two genotypes of genogroup iii bovine noroviruses (bonvs) and capsid sequence diversity among bonvs and nebraska-like bovine enteric caliciviruses sequence and genomic organization of norwalk virus characterization of a novel human calicivirus that may be a naturally occurring recombinant design and evaluation of a primer pair that detects both norwalk-and sapporo-like caliciviruses by rt-pcr coexistence of multiple genotypes, including newly identified genotypes, in outbreaks of gastroenteritis due to norovirus in japan phylogenetic analysis of the complete genome of 18 norwalk-like viruses sequence and genome organization of a human small round-structured (norwalk-like) virus european brown hare syndrome virus: molecular cloning and sequencing of the genome human enteric caliciviruses have a unique genome structure and are distinct from the norwalk-like viruses molecular characterization of a bovine enteric calicivirus: relationship to the norwalk-like viruses snow mountain virus genome sequence and virus-like particle assembly astroviruses expression and processing of the canine calicivirus capsid precursor complete nucleotide sequence, genome organization and phylogenic analysis of the canine calicivirus rabbit hemorrhagic disease virusmolecular cloning and nucleotide sequencing of a calicivirus genome genetic relatedness of the caliciviruses: san miguel sea lion and vesicular exanthema of swine viruses constitute a single genotype within the caliciviridae the capsid protein of vesicular exanthema of swine virus serotype a48: relationship to the capsid protein of other animal caliciviruses molecular epidemiology and phylogenetic analysis of sapporo-like viruses molecular characterization of bovine enteric caliciviruses: a distinct third genogroup of noroviruses (norwalk-like viruses) unlikely to be of risk to humans a chimeric bovine enteric calicivirus: evidence for genomic recombination in genogroup iii of the norovirus genus of the caliciviridae treeview: an application to display phylogenetic trees on personal computers x-ray crystallographic structure of the norwalk virus capsid hepatitis e virus picornaviridae: the viruses and their replication microbiology of calf diarrhoea in southern britain tree-puzzle: maximum likelihood phylogenetic analysis using quartets and parallel computing human rhinovirus 2: complete nucleotide sequence and proteolytic processing signals in the capsid protein region characterization of an enteropathogenic bovine calicivirus representing a potentially new calicivirus genus reverse transcription-pcr assays for detection of bovine enteric caliciviruses (bec) and analysis of the genetic relationships among bec and human caliciviruses aetiology of diarrhoea in young calves rna transcripts derived from a cloned fulllength copy of the feline calicivirus genome do not require vpg for infectivity cleavage of the feline calicivirus capsid precursor is mediated by a virus-encoded proteinase the staden package the costs of bovine enteritis in suckled calves the clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools molecular characterization of noroviruses detected in diarrheic stools of michigan and wisconsin dairy calves: circulation of two distinct subgroups isolation of small viruses resembling astroviruses and caliciviruses from acute enteritis of calves equine rhinovirus serotypes 1 and 2: relationship to each other and to aphthoviruses and cardioviruses key: cord-253351-b36g09r0 authors: luo, zongli; weiss, susan r. title: roles in cell-to-cell fusion of two conserved hydrophobic regions in the murine coronavirus spike protein date: 1998-05-10 journal: virology doi: 10.1006/viro.1998.9121 sha: doc_id: 253351 cord_uid: b36g09r0 abstract the spike (s) protein of coronavirus, mouse hepatitis virus (mhv), mediates attachment and fusion during viral entry and cell-to-cell fusion later in infection. by analogy with other viral proteins that induce cell fusion the mhv s protein would be expected to have a hydrophobic stretch of amino acids that serves as a fusion peptide. sequence analysis suggests that the s protein falls within the group of fusion proteins having internal rather than n-terminal fusion peptides. based on the features of known viral fusion peptides, we identified two regions (pep1 and pep2) of mhv-a59 s2 as possible fusion peptides. site-directed mutagenesis and anin vitrocell-to-cell fusion assay were used to evaluate the roles of pep1 and pep2, as well as a third previously identified putative fusion domain (pep3) in membrane fusion. substitution of bulky hydrophobic residues with charged residues within pep1 affects the fusion activity of the s protein without affecting processing and surface expression. similar substitutions within pep2 result in a fusion-negative phenotype; however, these mutant s proteins also exhibit defects in protein processing and surface expression which likely explain the loss of the ability to induce fusion. thus pep1 remains a candidate fusion peptide, while pep2 may play a significant role in the overall structure or oligomerization of the s protein. pep3 is an unlikely putative fusion peptide since it is not conserved among coronaviruses and nonconservative amino acid substitutions in pep3 have minimal effects on cell-to-cell fusion. mouse hepatitis virus strain a59 (mhv-a59) is a murine coronavirus with a positive-stranded rna genome of approximately 31 kb (siddell et al., 1983) . the coronavirus spike (s) protein forms the peplomer structure on the viral envelope; each spike is thought to be a dimer or trimer of s (cavanagh, 1983) . the s protein mediates binding of virions to the host cell receptor (collins et al., 1982) , virus cell fusion during entry, and cell-to-cell fusion at later times postinfection (vennema et al., 1990) . the mhv s protein is cotranslationally glycosylated to a 150-kda form, which is later processed to a 180-kda form during intracellular maturation (spaan et al., 1988) . the 180-kda mature form is cleaved in the golgi apparatus, by a host cell protease, into two similarly sized subunits: amino terminal s1 and carboxy terminal s2 (frana et al., 1985; luytjes et al., 1987; . it is believed that the s1 subunit forms the globular head of the spike, whereas the s2 subunit forms the membranebound stalk portion (de groot et al., 1987a) . sequence analysis suggests that the coronavirus spike protein has the structural features of a type i membrane protein (spaan et al., 1988) , including a transmembrane domain near the carboxy terminus of s2 and a hydrophobic signal peptide at the n-terminus of s1. other structural motifs include two heptad repeat domains in s2, the shorter of which is adjacent to the transmembrane domain and is a leucine zipper motif (britton, 1991) . the s1 subunit is believed to interact with receptor (cavanagh et al., 1986; taguchi, 1995) . a receptor binding activity has been demonstrated using a recombinant protein containing the amino terminal 330 residues of the s1 subunit of mhv-jhm (kubo et al., 1994) . recombinant s protein, expressed in tissue culture using a vaccinia virus-based expression system, is capable of inducing cell-to-cell fusion (bos et al., 1995; de groot et al., 1989) , demonstrating that the s protein alone is sufficient for the induction of cell-to-cell fusion. in spite of the important role played by the s protein in viral entry and cell-to-cell fusion, little is known about the fusion domain which is directly responsible for the fusion event. a common feature of viral fusion proteins is the presence of a fusion peptide, which is believed to participate directly in the fusion process (white, 1992) . fusion peptides are typically composed of 16 to 26 amino acid residues and conserved within, but only rarely among, virus families. they are relatively hydrophobic and generally show an asymmetric distribution of hydrophobicity when modeled into an ␣ helix; they are also rich in alanine and glycine. the majority of known fusion peptides are found at the n-terminus of the membraneanchored subunit of viral fusion proteins that undergo proteolytic cleavage during their maturation (white, 1990) . the cleavage is believed to be necessary in order to expose the peptide itself to facilitate the fusion process. however, not all fusion proteins undergo proteolytic cleavage and there are examples of fusion peptides that exist internally in the membrane-anchored subunit (white, 1990) . besides having the above common features, these internal fusion peptides are bounded by charged residues on both ends and may contain a proline residue in the center. although the mhv s protein is cleaved during processing, the n-terminus of the membrane-anchored s2 subunit does not contain a hydrophobic, conserved region. moreover, not all coronavirus s proteins (for example, feline infectious bronchitis virus and transmissible gastroenteritis virus) undergo cleavage during maturation (cavanagh, 1995) . thus it is likely that the coronavirus s protein has an internal fusion peptide. using the properties common to other fusion peptides (discussed above), we have detected two fusion peptide-like regions, pep1 and pep2, in the s2 subunit of the mhv-a59 s protein (fig. 1) . we performed mutational analysis of these two regions as well as of a third peptide (pep3) that was previously proposed as a possible fusion domain (chambers et al., 1990) . the effects of amino acid substitutions within these regions on sinduced cell-to-cell fusion, protein processing, and cell surface expression were examined. the data suggest that mutations in both pep1 and pep2 have a dramatic effect on the ability of s to induce cell-to-cell fusion. while mutations in pep1 have little effect on processing of the s protein, mutations within pep2 result in the loss of the ability of s to be processed and transported to the plasma membrane. therefore pep1 remains a likely candidate fusion peptide, whereas pep2 may play a role in maintaining the overall structure of s or oligomerization. pep3 appears unlikely to be a fusion peptide candidate as amino acid substitutions within this peptide have little effect on fusion. using known characteristics of fusion peptides, including sequence conservation, hydrophobicity, and the ability to be modeled as a sided ␣ helix, we identified two putative fusion peptides, pep1 and pep2, in the s2 subunit of the mhv-a59 s protein. their positions within the s2 subunit are shown in fig. 1a along with identified functional domains such as the two heptad repeats and the transmembrane domain. pep1 is located within the longer heptad repeat and pep2 is located between the two heptad repeats. a third candidate fusion domain, pep3, previously identified based on its hydrophobicity and its proximity to the heptad repeats (chambers et al., 1990) , is also shown. the mhv-a59 s protein sequence was aligned with those from other coronaviruses using the computer program clustal v (higgins et al., 1992) . regions corresponding to pep1, pep2, and pep3 are shown in fig. 1b . since coronaviruses are divided into three antigenic groups (cavanagh, 1995) , two representative coronaviruses from groups i (fipv, tgev) and ii (mhv, bcv) were shown as well as ibv, which is the sole member of group iii. pep1 shows several of the properties of known fusion peptides. it is conserved among coronaviruses and bordered by charged or polar residues. most of the unmatched amino acid residues in pep1 are conservative substitutions. besides sequence conservation, modeling pep1 into an ␣ helix shows a clear asymmetric distribution of bulky hydrophobic residues ( fig. 2a) . analysis of the hydrophobicity showed that pep1 has an overall hydrophobicity index (h.i.) of 0.73. the h.i. for the hydrophobic side is 1.7, while the h.i. for the hydrophilic side is ϫ0.5. pep1 is rich in alanine and glycine residues (35%). pep2 also has some features typical of fusion peptides in addition to sequence conservation. for example, pep2 is bordered by charged residues and showed an asymmetric distribution of hydrophobicity when modeled into an ␣ helix (fig. 2b) . the h.i.s for the hydrophobic and hydrophilic sides are 0.82 and ϫ0.05, respectively. while pep2 does not have alanine and glycine residues, it contains a central proline residue, another typical feature of internal fusion peptides (white, 1990) . alignment of pep3 indicates that this region is less conserved than pep1 and pep2 (fig. 1b) among the three antigenic groups of coronaviruses. furthermore, it is not bordered by charged residues and not predicted to form an ␣ helix with an asymmetric distribution of bulky hydrophobic residues. quantitative cell-to-cell fusion assay for the mhv s protein using the quantitative fusion assay described in detail under materials and methods, we examined the effects of amino acid substitutions on the membrane fusion activity of the s protein. the s gene was expressed in murine dbt cells by infection with vaccinia virus vtf7-3 to supply the t7 rna polymerase, followed by transfection with a plasmid containing the s gene downstream of a t7 rna polymerase promoter. another group of dbt cells was transfected with a plasmid containing the escherichia coli lacz gene, also downstream of the t7 rna polymerase promoter. only when cell fusion occurs between a cell expressing the s gene and a cell containing the lacz plasmid is the lacz gene transcribed and ␤-galactosidase subsequently produced. syncytia were observed and ␤-galactosidase activities were measured either by an in situ assay in which syncytia were stained blue in the presence of 5-bromo-4-chloro-3-indolyl-␤-dgalactopyranoside (x-gal) or by a quantitative assay using cprg (chlorophenol red-␤-d-galactopyranoside) as the substrate (see materials and methods). such an assay is illustrated in fig. 3 . in cells infected with vtf7-3 and mock transfected, only tiny blue background stains were detected (fig. 3a) . however, in cells infected with vtf7-3 and transfected with plasmid containing the wildtype s gene, fusion of donor cells and the surrounding recipient cells were observed and the resulting syncytia stained blue in the presence of x-gal (fig. 3b ). the size of the blue stains was equivalent to the size of the induced syncytia (fig. 3b ). the mutant s protein a974d-a976d (described further below) failed to induce fusion, resulting in a fusion phenotype similar to that of mock-transfected cells (fig. 3c) . the fusion-positive mutant s protein s975d (described further below) exhibited a phenotype similar to that of the wild-type s protein (fig. 3d ). the levels of the ␤-galactosidase activities for the parallel samples were also quantitated to demonstrate that the in situ assay reflects the levels of fusion (fig. 3e) . values indicating the level of fusion were expressed as the percentage of wild-type ␤-gal activity after subtracting the background value of mock-transfected cells. the (luytjes et al., 1987) , bcv strain f15 (boireau et al., 1990) , ibv strain m41 (binns et al., 1986) , fipv strain 79-1146 (de groot et al., 1987b) , and tgev strain tfi (chen et al., 1993) were aligned by the computer program clustal v. the regions corresponding to pep1, pep2, and pep3 are shown. their corresponding positions in the primary sequence are shown in brackets. at the bottom of each alignment, asterisks are used to indicate positions with identical amino acid residues, and a dot for positions with conservative amino acid changes. the positions with nonconservative changes are not indicated by any symbols. average size of syncytia usually corresponded to the ␤-gal percentage values. a percentage of 0 to 10% indicated a fusion-negative phenotype, while values over 70% of the wild type indicated a fusion phenotype similar to that of the wild type. values between 10 and 70% indicated intermediate fusion phenotypes. to determine the importance of the asymmetric distribution of hydrophobicity in the induction of cell-to-cell fusion, as predicted by the ability to model this peptide as a sided ␣ helix ( fig. 2a) , nonconservative and conservative amino acid substitutions were introduced in pep1 to target representative bulky hydrophobic residues (f977k, f977l, l981k, l981i) and polar or charged residues (s975d, n978d, n978l, d986v-d989v). the importance of the presence of small amino acid residues, such as alanine or glycine, in the mechanism of fusion was tested by substituting them with charged residues (a974d, a976d), bulky hydrophobic residues (a976v), or both at two different positions (a974d-a976d, a974d-a974v). since viral fusion proteins undergo complex posttranslational processing before arriving at the cell surface, it is possible that any alteration of the fusogenic ability of the s protein may be caused by defects in protein processing and/or transport rather than to the fusion process itself. proteins that are defective in processing, for example either misfolded or misassembled, are often retained in the er and thus not expressed on the cell surface (doms et al., 1993) . therefore, the level of surface expression is a good indicator as to whether mutant proteins are generally folded correctly. we examined the surface expression of the above pep1 mutant proteins by flow cytometry analysis to determine whether they were indeed processed and transported to the cell surface. as shown in fig. 4a , the flow cytometry histogram of the pep1 mutant a974d-a976d is similar to that of the wildtype s protein. the histograms of all the other pep1 mutants were similar as well (data not shown). quantitation of the levels of surface expression (table 1) indicates that all of the pep1 mutant s proteins were expressed on the cell surface at levels similar to that of the wild-type s protein, suggesting that their overall conformation was not altered. thus any alteration of fusion caused by these pep1 mutations is not likely due to overall conformational changes, but rather likely attributed to a direct influence on the fusion process. all mutant pep1 s proteins were assayed for fusion using both in situ and quantitative fusion assays, performed as shown in fig. 3 , using the vtf7-3-infected and wild-type s gene-transfected cells as positive controls and vtf7-3-infected and mock-transfected cells as negative controls. the results were summarized in table 1 . substitution of hydrophobic residues with charged residues generated the fusion negative phenotype. both f977k and l981k mutations reduced the ␤-galactosidase activity to the background level. however, replacement of the same residues with other hydrophobic amino acids (f977l, l981i) did not reduce fusion. these results suggest that maintenance of the hydrophobicity at the nonpolar side is important for fusion. substitution of polar or charged residues on the hydrophilic side of the predicted sided helix had less impact on fusion (table 1, pep1) . increasing the hydrophobicity on the polar side (n978l, d986v-d989v) resulted in a partially impaired fusion phenotype. increasing the hydrophilicity by substituting serine at position 975 with an aspartic acid residue (s975d) did not affect fusion. however, this was not true for the n978d mutant, which failed to maintain the wild-type fusion phenotype. unlike the serine residue at position 975, this asn residue is conserved among all coronavirus spike proteins (fig. 1b) . substitutions of ala48 nd a reported as percentage of ␤-galactoside produced in samples using wild-type s protein (see materials and methods). all data are averages of triplicates from one experiment. the experiments were repeated five times with a standard deviation of less than 25%. b reported as the percentage of the mean fluorescent intensity values measured for samples expressing the wild-type s protein after subtracting background values obtained for mock-transfected samples. experiments were repeated twice with a standard deviation of less than 20%. nd, not determined. nine residues with either a charged residue (a974d or a976d) or a bulky hydrophobic residue (a976v) resulted in a partially reduced fusion phenotype regardless of hydrophobicity. double mutants a974d-a976d and a974d-a976v both displayed a fusion-negative phenotype, suggesting that the effects of the separate mutation may be additive in the double mutants. the reduced fusion level in a976v mutant s proteins suggests that the alanine residue is required not merely for hydrophobicity. the reduction in fusion observed with the n978d mutant also suggests that conservation at some residues is more of a factor in fusion than hydrophilicity alone. pep2 contains a high percentage of bulky hydrophobic residues and may also be modeled as a sided helix (fig. 2b ). thus we determined the effect on fusion of substitution of hydrophobic residues on the nonpolar face with the charged lysine residue. all such mutant s proteins appear to have lost the ability to induce fusion (table 1) . consistent with this, no syncytia were observed in the in situ fusion assay. we examined the cell surface expression of pep2 spike mutants using flow cytometry analysis. figure 4b demonstrates that, for one representative pep2 mutant, spike protein is not expressed on the cell surface. similar results were obtained for all the pep2 mutants (data not shown); these results are summarized in table 1 . thus, pep2 mutant s proteins differed from the pep1 mutant proteins in that the former were not transported to the cell surface. therefore, the fusion-negative phenotype of these pep2 mutants is likely to be an indirect effect of overall conformational changes which prevents transport and expression on the cell surface (table 1) . to investigate whether the pep2 mutants were defective in protein transport, they were assayed for endoglycosidase h (endo h) resistance (fig. 5) . during transport from the endoplasmic reticulum (er) to the golgi apparatus, glycoproteins are modified by acquisition of oligosaccharides that are resistant to endo h digestion (kornfeld and kornfeld, 1985) . proteins that are not processed properly usually fail to gain this resistance. cells transfected with the plasmids expressing wild-type and pep2 mutant proteins were labeled with [ 35 s]methionine and cysteine for 1 h and then lysed or labeled and chased with an excess of unlabeled methionine and cysteine before lysis. proteins were immunoprecipitated with the ao4 anti-s sera, treated with endo h, and analyzed by sds±page. endo h resistance was not detectable after 1 h labeling of either wild-type or mutant proteins (fig. 5a) . however, after a 2-h chase, a portion of the wild-type s proteins were processed into an endo h-resistant form (fig. 5b) . these results are consistent with the observation that the processing of s expressed using a vaccinia expression system has a half-time of approximately 3 h (vennema et al., 1990) . interestingly, the fraction of the wild-type s protein that became endo h resistant is not particularly high. this is similar to the observation made in another study of the mhv-a59 s protein (bos et al., 1995) . endo h-resistant forms of the pep2 mutants were not detected even after a 2-h chase, indicating that these mutant proteins were unable to be transported from the er to the medial golgi complex. in contrast, an endo h-resistant form of the fusion-negative pep1 mutant a974d-a976d was detected after a 2-h chase. this protein was also expressed on the cell surface as described above (table 1 ), suggesting that correct processing of s correlates with its surface expression. (fig. 1) was previously proposed as a possible fusion domain based on its hydrophobicity and location adjacent to the heptad repeat domains (chambers et al., 1990) . however, this region is less conserved than pep1 and pep2. we performed a limited amount of mutagenesis within this region to determine the effects on fusion. substitution of the methionine residue at position 936 with lysine (m936k) or leucine (m936l) did not effect fusion. however, while substitution of the proline residue at position 938 with lysine (p938k) partially impaired fusion, replacing the same proline residue with a leucine residue did not have any effect on fusion. the results suggest that pep3 is unlikely to be a putative fusion peptide. although studies of the coronavirus s protein reveal that s alone is sufficient to induce cell-to-cell fusion in the absence of other coronavirus proteins, little is known about the fusion peptide domain of the s protein. using the characteristics of known fusion peptides including the observation that they may be modeled as ␣ helices with an asymmetric distribution of hydrophobicity, we identified two conserved hydrophobic regions, pep1 and pep2, in the mhv-a59 s protein s2 subunit, as candidate fusion peptides. site-directed mutational analysis was used to examine the significance of individual amino acid residues within each of these peptides in cell-to-cell fig. 4 . flow cytometry analysis of surface expression of wild-type and mutant s proteins. bhk-21 cells were infected with vtf7-3 and transfected with plasmids containing wild-type or mutant s genes. four hours after transfection, cells were subject to flow cytometry analysis for surface expression of s proteins (see materials and methods). fluorescent histograms of a pep1 mutant (a974d-a976d) and a pep2 mutant (p1107k) are shown side by side with the corresponding wild-type controls. two wild-type controls were shown because a and b represent different experiments. the histogram obtained from a mock-transfected negative control (shown in gray lines) was embedded in each histogram. the x axis indicates the arbitrary fluorescent intensity values shown in log scale. the y axis indicates the number of cells. the mean fluorescent intensity values (mfiv) were calculated and the surface expression of each mutant relative to wild type was determined using the following formula: (mfiv sample ϫ mfiv mock )/ mfiv wild type ϫ mfiv mock ) ϫ 100. fusion. a third previously identified peptide was also examined as a possible fusion domain. the mutagenesis results indicate that pep1 remains the most likely candidate fusion peptide. all pep1 mutants we analyzed, including the ones that displayed a fusion-negative phenotype, were expressed on the cell surface at a level similar to that of the wild-type protein. in contrast, the group of pep2 mutants were defective in a step in processing or intracellular transport; thus the negative fusion phenotype was likely due to the lack of expression on the cell surface rather than to a direct effect on the ability to induce cell-to-cell fusion. mutations in pep3 had less effect on the fusogenic ability of s; this along with the fact that this domain is not well conserved among coronaviruses makes it an unlikely candidate fusion peptide. a current hypothesis on how viral fusion proteins induce membrane fusion is that fusion involves a conformational change of fusion proteins which is triggered by low ph or another factor(s) (white, 1992) . the altered conformational change allows the hydrophobic fusion peptide to interact with the target membrane. the fusion peptide is postulated to act as a sided insertional helix. the hydrophobic face of the helix inserts into the membrane with an oblique orientation, leading to the disruption of the membrane structure (harter et al., 1989; white, 1990 white, , 1992 . alteration of this hydrophobic face would affect this interaction. the perturbation of the hydrophobic face in pep1 by substitution of bulky hydrophobic residues with charged ones may prohibit the insertion, thus abrogating the fusogenic activity. other less drastic amino acid substitutions may result in less severe obstruction of this type of interaction, thus having less drastic effect on fusion. pep1 is located within the longer of the two heptad repeat regions in s2. since heptad repeat motifs are typical of regions that form ␣ helical coiled coil structures (cohen and parry, 1986) , it is likely that pep1 adopts an ␣ helical conformation as part of this coiled coil structure. pep1 is distinguished from the rest of the heptad repeat region in that it possesses several features common to known viral fusion peptides. those include sequence conservation among coronaviruses, hydrophobicity, richness in alanine and glycine residues, and the ability to be modeled into a sided helix and bounded by charged residues. studies of heptad repeat regions in many viral fusion proteins have revealed that they are also essential for membrane fusion (buckland et al., 1992; dubay et al., 1992; sergel-germano et al., 1994) . although fusion peptides are generally found adjacent to heptad repeats but not within the heptad repeat, there is no indication that fusion peptides cannot reside in a heptad repeat. it is postulated that heptad repeats may also be able to insert into membranes to help elicit fusion because of the amphipathic nature of their helices (segrest et al., 1992; sergel-germano et al., 1994) . synthetic peptides representing part of the influenza virus ha heptad repeat region have been shown to insert reversibly into phospholipid vesicles under endosomal ph conditions (yu et al., 1994) . amphipathic ␣ helices of a protein molecule may associate with membranes spontaneously (degrado, 1993; segrest et al., 1990) . melittin, an amphipathic helical peptide, is capable of inducing membrane fusion due to a local disruption of the lipid bilayer (dempsey, 1990) . thus, fusion peptides and heptad repeats may be indispensable parts of an integrated fusion machinery whereby fusion peptides may or may not be located independently from heptad repeats. pep1 has 35% alanine and glycine residues. although substitution of one alanine residue by another charged one (a976d) reduced fusion, substitution with a more hydrophobic valine residue (a976v) also reduced fusion, suggesting that alanine residues may have roles in fusion other than the maintenance of hydrophobicity. replacement of glycine residues by valine residues in the hiv gp41 fusion peptide either abrogated or reduced fusion (delahunty et al., 1996) . it is postulated that the presence of small glycine/alanine residues in the fusion peptide provide the right balance of amphipathicity necessary in mediating fusion for influenza virus (white, fig. 5 . endoglycosidase h analysis of the pep2 mutants. cells expressing the wild-type or the pep2 mutant s proteins were metabolically labeled for 1 h with 35 s express labeling mix and either analyzed directly (a) or incubated further for 2 h in the presence of excess methionine and cysteine (b). cells were lysed and s proteins were immunoprecipitated using anti-s ao4 serum. half of each sample was digested with endo h f (ϩ) or incubated without the enzyme (ϫ). wildtype s gene (lanes 1, 2), l1102k mutant (lanes 3, 4), v1103k mutant (lanes 5, 6), p1107k mutant (lanes 7, 8), l1110k mutant (lanes 9, 10), pep1 mutant a974d±a976d (lanes 11, 12). the little black spot above the endo h-sensitive spike band in lane 6 was caused by a slight gel crack. 1992). furthermore, studies on the siv gp32 fusion peptide suggest that the presence of these residues may be important for oblique insertion of the fusion peptide into the target membrane (horth et al., 1991) . substitution of bulky hydrophobic residues with charged lysine residues in all constructed pep2 mutants abolished the fusogenic ability of s. however, this is likely due to a defect in processing or transport to the cell membrane rather than to a direct effect on the fusion process. the failure to detect an endo h-resistant form of s for the pep2 mutants (fig. 5) suggests that this region plays a role in the maturation of s. for example, mutations in pep2 may cause individual molecules to be misfolded, leading them to form aggregates even before appropriate oligomerization occurs. such aggregates are normally retained in the er (marquardt and helenius, 1992) . consistent with our data are studies in which gallagher (1996) demonstrated that membrane fusion induced by the mhv-jhm s protein was inhibited by modification of cys-1163 and suggested that this was likely due to dramatic changes in s protein structure. sequence alignment of mhv-jhm and mhv-a59 indicates that this cysteine residue corresponds to a tyrosine residue in mhv-a59, which is located in the pep2 region. furthermore, studies of two temperature-sensitive mhv-a59 mutants in which the endo h-resistant form of s was not detectable at nonpermissive temperatures was caused by the lack of oligomerization (luytjes et al., 1997) , albeit the location of these mutations is not known. appropriate oligomerization of many other viral proteins is required for them to be transported from er to golgi, where endo h resistance is obtained (doms et al., 1993) . we have analyzed the roles of three hydrophobic regions in the mhv-a59 s protein in s-induced cell-to-cell fusion. the results of the mutational analysis indicate that pep1 is likely to be directly involved in fusion, while pep2 may play a role in maintenance of structure and/or processing of s. although this study provides insights into identifying the structural elements of s involved in fusion, the molecular details of the fusion mechanism remains unclear. mutational analysis of pep1 and pep2 indicates only that these regions are necessary for the s protein to assume a fusion competent conformation. whether either of these two regions is a fusogenic peptide needs to be determined by biophysical studies using liposomes. similar studies have been conducted on fusion peptides for other proteins such as hiv gp41 (nieva et al., 1994) , influenza virus ha2 (luneberg et al., 1995) , and sperm surface protein ph-30 . such biophysical studies of the s protein, together with the elucidation of the three-dimensional structure of s, would greatly help the understanding of the fusion mechanism. plasmid pint2 contains a cdna copy of the entire wild-type mhv-a59 s gene, cloned into the saci and bamh i sites of pbluescript ii (ks ϩ ) (stratagene), the expression of which is under the control of the t7 rna polymerase promoter. the hindiii site of the s gene in pint2 was modified to an asei site by the introduction of silent mutations in codons 173 and 174. the mutant s plasmid clones were generated by oligonucleotide-directed pcr mutagenesis (ausubel et al., 1989) , using pint2 as the template. for the pep1 or pep3 region, desired nucleotide changes were introduced into a pcramplified fragment using the 5ј flanking primer wz157 (5ј-aacactgcatgcaggcag-3ј), the 3ј flanking primer wz175 (5ј-attaatacgcgtggtttggc-3ј), and the corresponding mutagenic primers listed in table 2 . the pcr fragments were digested with bbsi and mlui and cloned into the corresponding sites of pint2. for the pep2 mutants, primers wz115 (5ј-agcaaaagcccagataga-3ј) and wz11 (5ј-gggggatccaggtagc-3ј) were used as the 5ј and 3ј flanking primers, respectively, along with the corresponding mutagenic primers listed in table 2 . the resulting mutant plasmids were generated by subcloning the mlui±ndei-digested mutant pcr fragments into the corresponding sites of pint2. the presence of specific mutations in all constructs was verified by dna sequencing. spike-induced cell-to-cell fusion was examined by using expression of the e. coli lacz gene to monitor the level of fusion, in an assay adapted from nussbaum et al. (1994) . briefly, one group of dbt cells was seeded onto a 96-well plate (falcon) to be used as donor cells. the second group of dbt cells was seeded into a 80-cm 2 tissue culture flask (nunclon) as recipient cells. after overnight incubation at 37°c, cells from the donor cell group were infected with vaccinia virus vtf7-3 (fuerst et al., 1986) at 5 pfu/cell for 1 h at 37°c and then transfected with plasmids (0.2 g/well) containing either wildtype or mutant s genes using lipofectin (gibco/brl) according to the manufacturer's protocol. the recipient cells were transfected with the plasmid pg1nt␤gal (10 g/flask) containing the e. coli lacz gene under the control of the t7 rna polymerase promoter (nussbaum et al., 1994) . the efficiency of lipofection in this and all the other experiments described below was 20 ϯ 5%, as determined by the percentage of blue cells visualized by staining with x-gal after cells were infected with vtf7-3 and then transfected with pg1nt␤gal. after 4 h, the donor cells were washed once with dulbecco's modified eagle's medium (dmem) and resupplied with dmem. the recipient cells were trypsinized, washed once with dmem, resuspended in dmem, plated on top of the donor cell monolayer, and incubated overnight. a 3:1 excess of recipient cells to donor cells was used to ensure that donor cells fuse with the surrounding recipient cells. for the in situ staining assay, cells were fixed at 4°c for 5 min with 2% formaldehyde and 0.2% glutaraldehyde. the monolayers were overlaid with x-gal solution (5 mm potassium ferricyanide, 5 mm potassium ferrocyanide, 2 mm mgcl 2 , 1 mg/ml x-gal) and blue stain was observed after incubation at 37°c for 4 h. for quantitation of ␤-galactosidase activity, cell monolayers were lysed with 1% np-40. equal amounts of lysates and cprg substrate solution (16 mm cprg, 120 mm na 2 hpo 4 ⅐ 7h 2 o, 80 mm nah 2 po 4 ⅐ h 2 o, 20 mm kcl, 2 mm mgso 4 ⅐ 7h 2 o, 10 mm ␤-mercaptoethanol) were mixed and substrate hydrolysis rates were measured at 570 nm using a microplate absorbance reader (molecular devices). the amount of ␤-galactosidase was calculated using purified e. coli ␤-galactosidase (boehringer mannheim) as standard. dbt cell monolayers were infected with vtf7-3 at 5 pfu/cell. after 1 h at 37°c, cells were washed once with dmem and transfected with 2 g of plasmid containing either wild-type or mutant s genes as described above. after 4 h, cells were washed once with methionine-free dmem and incubated for 1 h with 35 s express protein labeling mix (110 ci/ml; nen/dupont) in methioninefree dmem. cells were either lysed immediately or incubated further for 2 h with dmem supplemented with excess unlabeled methionine and cysteine (2 mm each) before lysis with ice-cold lysis buffer [50 mm tris±hcl (ph 7.5), 150 mm nacl, 0.1% sds, 1% nonidet p-40, 0.5% sodium deoxycholate, 10 mm phenylmethylsulfonyl fluoride]. lysates were centrifuged for 10 min at 13,000 g at 4°c to pellet cell debris and nuclei. the supernatants were stored in a ϫ80°c freezer until further analysis. for immunoprecipitation, lysates containing equal amounts of radioactive label (4 ϫ 10 6 tca-precipitable cpm) were diluted with 1 ml of immunoprecipitation (ip) buffer [50 mm tris±hcl (ph 7.5), 150 mm nacl, 0.1 mm edta, 0.5% tween 80, 1 mm phenylmethylsulfonyl fluoride] containing 5 l of anti-s ao4 goat serum (kindly provided by dr. k. holmes, denver, colorado) and 30 l of protein a±sepharose 6mb beads (pharmacia biotech; diluted 1:1 by the ip buffer). the mixture was incubated overnight at 4°c. beads were collected by centrifugation and washed twice with 1 ml of ice-cold ip buffer, resuspended in 20 l of 10 mm tris ⅐ hcl (ph 6.8), 0.4% sds, a the region of the mhv-a59 s sequence covered by each oligonucleotide is marked by the positions of the 5ј and 3ј nucleotides separated by a dash. the position of the 5ј nucleotide is shown first for each oligonucleotide. the positions are numbered in reference to the first nucleotide for the s coding sequence. b oligonucleotide sequences are shown starting with the 5ј nucleotide. capital letters indicate that the sequence is exactly the same as the pint2 sequence. lowercase letters indicate the mismatched nucleotides designed for specific mutations. c the designated amino acid substitutions resulted from using each mutagenic primer are listed. and heated at 95°c for 5 min. samples were divided into two 10-l aliquots and mixed with either 10 l of 50 mm of sodium citrate (ph 5.5) containing 1 mu/l of endo h f (new england biolabs) or buffer alone. samples were incubated at 37°c overnight and analyzed by sds±page (sambrook et al., 1989) . gels were stained with coomassie brilliant blue r-250, destained, treated with sodium slicylate (chamberlain, 1979) , dried, and exposed to xray film at ϫ80°c. infection with vtf7-3 followed by transfection with 4 g of plasmid was carried out as above except that bhk-21 cells were used. four hours after transfection, cells were washed once with dmem and incubated overnight with dmem supplemented with 2% fetal bovine serum. cells were then detached by edta, washed once with ice-cold facs buffer (2% fetal bovine serum diluted in phosphate buffer saline), and resuspended in 100 l of facs buffer containing 2 l of anti-s antibody ao4. after 1 h incubation on ice, the cells were washed twice with cold facs buffer and then resuspended with 100 l of facs buffer containing fluorescein-conjugated rabbit anti-goat igg (cappel). the cells were incubated on ice for 1 h, washed twice with cold facs buffer, and analyzed on a facsscan. the mean fluorescent intensity value for each sample was measured. the surface expression levels of mutant s proteins were expressed as the percentage of the mean fluorescent intensity values for the wild-type s protein after subtracting the background. current protocols in molecular biology comparison of the spike precursor sequences of coronavirus ibv strains m41 and 6/82 with that of ibv beaudette nucleotide sequence of the glycoprotein s gene of bovine enteric coronavirus and comparison with the s proteins of two mouse hepatitis virus strains mutational analysis of the murine coronavirus spike protein: effect on cell-to-cell fusion coronavirus motif a leucine zipper structure present in the measles virus fusion protein is not required for its tetramerization but is essential for fusion coronavirus ibv: structural characterization of the spike protein the coronavirus surface glycoprotein coronavirus ibv: virus retaining spike glycopolypeptide s2 but not s1 is unable to induce virus-neutralizing or hemagglutination-inhibiting antibody, or induce chicken tracheal protection fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate heptad repeat sequences are located adjacent to hydrophobic regions in several types of virus fusion glycoproteins genomic organization of a virulent taiwanese strain of transmissible gastroenteritis virus alpha helical coiled coilsða widespread motif in proteins monoclonal antibodies to murine hepatitis virus-4 (strain jhm) define the viral glycoprotein responsible for attachment and cell-cell fusion cdna cloning and sequence analysis of the gene encoding the peplomer protein of feline infectious peritonitis virus stably expressed fipv peplomer protein induces cell fusion and elicits neutralizing antibodies in mice peptide engineering: catalytic molten globules mutational analysis of the fusion peptide of the human immunodeficiency virus type 1: identification of critical glycine residues the actions of melittin on membranes folding and assembly of viral membrane proteins mutations in the leucine zipper of the human immunodeficiency virus type 1 transmembrane glycoprotein affect fusion and infectivity proteolytic cleavage of the e2 glycoprotein of murine coronavirus: host-dependent differences in proteolytic cleavage and cell fusion eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage t7 rna polymerase murine coronavirus membrane fusion is blocked by modification of thiols buried within the spike protein hydrophobic binding of the ectodomain of influenza hemagglutinin to membranes occurs through the``fusion peptide clustal v: improved software for multiple sequence alignment theoretical and functional analysis of the siv fusion peptide assembly of asparagine-linked oligosaccharides localization of neutralizing epitopes and the receptor-binding site within the amino-terminal 330 amino acids of the murine coronavirus spike protein structure and topology of the influenza virus fusion peptide in lipid bilayers characterization of two temperature-sensitive mutants of coronavirus mouse hepatitis virus strain a59 with maturation defects in the spike protein primary structure of the glycoprotein e2 of coronavirus mhv-a59 and identification of the trypsin cleavage site misfolding and aggregation of newly synthesized proteins in the endoplasmic reticulum membrane interaction and conformational properties of the putative fusion peptide of ph-30, a protein active in sperm-egg fusion interaction of the hiv-1 fusion peptide with phospholipid vesicles: different structural requirements for fusion and leakage fusogenic mechanisms of enveloped-virus glycoproteins analyzed by a novel recombinant vaccinia virus-based assay quantitating cell fusiondependent reporter gene activation molecular cloning: a laboratory manual amphipathic helix motif: classes and properties the amphipathic helix in the exchangeable apolipoproteins: a review of secondary structure and function mutations in the fusion peptide and heptad repeat regions of the newcastle disease virus fusion protein block fusion the biology of coronaviruses proteolytic cleavage of the e2 glycoprotein of murine coronavirus: activation of cell-fusing activity of virions by trypsin and separation of two different 90k cleavage fragments the s2 subunit of the murine coronavirus spike protein is not involved in receptor binding intracellular transport of recombinant coronavirus spike proteins: implications for virus assembly viral and cellular membrane fusion proteins membrane fusion insertion of a coiled-coil peptide from influenza virus hemagglutinin into membranes this work was supported by nih grants ns-21954 and ns-30606. we thank dr. kathryn holmes for the ao4 antiserum and dr. bernard moss for the vtf7-3 vaccinia virus and plasmid pg1nt␤gal. we thank dr. francisco gonzalez-scarano, dr. paul bates, and joanna philips for comments on the manuscript. key: cord-260695-qwepi0we authors: postler, thomas s.; pantry, shara n.; desrosiers, ronald c.; ghosh, sankar title: identification and characterization of a long non-coding rna up-regulated during hiv-1 infection date: 2017-11-01 journal: virology doi: 10.1016/j.virol.2017.08.006 sha: doc_id: 260695 cord_uid: qwepi0we long non-coding rnas (lncrnas) are rapidly emerging as important regulators of a diverse array of cellular functions. here, we describe a meta-analysis of two independent rna-seq studies to identify lncrnas that are differentially expressed upon hiv-1 infection. only three lncrna genes exhibited altered expression of ≥2-fold in hiv-1-infected cells. of these, the uncharacterized lncrna linc00173 was chosen for further study. both transcript variants of linc00173 (lnc173 tsv1 and 2) could be detected by qpcr, localized predominantly to the nucleus and were reproducibly up-regulated during infection. knock-out of the linc00173 locus did not have detectable effects on hiv-1 replication. interestingly, however, stimulation of jurkat t cells with pma/ionomycin resulted in a decrease of lnc173 expression, and jurkat cells deficient for lnc173 on average expressed higher levels of specific cytokines than control cells. these data suggest that lnc173 may have a role in the regulation of cytokines in t cells. long non-coding rnas (lncrnas) are defined as rna transcripts that are not translated and are at least 200 nucleotides long. the current gencode release (version 26) has identified 15,787 lncrna genes in the human genome, encoding 27,720 different transcripts, excluding transcribed pseudogenes (https://www.gencodegenes.org/ stats/current.html) . only a few hundred of these lncrnas have been functionally characterized to date, but it has become evident that this class of biomolecules includes central regulators of varied biological processes, such as the regulation of cellular proliferation, immunity, development and even nuclear organization (atianand et al., 2017; chaudhary and lal, 2016; goff and rinn, 2015; perry and ulitsky, 2016; pircher et al., 2014) . based on their genomic context, lncrnas can be grouped into four simple classes: antisense rnas, which are transcribed in the opposite direction of overlapping protein-coding genes; large intergenic noncoding rnas (lincrnas), which are not flanked by other genes in close proximity; intronic lncrnas, which are encoded within an intron of a protein-coding gene; and overlapping lncrnas, which contain a protein-coding gene within a lncrna intron (atianand et al., 2017; spurlock et al., 2016) . more complex classification schemes have also been proposed (chen, 2016) . most lncrnas are thought to be capped, polyadenylated, and spliced using the same cellular machinery as protein-coding transcripts, although distinct differences have been noted (mukherjee et al., 2017; schlackow et al., 2016; ulitsky and bartel, 2013) . depending on their mechanism of action, lncrnas are located in the cytoplasm or in the nucleus. while cytoplasmic lncrnas tend to function as scaffolds for protein complexes or as microrna "sponges", nuclear lncrnas often aid or interfere with transcription in cis or in trans . for instance, nuclear lncrnas have been reported to bind protein regulators of transcription and either guide them to their intended target site or act as a decoy to sequester them (atianand et al., 2017; goff and rinn, 2015) . recent studies have only begun to elucidate the role of lncrnas during viral infections. several viruses have been shown to encode lncrnas in their genome, including both dna and rna viruses (tycowski et al., 2015) . multiple groups have reported that the genome of hiv-1 is transcribed in the antisense direction to yield various lncrna species (kobayashi-ishihara et al., 2012; landry et al., 2007; ludwig et al., 2006; saayman et al., 2014) . while the length of the identified transcripts differed between studies, there is evidence to suggest that at least some of these antisense rnas may interfere with viral transcription by establishing repressive histone methylation at the 5′ ltr (kobayashi-ishihara et al., 2012; saayman et al., 2014; zapata et al., 2017) . beyond virally encoded lncrnas, the expression levels of lncrnas encoded by the host have been shown to be altered upon infection with a variety of viral pathogens (fortes and morris, 2015) . specific examples include influenza a virus (iav), hepatitis c virus, severe acute respiratory syndrome-related coronavirus, adenovirus, herpes simplex virus 1 (hsv-1), human cytomegalovirus, and hiv-1 (carnero et al., 2016; chang et al., 2011; hu et al., 2016; ouyang et al., 2015; peng et al., 2010 peng et al., , 2014 trypsteen et al., 2016; winterling et al., 2014; zhang et al., 2016; zhao et al., 2016) . of the host-encoded lncrnas reported to be differentially expressed upon hiv-1 infection, only nuclear enriched abundant transcript 1 (neat1) and noncoding repressor of nuclear factor of activated t cells (nron) have been functionally characterized (lazar et al., 2016) . neat1 is a nuclear lncrna which stabilizes paraspeckles and is believed thereby to sequester unspliced and singly spliced hiv-1 transcripts (clemson et al., 2009; sasaki et al., 2009; west et al., 2014; zhang et al., 2013; zolotukhin et al., 2003) . of note, neat1 expression has also been shown to enhance the expression of antiviral genes during infection with iav and hsv-1, as well as after poly(i:c) treatment (imamura et al., 2014) . nron, by contrast, is a cytoplasmic lncrna that forms part of a high-molecular-weight complex including nuclear factor of activated t cells (nfat), a transcription factor of central importance to t-cell activation. nron stabilizes the inactive form of nfat, and correspondingly loss of nron results in the hyperactivation of t cells (macian, 2005; sharma et al., 2011; willingham et al., 2005) . a complex interplay of hiv-1 and nron has been described, where nron levels are decreased early during infection by the protein nef and increased by the late gene product vpu (imam et al., 2015) . recently, li et al. (2016) demonstrated that nron specifically induces the degradation of tat and thus contributes to hiv-1 latency. other lncrnas that have been documented to be up-regulated in hiv-1infected cells, such as malat1 and miat, have not been functionally analyzed in the context of infection. most prior reports specifically investigating the effect of hiv-1 on lncrna expression have focused on a small panel of previously characterized lncrnas rather than employing a genome-wide screen. furthermore, there was only limited overlap between the results of these studies, likely reflecting different experimental conditions (imam et al., 2015; zhang et al., 2013) . in this report, we describe a meta-analysis of two independent rna-seq studies of hiv-1-infected cells and show that, unexpectedly, only three lncrnas are differentially expressed in both of these datasets (chang et al., 2011; mohammadi et al., 2013) . of those three candidates, linc00173 exhibits the highest degree of evolutionary conservation. we demonstrate that both transcript variants of linc00173 are indeed detectable in a variety of human cell lines, are located predominantly in the nucleus, and are up-regulated upon infection with hiv-1. while, somewhat surprisingly, loss of linc00173 did not affect the viral replication cycle directly in cell culture, jurkat t cells deficient for linc00173 on average expressed higher levels of a subset of cytokines than control cells, while other cytokines remained unaffected. these data indicate a possible involvement of linc00173 in the regulation of cytokine expression. after filtering out transcripts representing snornas, mirnas and non-coding variants of protein-coding genes, the remaining transcripts were categorized as large intergenic non-coding rnas (lincs), uncharacterized long non-coding rnas (lncrnas), antisense transcripts, pseudogenes, and others (transcripts that do not fall in any of the above categories). the pie chart represents total numbers of transcripts in each category. t.s. postler et al. virology 511 (2017) 30-39 2. results to obtain an unbiased understanding of any changes in lncrna expression during hiv-1 infection, we performed a meta-analysis of two independent rna-seq studies conducted by two different groups using two different virus strains ( supplementary fig. s1 ) (chang et al., 2011; mohammadi et al., 2013) . chang et al. (2011) infected sup-t1 cells with the fully replication-competent hiv-1 isolate lai and performed rna-seq with samples harvested at 12 and 24 h p.i.. using this dataset, we identified non-coding transcripts based on their genbank classification. transcripts with a fold change of ≥ 2 were considered differentially expressed. twelve hours after infection, 21 non-coding transcripts were down-regulated, 3 were up-regulated ( fig. 1a) . at 24 h, 51 transcripts were down-regulated, while 71 had increased expression levels (fig. 1b) . unexpectedly, only 10 transcripts were differentially expressed at both time points (fig. 1c ). next, we filtered out snornas, mirnas, and non-coding transcript variants of protein-coding genes. the remaining transcripts comprised 8 large intergenic non-coding rnas (lincs), 9 uncharacterized lncrnas, 9 antisense transcripts, 16 pseudogenes and 6 other transcripts, such as a mirna polycistron (fig. 1d ). we further filtered these results using the findings of mohammadi et al. (2013) , who infected sup-t1 cells with vsv-g-pseudotyped hiv-1 nl4-3 δenv/egfp and performed rnaseq with samples harvested every 2 h for 24 h. the processed rna-seq data from that study have been made available via the dedicated online platform patterns of expression and analysis of clusters of hiv/host interactions (peachi, http://www.peachi.labtelenti.org). after excluding pseudogenes and the transcripts in the "other" category, we queried the remaining 26 lncrna transcripts from our analysis of the study by chang et al. in peachi. of these, only four were also identified as differentially expressed in peachi (11 were not differentially expressed and 11 were not catalogued). two of those 4 transcripts were variants of one gene, linc00173, identified by genbank as a large intergenic non-coding rna. the remaining 2 transcripts were both antisense rnas, ccdc18-as1 and mcm3ap-as1 (table 1) . we chose to further investigate linc00173 as both of its transcript variants were differentially expressed after hiv-1 infection in the rnaseq studies and it showed the highest degree of conservation across vertebrate species among the 3 candidate genes (table 1) . transcript variant 1 (lnc173 tsv1) is encoded by 2 exons and 1597 nt long, transcript variant 2 (lnc173 tsv2) is encoded by 3 exons and reaches a length of 435 nt. the central section of tsv1 is particularly well conserved across species, with a substantially similar region present even in the chicken genome ( fig. 2a ). rna-seq data from gm78 and k562 cells available in the ucsc genome browser indicate robust transcription and splicing ( fig. 2a ) (kent et al., 2002) . recorded histone modifications in multiple cell types are also consistent with expression, including histone h3k27 acetylation and h3k4 methylation marks near the transcriptional start site ( supplementary fig. s2 ). dnase i hypersensitivity levels suggest an open chromatin conformation at this locus ( supplementary fig. s2 ). all of these data point to active transcription of linc00173. the predicted secondary structures of lnc173 tsv1 and tsv2 are drastically different, reflecting the minimal sequence overlap restricted to the first exon ( fig. 2b , c). 2.3. both transcript variants of lnc173 are detectable in several human cell lines, are polyadenylated and localize predominantly to the nucleus to validate the results of the in silico analysis of lnc173 expression, we isolated rna from 4 human cell lines and probed for the presence of both transcript variants by quantitative rt-pcr (qrt-pcr; fig. 3a -d). tsv1 and tsv2 were readily detectable in jurkat t cells after reverse transcription with random-hexamer or oligo(dt) primers, demonstrating that both transcript variants are expressed and polyadenylated ( fig. 3a ). control samples without reverse transcriptase yielded no signal, confirming specificity of the assay for rna rather than genomic dna. similarly, lnc173 tsv1 and tsv2 were present in the monocyte-derived cell lines u-937 and thp-1, as well as in the renal epithelial cell line hek 293t ( fig. 3b -d). both transcript variants were highly enriched in the nuclear fraction of unstimulated jurkat cells, identifying lnc173 as a nuclear lncrna (fig. 4) . to confirm the rna-seq data by chang et al. and mohammadi et al., we infected hek 293t cells with serial 4-fold dilutions of vsv-gpseudotyped hiv-1 nl4-3 δenv/egfp and performed qrt-pcr with samples harvested 24 h p.i. (zhang et al., 2004) . both lnc173 transcript variants were indeed up-regulated in a dose-dependent manner compared to uninfected control cells. the lncrna malat1, which has also been reported to be induced upon hiv-1 infection, showed a dose-dependent increase of similar magnitude . stat1 mrna levels remained unchanged (fig. 5a ). infection of the monocytic thp-1 cell line mirrored the results of hek 293t infection in magnitude and dose dependence (fig. 5b ). infection of jurkat cells resulted in a reproducible but less pronounced increase in lnc173 expression (fig. 5c) . this was expected, as the efficiency of infection for this cell type ranged only from 50% to 70%, whereas the qrt-pcr table 1 lncrna transcripts with differential expression (fold change ≥ 2) in the rna-seq data sets of both chang et al. (2011) and mohammadi et al. (2013) . refseq id transcript length (nt) analysis reflected the average mrna levels of both uninfected and infected cells (cf. supplementary fig. s3c ). infection efficiency for hek 293t cells, by contrast, reached nearly 100% (cf. supplementary fig. s4b ). to determine the temporal kinetics of lnc173 expression during viral replication, we infected hek 293t cells in a 48-h time course and harvested samples every 12 h. levels of lnc173 tsv1 and tsv2 were increased compared to time-matched controls at 12 h and 24 h p.i., followed by a decline towards control levels. expression levels of malat1, by contrast, continued to increase throughout the entire time course (fig. 5d ). exposure to heat-inactivated viral particles did not result in an increase of lnc173 expression (fig. 5e ). taken together, these data establish that hiv-1 infection does induce an increase in the cellular levels of lnc173 tsv1 and tsv2 in a time-and dose-dependent manner and in multiple cell lines representing several cell types. to determine whether either transcript variant of lnc173 is involved in an important part of the hiv-1 life cycle, we used crispr-cas9mediated deletion to remove the entire linc00173 gene in jurkat cells. specifically, we targeted both ends of the gene with separate guide rnas simultaneously, derived monoclonal lines from the population and identified clones with no remaining copies of linc00173 by pcr. two separate combinations of guide rnas were used, set 4 and set 12 (supplementary table s1 ). a total of 6 lnc173 knock-out (ko) clones was obtained, 2 with set 4 and 4 with set 12. additionally, 4 control clones were derived from jurkat cells transfected with an sgrna-free cas9 vector. the absence of lnc173 transcripts was confirmed by qrt-pcr (fig. 6a , b and supplementary fig. s3a, b) . the residual signal detected with lnc173 ko clone 12-17 was close to the limit of detection but may reflect incomplete deletion of the gene. we then proceeded to systematically probe all parts of the hiv-1 replication cycle. to assess the post-entry steps of replication up to viral gene expression, we infected lnc173 ko cells or controls with vsv-g-pseudotyped hiv-1 nl4-3 δenv/egfp. this construct carries the gene encoding egfp in the open reading frame of the env gene and thus serves as a reliable reporter for the efficiency of reverse transcription, nuclear import, integration and expression of viral gene products (zhang et al., 2004) . a defect at any of these steps would result in a lower number of gfp+ cells and/or a lower level of gfp expression overall, and enhancement would result in the opposite phenotype. as expected with a collection of monoclonal lines derived from a heterogeneous parent population, the percentage of gfp+ cells and the intensity of gfp expression varied considerably between the clones ( supplementary fig. s3c, d) . however, the average values obtained for the 6 lnc173 ko clones and for the 4 control clones were remarkably similar and gave no indication of a defect or enhancement (fig. 6c, d) . analogously, we created two monoclonal hek 293t-derived cell lines with complete deletion of linc00173 (supplementary fig. s4a ). when these lines were infected with vsv-g-pseudotyped hiv-1 nl4-3 δenv/egfp, the percentage of gfp+ cells and gfp levels were also indistinguishable from two control lines ( supplementary fig. s4b, c ). next, we tested whether viral infectivity or particle release might be affected by the absence of lnc173. we produced fully infectious hiv-1 nl4-3 particles in the hek 293t lnc173 ko clone 11-2b3 and compared yield and infectivity to virus produced in hek 293t control clone 1b5. stocks produced from both cell lines yielded similar concentrations of viral particles, demonstrating that virion production and release were unaffected by deletion of linc00173 (supplementary fig. s5a ). when virus from these stocks, normalized for p24 content, was used to infect the tzm-bl reporter cell line to determine infectivity, no difference was detectable (fig. 6e) (kent et al., 2002) . (b-c) prediction for secondary structure of (b) lnc173 transcript variant 1 (tsv1) and (c) lnc173 tsv2. data obtained from lncipedia (www.lncipedia.org) (volders et al., 2015 (volders et al., , 2013 . t.s. postler et al. virology 511 (2017) 30-39 1998). to understand whether the presence of lnc173 influences viral growth over multiple rounds of replication, we infected 4 jurkat lnc173 ko clones and 4 control clones with fully replication-competent hiv-1 nl4-3 and tracked viral replication over a period of three weeks. while results varied between the clones of each group, no clear defect or enhancement was associated with the deletion of linc00173 (fig. 6f) . infection of the same clones with virus produced in lnc173-deficient hek 293t cells yielded a highly similar growth curve ( supplementary fig. s5b ). these results demonstrate that both transcript variants of lnc173 are dispensable for replication of hiv-1 in cell culture. hiv-1 replicates efficiently only in activated cells, and consequently has evolved multiple redundant mechanisms to increase the state of cellular activation in its host cell (alexander et al., 1997; fenard et al., 2005; mcdougal et al., 1985; postler and desrosiers, 2012; simmons et al., 2001; stevenson et al., 1990; zack et al., 1990; zagury et al., 1986) . we therefore investigated whether the increase in lnc173 levels was associated with general t-cell activation. surprisingly, stimulation of jurkat t cells with pma and ionomycin resulted in a marked decrease in the levels of lnc173 tsv1 and tsv2 (fig. 7a) . similarly, rna levels of a previously unknown, likely lnc173 ortholog in the mouse genome decreased upon stimulation with pma and ionomycin in el4 cells, indicating at least some degree of evolutionary conservation of this mechanism (supplementary fig. s6) . interestingly, the lnc173 ko jurkat clones on average expressed higher levels of mrna encoding a subset of cytokines than the cognate control lines. specifically, average mrna levels of ifng, ccl3 and cxcl8 upon stimulation with pma and ionomycin were significantly higher in lnc173 ko clones than in control clones, whereas mrna levels of il2 and tnf did not differ ( fig. 7b and supplementary fig. s7a-e) . due to the central role of ifn-γ in the differentiation of naive t cells into t h 1 cells during the immune response to viral and other intracellular infections, we tested whether the observed increase in ifng mrna translated into an increase in ifn-γ protein levels secreted by lnc173 ko clones (schoenborn and wilson, 2007) . indeed, the average concentration of ifn-γ in the supernatant of lnc173 ko jurkat clones was modestly but reproducibly elevated compared to the average of control clones, consistent with the mrna results ( fig. 7c and supplementary fig. s7f ). although there was considerable variability between the clones of each group, these results raise the intriguing possibility that lnc173 may be involved in the transcriptional regulation of a subset of cytokines. further studies are under way to address this question in detail. of the thousands of lncrnas expressed in the human body, the vast majority remains uncharacterized, in particular in the context of viral infections. until the recently published microarray study by trypsteen et al., all reports investigating the effect of hiv-1 infection on lncrna expression scrutinized only a small panel of previously characterized lncrnas and thus, while yielding important insights, were limited in scope (imam et al., 2015; trypsteen et al., 2016; zhang et al., 2013) . to our knowledge, this is the first study using rna-seq data to address specifically this aspect of the hiv-1 life cycle, although others have touched on the subject (peng et al., 2014) . to minimize the effect of interexperimental variability, we utilized two separate data sets created by two independent groups and employing two different strains of hiv-1 (chang et al., 2011; mohammadi et al., 2013) . the finding that only three lncrnas exhibited differential expression in both studies is somewhat surprising, especially considering that trypsteen et al. (2016) found 328 lncrna-coding genes to be altered (including those identified in this study, namely linc00173, ccdc18-as1 and mcm3ap-as1). the reason for this discrepancy is not clear, but it may stem from the inherent differences between rna-seq and microarray technology. individual lncrna expression levels tend to be lower than mrna levels, and while microarrays afford a lower dynamic range than rna-seq, they provide higher accuracy of quantification for lowabundance transcripts jiang et al., 2011; łabaj et al., 2011; ravasi et al., 2006; toung et al., 2011; xu et al., 2011) . additionally, using two independent data sets is likely to reduce the number of false positives but increase the number of false negatives. linc00173, a previously uncharacterized lncrna, is conserved amongst mammals and at least a partial homolog appears to be present in chickens. as cross-species conservation is often an indicator of functional importance, we chose to further investigate the role of linc00173 during hiv-1 infection. the experimental results presented here clearly demonstrate that its two transcript variants are robustly expressed in various human cell types, in accordance with the available in silico data. consistent with the data mined from the studies by chang et al. (2011) , mohammadi et al. (2013) , and trypsteen et al. (2016) , lnc173 tsv1 and tsv2 are up-regulated during the course of hiv-1 infection. this effect is dose-and, more importantly, timedependent. the observation that lnc173 tsv1 and tsv2 levels increase within the first 12 h after infection, remain increased at 24 h, but then decrease progressively towards baseline at 36 and 48 h, indicates targeted control of expression. nonetheless, loss of the linc00173 locus does not affect any aspect of the hiv-1 replication cycle in cell culture, from entry to particle production. based on their nuclear localization, lnc173 tsv1 and/or tsv2 are likely involved in transcriptional regulation, but their precise role in this process, if any, remains unclear. it is conceivable that lnc173 may contribute to the regulation of viral transcription only under specific conditions, such as entering or exiting viral latencyan intriguing possibility that remains to be explored. t.s. postler et al. virology 511 (2017) 30-39 it is also possible that lnc173 affects the expression or function of proteins that are not directly involved in viral replication on a cellular level but are important on an organismal level, such as cytokines that coordinate the immune response. activation of t cells by exposure to pma and ionomycin, which increases the expression of several cytokines, decreases the levels of both lnc173 transcript variants, opening up the possibility that lnc173 tsv1 and/or tsv2 may be negative regulators of cytokine expression. indeed, upon activation the monoclonal jurkat cell lines deficient for the linc00173 locus on average produced higher mrna levels of several cytokines than cognate controls, lending preliminary support to this hypothesis. ongoing experiments outside the scope of this study will further explore the role of linc00173 in cytokine regulation. in this context, it is also interesting to note that the expression levels of both lnc173 transcript variants are increased during hiv-1 infection but decreased during t-cell activation, even though hiv-1 has evolved several independent mechanisms to induce the activation of host cells (alexander et al., 1997; fenard et al., 2005; postler and desrosiers, 2012; simmons et al., 2001) . this unexpected antagonistic phenotype may imply a specific function of lnc173 to be exploited or blunted by hiv-1. of note, the cytokines exhibiting increased expression in the lnc173 ko jurkat clones include ifn-γ, a cytokine of central importance to the development and maintenance of the adaptive immune response to intracellular pathogens, including viruses (schoenborn and wilson, 2007) . it is therefore tempting to speculate that hiv-1 has evolved the ability to increase levels of lnc173 to impede the immune response. (barrett et al., 2013; chang et al., 2011; edgar et al., 2002) . gene information was analyzed and filtered by individual evaluation of associated refseq identifiers and records. all lncrna candidates identified in this data set were queried in the online platform created for the rna-seq data by mohammadi et al. (2013) , patterns of expression and analysis of clusters of hiv/host interactions (peachi, http://www.peachi.labtelenti.org). to that end, refseq identifiers of candidate genes were matched to corresponding ensembl identifiers (ensembl release 75, www.ensembl.org), which could be entered into peachi (yates et al., 2016) . phylogenetic conservation, expression data, chip-seq data and dnase hypersensitivity data were obtained from publicly accessible tracks on the ucsc genome browser (grch38, hg38; http://genome.ucsc.edu) (kent et al., 2002) . phylocsf score, number of bazzini small orfs and secondary structure prediction were obtained from lncipedia (www. lncipedia.org) (volders et al., 2013 (volders et al., , 2015 . jurkat cells (a kind gift from dr. stephen goff, columbia university), el4 cells, u937 cells and thp-1 cells (both atcc, manassas, va, usa) were maintained in rpmi-1640 medium (gibco/thermo fisher scientific, waltham, ma, usa) supplemented with 10% fetal bovine serum (fbs; atlanta biologicals, flowery branch, ga, usa). hek293t/17 (atcc) and tzm-bl cells (obtained through the nih aids reagent program, division of aids, niaid, nih) were maintained in dmem (gibco/thermo fisher scientific) with 10% fbs (derdeyn et al., 2000; platt et al., 1998) . the plasmids encoding hiv-1 nl4-3 δenv/egfp (pnl4-3-deltae-egfp) and vsv-g (pvsv-g) also were generous gifts from dr. stephen goff (haili zhang et al., 2004) . to produce pseudotyped virions, hek 293t/17 cells were co-transfected with pnl4-3-deltae-egfp and pvsv-g at a mass ratio of 3:1 using fugene 6 (promega, madison, wi, usa). transfection reagent was washed out after 6 h, and virion-containing supernatant was harvested 48 h after transfection. fully replication-competent hiv-1 nl4-3 was produced from plasmid pnl4-3, also obtained through the nih aids reagent program (genbank id af324493) (adachi et al., 1986) . nl4-3 stocks were produced from two monoclonal derivates of hek293t/17, linc00173 knock-out clone 11-2b3 and wild-type control clone 1b5. these cells were transfected using jetprime (polyplus transfection, illkirch, france) and supernatant was harvested after 72 h. the concentration of p24 in fully replication-competent virus stocks was determined by p24 antigen capture assay (advanced bioscience laboratories, rockville, md, usa). to isolate nuclear rna, 10 7 jurkat cells were resuspended in 1 ml phosphate-buffered saline (pbs), 1 ml of lysis buffer c1 (1.28 m sucrose, 40 mm tris-hcl ph 7.5, 20 mm mgcl 2 , 4% triton x-100) and 3 ml ddh 2 o. after incubation on ice for 15 min, cells were centrifuged at 4°c and 580×g for 15 min. supernatant containing the cytoplasmic fraction was discarded. the pellet containing the nuclear fraction was resuspended in 350 µl buffer rlt + β-mercaptoethanol (qiagen, germantown, md, usa). in parallel, 10 7 jurkat cells were resuspended directly in 350 µl buffer rlt + β-mercaptoethanol to obtain whole-cell rna. rna was isolated from both sample sets using the rneasy mini kit (qiagen), following the manufacturer's instructions. this included an on-column dnase i digest during purification. cells were lysed directly in buffer rlt + β-mercaptoethanol and rna was isolated using the rneasy mini kit (qiagen), according to the manufacturer's instructions, with on-column dnase i digest. reverse transcription of usually 1 µg of purified rna into cdna was performed with superscript iii reverse transcriptase and oligo(dt) 12-18 primers, unless noted otherwise. veriquest fast sybr green qpcr master mix was used for qrt-pcr (all thermo fisher scientific). relative cdna levels were determined using the δδc t method (livak and schmittgen, 2001) . see supplementary table s2 for primer sequences. in hek 293t cells, the linc00173 locus was deleted using plasmid pspcas9(bb)-2a-puro (px459), provided by dr. feng zhang (massachusetts institute of technology) through addgene (plasmid error bars indicate standard deviation. (b) mrna levels of the cytokine-encoding genes ifng, ccl3, cxcl8, il2 and tnf after stimulation with 25 ng/ml pma and 500 ng/ml ionomycin for 4 h in lnc173 knock-out and control jurkat cells, as quantified by qrt-pcr. rplp0 was used as reference gene. shown is the average of 6 monoclonal knock-out lines (lnc173 ko) and 4 monoclonal vector control lines, including every data point from 5 to 8 independent experiments. error bars indicate sem. (c) concentration of ifn-γ in the supernatant of lnc173 knock-out and control jurkat cells after stimulation with 25 ng/ml pma and 500 ng/ml ionomycin for 24 h, as determined by elisa. shown is the average of 6 monoclonal knock-out lines (lnc173 ko) and 4 monoclonal vector control lines, including every data point from 5 independent experiments. error bars indicate sem. p-values were determined with welch's t-test. ***, p ≤ 0.001; **, p ≤ 0.01; *, p ≤ 0.05; n.s., not significant. t.s. postler et al. virology 511 (2017) 30-39 #48139) (ran et al., 2013) . specifically, cells were transfected with pairs of plasmids targeting each end of the transcribed sequence, using lipofectamine 2000 (thermo fisher scientific). supplementary table s1 lists the sgrna sequences and combinations used. cells transfected with px459 without an sgrna sequence served as control. successfully transfected cells were selected with 4 µg/ml puromycin for three days. surviving cells were separated into monoclonal cultures by limiting dilution, which were then screened for successful deletion of the linc00173 locus with the primers listed in supplementary table s3 . for deletion of linc00173 in jurkat cells, the same sgrna sequences were introduced into plasmid pspcas9(bb)-2a-gfp (px458), also provided by dr. feng zhang through addgene (plasmid #48138) (ran et al., 2013) . jurkat cells were transfected with the same pairs of plasmids detailed in supplementary table s1 using an amaxa nucleofector ii device and the cell line nucleofector kit v (both from lonza, basel, switzerland). after 48 h, cells with high levels of gfp expression were identified by facs and sorted into monoclonal cultures. screening for successful deletion of linc00173 was performed as described above. hek 293t cells were infected by incubation with vsv-g-pseudotyped hiv-1 nl4-3 δenv/egfp for 4 h at 37°c, while thp-1 and jurkat cells were usually infected by spin inoculation for 2 h at 34°c and 1000×g. polybrene was added at a 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phenotypic assay for residual drug susceptibility and reduced replication capacity of drugresistant human immunodeficiency virus type 1 transcriptome altered by latent human cytomegalovirus infection on thp-1 cells using rna-seq neat1 long noncoding rna and paraspeckle bodies modulate hiv-1 posttranscriptional expression distinct temporal changes in host cell lncrna expression during the course of an adenovirus infection psf acts through the human immunodeficiency virus type 1 mrna instability elements to regulate virus expression the authors wish to thank drs. yosef sabo and stephen goff (both columbia university) for the generous gifts of their reagents, time and advice. this work was supported by nih grants r01ai068977 (to sg) and r01ai104523 (to rcd), as well as institutional support from columbia university. supplementary data associated with this article can be found in the online version at doi:10.1016/j.virol.2017.08.006. key: cord-265156-u1re7983 authors: de wilde, adriaan h.; zevenhoven-dobbe, jessika c.; beugeling, corrine; chatterji, udayan; de jong, danielle; gallay, philippe; szuhai, karoly; posthuma, clara c.; snijder, eric j. title: coronaviruses and arteriviruses display striking differences in their cyclophilin a-dependence during replication in cell culture date: 2017-12-15 journal: virology doi: 10.1016/j.virol.2017.11.022 sha: doc_id: 265156 cord_uid: u1re7983 cyclophilin a (cypa) is an important host factor in the replication of a variety of rna viruses. also the replication of several nidoviruses was reported to depend on cypa, although possibly not to the same extent. these prior studies are difficult to compare, since different nidoviruses, cell lines and experimental set-ups were used. here, we investigated the cypa dependence of three distantly related nidoviruses that can all replicate in huh7 cells: the arterivirus equine arteritis virus (eav), the alphacoronavirus human coronavirus 229e (hcov-229e), and the betacoronavirus middle east respiratory syndrome coronavirus (mers-cov). the replication of these viruses was compared in the same parental huh7 cells and in cypa-knockout huh7 cells generated using crispr/cas9-technology. cypa depletion reduced eav yields by ~ 3-log, whereas mers-cov progeny titers were modestly reduced (3-fold) and hcov-229e replication was unchanged. this study reveals that the replication of nidoviruses can differ strikingly in its dependence on cellular cypa. the order nidovirales is currently comprised of the arterivirus, coronavirus, ronivirus, and mesonivirus families (https://talk. ictvonline.org/taxonomy/) and includes agents that can have major economic and societal impact. this was exemplified by the 2002-2003 severe acute respiratory syndrome coronavirus (sars-cov) epidemic and the ongoing outbreaks of the middle east respiratory syndrome coronavirus (mers-cov) . both these coronaviruses were introduced into the human population following zoonotic transmission, revealing the potentially lethal consequences of nidovirus-induced disease in humans. within a few months, the emergence of sars-cov led to more than 8000 laboratory-confirmed cases (mortality rate of~10%). the mers-cov outbreaks thus far resulted in over 2000 confirmed human cases and a~35% mortality rate within that group (http://www.who. int/emergencies/mers-cov/en/). in addition, the porcine epidemic diarrhea coronavirus and the arterivirus porcine reproductive and respiratory syndrome virus (prrsv) are among the leading veterinary pathogens, having caused high economic losses in the swine industry (holtkamp et al., 2013; lin et al., 2016) . the economic and societal impact of nidovirus infections, and the lack of effective strategies to control them, highlight the importance of advancing our knowledge of the replication of these viruses and their interactions with the host cell. nidoviruses are positive-stranded rna (+rna) viruses with large to very large genomes, ranging from 13 to 16 kb for arteriviruses to 26-34 kb for coronaviruses (gorbalenya et al., 2006; nga et al., 2011) . their complex genome expression strategy involves genome translation to produce the polyprotein precursors of the nonstructural proteins (nsps) as well as the synthesis of a nested set of subgenomic (sg) mrnas to express the structural proteins (reviewed in de wit et al., 2016; snijder et al., 2013) . nidoviral nsps, presumably together with various host factors, assemble into replication and transcription complexes (rtcs) that drive viral rna synthesis (gosert et al., 2002; hagemeijer et al., 2012; pedersen et al., 1999; van hemert et al., 2008a van hemert et al., , 2008b . these rtcs are thought to be associated with a virus-induced network of endoplasmic reticulum (er)-derived membrane structures, including large numbers of double-membrane vesicles (gosert et al., 2002; knoops et al., 2012 knoops et al., , 2008 maier et al., 2013; pedersen et al., 1999; ulasli et al., 2010) . nidovirus replication thus depends on a variety of host cell factors and processes, including cellular proteins and membranes, membrane trafficking, and host signaling pathways (reviewed in de wilde et al., 2017b; van der hoeven et al., 2016; zhong et al., 2012) . among these, members of the cyclophilin (cyp) protein family previously have been implicated in nidovirus replication. cyclophilins are a family of peptidyl-prolyl isomerases (ppiases) that act as chaperones to facilitate protein folding, as well as protein trafficking and immune cell activation (reviewed in naoumov, 2014; nigro et al., 2013) . cyclophilins, and in particular the ubiquitously expressed cypa, have also been implicated in the replication of various other groups of rna viruses. the role of cypa in hepatitis c virus (hcv) and human immunodeficiency virus-1 (hiv-1) infection has been studied in most detail. for example, cypa assists hcv polyprotein processing, interacts with hcv ns5a to ensure remodelling of cellular membranes into hcv replication organelles, and stabilizes hiv-1 capsids to promote nuclear import of the hiv-1 genome (reviewed in hopkins and gallay, 2015) . cyclophilins were initially implicated as host factors in nidovirus replication during studies with general cyp inhibitors such as cyclosporine a (csa). in cell culture, the replication of a variety of coronaviruses and arteriviruses was found to be strongly inhibited by lowmicromolar concentrations of csa and the non-immunosuppressive csa analogs alisporivir (alv) and nim-811 (carbajo-lozoya et al., 2014 de wilde et al., 2017a de wilde et al., , 2013b de wilde et al., , 2011 kim and lee, 2014; tanaka et al., 2012; von brunn, 2015; von brunn et al., 2015) . subsequently, it was established that nidovirus replication can depend specifically on cypa and/or cypb. the replication in cell culture of the arterivirus equine arteritis virus (eav; de wilde et al., 2013a) and the alphacoronaviruses feline coronavirus (fcov; tanaka et al., 2017) , human coronavirus (hcov) nl63 (carbajo-lozoya et al., 2014), and hcov-229e (von brunn et al., 2015) was reported to be affected by cypa knockdown (kd) or knockout (ko), although the level of cypa dependence of these viruses, which was not compared directly, appeared to be quite different. finally, upon ultracentrifugation, the normally cytosolic cypa was found to co-sediment with membrane structures containing eav rtcs, suggesting a direct association with the arteriviral rna-synthesizing machinery (de wilde et al., 2013a) . the abovementioned studies differed in terms of the nidoviruses and cell lines tested, cypa expression levels, and readouts used to measure viral replication efficiency, which hampered a direct comparison of the cypa dependence of different nidoviruses. therefore, in this study, we investigated the cypa-dependence of the replication of three distantly related nidoviruses in the same cell line (huh7), in which cypa expression was knocked-out using crispr/cas9 gene editing technology (huh7-cypa ko cells). using different cell lines, the replication of two of these viruses, the arterivirus eav (de wilde et al., 2013a) and the alphacoronavirus hcov-229e , was previously concluded to depend on cypa. the cypa dependence of betacoronaviruses like mers-cov has not been documented before. infection of huh7-cypa ko cells with mers-cov revealed that its replication was only modestly affected by the absence of cypa, as opposed to eav which was strongly inhibited. strikingly, and in contrast to a previous report, hcov-229e replication was not affected at all in the huh7-cypa ko cells. our study thus reveals major differences in the magnitude of cypa dependence of the arterivirus eav when compared to two different coronaviruses, and calls for a more extensive evaluation of the role of cypa in the replication of members of the latter virus family. 293t (van kasteren et al., 2012) , bhk-21 (nedialkova et al., 2010) , huh7, and vero cells (de wilde et al., 2013b) were cultured as described previously. a cell culture-adapted derivative of the eav bucyrus isolate (bryans et al., 1957; den boon et al., 1991) , hcov-229e (atcc vr-740; hamre and procknow, 1966) , or mers-cov (strain emc/2012; van boheemen et al., 2012; zaki et al., 2012) were used to infect monolayers of huh7 cells (and derived cell clones) as described previously (cervantes-barragan et al., 2010; de wilde et al., 2013b; oudshoorn et al., 2016) . eav, hcov-229e, and mers-cov titers in cell culture supernatants were determined by plaque assays on bhk-21, huh7, or vero cells, respectively (de wilde et al., 2013b; nedialkova et al., 2010; zust et al., 2011) . to obtain huh7 cells that lack the expression of cypa, cypb, cypc or cypd, we used clustered regularly interspaced short palindromic repeat (crispr)/cas9 gene editing (cong et al., 2013) . the plenti-crispr v2 plasmid (a kind gift from feng zhang; addgene plasmid #52961; sanjana et al., 2014) was used to construct plasmids with guide rnas (grnas) specific for ppia, ppib, ppic or ppid, or non-targeting grna (origene; for sequences, see table 1 ), according to the protocol provided by addgene (https://www.addgene.org/52961/). grna sequences were selected using the atum crispr grna design tool (https://www.atum.bio/ecommerce/cas9/input). pseudo-infectious, third-generation lentivirus particles expressing gene-specific grnas were produced by transfection of 293 t cells with the plenti-crispr v2 plasmid and three "helper" plasmids (encoding hiv-1 gag-pol, hiv-1 rev, and vsv-g; carlotti et al., 2004) . after overnight transfection of 293t cells using the polyethyleneimine dna co-precipitation method, the culture medium was replaced with fresh medium, which was harvested at 72 h post-transfection (h p.t.), passed through a filter with 0.45 µm pore-size, and stored at −80°c. huh7 cells were transduced in 10-cm 2 dishes containing 1 ml of lentivirus harvest and 1 ml of fresh huh7 culture medium supplemented with 8 µg/ml polybrene (sigma). from 3 days post transduction onwards, lentivirus-transduced cells were selected by culturing in the presence of 3 µg/ml puromycin. loss of cyclophilin expression was verified by western blot analysis (see below). to clone huh7-cypa ko cells, lentivirus-transduced cells were trypsinized and seeded in 96-well culture plates at a density of one cell per well, which was verified by microscopy. cell proliferation was stimulated by supplementing the culture medium with 15% fcs. following their expansion, cypa ko clones were tested for lack of cypa expression by western blot analysis. the huh7-cypa ko clones constitutively expressed the cas9 nuclease as the gene is incorporated in the plenticrispr v2 vector, and integrated into the genome upon lentivirus transduction. using an alternative approach, we also generated of huh7-cypa ko clones lacking cas9 expression by direct transfection of the plenticrispr v2 plasmid into huh7 cells. cells (5 × 10 5 cells in a 10-cm 2 dish) were transfected with 2.5 µg plasmid dna and 6 µl lipofectamine 2000 (thermo fischer scientific). after 24 h at 37°c, the medium was replaced with fresh medium containing 3 µg/ml puromycin. at 4 d p.t., cells were harvested and seeded in 96-well culture plates at a density of one cell per well, to obtain clones as described above. for western blot analysis, we used rabbit polyclonal antibodies against cypa (sc-20360-r) or cypd (sc-66848) and a goat polyclonal antibody against cypb (sc-20361) (all from santa cruz biotechnology), a rabbit monoclonal antibody against cypc (ab184552, abcam), and mouse monoclonal antibodies against β-actin (ac-74, sigma) or transferrin receptor (tfr; h68.4, thermo fisher scientific). cells were lysed in laemmli sample buffer and after sds-page, proteins were transferred to hybond-lfp membranes (ge healthcare) by semi-dry blotting. membranes were blocked with 1% casein in pbs containing 0.1% tween-20 (pbst), and were incubated in pbst with 0.5% casein with anti-cypa (1:500), anti-cypb (1:1000), anti-cypc (1:1000), anti-cypd (1:500), anti-tfr (1:4000), or anti-β-actin (1:50,000) antisera. biotin-conjugated swine-anti-rabbit igg (1:2000) or goat-anti-mouse igg (1:1000) antibodies (dako) and cy3conjugated mouse-anti-biotin (1:2500) were used for detection. blots were scanned with a typhoon 9410 imager (ge healthcare) and analyzed with imagequant tl software. genomic dna was isolated from five different huh7 cypa ko clones to verify the generation of huh7 ppia knockout cells. approximately 3 × 10 6 cells were lysed in 3 ml of 75 mm nacl, 25 mm edta ph 8.0, 1% sds, and 100 µg/ml proteinase k, and incubated at 37°c for 16 h. one ml of 6 m (saturated) nacl was added and after mixing for 30 s, the lysate was centrifuged for 15 min at 4000×g and 4°c. to remove remaining cellular debris, the supernatant was transferred to a new tube and centrifugation was repeated. dna was precipitated in 70% etoh, pelleted by centrifugation and washed in 70% etoh. the dried pellet was dissolved in 10 mm tris, 1 mm edta ph 7.5. for sequence analysis of the ppia-gene, intron-specific primers (5′-accttgcagatttggca cac-3′ and 5′-agtgtttgttccgttccccc-3′) were used to amplify exon 4 of the ppia gene that is targeted by crispr/cas9-mediated cleavage (fig. 2d ). pcr products were cloned into pcr2.1-topo (thermo fisher scientific) according to the manufacturer's instructions and individual clones were analyzed by sanger sequencing to identify crispr/cas9-directed out-of-frame insertions or deletions ('indels'). in this manner, two huh7 cypa ko clones (clones #1 and #2) were obtained, which were derived from lentiviral transduction and direct transfection of huh7 cells, respectively. chromosome spreads of metaphase cells were prepared essentially as described previously (de graaff et al., 2015) and used for karyotyping by combined binary ratio labeling of nucleic-acid probes for multi-color fluorescence in situ hybridization (cobra-fish) staining. the analysis used whole chromosome paint probe sets to discriminate individual chromosomes and specific staining to identify short and long arms of chromosomes, as described previously (szuhai and tanke, 2006) . after hybridization, image capture and image analysis was performed using in-house written software and chromosomes were identified based on their specific coloring (szuhai and tanke, 2006) . based on the chromosome count, ploidy (numerical aberrations) was established and structural chromosomal aberrations including breaks, deletions, translocations and insertions, were identified based on chromosomal structure. parental huh7 cells and cypa kd huh7 cells were cultured overnight in triplicate wells at a density of 5 × 10 4 cells in a 24-well cluster. intracellular rna was isolated using the nucleospin rna ii kit (machery-nagel) according to the manufacturer's instructions and used as a template for cdna synthesis using revertaid h minus reverse transcriptase (thermo fisher scientific) and oligo(dt)20 primer. finally, samples were assayed by quantitative real-time pcr analysis (qrt-pcr) using primers for ppia (forward primer: ttcatctgcactgccaagac; reverse primer: cagacaaggt cccaaagacag), and the housekeeping genes rpl13 (forward primer: aaggtggtggtcgtacgctgtg; reverse primer: cg ggaagggttggtgttcatcc) and gapdh (forward primer: gcaa atttccatggcaccgt; reverse primer: gccccacttgattttggagg). pcr was performed using the iq sybr green super mix (bio-rad) and the cfx384 touch™ real-time pcr detection system. data was analyzed with cfx manager 3.1 software (bio-rad). previously, cypa and/or cypb were reported to play a role in the replication of a number of arteri-and coronaviruses (carbajo-lozoya et al., 2014; de wilde et al., 2013a; tanaka et al., 2017; von brunn et al., 2015) . the degree of sensitivity to cyp depletion appeared to differ between these viruses, but was not compared directly. as we set out to assess the cyp dependence of the recently emerged mers-cov, which had not been evaluated thus far, the fact that this betacoronavirus replicates in huh7 cells provided an excellent opportunity for a direct comparison with the arterivirus eav and the alphacoronavirus hcov-229e, which can also replicate in this cell line. therefore, using crispr/cas9 gene editing technology, we first generated cypa ko , cy-pb ko , cypc ko , and cypd ko huh7 cell pools. to this end, huh7 cells were transduced with lentiviruses co-expressing the nuclease cas9 as well as a grna that binds a specific sequence in the ppia, ppib, ppic, or ppid gene. cypc and cypd were included given that both these enzymes are also sensitive to csa treatment (davis et al., 2010) . the fact that it previously was implicated as a host factor in the replication of the betacoronavirus hcov-oc43 (favreau et al., 2012) was an additional reason to include cypd. western blot analysis of the cyp ko cell pools established that cypa and cypb were no longer detectable in huh7-cypa ko,pool and huh7-cypb ko,pool cells, respectively. for the cypc ko or cypd ko cell pools, however, some residual expression of the target gene was detected (fig. 1a) . next, the replication of eav, hcov-229e, and mers-cov in each of the four cyp ko cell pools was compared to their replication in huh7 control cells expressing an unrelated (non-targeting) grna. to allow multiple cycles of replication before analyzing virus yields, cell pools were infected with a low infectious dose (moi 0.01). the production of infectious mers-cov and hcov-229e progeny was found to be unchanged in all four cyp ko cell pools ( fig. 1b and c) . in contrast, eav virus titers were reduced by 2-logs after infection of the huh7-cypa ko cell pool, but not in any of the other knockout cell pools (fig. 1d) . this strengthened the case for an important role of specifically cypa as a host factor in eav replication. previously, sirnamediated knockdown of cypa expression could only reduce eav titers by about 4-fold (de wilde et al., 2013a) , suggesting that relatively small amounts of cypa may suffice to support normal levels of eav replication. for hcov-nl63, it was previously reported that virus production was inhibited only when the residual ppia mrna level was reduced to a.h. de wilde et al. virology 517 (2018) 148-156 3% of that in control cells (carbajo-lozoya et al., 2014) . also the results with eav (see above) suggested that major effects on virus replication may only be observed when cypa knockdown is highly efficient. as the huh7-cypa ko cell pools might exhibit low levels of residual cypa expression, which might still suffice to support normal levels of mers-cov and hcov-229e replication, we generated clonal huh7-cypa ko cell lines (as described in material and methods) for use in follow-up experiments. potential knockout clones were first tested for cypa expression by western blot analysis. subsequently, the presence of out-of-frame 'indels' was established by sequence analysis of exon 4 of the ppia gene. in total, five individual cypa ko cell clones were analyzed in detail. to this end, the exon that was targeted by the ppia-specific grna was pcr amplified using primers targeting the adjacent introns (fig. 2d , white triangles) and the pcr product was cloned in a plasmid vector. per cypa ko cell clone, over twenty plasmid clones were analyzed by sanger sequencing. remarkably, sequence analysis of these clones revealed six to eight different indel sequences ( fig. 2e ; see also below), establishing that these huh7 clones carried more than the anticipated two copies (alleles) of the ppia gene. for three out of five cell clones, some of the indels constituted in-frame deletions, indicating that (low amounts of) cypa variants lacking one or more amino acids might still be expressed (data not shown). two of the huh7-cypa ko cell clones were found to contain only out-of-frame indels (fig. 2e , clones #1 and #2), which was supported by a lack of detectable cypa expression (fig. 3a) . of note, one of these clones was obtained after lentivirus transduction (clone #1), whereas the other resulted from transfection of the plenticrispr plasmid (clone #2). to investigate the unexpectedly large number of different ppia-gene specific sequences in more detail, we performed karyotyping of the parental huh7 cells and huh7-cypa ko clone #1. for both cell lines, cobra-fish established a 4n+ chromosome content, with more than 100 chromosomes per cell and exceedingly large numbers of translocations involving all chromosomes (results from the parental huh7 cells are shown in fig. 2a) . the ppia gene is located on the short arm of chromosome 7 (chr7p) and karyotyping using the short-and long armspecific whole chromosome paint probe set (szuhai and tanke, 2006) revealed four copies of that chromosome, with one appearing normal (see chromosome 1; fig. 2b ), one carrying a translocation with a piece of chromosome 5 in the long arm (see chromosome 2; fig. 2b ), and two representing isochromosomes (i.e. containing two short arms of chromosome 7; see chromosomes 3 and 4; fig. 2b ). staining of the short arm of chromosome 7 (chr7p) revealed six copies of this chromosome in the parental huh7 cells (fig. 2c, white arrows) and huh7-cypa ko clone #1 (data not shown). overall, eight (clone #1) and six (clone #2) different ppia-specific indels were identified. based on the karyotyping analysis, this could be explained by the presence of (at least) six copies of the short arm of a.h. de wilde et al. virology 517 (2018) 148-156 chromosome 7. as the resolution of this techniques is limited to the detection of large (>1000 kb) translocations (szuhai and tanke, 2006) , an even higher number of ppia gene copies in the huh7 genome cannot be excluded, and would in fact be in line with the results obtained for clone #1. since the analysis of >20 cloned ppia-specific pcr products from cypa ko clones #1 and #2 exclusively revealed out-of-frame indels, both clones were assumed to have lost all expression of functional cypa and were used for further infection experiments. parental huh7 cells and huh7-cypa ko clones #1 and #2 were all infected with mers-cov, hcov-229e, or eav at an moi of 0.01. the inactivation of cypa expression in these cells was found to significantly reduce mers-cov replication by~3-fold in both cypa ko cell clones (fig. 3b ). however, virus yields released from control cells and cypa ko cell clone #2 were similar upon high moi inoculation (moi of 5; fig. 3c ). interestingly, in both clones, no effect of a lack of cypa was seen for hcov-229e (fig. 3d ). for eav, however, a~3-log reduction of virus yields was observed (fig. 3e) , representing a 10-fold stronger inhibition compared to the huh7-cypa ko,pool cells used previously (fig. 1d) . the importance of cypa in virus replication has been reported for a variety of viruses, including hcv and hiv-1. also nidovirus replication has been shown to depend on cypa, but the role of this host factor is poorly understood thus far. by analogy with the role of cypa in hcv fig. 2 . karyotyping of huh7 cells and sequencing of huh7-cypa ko clones. a) representative cobra fish karyogram of a huh7 cell. a complex karyotype with both numerical and large number of structural rearrangements was observed. b) the short and long arm-specific whole chromosome paint probe set (for details, see szuhai and tanke, 2006) identified four copies of chromosome 7. c) staining of the chromosomes' short arms revealed the presence of two copies of isochromosome 7p (indicated as nr 3 and 4) and at least six copies of chromosome 7p (indicated by white arrows). d) overview of the ppia gene and ppia-specific mrna splice variants, including ppia exons 1-5. the grna binding site is indicated by a grey triangle. e) sequence analysis of cloned pcr products covering the ppia-gene region targeted by the grna revealed eight (clone #1) or six (clone #2) out-of-frame mutations. sequence analysis of clones derived from the parental huh7 cells only yielded the reference ppia gene sequence. the protospacer adjacent motif (pam) sequence, which served as cas9 docking site, is highlighted in grey. a.h. de wilde et al. virology 517 (2018) 148-156 replication (reviewed in hopkins and gallay, 2015) , and supported by the reduced replication of eav in sirna-treated cypa kd cells, we hypothesized cypa to be an integral component of the membrane-associated replication machinery of arteriviruses (de wilde et al., 2013a; de wilde et al., in preparation) . previously, the role of cypa in nidovirus replication has been studied by multiple laboratories, with different outcomes. however, the use of a range of nidoviruses and cell lines, and variable experimental set-ups and read-outs likely contributed to these differences. by infecting the same huh7-cyp ko cell pools (fig. 1) and huh7-cypa ko clones (fig. 3) with the three nidoviruses compared in this study, we could now eliminate a number of experimental variables. our data revealed striking differences between the arterivirus eav and the two coronaviruses in terms of their sensitivity to cypa-depletion in huh7 cells. whereas eav replication was strongly inhibited (fig. 3) , coronavirus replication was affected only modestly (mers-cov) or not at all (hcov-229e). interestingly, the relatively small difference in mers-cov progeny titers from cypa ko and control huh7 cells was not observed when infections were carried out with a high moi (fig. 3c) . a possible explanation for this difference could be the presence of cypa in mers-cov virions. virion-associated cypa has been reported previously for hiv-1 and sars-cov (braaten et al., 1996; hatziioannou et al., 2005; neuman et al., 2008) , and although its role and importance are unclear, the virion-mediated transfer of cypa to cypa-deficient cells may initially suffice to support efficient replication in these cells. like sars-cov particles, mers-cov virions may contain cypa, which could explain complementation of the lack of cypa in knockout cells during a high-moi, one-cycle replication assay. during a multi-cycle infection experiment, however, the virus produced during the first round of replication may lack cypa, which may negatively influence virus yields during subsequent rounds of replication. in the case of hcov-229e, the comparable replication efficiency in (chatterji et al., 2009 ), huh7-cypa ko,pool cells (used in fig. 1) , and huh7-cypa ko cell clone #1 (used in fig. 3) , presented as the difference in virus yields between huh7 control cells and cypa ko or cypa kd cells (in log 10 reduction in eav titer). results are the mean ± sd from two independent experiments. a.h. de wilde et al. virology 517 (2018) 148-156 huh7-cypa ko cells and control huh7 cells appears to be at odds with published studies for three different alphacoronaviruses (including hcov-229e itself) in which a modest to strong inhibition of virus replication was reported (see below). likewise, the insensitivity (fig. 1 ) of the alphacoronavirus hcov-229e and betacoronavirus mers-cov to depletion of cypb and cypd, contrasts with previous reports on the cypb and cypd dependence of fcov (tanaka et al., 2017) and hcov-oc43 (favreau et al., 2012) , an alpha-and a betacoronavirus respectively. significant efforts were made to characterize two of the huh7-cypa ko clones that were generated using crispr/cas9-technology: clone #1 was derived from transduction with a ppia grna-expressing lentivirus, whereas clone #2 resulted from transfection of a plasmid coexpressing the ppia-specific grna and the cas9 nuclease. consequently, clone #1, in contrast to clone #2, ubiquitously expressed cas9 and the ppia-gene directed grna. in theory, this could lead to offtarget cleavage events in the genome, and thus to unwanted side-effects. however, using both clones, we observed very similar effects of cypa depletion on the replication of the three nidoviruses tested. previously documented differences in terms of sensitivity to csa treatment appear to coincide with similar sensitivity differences to cypa knockdown as observed in this study. while eav replication in cell culture is completely blocked at a dose of 1 µm csa, concentrations of 9 and 16 µm are required to achieve a similar block of the replication of mers-cov (de wilde et al., 2013b) and hcov-229e (de wilde et al., 2011; pfefferle et al., 2011) , respectively. an unexpected result was the lack of an effect of cypa depletion on hcov-229e replication, since a previous study reported a 10-fold reduction of the hcov-229e-driven expression of a luciferase reporter gene in huh7.5-shrna-cypa kd cells . in our hands, however, hcov-229e replication was unchanged when comparing parental huh7 cells, huh7-cypa ko,pool cells, and the two huh7-cypa ko clones (figs. 1c and 3c). these contradictory results may (in part) be explained by the use of different viruses (wild-type versus recombinant hcov-229e), the use of different readouts of virus replication (hcov-229e progeny titers versus hcov-229e-driven luciferase expression), or the use of huh7 versus huh7.5 cells. the latter is a sub-clone of huh7 cells that supports hcv replication more efficiently, most likely because huh7.5 cells express an inactive form of the retinoic acid-inducible gene-i (rig-i), resulting in a reduced antiviral response to virus infection (blight et al., 2002) . even more striking, is the difference with previous results obtained for two other alphacoronaviruses, fcov and hcov-nl63, for which progeny titer reductions of >5-fold and 5-log were reported upon cypa knockdown, respectively (carbajo-lozoya et al., 2014; tanaka et al., 2017) . cypa is one of the most abundant cytosolic proteins constituting 0.1-0.4% of the total cellular protein content (harding et al., 1986) . data from different labs suggest that relatively low levels of cypa may suffice to support efficient coronavirus replication. for example, we have previously shown that sars-cov replication was not affected when sirna transfection was used to reduce the cypa expression level to 25% of that in parental cells (de wilde et al., 2013a (de wilde et al., , 2011 . hcov-nl63 yields in caco-2 cells were reduced only when a~97% reduction in cypa mrna expression was achieved (carbajo-lozoya et al., 2014) . subsequently, fcov replication was found to be completely abolished in crispr/cas9-mediated cypa ko fcwf-4 (feline) cells, while only partial inhibition of fipv replication was observed when cypa knockdown was mediated by short-hairpin (sh)rnas (tanaka et al., 2017) . we observed a similar correlation between remaining cypa expression levels and eav replication. this study was initiated using huh7 cells in which cypa knockdown was mediated by constitutive expression of shrnas ( fig. 3e ; chatterji et al., 2009) . in these cells, cypa protein expression was undetectable and the cypa mrna level was only 2% of that in control huh7 cells expressing a non-targeting shrna (data not shown). in these cells, eav replication was reduced by about 0.5-log (fig. 3f, left bar) . also in our huh7-cypa ko cell pools (figs. 1d and 3f) residual cypa expression could not be detected by western blot analysis, but now a~2-log reduction in eav yield was observed. the stronger inhibition of eav suggests that cypa levels in these cells were lower than those in shrna-expressing cypa kd cells, but due to the nature of the gene editing method used (targeting the ppia gene instead of the mrna) cypa depletion could not be readily compared by measuring mrna levels. finally, the largest reductions of eav progeny yields, by about~3-logs, were observed using our huh7-cy-pa ko cell clones (fig. 3b-f) . interestingly, the characterization of our huh7-cypa ko clones and the parental huh7 cell line revealed a severely disordered chromosome composition with multiple translocations and duplications involving all chromosomes, including chromosome 7 that contains the ppia gene. such structural rearrangements, leading to gene expression abnormalities, are common in tumor-derived cell lines (lin et al., 2014; mertens et al., 2015) , but are not always taken into account during studies of virus-host interactions. since huh7 cells originated from a hepatocellular carcinoma (nakabayashi et al., 1982) , these abnormalities were to be expected, also on the basis of a previous study that established chromosomal imbalances in liver carcinoma-derived cell lines, including huh7 (wilkens et al., 2012) . interestingly, this study describes a gain of, among other regions, the part of short arm of chromosome 7 that includes the ppia gene. polyploidy and chromosome rearrangements may hamper the generation of bona fide knockout cell lines. therefore, our observations underline the importance of an in-depth genomic characterization of knockout cell lines obtained by (crispr/ cas9-mediated) gene editing. this is further emphasized by the relatively low success rate of our efforts to obtain true cypa ko cell clones, since more gene editing events are needed to achieve complete knockout. several of our other clones were found to carry in-frame deletions in ppia and (potentially) expressed cypa variants lacking one or multiple amino acid residues, with unpredictable consequences for the protein's function. the replication of mers-cov, hcov-229e, and eav in cypb ko , cypc ko , or cypd ko cell pools was unaltered compared to control cells, suggesting that in the huh7 setting these viruses do not depend on the presence of cypb, cypc, or cypd. however, as in the case of cypa, we cannot exclude the possibility that (very) low levels of these cyps may still suffice to support efficient virus replication. this may be exemplified by the dependence of fcov on cypb. whereas fcov replication in feline cypb ko fcwf-4 cells was strongly reduced, replication in shrna-mediated cypb kd fcwf-4 cells was only marginally affected, suggesting that a very strong reduction of cypb levels is needed to block replication efficiently (tanaka et al., 2017) . unfortunately, in the present study, we were unable to generate true huh7-cypb ko , huh7-cy-pc ko , and huh7-cypd ko cell clones, but the analysis of nidovirus replication in the context of cloned cells lacking these cyps definitely warrants further efforts. likewise the quantitative and functional aspects of the role of cypa in nidovirus replication needs to be followed up, particularly in the light of the quite different results that can apparently be obtained with the same coronavirus in different experimental settings. highly permissive cell lines for subgenomic and genomic hepatitis c virus rna replication cyclophilin a is required for an early step in a.h. de wilde et al the life cycle of human immunodeficiency virus type 1 before the initiation of reverse transcription isolation of a filterable agent causing arteritis of horses and 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rig-i to control innate immune signaling editorial overview: engineering for viral resistance genetic deficiency and polymorphisms of cyclophilin a reveal its essential role for human coronavirus 229e replication cholangiolar carcinoma-derived cell lines reveal distinct sets of chromosomal imbalances isolation of a novel coronavirus from a man with pneumonia in saudi arabia recent progress in studies of arterivirus-and coronavirus-host interactions ribose 2′-o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda5 we thank diede oudshoorn for helpful discussions and technical assistance on creating crispr/cas9-mediated cyp knockout cells, dr. ron fouchier (erasmus medical center rotterdam, the netherlands) for sharing mers-cov isolate emc/2012, and dr. volker thiel for providing hcov-229e. key: cord-259717-e8ljkv2y authors: holtz, lori r.; cao, song; zhao, guoyan; bauer, irma k.; denno, donna m.; klein, eileen j.; antonio, martin; stine, o. colin; snelling, thomas l.; kirkwood, carl d.; wang, david title: geographic variation in the eukaryotic virome of human diarrhea date: 2014-11-01 journal: virology doi: 10.1016/j.virol.2014.09.012 sha: doc_id: 259717 cord_uid: e8ljkv2y little is known about the population of eukaryotic viruses in the human gut (“virome”) or the potential role it may play in disease. we used a metagenomic approach to define and compare the eukaryotic viromes in pediatric diarrhea cohorts from two locations (melbourne and northern territory, australia). we detected viruses known to cause diarrhea, non-pathogenic enteric viruses, viruses not associated with an enteric reservoir, viruses of plants, and novel viruses. viromes from northern territory children contained more viral families per sample than viromes from melbourne, which could be attributed largely to an increased number of sequences from the families adenoviridae and picornaviridae (genus enterovirus). qrt-pcr/pcr confirmed the increased prevalence of adenoviruses and enteroviruses. testing of additional diarrhea cohorts by qrt-pcr/pcr demonstrated statistically different prevalences in different geographic sites. these findings raise the question of whether the virome plays a role in enteric diseases and conditions that vary with geography. little is known about the population of eukaryotic viruses in the human gut ("virome") or the potential role it may play in disease. we used a metagenomic approach to define and compare the eukaryotic viromes in pediatric diarrhea cohorts from two locations (melbourne and northern territory, australia). we detected viruses known to cause diarrhea, non-pathogenic enteric viruses, viruses not associated with an enteric reservoir, viruses of plants, and novel viruses. viromes from northern territory children contained more viral families per sample than viromes from melbourne, which could be attributed largely to an increased number of sequences from the families adenoviridae and picornaviridae (genus enterovirus). qrt-pcr/pcr confirmed the increased prevalence of adenoviruses and enteroviruses. testing of additional diarrhea cohorts by qrt-pcr/pcr demonstrated statistically different prevalences in different geographic sites. these findings raise the question of whether the virome plays a role in enteric diseases and conditions that vary with geography. & 2014 elsevier inc. all rights reserved. it is well established that disease prevalence varies in different geographical regions of the world. improvements in hygiene and decreased microbial exposure in childhood have been hypothesized to be responsible for the increased occurrence of allergies, autoimmune disorders, and inflammatory bowel disease in the westernized world (strachan, 1989) . on the other hand, conditions such as environmental enteropathy and decreased oral vaccine efficacy are seen in the developing world. environmental enteropathy is a diffuse villous atrophy of the small bowel, which is ubiquitous in children in the developing world (campbell et al., 2003; menzies et al., 1999) . it has been observed that environmental enteropathy reverses on transfer to an environment with improved hygiene and sanitation (lindenbaum, 1968; lindenbaum et al., 1971) . clearly, the environment is an important factor in the development of human disease. the human gut contains a diverse microbial community, and it is postulated that many disorders of digestion and growth including diarrhea, inflammatory bowel disease, environmental enteropathy, and malnutrition could be related to perturbations in this biomass. significant effort has been made to understand the bacterial component of the human stool microbiome and the role it may play in human disease. for example, a dysbiosis or shift in the relative abundances of the bacterial taxa has been associated with obesity (turnbaugh et al., 2009) , inflammatory bowel disease (frank et al., 2007; sartor, 2008) , diabetes (larsen et al., 2010) , and necrotizing enterocolitis (mai et al., 2011) . the bacterial microbiome can be influenced by several factors including diet, geography, and phage populations. studies comparing the bacterial gut communities from healthy populations in different parts of the world show that the bacterial microbiome varies with geography (de filippo et al., 2010; yatsunenko et al., 2012) . a few studies have examined the human bacteriophage virome in stool. all of the studies reported to date have been done in healthy humans. these findings have shown that the phage virome changes rapidly in the first week of life (breitbart et al., 2008) , interpersonal variation is high while intrapersonal diversity is low (reyes et al., 2010) , diet plays an important role (minot et al., 2011) , and that the phage virome evolves quickly (minot et al., 2013) . by contrast, little is known about the enteric eukaryotic virome or the potential role it, and variation within it, may play in human disease. in monkeys, pathogenic infection with simian immunodeficiency virus is associated with expansion of the enteric virome, including both rna viruses and dna viruses (handley et al., 2012) . a limited number of studies have begun to define the eukaryotic stool viromes of people with diarrhea (finkbeiner et al., 2008; nakamura et al., 2009; phan et al., 2012; smits et al., 2014; van leeuwen et al., 2010) , non-polio acute flaccid paralysis (victoria et al., 2009) , and healthy adults (zhang et al., 2006) . however, a limitation of the previous human studies is that none have explicitly compared eukaryotic viromes between geographic sites, disease states or age groups, and thus it is not known what factors might influence the composition of the human virome. acute diarrhea is one of the leading causes of mortality worldwide, and viruses are known to play a major etiologic role. as a significant fraction of cases are of unexplained etiology, many metagenomic studies have focused upon identifying novel candidate microbial agents. because previous metagenomic studies demonstrated significant virus diversity in patients with diarrhea (finkbeiner et al., 2008) we focused this study on comparing the eukaryotic virus populations in stools of children with diarrhea collected from two different locations, melbourne, australia and the northern territory, australia. metagenomic sequencing was performed on 43 stool samples from melbourne and 44 stool samples from northern territory. the melbourne and northern territory cohorts were 53% and 50% female, respectively (p¼non-significant). the average age of the melbourne cohort was 25.4 months, while the average age of the northern territory cohort was 15.7 months (p¼0.002 (t-test)). 454 sequencing of the melbourne and northern territory samples yielded 646,630 and 406,252 total reads and 76,347 and 118,420 unique reads respectively. the average sequence length of the melbourne and the northern territory samples was 339 and 329 respectively. 83.6% of the unique reads possessed detectable similarity to sequences in genbank while the remaining 16.4% of reads had no significant similarity to sequences in genbank. of the 162,784 classifiable sequences 2.5% were viral, 75.8% bacteria, 4.4% phage, 8.9% fungi, 7.0% human, and 1.4% other (plant, fish, etc.) . further breakdown of these statistics by cohort is shown in table 1 . since all of the stool samples sequenced were from children with acute diarrhea, it was anticipated that a known diarrhea virus would be detected in many of the samples. twenty-four (28%) of the 87 samples contained sequences derived from the families reoviridae, caliciviridae, adenoviridae or astroviridae. strikingly, sequences from many additional virus families were detected in these samples as well as in samples that did not contain sequences from the four canonical diarrheagenic virus families. the most commonly detected virus family was the anelloviridae, which was present in 37 samples. in total, viral sequences were identified in 57 (66%) of the 87 australian samples while 35/87 (40%) of the samples had sequences from 2 or more viral families (supplementary table 1 ). thirty-five of the 44 (80%) samples from the northern territory had one or more viral families detected which was more than the 22 of the 43 (51%) samples from melbourne that had virus detected. one sample from northern territory contained sequences from 8 different viral families (fig. 1a) . overall, sequences from 22 different viral families were detected in these 87 samples. these included viruses from families known to reside in the gastrointestinal tract such as picornaviridae (pallansch and roos, 2007) , anelloviridae (okamoto et al., 1998) , circoviridae (li et al., 2010) , and orthomyxoviridae (wootton et al., 2006) and known plant viruses, including members of the families betaflexiviridae, bromoviridae, endornaviridae, and virgaviridae, consistent with previous studies that reported detection of plant viruses (finkbeiner et al., 2008; victoria et al., 2009; zhang et al., 2006) . in addition, viruses not previously thought to have an enteric reservoir were detected. for example, human parainfluenza 3 was detected in one sample. the most common viral families detected were anelloviridae (37 samples), picornaviridae (26 samples) and adenoviridae (10 samples). as with all sequencing based studies, our findings are limited by the depth of sequencing achieved. it is possible that deeper sequencing may detect additional viruses present at low abundance. a number of samples contained what are likely to be novel viruses that shared only very limited sequence identity with known viruses. for example, one sample contained 110 unique sequences with limited similarity to viruses in the order of picornavirales. assembly of these reads and sequences that did not share detectable sequence similarity with anything in the database yielded 9 contigs that shared highest sequence similarity with viruses in the order picornavirales. the longest contig of 9898 nt (601 reads, 18 â coverage) shared only 28% amino acid identity to the israel acute paralysis virus of bees and is likely to be almost the complete genome. a second contig of 7748 â nt (322 reads, 12 â coverage) shared 28% amino acid identity to the kashmir bee virus. the two novel viruses shared 82% nt identity with each other. these contigs have been deposited in genbank [accession numbers kj420969-kj420970] . in other samples, sequences were also identified from divergent dna and rna viruses, including small dna viruses from the family anelloviridae and rna virus families including endornaviridae, betaflexiviridae, partitiviridae, and virgaviridae. it is unknown if these newly described viruses are capable of infecting humans or if they are dietary passengers that infect food ingested by the individual. for each stool specimen, it would be ideal to define the number of distinct virus species present and the relative abundance of each species. however, a number of technical challenges and limitations precluded this form of analysis. first, highly variable coverage levels of the viruses ranging from a single read to hundreds of reads of a virus were obtained, presumably due to variations in viral load as well as the relative abundance of the virus compared to other non-viral nucleic acid molecules in the sample. for samples with low coverage levels, it is impossible to determine whether multiple reads that align to different regions of the same reference virus are derived from the same or related but distinct viruses; therefore, it is challenging to accurately define the number of virus species present. furthermore estimation of the viral load or copy number from the number of reads obtained is complicated by the sequence independent pcr step, which may introduce amplification biases. due to these considerations, we conservatively chose to define viral diversity based on the presence or absence of sequences derived from each of the 95 virus families defined by the ictv. therefore, the virome of an individual sample was defined as the number and type of viral taxa with at least one representative sequence present in the sample. these conservative criteria will likely underestimate the true viral diversity of the sample. similar analyses were also performed by quantifying the presence or absence of sequences from each virus genus. to assess whether gender influences the virome, we compared the number of viral families detected per sample by gender in the northern territory and melbourne cohorts and as an aggregate cohort. there was no statistically significant difference (wilcoxon) in the number of viral families per sample by gender (p ¼0.912 (regardless of location), p ¼0.444 (melbourne), p¼ 0.952 (northern territory)). we then assessed if there were gender differences at the individual virus family level. no individual virus family had a statistically significant difference. to determine if the virome differs by geographic location we compared the number of viral families and genera per sample in each location (fig. 1 ). diarrhea samples from the northern territory contained more viral families per sample than diarrhea from melbourne (p¼ 0.0002 (wilcoxon)). this was also true at the genus level (p ¼ 0.0003 (wilcoxon)). to control for differences for the larger number of unique reads generated in the northern territory samples compared to the melbourne samples (table 1) , we randomly selected unique reads from the northern territory samples to achieve the same sampling depth of the melbourne samples 100 times. all 100 iterations showed that samples from the northern territory contained more viral families (and viral genera) per sample than the samples from melbourne (p o0.05 (wilcoxon)). this result demonstrates that the significant difference between northern territory and melbourne is not due to the higher number of unique reads obtained for northern territory samples. to determine if a particular viral family drove this difference we compared the prevalence of each individual virus family in the two cohorts. the highly abundant anelloviruses were detected with similar frequencies in the two cohorts (northern territory, 52.3%, melbourne, 32.6%; (p ¼0.083 (fisher's exact))). two families, the picornaviridae (p¼ 0.0003 (fisher's exact)) and the adenoviridae (p¼ 0.015 (fisher's exact)) were statistically different between the two locations. as there are many picornaviruses known to infect humans, we then determined which picornavirus genera were present. sequences from the following genera in the family picornaviridae were detected: cardiovirus, enterovirus, klassevirus, kobuvirus, and parechovirus ( table 2 ). the genus enterovirus was the most frequently detected and was the only one that was statistically different between the two locations (p o0.001 (fisher's exact)). as the average age of the two cohorts was statistically significantly different (25.4 months (melbourne) vs. 15.7 months (northern territory)), we evaluated if the difference in age was confounding the difference seen by location. we assessed if there was a correlation between age and the total number of viral families present. no correlation was detected in either northern territory (spearman, rho à 0.271 (p ¼0.075)) or melbourne (spearman, rho à 0.113 (p¼ 0.472)). furthermore, to assess if the relationship between location and total number of viral families per sample was stable regardless of age, we performed a semiparametric analysis of covariance in which the number of total viral families per sample was rank transformed. after adjusting for age, both the number of viral families (p ¼ 0.001) and the number of viral genera (p ¼0.001) between the two sites were still statistically different. therefore, age differences between the northern territory and melbourne cohorts do not contribute significantly to the virome differences between the two regions. pcr of adenovirus, astrovirus, coronovirus, enterovirus, norovirus, and rotavirus in order to independently confirm the sequencing results, we used pcr to define the prevalence of the most frequently detected viruses for which pan-family or pan-genus primers could be used including: adenovirus, astrovirus, enterovirus, norovirus, and rotavirus. we additionally screened the samples for a family not detected by sequencing, the coronaviridae. there was good concordance between the sequencing results and the qpcr for adenovirus. all ten of the positive samples by sequencing were confirmed by our qpcr. additionally, there were two samples that were negative by sequencing, but positive by qpcr. the adenovirus qpcr was capable of detecting five copies per reaction (supplementary fig. 1a) . the two samples that sequencing failed to identify as adenovirus positive were the two with the lowest copy number by qpcr (6.9 â 10 2 and 1.2 â 10 3 copies/g stool). there were three samples sequencing positive for astrovirus. one of these samples was pcr positive. no samples were pcr positive and sequencing negative. one of the samples positive by sequencing and negative by pcr had 165 reads whose top blast hits were astroviruses. close inspection of the consensus sequence of these reads showed 3 and 4 mismatches in the forward and reverse primer regions, respectively, which is a likely explanation for why it was not detected by pcr. the other sample positive by sequencing had only a single read, which was not in the region targeted by the primers, so it is unknown if sequence variation in the primer binding sites could be an explanation for this sample as well. nineteen samples were positive by sequencing for the enterovirus genus, including 3 that contained rhinovirus species and 16 that contained enterovirus species. we utilized a pan-enterovirus assay (verstrepen et al., 2001 ) that does not detect rhinovirus species to evaluate concordance. twelve of the 16 sequencing positive samples were qrt-pcr positive. the sensitivity limit of the enterovirus qrt-pcr assay was 500 copies as defined by its standard curve ( supplementary fig. 1b) , so it is possible that these four discordant samples were below the limit of detection. in addition, there was one sample that was qrt-pcr positive, but sequencing negative. six samples were positive by sequencing for norovirus gii. all of these samples were detected by qrt-pcr. there was one additional sample that was qrt-pcr positive but sequencing negative. no samples were sequencing or qrt-pcr positive for norovirus gi. there were eight samples sequencing positive for rotavirus. all eight of these samples were rt-pcr positive as well. additionally, there were four samples from the northern territory that were positive for rotavirus by rt-pcr, but negative by sequencing. the sequencing based analysis of rotaviruses was not statistically different between the two sites (p ¼0.2658). however, because the rt-pcr assay detected four additional positive samples, rotavirus prevalence as measured by rt-pcr was statistically significant (p ¼0.0264). one factor that complicates interpretation of this difference is that fact that different vaccines were used in the two different regions. in melbourne rotateq (merck), a live human-bovine pentavalent reassortant vaccine is used, while rotarix (glaxosmithkline), a live attenuated human vaccine strain, is used in the northern territory. the stool viral load of rotarix has been found to be 100 fold higher than children receiving rotateq (hsieh et al., 2014) which may have impacted the differences seen. no samples were positive by sequencing for coronaviruses. one sample was positive by rt-pcr. this amplicon was cloned and sanger sequenced and confirmed to be coronavirus 229e, which has been reported in diarrhea samples previously (risku et al., 2010) . overall there was good concordance between the sequencing data and the pcr screening. the level of concordance observed was similar to that observed in other studies comparing next generation sequencing to pcr (wylie et al., 2012) . to explore whether the differences seen in adenovirus and enterovirus, could be seen when examining a larger number of samples, we tested additional samples from melbourne and northern territory. further testing for rotavirus was not pursued as the vaccination status of each subject was unknown and could be a potential confounder. we tested samples from melbourne (n ¼159) and northern territory (n ¼165), including all of the samples that were analyzed by metagenomic sequencing. enteroviruses were more prevalent in the northern territory (18.0%) compared to melbourne (0.6%) by qrt-pcr and adenoviruses were more abundant in the northern territory (18.8%) than in melbourne (6.3%) by pcr confirming the metagenomic results. to explore the generalizability of these results, we assessed the prevalence of enteroviruses and adenoviruses in additional diarrhea cohorts from seattle (n ¼80) and the gambia (n¼ 160) (table 3a -c), which yielded enterovirus positivity rates of 0% and 28.8%, respectively. adenovirus positivity was even higher in the gambia (41.9%), while comparable adenovirus frequencies were detected in seattle (21.3%), and northern territory (18.8%). we examined the diversity of viral communities in stools from 87 children with diarrhea from two different geographic locations collected in the same time period using standardized inclusion criteria at both sites. this study design enabled us to minimize variables while defining and comparing the viromes of patients collected from the two sites. many virus families were equally prevalent in the two cohorts, including common diarrhea causing viruses such as norovirus (caliciviridae) and astrovirus (astroviridae). in addition, some viruses that have no known pathogenic properties, such as anelloviruses, were frequently detected in both cohorts. viruses with limited identity to non-human viruses (ex. israeli acute paralysis virus) were detected. it is unknown whether these represent novel human pathogens or are dietary passengers. other studies of reclaimed water (rosario et al., 2009) , raw sewage (cantalupo et al., 2011; ng et al., 2012) , and stool (victoria et al., 2009 ) have also found invertebrate viruses. moreover, it is striking that many samples had multiple viral families detected beyond the viruses typically associated with diarrhea. most notable was the sample with 8 different viral families detected which raises the question of whether there may be some synergy between viruses (i.e. do some infections make the gut more permissive to infection by additional viruses?). furthermore, these observations suggest the possibility that viruses are present in the human gut in a fashion analogous to the well-established bacterial microbiome. longitudinal studies are needed to shed further light on the nature of the eukaryotic virome. we demonstrated that the diarrhea samples from the northern territory had more viral families per sample than the samples from melbourne. further analysis demonstrated that the families adenoviridae, picornaviridae, and reoviridae were more common in the northern territory than in melbourne. furthermore, we found that within the picornaviruses the genus enterovirus was the most commonly detected and was preferentially found in the northern territory. these results clearly demonstrate that enteric viromes in patients with diarrhea can differ between two different geographic sites. there are many possible explanations for the observed population-specific differences in the human diarrhea stool viromes. it is plausible that environmental factors such as diet, living conditions, water quality, hygiene and/or socioeconomic status could dictate the composition of the stool virome. the samples from melbourne mainly represent patients living in a westernized and urban setting, while the samples from the northern territory represent patients largely from remote communities scattered across a large area. health indicators among children in the northern territory, including for a range of infectious diseases, are worse than for children elsewhere in australia (gracey and king, 2009; ruben and walker, 1995) . the paucity of enteroviruses in the seattle cohort and their comparative abundance in children from the gambia is consistent with the model that these viruses are associated with children living in impoverished conditions. culture-based studies from several decades ago of healthy children also demonstrated a greater frequency of excretion of enteroviruses among persons of lower socioeconomic status (honig et al., 1956; otatume and addy, 1975) . additionally, a recent study in seattle showed no enterovirus detection in stools from children with diarrhea (braun et al., 2012) . from the sequencing data there was not a statistically significant difference in the prevalence of rotavirus between melbourne and the northern territory. rt-pcr confirmed the presence of rotavirus in these samples and identified four additional positive samples in the northern territory. these additional positive samples then made rotavirus statically different between melbourne and the northern territory. it is possible that these differences are related to the different rotavirus vaccinations given, the proportion of children that were vaccinated, or differences in vaccine efficacy. while we observed differences in the prevalence of adenoviruses between melbourne and the northern territory, broader analysis of additional diarrhea cohorts (seattle and the gambia) yielded a more complex picture. as with the enteroviruses, the highest rate of adenovirus positivity was in the gambia (42%) and the lowest was in melbourne (6%). the 42% adenovirus prevalence in the gambia is similar to previously described reports from some other locations (france and kenya) (berciaud et al., 2012; magwalivha et al., 2010) . unlike the situation with enteroviruses, adenoviruses were detected in approximately equal frequencies in northern territory and seattle. further studies are needed in larger populations, over multiple years, in these sites to ascertain whether these observations truly reflect geographic differences, or whether additional factors such as seasonality, year-to-year variation, or community outbreaks contribute to these findings. this study demonstrates major inter-population differences in the human stool eukaryotic virome. the potential etiologic role of the human stool virome in disorders with profound geographic differences remains to be seen. in one hypothesis, these disorders, which include environmental enteropathy, oral vaccine failures, autoimmune diseases, inflammatory bowel disease, and allergic conditions, could be consequences of perturbations in the stool virome. to evaluate this and other hypotheses, it will be important to define the childhood stool virome in other geographic locations as well in health and other states of disease. all stool samples were obtained with parental/guardian consent under protocols approved by human studies committees from seattle children's hospital, university of maryland baltimore, the central australian human research ethics committee, the human research ethics committee of the menzies school of health research, the northern territory department of health and families, the royal children's hospital, and the joint ethics committee of the gambia government/medical research council unit. the use of deidentified samples from the previous studies was approved by the human studies committee of washington university in st. louis. stool samples were collected from children less than 5 years old admitted to the royal children's hospital, melbourne, victoria, australia and alice springs hospital or royal darwin hospital northern territory, australia, with acute diarrhea between december 2009 and september 2010. these samples were collected under the same inclusion criteria and were archived at à 70 o c in melbourne. rotavirus vaccine was part of the routine childhood vaccinations throughout the collection period. in melbourne rotateq (merck), a live human-bovine pentavalent reassortant vaccine is used, while rotarix (glaxosmithkline), a live attenuated human vaccine strain is used in the northern territory. 165 samples from the northern territory and 159 samples from melbourne were randomly selected for this study. of these, 44 samples from northern territory and 43 samples from melbourne were sequenced. stool samples were collected from children less than 5 years old who were evaluated for self-defined diarrhea at the seattle children's hospital emergency department from 2003 to 2005 as part of a prospective study (denno et al., 2012) . eighty samples were randomly selected from this cohort for the adenovirus and enterovirus qpcr prevalence phase of this study. stool samples were collected from children with moderate-tosevere diarrhea less than 5 years old from 2008 to 2009 as part of the global enteric multi-center study (gems) (kotloff et al., 2012) . diarrhea was defined as new (onset after 47 diarrhea free days), acute (onset within the previous 7 days) and having one of the following criteria: sunken eyes, loss of skin turgor, intravenous hydration administered or prescribed, visible blood in loose stools, or admission to hospital with diarrhea (kotloff et al., 2013) . samples were collected from children located in the upper river division (urd), a rural region in the gambia. 160 diarrhea stools were randomly selected from this cohort for the adenovirus and enterovirus qpcr prevalence phase of this study. frozen stool was resuspended in 6 volumes of sterile pbs and centrifuged. resulting supernatants were passed through a 0.45 um filter. total nucleic acids (rna and dna) were isolated from the filtrate using the ampliprep dna extraction machine (roche). in order to evaluate samples for both rna and dna viruses the total nucleic acids were randomly amplified. specifically, rna present in the sample was converted to cdna by reverse transcription. following second strand synthesis, the doublestranded cdna and any single-stranded or double-standed dna were then pcr amplified using unique sequence primers which were used as a barcode to assign sequences to their corresponding sample (wang et al., 2003) . the products were then pooled, adapter ligated and sequenced using the 454 gs flx titanium platform (454 life science). sequences were analyzed using a custom bioinformatics pipeline (virushunter) (zhao et al., 2013) . briefly, sequences were deconvoluted based on their barcode (supplemental table 2 ). to reduce the risk of cross contamination between samples barcodes were required to be present at both ends of the read with a perfect match and was trimmed before any further analysis. to reduce the number of near identical sequences introduced from pcr bias, sequences were clustered using cd-hit (li and godzik, 2006) requiring 95% identity over 95% of the sequence. the longest sequence from each cluster was chosen as the representative unique sequence. sequences were then masked by repeatmasker (http://www.repeatmasker.org). if a sequence did not contain a stretch of at least 50 consecutive non-"n" nucleotides or if greater than 40% of the total length of the sequence was masked, it was removed from further analysis. the remaining high quality unique sequences were then sequentially compared to (1) human genome using blastn, (2) nt database by blastn and (3) nr database using blastx. minimum e-value cutoffs of 1 â 10 à 10 for blastn and 1 â 10 à 5 for blastx were used. sequences were then binned based on the top blast hit to human, mouse, fungal, bacterial, phage, viral, and other. sequences aligning to both a virus and another kingdom with the same e-value were classified as ambiguous and not subjected to further analysis. viral sequences were binned into families based on the taxonomical identity of the best blast hit as defined by the ncbi taxonomy database. results from this approach have been previously compared directly to other similar blast-based taxonomic classifiers such as megan (handley et al., 2012) . samples were considered positive for a family (or genus) if at least one unique read was present. from previous experience with stool virome analysis, alignments to particular viral families in the ncbi taxonomy database (herpesviridae, iridoviridae, mimiviridae, phycodnaviridae, poxviridae, unclassified dsdna viruses, and environmental samples) have a high frequency of false positives, usually due to low complexity and/or repetitive sequence motifs yielding artefactual alignments. thus, all sequences aligning to these families were manually evaluated and those deemed to be false positives were removed from further analysis. assembly was performed on one sample, which was noted to contain 110 unique sequences with limited similarity to viruses in the order of picornavirales. sequences identified as viral as well as sequences that had no significant hit to any sequence in the nr and nt databases were assembled using newbler (454 life sciences, branford, ct) with default parameters. a previously described pan-enterovirus taqman assay targeting conserved sequences in the 5 0 utr region of the genome (verstrepen et al., 2001 ) was used to assess enterovirus prevalence in all cohorts. the qrt-pcr was performed using the one-step rt-pcr taqman kit (applied biosystems). the 25 μl reaction included 5 μl of extracted sample, 12.5 pmol of each primer, and 6.25 pmol of probe. the following cycling conditions were used: 48 1c for 30 min, 95 1c for 10 min, 40 cycles of 95 1c for 15 s and 60 1c for 1 min. to generate a standard curve for this assay, in vitro transcribed rna was generated from a plasmid containing the region of interest using maxiscript (ambion) per the manufacturer's protocol. serial dilutions of this in vitro transcribed rna from 5 â 10 6 to 5 copies were used to generate a standard curve and a limit of detection of 500 copies was defined (supplemental fig. 1b) . samples were tested in a 96-well plate format with 8 negative controls and 1 positive control per plate. the threshold of all plates was set at a standard value, and samples were counted as positive if their cycle threshold was o38.00. a pan-adenovirus taqman assay that detects all adenovirus species was used to assess adenovirus prevalence in all cohorts (jothikumar et al., 2005) . qpcr was performed with the taqman universal pcr master mix (applied biosystems). the 25 μl reaction included 5 μl of extracted sample, 12.5 pmol of each primer, and 4 pmol of probe. the following cycling conditions were used: 50 1c for 2 min, 95 1c for 10 min, 45 cycles of 95 1c for 15 s and 60 1c for 1 min. to establish a standard curve for this assay, a plasmid containing the region of interest was used in serial dilutions of 5 â 10 6 to 5 copies and a limit of detection of 5 copies was defined ( supplementary fig. 1a ). samples were tested in a 96-well plate format with 8 negative controls and 1 positive control per plate. the threshold of all plates was set at a standard value, and samples were counted as positive if their cycle threshold was o38.00. we modified a previously described assay to detect norovirus gi and norovirus gii separately (rolfe et al., 2007) . this was used to independently confirm our sequencing results. the qrt-pcr was performed using the one-step rt-pcr taqman kit (applied biosystems). the primers and probes were as previously described (rolfe et al., 2007) except ring2p was labeled with vic instead of joe. the 25 μl reaction included 5 μl of extracted sample, 12.5 pmol of each primer, and 6.25 pmol of probe. the following cycling conditions were used: 50 1c for 30 min, 95 1c for 10 min, 50 cycles of 95 1c for 15 s and 56 1c for 1 min. to generate a standard curve for this assay, in vitro transcribed rna was generated from plasmids containing the regions of interest using megascript (ambion) per the manufacturer's protocol. serial dilutions of these in vitro transcribed rna from 5 â 10 6 to 5 copies were used to generate a standard curve and a limit of detection of 5 copies was defined ( supplementary fig. 1c and d) for both assays. samples were tested in a 96-well plate format with 8 negative controls and 1 positive control per plate. the threshold of all plates was set at a standard value, and samples were counted as positive if their cycle threshold was o38.00. a previously described astrovirus consensus rt-pcr was used to independently confirm our sequencing results (finkbeiner et al., 2009) . qiagen onestep rt-pcr kit was used to screen 3 μl of extracted material from each sample using the consensus primers sf0073 (5 0 -gattggactcgatttgatgg-3 0 ) and sf0076 (5 0 -ctggcttaacccacattcc-3 00 ) which target the orf 1b (rna polymerase) of astroviruses under the following cycling conditions: 30 min rt step, 94 1c hold for 10 min, followed by 40 cycles of 94 1c for 30 s, 52 1c for 30 s, and 72 1c for 50 s. to independently confirm our sequencing results a previously described rt-pcr to detect group a rotavirus was used (rao et al., 1995) . rt-pcr screening was done using the qiagen onestep rt-pcr kit. each 25 μl reaction used 3 μl of extracted material from each sample and forward (5 0 -atgctcaagatggagtct-3 0 ) and reverse (5 0 -ggtcacataacgcccctat-3 0 ) primers which target nonstructural protein (nsp) 3 under the following cycling conditions: 30 min rt step, 94 1c hold for 10 min, followed by 40 cycles of 94 1c for 30 s, 56 1c for 30 s, and 72 1c for 1 min. a previously described pan-coronavirus rt-pcr was used to independently confirm our sequencing results (vijgen et al., 2008) . qiagen onestep rt-pcr kit was used to screen 3μl of extracted material from each sample using the primers cor-fw (5 0 -acw-carhtvaayytnaartaygc-3 0 ) and cor-rv (5 0 -tcrcayttdggr-tartccca-3 0 ) which target a 251 bp region of the rna polymerase of coronaviruses under the following cycling conditions: 30 min rt step, 94 1c hold for 10 min, followed by 50 cycles of 94 1c for 30 s, 48 1c for 30 s, and 72 1c for 1 min. pcr amplicons were cloned into pcr4 (invitrogen) and sequenced using standard sanger sequencing technology. two-tailed p values were derived using wilcoxon rank-sum, chi-square, fisher's exact, or t-tests, as indicated. wilcoxon ranksum test, a non-parametric test, was used to compare ordinal data. fisher's exact test was used for categorical data (ex. presence or absence). assessment of correlation was done using spearman rank. for the site comparisons of qpcr results, chi-square with sidak adjustment for multiple comparisons was used. statistical analyses were performed using sas (cary, nc) software version 9.2. sequences (human reads removed) have been uploaded to the mg-rast (http://metagenomics.anl.gov/) server under the project name australian pediatric diarrhea [mg-rast id 4529846.3-4529932.3 ]. adenovirus infections in bordeaux university hospital 2008-2010: clinical and virological features human parechovirus and other enteric viruses in childcare attendees in the era of rotavirus vaccines viral diversity and dynamics in an infant gut chronic t cell-mediated enteropathy in rural west african children: relationship with nutritional status and small bowel function raw sewage harbors diverse viral populations impact of diet in shaping gut microbiota revealed by a comparative study in children from europe and rural africa diarrhea etiology in a pediatric emergency department: a case control study metagenomic analysis of human diarrhea: viral detection and discovery detection of newly described astrovirus mlb1 in stool samples from children molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases 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circoviruses infect farm animals and are commonly found in human and chimpanzee feces cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences small intestine dysfunction in pakistanis and americans resident in pakistan recovery of small-intestinal structure and function after residence in the tropics. i. studies in peace corps volunteers high prevalence of species d human adenoviruses in fecal specimens from urban kenyan children with diarrhea fecal microbiota in premature infants prior to necrotizing enterocolitis geography of intestinal permeability and absorption rapid evolution of the human gut virome the human gut virome: inter-individual variation and dynamic response to diet direct metagenomic detection of viral pathogens in nasal and fecal specimens using an unbiased high-throughput sequencing approach high variety of known and new rna and dna viruses of diverse origins in untreated sewage fecal excretion of a nonenveloped dna virus (ttv) associated with posttransfusion non-a-g hepatitis ecology of enteroviruses in tropics. i. circulation of enteroviruses in healthy infants in tropical urban area enteroviruses: polioviruses, coxsackieviruses, echoviruses, and newer enteroviruses acute diarrhea in west african children: diverse enteric viruses and a novel parvovirus genus comparative nucleotide and amino acid sequence analysis of the sequence-specific rnabinding rotavirus nonstructural protein nsp3 viruses in the faecal microbiota of monozygotic twins and their mothers detection of human coronaviruses in children with acute gastroenteritis an internally controlled, one-step, real-time rt-pcr assay for norovirus detection and genogrouping metagenomic analysis of viruses in reclaimed water malnutrition among rural aboriginal children in the top end of the northern territory microbial influences in inflammatory bowel diseases new viruses in idiopathic human diarrhea cases, the netherlands hay fever, hygiene, and household size a core gut microbiome in obese and lean twins human picobirnaviruses identified by molecular screening of diarrhea samples rapid detection of enterovirus rna in cerebrospinal fluid specimens with a novel single-tube real-time reverse transcription-pcr assay metagenomic analyses of viruses in stool samples from children with acute flaccid paralysis a pancoronavirus rt-pcr assay for detection of all known coronaviruses viral discovery and sequence recovery using dna microarrays detection of human influenza virus in the stool of children sequence analysis of the human virome in febrile and afebrile children human gut microbiome viewed across age and geography rna viral community in human feces: prevalence of plant pathogenic viruses identification of novel viruses using virushunter-an automated data analysis pipeline this work was funded in part by national institutes of health [grant u54 ai057160] supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2014.09.012. key: cord-267532-5rnqd9mb authors: zhang, xuming; hinton, david r.; park, sungmin; parra, beatriz; liao, ching-len; lai, michael m.c.; stohlman, stephen a. title: expression of hemagglutinin/esterase by a mouse hepatitis virus coronavirus defective–interfering rna alters viral pathogenesis date: 1998-03-01 journal: virology doi: 10.1006/viro.1997.8993 sha: doc_id: 267532 cord_uid: 5rnqd9mb abstract a defective-interfering (di) rna of mouse hepatitis virus (mhv) was developed as a vector for expressing mhv hemagglutinin/esterase (he) protein. the virus containing an expressed he protein (a59-de-he) was generated by infecting cells with mhv-a59, which does not express he, and transfecting thein vitro-transcribed di rna containing the he gene. a similar virus (a59-de-cat) expressing the chloramphenicol acetyltransferase (cat) was used as a control. these viruses were inoculated intracerebrally into mice, and the role of the he protein in viral pathogenesis was evaluated. results showed that all mice infected with parental a59 or a59-de-cat succumbed to infection by 9 days postinfection (p.i.), demonstrating that inclusion of the di did not by itself alter pathogenesis. in contrast, 60% of mice infected with a59-de-he survived infection. heor cat-specific subgenomic mrnas were detected in the brains at days 1 and 2 p.i. but not later, indicating that the genes in the di vector were expressed only in the early stage of viral infection. no significant difference in virus titer or viral antigen expression in brains was observed between a59-de-heand a59-de-cat-infected mice, suggesting that virus replication in brain was not affected by the expression of he. however, at day 3 p.i. there was a slight increase in the extent of inflammatory cell infiltration in the brains of the a59-de-he-infected mice. surprisingly, virus titers in the livers of a59-de-he-infected mice were 3 log10lower than that of the a59-de-cat-infected mice at day 6 p.i. also, substantially less necrosis and viral antigen were detected in the livers of the a59-de-he-infected mice. this may account for the reduced mortality of these mice. the possible contribution of the host immune system to this difference in pathogenesis was analyzed by comparing the expression of four cytokines. results showed that both tumor necrosis factor-α and interleukin-6 mrnas increased in the brains of the a59-de-he-infected mice at day 2 p.i., whereas interferon-γ and interleukin-1α mrnas were similar between a59-de-heand a59-de-cat-infected mice. these data suggest that the transient expression of he protein enhances an early innate immune response, possibly contributing to the eventual clearance of virus from the liver. this study indicates the feasibility of the di expression system for studying roles of viral proteins during mhv infection. the development of infectious, full-length cdna clones of viral rna has provided a powerful tool for understanding the structure and function of individual viral genes and for studying their roles in viral pathogenesis. unfortunately, an infectious cdna clone is currently not available for some rna viruses with a large genome, such as coronaviruses. we have recently developed an alternative approach, namely, a defective±interfering (di) rna (makino et al. 1985 (makino et al. , 1991 , which functions like a minigenome, as an expression vector . such a vector can express viral or foreign proteins, including cytokines (liao et al. 1995; zhang et al. 1997) , thus allowing more critical analysis of the functional role of these proteins in coronavirus pathogenesis. mouse hepatitis virus (mhv), a murine coronavirus, is a member of the coronaviridae. it contains a singlestrand, positive-sense rna genome of 31 kb (lee et al. 1991) . in addition to the genomic-length rna, six or seven subgenomic mrnas are synthesized in infected cells. these rnas are conested at the 3ј end and extend toward the 5ј end to different lengths at intergenic (ig) regions (lai et al. 1981) . each mrna contains a leader rna of approximately 70 nt at the 5ј end, which is identical to the leader of the genomic rna (lai et al. , 1984 . the ig sequence serves as an initiation site and is required for the synthesis of a subgenomic mrna. in general, each mrna translates only one protein from the 5ј-most open reading frame (orf) (lai and cavanagh, 1997) . mhv contains four or five structural proteins. the spike (s) protein is the major surface glycoprotein of the virion. it facilitates the attachment of virus to the cellular receptors, elicits the production of neutralizing antibodies, and induces cell fusion (sturman and holmes, 1983) . the membrane (m) glycoprotein is also associated with the viral envelope and, together with another viral envelope (e) protein, is required for virus assembly (bos et al. 1996; vennema et al. 1996) . the nucleocapsid (n) protein is a phosphorylated protein which together with the viral genomic rna forms the nucleocapsid. the hemagglutinin/esterase (he) protein is an optional envelope protein, present only in some mhv strains . it contains hemagglutination (receptor-binding) and acetylesterase (receptor-destroying) activities, similar to the he protein of influenza c virus (herrler et al. 1985 (herrler et al. , 1888 yokomori et al. 1989 ). the he protein binds sialic acidcontaining cell surface molecules. the precise role of the he protein in coronaviral infection is not clear. it has been shown that the he protein of bovine coronavirus induced the production of neutralizing antibodies and that inhibition of the esterase activity resulted in a 100-to 400-fold reduction in viral infectivity, suggesting an important role of the he protein in viral infectivity (deregt and babiuk, 1987; vlasak et al. 1988) . in contrast, no neutralizing antibody specific for the he protein of mhv has been identified , and expression of the he protein alone, without the s protein, is not sufficient to initiate mhv infection (gagneten et al. 1995 ). an mhv variant that expresses abundant he appeared to be more neurovirulent than the one that expresses little he (yokomori et al. 1995) . consistent with these data, passive immunization with he monoclonal antibodies protected mice from lethal infection and altered the pathogenicity . these findings suggest that the he protein contributes to viral neuropathogenicity by influencing either the rate of virus spread, viral tropism, or both (yokomori et al. 1995) . although five separate clonal populations expressing either high or low levels of he protein yielded identical results, no precise role of the he protein could be unequivocally determined because these virus variants may contain additional mutations in other viral genes. in a previous study (liao et al. 1995) , we expressed the he protein of mhv in cell culture using the mhv-di rna expression vector. in this system, the expression of the he-containing di rna requires the presence of a helper mhv, and the expressed he protein was shown to be at least in part incorporated into the virions. however, due to the lack of a specific packaging signal, the di rna cannot be efficiently packaged into virus particles; it can only be copackaged nonspecifically with the helper virus particles for a limited number of cell culture passages. the di rna eventually loses its association with the helper virus and its ability to replicate during serial passages in cell culture. therefore, the expression of the di rna is transient and does not involve a recombination between the helper virus genome and the di rna. in the present study, we used this di rna vector system to express the viral he gene in the cns of mice in the presence of an he-deficient helper virus. we found that the transient expression of the he protein by this vector altered viral pathogenesis. this study thus demonstrates unequivocally that he protein plays a role in mhv pathogenesis and further demonstrates the feasibility of using this di rna vector for in vivo expression of a foreign protein. it has previously been shown that mhv-a59 virus does not express he protein due to mutations in the transcription start signal and coding sequence shieh et al. 1989) . to assess the function of he protein, we were interested in determining if the expression of a functional he gene derived from jhm virus by the di rna vector could influence the pathogenesis of a59 virus (fig. 1 ). mice were infected ic with 1 ϫ 10 5 pfu each of a59-de-he, a59-de-cat, or parental a59. figure 2 shows that 100% of mice infected with a59-de-cat succumbed to infection by day 9 postinfection (p.i.), similar to mice infected with the parental a59 alone. these data demonstrate that inclusion of di rna that expresses a cat gene did not by itself alter the outcome of a59 infection. in contrast, 60% of mice infected with a59-de-he virus survived infection. mice infected with all three virus preparations showed similar signs of encephalitis (hunched back and ruffled fur) for the first 5 to 6 days p.i. subsequently, clinical symptoms gradually improved in those a59-de-he-infected mice that had survived infection; by 12 to 14 days p.i., the clinical signs were completely resolved and no mice subsequently succumbed to infection. these data demonstrate that the expression of the jhm virus he gene by the di rna vector substantially alters the pathogenesis of a59 virus infection. we have previously shown that the expression of genes from the di vector can be maintained for only a few passages in vitro (liao et al. 1995) . thus, it was not clear whether the he protein could be expressed from the di vector within the cns. to address this issue, rna was extracted from brains of infected mice at various times p.i. and analyzed by rt±pcr using primers which specifically detect he-containing rna (see materials and methods). since the he-containing subgenomic mrna is transcribed from the ig site within the vector rna and contains a leader sequence at the 5ј end, the pcr product corresponding to the subgenomic he mrna is distinguishable from that of the input di rna by their size (fig. 1) . the result shows that the subgenomic he mrna was undetectable in rna extracted from the brains of infected mice at 12 h p.i. (data not shown); however, it became detectable in rna samples prepared from the brains of a59-de-he-infected mice at 24, 36, and 48 h p.i. (table 1 shows data at 24 and 48 h p.i. only). no he mrna was detected in rna samples obtained on day 4 or 6 p.i. similarly, the mrna encoding cat could be detected in a59-de-cat-infected mice only at 24, 36, and 48 h p.i. (table 1 ). these data suggest that the he or the cat gene indeed could be expressed from the di rna in the brains during in vivo infection but that their expression was limited to the first 2 days p.i., consistent with its limited ability to be serially passaged in vitro (liao et al. 1995 ). because of the low level of mrna expression, the amounts of he-or cat-specific mrna were not quantitatively compared. to determine the basis for the reduction in mortality following a59-de-he infection compared to other viruses ( fig. 2) , virus titers in the brains of mice infected with a59-de-he were initially compared to mice infected with the parental a59 or a59-de-cat at 6 days p.i. based on the survival of most mice infected with a59-de-he, reduced viral replication in the cns was anticipated. however, only a slight (approximately 1 log 10 ) reduction in virus titer within the cns was noted at 6 days p.i. in a59-de-he-infected mice compared to mice infected with either a59-de-cat or parental a59 (table 1) . we reasoned that, since he protein expression was detectable only early in infection, a more significant reduction of virus titer in cns might be detectable at an earlier time of infection; thus, virus titers in the cns of mice infected with a59-de-he and a59-de-cat were compared at 1, 2, and 4 days p.i. similar to the titers found at 6 days p.i., no significant difference was detected at any of the early time points (table 1) . therefore, although he expression in the cns was detected for the first 2 days p.i., there appeared to be no significant difference in virus replication in the cns between a59-de-he-and a59-de-catinfected mice. the pathogenicity of a59-de-he and a59-de-cat in the cns was then examined to determine the basis for the difference in mortality between the two groups. brains and spinal cords were examined at 1, 2, 3, 4, and 6 days p.i. for mononuclear cell infiltrates and distribution of virus-infected cells. consistent with the similar amount of virus replication, no differences were detected in either the extent or the distribution of either mononuclear cell infiltration or viral antigen at 1 or 2 days p.i. in two separate experiments. however, at 3 days p.i., there was a slight but reproducible increase in the extent of inflammatory cell infiltration and reduced viral antigen in the mice infected with a59-de-he compared to mice infected with a59-de-cat (fig. 3) . quantitation of viral antigen-positive cells revealed approximately one-third fewer positive cells in the a59-de-he-infected mice than in those in a59-de-cat-infected mice. this difference was no longer noticeable by 6 days p.i. these data suggest that expression of the he gene within the cns resulted in a transient increase in cns inflammation and reduction in viral antigen. this difference was transient and minor and did not appear sufficient to explain the increased survival of mice infected with a59-de-he. survivors of a59-de-he infection examined at 21 days p.i. showed mononuclear cell infiltrations, presence of viral antigen, and extensive demyelination, consistent with the pathology reported for mice infected with sublethal doses of parental a59 (lavi et al. 1984) . to ensure that the altered pathogenesis of a59-de-he was due to transient he expression but not to the selection of a variant a59 virus, virus was recovered from a59-de-he-infected mice at 6 days p.i. this time point was chosen based on the absence of detectable he gene expression in the cns. virus isolated (designated a59-r) was propagated once in dbt cells and used to infect mice using the same dose as that in the previous experiments (1 ϫ 10 5 pfu). all mice infected with this virus succumbed to infection by day 9. furthermore, the amount of virus present in the cns of these mice at day 6 was equivalent to the virus detected in mice infected with a59-de-he, a59-de-cat, or parental a59 (table 1) . these combined data suggest that the difference in mortality following ic infection with a59-de-he was not due to alterations in virus replication within the cns, altered cellular tropism, or selection of an attenuated variant. the a59 strain of mhv is both neurotropic and hepatotropic (lavi et al. 1986 ). since differences in virus replication and pathogenesis of a59-de-he in the cns did not appear to be sufficient to account for the differences in mortality, we explored the possibility that the decreased mortality in a59-de-he-infected mice was due to altered hepatotropism. therefore, virus replication and pathological changes in the livers of mice infected with a59-de-he and a59-de-cat were compared. table 2 shows that at day 1 p.i., there was approximately 1 log 10 less infectious virus recovered from the liver of mice infected with a59-de-he than from mice infected with a59-de-cat. however, no differences in virus replication were found at either day 2 or 4 p.i. surprisingly, by day 6 p.i., when the a59-de-cat-infected mice were beginning to succumb to infection, there was a substantial reduction (3 log 10 ) in the amount of infectious virus in the liver of a59-de-he-infected mice (table 2 ). this difference was reproducible in three independent experiments. histological examination of the livers of mice infected with a59-de-cat showed increasing viral antigen and inflammatory changes with time throughout the infection. by day 6 p.i., the livers of these mice showed evidence of extensive necrosis with prominent viral antigen (figs. 4a and 4b). similar hepatic necrosis was found following ic infection with both parental a59 and a59-r (data not shown). a59-de-he-infected mice also showed similar inflammatory changes, hepatic necrosis, and viral antigen in the livers at day 1 or 2 p.i., compared to a59-de-cat-infected mice. however, by day 3 p.i., fewer viral antigen-positive cells and reduced necrosis were noted in mice infected with a59-de-he. by day 6 p.i., there was a significant reduction in the amount of viral antigen (fig. 4d) , consistent with decreased titer of infectious virus (table 2 ) and substantially less necrosis (fig. 4c) , in contrast to the almost confluent necrosis found in the mice infected with either parental a59 or a59-de-cat (figs. 4a and 4b) . again, the a59 virus (a59-r) recovered from the cns at day 6 p.i. did not differ from the original a59 virus. these data suggest that the transient expression of the he gene in the cns affected the virus replication and subsequent pathology in the liver. this is likely the basis for the reduced mortality of a59-de-he-infected mice. to determine if the activation of the mouse fibrinogen-like protein (fgl-2) gene was associated with the fulminent hepatitis induced by ic infection with the a59-de-cat virus, as has been shown to be the case in the hepatitis induced by mhv-3 (parr et al. 1995) , livers were examined for the presence of fgl-2 gene mrna by rt±pcr. no difference in expression of fgl-2 mrna was detected between a59-de-he-and a59-de-cat-infected mice (data not shown). histologic examination of the livers did not reveal coagulopathy typically induced by mhv-3 infection (parr et al. 1995) . we next examined the virus titers in the spleen following ic infection to determine if the difference in virus titer in the livers of a59-de-he-infected mice was due to a difference in virus spread from the cns. a slight reduction in virus titer in the spleens of mice infected with a59-de-he on day 1 p.i. was found compared to mice infected with a59-de-cat (table 3) . however, no difference was detected at day 2 or 4 p.i., and no infectious virus could be recovered from the spleens of either group by day 6 p.i. (table 3 ). these data suggest that a59 replicated poorly and only transiently in the spleen following ic infection; nevertheless, this small amount of virus in the spleen may have spread to the liver via a hematogenous route. therefore, a59-de-he and a59-de-cat appeared to spread similarly from the cns to spleen and liver. to determine if a59-de-he exhibited reduced hepatotropism, compared to a59-de-cat and parental a59, we examined virus replication in the liver following intraperitoneal (ip) infection. groups of four mice were infected with 1 ϫ 10 5 pfu of each virus ip. surprisingly, at 6 days p.i., no infectious virus was recovered from the livers or cns of any of the three groups and no evidence of hepatitis or viral antigen was detected (data not shown). these data indicate that in contrast to previous data (lavi et al. 1986 ), none of these three virus preparations are directly hepatotropic following ip infection. to correlate early, transient he protein expression with the observed alterations in mononuclear cell infiltrates, expression of various cytokine mrnas in the cns and liver were examined at 2 days p.i. this time point was chosen since it was the last time point p.i. when he mrna could be detected (table 1 ). increased expression of the mrnas encoding tnf-␣ was detected in the cns of a59-de-he-infected mice compared to samples from mice infected with the a59-de-cat virus (fig. 5a) . the tnf-␣ mrna expression in the liver showed a similar trend, although the level of this mrna in the liver was lower than that in the cns (fig. 5a ). il-6 mrna was also increased in the cns of mice infected with a59-de-he virus (fig. 5b) ; however, no differences were detected in the liver between the two groups. although the level of ifn-␥ mrna was very low in the tissues from both groups, a slight decrease in ifn-␥ mrna was detected in the cns but not in the livers of mice infected with the a59-de-he virus (fig. 5c) . no difference was found in the expression of il-1␣ mrna in either cns or liver (fig. 5d) . these data indicate that the transient expression of the he protein resulted in the early induction of both tnf-␣ and il-6 mrna in the cns. the he protein is expressed only by some strains of mhv (yokomori et al. 1991) ; thus, it is not required for viral replication. during virus passage in tissue culture or in animals, the he gene often undergoes mutation so that the virus loses the ability to synthesize he protein (yokomori et al. 1991) . however, reverse changes have also occurred; for example, a jhm variant, which has three repeats of the ucuaa pentanucleotide at the 3ј end of the leader sequence and synthesized a very small amount of he, generated, upon passage, a jhm variant containing two repeats of the ucuaa pentanucleotide, resulting in expression of a large amount of he protein shieh et al. 1989; yokomori et al. 1989 ; la monica et al. 1992) . (3) a day(s) postinfection. mice were infected ic on day 0 with 1 ϫ 10 5 pfu. b mean titer in pfu/g liver, in log 10 ϯ standard deviation (number/group). c not done. initial demonstration that he may play a role in viral neuropathogenesis was obtained from studies showing that passive transfer of anti-he monoclonal antibodies protected mice from lethal infection by jhm viruses expressing various amounts of he, but not a jhm variant which expressed no he . histopathological studies of the he-protected mice suggested that an early mononuclear infiltration into the cns was associated with the protection by this monoclonal antibody. subsequent comparisons of the pathogenicity of the jhm variants expressing different levels of he demonstrated that the jhm variant which expresses high levels of he replicated less efficiently in glial cells in vitro and preferentially infected neurons in vivo (yokomori et al. 1995) . together, these data suggested that he protein is at least one of the factors influencing the neuropathogenesis of jhm in the cns. however, the effect of he protein on viral pathogenesis may depend on the genetic background of the virus. this question was difficult to assess in the previous studies since jhm variants used in those studies may differ not only in he gene expression but also in other unscrutinized viral genes as well. in this report, we examined the influence of he protein on neuropathogenesis of the a59 strain of mhv using a di vector approach, so that the presence or the absence of he can be studied on the same virus background. a59 was chosen because its pathogenesis in mice has been well described (lavi et al. 1984 (lavi et al. , 1986 and it does not produce any background level of the he protein due to a mutation in the transcription start signal and a premature stop codon in the he gene shieh et al. 1989) . our data showed that expression of the he gene from the di vector did indeed alter the pathogenesis of a59 infection by increasing the survival rate of the infected mice when compared to either parental a59 or a59 containing a cat-expressing di rna (a59-de-cat). however, this higher survival rate was apparently due to the reduction in liver pathology. in the cns, the expression of he by di rna resulted in only a slight and transient increase of mononuclear cell infiltration and no alteration in cellular tropism. the apparent differences between this di-expressed he and the he expressed directly from the virus was probably due to the transient nature of he expression by the di rna, while the latter was expressed continuously throughout the infection. nevertheless, this study shows that even transient expression of the he protein can alter viral pathogenicity. these data removed two constraints upon the use of di vector for expressing foreign proteins during viral infections. first, expression of an irrelevant gene from the di rna during infection did not alter pathogenesis of the virus. this suggests that the weak interference activity exhibited by di rna in vitro (makino et al. 1985) plays very little role during virus infection in vivo. second, the conservation of wild-type virus after the di rna had been lost suggested that the presence of di rna did not result in the selection of a variant virus, which potentially could have been derived by rna recombination, either during in vitro preparation of the di rna-containing virus or during subsequent in vivo replication. the a59 strain of mhv is a dual tropic virus, infecting both the cns and liver following ic or intranasal infection (lavi et al. 1986) . at low infectious doses, the hepatitis usually resolves and survivors develop extensive chronic demyelination within the cns (lavi et al. 1984) . our data using a relatively large infecting dose show that mice infected with the parental a59 or a59-de-cat or a59-de-he virus succumb to the infection most likely due to hepatic necrosis, since there was only relatively mild cns pathology at the time of death of the infected mice. because virus replication in the liver and spleen early in infection was similar between a59-de-he-and a59-de-cat-infected mice, it is unlikely that there was a fundamental difference in hepatotropism between these two viruses. therefore, the striking resolution of hepatitis and the reduction in infectious virus in mice infected with a59-de-he at day 6 p.i. suggests that an early function of the innate immune response is most likely responsible for this phenomenon. it is worthy of note that no cd4 ϩ or cd8 ϩ t cell epitopes have been detected within the he gene (bergmann et al. 1996; van der veen, 1996) . in addition, there is no detectable anti-mhv antibody in the serum at the time when the hepatitis resolves (parra et al. 1997) . together, these data suggest that he gene expression may activate or enhance a function of nonspecific innate immunity. consistent with this notion, increased levels of tnf-␣ and il-6 mrna were detected in the cns of mice infected with a59-de-he. although tnf-␣ mrna has previously been demonstrated to increase in the cns following infection with the jhm strain of mhv (pearce et al. 1994; parra et al. 1997) , inhibition of tnf has no effect on either virus clearance or cns mononuclear cell infiltration . therefore, the potential role of increased tnf-␣ mrna expression following expression of the he protein is not clear. increased il-6 mrna, another pleotropic cytokine (van snick, 1990) , was also detected in the cns of a59-de-he-infected mice. il-6 is secreted from both endothelial cells and astrocytes following mhv infection (joseph et al. 1993) and il-6 mrna peaks rapidly in the cns following mhv infection as a direct consequence of the immune response to infection (parra et al. 1997) . based on a kinetic analysis of il-6 mrna induction in the cns following mhv infection (parra et al. 1997) , il-6 has been suggested to play a role in the inflammatory response via enhanced recruitment of mononuclear cells and activation of acute phase responses in addition to its role as a cofactor for ctl induction (romano et al. 1997; kopf et al. 1994) . increased il-6 mrna present in the cns of mice infected with a59-de-he virus is consistent with the increased mononuclear cell infiltration at a time during infection (day 3 p.i.) prior to detectable virus-specific immunity. these data support the suggestion that he protein expression enhances the innate immune re-sponse, contributing to the eventual clearance of virus from the liver. this study and the previous report of the expression of ifn-␥ (zhang et al. 1997 ) using a mhv-di rna vector fig. 4ðcontinued system indicate the feasibility of this system for studying roles of viral proteins or cytokines during mhv infection. although expression of proteins is transient and localized at the sites of viral infection, these features may actually offer advantages in studying viral pathogenesis. mice c57bl/6 mice were purchased from the jackson laboratories (bar harbor, me) at 6±7 weeks of age. mice were housed locally in microisolator cages and were used within 7 days of receipt. none were found to have preexisting anti-mhv antibodies prior to use. mice were inoculated intracranially (ic) with 1 ϫ 10 5 plaque-forming units (pfu) of virus in 30 l of dulbecco's phosphatebuffered saline (dpbs). mhv strain a59 was used throughout the study. it was originally obtained from dr. cliff bond of university of california, san diego (leibowitz et al. 1981) , plaquepurified four times, and used within eight passages of purification. the murine astrocytoma cell line dbt (hirano et al. 1974 ) was used for virus propagation, plaque assay, and preparation of di stocks. to construct a di vector containing the he gene, we used p25cat , which contains a cat gene behind the (ig) sequence of gene 7 (ig7) in the cdna of disse rna ) as a starting plasmid. the cdna of the he gene was generated by reverse transcription±polymerase chain reaction (rt± pcr) amplification using the cytoplasmic rnas from mhv-jhm-infected cells as templates and two primers, 5ј-he (5ј-ggg act agt att gtt gag aat cta atc taa act tta agg aat ggg cag tac gtg c-3ј) and 3ј-he (5ј-ggg ctg cag tta tgc ctc atg caa tct-3ј), corresponding to the ends of the he gene plus spei and psti (italicized sequences). following restriction enzyme digestion with spei and psti, a 1.4-kb cdna containing the complete he gene sequence was purified from low-melting-point agarose gel and cloned into the spei and psti sites of p25cat to replace the cat gene , resulting in pde-he (fig. 1) . for making di stock viruses, plasmid dnas of pde-he and p25cat were linearized with restriction enzyme xbai and subjected to in vitro transcription. the in vitro transcription was carried out in a reaction containing 1ϫ transcription buffer (promega, madison, wi), 10 mm dithiothreitol (dtt), 1 u/l rnasin, 0.5 mm each atp, ctp, utp, and gtp, 5 g linearized dna of pde-he or p25cat, and 40 u t7 rna polymerase at 37°c for 2 h according to the procedure recommended by the manunot detectable not detectable a day(s) postinfection. mice were infected ic with 1 ϫ 10 5 pfu of various viruses on day 0. b mean titer in pfu/g spleen, in log 10 ϯ standard deviation (number/ group). facturer (promega). the in vitro-transcribed rna was transfected into mhv-a59-infected dbt cells using the dotap method (boehringer-mannheim, indianapolis, in) . briefly, monolayers of dbt cells at approximately 80% confluence in 60-mm petri dishes were infected with a59 at a m.o.i. of 5. at 1 h postinfection (p.i.), virusinfected cells were washed once with serum-free eagle's minimal essential medium (mem) and then covered with 2 ml of mem containing 1% newborn calf serum. ten micrograms of in vitro-transcribed rna was mixed with dotap (boehringer-mannheim) in 1ϫ hbs buffer (20 mm hepes; 150 mm nacl; ph 7.4). the rna±dotap mixture was incubated at room temperature for 10 min and then added slowly to the cell culture. at 12 to 14 h posttransfection, culture medium was harvested and cell debris removed by low-speed centrifugation. supernatants were used as di stocks. each di stock consisted of a mixture of virus populations (helper virus alone, helper virus containing he protein on the surface, and helper virus containing a copackaged di genomic rna); only the virus with a copackaged di rna could synthesize the he protein. prior to inoculation into animals, virus titers of the di stocks were determined by plaque assay using dbt cells as previously described (fleming et al. 1983 ). the titers were 7.5 ϫ 10 7 for a59-de-cat and 8.0 ϫ 10 7 for a59-de-he. the replication of the di rnas of these stock viruses in dbt cells was determined by rt±pcr (zhang et al. 1997) and was shown to be similar. the expression of the he and cat proteins in dbt cells was confirmed by immunoprecipitation using an anti-he antibody ) and cat assay , respectively. these di stocks were then used to inoculate mice intracerebrally at 1 ϫ 10 5 pfu per animal. virus titers in mouse tissues were determined by homogenization of one half of the brain or liver lobe in 4.0 ml of dpbs, ph 7.4, using tenbrock tissue homogenizers. the remainder was processed for histopathology (see below). following centrifugation of the tissue homogenates at 1500 g for 20 min at 4°c, supernatants were assayed immediately or frozen at ϫ70°c. virus titers were determined by plaque assay using monolayers of dbt cells as previously described (fleming et al. 1983 ). data were presented as the average log 10 titer per gram of tissue for groups of three or more mice. rnas were isolated from tissues with the trizol reagent according to the manufacturer's instruction (life technologies, grand island, ny). briefly, approximately 0.1 g of tissue was homogenized in 1 ml trizol reagent using a glass±teflon homogenizer. following incubation of the homogenized sample for 5 min at room temperature, 0.2 ml of chloroform was added to each sample. rnas were separated by centrifugation at 12,000 g for 15 min at 4°c, precipitated with ethanol, and resuspended in water. for detection of the mrna species representing he and cat genes, rna samples were subjected to reverse transcription (rt) using primers 3ј-he and 3ј-cat542 , respectively. cdnas were amplified by nested pcr (95°c, 1 min for denaturing; 59°c, 30 s for annealing; 72°c, 2 min for extension). the primer pairs 5ј-l9 (specific to the leader rna, )/ 3ј-he for the he gene and 5ј-l9/3јcat542 for the cat gene were used for the first pcr and 5ј-l9/3ј-he74 or 5ј-l9/3ј-cat106 , respectively, for the second pcr. each pcr was carried out for 25 cycles. these pcrs would amplify both the full-length di rna (3.6 kb for de-he and 2.9 kb for de-cat) and the subgenomic mrnas representing he and cat genes (2.1 kb for de-he and 1.4 kb for de-cat). the pcr products were analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining. the nature of the pcr product representing the subgenomic mrna was confirmed by the size and the restriction patterns of the pcr products. for detection of the gene coding for the mouse fibrinogen-like protein (fgl-2, formerly named musfiblp), rt± pcr was carried out using the antisense primer 1423b (5ј-taa aca gta agt gaa tta cca c-3ј, complementary to nucleotide positions 2063±2084) and the sense primer 1423a (5ј-atg ggt gga ggc tgg acg gt-3ј, corresponding to nucleotide positions 861±880) (parr et al. 1995) . primers were kindly provided by dr. julian leibowitz (university of texas a & m). for cytokine mrna expression, rnas were reverse-transcribed using amv reverse transcriptase and oligo(dt) primers (promega, madison, wi). the cdnas were amplified by pcr using oligonucleotide primers specific for interferon-␥ (ifn-␥), tumor necrosis factor (tnf)-␣, interleukin (il)-6, and il-1␣ parra et al. 1997) . pcr products were quantitated by dot blot assays of serial dilution using specific [␥-32 p]atp-labeled oligonucleotide probes as previously described parra et al. 1997) . signal intensities were measured using a phosphor imager scanner (molecular dynamics, sunnyvale, ca). values obtained for the cytokine mrnas were normalized to the housekeeping enzyme hypoxanthine phosphoribosyltransferase (hprt) mrna values obtained in parallel experiments to compensate for variations in input cdna. data are expressed as the relative values of the maximum obtained for individual samples. for routine histopathological analysis, mice were sacrificed by co 2 asphyxiation. brains were removed and dissected in the midcoronal plane. the side of the brain opposite the needle track was taken for immunohisto-chemical study in all cases. brain hemispheres, spinal cords, and livers were fixed for 3 h in clark's solution (75% ethanol and 25% glacial acetic acid) and embedded in paraffin. for routine analysis of brain sections a single coronal section including cerebellum, brain stem, hippocampus, and olfactory cortex was used. the same region of the brain was examined in all animals. at critical time points (e.g., day 3 p.i.) five step sections were taken through each hemisphere and stained for viral antigen. all viral antigen-positive cells were counted in each section and summed for each animal. sections were stained with hematoxylin and eosin for microscopic examination. the distribution of viral antigen was examined using immunoperoxidase staining (vectastain-abc kit, vector laboratories, burlingame, ca) and anti-jhmv monoclonal antibody mab j.3.3 specific for the carboxy terminus of the n protein (fleming et al. 1983 ). all slides were read in a blinded manner. the jhm strain of mouse hepatitis virus induces a spike protein-specific d b -restricted cytotoxic t cell response the production of recombinant infectious di-particles of a murine coronavirus in the absence of helper virus self-antigeninduced th2 responses in experimental allergic encephalomyelitis (eae)-resistant mice 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acute encephalomyelitis cytokine induction during t-cell mediated clearance of mouse hepatitis virus from neurons in vivo role of il-6 and its soluble receptor in induction of chemokines and leukocyte recruitment identification of a new transcriptional initiation site and the corresponding functional gene 2b in the murine coronavirus rna genome tumor necrosis factor expression during mouse hepatitis virus-induced demyelinating encephalomyelitis immunogenicity of jhm virus proteins: characterization of a cd4 ϩ t cell epitope on nucleocapsid protein which induces different t-helper cell subsets. virology 225, 339±346. van snick nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes the e3 protein of bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity neuropathogenicity of mouse hepatitis virus jhm isolates differing in hemagglutinin-esterase protein expression heterogeneity of gene expression of the hemagglutinin-esterase (he) protein of murine coronaviruses biosynthesis, structure, and biological activities of envelope protein gp65 of murine coronavirus expression of interferon-gamma by a coronavirus defectiveinterfering rna vector and its effect on viral replication, spread, and pathogenicity unusual heterogeneity of leader-mrna fusion in a murine coronavirus: implications for the mechanism of rna transcription and recombination coronavirus leader rna regulates and initiates subgenomic mrna transcription both in trans and in cis we thank wen-qiang wei for preparing histological slides, steve ho for plaque assays, and daphne shimoda for helping with the preparation of the manuscript. this work was supported by grant ns 18146 from the national institute of health. b.p. was supported by a training grant from colciencias and universidad del valle, columbia. m.m.c.l. is an investigator of the howard hughes medical institute. key: cord-260782-1lm8tzbc authors: giles, julia; perrott, matthew; roe, wendi; shrestha, kshitiz; aberdein, danielle; morel, patrick; dunowska, magdalena title: viral rna load and histological changes in tissues following experimental infection with an arterivirus of possums (wobbly possum disease virus) date: 2018-07-14 journal: virology doi: 10.1016/j.virol.2018.07.003 sha: doc_id: 260782 cord_uid: 1lm8tzbc tissues from australian brushtail possums (trichosurus vulpecula) that had been experimentally infected with wobbly possum disease (wpd) virus (wpdv) were examined to elucidate pathogenesis of wpdv infection. mononuclear inflammatory cell infiltrates were present in livers, kidneys, salivary glands and brains of wpd-affected possums. specific staining was detected by immunohistochemistry within macrophages in the livers and kidneys, and undefined cell types in the brains. the highest viral rna load was found in macrophage-rich tissues. the detection of viral rna in the salivary gland, serum, kidney, bladder and urine is compatible with transmission via close physical contact during encounters such as fighting or grooming, or by contact with an environment that has been contaminated with saliva or urine. levels of viral rna remained high in all tissues tested throughout the study, suggesting that on-going virus replication and evasion of the immune responses may be important in the pathogenesis of disease. wobbly possum disease (wpd) is a fatal neurological disease of the australian brushtail possum (trichosurus vulpecula) that has been thus far reported only in new zealand, both in captive and free-living possums (mackintosh et al., 1995; o'keefe et al., 1997; perrott, 1998; perrott et al., 2000a perrott et al., , 2000c . the partial genomic sequence of wpd virus (wpdv) was first identified in archival tissues from wpd-affected possums (dunowska et al., 2012) , and a full genomic sequence has been reported recently (gulyaeva et al., 2017) . based on the analysis of this sequence, wpdv has been classified within the family arteriviridae as a sole member of the newly established genus dipartevirus (anonymus, 2017; kuhn et al., 2016) . aetiological involvement of the virus in the development of wpd has been confirmed by induction of disease by experimental infection of wild-caught possums with a purified wpdv isolate (giles et al., 2016) . whilst possums are native to australia and protected in some states, in new zealand they are an introduced pest species causing substantial destruction of the country's unique ecosystem and wildlife (cowan, 1990; cowan and waddington, 1990; king, 1990) . as such, interest in understanding pathogens of possums was originally driven by their potential to be used as biological control in new zealand (perrott, 1998) , and conversely, from conservation efforts in australia. very limited numbers of viruses have been identified from the possum host. in addition to wpdv, these include possum enteroviruses (zheng, 2007) , possum adenovirus (thomson et al., 2002) , and possum papillomavirus (perrott et al., 2000b) . herpesvirus-and coronavirus-like particles have also been observed in fecal samples from possums by electron microscopy (rice and wilks, 1996) . of these, only wpdv has been linked to severe systemic disease characterised by signs of multifocal neurological disturbances, clinically appearing to involve the vestibular system and resulting in ataxia, a decreased ability to climb, head tremor, and behavioural changes (giles et al., 2016; perrott et al., 2000c) . under experimental conditions, decreased appetite precedes the development of neurological signs and significant weight loss is also observed (giles et al., 2016) . the histological hallmark of the disease is mononuclear perivascular infiltrates in multiple organs, including the liver, spleen, brain and kidney (o'keefe et al., 1997; perrott, 1998) . the disease has been reproduced following the inoculation of healthy wild-caught possums with blood, urine, tissue homogenates and homogenized hematophagus skin mites (trichosurolaelaps crassipes) derived from wpd-affected possums and administered by multiple routes (o'keefe et al., 1997; perrott, 1998; perrott et al., 2000c) . however, the infectious dose and mechanisms of natural transmission of wpdv are currently unknown. under experimental conditions, healthy wild-caught possums sharing an enclosure with wpd-affected possums developed wpd whereas possums housed in adjacent separate cages remained healthy (perrott, 1998; perrott et al., 2000c) . this suggests that the virus is transmitted via direct contact or fomites and that aerosol or droplet transmission does not occur. the interest in arteriviruses has been driven predominantly by the desire to improve control measures against porcine reproductive and respiratory syndrome virus (prrsv), which is considered to be one of the most economically important pathogens of swine (perez et al., 2015) . while wpdv shares some features common to arteriviruses, it is only distantly related to other members of the family. phylogenetically, wpdv comprises a single-virus lineage separate to another lineage that includes all other arteriviruses from different placental hosts (gulyaeva et al., 2017; kuhn et al., 2016) . its basal position in the phylogenetic tree suggests that wpdv is the closest to the ancestral arterivirus. alternatively, the separation between wpdv and other arterivirus sequences may reflect a convergent evolution of their hosts, marsupials and placental mammals (luo et al., 2011) . hence, deciphering pathogenesis and biological properties of wpdv is interesting not only from the point of view of possum health, but also from the evolutionary perspective. the aim of the current study was to describe histological lesions and viral rna levels in tissues from possum experimentally infected with wpdv. material for the current study comprised archival tissue and body fluid samples from 16 experimental possums used in a previous liveanimal transmission study (giles et al., 2016) . briefly, wild-caught possums were individually housed and assigned consecutive id numbers, before being inoculated via intraperitoneal (ip) injection with purified wpdv isolate (possums 5-8), infected cell culture lysate (possums 9-12), or standard inoculum that comprised a filtered 10% tissue suspension of brain, liver and spleen from seven wpd-affected possums (possums 13-16). possums 1 and 2 received nothing and possums 3 and 4 received control inoculum. the latter comprised iodixanol medium (optiprep, sigma-aldrich) equivalent to the position of the virus band from density-gradient purification, but obtained after processing of uninfected cultures of primary possum macrophages. following the development of neurological signs consistent with wpd, possums were euthanized and two sets of tissue samples from all major internal organs, including multiple regions of the brain, were collected from each possum on post-mortem examination. one piece of each tissue was stored at − 80°c and another was fixed in 2% paraformaldehyde in phosphate buffer ph 7.4 with added lysine (0.15 m) and sodium-m-periodate (0.01 m) following the method of mcclean and nakane (1974) . urine, where possible, was collected by cystocentesis and stored at − 80°c. blood was collected into red-top vacutainer tubes (bd vacutainer®) by cardiocentesis or venipuncture of the great vessels. serum was separated from the clot by centrifugation at 1500 ×g for 10 min and stored at − 80°c. all animal manipulations were approved by the massey university animal ethics committee (muaec protocol 12/15). total rna was extracted from~20 mg of tissue (liver, spleen, kidney, forebrain, hindbrain, lumbar spinal cord, salivary gland, retropharyngeal lymph node and bladder) using total rna mini kit for tissue (geneaid biotech ltd) and from 100 µl samples of body fluids (urine and serum) using total rna mini kit for blood and cultured cells (geneaid biotech ltd) according to the manufacturer's instructions, with the exception that body fluids were used instead of cell culture media or lysates. full sets of tissues were available for seven wpd-affected possums. urine was the only missing sample for two possums (possums 10 and 16) and lymph node was missing for one possum (possum 14). for the remaining two possums (6 and 11) only liver, spleen and sera were available for testing. rna from tissues and body fluids were eluted with 50 µl of water and nanodrop™ (thermo fisher scientific) was used to determine the concentration and quality of rna. up to 1 µg rna was used for cdna synthesis using qscript™ supermix (quanta biosciences) according the manufacturer's instructions, with the exception that reactions were scaled down to a total volume of 10 µl. the viral rna load was determined using wpdv-specific reverse transcriptase quantitative pcr (rt-qpcr) as previously described (dunowska et al., 2013) with the modification that the assay was performed using mic instrument (bio molecular systems) and luminaris higreen qpcr mastermix (thermo fisher scientific) with the following conditions: 50°c for 2 min (uracil dna glycosylase activation), 95°c for 10 min (initial denaturation and activation of dna polymerase), 40 cycles of 95°c for 5 s, 60°c for 5 s and 72°c for 15 s, followed by melt from 72°c to 95°c at 0.3°c/s. several dilutions of a plasmid containing wpdv pcr fragment and water were used in each rt-qpcr run as positive and negative controls, respectively. all available tissues from wpd-affected possums were tested. in addition, tissues from possums 1 and 3 were included as negative controls. viral loads were expressed as viral copy numbers per µg of rna (solid tissues) or per µl of template cdna (body fluids). differences in viral load between tissue types for infected possums were determined by least mean squares using sas software. two possums (possums 6 and 11) were removed from the analysis due to multiple missing data points. two negative data points from wpd-affected possums (spinal cord from possum 7 and hindbrain from possum 8) were also removed from the analysis. values were log transformed to be normally distributed and a mixed model with tissue as fix effect and possum as random effect was fitted to the data (proc mixed, sas). the significance level was set at p < 0.05. histological examination was performed on the following tissues: liver, spleen, brain, kidney, bladder and salivary gland from all possums, and a lymph node from the head region from all possums except possums 9,10,14 and 15. all tissue samples were fixed in paraformaldehyde in phosphate buffer as described above, then transferred and held in 60% ethanol prior to processing and embedding into paraffin blocks. sections (4 µm) were stained using gill's haematoxylin and eosin method (bancroft and stevens, 2001 ) and permanently mounted with entellan (merck). kidney slides were also stained for collagen using masson's trichrome and permanently mounted with entellan (merck). slides were observed using a light microscope (olympus bx 51) and images captured using an olympus xc 50 digital camera. slides were examined by four of the authors (jg, mp, wr, da) blinded. the severity of lesions within the renal interstitium, glomeruli and tubules were individually graded on a scale of 0-3, where 0 represented no discernible lesions and 3 represented severe lesions. a total score was given by summation of individual lesion scores. statistical significance for individual parameters and total grade between inoculated and control possums were determined by kruskal-wallis test (sas). the significance level was set at p < 0.05. testing for urea and creatinine levels in stored serum samples was performed by idexx laboratories (palmerston north, new zealand). sera from all four control possums and 11/12 wpdv-infected possums were tested. insufficient serum from possum 15 was available for testing. possums were considered to have elevated urea if it exceeded 15.8 mmol/l and elevated creatinine if it exceeded 100 µmol/l (viggers and lindenmayer, 1996) . mild azotaemia was considered as a serum creatinine of 100-150 µmol/l and urea of 15.8-45 mmol/l. severe azotaemia was considered as a serum creatinine > 400 µmol/l and urea of > 45 mmol/l. to further investigate the aetiology of the rental lesions, leptospira qpcr targeting the dna gyrase subunit b gene was performed using dna extracted from frozen kidney tissues according to the method of slack et al. (slack et al., 2006) . samples from 14/16 experimental possums were tested. frozen kidneys from possums 6 and 11 were not available. immunostaining for wpdv antigen was performed on liver, brain and kidney tissues from all 16 possums. briefly, sections (5 µm) were cut and mounted onto positively charged slides (hdd thermo scientific) and oven-dried at 65°c for approximately one hour. after deparaffinization with xylene, sections were rehydrated through graded ethanol to water. formalin pigment was removed by incubation with saturated picric acid in alcohol for five minutes. slides were rinsed in water for five minutes, quenched with 3% h 2 o 2 in methanol for 10 min and rinsed in water for two minutes. heat-based antigen retrieval (tris edta ph 8.5 for 30 min at 95°c) was followed by permeabilization (0.3% triton x in pbs ph 7.0 for five minutes). slides were then washed in pbs ph 7.0 containing 0.2% tween 20 (pbs/tween20), blocked with superblock blocking buffer (thermo scientific) containing 0.1% tween 20, and incubated overnight (4°c) with anti-wpdv antibody diluted 1:500 (0.7 µg/ml) in superblock blocking buffer. affinity purified polyclonal rabbit igg raised against e.coli-expressed recombinant nucleocapsid (rn) protein (anti-wpdv) was commercially produced (lifeome). the rn protein had previously been shown to be recognised by sera from wpdv infected possums by enzyme-linked immunosorbent assay and western blot (giles et al., 2018) . the following morning, the slides were washed three times in pbs/tween20. the presence of bound primary antibody was detected using the abc universal detection system (vector laboratories) according to the manufacturer's instructions and visualised with immpact™ dab substrate (vector laboratories). after approximately five minutes of incubation with the chromogen, the slides were washed in running tap water for five minutes and counterstained with haematoxylin. to assess nonspecific binding, rabbit polyclonal anti-epidermal growth factor receptor antibody (ab-3, calbiochem®, merck millipore) was used as irrelevant antibody. on average, the highest levels (> 10 6 copies/µg rna) of viral rna were detected in the retropharyngeal lymph node, spleen and liver of wpdv-infected possums (fig. 1) . moderately high levels of viral rna (approximately 10 4 -10 5 copies/µg rna) were found in the salivary gland, kidney, bladder and forebrain. comparatively low levels of viral rna (< 10 3 copies/µg rna) were found in the spinal cord and hindbrain. the mean viral load in the liver was significantly higher than the mean viral load in the kidney, forebrain, hindbrain, salivary gland and bladder (p < 0.05) but was not significantly different from the mean viral rna levels in the spleen and lymph node (p > 0.05). the mean viral load in the forebrain was significantly higher than the mean viral load in the hindbrain and spinal cord (p < 0.05). moderately high levels of viral rna were detected in sera from all but one infected possums (average 2.76 × 10 5 copies/µl), however, there were marked differences between individual possums. lower levels of viral rna were detected in the urine than the serum (1.02 × 10 3 copies/µl), with similar variability between individual animals. histological examination was performed on possums 1-4 (controls) and all wpdv-infected possums (possums 5-16). small, multifocal inflammatory aggregates were disseminated throughout the hepatic parenchyma in 11/12 wpd-infected possums. inflammatory aggregates comprised predominantly plasma cells with fewer macrophages, and frequently appeared to be clustered around macrophages with increased amounts of vacuolated cytoplasm. moderate-sized aggregates of the same cell population were also observed surrounding vessels in periportal ( fig. 2a) and centrilobular regions. inflammatory aggregates were not obvious in the liver from possum 15, which died on day 6 post infection. there was a marked increase in inflammatory cells in the hepatic parenchyma and sinusoids observed in the liver of this possum, but these changes were more diffuse/uniform than those observed in other wpd-affected possums. small foci of perivascular mononuclear inflammation around the central veins were also observed in sections of liver from two control possums (possums 1 and 3), however, control possums lacked mononuclear cell infiltration in the hepatic parenchyma. kidney lesions are summarised in table 1 . mild to severe interstitial nephritis was present in 2/4 (50%) control possums and 11/12 (91.6%) wpdv-infected possums. additionally, mild to severe tubular lesions were present in 10/12 (83.3%), mild to moderate glomerular lesions were present in 5/12 (41.7%) and mild to severe interstitial fibrosis were present in 4/12 (33.3%) wpdv-infected possums. scores for tubular lesions (p = 0.009), interstitial nephritis (p = 0.032) and total scores (p = 0.014) were significantly higher for wpdv-infected possums than control possums. the extent of interstitial fibrosis and glomerular lesions did not differ significantly between infected and control possums (p > 0.05). interstitial nephritis was present in 11 wpdvinfected possums, but mild interstitial nephritis was also present in two control possums (possums 3 and 4). in 10/11 (90.9%) wpdv-infected possums and 2/4 (50%) control possums, the interstitial infiltrate was fig. 1 . quantity of viral rna in tissues and body fluids from wobbly possum disease virus (wpdv)-infected possums. viral rna was extracted and quantified using a previously described wpdv-specific rt-qpcr (dunowska et al., 2013) . the results were expressed as wpdv copies/µg rna for tissues (liver, spleen, lymph node, bladder, kidney, forebrain, hindbrain, spine and salivary gland) or as wpdv copies/µl cdna for body fluids (serum and urine). lymphoplasmacytic, while a mixed infiltrate (predominantly lymphocytes and plasma cells with fewer neutrophils) was present in one (8.3%) wpdv infected animal (possum 5). in possums with moderate to severe interstitial nephritis (grade 2-3; n = 7), renal tubules were often lined by degenerating or proliferating epithelial cells and contained eosinophilic material (proteinaceous fluid) (fig. 2b) . glomerular changes were variable and mild in 3/12 (25%) wpdv-infected possums and moderate in 2/12 (16.7%) wpdv-infected possums. where present, glomerular changes comprised one or more of the following: periglomerular fibrosis, hyperplasia or hypertrophy of parietal epithelium, synechiae, and increased glomerular cellularity. in 1/12 (8%) cases there were perivascular aggregates of moderate numbers of mononuclear cells in the peri-renal adipose tissue (possum 5), but these were also present in one control animal (possum 3). masson's trichrome was strongly positive for collagen in the interstitium of both of these possums. lesions are summarised in table 2 . mild to severe non-suppurative encephalitis (n = 1; control possum) or meningoencephalitis (n = 10; wpdv-infected possums) was present in 11 possums. meningoencephalitis in these cases was characterised by infiltration of the subarachnoid space by few to moderate numbers of lymphocytes, plasma cells and macrophages, with perivascular cuffs of mononuclear cells 1-4 layers thick surrounding vessels in the superficial cortex and periventricular grey matter (fig. 2c) . the inflammatory infiltrate was partially (possum 9) or predominantly (possum 7) neutrophilic in two possums. in 2/12 (16.7%) infected possums (possums 5 and 7) the perivascular inflammation extended into the surrounding neuropil. inflammatory changes were often more apparent in subependymal regions. lymphoplasmacytic perivascular inflammation was present within the choroid plexus of 6/12 (50%) infected possums (possums 5, 7, 12, 13, 14 and 16) . multifocal dense aggregates of glial cells (glial nodules) involving both grey or white matter were present in 5/12 (41.7%) infected possums (possums 5, 7, 8, 12, 16) and 1/4 (25%) control possum. occasional foci of malacia were present in the forebrain of possum 9, while multiple suppurative foci and necrotising vasculitis were present within the forebrain of possum 7. in all wpd possums, lymphoid follicles were poorly organised, with few obvious germinal centres. variable numbers of foamy macrophages were also present in the red pulp of the spleens of all wpd possums. changes were not present within spleens of control possums. variable degrees of lymphoid necrosis and an increased number of foamy macrophages were present in the lymph nodes of 7/8 (88%) wpd-infected possums examined. the lymph nodes of control possums did not contain lesions. moderate numbers of mononuclear cells were present in the periacinar and perivascular regions in salivary glands from all 12 wpdinfected possums but not from control possums. occasional mild perivascular infiltrates comprising several plasma cells and lymphocytes were observed in the submucosa of the bladder of 6/12 (50%) wpd-infected possums and in none of the control possums. the wpdv antigen appeared as granular staining within the cytoplasm of cells. it was detected in macrophages in the livers of all experimentally infected possums (fig. 3a) , including possum 15 with atypical histological changes, but not in control possums. inflammatory aggregates often appeared to be clustered around immunopositive macrophages. immunostaining was also detected in the kidneys of 10/ 12 (83.3%) wpdv-infected possums and none of the control possums (table 1, fig. 3c ). here, immunostaining was detected in macrophages within inflammatory aggregates in the renal interstitium. immunostaining was also detected in the brains of 8/12 wpdv-infected possums and none of the control possums. where present, immunostaining with a predominantly cytoplasmic distribution was seen in one or more of the following locations: glial nodules (fig. 3e) , the choroid plexus, the subarachnoid space and various subependymal locations. mild azotaemia was present in two wpd-affected possums (possums 6 and 8) and severe azotaemia in another two possums (possums 10 and fig. 2 . histology of brain, kidney and liver tissue from wobbly possum disease virus (wpdv)-infected possums. a) possum 16 liver viewed under 10 × objective. inflammatory aggregates surrounding hepatic veins. aggregates comprise predominantly plasma cells with some macrophages. b) possum 12 kidney, viewed under 20 × objective. severe interstitial nephritis and tubular damage. the interstitial infiltrate is lymphoplasmacytic. renal tubules are dilated, and proteinacious fluid and cellular debris are present in the tubules. c) possum 13 brain, viewed under 20 × objective. mononuclear cell perivascular cuff in the forebrain. slides were observed using a light microscope (olympus bx 51) and images captured using an olympus xc 50 digital camera. 14). urea was mildly elevated in one additional possum (possum 7), and both values were within references ranges in the remaining experimental possums including control possums (table 1) . two of the 14 possum kidneys tested were positive for leptospira dna by qpcr (table 1) with ct values of 19.89 (possum 1, uninoculated control) and 31.56 (possum 8, wpd). whilst rna levels in selected tissues (liver, spleen, brain and kidney) from wpd-infected possums have been previously reported (dunowska et al., 2013) , we have expanded both the number of possums and the tissue types tested to provide a greater understanding of the tissues targeted by the virus in-vivo. we have also demonstrated for the first time the presence and localisation of the viral antigens within lesions in selected tissue types. to allow for comparison of rna levels between tissues, qpcr results were normalised to µg of total rna based on amount of rna used for the cdna synthesis step, while rna levels in body fluids (urine and serum) were expressed as copy numbers/µl cdna per rt reaction. because total rna levels extracted from body fluids were extremely low, normalisation to total rna would have resulted in significant inaccuracies. as such, a direct comparison between viral load in solid tissues and body fluids was not possible. consistent with results of the previous study (dunowska et al., 2013) , high levels of viral rna were present in livers and spleens of all wpd-affected possums. in addition, high levels of viral rna were also detected in lymph nodes examined. these results suggest that wpdv, similarly to other arteriviruses (teifke et al., 2001) , has in-vivo tropism for macrophage-rich tissues, which may result in generalized infection of the mononuclear-phagocyte system. this is further supported by the ihc data, with wpdv antigen detected within macrophages in the liver and kidney of infected possums. these results also correspond well with the in-vitro growth requirements for wpdv, as the virus has been cultivated so far only in primary possum macrophages (giles et al., 2015) . based on these results, macrophage-rich tissues should be considered as the samples of choice for detection of the virus or viral rna in diagnostic settings. detection of moderate to high levels of viral rna from the sera of all but one infected possum in this study also supports the use of blood or serum samples for detection of wpdv. finally, detection of moderately high levels of viral rna in salivary glands raises the possibility of virus excretion in saliva. the latter two sample types (blood and saliva) may provide a means of diagnosis of active infection in live animals, without the necessity of a retrospective diagnosis following death or euthanasia. moderate to high levels of viral rna in serum samples in all but one infected possum indicate that cell-free viraemia is a feature of wpd. high levels of viral rna were still detected in the serum nearly four weeks post infection, suggesting a failure of the immune system to clear the virus within this time-frame. a feature of arteriviruses is the ability to evade the immune response and induce persistent infections (kimman et al., 2009; sun et al., 2012) . for example, mice infected with lactate dehydrogenase-elevating virus (ldv) develop persistent lifelong viraemia which is believed to be facilitated by the fact that the primary target cell for ldv infection is a renewable, continuously regenerated and apparently 'non-vital' subpopulation of macrophages . persistent viraemia (months to years) has been also described for pigs infected with prrsv (benfield et al., 1999) , and for patas monkeys persistently infected with low-virulence isolates (p-248 and p-741) of simian haemorrhagic fever virus (gravell et al., 1986) . the presence of multi-organ lymphoplasmacytic inflammation in four possums that were euthanased or died within six to nine days following infection indicates that immune response to viral antigens begins early in the disease course. this is supported by detection of rising levels of wpdv antibody in two (possums 6 and 15) of those possums. the wpdv antibody levels in the remaining two possums (10 and 14) were already high at the time of experimental infection (giles et al., 2018) . this response, however, appears to be ineffective at containing virus replication within the time-period studied, as high levels of viral rna continued to be detected in tissues from possums that were euthanased approximately three weeks later. an on-going inflammatory response may promote egress of circulating monocytes into tissues. this may be an undesirable consequence as, in addition to contributing to tissue damage, they could serve as new targets for virus infection. the presence of the virus in serum, urine and salivary glands provides indicators on possible routes of transmission of the virus. fighting between possums with exchange of saliva and/or blood through bite wounds may facilitate transmission. grooming of juvenile possums (joeys) by mothers, face-to-face contact, and hissing during encounters could also facilitate spread of the virus via saliva. despite comparatively low levels of viral rna in the urine, urinary excretion of wpdv could provide a source of virus contamination in the environment. direct contact with urine from infected possums may also occur during sharing of dens by multiple possums. moderately high levels of viral rna in the serum raise the possibility that wpdv could be spread by blood-sucking ectoparasites. this is supported by successful induction of wpd by injection of macerated hematophagus mites (trichosurolaelaps crassipes) derived from wpd-affected possums into healthy possums in one of the previous studies (perrott et al., 2000c) . further studies are required to provide experimental support for the main routes of transmission during natural infection as well as the infectious dose of the virus required to elicit compatible clinical signs. higher levels of viral rna were consistently detected in the forebrain in comparison with the hindbrain. one possible explanation of these results is that location-specific differences in the numbers of cells supportive of viral replication exist. regional variations in the distribution of microglia have been described in the brain of mice, with denser populations of microglia in the forebrain as compared to the hindbrain (lawson et al., 1990; perry et al., 1985) . given that macrophages are the target cell of arteriviruses (duan et al., 1997; onyekaba et al., 1989; plagemann and moennig, 1992) , it is possible that microglia which are similarly derived from myeloid precursors, may support wpdv replication. whilst ihc for wpdv-antigen was positive in 8/12 brains from wpdv-infected possums, the cell types supporting viral replication were not unequivocally identified. further investigation into the cell types that support viral replication within the cns is thus warranted. the histological lesions observed in the wpd-infected possums were, in general, similar to those described previously (o'keefe et al., 1997; perrott, 1998) . lesions in the brain included varying degrees of non-suppurative meningoencephalitis, choroiditis, gliosis and glial nodules, the number of which varied between possums. the cause of the glial nodules in one control possum is unknown, however, it is unlikely to be related to active wpdv infection as this possum was clinically normal and viral rna was not detected in its brain. the preponderance of inflammatory infiltrates in the subependymal region of the brain in some infected possums is intriguing. similar periventricular lesion location has been reported for neurotropic coronaviruses such as feline infectious peritonitis virus and mouse hepatitis virus (diaz and poma, 2009 ) and occasionally for neurotropic arterivirus variants, for example during field outbreaks of prrsv (thanawongnuwech et al., 1997) . given the lack of overt endothelial cell damage and lack of immunopositivity for wpdv antigen within endothelial cells in the central nervous system (cns) and elsewhere, it is unlikely that wpdv traverses the blood-brain barrier within the cns through infection of endothelial cells. other possible mechanisms of transport into the cns could include passive transfer across the endothelium at pinocytotic junctions of the choroid plexus or transport across endothelium within infected blood-borne monocytes (whitley and gnann, 2002) . neurological signs are atypical with infection with other arteriviruses but can occur with neurotropic variants of prrsv and ldv (anderson et al., 1995; rossow et al., 1999; tian et al., 2007) . because of the multi-organ distribution of histological lesions and high viral rna levels in multiple tissues, the nervous system is not the sole target for wpdv replication. whilst the most clinically obvious signs in the experimentally infected possums were neurological, it is possible that disturbances in other organ systems, general malaise and severe acute wasting contributed in part to the clinical picture, both during the current and previous studies (giles et al., 2016; mackintosh et al., 1995; o'keefe et al., 1997; perrott, 1998; perrott et al., 2000c) . in contrast to previous reports (o'keefe et al., 1997; perrott, 1998) , renal lesions were consistently observed in the vast majority (11/12, 92%) of experimentally infected possums in the current study, which prompted further investigation into the aetiology of these lesions and their contribution to the clinical signs observed. mild azotaemia was present in two and severe azotaemia in another two of 12 infected possums suggesting that despite gross and pathological changes observed in the kidneys, acute severe kidney injury was not a universal feature of wpdv infection. whilst histological features of nephritis support the possibility that azotaemia in the four wpdv-infected possums was of renal origin, pre-renal causes such as dehydration cannot be ruled out, particularly in possums with milder azotaemia. the determination of urine specific gravity (usg) could have helped in the differentiation of pre-renal and renal azotaemia, but was not performed. leptospirosis was also considered as a possible cause of the renal changes. this disease is endemic in new zealand and infection with pathogenic serovars can result in kidney disease. however, only one control possum and one wpd-affected possum were positive for leptospiral dna and it is unlikely that pre-existing infection with pathogenic leptospira spp. resulted in the biochemical or histological changes observed in the kidneys of leptospira-negative possums. to help provide a further understanding of the pathogenesis of the renal lesions, ihc for wpdv antigen was performed. immunopositivity was predominantly observed within the cytoplasm of macrophages in interstitial inflammatory aggregates and to a lesser extent in cells within glomeruli. whilst the cellular populations supporting the virus replication within these locations are currently unknown, we speculate that these are cells of monocyte-macrophage lineage, such as interstitial macrophages and/or mesangial cells. this is based on the fact that arteriviruses are generally tissue-type and cell-type specific, primarily replicating in macrophages (duan et al., 1997; onyekaba et al., 1989; plagemann and moennig, 1992) . however, expanded cell and tissue tropism has been described for highly pathogenic arteriviruses, including a highly pathogenic prrsv that is capable of replication within distal renal tubular epithelium in pigs (li et al., 2012) . thus, to further understand the pathogenesis of renal lesions the exact cell types supporting viral replication within kidney tissues need to be further investigated. one important limitation of this study is that possums were inoculated with wpdv via an unnatural route, using a high dose of the infectious virus (giles et al., 2016) . hence, the pathogenicity of the virus following a natural exposure to a potentially lower infectious dose may differ from that described in this paper. however, healthy possums infected with various infectious inocula during one of the previous studies (perrott et al., 2000c) developed disease with indistinguishable clinical and pathological features irrespective of the type of inoculum and the route of exposure used. the latter included ip, intra-gastric, intra-tracheal and intra-dermal infections. in addition to the si, blood, urine and minced mites collected from wpd-affected possum were used as infectious inocula. importantly, five possums infected via direct contact with two experimentally infected possums also developed wpd. with the exception of an apparently longer incubation period, suggesting that it took some time for in-contact possums to acquire wpdv infection, the clinical disease and pathological changes were the same for all affected possums. altogether, this suggests that the tissue distribution of the virus and histopathological features 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pathological studies of wobbly possum disease in new zealand brushtail possums (trichosurus vulpecula) extensive cytocidal replication of lactate dehydrogenase-elevating virus in cultured peritoneal macrophages from 1-2-week-old mice lessons learned and knowledge gaps about the epidemiology and control of porcine reproductive and respiratory syndrome virus in north america viruses of the common brushtail possum (trichosurus vulpecula) (thesis) a neurological syndrome in a free-living population of possums (trichosurus vulpecula) a new papillomavirus of possums (trichosurus vulpecula) associated with typical wartlike papillomas routes of transmission of wobbly possum disease immunohistochemical localization of macrophages and microglia in the adult and developing mouse brain lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus: a new group of positive-strand rna viruses lactate dehydrogenaseelevating virus: an ideal persistent virus? springer seminars in immunopathology virus and virus-like particles observed in the intestinal contents of the possum, trichosurus vulpecula porcine reproductive and respiratory syndrome virus infection in neonatal pigs characterised by marked neurovirulence identification of pathogenic leptospira species by conventional or real-time pcr and sequencing of the dna gyrase subunit b encoding gene interplay between interferonmediated innate immunity and porcine reproductive and respiratory syndrome virus detection of european porcine reproductive and respiratory syndrome virus in porcine alveolar macrophages by two-colour immunofluorescence and in-situ hybridization-immunohistochemistry double labelling immunohistochemical detection of porcine reproductive and respiratory syndrome virus antigen in neurovascular lesions molecular confirmation of an adenovirus in brushtail possums (trichosurus vulpecula) emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark variation in hematological and serum biochemical values of the mountain brushtail possum, trichosurus caninus ogilby (marsupialia: phalangeridae) viral encephalitis: familiar infections and emerging pathogens characterisation of two enteroviruses isolated from australian brushtail possums (trichosurus vulpecula) in new zealand leptospira qpcr was kindly performed by dr julie collins-emerson, school of veterinary science, massey university, palmerston north, new zealand. histology sections and staining support was kindly provided by saritha gils and evelyn lupton. the study was supported by the following grants: massey university research fund, ivabs postgraduate research fund, and mcgeorge research fund. jg was supported by the colin holmes dairy scholarship, the george mason sustainable land use scholarship and the massey university doctoral scholarship. the authors declare that they have no conflict of interest. key: cord-267027-diwm1940 authors: le, shu-yun; chen, jih-h.; sonenberg, nahum; maizel, jacob v. title: conserved tertiary structure elements in the 5′ untranslated region of human enteroviruses and rhinoviruses date: 1992-12-31 journal: virology doi: 10.1016/0042-6822(92)90261-m sha: doc_id: 267027 cord_uid: diwm1940 abstract a combination of comparative sequence analysis and thermodynamic methods reveals the conservation of tertiary structure elements in the 5′ untranslated region (utr) of human enteroviruses and rhinoviruses. the predicted common structural elements occur in the 3′ end of a segment that is critical for internal ribosome binding, termed “ribosome landing pad” (rlp), of polioviruses. base pairings between highly conserved 17-nucleotide (nt) and 21-nt sequences in the 5′ utr of human enteroviruses and rhinoviruses constitute a predicted pseudoknot that is significantly more stable than those that can be formed from a large set of randomly shuffled sequences. a conserved single-stranded polypyrimidine tract is located between two conserved tertiary elements. r. nicholson, j. pelletier, s.-y. le, and n. sonenberg (1991, j. virol. 65, 5886–5894) demonstrated that the point mutations of 3-nt uuu out of an essential 4-nt pyrimidine stretch sequence uuuc abolished translation. structural analysis of the mutant sequence indicates that small point mutations within the short polypyrimidine sequence would destroy the tertiary interaction in the predicted, highly ordered structure. the proposed common tertiary structure can offer experimentalists a model upon which to extend the interpretations for currently available data. based on these structural features possible base-pairing models between human enteroviruses and 18 s rrna and between human rhinoviruses and 18 s rrna are proposed. the proposed common structure implicates a biological function for these sequences in translational initiation. poliovirus, coxsackievirus, and human rhinovirus are members of the picornaviridae family. they contain an unusually long, conserved 5' utr. all picornavirus mrnas, unlike most eukaryotic mrnas, lack a 5' cap structure (hewlett eta/., 1976; nomoto et a/., 1976) . ln vitro and in vivo experiments have shown that the internal initiation of cap-independent translation requires a large segment (nt 140 to 630) of the 5' utr of poliovirus (pelletier and sonenberg, 1988; trono et a/., 1988a,b) . the importance of several stem-loop structures in the 5'-end of the segment termed "ribosome landing pad" (rlp) were demonstrated in poliovirus type 2 (lansing strain) in viva and in vitro (meerovitch et a/., 1991) . the exact ribosome binding sequence, however, is not known. genetic analysis (trono et a/., 1988b; kuge and nomoto, 1987) of poliovirus mutants suggests that the rlp assumes a distinct tertiary structure that can be recognized by trans-acting factor(s) and/or 40 s ribo-somes. although some common rna secondary structures in the 5' utr of the enterovirus and rhinovirus had been derived by phylogenetic comparative sequence analysis (pilipenko et a/., 1989; rivera et al., 1988; skinner et a/., 1989) or by combining comparative sequence analysis and a thermodynamic suboptimal folding method (le and zuker, 1990) , the existence of a three-dimensional structure was not proposed directly. a pseudoknot is an important structural element that can significantly contribute to tertiary interactions in rna folding (puglisi et a/., 1988) . experimental evidence has implicated an important role for pseudoknots in protein recognition, translational mediation and the replication of the plant viruses (tang and draper, 1989; mcpheeters et a/., 1988; brierley et al., 1989 brierley et al., , 1991 pleij et a/., 1985; pot-tier et a/., 1990; bredenbeek et al., 1990) . mutational analyses in the (y operon mrna (tang and draper, 1989) and rps0 mrna (portier et a/., 1990) of escherichia co/i, as well as in the autogenous regulation of gene 32 protein expression in bacteriophage t4 (mcpheeters et a/., 1988) have also suggested that pseudoknot structures are specifically recognized by components of the translation apparatus. to understand the mechanism of internal initiation of cap-independent translation in the poliovirus, coxsackievirus, and human rhinovirus. the determina-tion of tertiary structure of the rlp in their 5' utrs is necessary. presently available programs of energy minimization for predicting secondary structure exclude the pseudoknot motif and other possible tertiary interactions because thermodynamic parameters for the structural elements of tertiary interactions are unknown. in this paper, we use a new method (chen et al., 1992) to predict possible pseudoknots or tertiary structures formed in the 5' utr. the method is a combination of the prediction of significantly stable rna secondary structures and evaluation of statistical significance for tertiary interactions. the validity of the predicted tertiary interactions in the sequence is then examined by conservation in all homologous sequence rnas in the 5' utrs of the poliovirus, coxsackievirus, and human rhinovirus. based on the theoretical structure model, potential base pairings between rlp and human 18 s rrna are determined. a functional role of the proposed common tertiary structure of rlp is suggested in the recognition of trans-acting factors and/or 40 s ribosomes to the rlp. it is proposed that the pyrimidine stretch downstream from the conserved 21-nt sequence contributes to an efficient translation of viral mrna in a manner analogous to that of the shine-dalgarno sequence in prokaryotic systems (pilipenko et al., 1992) . the sequence data of 1 1 polioviruses, 6 coxsackieviruses, and 5 human rhinoviruses were obtained from the genbank and embl databases. except for pv2w (poliovirus type 2, strain w-2) (pevear et al., 1990) ca21 (coxsackievirus a21) (hughes et al., 1989 ) ca9 (coxsackievirus a9) (chang et a/., 1989) and cvb3 (coxsackievirus b3) (klump et al., 1990) , the other 18 sequences are the same as those reported in a previous paper (le and zuker, 1990) . the multiple alignment of 22 sequences was performed using zuker's multiple alignment program, mal (le and zuker, 1990) . mal was written in fortran 77 and could run under iris/unix or vaxivms. the pseudoknot structures in these sequences were predicted using the rnaknot program (chen et a/., 1992) . rnaknot was written in fortran 77 on a cray-ymp/8128 (unicos) system. the method for predicting pseudoknots or tertiary structures in a given rna sequence can be summarized as follows: (1) construct a list (list a) of nonoverlapped regions that are thermodynamically and statistically significant in the given sequence . (2) create a list (list b) of nonoverlapped regions that are thermodynamically significant by comparing only their stability scores for each segment in the sequence. each item in list b is nonoverlapped with the regions in list a. (3) search and build a list of all possible pseudoknots that satisfy steric constraints and would not overlap with regions in list a and list b from both the 5' and 3' sides of the hairpin loop structure under consideration for each item in list a. (4) eliminate less stable pseudoknots from the list mentioned in the step 3. (5) evaluate the statistical significance of these computed potential pseudoknots using three scores z, n,, and q. in the evaluation, the z score is defined as z = (nobs -rmean)/sd for each tertiary interaction pattern in the sequence, where nobs is the number of times the pattern occurs in the real sequence, rmean is the average number of times the occurrence of the pattern in a set of scrambled sequences whose composition and length are identical to the actual sequence, and sd is the standard deviation. the scores n, and n2 are defined as the numbers of randomized sequences that have a pseudoknot thermodynamically more stable than the real sequence. n, and n2 differ in that the free energy contributed by a pseudoknot is calculated using different simulation rules. thus, a large value of z and/or a small value of n, or n2 might be an indication of nonrandomness of the occurrence of the pseudoknot folded in the sequence. it provides supporting evidence for the existence of the pseudoknot in rna molecules. the possible pseudoknot interactions in the 5' utr of pv2l (poliovirus type 2, lansing), hrv89 (human rhinovirus type 89) and cb4 (coxsackievirus 84) were searched throughout the sequence by the program rnaknot. by extensive simulations five potential pseudoknots in pv2l were selected. two were located at the 5'-end of the utr and three other tertiary interactions were found in the rlp of polioviruses and its downstream region. the two potential pseudoknots predicted in the 5'-end of pv2l utr are highly conserved in 11 polioviruses. the occurrence of the two pseudoknots, however, will destroy a significantly stable rna secondary structure formed in the folding region 2-88 (le and zuker, 1990) . furthermore, the two pseudoknots are not conserved in human rhinoviruses and coxsackieviruses. therefore, the two tertiary structures cannot be considered significant in enteroviruses and rhinoviruses. the predicted pseudoknots, k2 (tertiary interactions: between the region 497-501 and 550-554) and k3 (579-581 and 600-602) in the rlp of pv2l were found to be totally conserved in all 22 human enteroviruses and rhinoviruses. the hairpins involved in the two common pseudoknots k2 and k3 correspond to two significant structure motifs designated as sm2 (484-514) and sm3 (585-620) in a previous paper (le and zuker, 1990) . the 5-nt ugggu in totally conserved 21 -nt sequence of the 22 picornaviruses participates in the tertiary interaction of pseudoknot k2. also, compensatory base changes were detected in the computed pseudoknot k3. that is, two or more independent covariations occur in the complementary sequences such that the predicted pseudoknot k3 is preserved in all sequences. for instance, 5'-gug/ cac-3' base pairings in poliovirus type 1 vary to 5'-aug/cau-3' in pv2l and poliovirus type 3, to 5'-auu/ agu-3' in hrv89, and to 5'-auawauau-3 in hrvpi and hrv14 (see fig. 1 ). based on comparative sequence analysis, another well-conserved tertiary structural element, kl, was detected (459-464 and 559-564 in pv2l) in all 22 picornaviruses. the involved hairpin in the conserved tertiary structure element is a small hairpin of 4 bp that is thermodynamically significant. this small hairpin is a structural element in the common rna secondary structure for these viruses (le and zuker, 1990) . interestingly, the perfectly conserved 6-nt sequence gug-uuu (559 to 564) is included in the distinct tertiary interaction. the conservation of these three conserved tertiary interactions (kl , k2, and k3) in 22 sequences is shown in fig. 1 . the significance of the predicted conserved tertiary structure elements in 22 sequences of human enteroviruses and rhinoviruses was assessed individually. the three significance scores, z, n, , and n2, of these pseudoknots or tertiary structures (see materials and methods) were calculated from 1000 randomly shuffled sequences. the results are listed in table 1 . for most of the pseudoknots kl and k2 the values for n2 are not large (i.e., their ratios are less than 0.1). both ki and k2 have small n, values. for example, the three significance scores of z, n, and n2 of the pseudoknot kl in pv2l are 8.36, 31, and 84. it means that we can expect the average 31 (or 84) observations in 1000 random samples would form more stable pseudoknots than that of the predicted pseudoknot kl in pv2l. it indicates that the pseudoknot kl predicted in pv2l is highly stable and statistically significant. among these three significance scores, the parameter z may not be a sensitive measure from our experience (chen et a/., 1992) . the large values in z scores (say z > 4.5) for these pseudoknots shows that the occurrences of the specific base-pairing patterns in their tertiary interactions are not random. conserved tertiary structure elements in the 5' utr of human enteroviruses and rhinoviruses figures 2-4 show the common tertiary structures in the 5' utr of pv2l, hrv89, and cb4, respectively. in these common structural models, an 1 1-nt sequence of a 21 -nt conserved sequence (545 to 565) is involved in two conserved tertiary structure elements, kl and k2. the aug-7 (nt 588 to 590 in poliovirus type 2, whose position relative to the pyrimidine stretch is relatively constant in all picornaviruses; the number changes depend on the viral serotype) downstream from the 21 -nt sequence is embraced in the common tertiary structure element k3. based on the computed common tertiary structure, a new base-pairing model between pv2l and human 18 s rrna is proposed (fig. 2 ). in the model, the singlestranded purine rich region at the 3'-end of 18 s rrna (1823 to 1837) that is followed by a significantly stable stem-loop structure (le and maizel, 1989) can be complementary to the pyrimidine stretch between the conserved 21-nt sequence and aug-7. the singlestranded pyrimidine stretch is positioned between the two common tertiary structure elements. the basepairing models between hrv89 and human 18 s rrna and between cb4 and 18 s rrna are the same as that of pv2l (figs. 3 and 4) . the results show that the possible base pairings between the viral mrnas and human 18 s rrnas are well conserved in the human enteroviruses and rhinoviruses (fig. 5) . the possible interactions between these mrnas of picornaviruses and 18 s rrnas include most nt of the polypyrimidine tract downstream from the conserved 21 -nt sequence. the sequence complementary to the viral pyrimidine stretch is also evolutionary conserved among 18 s rrnas of eukaryotes. the chemical and rnase probing results also suggest that this complementary sequence is not base paired in the 18 s rrnas (rairkar et a/., 1988) . results from a number of laboratories (jang et al., 1988 (jang et al., , 1989 belsham and brangwyn, 1990; kuhn et a/., 1990; pelletier and sonenberg, 1988; bienkowska-szewczyk and ehrenfeld, 1988; jackson et a/., 1990) indicate that the translational initiation of picornaviruses involves a mechanism of internal ribosome entry. recently reported that the 5' border of the rlp core element of poliovirus type 2 was located between nt 134 and 155. deletions of 240-302, 350-380, and 450-480 in pv2l cause the complete abolition of internal initiation of translation. it demonstrates that a highly conserved and extensive stemloop structure (237-441 in pv2l) is essential for internal initiation of translation for polioviruses. small mutations within a 9-nt polypyrimidine sequence 5'-uuuccuuuu-3'(560 to 568) of pv2l showed that the 4-nt pyrimidine stretch sequence uuuc (560 to 563) was also required for the internal initiation . systematic mutation of this stretch of sequence revealed that the sequence uuuccuu constituted an important c&acting rna element for translation of poliovirus in vitro (pestova et al., 1991) . recently, nomoto and co-workers (lizuka et al., 1991) also demonstrated that the conserved 2 1 -nt sequence (546-566) was essential for in vitro translation of coxsackievirus bl (cbl). the experimental data strongly suggest that the 3'-end of rlp constitutes a highly ordered tertiary structure, which can be specifically an2 was computed by using parameter c = 2.0 (chen et al., 1992) . * if the stem 2 of the pseudoknot k3 in hrv2 is uug and aac instead of uauu and auaa. recognized by 40 s ribosomes and/or trans-acting factor(s). however, the details of the tertiary structure of internal ribosomal entry site have not been determined yet. in this paper we propose a common rna tertiary structural model for the 3' part of the 5'-utr of 22 picornaviruses. the conserved tertiary structure is situated just 3' to the functional stem-loop structure (237 to 441 in pv2l) for internal initiation of translation. the conserved 17-nt (445 to 461 in pv2l) and 2 1 -nt (543 to 563 in pv2l) sequences are involved in the superstructure. base pairings between the two highly conserved regions contribute two significant tertiary structure elements kl and k2 in the predicted structure model. the point mutations of 3 nt uuu out of an essential 4-nt pyrimidine stretch sequence uuuc would destroy the predicted tertiary interactions in the pseudoknot kl the proposed common tertiary structure can offer experimentalists a rationale upon which to extend interpretations of current data pestova et a/., 1991) . it is conceivable that the pseudoknot is specifically recog-nized by components of the translation apparatus as in the case of the translational control of the ribosomal s4 (tang and draper, 1989) and ribosomal protein s15 (pot-tier et a/., 1990) of the e. co/i. similarly, the conserved tertiary structure element k2 is suggested as a potential binding site for the interaction between by 40 s ribosomes and/or trans-acting factor(s) and viral mrnas. base complementarity between the pyrimidine stretch of the picornavirus mrna and 18 s rrna may participate in the interaction between viral mrna and the small ribosomal subunit (kuhn et al,, 1990; . the pyrimidine stretch uuucc has been suggested to be an analogue of the prokaryotic shine-dalgarno sequence by agol and co-workers (pilipenko et al., 1992) . based on the common rna secondary structures (le and zuker, 1990 ) of the 5' utr in 18 human enteroviruses and rhinoviruses, two sequences complementary to the highly conserved polypyrimidine sequence in all picornaviruses were identified in human 18 s rrna . they are located at nt 1194 to 1207 (3'-ggcaguuaaggpredicted base-pairing model between poliovirus type 2 (lansing, referred to as pv2l) and human 18 s rrna. the conserved tertiary structure folded in the rlp of pv2l is highly stable and statistically significant. the three conserved tertiary structure elements in 22 picornaviruses are denoted by kl , k2, and k3. the rna secondary structure of human 18 s rrna 3'-end is based on the published model (huysmans and wachter, 1986; chan et al., 1984) . an asterisk between two sequences denotes the base pairing between pvzl and human 18 s rrna. aaa-5') and nt 1822 to 1837 (3'-ggaacaaugcuga-aaa-5'). the two regions are not base-paired in the common rna secondary structural model of 18 s rrnas and are highly conserved throughout evolution. according to the new common tertiary structure model, the sequence 3'-ggaacaaugcugaaaa-5' spanning nt 1822 to 1837 in human 18 s rrna is a better complementary sequence to the polypyrimidine stretch of 22 picornaviruses. for polioviruses, at least 11 of 14 nt can be base pairing with the complementary sequence. it is known that the sequence upstream from the aug-7 (in pv2l) affects translational effi-ciency of poliovirus (pelletier et al., 1988a,b; pilipenko et a/., 1992) . structural analyses for these variants described by pilipenko et a/. (1992) and pestova et a/. (1991) reveal that the base-pairing interaction between viral mrna and 18 s rrna would be destabilized in the mutated transcripts. the template activity of the mutated transcripts showed markedly lower than that of the wild type (pilipenko et al., 1992; pestova et al., 1991) . the pseudoknot k3, which contains an aug-7 and aug-6 (in pv2l) is conserved in all 22 tested picornavirus sequences. this tertiary interaction in coxsackieviruses is extremely weak (at least two g-u base pairings in the structure) and is not supported by the statistical analysis (large n, and n, values) in our procedure. however. it can be supported by compensatory base predicted base-pairing model between human rhinovirus type 89 (hrv89) and human 18 s rrna. for further details see the caption to fig. 2 . changing found in these predicted structural models. it is worth noting that the single-stranded sequence spanning between pseudoknots kl and k3 is partially complementary to the 3'-end of human 18 s rrna. for human rhinovirus rna, borman and jackson (1992) recently demonstrated that the ribosome entry site included the single-stranded sequence. several lines of evidence indicated that pseudoknot motifs might be involved in protein binding to a mrna (mcpheeter et a/., 1988; tang and draper, 1989; portier et al., 1990) . recently, brown et a/. (1991) proposed a putative pseudoknot formed in the 3'-end of the 5' utr of hepatitis a virus. although our theoretical model awaits verification by experiments, our model does provide a reasonable explanation for current mu-tagenesis results in the translational initiation of human enteroviruses pilipenko et al., 1992; pestova et a/., 1991) . brierley et a/. (1991) recently demonstrated that the rna pseudoknot in coronavirus mrna could not be functionally replaced by a hairpin structure that was composed of an equivalent set of base pairs. also, the removal of a single basepair contact in either stem of the pseudoknot reduced or abolished the ribosomal frameshifting. thus, it is conceivable that two helical stems have to be in proximity to generate a specific functional structure, perhaps stacked coaxially. it is such a structural feature that the rna pseudoknot can provide as a recognition site for protein binding. sonenberg and co-workers (meerovitch et al., 1989) reported that a novel protein (~52) could specifically bind to nt 559 to 624 in 5' utr. wimmer and coworkers (pestova et a/., 1991) also indicated that a ribosome-bound protein p57 was an important cofactor for the translation of poliovirus rna in vitro and it could bind upstream of the pyrimidine stretch. thus, we suggest that the common superstructure formed in the sequence (nt 450 to 616 in pv2l) is essential for the translational initiation of human enteroviruses and rhinoviruses. we conjecture that the 40 s ribosome subunit can recognize the pseudoknot kl, and probably k2, in translation initiation of poliovirus mrnas. the 3' -ggmxaugcugaaa-5' l *** **** *** 5'-ccw.wau. .wuauu-3' 5'-ccw.wau..wuauu-3' 5'-ccw.wau. .wuauu-3' 5'-ccu.gwau..wuuauc-3' 5'-ccw.wau. .wwaca-3' 5'-ccw.wau. .uwuaca-3' 5'-ccw.wau.ucwga-3' 5'-ccw.wau.ucucauu-3' 5'-ccw.wau.ucwga-3' 5'-ccw.wau.ucwga-3' 5'-ccw.wau.ucwga-3' 5'-caw.wauuccuauac-3' 5'-caw.wauuccuauac-3' 5'-caw.wauuccuauac-3' 5' -ccw. wau .ucwaccu-3' 5' -cwu. wauuccuauau-3' 5' -cccu. wauauucauac-3' 5'-cuca.wuu.ucwc-3' 5'-cuca.wuu.ucwc-3' 5'-cacu.wuu.ccw-3' 5'-cacu.wuu.ccwuau-38 5'-ccu.gwuu.ucwuug-3' l ** **it l l *** human 18s rrna 3'-ggaacaaugcugaaa-5' (1823-1837) fig. 5 . base pairings between human 18 s rrna 3'-end and polypyrimidine stretches in the 5' utr of human enteroviruses and rhinoviruses. an asterisk between two sequences denotes the base pairing between these virus mrnas and human 18 s rrna. binding interaction of pseudoknot kl (and/or k2) and 40 s ribosome subunit and/or trans-acting factors can be strengthened by the strong base pairing between the sequences in the polypyrimidine stretch and 3'-end of 18 s rrna. these plausible structural predictions add to the known intriguing properties of the long untranslated 5' regions of picornavirus genome/messages. the project was started when s.-y. le was working at the institute for biological science, national research council of canada. research was sponsored, at least in part, by the national cancer institute, dhhs, with program resources, incorporated. the content of this publication does not necessarily reflect the views or policies of the department of health and human services, nor does mention of trade names, commercial products, or organizations imply endorsement by the u.s. government. a region of the 5' noncoding region of 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cap-independent translation of poliovirus mrna is conferred by sequence elements within the 5' noncoding region mutational analysis of upstream aug codons of poliovirus rna translation of poliovirus rna: role of an essential cis-acting oligopyrimidine element of a cellular 57 localization of genomic regions specific for the attenuated, mouse adapted poliovirus type 2 strain w-2 conserved structural domains in the 5'-untranslated region of picornaviral genomes: an analysis of the segment controlling translation and neurovirulence prokatyotic-like cis elements in the cap-independent internal initiation of translation on picornavirus rna a new principle of rna folding based on pseudoknotting translational control of ribosomal protein s15 a pseudoknotted rna oligonucleotide chemical probing of adenine residues within the secondary structure of rabbit 18 s ribosomal rna comparative sequence analysis of the 5'noncoding region of enteroviruses and rhinoviruses new model for the secondary structure of the 5' non-coding rna of poliovirus is supported by biochemical and genetic data that also show that rna secondary structure is important in neurovirulence unusual mrna pseudoknot structure is recognized by a protein translational repressor translation in mammalian cells of a gene linked to the poliovirus 5' noncoding region an rna sequence of hundreds of nucleotides at the 5'end of poliovirus rna is involved in allowing viral protein synthesis key: cord-263334-wwkdum94 authors: li, chen; ge, ling-ling; li, peng-peng; wang, yue; dai, juan-juan; sun, ming-xia; huang, li; shen, zhi-qiang; hu, xiao-chun; ishag, hassan; mao, xiang title: cellular ddx3 regulates japanese encephalitis virus replication by interacting with viral un-translated regions date: 2014-01-20 journal: virology doi: 10.1016/j.virol.2013.11.008 sha: doc_id: 263334 cord_uid: wwkdum94 japanese encephalitis virus is one of the most common causes for epidemic viral encephalitis in humans and animals. herein we demonstrated that cellular helicase ddx3 is involved in jev replication. ddx3 knockdown inhibits jev replication. the helicase activity of ddx3 is crucial for jev replication. gst-pulldown and co-immunoprecipitation experiments demonstrated that ddx3 could interact with jev non-structural proteins 3 and 5. co-immunoprecipitation and confocal microscopy analysis confirmed that ddx3 interacts and colocalizes with these viral proteins and viral rna during the infection. we determined that ddx3 binds to jev 5′ and 3′ un-translated regions. we used a jev-replicon system to demonstrate that ddx3 positively regulates viral rna translation, which might affect viral rna replication at the late stage of virus infection. collectively, we identified that ddx3 is necessary for jev infection, suggesting that ddx3 might be a novel target to design new antiviral agents against jev or other flavivirus infections. japanese encephalitis virus (jev), a mosquito-borne flavivirus, is one of the most common causes for epidemic viral encephalitis in humans and animals (solomon, 2003; tsai, 2000) . its genome encodes a single polyprotein composed of three structural proteins [core, membrane (prm/m), and envelope (e)] and seven nonstructural proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b, and ns5). the mature viral proteins are produced via proteolytic processing of the single polyprotein by viral serine protease (ns2b-ns3) and cellular proteases rice, 2001, 2003) . the viral replication is initiated by the replication complex through a process of rna-dependent rna polymerization in the perinuclear endoplasmic reticulum membranes (westaway et al., 2003) . nonstructural proteins 3 and 5 are components of the replication complex, which associates with the 3′ noncoding region of genomic rna to initiate viral replication (chen et al., 1997; edward and takegami, 1993) . ns5, the largest and most conserved viral protein, contains methyltransferase (mtase) and rna-dependent rna polymerase (rdrp) domain. the mtase is involved in methylation of the 5′-cap structure of genomic rna, while rdrp is important for viral genome replication (ackermann and padmanabhan, 2001; egloff et al., 2002; guyatt et al., 2001; koonin, 1993) . until now there is no specific and effective treatment available for japanese encephalitis virus infection; vaccination is the only effective control measure. although vaccination is widely used, some public health concerns exist (takahashi et al., 2000) . cellular dead-box helicases reside in both nucleus and cytoplasm (chao et al., 2006; yedavalli et al., 2004) ; they are involved in a variety of cellular processes related to rna, such as splicing, mrna export, transcriptional and translational regulation, rna decay and ribosome biogenesis (beckham et al., 2008; choi and lee, 2012; garbelli et al., 2011; rocak and linder, 2004; shih et al., 2007; soulat et al., 2008) . ddx3(x), identified in 1997, is a member of the dead (asp-glu-ala-asp)-box helicase family (park et al., 1998) . it is ubiquitously expressed in a wide range of tissues (kim et al., 2001) . ddx3 has orthologous candidates in many eukaryotic organisms from yeast and plants to animals; they share significant identities between each other . ddx3 is a nucleo-cytoplasmic shuttling protein characterized by nine conserved motifs. it displays rna-dependent atpase and atp-dependent rna helicase activities (rocak and linder, 2004; schröder, 2010) . recently, ddx3 attracts great interest because several studies have shown that it is involved in the replication of several viruses that have major global health threats, such as human immunodeficiency virus (hiv) (owsianka and patel, 1999) , hepatitis b virus (wang et al., 2009 ), hepatitis c virus (hcv) (ariumi et al., 2007; mamiya and worman, 1999; owsianka and patel, 1999; you et al., 1999) and poxviruses (kalverda et al., 2009) . the other cellular helicases such as ddx1, ddx5, dhx15, ddx28, ddx56 and ddx42 are the cofactors for the replication of hiv, coronavirus, hepatitis b virus, hcv, jev and wnv (ariumi et al., 2007; fang et al., 2005 fang et al., , 2004 li et al., 2013; wang et al., 2009; xu et al., 2010 xu et al., , 2011 xu and hobman, 2012) . two recently published papers report that the novel ddx3 inhibitors possess the anti-hiv activity which highlights ddx3 as a novel target for the development of antiviral agents radi et al., 2012) . in order to determine whether ddx3 is involved in the jev infection, we first silenced the endogenous ddx3 expression and found that jev replication was significantly reduced. we then determined that the helicase activity is crucial for jev replication by studying the effects of the helicase-dead ddx3 mutants and ddx3 inhibitor on virus replication. furthermore, we also demonstrated that ddx3 could interact with jev ns3, ns5-mtase and ns5-rdrp proteins. meanwhile, we found that ddx3 could bind to the jev 5′ and 3′ un-translated regions (utr) and colocalize with viral rna during viral infection. finally, we showed that ddx3 plays an important role in viral protein translation, which might affect the viral rna replication at the late stage of viral infection. collectively, our studies demonstrated that cellular ddx3 plays critical roles in regulating jev infection. it has been reported that ddx3 plays important roles in the replication of several viruses schröder, 2010) . to determine whether ddx3 is required for jev replication, we western blotting analysis of cellular lysate using anti-ddx3 or anti-β-actin antibody after ddx3 knockdown by ddx3 shrna. (b) viral titers determined by plaque formation assay after ddx3 knockdown. bhk-21 cells transfected with the ddx3 shrna were infected with jev (moi¼ 0.01) for 48 h, viral titers determined by plaque formation assay at 48 hpi. (c) the jev genomic rna levels in jev infected bhk-21 cells monitored by q-pcr after ddx3 knockdown. bhk-21 cells transfected with the ddx3 shrna were infected with jev (moi¼ 0.01) for 48 h, jev rna copy number was determined by q-pcr at 48 hpi. (d) western blot analysis on the jev envelope (e protein) and ns5 protein expression levels in jev infected bhk-21 cells after ddx3 knockdown. (e) bhk-21 cells transfected with the ddx3 shrna were infected with jev (moi¼ 0.01) for 48 h. the amount of virus released into the medium was determined by plaque formation assay at different time points. (f) bhk-21 cells transfected with different amounts of ddx3 shrna plasmid were infected with jev (moi ¼0.01), 48 h later, the amount of virus released into the medium was determined by plaque formation assay. the differences between means were considered significant at *p o 0.05, very significant ** p o 0.01. first used ddx3 shrna to reduce the expression level of endogenous ddx3 before the cells were infected with jev (moi ¼0.01). western blot analysis using anti-ddx3 antibody demonstrated that 90% reduction of protein expression level was achieved upon gene silencing compared with the control (fig. 1a) . a reduction of the endogenous ddx3 expression resulted in more than 15-fold decrease in viral titers (fig. 1b) and a significant decrease in jev genomic rna level (fig. 1c ). the viral protein expression level was also decreased after ddx3 silencing as detected by western blot analysis using anti-jev-envelope monoclonal antibody and anti-jev-ns5 polyclonal antibody (fig. 1d) . as knockdown of ddx3 can influence the replication of jev at 48 hpi, we further determined the effect of knockdown of ddx3 on jev replication at different time points. the cells were infected with jev (moi ¼0.01) after being transfected with ddx3 shrna plasmid; the virus loads were determined at different time points. as shown in fig. 1e , the titers of jev in ddx3 knockdown cells were lower than that in the control cells (p o0.01). at the same time, we determined whether the effect of ddx3 knockdown on jev replication is dose-dependent; we transfected the bhk-21 cells with different amounts of ddx3 shrna plasmid before infecting the cells with jev (moi ¼0.01). the virus load analysis showed that ddx3 knockdown reduced jev titers in a dose-dependent manner. the ddx3 shrna interference efficiency of different amounts of ddx3 shrna plasmid was detected using anti-ddx3 polyclonal antibody (fig. 1f ). in order to determine whether cellular ddx3 is involved in virus assembly or release, the bhk-21 cells were transfected with ddx3 shrna plasmid before being infected with jev (moi ¼0.01). 48 h later, the jev rna copy numbers between the supernatants and the cell lysates were detected; although the rna copy numbers in the cell lysates were nearly 15-fold higher than that in the supernatants, the ratios between them were almost the same between the ddx3 knockdown cells and the control cells (p 40.05), demonstrating that ddx3 silencing did not affect jev assembly ( fig. 2a) . meanwhile, the ratios of the viral titers in the supernatants and cell lysates were also similar between ddx3 knockdown cells and the control cells (p 4 0.05) as determined by plaque formation assay (p 40.05) (fig. 2b) , indicating that ddx3 knockdown did not affect the virus release. in order to determine whether the helicase activity of ddx3 was important for jev replication, the ddx3 helicase-dead mutant lacking either atpase activity (k230e) or rna unwinding activity (s382l) (cordin et al., 2006; garbelli et al., 2011) was used. bhk-21 cells were first transfected with ddx3 shrna plasmid to decrease the endogenous ddx3 expression level. 24 h later, the cells were transfected with rna interference-resistant plasmid (to exclude the influence of ddx3 shrna plasmid) pcdna3.1-ddx3r-ha, pcdna3.1-ddx3r-k230e-ha or pcdna3.1-ddx3r -s382l-ha before the cells were infected with jev (moi ¼0.01). the virus titers were detected 2 days later by plaque formation assay, the results showed that overexpression of ddx3r-k230e, ddx3r-s382l and the control plasmid pcdna3.1 after ddx3 knockdown resulted in the reduction of jev replication for 13-fold (p o0.01), 12-fold (p o0.01) and 15-fold (p o0.01). meanwhile overexpression of ddx3r after ddx3 knockdown did not reduce jev replication when compared with the control cells (fig. 3a) , suggesting that overexpression of the exogenous ddx3r could restore jev replication. the q-pcr results of the ddx3r, ddx3r-k230e or ddx3r-s382l transfected cells after ddx3 knockdown were consistent with the results of plaque formation assay ( fig. 3b ) (p o0.01). the expression of endogenous ddx3 or ddx3 mutants was confirmed by western blot analysis using anti-ha monoclonal antibody and anti-ddx3 polyclonal antibody (fig. 3c) . both of the results demonstrated that ddx3 helicase activity was crucial for jev infection. the cell viability of all ddx3 constructs and control plasmids transfected bhk-21 cells was not significantly affected as determined by using the cytotoxicity test using cytotox 96 s nonradioactive cytotoxicity assay kit (fig. 3d) . it has been shown that ddx3 is involved in hiv-1 replication by exporting un-spliced or partially spliced viral rnas from the nucleus to the cytoplasm (yedavalli et al., 2004) , and one recently published paper reported that two chemical compounds (cmp6 and cmp8) can specifically inhibit the helicase activity of ddx3, and therefore inhibit hiv replication in pbmcs (radi et al., 2012) . we synthesized cmp6 to further confirm whether ddx3 helicase activity is critical for jev infection. we infected bhk-21 cells with jev virus (moi ¼0.01) at 37 1c for 2 h before cmp6 was added (0-50 μm). the plaque formation assay at 48 hpi showed that cmp6 significantly inhibited jev replication by 20-fold at the concentration of 50 μm (fig. 4a ). the q-pcr also confirmed the results (fig. 4b) , and the results were consistent with the ddx3 helicase-dead mutants experiment. meanwhile, we confirmed that cmp6 did not stimulate significant cytotoxicity in bhk-21 cells at the concentration of 50 μm, while significant cytotoxicity was detected at the concentration of 100 or 200 μm by ldh assay (fig. 4c) . therefore, 0-50 μm of cmp6 was used in our study. in summary, our results suggested that the helicase activity of ddx3 was necessary for jev infection. the influence of ddx3 knockdown on virus assembly. the ratio of jev rna copy numbers between the supernatants and the cell lysates were detected by q-pcr at 48 hpi. (b) the influence of ddx3 knockdown on virus release. the bhk-21 cells was infected with jev (moi¼ 0.01) after ddx3 silence, the ratio of the virus titers in supernatants and cell lysates were determined by plaque formation assay at 48 hpi. the differences between means were considered significant at *p o 0.05, very significant **p o0.01. it has also been shown that both jev ns3 and ns5 are components of the replication complex (uchil and satchidanandam, 2003) . we next determined whether ddx3 was involved in jev replication by interacting with jev proteins responsible for viral rna replication. we used gst-pulldown assay to monitor the interactions between ns3, ns5-mtase, ns5-rdrp and ddx3 or vice versa. the western blot analysis showed gst-ns3, gst-ns5-mtase or gst-ns5-rdrp could interact with ddx3 ( fig. 5a) , and gst-ddx3 could also interact with his-tagged jev ns3, ns5-mtase or ns5-rdrp (fig. 5b ). ns5 and ns3 has been hypothesized to seed the formation of the replication complex, and they may form a replication complex together with 3′ noncoding region of jev genomic rna (uchil and satchidanandam, 2003) . therefore, we examined the interactions between ddx3 and viral proteins in bhk-21cells. we first infected the bhk-21 cells with jev (moi ¼0.01). 48 h later, the anti-ddx3 polyclonal antibody was used to co-immunoprecipitate the proteins from the whole cell lysate. the results showed jev ns3 and ns5 proteins could be co-immunoprecipitated by the endogenous ddx3 (fig. 5c ). since ddx3 is a nucleic acid-binding protein, it is possible that the interactions might be mediated by rna or dna, and then we determined the interactions between ddx3 and viral proteins in the presence of rnase (100 μg/ml) and dnase (100 μg/ml). western blot analysis showed that the interaction between ddx3 and jev ns3 or ns5 proteins was not mediated by rna or dna (fig. 5d) . several studies reported that ddx3 can be incorporated into hbv, herpes simplex virus type 1(hsv-1) and human cytomegalovirus (hcmv) (stegen et al., 2013; varnum et al., 2004; wang et al., 2009 ), so we also determined whether ddx3 was incorporated into the jev virions. we infected the bhk-21 cells with jev (moi¼ 0.01) for 48 h, the anti-e monoclonal antibody was used to co-immunoprecipitate the mature virions in the medium, the virions were then detected using anti-ddx3 polyclonal antibody by western blot analysis. we did not detect the presence of ddx3 in our experiment, suggesting that ddx3 is not incorporated into the jev virions (fig. 5e ). ddx3 colocalized with the jev ns3 and ns5 proteins and viral rna during virus infection as previously reported, ddx3 is primarily localized in the cytoplasm (choi and lee, 2012; lai et al., 2008) . to find out whether the cellular localization of the endogenous ddx3 was changed during jev infection and whether ddx3 colocalized with jev ns3 and ns5 proteins, we infected bhk-21 cells with jev (moi¼ 0.01) for 48 h, and studied the localization of ddx3 and viral proteins by confocal microscopy. the results demonstrated that ddx3 colocalized with jev ns3 and ns5 proteins in the cytoplasm (fig. 6a) . we did not observe any changes in the distribution of ddx3 in the cells infected with jev (moi¼ 0.01) compared with mock-infected cells. the monoclonal antibody against jev envelope protein was used to confirm that the cells were not infected. meanwhile, we also found that endogenous ddx3 colocalized with jev rna in the cytoplasm (fig. 6b) , implying that ddx3 might be involved in jev rna replication. ddx3 interacts with jev 5′ and 3′ utr and positively regulate jev protein translation and viral rna replication since jev ns3 and ns5 bind to the 3′ utr of the viral rna during virus replication (chen et al., 1997) , we next determined whether ddx3 could bind to jev 5′or/and 3′-utr. we labeled jev 5′ and 3′ utr with tobramycin affinity tag to enable the rnas to bind to tobramycin matrix beads to pulldown the endogenous ddx3. the western blot results showed that ddx3 could bind to both jev 5′ and 3′ utrs (fig. 7a) , which was consistent with the fact that ddx3 colocalized with viral rna during the infection (fig. 6b ). it has been suggested that 5′ and 3′ utr of jev are both involved in the first round of viral protein translation and also in the virus rna replication (chien et al., 2011) , so we constructed a jev replicon reporter system to determine whether ddx3 could regulate viral translation and/or rna replication according to the previous study (chien et al., 2011) . bhk-21 cells were first transfected with ddx3 shrna to knockdown the expression of ddx3, the non-targeting shrna was used as the control. the cells were then transfected with in vitro-transcribed jev replicon and firefly luciferase rna was used as the internal control. the cells were harvested for dual luciferase assays at 3, 6, 9, 24, 48, and 72 h post transfection. the results showed that there were two peaks of replicon luciferase activity, which represented the first translation and the second replication peak. in ddx3 knockdown cells, lower luciferase activities were observed for all of the time points in both peaks (fig. 7b) , indicating that viral protein translation and viral rna replication were all hampered by the knockdown of ddx3. because jev rna replication depends on viral proteins, we next confirmed the roles of ddx3 on viral translation using a replication-deficient replicon, which contains a gdd-aag mutation in ns5 polymerase (blight et al., 2003) . the results showed that luciferase activity derived from deficient-replicon rna was lower in ddx3 knockdown cells when compared with the control cells (fig. 7c) . however, the luciferase activities derived from a control reporter, renilla luciferase flanked by cellular gapdh 5′ and 3′ utrs were not significantly changed (fig. 7d ). in conclusion, all the data demonstrated that ddx3 could positively regulate jev infection by promoting viral protein translation. because ddx3 can bind to viral 3′ utr and viral ns3 and ns5 proteins, ddx3 might promote viral rna replication. the prevalence of the disease caused by jev and the limited efficacy of therapies stimulate the search for safer and more nawa et al., 2003; ray and shi, 2006; yoo et al., 2009 ). however, the virus resistance to these kinds of inhibitors has been a problem. one possible solution to overcome virus resistance mutations is to target host cellular factors . theoretically, a drug targeting the cellular factors important for the viral life cycle can be used to inhibit the viral infection (kwong et al., 2005) . various host proteins are known to interact with ddx3, such as several translation initiation factors, transcription factor sp1, spliced mrna in an exon junction complex, mrnp complex, nucleo-cytoplasmic shuttling proteins (crm1 and tap) lai et al., 2008; schröder, 2010; yennamalli et al., 2009 ) and eif4e (shih et al., 2007) . several viral proteins have also been reported to interact with ddx3, including hepatitis b virus dna polymerase, hepatitis c virus core protein, vaccinia virus k7, hiv-1 and poxviruses proteins (angus et al., 2010; ariumi et al., 2007; mamiya and worman, 1999; owsianka and patel, 1999; schröder, 2010; sun et al., 2010; wang et al., 2009; wang and ryu, 2010) . given that ddx3 is required for replication of hbv, hcv, hiv and poxviruses, it can be suggested that it is a promising target for drug development against these viruses that pose major global health threats kwong et al., 2005) . many single-stranded positive-sense rna viruses have been reported to use the host intracellular membranes as platforms for viral replication (strauss and strauss, 1994; wimmer et al., 1993) . the viral genome replication is thought to utilize viral proteins as well as host proteins (kuo et al., 1996; lindenbach et al., 2005; lindenbach and rice, 1997; mackenzie et al., 1998; uchil and satchidanandam, 2003; westaway et al., 1997) . in our studies, we demonstrated that ddx3 was required for jev replication, the infectious virus production was strongly inhibited by ddx3 knockdown. we also confirmed that helicase activity of ddx3 was crucial for jev replication. it has been predicted that jev 5′ and 3′ utr contain highly conserved rna secondary structures involved in the regulation of viral translation and rna replication (brinton and dispoto, 1988; song et al., 2008) . the minus-strand rna synthesis requires the presence of both the 5′ and 3′ utr (you et al., 2001) , and genomic rna cyclization mediated by 5′ and 3′-utr is essential for viral replication in all mosquito-borne flaviviruses (fan et al., 2011; ivanyi-nagy and darlix, 2012) . ddx3 also has been reported to be involved in translation initiation (geissler et al., 2012) and ddx3 can promote hiv proteins translation by binding to viral 5′ utr. it is most likely that ddx3 destabilizes the rna structure locally to form a region large enough to allow the recognition of the m 7 gtp cap or loading onto the 40s subunit to initiate protein translation (soto-rifo et al., 2012) . a recently published paper suggests that hcv 3′ utr retains ribosome complexes during translation termination to facilitate efficient initiation of subsequent rounds of translation (bai et al., 2013) . our jev replicon experiment confirmed that ddx3 positively regulates jev protein translation. jev ns3 and ns5 proteins have been shown to play important roles in viral genome replication (chen et al., 1997) . although we confirmed the role of ddx3 in jev protein translation, we could not rule out its role in viral rna replication in our studies. since ddx3 could bind to jev 5′ and 3′ utr and the helicase activity was necessary for jev replication, we suspected that ddx3 might help the unwinding of the secondary structure viral rna to enable viral rna replication more efficiently; especially our gst-pulldown, gst-fused jev, ns3, ns5-mtase or ns5-rdrp protein was first incubated with gst beads. after washing five times with pbs, the beads were incubated with his-tagged ddx3. the target proteins were detected by anti-his or anti-gst monoclonal antibody. (b) gst-fused ddx3 was incubated with gst beads first. the beads were then incubated with either his-tagged jev ns3, ns5-mtase or ns5-rdrp protein after extensive washing with pbs. the target proteins were detected by western blot analysis using anti-his or anti-gst monoclonal antibody. the gst protein was used as the control. (c) ddx3 interacted with jev ns3, ns5 proteins during virus infection. bhk-21 cells was infected with jev (moi ¼0.01). 48 h later, the anti-ddx3 polyclonal antibody was used to co-immunoprecipitate proteins from the whole cell lysate either in the absence or presence of rnase (100 μg/ml) and dnase (100 μg/ml). the jev-ns3 and ns5 proteins were detected using their polyclonal antibodies respectively. the cell lysates (2% input) were analyzed in parallel by western blot. (d) ddx3 was not incorporated into the virions. bhk-21 cells were infected with jev (moi¼ 0.01) for 48 h, the anti-e monoclonal antibody was used to immunoprecipitate the mature virions in the medium, the proteins in the immunoprecipitated virions (ip) were detected with anti-ddx3, anti-envelope (e protein) or anti-core protein antibody. co-immunoprecipitation and confocal microscopy analyses demonstrated that ddx3 could interact with jev ns3, ns5 and viral rna. collectively, our results suggested that ddx3 regulates jev replication by modulating jev protein expression. therefore, the drugs targeting ddx3 might be used for treatment of japanese encephalitis virus or other flaviviruses infection. bhk-21 cells (c-13, american type culture collection) were maintained in dulbecco's minimal essential medium supplemented with 10% fetal bovine serum and penicillin (100 u/ml) and streptomycin (100 mg/ml) at 37 1c in 5% co 2 . sa14-14-2 strain of jev was propagated in bhk-21 cells, and the viral titers were determined by plaque formation assay. the cells were transfected using polyethylenimine (25 kda; sigma-aldrich). the amount of plasmid dna with which the cells were transfected (1 μg per well of 24-well plate and 10 μg per 100 mm plate) was kept constant by the inclusion of empty expression vector. transfection efficiencies around 70% were routinely obtained by using pegfp-n2 (clontech) plasmid to monitor the transfection efficiency. to construct ha-tagged pcdna3.1-ddx3, pgex-4t-3-ddx3, pcold-i-ddx3, and ddx3 gene were amplified by pcr using ha-ddx3 plasmid (as a gift from prof. k.t. jeang) as the template with speci-fic primers and cloned into pcdna3.1-(þ ) (invitrogen), pgex-4t-3 (ge healthcare) or pcold-i (takara) vector respectively. the genes encoding jev core, ns3, ns5-mtase (1-268) or were also subcloned into pet-24a (þ ) vector (novagen). the oligonucleotides gtgccgtcttggttagaaa was cloned into pgpu6/neo vector as short hairpin rna (shrna) plasmid against ddx3 (lai et al., 2008) . the oligodeoxyribonucleotide encoding nontargeting shrna (gttctccgaacgtgtcacgt) was cloned into pgpu6/ neo vector by genepharma (shanghai, china), which was used as the negative control. pcdna3.1-ddx3-k230e-ha, pcdna3.1-ddx3-s382l-ha mutants were made with quick-change site-directed mutagenesis kit (stratagene) according to the manufacturer's instructions using pcdna3.1-ddx3-ha as the template by pcr using specific primers. to construct rna interference resistant pcdna3.1-ddx3r, pcdna3.1-ddx3r-k230e-ha or pcdna3.1-ddx3r-s382l-ha mutant plasmid, the correspondent sequence to ddx3 shrna in these ddx3 mutants was mutated into gtcccatcatggctggag without introducing any residue change by using quikchange site-directed mutagenesis kit (stratagene) using pcdna3.1-ddx3-ha, pcdna3.1-ddx3-k230e-ha or pcdna3.1-ddx3-s382l-ha as the template. the nucleotide sequences of all the plasmids were confirmed by dna sequencing. to construct the templates for jev 5′ and 3′ utr rna transcription, the jev 5′ or 3′ utr gene was amplified by pcr with specific primers: jev5′-sense primer: taatacgactcactatagggagaagtttatctgtgt-gaacttcttggcttag, anti-sense primer: ggctcagcacgagtgtagc-taaacctcgctatact aagccggttatcttccgttctaa, jev 3′-sense primer: taatacgactca tagggtagtgtgatttaaggtagaaaagtag, jev 3′anti-sense:ggctca gcacgagtgtagctaaacctcgctatactaa-gccagatcttgtgttcttcct. an unrelated control rna was generated by pcr amplification of part of the ampicillin resistance gene from pcdna3.1 (þ) vector (1-250 bp) using ampicillin-sense fig. 7 . ddx3 bound to jev 5′ and 3′-utr and modulated jev protein translation and rna replication. (a) ddx3 bound to jev 5′ and 3′-utr. jev 5′ and 3′-utr or the control rna was transcribed in vitro, then incubated with tobramycin conjugated matrix beads. the beads were then incubated with bhk-21 cell lysate. ddx3 was detected by western blot using anti-ddx3 polyclonal antibody. (b) bhk-21 cells were co-transfected with a renilla luciferase jev replicon, a renilla luciferase jev-replication deficient replicon (c) and a renilla luciferase reporter flanked by gapdh 5′ and 3′ utrs plus firefly luciferase rna (d) as the control. at various time points post transfection, cell lysates were collected for dual-luciferase assays. renilla luciferase activity was normalized to that of firefly luciferase. the differences between means were considered significant at *p o 0.05, very significant **po 0.01. primer: taatacgactcactatagggatgagtattcaacatt tccgtg-tcg and ampicillin-anti-sense primer: ggctcagcac gagtgt-agctaaacctcgctatactaagcccgtcaatacgggataatac (vashist et al., 2012) . t7 promoter and tobramycin aptamer tag (hartmuth et al., 2004) sequences underlined were included in sense and antisense primer respectively. the pcr products were subcloned into pmd-18t vector (takara, china). the nucleotide sequences of all the constructs were confirmed by dna sequencing. the jev replicon with renilla luciferase gene was generated by cloning jev sa14-14-2 strain cdna into pbluescript ii ks (agilent technologies) under the control of a t7 promoter as the previous study (chien et al., 2011) . the renilla luciferase gene was inserted after the first 102 bp of the jev c gene, followed by the foot-andmouth disease virus 2a self-cleaving protease (fmdv-2a) to enable the cleavage of the luciferase away from downstream nonstructural proteins (lo et al., 2003; varnavski and khromykh, 1999) . fmdv 2a was fused to the last 90 bp of the e gene that is necessary for the proper topology of the following viral proteins. to ensure rna stability and processing, a hepatitis delta virus ribozyme was placed immediately adjacent to the 3′ end of the jev cdna followed by a bovine growth hormone (bgh) polyadenylation sequence. the jev-replication-deficient replicon was constructed by introducing a mutation (gdd-aag) in ns5 gene (blight et al., 2003) using quick-change site-directed mutagenesis kit (stratagene) with the specific primers. the control construct: gapdh 5′ and 3′utr-flanking luciferase reporter gene, was cloned into pmd-18t vector (takara, china). to express the proteins used in the experiments, the plasmid pcold-i-ddx3, pgex-4t-3-ddx3, pet-24a ( þ)-core, pet-24a (þ)-ns3, pet-24a (þ )-ns5-mtase, pet-24a (þ )-ns5-rdrp, pgex-4t-3-ns3, pgex-4t-3-ns5-mtase or pgex-4t-3-ns5-rdrp was transformed into e. coli expression strain rosetta 2 cells. a single colony was used to inoculate 10 ml of luria-bertani (lb) media containing either ampicillin (100 μg/ml) or kanamycin (50 μg/ml), and the culture was grown at 37 1c overnight. the cultures were then diluted into the lb media containing either ampicillin (100 μg/ml) or kanamycin (50 μg/ml) and grown to an a 600 of 0.6 at 37 1c; protein expression was then induced with 0.1 mm iptg for 16-20 h at 22 1c. the cells were harvested by centrifugation at 5000g for 20 min at 4 1c and re-suspended in lysis buffer (100 mm nacl, 20 mm tris-hcl, ph 8.0, 0.1 mm pmsf). the cells were disrupted by sonication, and the supernatant was collected by centrifugation. the supernatant was then subjected to affinity purification using bio-rad profinity™ imac or profinity™ gst column (bio-rad). the proteins were then eluted, pooled together and further purified using size exclusion chromatography (super-dex75, ge healthcare). the concentration of the purified protein was determined by bradford assay. the ddx3 rabbit polyclonal antibody, anti-β-actin, anti-gst, anti-his monoclonal antibodies and hrp conjugated secondary antibody were purchased from abmart company (shanghai, china). the monoclonal antibody against the jev envelope glycoprotein was described previously (ishag et al., 2013b) . to produce anti-core, ns3 and ns5 polyclonal antibodies of mice source, the balb/c mice free of pathogens was inoculated with 50 mg of ns5-rdrp protein emulsified with equal amount of freund's complete adjuvant (sigma-aldrich) via subcutaneous injection. booster doses were subsequently given at 1, 2 and 3 weeks later with 50 mg of ns3, ns5-rdrp emulsified with incomplete adjuvant. finally, the serum of the mice was collected. the serum of the mice without immunized with any protein was collected as the negative serum. both immunoblot and elisa were performed to evaluate the immunoreactivity between jev ns3 and ns5 proteins and their polyclonal antibodies. animal use was in compliance with the nanjing agricultural university institutional animal care and use committee. the viral culture supernatants with 10-fold dilutions (from 10 2 to 10 5 ) were added into 6-well plate with confluent monolayer of bhk-21. the plate was then incubated at 37 1c for 2 h, with gentle agitation at every 15-min interval. the excess virus inocula were removed by rinsing the cells with pbs for three times. subsequently, overlay medium (2% low melting-point agarose with dmem medium containing 2% fbs) was added to each well and further incubated at 37 1c with 5% co 2 for 3-5 days. the cells were stained with 0.5% crystal violet. the cell viability of bhk-21 cells transfected with the plasmids was detected by the lactate dehydrogenase (ldh) activity, a stable cytosolic enzyme that is released upon cell lysis using cytotox 96 s nonradioactive cytotoxicity assay (promega) at 72 h after transfection. the cytotoxicity of cmp6 on bhk-21 cells was detected by cytotox 96 s nonradioactive cytotoxicity assay at 48 h after being added to the cells with different the concentrations (0-200 ng/ml). gst pulldown assay 50 μl glutathione-sepharose 4b beads (ge healthcare) were resuspended in binding buffer (20 mm tris-hcl, ph 7.5, 500 mm nacl, 0.5% np-40, and 0.1 mm pmsf). 10 mg purified gst-ddx3, gst-ns3, gst-ns5-mtase or gst-ns5-rdrp was incubated with the beads for 2 h at 4 1c under gentle agitation. unbounded proteins were washed away using washing buffer (20 mm tris-hcl, ph 7.5, 150 mm nacl, 0.1% np-40, 0.1 mm pmsf). the purified ns3, ns5-mtase, ns5-rdrp or ddx3 protein was incubated with the beads overnight with gentle agitation at 4 1c respectively. the beads were washed four times with 1 ml of washing buffer. finally the beads were resuspended in 200 ml of sds-page sample buffer and boiled. 15 μl protein sample was separated on 12% or 15% sds-page gel and then transferred to pvdf membrane. the presence of the targeted protein was detected with either anti-ddx3 polyclonal antibody or anti-his monoclonal antibody. glutathione-sepharose 4b beads incubated with gst proteins were used as control. co-immunoprecipitation assay bhk-21 cells were infected with jev for 48 h. the cells were rinsed twice in cold phosphate-buffered saline and incubated for 3 h at 4 1c in lysis buffer (50 mm tris-hcl, ph 7.4, 150 mm nacl, 1 mm edta, 1 mm dithiothreitol, 0.2 mm pmsf, 1% np-40) with gentle agitation. cell debris was removed by centrifugation at 10,000g for 10 min at 4 1c. the protein a/g (abmart) agarose beads coupled with ddx3 polyclonal antibody were incubated with the supernatant either in the absence or presence of rnase (100 μg/ml) and dnase (100 μg/ml) for 2 h at 4 1c. the beads were washed five times with 1 ml washing buffer (20 mm tris-hcl, ph 7.5, 150 mm nacl, 0.1% np-40), resuspended in 200 ml sds-page sample buffer and boiled. the protein samples were then subjected to western blot analysis. the targeted proteins were analyzed by western blot analysis using anti-ddx3, anti-ns3 or anti-ns5 polyclonal antibody. the protein mixtures were separated on 12% or 15% sds-page gel and transferred to pvdf membrane (millipore, usa) in a trans-blot sd semidry transfer cell (bio-rad, usa). the membrane was blocked with 5% non-fat milk powder in tbst buffer (20 mm tris-hcl, ph 7.4, 150 mm nacl, 0.1% tween 20), and then probed with anti-jev-envelope monoclonal antibody, anti-β-actin antibody the bound antibody was detected using hrp-conjugated secondary antibody and visualized using enhanced chemiluminescence (ge healthcare). the intensity band ratio of grayscale for jev envelope, ns5 and ddx3 proteins were analyzed by imagej software using β-actin as the internal control. jev-specific rna copy number was quantified using quantitative pcr (q-pcr). the total rna was extracted from jev-infected bhk-21 cells with trizol reagent (invitrogen), and purified according to the manufacturer's recommendations. for cdna preparation, total rna (1 mg) was reverse transcribed with first strand cdna synthesis kit (takara, china). cdna samples were amplified with the rt-pcr kit. intracellular jev genome levels were quantified with the sybr green probe 3-step q-pcr kit (takara, china) and fluorescent quantization machine (abi prism 7300 sequence detection system, applied biosystems). the primer for the jev ns1 and β-actin were the following: ns1 sense primer: 5′-acactcgtcagatcacaggttca-3′; antisense primer: 5′-gccagaaacatcaccagaagg-3′, β-actin sense primer: 5′catccgtaaagacctctatgccaac-3′, antisense primer, 5′-atggagccaccgatccaca-3′ and cellular β-actin mrna from the same rna extract was used as an internal control in bhk-21 cells as the previous study (ishag et al., 2013b) . to synthesize rna in vitro, purified plasmid was first linearized with bamhi or kpni enzyme. in vitro transcription of jev replicon, replication-deficient replicon, gapdh-luciferase, jev 5′, 3′ utr and control rnas were performed using riboprobe s system-t7 kit (promega, china) in a 20 ml reaction mixture containing 4 ml 5 â transcription buffer, 2 ml rnasin s rna inhibitor (40 u/μl), 1 ml each ntp (10 mm), 1 μg of linearized dna template and 1 ml t7 rna polymerase (20 u/μl). the mixture was incubated at 37 1c for 2 h. 10 ml of dnasei (1 u/ul) was added to the mixture and incubated at 37 1c for 30 min. the synthesized rnas were purified using the rneasy mini kit (qiagen) and analyzed by 1% agarose gels. jev 5′, 3′ utr and control rnas were added to the matrix beads conjugated with tobramycin (hartmuth et al., 2004) and incubated by head-over-tail rotation for 1-1.5 h. the matrix beads were washed three times with pbs (prepared with 0.1% diethyl pyrocarbonate-treated water). the supernatants of bhk-21 cell lysate were mixed with beads and incubated by head-over-tail rotation for 2 h. the mixture was subjected to western blot analysis using anti-ddx3 polyclonal antibody. the jev 5′ and 3′ utr rna were detected on 1% agarose and stained with ethidium bromide (eb). bhk-21 cells were plated onto cover slips in a 6-well plate. in the following day, the cells were infected with jev. 48 h later, the cells were fixed with ethanol for 30 min at 4 1c. viral proteins and endogenous ddx3 were firstly stained with their polyclonal antibodies. the cells were then washed with pbs and treated for 60 min at room temperature with rhodamine-conjugated or fitcconjugated secondary antibody (invitrogen, china). the coverslip was mounted onto the slide glass using pbs containing 50% glycerol. the cell nucleus was stained using 4′,6-diamidino-2phenylindole (dapi) (sigma, china). for viral rna staining, cells were first infected with jev (moi ¼0.01). 3 h later, the cells were treated with actinomycin d (sigma, china) for 4 h (15 mg/ml) (xu et al., 2010) to inhibit cellular rna transcription before 5-ethynyl uridine (eu) was supplied. the viral rna was detected using the click-it s rna imaging kits (invitrogen, china) according to the manufacturer's instructions. the coverslip was mounted onto the slide glass using pbs containing 50% glycerol. the cell nucleus were stained using 4′,6-diamidino-2-phenylindole (dapi) (sigma, china). confocal images were obtained using a zeiss lsm 710 scanning confocal microscope. for the replicon luciferase reporter assay, bhk-21 cells were seeded in 24-well plates and transfected with ddx3 shrna plasmid. 24 h later, the cells were transfected with 0.6 mg in vitrotranscribed jev replicon rna plus 0.2 mg of a firefly luciferase rna in vitro-transcribed as an internal control using transfast™ (promega). at the indicated time points post transfection, cell lysates were collected for the dual-luciferase assay using dual-luciferase s reporter assay system kit and glomax s 20/20 luminometer instrumentation (promega). all data were determined in triplicate and were representative of at least two separate experiments. the results represented the means 7standard deviations of triplicate determinations. the differences between means were considered significant at n po 0.05 and very significant nn p o0.01. all statistical analyses were performed by one-way anova using a spss 16.0 software package (version 16.0, spss inc., chicago, il, usa). de novo synthesis of rna by the dengue virus rna-dependent rna polymerase exhibits temperature dependence at the initiation but not elongation phase requirement of cellular ddx3 for hepatitis c virus replication is unrelated to its interaction with the viral core protein ddx3 dead-box rna helicase is required for hepatitis c virus rna replication hepatitis c virus 3′utr regulates viral translation through direct interactions with the host translation machinery the dead-box rna helicase ded1p affects and accumulates in saccharomyces cerevisiae p-bodies efficient replication of hepatitis c virus genotype 1a rnas in cell culture sequence and secondary structure analysis of the 5′-terminal region of flavivirus genome rna ddx3, a dead box rna helicase with tumor growth-suppressive property and transcriptional regulation activity of the p21waf1/cip1 promoter, is a candidate tumor suppressor rna-protein interactions: involvement of ns3, ns5, and 3′noncoding regions of japanese encephalitis virus genomic rna fuse binding protein 1 interacts with untranslated regions of japanese encephalitis virus rna and negatively regulates viral replication bovine lactoferrin inhibits japanese encephalitis virus by binding to heparan sulfate and receptor for low density lipoprotein the dead-box rna helicase ddx3 interacts with ddx5, co-localizes with it in the cytoplasm during the g2/m phase of the cycle, and affects its shuttling during mrnp export the dead-box protein family of rna helicases inhibition of flavivirus infections by antisense oligomers specifically suppressing viral translation and rna replication localization and functions of japanese encephalitis virus nonstructural proteins ns3 and ns5 for viral rna synthesis in the infected cells an rna cap (nucleoside-2′-o-)-methyltransferase in the flavivirus rna polymerase ns5: crystal structure and functional characterization small noncoding rna modulates japanese encephalitis virus replication and translation in trans the rna helicase ddx1 is involved in restricted hiv-1 rev function in human astrocytes a dead box protein facilitates hiv-1 replication as a cellular co-factor of rev targeting the human dead-box polypeptide 3 (ddx3) rna helicase as a novel strategy to inhibit viral replication the dead-box helicase ddx3 supports the assembly of functional 80s ribosomes expression and purification of enzymatically active recombinant rna-dependent rna polymerase (ns5) of the flavivirus kunjin tobramycin affinity tag purification of spliceosomes inhibition of japanese encephalitis virus infection in vitro and in vivo by pokeweed antiviral protein griffithsin inhibits japanese encephalitis virus infection in vitro and in vivo core protein-mediated 5′-3′ annealing of the west nile virus genomic rna in vitro poxvirus k7 protein adopts a bcl-2 fold: biochemical mapping of its interactions with human dead box rna helicase ddx3 gene structure of the human ddx3 and chromosome mapping of its related sequences computer-assisted identification of a putative methyltransferase domain in ns5 protein of flaviviruses and lambda 2 protein of reovirus characterizationof the ntpase activity of japanese encephalitis virus ns3 protein viral and cellular rna helicases as antiviral targets the dead-box rna helicase ddx3 associates with export messenger ribonucleoproteins as well as tip-associated protein and participates in translational control rana catesbeiana ribonuclease inhibits japanese encephalitis virus (jev) replication and enhances apoptosis of jev-infected bhk-21 cells the dead-box rna helicase ddx5 acts as a positive regulator of japanese encephalitis virus replication by binding to viral 3′ utr inhibition of japanese encephalitis virus entry into the cells by the envelope glycoprotein domain iii (ediii) and the loop3 peptide derived from ediii complete replication of hepatitis c virus in cell culture flaviviridae: the viruses and their replication trans-complementation of yellow fever virus ns1 reveals a role in early rna replication molecular biology of flaviviruses functional analysis of mosquitoborne flavivirus conserved sequence elements within 3′ untranslated region of west nile virus by use of a reporting replicon that differentiates between viral translation and rna replication subcellular localization and some biochemical properties of the flavivirus kunjin nonstructural proteins ns2a and ns4a toward the discovery of novel anti-hiv drugs. second-generation inhibitors of the cellular atpase ddx3 with improved anti-hiv activity: synthesis, structure-activity relationship analysis, cytotoxicity studies, and target validation hepatitis c virus core protein binds to a dead box rna helicase interference in japanese encephalitis virus infection of vero cells by a cationic amphiphilic drug, chlorpromazine hepatitis c virus core protein interacts with a human dead box protein ddx3 assignment of a human putative rna helicase gene, ddx3, to human x chromosome bands p11. 3-p11. 23 discovery of the first small molecule inhibitor of human ddx3 specifically designed to target the rna binding site: towards the next generation hiv-1 inhibitors recent advances in flavivirus antiviral drug discovery and vaccine development dead-box proteins: the driving forces behind rna metabolism human dead-box protein 3 has multiple functions in gene regulation and cell cycle control and is a prime target for viral manipulation candidate tumor suppressor ddx3 rna helicase specifically represses cap-dependent translation by acting as an eif4e inhibitory protein recent advances in japanese encephalitis a complex rna motif defined by three discontinuous 5-nucleotide-long strands is essential for flavivirus rna replication dead-box protein ddx3 associates with eif4f to promote translation of selected mrnas the deadbox helicase ddx3x is a critical component of the tank-binding kinase 1-dependent innate immune response analysis of virion-incorporated host proteins required for herpes simplex virus type 1 infection through a rna interference screen the alphaviruses: gene expression, replication, and evolution hepatitis c virus core-derived peptides inhibit genotype 1b viral genome replication via interaction with ddx3x adverse events after japanese encephalitis vaccination: review of post-marketing surveillance data from japan and the united states new initiatives for the control of japanese encephalitis by vaccination: minutes of a who/cvi meeting architecture of the flaviviral replication complex protease, nuclease, and detergents reveal encasement within doublelayered membrane compartments noncytopathic flavivirus replicon rna-based system for expression and delivery of heterologous genes identification of proteins in human cytomegalovirus (hcmv) particles: the hcmv proteome identification of rnaprotein interaction networks involved in the norovirus life cycle ddx3 dead-box rna helicase inhibits hepatitis b virus reverse transcription by incorporation into nucleocapsids hepatitis b virus polymerase blocks pattern recognition receptor signaling via interaction with ddx3: implications for immune evasion ultrastructure of kunjin virus-infected cells: colocalization of ns1 and ns3 with double-stranded rna, and of ns2b with ns3, in virus-induced membrane structures kunjin rna replication and applications ofkunjin replicons genetics of poliovirus the cellular rna helicase ddx1 interacts with coronavirus nonstructural protein 14 and enhances viral replication the capsid-binding nucleolar helicase ddx56 is important for infectivity of west nile virus the helicase activity of ddx56 is required for its role in assembly of infectious west nile virus particles requirement of ddx3 dead box rna helicase for hiv-1 rev-rre export function identification of novel target sites and an inhibitor of the dengue virus e protein inhibition of japanese encephalitis virus replication by peptide nucleic acids targeting cis-acting elements on the plus-and minus-strands of viral rna hepatitis c virus core protein interacts with cellular putative rna helicase in vitro rna synthesis from exogenous dengue viral rna templates requires long range interactions between 5′-and 3′-terminal regions that influence rna structure we thank prof. k.t. jeang (nih) for plasmids ha-ddx3. this project was funded by the priority academic program development of jiangsu higher education institutions. key: cord-255828-jrqdyfbg authors: du, yijun; pattnaik, asit k.; song, cheng; yoo, dongwan; li, gang title: glycosyl-phosphatidylinositol (gpi)-anchored membrane association of the porcine reproductive and respiratory syndrome virus gp4 glycoprotein and its co-localization with cd163 in lipid rafts date: 2012-03-01 journal: virology doi: 10.1016/j.virol.2011.12.009 sha: doc_id: 255828 cord_uid: jrqdyfbg the porcine reproductive and respiratory syndrome virus (prrsv) glycoprotein 4 (gp4) resembles a typical type i membrane protein in its structure but lacks a hydrophilic tail at the c-terminus, suggesting that gp4 may be a lipid-anchored membrane protein. using the human decay-accelerating factor (daf; cd55), a known glycosyl-phosphatidylinositol (gpi) lipid-anchored protein, chimeric constructs were made to substitute the gpi-anchor domain of daf with the putative lipid-anchor domain of gp4, and their membrane association and lipase cleavage were determined in cells. the daf-gp4 fusion protein was transported to the plasma membrane and was cleaved by phosphatidylinositol-specific phospholipase c (pi-plc), indicating that the c-terminal domain of gp4 functions as a gpi anchor. mutational studies for residues adjacent to the gpi modification site and characterization of respective mutant viruses generated from infectious cdna clones show that the ability of gp4 for membrane association corresponded to virus viability and growth characteristics. the residues t158 (ω − 2, where ω is the gpi moiety at e160), p159 (ω − 1), and m162 (ω + 2) of gp4 were determined to be important for virus replication, with m162 being of particular importance for virus infectivity. the complete removal of the peptide–anchor domain in gp4 resulted in a complete loss of virus infectivity. the depletion of cholesterol from the plasma membrane of cells reduced the virus production, suggesting a role of lipid rafts in prrsv infection. remarkably, gp4 was found to co-localize with cd163 in the lipid rafts on the plasma membrane. since cd163 has been reported as a cellular receptor for prrsv and gp4 has been shown to interact with this receptor, our data implicates an important role of lipid rafts during entry of the virus. porcine reproductive and respiratory syndrome (prrs) is a recently emerged viral disease and causes significant economic losses to the swine industry today. the etiological agent is the prrs virus (prrsv), which is a member of the family arteriviridae (meulenberg et al., 1993 (meulenberg et al., , 1994 wensvoort et al., 1991; nelson et al., 1993) that includes other viruses such as equine arteritis virus (eav), lactate dehydrogenaseelevating virus of mice (ldv), and simian hemorrhagic fever virus (shfv). the family arteriviridae together with the families coronaviridae and roniviridae form the order nidovirales (cavanagh, 1997; cowley et al., 2000; gonzález et al., 2003; smits et al., 2003) . prrsv contains a single-stranded positive-sense rna genome of approximately 15 kb that encodes two large non-structural polyproteins (pp1a and pp1a/1b) in the 5′-terminal 12 kb region and 8 structural proteins in the 3′-terminal 3 kb region: gp2 (glycoprotein 2), e (small envelope), gp3, gp4, gp5, orf5a, m (membrane), and n (nucleocapsid) proteins in order (firth et al., 2011; johnson et al., 2011; meulenberg et al., 1993; nelsen et al., 1999; snijder and meulenberg, 1998; snijder et al., 1999; wootton et al., 2000) . while n protein associates with the genomic rna and makes up the viral capsid, the other proteins are membrane-associated. of these, gp5 and m form a disulfide-linked heterodimer (mardassi et al., 1996) that is essential for virus infectivity (faaberg et al., 1995; snijder et al., 2003) . the e protein is a myristoylated protein and likely functions as an ion-channel protein embedded in the viral envelope facilitating the uncoating of virus and release of the genome into the cytoplasm (lee and yoo, 2006) . gp2, gp3, and gp4 are minor glycoproteins and form a disulfide-linked heterotrimer essential for viral infectivity (wieringa et al., 2003a (wieringa et al., , 2003b . co-expression of e, gp2, gp3, and gp4 results in the transport of these proteins from the endoplasmic reticulum (er) through the golgi, suggesting an important role of the hetero-multimerization for virus assembly and maturation (wissink et al., 2005) . orf5a is a newly identified membrane protein encoded in the internal orf within orf5 with an unknown function (firth et al., 2011; johnson et al., 2011) . gp4 protein is of 178 and 183 amino acids for the north american (type ii) and european (type i) prrsv, respectively (meulenberg et al., 1995; murtaugh et al., 1995) . amino acid sequence analysis of gp4 reveals two distinct hydrophobic domains, one at the extreme n-terminus at positions 1-17 and the other at the c-terminus at 165-183, which likely functions as the signal peptide and a membrane anchor, respectively (meulenberg et al., 1995) . gp4 however has a unique structural feature, not commonly seen in the class i-type integral membrane protein since gp4 does not contain a cytoplasmic tail which normally protrudes into the lumen once it associates with the membrane. the reason(s) for the lack of the cytoplasmic tail in gp4 is unknown. glycosyl-phosphatidylinositol (gpi) anchor is a c-terminal posttranslational modification found in some eukaryotic proteins residing in the outer leaflet of the cell membrane. genes encoding gpi-anchored proteins specify two signal sequences in the primary translation product: an n-terminal signal sequence for er targeting and a c-terminal hydrophobic sequence that directs its association to the membrane via the lipid (orlean and menon, 2007) . the process for gpi biosynthesis takes place in the er (takeda and kinoshita, 1995) , and the proteins subjected to gpi-modification enter the er lumen via the n-terminal er targeting signal which is cleaved off by a signal peptidase in the lumen (gerber et al., 1992) . in addition to the n-terminal signal sequence, gpi-anchored proteins contain a c-terminal hydrophobic sequence that directs the cleavage of the signal sequence from the protein and the replacement with a preformed gpianchor by action of the transamidase complex (ikezawa, 2002) . the gpi-linked proteins are then targeted to the membranes. the structural feature of prrsv gp4 resembles that of a gpi-anchored protein. hundreds of functionally and structurally diverse proteins have been identified as gpi-anchored proteins including the ns1 protein of dengue virus (jacobs et al., 2000) , prion proteins for transmissible spongiform encephalopathies (taylor and hooper, 2006) , adhesion molecules (dustin et al., 1987) , decay accelerating factor (daf; nickells et al., 1994) , just to name a few (see a review; ikezawa, 2002) . gpi modification serves a variety of functions including directing proteins to the cell surface (lisanti et al., 1988) , association with lipid rafts (taylor and hooper, 2006) , lymphocytes activation (robinson et al., 1989) , and signal transduction (cary and cooper, 2000; jacobs et al., 2000) . the gpi anchor in particular has the propensity to target proteins to lipid rafts (brown and london, 1998; metzner et al., 2008) . lipid rafts are dynamic assemblies of the lipid-ordered phase of microdomains that are highly enriched with cholesterol and sphingolipids in the exoplasmic leaflet of the plasma membrane (ikonen, 2001; simons and toomre, 2000) . lipid rafts compartmentalize cellular processes by serving as organizing centers for assembly of signaling molecules, influencing membrane fluidity and protein trafficking, endocytosis, transcytosis as well as for hostpathogen interactions (ikonen, 2001; pike, 2009; simons and toomre, 2000; van der goot and harder, 2001) . for viruses, lipid rafts play an important role in viral entry (ewers and helenius, 2011); norkin, 1999; parton and lindsay, 1999; vieira et al., 2010 , assembly (manié et al., 2000 ono and freed, 2001) and budding (chazal and gerlier, 2003; scheiffele et al., 1999) . some viral membrane-associated and also envelope proteins such as the gag protein of human immunodeficiency virus type 1 (hiv-1) (ono and freed, 2001) , the hemagglutinin of influenza virus (takeda et al., 2003) , the tegument protein of herpes simplex virus (lee et al., 2003) , the ns1 protein of dengue virus (noisakran et al., 2008) , and the spike protein of severe acute respiratory syndrome coronavirus (sars-cov) (lu et al., 2008) associate with the lipid rafts. the involvement of lipid rafts in prrsv infection has not been examined. in the present study, we provide evidence that the viral gp4 protein is a gpi-anchored protein, which co-localizes with the prrsv receptor, cd163 in the lipid rafts and may be involved in the viral entry process. the topology of gp4 predicts two hydrophobic domains on the protein; one at residues 1-17 and the other at 164-178 (http:// mobyle.pasteur.fr/cgi-bin/portal.py?#forms::toppred; claros and von heijne, 1994) . the n-terminal hydrophobic domain likely functions as the signal peptide to direct the protein to er membrane and the c-terminal hydrophobic domain to anchor the protein to membrane, but unlike the common structure seen in type i membrane glycoproteins, gp4 does not possess a hydrophilic cytoplasmic tail following the hydrophobic anchor domain at the c-terminus (fig. 1) . the cytoplasmic tail generally protrudes into the lumen when the protein is associated with the er membrane, and the unusual feature of gp4 resembles the topology of gpi-anchored protein, for example, daf which is a well-characterized gpi-anchored protein (nickells et al., 1994) . when gp4 sequences were analyzed using the gpi prediction program (http://gpi.unibe.ch; fankhauser and mäser, 2005) , a gpi anchor signal was readily detectable. when a large number of gp4 sequences were examined including the european and north american genotypes, the european-like prrsv circulating in the usa, and the highly virulent prrsv emerged in china (fig. 1a) , the gpi anchor signal became more prominent and appeared to be highly conserved across the genotypes including ldv and eav. thus, we hypothesized that the prrsv gp4 protein might be modified by gpi attachment and anchored with the membrane through the gpi. since gp4 is a minor protein and thus its expression level is low, and because a suitable antibody for gp4 is not available, a gp4-egfp fusion construct was made using a linker of five glycine residues inserted between the egfp and gp4 sequences to facilitate the detection of gp4 in cells (fig. 1b; tsuneki et al., 2007) . when hela cells were transfected with the pegfp-gp4 plasmid, the gp4-egfp fusion protein of about 56 kda was expressed ( fig. 2a, lane 4) . in addition, a minor band of 30 kda which was slightly larger than egfp alone was identified (fig. 2a, lane 4, δgp4) , suggesting that the c-terminal portion of gp4 might have been cleaved and the cleaved portion which was fused with egfp was detectable by egfp antibody. such band was absent in cells transfected with pegfp-n which is a fusion construct of the prrsv n protein with egfp ( fig. 2a, lane 5) . this result suggests that gp4 is possibly a gpi-anchored protein and not all but some of the gp4 molecules are modified by gpi attachment. a gpi-anchored protein can be distinguished from its un-modified form by its mobility (ikezawa, 2002) , and thus the migration pattern of gp4 was examined by sds-page and western blot. hela cells naturally express human daf as a 41 kda protein before gpimodification (karnauchow et al., 1996) and after glycosylation and gpi-modification, it becomes approximately 82-88 kda (nickells et al., 1994) , which can readily be detectable by daf mab from cell lysates (fig. 2b, lane 4) . to facilitate the detection of gp4 while maintaining its structural integrity as much as possible, the flag sequence tag of 'ykddddk' was inserted between positions d87 and e88 of gp4, and pxj41-flag-gp4 was constructed (fig. 1c) . hela cells transfected with pxj41-flag-gp4 produced the gp4 protein of approximately 29 kda (fig. 2b , lane 3, lower arrow). this form of gp4 was predominant among two other forms of gp4 produced in these cells. gp4 is an integral membrane protein with multiple glycans added onto it (meulenberg et al., 1995; wissink et al., 2005) , and the 29 kda protein and two additional bands of smaller sizes likely represent the fully mature and the partially glycosylated forms of gp4 as previously observed (das et al., 2010) . a 50 kda band was identified (fig. 2b, lane 3, upper arrow) , and this is likely the fully modified gpi-anchored form of gp4. to determine the gpi modification of gp4, phosphatidylinositolspecific phospholipase c (pi-plc) digestion was conducted. pi-plc is a lipase known to cleave and release a gpi-linked protein from the cellular membrane, and therefore the release of a soluble protein by pi-plc digestion is considered standard biochemical evidence for gpi-anchored proteins (ikezawa, 2002) . for this experiment, hek-293 cells were transfected with pxj41-flag-gp4 and at 24 hpt, trypsinized and digested with pi-plc in suspension. the supernatants were then analyzed by western blot using anti-flag ab. in parallel, pi-plc-digested cells were subjected to facs analysis after staining with anti-flag ab. unexpectedly, no protein band equivalent to digested gp4 was observed from the supernatants, and the intensity of fluorescence was not diminished in pi-plc-digested cells as compared to the untreated cells (data not shown). total lysates of pi-plc-digested cells and untreated cells were also examined by western blot, but no difference was found in their migration patterns. the 50 kda band did not change its migration after pi-plc digestion (data not shown), and we concluded that the gpi modification of gp4 was resistant to pi-plc digestion. when intact cells are treated with pi-plc, partial or total resistance to digestion may occur due to the inaccessibility of an enzyme to the cleavage site, expression of the protein on the cell surface in both gpi-modified and gpi-unmodified hydrophobic peptide-anchored forms, or tight association of the gpi-modified protein with a plcnon-susceptible protein on the cell surface (rosenberry, 1991) . the prrsv gp4 protein may exist as the gpi-modified form and the gpiunmodified hydrophobic peptide-anchored form, which may have caused the inability of pi-plc digestion. thus to further determine if gp4 was a gpi-modified protein, two chimeric constructs were made such that the known gpi-anchor domain of human daf was substituted with the putative gpi-modification domain of gp4 protein (figs. 3a, b) and the c-terminal hydrophobic region of gp2 protein of prrsv (fig. 3c ). the residue cross-linked to the lipid moiety is termed ω site, and upstream residues are designated ω-minus while downstream residues are designated ω-plus with respect to their positions from the ω site (ikezawa, 2002) . the ω site of human daf is serine at position 353 (s353) (nickells et al., 1994; fig. 3a) , which is identical to the computer prediction (gpi modification prediction; http:// mendel.imp.ac.at/gpi/gpi_server.html). by using the same program, the potential ω site for gp4 was predicted to be at e160, and based protein of prrsv pa8. gp4 contains two hydrophobic domains (hd), one at the n-terminus and the other at the c-terminus. gp4 was cloned to express as a fusion protein with egfp using a linker of five glycine (5g) residues. 'ω' at position 160 (e160) indicates the putative gpi modification site, and the arrow between positions 160 and 161 indicates the potential cleavage site specific for pi-plc lipase. numbers indicate amino acid positions. (c) the flag-tag sequence of ykddddkgs was inserted between positions d87 and e88 (nucleotide sequence positions 261 and 262) of gp4 gene cloned in pxj41, and pxj41-flag-gp4 was constructed. on this prediction the region of 144-178 of gp4 was chosen to replace with the 349-381 fragment of human daf to construct pxj41-daf/4 (fig. 3b ). the prrsv gp2 protein was chosen to serve as a gpinegative control and the 204-256 fragment of gp2 was used to replace the 349-381 region of daf. this construct was designated pxj41-daf/ 2 (fig. 3c ). using the daf/4 fusion construct, gpi modification of gp4 was reexamined by pi-plc digestion. the endogenous expression of human daf was high in hela cells whereas negligible and undetectable in hek-293 cells (data not shown). thus, hek-293 cells were used in this study to express daf/4. daf of orangutan erythrocytes (e or ) migrates as an 88 kda protein, and after digestion with pi-plc, its . for gp4, the ω site is predicted at e160, and the putative lipase cleavage site is predicted between e160 and t161 (arrow). (c) gp2 is type i integral membrane protein, and the c-terminal 33 amino acids of human daf were replaced with the c-terminal 53 amino acids representing positions 204 and 256 of prrsv gp2 to generate daf/2. this region of gp2 contains a hydrophobic trans-membrane sequence plus a short hydrophilic cytoplasmic tail. numbers for 204-256 indicate amino acid positions with respect to gp2. migration reduces to 67 kda (nickells et al., 1994) . in our study, the molecular migration of human daf in hela cells decreased from 82-88 kda to 65-70 kda when digested with pi-plc (fig. 4a , lanes 2, 4), which was consistent with the orangutan report. after pi-plc digestion, fluorescent cells by daf staining were also reduced by 96% in hela cells (figs. 4, ba, ca) , confirming the efficient digestion of daf by pi-plc. in contrast, hek-293 cells transfected with the empty vector pxj41 showed no fluorescence for daf (figs. 4, bb, cb) , and no cleavage product was released by pi-plc digestion (fig. 4a, lane 6) . thus, hek-293 cells were transfected with pxj41-daf, pxj41-daf/4, or pxj41-daf/2, and analyzed by pi-plc digestion followed by western blot (fig. 4a) , fluorescence staining (fig. 4b) , and facs (fig. 4c) analyses. daf/2, daf, and daf/4 were all expressed on the cell surface and detectable by daf-specific mab evr1 (figs. 4b, c; panels c, d, e) . after digestion with pi-plc, the fluorescence was lost in daf and daf/4 expressing cells, and daf and daf/4 proteins cleaved by pi-plc were released to the supernatants, which were then detectable by western blot (fig. 4a, lanes 10, 12) . in contrast, the intensity of fluorescence and the percentage of fluorescence-positive cells were unaffected for daf/2 by pi-plc treatment (fig. 4b, panel c; fig. 4c , panel c), and the daf/2 protein was not detectable in the supernatant (fig. 4a, lane 8) . these results show that the c-terminal region of gp4 was modified for pi-plcspecific gpi-attachment, whereas gp2 protein did not undergo such a modification, which is consistent with previous reports that gp2 anchors on the cell membrane through the c-terminal hydrophobic transmembrane domain (meulenberg et al., 1995; wissink et al., 2005) . mutational studies on the gpi-anchor region since our data showed that prrsv gp4 was a gpi-modified protein, the importance of individual amino acids surrounding the gpi attachment site (ω site) was examined for pi-plc cleavage. using pxj41-daf/4 as the parental construct, six mutants were constructed such that the residues adjacent to e160 (the ω site) were individually changed to valine ( fig. 5a ; gerber et al., 1992; furukawa et al., 1997) . following expression in transfected cells and after pi-plc digestion of wild-type daf/4, the cleaved product released into the supernatant was identified as a 65-70 kda band (fig. 5b, lane 2) . mutants daf/ 4-m157v (ω − 3) and daf/4-e160v (ω) showed similar cleavage properties to those of wild-type daf/4 as inferred from the intensity of the cleaved product (fig. 5b, lanes 4, 10) and the percentage of positive cells (fig. 5c, panels a, d; fig. 5d , panels a, d). for daf/4-t161v (ω + 1), the fluorescence intensity was 6 × 10 2 as compared to 1 × 10 3 for daf/4 ( these results show that the residues surrounding the ω site of gp4 contribute to the ability of gpi to anchor to the membrane. these experiments were repeated three times for confirmation, and the representative results are shown in figs. 5c and d. in summary, all mutations but m162* retained the ability of gpi-anchorage to various extents. to determine the significance of residues adjacent to the ω site of gp4 for virus infectivity, the respective mutations were introduced into the prrsv infectious cdna clone and a total of 7 mutant viral genomic clones were generated. the prrsv infectious cdna clone was modified to place the full-length genomic sequence under the eukaryotic promoter and thus dna transfection of the full-length genomic clones can produce infectious progeny in transfected cells. thus, the wild type and mutant genomic clones were transfected into marc-145 cells and the cells were incubated for 5 days. cytopathic effects (cpe) were evident for prrsv-gp4-wt and some of the mutants. culture supernatants were collected and designated 'passage-1'. for mutants that did not produce visible cpe, extracellular and intracellular rnas were examined for the presence of viral genome by rt-pcr for n gene at passages 3 and 5. the mutant viruses prrsv-gp4-m157v (ω − 3), prrsv-gp4-e160v (ω), and prrsv-gp4-t161v (ω + 1) grew to titers similar to that of prrsv-gp4-wt (fig. 6a ), and these were the mutants that did not impair the ability for gpianchorage in hek-293 cells. these mutant viruses grew normally and induced cpe typical for prrsv. mutation at ω − 2 (t158v), ω − 1 (p159v), and ω + 2 (m162v) appeared to be important for virus growth as the titers for prrsv-gp4-t158v, prrsv-gp4-p159v, and prrsv-gp4-m162v decreased in 'passage 2' and 'passage 3' (fig. 6a ). their growth kinetics was also slower compared to that of prrsv-gp4-wt (fig. 6b) , and their characteristics were consistent with their fluorescence staining. in particular, prrsv-gp4-m162v (ω + 2) appeared to affect the virus infectivity most notably. prrsv-gp4-m162v grew slowly and its titer was also low. the truncation mutant prrsv-gp4-m162* did not exhibit any sign of infectivity (figs. 6a, b) . furthermore, no viral rna was detectable at passages 3 and 5 and thus it was concluded that the lack of gpi-anchor, thus the lack of membrane association, was lethal for prrsv infectivity. for mouse hepatitis coronavirus, a member virus in the order nidovirales, the cholesterol levels on the cell membrane determine the susceptibility of cells to virus infection, and lipid rafts are required for virus entry and membrane fusion (choi et al., 2005) . since cholesterol is a major component residing in the lipid rafts of the cell membrane, we examined whether lipid rafts were involved in the entry of prrsv in permissive cells. a cholesterol-depletion experiment was conducted using methyl-β-cyclodextrin (mβcd) in both marc-145 and pam cells. mβcd is not incorporated into membranes but extracts cholesterol selectively from membranes by binding it in the central non-polar cavity and thus depleting from the plasma membrane (ilangumaran and hoessli, 1998) . to avoid the possibility that the newly synthesized cholesterol and/or cholesterol from internal compartments may restore the rafts and affect the entry of virus, incubation of mβcdtreated cells with prrsv was limited to 1 h at 37°c. it was apparent that the mβcd treatments of marc-145 and pam cells impaired the production of prrsv in a dose-dependent manner (figs. 7a, b) , suggesting an essential role of cholesterol for prrsv entry. to confirm the involvement of cholesterol in prrsv infectivity, a depletion reversion study was conducted. cells were treated with mβcd for 1 h and then, the depletion was reversed by supplementing with cholesterol at various concentrations, followed by prrsv infection and examination of virus yield from these cells. after replenishment with cholesterol, the virus titer increased dramatically, and at 100 μg/ml of cholesterol supplementation, the production of virus was almost fully restored in both marc-145 and pam cells (figs. 7c, d) .these data indicate that the reduction of virus production by mβcd was due to the depletion of cholesterol from the cells and this effect was reversible, suggesting a role of lipid rafts in prrsv infection. the importance of cholesterol for prrsv infection suggests that viral proteins may interact with cellular proteins in the lipid rafts during entry. gpi modification has the propensity to target the gpiproteins to lipid rafts (brown and london, 1998) and since prrsv gp4 is a gpi-anchored protein, its localization in the lipid rafts was first examined by dual staining immunofluorescence. daf (a synonym of cd55) is a resident protein in the lipid rafts and thus is frequently used as a marker for lipid rafts (stuart et al., 2002) . since hela cells constitutively express daf which is readily detectable by daf-specific mab evr1 (fig. 2b, lane 4; fig. 8a ), hela cells were used to examine co-localization of gp4 and daf proteins. the gp4 protein fused with a flag-tag was detectable on the cell membrane (figs. 8e, h) and was co-localized with cd55 (figs. 8f, i), demonstrating that the prrsv gp4 protein is a membrane protein localized in the lipid rafts in hela cells. co-localization of gp4 with cd163 in the lipid rafts cd163 has been studied as a cellular receptor for prrsv, and cd163 renders prrsv non-permissive cells permissive for infection (calvert et al., 2007; lee et al., 2010; patton et al., 2009) . cd163 has been shown to localize and form high partition in the lipid rafts (wolf et al., 2007) . since gp4 co-localizes with daf (fig. 8) , colocalization of gp4 with cd163 was examined. for this study, cells of the porcine origin were used to co-express both gp4 and cd163. dulac cells are porcine kidney cells but non-permissive for prrsv infection, and thus using these cells, a cell line was generated to stably express porcine cd163 which was then designated dulac-cd163. dulac-cd163 cells were rt-pcr positive for cd163 transcription (fig. 9a, lane 4) and the protein was also expressed (figs. 9b, c). dulac-cd163 cells became permissive for prrsv infection and the gfp expression was evident when infected with prrsv-p129-gfp virus (fig. 9d) , confirming the expression of porcine cd163 and infection of prrsv in these cells. when dulac-cd163 cells were transfected with pxj41-flag-gp4, cd163 and gp4 were found to be co-localized on the plasma membrane (figs. 10, f, i). these results demonstrate the interaction of gp4 with cd163 in the lipid rafts and suggest that this interaction may participate in the initial stage of virus entry into cells. the signal for gpi modification of a protein consists of three parts: a stretch of three amino acids including the residue where gpi attaches (the ω site), a hydrophobic segment of 12-20 amino acids, and a hydrophilic spacer segment of usually less than 10 amino acids between them (furukawa et al., 1997) . for modification, the hydrophobic segment is first cleaved and the lipid is then attached (gerber et al., 1992) . the gp4 protein satisfies the above conditions with a hydrophobic segment of 15 residues at the c-terminus and a hydrophilic space of 3 amino acids between the hydrophobic segment and the ω site (fig. 1a) . in the present study, we provide evidence that the prrsv gp4 protein can undergo post-translational modification for gpi attachment and anchors to the membrane via the lipid. the gp4-egfp fusion protein was cleaved at the ω site and the 30 kda peptide was likely the cleavage product representing the c-terminal portion of gp4 fused with egfp. gp4 existed in two forms to anchor to the membrane, and this observation was consistent with other lipid-anchored proteins such as lfa-3 (lymphocyte functionassociated antigen 3) and ncam-1 (neural cell adhesion molecule 1) as they also exist in two forms, a hydrophobic peptide-anchored form and a lipid-anchored form (arai et al., 2004; cross, 1990; dustin et al., 1987) . these alternative forms behave differently. the routes (rothberg et al., 1990 ) and rates (keller et al., 1992) for endocytosis virus at a multiplicity of infection (moi) of 5, and their supernatants were harvested at indicated times. virus titers were determined by a microtitration infectivity assay and recorded as tissue culture infectious dose (tcid) 50 /ml. experiments were conducted in duplicate and repeated three times. the data are shown as mean titers ± standard error. of the gpi-anchored and hydrophobic peptide-anchored proteins differ. in addition to the transport differences, the lipid-anchored and peptide-anchored forms of a same protein can also have different biological properties. the lymphocyte surface molecules ly-6a/e and qa-2 only activate t cells when present as a gpi-anchored form (robinson et al., 1989; su et al., 1991) . the gp4 protein expressed as the flag-gp4 fusion protein appeared to be resistant to pi-plc digestion, and this was likely due to the co-existence of lipid-anchored and peptide-anchored forms of gp4. in contrast, the daf/4 construct became sensitive to pi-plc digestion, and constructs made in the similar way have been shown to be useful for some proteins including cd4 for the study of hiv. hiv efficiently infected human cells expressing the gpi-anchored cd4 receptor (diamond et al., 1990; jasin et al., 1991) and the cd4-gpi fusion protein was efficiently cleaved by pi-plc (anderson et al., 1996) . replacement of the gpi modification signal by a hydrophobic peptide segment leads to expression of the protein as a peptide-anchored membrane protein (takeda and kinoshita, 1995) , and this was consistent with our finding that daf/2, in which the gpi modification signal in daf was substituted with the hydrophobic segment of gp2, resulted in peptide-anchored membrane association and thus non-digestible by pi-plc. this finding confirms that unlike gp4, gp2 is a type i integral membrane protein (meulenberg et al., 1995; wissink et al., 2005) . in contrast to daf/2, daf/4 was successfully digested by pi-plc, demonstrating that the c-terminal region of gp4 indeed possessed the gpimodification signal. m157v (ω − 3), e160v (ω), and t161v (ω +1) mutations of gp4 did not affect the gpi-modification or the growth of mutant viruses, but t158v (ω − 2), p159v (ω − 1), and m162v (ω + 2) were important for both gpi modification and the virus infectivity. the residues at positions ω − 2, ω − 1, and ω + 2 were highly conserved among different prrsv isolates of the north american and european genotype, and among those three mutations, m162v (ω + 2) contributed the most to the gpi modification and virus infectivity. the m162* mutation, the c-terminal 16 amino acids truncation mutant, was lethal for infectivity, indicating the essential requirement of this hydrophobic segment for prrsv infection. this is the first report that the gpi-anchor of a viral membrane protein contributes to viral infectivity and growth rates. for viruses to infect target cells, they first bind to a specific receptor on the cell surface. for prrsv, cd163 is the cellular receptor (calvert et al., 2007) and has been recently shown to interact with gp2 and gp4 proteins (das et al., 2010) . cd163 is a macrophagespecific protein in the cysteine-rich scavenger receptor superfamily comprising a large number of cell surface and soluble glycoproteins involved in the recognition of various ligands of proteins, polyribonucleotides, polysaccharides, and lipids (sarrias et al., 2004) . the localization of a receptor in the lipid rafts is crucial for entry of some viruses (ewers and helenius, 2011) including sv40 and murine leukemia virus (lu and silver, 2000; pelkmans et al., 2001) . for nidoviruses, cd13 is the cellular receptor for human coronavirus 229e and is localized in the lipid rafts (nomura et al., 2004) , and the importance of lipid rafts for virus replication has also been documented (lorizate and kräusslich, 2011; lu et al., 2008; thorp and gallagher, 2004) . we show in the present study that gp4 localizes to the lipid rafts of the plasma membrane where it associates with cd163, the cellular receptor for prrsv. the lipid-modification of gp4 contributes to prrsv infectivity, suggesting an important role of gpi for prrsv entry and infection. the daf/4-m162* mutant was lethal for infectivity, which is probably due to the inability of gp4 to anchor to the membrane, and thus unable to associate with the lipid rafts, supporting the notion that the lipid rafts play an important role for prrsv infection. co-localization of gp4 and cd163 in the lipid rafts may mediate the entry of prrsv. compared to peptide-anchored gp4, lipid-anchored gp4 is likely to have the priority to locate in lipid rafts where it binds to cd163 to promote the entry of prrsv. this may explain why certain mutations affecting the ability of lipid-anchor formation of gp4 on the cell membrane affected the titers and growth of prrsv as seen in other studies (metzner et al., 2008) . taken together, we have shown here that the prrsv gp4 protein is a gpi-modified membrane-associated protein. co-localization of gp4 with cd163 in the lipid rafts on the fig. 8 . co-localization of prrsv gp4 and the daf (cd55) protein as a lipid raft marker on the plasma membrane of hela cell. cells were transfected with pxj41-flag-gp4 (d through f) and at 24 h post-transfection, washed with ice-cold pbs. cells were then co-stained with daf-specific mab evr1 (anti-cd55; a and d) and rabbit anti-flag antibody (b and e), followed by staining with alexa fluor 488® conjugated goat anti-mouse igg (h + l) and alexa fluor 594® conjugated goat anti-rabbit igg (h +l) secondary antibodies, respectively. images were visualized using a laser-scanning confocal fluorescence microscope (model bx50, olympus). panels g through i represent the enlargement of the indicated areas in panels d through f, respectively. cell membrane implicates an important role of the complex for prrsv entry and infection. these findings deepen our understanding on gp4 and establish the cholesterol and lipid rafts as potential targets in the development of control measures against prrsv infection. marc-145 cells (a subline of ma-104 african green monkey kidney cells; kim et al., 1993) were maintained as described elsewhere (lee et al., 2005) . porcine alveolar macrophages (pams) were kindly provided by dr. f. zuckermann (university of illinois at urbana-champaign, urbana, il) and grown in rpmi-1640 (invitrogen, carlsbad, ca) containing 10% fetal bovine serum (fbs; hyclone, logan, ut). hela and hek-293 cells (nih aids research and reference reagent program, germantown, md) were grown in dulbecco's modified eagle medium (dmem) supplemented with 10% fbs. dulac porcine kidney cells (obtained from dr. l. babiuk, vaccines and infectious disease organization, saskatoon, canada) were free of porcine circovirus type 1 and grown in modified eagle's medium (mem) supplemented with 10% fbs. stocks of prrsv (north american genotype strain pa8; wootton et al., 2000) were prepared in marc-145 cells. prrsv p129-gfp expressing gfp is described elsewhere (pei et al., 2009) . culture supernatants harvested at 5 days post-transfection with prrsv infectious clones were designated 'passage-1'. the 'passage-1' virus was used to inoculate fresh marc-145 cells, and the 5-day harvest was designated 'passage-2'. the 'passage-3' virus was prepared in the same way as for 'passage-2'. each passage virus was aliquoted and stored at − 80°c until use. virus titers of 'passage-2' and 'passage-3' were determined in marc-145 cells by a microtitration infectivity assay and recorded as 50% tissue culture infectious dose per ml (tcid 50 /ml). to determine the growth kinetics of the mutant viruses, marc-145 cells were infected in duplicate with 'passage-2' virus at a multiplication of infection (moi) of 5 and incubated for indicated times. culture supernatants were collected and titrated by a microtitration infectivity assay and recorded as tcid 50 /ml. monoclonal antibody (mab) against human daf (clone 1g3 anti-daf [aa:35-135]), anti-flag mab (clone m2), and anti-flag rabbit antibody were from sigma-aldrich (st. louis, mo); anti-gfp rabbit antibody from cell signaling technology (danvers, ma); β-actin mab (ac-15) from santa cruz biotechnology (santa cruz, ca); and anti-porcine cd163 mab (clone 2a100/11) from abd serotec (raleigh, nc). tissue culture supernatant containing mab evr1 raised against human daf was kindly provided by k. dimmock (university of ottawa, ottawa, canada) and described elsewhere (karnauchow et al., 1996) . alexa fluor 488®-conjugated goat anti-mouse igg (h + l), alexa fluor 594®-conjugated goat anti-rabbit igg (h+ l), and g418 (neomycin sulfate analog) were purchased from invitrogen (carlsbad, ca). horseradish peroxidase (hrp) conjugated-goat anti-mouse igg and -goat anti-rabbit igg were purchased from jackson immunoresearch laboratories (west grove, pa). phosphatidylinositol-specific phospholipase c (pi-plc) of bacillus cereus, methyl-β-cyclodextrin (mβcd), and water soluble cholesterol were purchased from sigma-aldrich. the eukaryotic expression vector pxj41 is a derivative of pxj40 in which the polylinker region was modified (xiao et al., 1991) . the plasmid pegfp-n contains the prrsv n gene fused to egfp . the prrsv infectious cdna clone pcmv-s-p129 is described elsewhere (lee et al., 2005; lee and yoo, 2006) . to construct pegfp-gp4, prrsv gp4 gene was pcr-amplified from pcmv-s-p129 using the gp4-gfp-fwd and gp4-gfp-rev primer pair (table 1 ) and inserted into ecori and bamhi sites in pegfp-n1 (clontech; mountain view, ca). to generate pxj41-flag-gp4, the flagtag sequence (ykddddkgs) was inserted between nucleotide positions 261 and 262 (corresponding amino acid positions d87 and e88, respectively) of gp4 by three-point ligations of pcr products generated using the primer pairs, flag-gp4-a and -b, and flag-gp4-c and -d (table 1) at ecori, bamhi, and hindiii sites (fig. 1f) . the human daf gene was amplified from plasmid pef6/v5-daf (provided by k. dimmock, university of ottawa, ottawa, canada) using daf-fwd and daf-rev primers ( table 1 ). the pcr product was digested with ecori and bamhi followed by cloning into pxj41 to generate pxj41-daf. pxj41-daf/4 was created by overlapping extension pcr (horton et al., 1989) . briefly, the region representing amino acid positions 1-348 of human daf was amplified from pef6/v5-daf using daf-fwd and daf/4-b primers. the c-terminal region of gp4 (gp4-c, positions 144-179; fig. 1 ) was amplified from plasmid pcmv-s-p129 using primers daf/4-c and daf/4-d (table 1) . then, overlapping extension pcr was conducted using the pcr products of daf (1-348 aa) and gp4-c (144-179 aa) as templates and primers daf-fwd and daf/4-d (table 1 ). the daf/4 fusion gene (1-348 aa fragment of daf and gp4-c fragment) was inserted at ecori and bamhi sites in pxj41 to generate pxj41-daf/4 (fig. 3) . plasmid pxj41-daf/2 was constructed in a similar way using gp2-c representing 204-256 aa of gp2 (fig. 3) . the primer sequences are listed in table 1 . the topology of gp4 (http://mobyle.pasteur.fr/cgi-bin/portal.py? #forms::toppred; claros and von heijne, 1994) and the gpi anchor attachment signal (http://gpi.unibe.ch; fankhauser and mäser, 2005; eisenhaber et al., 1998) were predicted using on-line programs available on the bioinformatics resource portal (http://expasy.org/ tool). specific mutations were introduced to plasmids pxj41-daf/4 and pcmv-s-p129 using the quikchange xl site-directed mutagenesis kit (stratagene, la jolla, ca) with modifications as described elsewhere . plasmid dna transfection was carried out using lipofectamine™ 2000 according to the manufacturer's instruction (invitrogen, carlsbad, ca). hela cells were seeded in 35-mm diameter dishes and grown to 70% confluency and transfected with 2 μg of dna diluted in opti-mem (invitrogen, carlsbad, ca). after 4 h incubation, transfection medium was replaced with dmem supplemented with 10% fbs. at 24 h post-transfection (hpt), cells were washed with pbs and lysed in the buffer (20 mm tris-hcl [ph 7.5], 150 mm nacl, 1 mm edta, 1 mm egta, 1% tritonx-100, 1% np-40 and 1 mm pmsf) for western blot analysis. for transfection of pxj41-daf, pxj41-daf/2, pxj41-daf/ 4, and their mutant derivatives, hek-293 cells grown in 60-mm diameter dishes were transfected using 10 μl lipofectamine 2000 and 4 μg individual plasmid in 2 ml opti-mem. after 6 h incubation, the transfection mix was replaced with dmem supplemented with 10% fbs. at 24 hpt, cells were washed once with pbs and trypsinized for pi-plc digestion and facs analysis. for transfection of prrsv infectious clone pcmv-s-p129 and its derivatives, marc-145 cells grown in 35-mm diameter dishes were used. marc-145 cells were transfected with 2 μg of an infectious clone using lipofectamine 2000 as described above. transfected cells were incubated at 37°c for 5 days in dmem supplemented with 5% fbs prior to collection of supernatant for virus recovery. hela cells or hek-293 cells were transfected with pxj41-daf, pxj41-daf/2, pxj41-daf/4 or their derivatives and incubated for 1 day. cells were washed with pbs, trypsinized, and collected by centrifugation at 2000 rpm for 10 min (eppendorf 5415r). then, the cells were washed twice using pi-plc buffer (10 mm tris-hcl [ph 7.4], 144 mm nacl, 0.05% bovine serum albumin [bsa] ) and resuspended in pi-plc buffer. cells were divided into two equal fractions: one for pi-plc treatment (1.5 u/ml; sigma-aldrich) and another as a control without treatment. following incubation at 37°c for 30 min with pi-plc, both fractions of cells were centrifuged at 2000 rpm for 10 min (eppendorf 5415r). the supernatants were subjected to western blot analysis whereas the cell pellets were washed twice in ice-cold facs buffer (1.0% bsa, 0.01% sodium azide in pbs) followed by fluoresceinactivated cell sorter (facs) analysis. pi-plc-treated or untreated cells were stained with daf-specific mab evr1 at 4°c for 30 min. cells were washed twice in ice-cold facs buffer and fixed with 4% paraformaldehyde in pbs (ph 7.4) for 1 h at 4°c. after wash with facs buffer, cells were incubated with alexa fluor 488®-conjugated goat anti-mouse igg (h + l) secondary antibody for 1 h, washed twice with facs buffer, and resuspended in 1% paraformaldehyde in pbs (ph 7.4) for flow cytometry (bd biosciences lsr ii analyzer). the data were analyzed using the facs express software (ver. 3 research edition, de novo software; los angeles, ca). the supernatants obtained from pi-plc digested cells were centrifuged to remove cell debris at 13,000 rpm for 20 min in a microcentrifuge (eppendorf 5415r). the supernatants were collected and boiled in sds-page loading buffer (60 mm tris-hcl [ph 6.8], 2% sds, 0.1% bromophenol blue, 25% glycerol, 5% β-mercapto ethanol) for 5 min, followed by 10% sds-page and transfer to polyvinylidenedifluoride (pvdf) membranes (millipore). the membranes were blocked with 5% non-fat dry milk in tbst (10 mm tris-hcl [ph8.0], 150 mm nacl, 0.1% tween 20) for 4 h at room temperature (rt) and incubated with rabbit anti-gfp antibody, anti-flag mab, or anti-daf mab overnight at 4°c. after 5 washes for 10 min each with tbst, the membranes were incubated with hrp-conjugated goat anti-rabbit or goat antimouse igg for 1 h, washed five times for 10 min each with tbst, and developed using supersignal® west pico chemiluminescent substrate according to the manufacturer's instruction (pierce, rockford, il). cells resuspended in 1% paraformaldehyde in pbs for facs analysis were spotted on microscope slides and visualized using a fluorescent microscope (olympus, model ax70). dulac-cd163 cells infected with prrsv p129-gfp were also examined directly by fluorescent microscopy. cell cytotoxicity of mβcd was determined for both marc-145 cells and pams in 96-well plates at concentrations of 0, 0.25,0.5, 1, 2, 5, 10, and 20 mm. ten millimolar or higher concentration of mβcd was found to be toxic for marc-145 cells and pams, and thus 0.25-2 mm concentrations were chosen for subsequent studies. marc-145 cells and pams were treated with variable concentrations of mβcd for 1 h at 37°c and infected with prrsv pa8 at a moi of 5. after 1 h incubation, cells were washed twice and cultivated in dmem or rpmi-1640 supplemented with 2% fbs. for cholesterol replenishment, cells were pretreated with 2 mm mβcd for 1 h at 37°c and then supplemented with variable concentrations of water soluble cholesterol for 1 h at 37°c (popik et al., 2002) . after two washes with dmem for marc-145 cells or rpmi-1640 for pams, the cells were infected with prrsv as above. marc-145 cells and pams were incubated for 3 days and 20 h, respectively, and the culture supernatants were collected and titrated in marc-145 cells as described above. dulac cells were transfected with pcdna-cd163 which contained the porcine cd163 gene, and selected for neomycin resistance using 1 mg/ml of g418 in the culture medium. g418-resistant cell colonies were picked using cloning cylinders and amplified for screening as described elsewhere (lee et al., 2004) . the gene expression was confirmed by pcr, rt-pcr, and protein assays by fa and western blot, and the cells expressing porcine cd163 were designated dulac-cd163. hela, dulac, and dulac-cd163 cells were plated on microscope cover slips and transfected with pxj41-flag-gp4. at 24 hpt, cells were washed once with ice-cold pbs and incubated with respective primary antibodies at 4°c for 30 min as follows: daf-specific mab evr1 and anti-flag rabbit antibody for hela cells transfected with pxj41-flag-gp4; cd163-specific mab and anti-flag rabbit antibody for dulac-cd163 cells transfected with pxj41-flag-gp4. cells were washed twice in ice-cold pbs and fixed with 4% paraformaldehyde in pbs at 4°c for 1 h. cells were then washed again three times with ice-cold pbs and incubated with alexa fluor 488®-conjugated goat anti-mouse igg (h + l) or alexa fluor 594®-conjugated goat anti-rabbit igg (h + l) antibodies. the coverslips were washed five times in pbs, mounted on microscope slides in mounting buffer (60% glycerol and 0.1% sodium azide in pbs), and visualized under a laser-scanning confocal fluorescence microscope (model bx50, olympus). intercellular transfer of a glycosylphosphatidylinositol (gpi)-linked protein: release and uptake of cd4-gpi from recombinant adeno-associated virus-transduced hela cells association of neural cell adhesion molecule 1 gene polymorphisms with bipolar affective disorder in japanese individuals functions of lipid 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detergent resistant membrane microdomains full-length sequence of a canadian porcine reproductive and respiratory syndrome virus (prrsv) isolate cloning, expression, and transcriptional properties of the human enhancer factor tef-1 the authors thank aimee bachand for construction of daf/4 and daf/2 and federico zuckermann for providing z-mac cells. this study was supported by the national research initiatives of the us department of agriculture cooperative state research education and extension service, grant number 2008-35204-04634 awarded to dy. key: cord-268139-tgpsu4qz authors: brockway, sarah m.; denison, mark r. title: mutagenesis of the murine hepatitis virus nsp1-coding region identifies residues important for protein processing, viral rna synthesis, and viral replication date: 2005-09-30 journal: virology doi: 10.1016/j.virol.2005.06.035 sha: doc_id: 268139 cord_uid: tgpsu4qz despite ongoing research investigating mechanisms of coronavirus replication, functions of many viral nonstructural proteins (nsps) remain unknown. in the current study, a reverse genetic approach was used to define the role of the 28-kda amino-terminal product (nsp1) of the gene 1 polyprotein during replication of the coronavirus murine hepatitis virus (mhv) in cell culture. to determine whether nsp1 is required for mhv replication and to identify residues critical for protein function, mutant viruses that contained deletions or point mutations within the nsp1-coding region were generated and assayed for defects in viral replication, viral protein expression, protein localization, and rna synthesis. the results demonstrated that the carboxy-terminal half of nsp1 (residues k(124) through l(241)) was dispensable for virus replication in culture but was required for efficient proteolytic cleavage of nsp1 from the gene 1 polyprotein and for optimal viral replication. furthermore, whereas deletion of nsp1 residues amino-terminal to k(124) failed to produce infectious virus, point mutagenesis of the nsp1 amino-terminus allowed recovery of several mutants with altered replication and rna synthesis. this study identifies nsp1 residues important for protein processing, viral rna synthesis, and viral replication. mutagenesis of the murine hepatitis virus nsp1-coding region identifies residues important for protein processing, viral rna synthesis, and viral replication coronaviruses belong to a family of enveloped positivestrand rna viruses that are responsible for devastating illnesses in livestock and domestic animals. the identification of a novel human coronavirus as the etiological agent of severe acute respiratory syndrome (sars) in 2003 highlighted the potential of this virus family to also cause severe human disease (kuiken et al., 2003) . even with continuing research addressing how coronaviruses replicate and cause disease, the functions of many viral proteins remain to be elucidated. for example, 14 -16 nonstructural proteins (nsps) are expressed from coronavirus gene 1 polyproteins, but at least seven of these nsps have no known roles in viral replication. in the past, gene 1 has been referred to as the ''replicase gene'' and gene 1 nsps as ''replicase proteins'' named by their molecular weight in kilodaltons. more recently, nsps have been named based on their order in gene 1 and are numbered consecutively beginning at the amino-terminus of the polyprotein (i.e., nsp1 through nsp16) (harcourt et al., 2004; prentice et al., 2004b; snijder et al., 2003) . the gene 1 nsps of the coronavirus murine hepatitis virus (mhv) are similar in number, size, and organization to those of sars coronavirus (sars-cov) (marra et al., 2003; snijder et al., 2003) . this resemblance suggests that mhv is an excellent model for studies of coronavirus nsp function and may 0042-6822/$ -see front matter d 2005 elsevier inc. all rights reserved. doi:10.1016/j.virol.2005.06.035 increase our understanding of sars-cov replication and pathogenesis. following entry of mhv into a host cell, the first event in the virus life cycle is translation of two polyproteins from gene 1 of the input rna genome. gene 1 is comprised of two overlapping open reading frames (orf1a and orf1b) that are connected by a à1 ribosomal frameshift (fig. 1a ) (bonilla et al., 1994; bredenbeek et al., 1990; brierley et al., 1989; lee et al., 1991; pachuk et al., 1989) . translation of either orf1a or the orf1ab fusion results in possible 495-kda or 803-kda polyproteins, respectively. these co-aminoterminal polyproteins are proteolytically processed by three virus-encoded proteinases, including two papain-like proteinases (plp1 and plp2 within nsp3) and a picoronavirus 3c-like proteinase (nsp5), to yield at least 16 mature gene 1 nsps as well as intermediate precursors. to date, all mhv gene 1 nsps tested co-localize with sites of active viral rna synthesis at cytoplasmic viral replication complexes on intracellular double-membrane vesicles (bost et al., 2000 (bost et al., , 2001 brockway et al., 2003 brockway et al., , 2004 gosert et al., 2002; prentice et al., 2004a; shi et al., 1999; van der meer et al., 1999) . nsps are thought to mediate replication of the mhv rna genome and subgenomic rna synthesis at these membrane-bound complexes. however, the viral proteinases are the only mhv nsps with experimentally confirmed functions (baker et al., 1989; bonilla et al., 1995; lu et al., 1996; lu et al., 1995) . based on homology to proteins with known functions, roles in viral rna synthesis have been predicted for several other mhv gene 1 nsps. these proteins include two trans-membrane scaffolding proteins (nsp4 and nsp6) (gosert et al., 2002) , an rna-dependent rna polymerase (nsp12) (cheng et al., 2005; gorbalenya et al., 1989; lee et al., 1991) , an rna helicase (nsp13) (seybert and ziebuhr, 2001; seybert et al., 2000) , and several rna processing enzymes (nsp14, nsp15, and nsp16) (bhardwaj et al., 2004; ivanov et al., 2004; thiel et al., 2003; ziebuhr, 2005) . currently, there are no known or envisaged functions in replication for at least seven mhv gene 1 proteins (nsp1, nsp2, and nsp7 -11) . previous studies have provided intriguing evidence about the potential functions of the 28-kda amino-terminal protein nsp1 (previously referred to as p28) in mhv replication. the kinetics of nsp1 expression suggest that it might have an early regulatory role during the viral life cycle. nsp1 is the first mature protein processed from the gene 1 polyprotein and is likely cleaved quickly following transorf1a -orf1b fusion polyprotein is illustrated with mature nonstructural protein (nsps) represented as numbered boxes. the gray box represents the aminoterminal cleavage product (nsp1). nsps with confirmed or predicted functions include: two papain-like proteinases (plp1 and plp2 within nsp3), the 3c-like proteinase (3cl pro ; nsp5), two trans-membrane proteins (mp1 and mp2; nsp4 and nsp6, respectively), the rna-dependent rna polymerase (pol; nsp12), the rna helicase (hel; nsp13), the 3v-to-5v exonuclease (exo; nsp14), the endoribonuclease (endo; nsp15), and the rna methyltransferase (mt; nsp16). (b) nsp1 mutant proteins. the schematics illustrate the engineered deletions and point mutations within nsp1. nsp1 amino acid numbers are listed below each protein, and the predicted protein size (in kilodaltons) is listed to the right of each. the amino-terminal charge-to-alanine mutations for each vusb mutants are listed below the bottom nsp1 protein. the asterisks (*) indicate mutants that did not establish productive infections as determined by lack of recovered virus from electroporated cells. lation of plp1 within nsp3 (baker et al., 1989 (baker et al., , 1993 denison and perlman, 1987; denison and perlman, 1986; denison et al., 1992 denison et al., , 1995 . mhv mutants that are incapable of liberating nsp1 from the nascent polyprotein exhibit delayed replication, diminished peak titers, small plaques, and reduced rna synthesis compared to wild-type controls . these results emphasize the importance of nsp1 cleavage for optimal viral rna synthesis and suggest that nsp1 might play an important role at mhv replication complexes. in support of this notion, nsp1 localizes to replication complexes in the infected cell cytoplasm during times of peak viral rna synthesis, and biochemical experiments demonstrate interactions between nsp1 and two other replication complexassociated proteins (nsp7 and nsp10) . however, later in infection, nsp1 is distinct from replication complexes and instead co-localizes with mhv structural proteins at virion assembly sites . it has also been reported that exogenous mhv nsp1 expression induces cell-cycle arrest (chen et al., 2004) . together, these results led to the hypothesis that nsp1 may participate in multiple stages of the mhv life cycle. still, the function of this protein remains a mystery as analysis of nsp1 primary amino acid sequence does not reveal common motifs or protein homologs, and the nsp1 crystal structure has not been solved. the goal of the current study was to determine the role of nsp1 during mhv replication in cell culture and to identify regions of the protein that are critical for its function. using the reverse genetic system for mhv developed by yount et al. (2002) , genomic rna molecules that contained deletions or point mutations within the nsp1-coding region were generated and tested for their capacity to produce infectious virus following transfection into permissive cells. the results demonstrate that the carboxy-terminal half of nsp1 is dispensable for replication in culture but is important for efficient proteolytic cleavage of the protein and optimal viral replication. in contrast, deletion of the entire nsp1 protein or a middle region of the protein resulted in the lack of virus recovered from electroporated cells. analysis of nsp1 point mutants identified residues important for viral replication and rna synthesis. the results of this study provide valuable information regarding the possible functions of the amino-terminal nsp1 protein in coronavirus replication. the carboxy-terminal half of nsp1 is dispensable for mhv replication to determine whether nsp1 is required for mhv replication, viruses were engineered to lack nsp1 residues a 2 through l 241 (nsp1dfl) or to express truncated forms of the protein lacking either q 87 through n 164 (nsp1dmid) or k 124 through l 241 (nsp1dc) (fig. 1b) . all deletion constructs were designed to maintain the start codon, the translational reading frame, and the minimal residues thought to be required for cleavage of nsp1 from the polyprotein by plp1 (nsp1 residues k 243 through g 247 ) . mutations were engineered into the nsp1-coding region within the mhv infectious cdna (icmhv) fragment a plasmid. the mutagenized plasmids were then used to assemble full-length mhv cdna, which was transcribed in vitro to yield mhv genomic rna (yount et al., 2002) . bhk-mhvr cells were electroporated with either assembled wild-type (icwt) or nsp1 mutant genomic rna transcripts. cells were incubated at 37 -c and were monitored for syncytia formation, a cytopathic effect (cpe) of mhv replication. virus-induced syncytia were detected at 24 h postelectroporation (p.e.) in cells transfected with icwt genomic rna and at 72 h p.e. in cells transfected with genomic rna for the carboxy-terminal truncation mutant (nsp1dc). clarified cell culture media harvested from cells electroporated with either icwt or nsp1dc rna were capable of initiating a productive infection in freshly inoculated dbt-9 cells, demonstrating the presence of viable virus. in contrast, cells electroporated with nsp1dfl or nsp1dmid rna showed foci containing 5 -10 cell nuclei at 72 -96 h p.e., but these multi-nucleated foci did not progress or spread over time like wild-type mhv syncytia. the clarified media from cells electroporated with nsp1dfl or nsp1dmid genomes were not capable of producing cpe in freshly inoculated dbt-9 cells. this result indicated that nsp1dfl and nsp1dmid were not capable of completing a productive life cycle, defined by the lack of infectious virus particles in the cell media supernatant following electroporation. nsp1dfl and nsp1dmid rna molecules were tested for their capacity to produce viruses in three independent experiments, all of which yielded this same result. the nsp1dc virus was purified by three rounds of plaque isolation, and rt-pcr was used to sequence across the entire nsp1-coding region. the mutant virus maintained the engineered deletion and had no other mutations within nsp1. these results demonstrate that the carboxy-terminal half of nsp1 is dispensable for mhv replication and suggest that deletion of residues in the amino-terminal half is not tolerated for a productive infection. charge-to-alanine mutagenesis of the nsp1 amino-terminus the fact that nsp1dfl and nsp1dmid did not generate productive infections suggests that this region of the protein has a critical function in mhv replication. however, it is also possible that the deletions removed required rna elements within the 5v end of the nsp1coding sequence. to investigate the function of the nsp1 amino-terminus with minimal disruption of the correspond-ing rna sequence, viruses were engineered to contain point mutations within this region. mutations were introduced into the icmhv fragment a plasmid using sitedirected mutagenesis. single or paired charged residues (d, e, r, k, and h) within the amino-terminal half of mhv nsp1 that are conserved across all group 2 coronaviruses were substituted with alanine (fig. 1b) . mutated fragment a cdnas were used as before to generate full-length genomic viral rna. virus-induced syncytia were detected within 24 h in bhk-mhvr cells electroporated with either icwt rna or with rna for nsp1 mutants vusb1, vusb2, vusb3, or vusb7. the timing and extent of cpe following transfection with these mutants were indistinguishable from icwt. cells transfected with rna for vusb4 exhibited cpe at 72 h p.e., similar to the kinetics observed with nsp1dc. the clarified media supernatants from cells transfected with vusb1, vusb2, vusb3, vusb4, or vusb7 rna were capable of infecting dbt-9 cells, indicating that they contained nsp1 mutant viruses. as with nsp1dc, the nsp1 point mutants were purified by three rounds of plaque isolation, and rt-pcr was used to sequence across the entire nsp1-coding region. the mutant viruses maintained the engineered changes and lacked other mutations within nsp1. similar to the results with nsp1dfl and nsp1dmid, cells transfected with vusb5 or vusb6 rna produced foci containing 5 -10 cell nuclei at 72 -96 h p.e, but the extent of these multi-nucleated foci did not increase over time. moreover, clarified media supernatant harvested from cells transfected with vusb5 or vusb6 rna did not generate a productive infection following incubation with dbt-9 cells, indicating that vusb5 and vusb6 were blocked at some stage in the viral life cycle. vusb5 and vusb6 rna molecule were assayed for their capacity to produce viruses in two independent experiments, both of which showed the same result. it has been reported that charge-to-alanine mutagenesis of viral proteins may generate temperaturesensitive mutants (hanley et al., 2002; hassett and condit, 1994; tang et al., 2002) . in the current study, nsp1 mutants vusb5, vusb6, and nsp1dmid did not exhibit temperature-sensitive phenotypes as no infectious virus was recovered at either 37 -c or 32 -c. the nsp1dfl virus was not tested at 32 -c. experiments were next performed to determine if viral gene expression occurred in cells electroporated with rna for mutants that did not produce infectious virus (vusb5, vusb6, nsp1dfl, and nsp1dmid). using antisera against mhv virions (a-mhv) or against nsp2 (a-nsp2), no viral proteins could be detected by immunofluorescence or immunoprecipitation (data not shown). furthermore, no specific products were amplified from electroporated cells using rt-pcr and a primer that detects subgenomic rnas (data not shown). these results suggest that, if viral protein or rna expression occurred, it was below the level of detection using these assays. to determine whether mutagenesis of nsp1 results in viral replication defects, single-cycle replication assays were performed with the nsp1 mutants (fig. 2) . dbt-9 cells were infected with icwt or nsp1 mutants (vusb1, vusb2, vusb3, vusb4, vusb7, or nsp1dc). samples of infected cell media supernatant were taken at various times from 1 -24 h post-infection (p.i.), and viral titers in each sample were determined by plaque assays. wild-type (icwt) or nsp1 mutant viruses were used to infect dbt-9 cells at an moi of 5. cells were rinsed three times with pbs, incubated under medium at 37 -c, and samples of medium were obtained at the indicated times post-infection. viral titers in each sample were determined using plaque assays on dbt-9 cells at 37 -c. the graph shows the results of a representative experiment. values are the averages obtained from duplicate media samples. (b) nsp1 mutant viral yield. to determine viral yield, viral titer at 1 h p.i. was subtracted from peak titer for each virus. bars represent average viral yield calculated from the experiments (n = 3). lines represent standard error from the experiments. statistical analysis software was used to determine p values using a two-sample t test. asterisks (*) indicate p values 0.05. (c) relative plaque size of icwt, vusb1, and vusb4 (15 h p.i.). images were obtained at the same resolution (10â) on a nikon eclipse te2000-e microscope. white circles were drawn to facilitate visualization of the plaque boundary. the nsp1 mutants exhibited viral replication kinetics similar to icwt with peak virus release occurring between 12 -16 h p.i. (fig. 2a) . however, several of the nsp1 mutants (vusb1, vusb 4, and nsp1dc) showed a reduction in viral yield compared with icwt ( fig. 2b ). whereas icwt gave an average yield of approximately 4.9 â 10 6 pfu/ml, vusb1, vusb4, nsp1dc viruses exhibited viral yields of 2.4 â 10 5 , 1.4 â 10 6 , 1.0 â 10 6 pfu/ml, respectively. all of the nsp1 mutants exhibited decreased yields; however, only the vusb1, vusb4, and nsp1dc yields were statistically significant ( p 0.05 using a twosample t test). vusb1 and vusb4 also demonstrated small plaque phenotypes (fig. 2c ). these results indicate that nsp1 mutants have replication defects in cultured dbt-9 cells. nsp1 mutant viruses were evaluated to determine whether they are defective in the expression or processing of viral proteins (fig. 3) . immunoprecipitation experiments were performed to analyze processing of nsp1 and nsp2 or 3clpro-mediated processing of nsp8. to confirm expression of the structural proteins, antisera raised against mhv virions (a-mhv) were used. dbt-9 cells were either mockinfected or infected with icwt or nsp1 mutants, radiolabeled from 5-8 h p.i. with [ 35 s]met/cys, and used to prepare cytoplasmic lysates for immunoprecipitations. antisera against nsp1 (a-nsp1) immunoprecipitated a 28-kda protein (nsp1) from cells infected with icwt or the nsp1 point mutants (vusb1, vusb2, vusb3, vusb4, or vusb7) (fig. 3a) . migration of vusb7-nsp1 differed from icwt and all other point mutants, suggesting differences in protein folding or charge. no 28-kda proteins were immunoprecipitated from mock-infected cell lysates or from lysates generated from cells infected with the nsp1dc virus ( fig. 3a) . rather, from nsp1dc-infected cell lysate, a-nsp1 immunoprecipitated a unique 14-kda protein, the predicted size of nsp1dc, as well as a previously undescribed 80-kda protein (fig. 3a) . in both vusb4-and nsp1dc-infected cells, several additional proteins (30 to 60 kda) of unknown identity were also precipitated using a-nsp1 (fig. 3a ). antisera against nsp2 (a-nsp2) immunoprecipitated a 65-kda protein (nsp2) from all infected cell lysates, but not from mock-infected lysates (fig. 3b ). similar to the result using a-nsp1, an 80-kda protein was also detected by a-nsp2 in nsp1dc-infected cells (fig. 3b ). antisera against nsp8 (a-nsp8) immunoprecipitated a 22-kda protein (nsp8) from all infected cell lysates, but not from mock-infected cell lysates (fig. 3c ). the viral structural protein s, n, and m were detected in all infected cell lysates using antisera against virions (a-mhv) (fig. 3d ). the nsp1 mutants showed band intensities slightly increased compared with icwt. the reason for this difference in band intensity is unknown but might be a reflection of more protein synthesis or processing with the mutants at the time of label. still, although the amount of protein detected differed slightly, the results show that amino-terminal nsp1 point mutants do not have defects in their capacities to express or process viral proteins. nonetheless, the detection of an 80-kda protein from nsp1dc-infected cells using either a-nsp1 or a-nsp2 suggests that cleavage of nsp1dc from nsp2 may be inefficient. fig. 3 . nsp1 mutant viral protein expression and processing. cytoplasmic lysates were generated from radiolabeled dbt-9 cells that were either mock-infected (m) or infected with icwt or nsp1 mutant viruses. labeled proteins were immunoprecipitated from cytoplasmic lysates with the indicated polyclonal antisera. proteins were resolved by sds-page in 5 -18% polyacrylamide gradient gels and visualized following fluorography. images were obtained following 4-day film exposure. bands corresponding to unique or predicted proteins are indicated on the right of the fluorograms, and molecular weight standards (in kilodaltons) are shown on the left. the mhv structural proteins are designated as follows: spike (s), nucleocapsid (n), and membrane (m). the antisera used for immunoprecipitation are indicated: the carboxy-terminal half of nsp1 is required for efficient cleavage at cs1 previous studies have shown that nsp1 is processed from the gene 1 polyprotein very rapidly, and nsp1-containing precursors have not been detected (denison et al., 1992) . in contrast, nsp2 is processed with slower kinetics likely from a 275-kda nsp2 -nsp3 precursor protein (denison et al., 1992 harcourt et al., 2004; schiller et al., 1998) . to determine the expression kinetics of the 80-kda protein in nsp1dc virus-infected cells and to determine whether this protein is capable of being processed into 14-kda (nsp1dc) and 65-kda (nsp2) proteins, pulse-chase translation experiments were performed (fig. 4) . dbt-9 cells were infected with either icwt or the nsp1dc virus, and proteins were radiolabeled from 6 -6.5 h p.i. with [ 35 s]met/cys. infected cells were then chased in medium without radiolabel but containing cyclohexamide to inhibit new protein synthesis. cells were harvested at various times post-chase (p.c.), and cytoplasmic lysates were generated for immunoprecipitations using a-nsp1 and a-nsp2. in the current study, the kinetics of nsp1 and nsp2 expression and processing in icwt-infected cells were similar to published reports (fig. 4a ). nsp1 was detected as a mature 28-kda protein at 0 min p.c., suggesting that this protein was processed during the 30 min radiolabeling period. although nsp2 was detected as a mature 65-kda protein at 15 min p.c., nsp2 detection was increased at 60-360 min p.c. the result that nsp1 and nsp2 remained detectable even at 360 min p.c. suggested that these proteins were stable. in these experiments, the 275-kda nsp2 -nsp3 precursor was not detected by a-nsp2. like nsp1 from icwt, nsp1dc (14 kda) was detectable at 0 min p.c., indicating that processing at cs1 occurred within fig. 4 . pulse-chase translation in nsp1dc virus-infected cells. proteins in infected dbt-9 cells were radiolabeled for 30 min with [ 35 s]met/cys at 6 h p.i. and then incubated in medium containing cyclohexamide for 15 to 360 min as described in materials and methods. cells were lysed at the indicated times (min) post-chase (p.c.), and cytoplasmic lysates were generated for immunoprecipitation studies using a-nsp1 and a-nsp2. proteins were analyzed as in fig. 3 . the identities of proteins are indicated to the right of the fluorograms. the number of days the gels were exposed to film (d exp) to generate the image is listed next to the antisera used for immunoprecipitation. pulse-chase translation in cells infected with (a) icwt or (b) nsp1dc virus. . intracellular localization of mutant nsp1 proteins. dbt-9 cells grown on glass coverslips were infected with icwt or nsp1 mutant viruses for 7 h, fixed and permeablized with 100% methanol, and incubated with antibodies against nsp1 (red), nsp2 (green), and n (purple). cells were imaged using a zeiss lsm 510 confocal microscope at 546 nm (red), 488 nm (green), and 633 nm (purple). images are single confocal slices obtained using a 40â objective. co-localization of green and purple for vusb1 is shown in the merged image as light green pixels. co-localization of all three colors is shown in the merged images as white pixels. multi-nucleated cells are a cytopathic effect of mhv replication. the 30 min period of radiolabel. however, in contrast to icwt nsp1, nsp1dc was not detectable after 120 min p.c., suggesting that once processed the protein may be subject to degradation. the kinetics of nsp2 expression in nsp1dc virus-infected cells were similar to those of nsp2 from icwt (fig. 4b) ; nsp2 (65 kda) was first detected at 0-15 min p.c., but detection was increased at 60 -360 min p.c. using either a-nsp1 or a-nsp2, the 80-kda protein was detectable by 0 min p.c. and remained stable or increasing until 240 min p.c. at 360 min p.c., the 80-kda band was slightly less intense; however, at this time point, there was no concurrent increase in nsp1dc or nsp2 detection. this result suggests that the 80-kda protein may be degraded at late times of chase rather than processed. as with icwt, the high molecular weight nsp1dc viral precursor proteins were not detected by the antisera. during early times of infection (4 -7 h p.i.), nsp1 colocalizes with nsp2 and the viral nucleocapsid protein (n) at viral replication complexes in the cytoplasm . at these times, nsp1 is mostly distinct from the virion membrane protein (m), which localizes to sites of particle assembly . to determine the intracellular localization of viral proteins from the nsp1 mutants, immunofluorescence confocal microscopy was performed. dbt-9 cells on glass coverslips were infected for 7 h, fixed and permeabilized with methanol, and incubated with antisera against nsp1, nsp2, and either n or m (fig. 5) . the a-nsp1 staining pattern in cells infected with vusb2, vusb3, vusb4, vusb7, or nsp1dc was similar to icwt. nsp1 from these mutant viruses co-localized with nsp2 and n in punctate cytoplasmic replication complexes (fig. 5) . at this time point, the nsp1 proteins from vusb2, vusb3, vusb4, vusb7, nsp1dc, or icwt viruses were also distinct from sites of virion assembly as determined by lack of predominant nsp1 co-localization with m (data not shown). using the a-nsp1 antisera, no specific staining above background levels was detected in vusb1-infected cells, whereas nsp2, n, and m staining was indistinguishable from icwt ( fig. 5 and data not shown) . the nsp1 antiserum is capable of detecting vusb1-nsp1 by immunoprecipitation (fig. 3a) , demonstrating that the engineered substitutions did not abolish the epitope(s). therefore, the lack of staining during immunofluorescence assays might reflect alternative folding of vusb1-nsp1. nonetheless, these results suggest that nsp1 mutant viruses do not have defects in intracellular protein localization or replication complex formation. to investigate whether the nsp1 mutant viruses exhibit changes in the timing or levels of viral rna synthesis, metabolic labeling assays were performed (fig. 6 ). dbt-9 fig. 6 . nsp1 mutant viral rna levels. (a) nsp1 mutant viral rna levels represented as percent of icwt. dbt-9 cells were infected at an moi of 5 with icwt or nsp1 mutant viruses. actinomycin d was added to a final concentration of 20 ag/ml 30 min prior to the addition of [ 3 h]uridine. viral rna was radiolabeled from 5 -9 h p.i. and then precipitated from equal volumes of cytoplasmic lysates in replicate using trichloracetic acid. to quantitate [ 3 h]uridine incorporation as counts per minute (cpm), liquid scintillation was used. for each experiment (n = 5), labeled viral rna levels (cpm) for icwt were set to 100%, and nsp1 mutant viral rna was calculated as a percentage of the icwt value. the bars represent the average percent viral rna from all experiments, and lines indicate standard error. statistical analysis software was used to determine p values using a one-sample t test. asterisks (*) indicate p values 0.05. (b) gel analysis of viral rna. dbt-9 cells were mock-infected or infected with icwt or the indicated nsp1 mutants. rna was labeled as in panel (a) above, and cells were lysed using trizol. rna was isolated from cell lysates and normalized so as to electrophorese the same amount of radiolabeled viral rna for each mutant (approximately 400,000 cpm from a maximum of 10 6 cells). for the mock-infected control (m), rna from 10 6 cells was used. the rna was separated in an 0.8% formaldehyde/agarose gel, and individual rna species were visualized following fluorography. the image is from a 2-day exposure of the gel to film. individual viral rna species are numbered to the right of the fluorogram (rna 1 = genome and rna 2 -7 = subgenomic rnas). the asterisk (*) indicates a unique rna band detected in nsp1dc virus-infected cells. cells were infected with icwt or nsp1 mutants (vusb1, vusb2, vusb3, vusb4, vusb7, or nsp1dc). viral rnas were radiolabeled from 1 -13 h p.i. at intervals using 100 aci/ml of [ 3 h]uridine in the presence of actinomycin d, a drug that inhibits dna-dependent rna synthesis. rna was precipitated from cytoplasmic lysates using trichloroacetic acid, and 3 h levels were quantitated using liquid scintillation. the timing of rna synthesis for the nsp1 mutants was indistinguishable from icwt, with all viruses exhibiting maximal [ 3 h]uridine incorporation when cells were labeled between 5 -9 h p.i. (data not shown). however, the total levels of radiolabeled viral rna differed among the nsp1 mutants when compared with icwt. vusb1 and vusb4 rna levels were only 73% and 79% of icwt, respectively, whereas vusb3 rna levels were 129% if icwt (fig. 6a) . these data suggest that nsp1 may function at the replication complex in the synthesis of viral rna. to determine whether nsp1 mutants have specific defects in genome replication or subgenomic rna synthesis, radiolabeled viral rna was analyzed by gel electrophoresis (fig. 6b ). dbt-9 cells were either mock-infected or infected with icwt or nsp1 mutant viruses, and rna was labeled in the presence of actinomycin d from 5 -9 h p.i. using [ 3 h]uridine. total viral and cellular rna was extracted from cell lysates, and radioactivity in each sample was quantitated using liquid scintillation. equal amounts of radiolabeled viral rna (equal cpm) were separated by electrophoresis in formaldehyde/agarose gels and then visualized following fluorography. all seven species of mhv viral rna were detected with the nsp1 mutant viruses and icwt. interestingly, with the nsp1dc virus, an additional band that migrated above rna4 was consistently detected. the identity of this band is not known, but it might represent a more stable replicative intermediate or a new subgenomic rna species. still, the approximate ratio of rna1 (genome) to rna7 for each nsp1 mutant was similar to that of icwt, suggesting that nsp1 mutants do not have specific defects in either genome replication or subgenomic rna synthesis as detected by this assay. coronavirus gene 1 nsps are predicted to function in the synthesis of viral rna at cytoplasmic replication complexes; however, many of these proteins have no known roles during the viral life cycle. in the current study, a reverse genetic approach was used to investigate the role of nsp1 during mhv replication in cell culture and to identify residues critical for its function. it was shown that viruses containing deletions within nsp1 amino-terminal to k 124 are not capable of establishing productive infections, suggesting that residues essential for mhv viability reside within the amino-terminal half of the protein. several such residues were identified using point mutagenesis, and the importance of the nsp1 for viral replication and rna synthesis was characterized. moreover, the results show that the carboxyterminal half of nsp1 is not required for mhv to complete its life cycle but is necessary for efficient cleavage of nsp1 from the gene 1 polyprotein and for optimal viral replication. together, these data are consistent with the hypothesis that mhv nsp1 contains at least two domains important for virus replication: (1) an essential aminoterminal domain involved in viral rna synthesis and (2) a non-essential carboxy-terminal domain that influences cs1 cleavage efficiency. the analyses of mhv nsp1 deletion and point mutants emphasize the significance of the amino-terminus for virus viability, replication, and rna synthesis. deletion of different portions of the nsp1 amino-terminal half, as was done with nsp1dfl and nsp1dmid, was not tolerated for virus viability. this result is consistent with the idea that critical replication determinants reside within the region spanning residues m 1 through p 123 . charge-to-alanine mutagenesis identified four candidate residues (r 64 and e 69 with vusb5; r 78 and d 79 with vusb6), all or any of which may be vital for some stage of the mhv life cycle. in cells transfected with rna genomes for these nonproductive mutants (nsp1dfl, nsp1dmid, vusb5, or vusb6), foci containing 5 -10 cell nuclei were seen. how these foci form in electroporated cell monolayers is unknown, but the presence of this cpe suggests that transient viral gene expression may have occurred. however, no viral protein was detected in transfected cell monolayers by immunoprecipitation or immunofluorescence assays, and rt-pcr did not amplify subgenomic rna, indicating that, if gene expression occurred, it was below the limits of detection using these assays. consequently, the exact stage of the mhv life cycle at which these mutant viruses are blocked could not be determined. still, the result that nsp1dfl, nsp1dmid, vusb5, or vusb6 did not establish productive infections underscores the importance of the nsp1 amino-terminus and suggests that this protein has a critical function during viral replication. in support of nsp1 having a central role in the mhv life cycle, it was demonstrated that all of the viable nsp1 point mutants exhibited reduced viral yields during singlecycle replication assays. vusb1 and vusb4 were the most defective and resulted in viral titers approximately 0.2-to 1.3-log reduced compared with icwt. these two viruses also exhibited small plaque phenotypes, suggesting defects in cell-to-cell spread. the reduced replication and small plaques seen with vusb1 and vusb4 are most likely a reflection of reduced viral gene expression as both of these nsp1 point mutants showed significantly lower levels of viral rna. interestingly, compared with icwt, vusb3 had increased levels of viral rna associated with a slight reduction in viral replication, suggesting that any deviation from wild-type viral rna levels may negatively impact viral replication. there were no detectable differences in the ratios of genome rna versus subgenomic rnas for any of the viable nsp1 mutants. these results suggest that nsp1 might play a role in the regulation of total viral rna amounts rather than in species-specific viral rna synthesis. it has been previously shown that mhv mutants unable to liberate nsp1 from the gene 1 polyprotein (dcs1 mutants) exhibit phenotypes very similar to the nsp1 mutant viruses described in this study . because the dcs1 mutations resulted in a noncleaved nsp1-nsp2 fusion protein, it could not be concluded that the viral phenotypes were solely related to defects in nsp1 function. this study corroborates and extends these previous findings and directly correlates mutations in the mhv nsp1-coding region with changes in viral replication and rna synthesis. the results of this study raise important questions regarding the mechanistic basis for the described replication and rna synthesis defects with nsp1 mutants. why is the nsp1 amino-terminus required for mhv replication? what function does this domain have during mhv rna synthesis? do the deletion and point mutations change the nature of nsp1 -protein interactions? yeast two-hybrid and co-immunoprecipitation assays previously demonstrated that wild-type nsp1 directly binds to nsp7 and nsp10, two other gene 1 proteins . the interactions between nsp1, nsp7, and nsp10 do not require the nsp1 carboxy-terminus (f 167 through g 247 ) but do require residues amino-terminal to f 167 (i.e., residues e 84 through r 166 ). because the nsp1dmid virus was engineered to lack this putative interaction domain, it is interesting to speculate whether the lack of viability for this mutant correlates with a loss of nsp1 binding to either nsp7 and/or nsp10. furthermore, it is thought that charge-to-alanine mutagenesis alters a protein's capacity to participate in interand intra-molecular interactions (cunningham and wells, 1989) . this mutagenesis approach is supposed to minimize disruption of protein secondary structure; however, it is possible that some of the charge-to-alanine mutations caused nsp1 to misfold. in support of this possibility, vusb1-nsp1 was detected by a-nsp1 antisera during immunoprecipitation assays but not during immunofluorescence assays, suggesting that this mutant protein may have a different structural conformation. regardless, the introduced mutations may have altered the capacity of nsp1 to bind to nsp7, nsp10, or other proteins resulting in viral rna synthesis and viral replication defects. during wild-type mhv infection, nsp1, nsp2, and nsp3 are processed in an ordered manner (fig. 7a) . immediately following translation of plp1 within nsp3, cs1 is rapidly cleaved, releasing nsp1 as polyprotein synthesis continues (denison et al., 1992 harcourt et al., 2004; schiller et al., 1998) . next, cleavage site 3 (cs3) is cleaved (likely by plp2) liberating an nsp2 -nsp3 precursor, which is finally processed at cleavage site 2 (cs2) to release mature nsp2 and nsp3 products (denison et al., 1992 harcourt et al., 2004; schiller et al., 1998) . in nsp1dc virus-infected cells, nsp1dc (14-kda) and nsp2 are present, demonstrating that the carboxy-terminal half of nsp1 is not absolutely essential for cleavage at either cs1 or cs2. however, the identification of an 80-kda nsp1dc -nsp2 polypeptide in nsp1dc virus-infected cells reveals that this region of the protein (residues k 124 through l 241 ) is important for efficient cs1 processing. fig. 7 . model of icwt and nsp1dc virus protein processing. shown are schematics of wild-type and nsp1dc virus amino-terminal gene 1 nsps (boxes). plp1 and plp2 are shown as gray boxes within nsp3. the individual cleavage sites are labeled, and the order of processing is indicated above the closed arrowheads. lines below the protein schematics illustrate the size of mature proteins following cleavage at individual sites. (a) processing of wild-type nsp1, nsp2, and nsp3. during infection, nsp1 (28 kda) is cleaved rapidly at cleavage site 1 (cs1) as the polyprotein is translated (indicated by forward slashes). next, cleavage site 3 (cs3) is processed to yield a 275-kda nsp2 -nsp3 precursor. finally, cleavage site 2 (cs2) is cleaved to liberate nsp2 (65 kda) and nsp3 (210 kda). (b) wildtype pattern of processing for nsp1dc mutant. the order of cleavage for nsp1dc mutant polyprotein is identical to that of icwt. nsp1dc protein (dc; 14 kda) is liberated by cs1 cleavage. next, cs3 is processed to yield a 275-kda nsp2 -nsp3 precursor. finally, cs2 is cleaved to liberate nsp2 (65 kda) and nsp3 (210 kda). (c) alternative pattern of processing for nsp1dc mutant. if cs1 is not initially cleaved, a 290-kda nsp1dc -nsp2 -nsp3 precursor is made following cs3 processing. this precursor is then cleaved at cs2 to yield an 80-kda protein (nsp1dc -nsp2) and nsp3. the results described in this report are consistent with a model in which the nsp1dc gene 1 polyprotein exists in two different conformations: a cs1 cleavable form and a cs1 non-cleavable from. if the mutant polyprotein is folded in such a manner that cs1 is accessible by the proteinase, then this site is cleaved normally and the mutant polyprotein follows a wild-type pattern of processing (fig. 7b) . alternatively, if the mutant polyprotein is folded in a way that masks cs1, this site is not processed and nsp1dc remains fused to nsp2 (fig. 7c ). in this model, an nsp1dc-nsp2 -nsp3 precursor is the first processed protein following cs3 cleavage. this polypeptide is then processed at cs2 to yield the 80-kda protein (nsp1dc -nsp2) and nsp3. although the high molecular weight precursors were not detected in these studies, the kinetics of appearance for nsp1dc, nsp2, and the 80-kda protein are compatible with the proposed model. during pulse-chase experiments, nsp1dc and nsp2 are first detected in nsp1dc virus-infected cells with the same timing as the corresponding nsp1 and nsp2 proteins in icwt-infected cells. however, the expression kinetics for the 80-kda protein are unlike either nsp1 or nsp2, suggesting that the timing and order of polyprotein processing are different under circumstances where cs1 is not immediately cleaved. whether the 80-kda protein is capable of being subsequently processed into nsp1dc and nsp2 could not be determined from these studies. at extended times p.c. (360 min), the 80-kda protein is still detectable, albeit at slightly reduced levels, and there is no new accumulation of nsp1dc at this same time. these data are most consistent with the idea that the 80-kda protein is not further processed once it is generated. in addition to cs1 processing defects, the nsp1dc virus also exhibits subtle replication defects, but normal viral rna synthesis levels, compared with icwt. a unique rna species migrating above rna4 was detected in nsp1dcinfected cells. the identity of this rna band remains to be determined, but it might represent a stable replicative intermediate and implies that this mutant virus may have minor defects in discontinuous transcription. another interesting possibility is that this band might represent a new species of subgenomic rna. because some molecules of nsp1dc are fused to nsp2, defects in nsp2 function may contribute to the mutant viral phenotype. alternatively, the reduced replication of the nsp1dc virus might suggest that nsp1 plays an important role downstream of viral rna synthesis, such as in virion assembly. at late times of infection, wild-type nsp1 co-localizes with the mhv structural protein m at virion assembly sites . because the a-nsp1 antisera used in this study also detects the 80-kda nsp1dc -nsp2 protein, we were unable to precisely determine whether the 14-kda nsp1dc protein is capable of localizing to virion assembly sites late in infection. nonetheless, it will be valuable to determine whether nsp1dc, or any of the mutant nsp1 proteins, exhibit differences in protein localization during late times of infection. in summary, the phenotypes of these mhv mutant viruses reveal new information about the possible roles of nsp1 during viral replication. however, it is important to note the limitations of the reverse genetic approach used in this study and to explore alternative explanations of the available data. for example, it has been reported that exogenous mhv nsp1 expression induces cell-cycle arrest (chen et al., 2004) . therefore, it is also possible that the nsp1 mutations have an indirect effect on mhv replication as a result of cell biological changes during infection. moreover, while it is clear that some of the nsp1 mutants have differences in nsp1 confirmation and interactions, it is possible that uncharacterized rna structural elements have also been disrupted as a result of nsp1 mutagenesis resulting in changes in viral rna synthesis and replication. furthermore, in the absence of sequencing the entire 32-kb genomes of the nsp1 mutant viruses, the possibility that other mutations contribute to the described phenotypes cannot be excluded. to rule out the contributions of other mutations or rna elements, wild-type nsp1 expressed in cells must complement the defects of these viruses. while ideal, trans-complementation using protein expression has not been successful for coronaviruses. in fact, efficient complementation for other positive-strand rna viruses has required the development of replicon systems to facilitate viral replication complex formation (appel et al., 2005; grassmann et al., 2001; khromykh et al., 1998; lindenbach and rice, 1997; liu et al., 2002) . despite these limitations, this is the first report of detailed mutagenesis of an mhv gene 1 nsp-coding region within the context of a virus. these results will enhance our knowledge of mhv replication determinants, and future analysis of nsp1 mutant viruses will provide greater insight into the function of rna and protein elements within the 5v end of gene 1. even more, studies with these engineered mutant viruses are expected to contribute to understanding the replication strategies of other coronaviruses, such as sars-cov. delayed brain tumor cells selected for high-level expression of the mhv receptor carcino-embryonic antigen cell adhesion molecule-1 (dbt-9) (chen et al., 1997; hirano et al., 1976; yount et al., 2002) and baby hamster kidney-21 cells expressing the mhv receptor (bhk-mhvr) (chen et al., 1997; yount et al., 2002) were grown in dulbecco's modified eagle medium (dmem) (gibco) that was supplemented with 10% heat-inactivated fetal calf serum (fcs) (sigma) for all experiments. medium for bhk-mhvr cells was supplemented with g418 (800 ag/ ml) to select for cells expressing the mhv receptor. polyclonal antisera used for biochemical and immunofluorescence experiments have been previously described. these include guinea pig a-nsp1 antisera (gp3) ; rabbit a-nsp2 antisera (vu153) (sims et al., 2000) ; rabbit a-nsp8 antisera (p1a-22) (bost et al., 2000) ; and rabbit a-mhv antisera generated against intact virions . j. fleming (university of wisconsin, madison) kindly provided murine monoclonal antibodies specific for the structural proteins nucleocapsid (a-n, j.3.3) and membrane protein (a-m, j.1.3). to delete portions of the nsp1-coding sequence, pcr was performed using the icmhv-a59 fragment a plasmid (pcr-xl-topoa) as template (yount et al., 2002) . to generate nsp1dfl, deleting nearly the entire nsp1-coding sequence (nsp1 amino acids a 2 through l 241 ), a pcr product was amplified using oligodeoxynucleotide primers (sense) 5v-gtt taa acg aga cat aat acg-3v and (antisense) 5v-ctt aag cat tat gca acc tat-3v. to generate nsp1dmid, deleting the middle portion of nsp1 (q 87 through n 164 ), two separate pcr products were amplified. the first pcr reaction used primers (sense) 5v-ccg ccg gcc tgg tct tgt-3v and (antisense) 5v-gga tcc tca tct aca aa-3v to amplify sequences corresponding to the 5vutr and the amino-terminal 86 residues (m 1 through p 86 ) of nsp1. the second reaction used primers (sense) 5v-gat ccc ggc cgt ttt ata ggc-3v and (antisense) 5v-ctt aag aag agc ata-3v to amplify sequences corresponding to the carboxy-terminal 78 residues (g 165 through l 241 ). to generate nsp1dc, deleting the carboxy-terminus of nsp1 (k 124 through l 241 ), a pcr product was amplified using primers (sense) 5v-ccg ccg gcc tgg tct tgt-3v and (antisense) 5v-ctt aag ggg aag cac acc caa-3v. all pcr products were ligated into pgem-t-easy (promega) and sequenced to ensure the fidelity of pcr. nsp1 sequences were then subcloned into pcr-xl-topoa in place of wild-type nsp1 using primer-generated restriction sites: nsp1dfl (5vpmei to 3vaflii), nsp1dmid (first ligated at the bamhi site then subcloned into pcr-xl-topoa using 5v sacii and 3vaflii), and nsp1dc (5v sacii to 3vaflii). all fragment a plasmids were sequenced across the nsp1coding region to ensure proper ligation and maintenance of the translational reading frame. the mhv-a59 nucleotides (nt) 103 to 954 (restriction sites 5v sacii to 3vaflii) were pcr amplified using pcr-xl-topoa as template and cloned into pgem-t-easy to generate pgem-nsp1. charge-to-alanine substitutions were introduced into pgem-nsp1 using complementary pairs of mutagenic primers and the quikchange site-directed mutagenesis kit (stratagene). mutagenized pgem-nsp1 plasmids were sequenced to verify the incorporation of the appropriate substitutions and the fidelity of pcr. restriction enzyme sites 5v sacii and 3vaflii were then used to subclone mutant nsp1 sequences into pcr-xl-topoa in place of the wild-type nsp1 sequence. viruses containing nsp1 mutations were produced using the infectious clone strategy for mhv-a59 (icmhv) described by yount et al. (2002) and modified by denison et al. (2004) . all assembled viruses, including the wildtype control (icwt), were generated using the icmhv fragment f plasmid corresponding to the virulent vuss3 strain (sperry et al., 2005) . plasmids containing the cdna cassettes of the mhv genome were digested using mlui and bsmbi for fragment a, bgli and bsmbi for fragments b and c, ncii and bsmbi for fragments d and e, bsmbi for f, and sfii and bsmbi for fragment g. gel purified restriction fragments were ligated together using t4 dna ligase (new england biolabs) in a total reaction volume of 150 al at 16 -c overnight. following chloroform extraction and isopropanol precipitation of ligated cdna, full-length transcripts of icmhv rna were generated in vitro using the mmessage mmachine t7 transcription kit (ambion) according to the manufacturer's protocol with the following modifications. fifty microliter reactions were supplemented with 7.5 al of 30 mm gtp, and transcription was performed at 40.5 -c for 25 min, 37.5 -c for 50 min, and 40.5 -c for 25 min. in parallel, rna transcripts encoding the mhv nucleocapsid protein (n) were generated from n cdna. n transcripts and icmhv genomic rna were then mixed and electroporated into bhk-mhvr cells. briefly, bhk-mhvr cells were grown to sub-confluence, trypsinized, then washed twice with phosphate-buffered saline (pbs) and resuspended in pbs at 10 7 cells/ml. six hundred microliters cells were then added to rna transcripts in a 4-mm gap electroporation cuvette, and three electrical pulses of 850 v at 25 af were delivered with a gene pulser ii electroporator (bio-rad). transfected cells were then seeded on a monolayer of 10 6 uninfected dbt-9 cells in a 150 cm 2 flask and incubated at either 37 -c or 32 -c for 24 to 96 h. virus viability was determined by syncytia formation in the electroporated cell culture and by the capacity of the clarified transfected cell supernatant to induce syncytia in a monolayer of fresh dbt-9 cells. plaque purification, rt-pcr, and sequencing mutant viruses were subjected to three rounds of plaque purification, and reverse transcription (rt)-pcr was used to amplify the 5v end of the viral genome for sequence analysis. rna was harvested from infected dbt-9 cells using trizol (invitrogen) according to the manufacturer's protocol and used as template for rt-pcr. to generate viral cdna, reverse transcription was performed using superscript ii rt (invitrogen) and an antisense primer complimentary to nt 5531 -5500 of the mhv-a59 genome. the nsp1-coding region was then amplified using pcr with primers corresponding to nt 284-302 (sense) and nt 1150-1165 (antisense). the resulting amplicons were sequenced across the nsp1-coding region to confirm the retention of the introduced nsp1 mutations and the absence of second-site mutations. viable nsp1 mutant viruses were analyzed for replication using single-cycle replication assays as previously described . briefly, dbt-9 cells (approximately 10 6 cells) were infected at an moi of 5 pfu/cell with icwt or nsp1 mutant viruses. virus inoculum was removed following 30 min adsorption at 25 -c, and cells were washed three times with pbs. cells were then incubated at 37 -c in fresh pre-warmed dmem with 10% fcs, and samples of medium were collected at various times up to 24 h post-infection (p.i.). viral titers in the medium were determined by plaque assay (hirano et al., 1976) . to determine viral yield, titer at 1 h p.i. was subtracted from peak titer for each virus, and an average was calculated using data from three separate experiments. data analysis was performed using smith's statistical package-version 2.75. a two-sample t test was performed for each virus ( p value 0.05). for imaging relative plaque size, confluent dbt-9 cells in 60-mm culture dishes were infected with serial dilutions of icwt, vusb1, or vusb4 virus and then were overlaid with 1% agar -dmem supplemented with 10% fcs. at 15 h p.i., the agar medium was removed, and the cells were fixed for 30 min using à20 -c 100% methanol. cells were incubated in pbs for 30 min, and then images of plaques were obtained on a nikon eclipse te2000-e microscope using a 10â objective. images were prepared using adobe photoshop 8.0 (adobe). dbt-9 cells (approximately 3 â 10 6 cells) were either infected at an moi of 5 or mock-infected using dmem with 10% fcs. at 2.5 h p.i., the medium was replaced with fresh dmem lacking methionine and cysteine and supplemented with 5% fcs and actinomycin d (5 ag/ml). proteins were radiolabeled from 5 -8 h p.i. using 100 aci/ml of [ 35 s]met/ cys (translabel; icn). for pulse-chase analysis, proteins were radiolabeled from 6-6.5 h p.i. and then chased for various times with dmem supplemented with 10% fcs and cyclohexamide (250 ag/ml). cells were washed using 500 al of pbs and then lysed in 300 al of lysis buffer containing 150 mm nacl, 1% np40, 0.5% doc, 50 mm tris ph 8.0. lysates were subjected to centrifugation at 3500 â g to remove cell nuclei. immunoprecipitations were performed in a final volume of 500 al using protein a -sepharose beads (sigma), 100 al of radiolabeled cytoplasmic lysate (derived from approximately 10 6 cells) which was boiled for 5 min in 1% sds, and 2 -10 al of polyclonal antisera in immunoprecipitation buffer containing 300 mm nacl, 0.1% sds, 0.5% tx-100, 4 mm edta, 0.1% dtt, and 50 mm tris ph 7.4. immunoprecipitation reactions were incubated at 4 -c for 4 h with rotation. protein-bead conjugates were washed three times in immunoprecipitation buffer, and proteins were eluted from beads by boiling for 5 min in 2â protein loading buffer (200 mm dtt, 100 mm tris ph 6.8, 0.04% bromophenol blue, 20% glycerol). proteins were resolved by sds-page in 5 -18% polyacrylamide gradient gels and analyzed by fluorography. the [ 14 c] high molecular weight standard (gibco) and full-range rainbow marker (invitrogen) were used as molecular weight standards. images were prepared using adobe photoshop 8.0 (adobe). dbt-9 cells cultured on glass coverslips were either mock-infected or infected at an moi of 5 at 25 -c for 30 min. following virus adsorption, infected medium was replaced with pre-warmed dmem with 10% fcs, and cells were incubated at 37 -c. at 7 h p.i., cells were fixed and permeablized with à20 -c 100% methanol for a minimum of 30 min. indirect immunofluorescence assays were performed as previously described (bost et al., 2000) . gp3 was used at 1:2000 dilution, and all rabbit polyclonal antisera were used at 1:100 dilution. murine monoclonal antibodies were used at 1:1000 dilution. secondary antibodies conjugated to fluorophores (molecular probes) were used at 1:1000 and included a-guinea pig-alexa 546, arabbit-alexa 488, and a-murine-alexa 633. immunofluorescence was detected using a zeiss lsm 510 laser scanning confocal microscope with a 40â oil immersion objective. image analysis and merging were performed using adobe photoshop 8.0 (adobe). dbt-9 cells (approximately 10 6 cells) were infected at an moi of 5 with icwt or nsp1 mutant viruses. virus inoculum was removed following 30 min adsorption at 25 -c, and cells were washed three times with pbs. cells were then incubated for various times in fresh pre-warmed dmem with 10% fcs at 37 -c. at 30 min prior to addition of radiolabel, cell medium was supplemented with actinomycin d (5 ag/ml). viral rna was metabolically labeled in the presence of actinomycin d using 100 aci/ml [ 3 h]uridine. to harvest viral rna, cells were washed twice with pbs and then lysed with 300 al of cell lysis buffer (150 mm nacl, 1% np40, 0.5% doc and 50 mm tris ph 8.0). lysates were centrifuged at 3500 â g to remove nuclei, and rna in 100 al of cytoplasmic extract was precipitated in replicate using 10% trichloroacetic acid. precipitated rna was dried onto glass microfiber filters (whatman) using vacuum filtration, and radioactivity was measured as counts per minute (cpm) using a liquid scintillation counter (beckman). statistical analysis software (smith's statistical package-version 2.75) was used for data analysis. for each experiment (n = 5), icwt values (cpm) from the 5-9 h time intervals were set to equal 100%. and, nsp1 mutant values were calculated as percent of icwt. one-sample t tests were performed using a p value 0.05. analysis of [ 3 h] viral rna by gel electrophoresis was performed as described previously (kim et al., 1995) with a few modifications. viral rna was radiolabeled from 5-9 h p.i. as before and was isolated using trizol (invitrogen) according to the manufacturer's protocol. the amount of [ 3 h] incorporation was quantitated using liquid scintillation. equal amounts of radiolabeled viral rna (approximately 400,000 cpm from a maximum of 10 6 cells) were denatured in formamide gel loading buffer at 65 -c for 10 min and then electrophoresed in a 0.8% formaldehyde/ agarose gel at 144 v for 4 h. for mock-infected samples, rna from 10 6 cells was used. following electrophoresis, the gel was incubated in 100% methanol for 2 h then in 1% diphenyloxazole/methanol for 2 h prior to overnight incubation in distilled water. the gel was dried at 50 -c using vacuum filtration before exposing it to film. images were prepared using adobe photoshop 8.0 (adobe). efficient rescue of hepatitis c virus rna replication by trans-complementation with nonstructural protein 5a identification of a domain required for autoproteolytic cleavage of murine coronavirus gene a polyprotein identification of the catalytic sites of a papain-like cysteine proteinase of murine coronavirus the severe acute respiratory syndrome coronavirus nsp15 protein is an endoribonuclease that prefers manganese as a cofactor mouse hepatitis virus strain a59 rna polymerase gene orf 1a: heterogeneity among mhv strains characterization of the leader papain-like proteinase of mhv-a59: identification of a new in vitro cleavage site four proteins processed from the replicase gene polyprotein of mouse hepatitis virus colocalize in the cell 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identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins processing of the coronavirus mhv-jhm polymerase polyprotein: identification of precursors and proteolytic products spanning 400 kilodaltons of orf1a guanosine triphosphatase activity of the human coronavirus helicase the human coronavirus 229e superfamily 1 helicase has rna and dna duplexunwinding activities with 5v-to-3v polarity colocalization and membrane association of murine hepatitis virus gene 1 products and de novo-synthesized viral rna in infected cells mouse hepatitis virus replicase proteins associate with two distinct populations of intracellular membranes unique and conserved features of genome and proteome of sarscoronavirus, an early split-off from the coronavirus group 2 lineage single-amino-acid substitutions in open reading frame (orf) 1b-nsp14 and orf 2a proteins of the coronavirus mouse hepatitis virus are attenuating in mice clustered charge-toalanine mutagenesis of human respiratory syncytial virus l polymerase generates temperature-sensitive viruses mechanisms and enzymes involved in sars coronavirus genome expression localization of mouse hepatitis virus nonstructural proteins and rna synthesis indicates a role for late endosomes in viral replication systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a59 the coronavirus replicase we express our appreciation to xiao tao lu key: cord-267014-3vi7pgvr authors: vennema, h.; rossen, j.w.a.; wesseling, j.; horzinek, m. c.; rottier, p.j.m. title: genomic organization and expression of the 3′ end of the canine and feline enteric coronaviruses date: 1992-11-30 journal: virology doi: 10.1016/0042-6822(92)90174-n sha: doc_id: 267014 cord_uid: 3vi7pgvr abstract the genomic organization at the 3′ end of canine coronavirus (ccv) and feline enteric coronavirus (fecv) was determined by sequence analysis and compared to that of feline infectious peritonitis virus (fipv) and transmissible gastroenteritis virus (tgev) of swine. comparison of the latter two has previously revealed an extra open reading frame (orf) at the 3′ end of the fipv genome, lacking in tgev, which is currently designated orf 6b. both ccv and fecv possess 6b-related orfs at the 3′ ends of their genomes. the presence of orf 6b in three of four viruses in this antigenic cluster strongly suggests that tgev has lost this orf by deletion. the ccv orf 6b is collinear with that of fipv, but the predicted amino acid sequences are only 58% identical. the fecv orf 6b contains a large deletion compared to that of fipv, reducing the collinear part to 60%. the sequence homologies were highest between ccv and tgev on the one hand and between fecv and fipv on the other. previously, we showed that the expression product of the fipv orf 6b can be detected in infected cells by immunoprecipitation (vennema et al., 1992). in the present study we have performed similar experiments with ccv and fecv. in infected cells both viruses produced proteins related to but different from the fipv 6b protein. canine coronavirus (ccv), feline enteric coronavirus (fecv), feline infectious peritonitis virus (fipv), and transmissible gastroenteritis virus (tgev) of swine belong to one antigenic cluster within the family coronaviridae (siddel et al., 1983) . sequence analysis revealed a close genetic relatedness between fipv and tgev (jacobs et al., 1987; de groot et al., 1988; vennema et a/., 1991) . fipv contains an extra open reading frame (orf) in the 3'-terminal region of its genome (de groot et a/., 1988) . it is the second orf of mrna 6 which is currently designated orf 6b, according to the new nomenclature (cavanagh et al., 1990) . the first orf of mrna 6, designated orf 6a, is the counterpart of tgev orf 7, previously called orf-x3 (kapke and brian, 1986; rasschaert et al., 1987) or orf-4 (britton et a/., 1988) . comparison of orfs 6a and 7 revealed that the fipv genome contained an in frame insertion of 69 nucleotides (de groot et a/., 1988) . the protein product of tgev orf 7 was identified in tgev-infected cells (garwes et al., 1989) . recently, the 6b gene was shown to be expressed in fipv-infected cells (vennema et a/., 1992) . it is a glycoprotein which is released into the extracellular medium and is not stably associated with virus particles. in cats it induces antibodies during natural and experimental fipv infections. therefore, the 6b protein provides an antigenic distinction between fipv and tgev. the aim of the ' to whom reprint requests should be addressed. present study was to examine whether this distinction could be extended to ccv and fecv. we determined the genomic organization of the 3' end of the viral genomes and investigated whether these viruses produced proteins related to the 6b protein of fipv. fipv strain 79-l 146, fecv strain 79-l 683 (mckeirnan et a/., 1981; obtained from dr. j. evermann), ccv strain k378 (dutch field isolate, obtained from dr. h. flare), and ccv strain l-71 (binn et a/., 1975) were grown in fe/is catus whole fetus cells (fcwf-d; obtained from dr. n. c. pedersen) and crandell feline kidney cells (crfk). recombinant vaccinia virus vtf7-3 (fuerst eta/., 1986 ; obtained from dr. b. moss) infections were carried out in hela cells. cells were maintained in dulbecco's modified eagle's medium (gibco laboratories) containing 5% fetal bovine serum. cloning and sequence analysis of the 3' end of ccv cdna libraries were prepared of intracellular poly(a)containing rna from ccv-infected fcwf-d cells as described elsewhere (wesseling et al., manuscript in preparation) . clones containing sequences derived from the 3'end of the genome were selected by colony hybridization with restriction fragments of fipv cdna clone b12 (de groot eta/., 1988) as probes. nucleotide sequencing was performed by the dideoxy chain termi-nation procedure (sanger et a/., 1977) using doublestranded dna and a bacteriophage t7 dna polymerase based kit (pharmacia, lkb). sequence data were analyzed using the computer programs of devereux et a/. (1984). cdna synthesis and pcr amplification of the 3' end of fecv total intracellular rna was isolated from fecv-infected fcwf-d cells as described (chomczynski and sacchi, 1987) . synthesis of cdna on total rna was pet-formed as described (kawasaki and wang, 1989) by priming specifically with synthetic oligonucleotide 179 (y-ccagllltagacatcggg-3', reverse complement of nucleotides 1006-l 023; de groot et al., 1988) which binds to a sequence in the 3' noncoding region of fipv, downstream of orf 6b. oligonucleotide 185 (5'-gatccagacgttagctc-3', reverse complement of nucleotides 1202-l 218; de groot et a/., 1988), was used to prime cdna synthesis from a position closer to the 3' end. amplification of cdna was performed by the polymerase chain reaction (pcr) as described (kawasaki and wang, 1989) , after the addition of synthetic oligonucleotide 178, 5'-gatgacacacaggltgag-3', which is identical to the carboxyl-terminus of the nucleocapsid (n) protein gene of fipv (nucleotides 1945-l 962; vennema et a/., 1991) . the positions of the primers are indicated in fig. 5 . pcr-amplified fecv cdna fragments were isolated from agarose gel, oligo-dc tailed with terminal transferase and cloned after annealing with oligo-dg-tailed puc9 (pharmacia lkb). sequence analysis was performed as described above for ccv cdna clones, with puc/m 13 primers and with the primers used for pcr amplification. the ccv 6b gene was isolated from cdna clone cl6 as a /-/pall-pstl fragment and recloned in pbluescript sk-(stratagene) digested with accl and pstl, yielding pbsc6b. the 6b gene fragment was recloned from this construct as a xhol-psrl fragment into the vector part of ptfgb, (vennema et a/., 1992) digested with the same enzymes. the final construct was designated ptc6b. the fecv 6b gene was recloned from cdna-pcr clone fe1 as a spel-pstl fragment in the vector part of ptfgb, prepared by digestion with spel and pstl. this construct was designated pte6b. all constructs were used in the transient t7 expression system with recombinant vaccinia virus vtf7-3 producing the t7 rna polymerase (fuerst et a/., 1986 ; obtained from dr. b. moss). lysates from coronavirus-or vaccinia virus-infected cells were prepared after metabolic labeling with l-[35s]cysteine (icn biomedicals, inc.). lysis, ripa with ascites fluid from a field case of fip, and endo-p-nacetylglucosaminidase h (endo h; boehringer-mannheim biochemicals) treatment were carried out as described (vennema et a/., 1990) . the ascites fluid contains antibodies to all viral proteins of fipv identified so far, which are cross-reactive with proteins from ccv, fecv, and tgev. endo-p-n-acetylglucosaminidase f (endo f; boehringer-mannheim biochemicals) digestions were carried out for 16 hr at 30" in 50 mm potassium phosphate buffer, ph 6.8, 20 mll/l edta, 1% triton x-l 00, 0.2% sodium dodecyl sulphate (sds), 1% 2-mercaptoethanol. analysis by sds-polyacrylamide gel electrophoresis (sds-page) was performed as described (laemmli, 1970) . sequence analysis of the 3' end of the ccv genome ccv cdna clones were prepared and selected as described under materials and methods. most clones were derived from ccv strain k378, a field isolate from the netherlands (fig. 1) . a clone derived from the laboratory strain l-71 (binn et al., 1975) was partially analyzed. the nucleotide sequences from these strains were more than 99% identical. we obtained a contiguous sequence of strain k378, extending 2.5 kb from the poly(a)-tail in the 5' direction ( fig. 2) . translation of the nucleotide sequence revealed three orfs corresponding to the n protein and orfs 6a and 6b of fipv. the organization of the 3'end of the ccv genome was compared to that of fipv and tgev (fig. 5 ). ccv contains an orf 6b and an orf 6a with 69 additional nucleotides which are lacking in orf 7 of tgev. the amino acid sequences were aligned with the corresponding sequences of fipv and tgev to determine the percentages of identical amino acid residues (table 1). the n and 6a amino acid sequences have a higher level of identity when compared between ccv and tgev than between ccv and fipv. the same was found when the nucleotide sequences were compared. the 6b amino acid sequences of ccv and fipv were only 58% identical and several small insertions in the ccv sequence were found. nevertheless, the hydrophobicity plots of the putative 6b proteins were remarkably similar (data not shown), including a short hydrophobic amino-terminus, which may function as a signal sequence. in contrast to the fipv orf 6b, the ccv orf 6b contains no potential n-glycosylation site. pcr amplification of cdna derived from the 3' end of the fecv genome the nucleotide sequences flanking the orfs 6a and 6b of fipv and ccv and orf 7 of tgev were aligned to design primers for cdna synthesis and polymerase chain reaction (pcr) amplification. the positions of the primers are indicated in fig. 3 . synthetic oligonucleotide 179 was used to prime cdna synthesis on total rna isolated from fecv and ccv-infected cells. the cdna synthesis was followed by pcr amplification after addition of primer 178. the ccv product had the expected size of approximately 1 kbp (data not shown). the fecv product was considerably smaller, being approximately 750 bp. the controls with rnafrom mockinfected cells and without rna were both negative. the sequence analysis presented below revealed that the fecv orf 6b extended into the sequences used to design the pcr primers. therefore, cdna synthesis and pcr amplification were repeated with synthetic oligonucleotides 185 and 178, resulting in a fragment of approximately 950 bp (data not shown). the pcr products of fecv were cloned and sequenced. several independent clones were analyzed (fig. 3) leading to a contiguous sequence of 957 nucleotides which ends approximately 36 nucleotides upstream of the poly(a)-tail (fig. 4) . comparison with the corresponding sequence of the fipv genome showed a single deletion of 238 nucleotides and an overall sequence identity of 93.6%. translation of the nucleotide sequence revealed the presence of two orfs similar to the fipv orfs 6a and 6b. the 6a polypeptides are virtually identical, with only one amino acid difference. the deletion is located in orf 6b. the fecv and fipv 6b sequences are collinear for the amino-terminal 123 amino acid residues. in this part the identity is 89%. the deletion results in a shift to the -1 reading frame which extends 53 codons. the orf specifies a polypeptide with a total length of 176 amino acid residues and a predicted mol wt of 20,300. the protein sequence predicts a short hydrophobic aminoterminus, probably acting as a signal sequence and one n-glycosylation site. the genomic organization of the fecv 3' end is similar to fipv and ccv, which all contain an orf 6b and the extra 69 nucleotides in orf 6a as compared to tgev orf 7 (fig. 5) . paired alignments of the collinear parts of the amino acid sequences with the corresponding sequences of fipv, ccv, and tgev revealed that fecv is more closely related to fipv than to ccv and tgev (table 1) . similar homologies were found by comparing the nucleotide sequences (data not shown). the fipv 6b protein was readily detected in lysates of fipv-infected cells (vennema et a/., 1992) . it comigrated in sds-page with an expression product of the cloned 6b gene which was prepared by using the recombinant vaccinia virus t7 rna polymerase expression system (fuerst et a/., 1986) . to identify the ccv and fecv 6b proteins in a similar way, their 6b orfs were recloned in a t7 expression vector. the resulting constructs ptc6b and ptegb, respectively, and ptf6b containing the fipv 6b gene (vennema et a/., 1992) were used to transfect hela cells infected with recombinant vaccinia virus vtf7-3, which produces t7 rna polymerase (fuerst et a/., 1986) . the expression products were analyzed by metabolic labeling with [35s]cysteine, ripa, and endo h treatment followed by sds-page (fig. 6) . the ccv and fecv 6b proteins appeared to be slightly smaller than the fipv 6b protein, the fecv 6b protein being the smallest. digestion with endo h which cleaves high-mannose n-linked oligosaccharides, resulted in an approximately 2000 mol wt reduction of the fecv and fipv 6b protein. the ccv 6b protein, however, was not affected and was also insensitive to digestion by endo f, which cleaves complex n-linked sugars (data not shown). these observations indicate that fecv and fipv are glycoproteins while ccv 6b is not. the shift in molecular weight of the fecv and fipv 6b proteins is consistent with the removal of one sugar side chain (neuberger et a/., 1972) . this is in agreement with the predicted numbers of glycosylation sites in the amino acid sequences. the observed molecular weights of the ccv 6b protein and of the fecv 6b protein after deglycosylation are also in agreement with those predicted from the amino acid sequences. the recombinant expression products were compared to the proteins produced in ccv-, fecv-, and fipv-infected cells, which were analyzed similarly (fig. 6) . all three matched with a protein in the sample from the corresponding coronavirus-infected cell lysate. in the latter samples the three known structural proteins also appeared: the membrane (m), nucleocapsid (n), and spike (s) proteins. in all cases the m protein was partially resistant to endo h digestion. the same was observed for the 6b protein in fipv-infected cells. the lanes of fecv were overexposed to reveal the 6b protein band. this indicates that the expression level of the fecv 6b protein was much lower than that of ccv and fipv. the genomic organization of the 3' end of fipv differs from that of tgev in that it contains an additional orf (de groot et a/., 1988). recently, we identified the expression product of this extra gene, designated 6b (vennema et al., 1992) . these observations prompted us to study ccv and fecv of the same antigenie cluster. sequence analysis showed that their genomic organization in the 3'terminal region is similar to that of fipv. therefore, the presence rather than the absence of orf 6b appears to be the common theme, suggesting that tgev has lost the corresponding orf by deletion. the same inference probably holds true for the 69 nucleotides that are present in all 6a orfs but not in the corresponding orf 7 of tgev. deletions appear to happen frequently during coronavirus evolution. they occur, for example, in the s and he protein genes of murine hepatitis virus (mhv; parker et al., 1989; la monica et a/., 1991) in the s protein gene of porcine respiratory coronavirus (prcv; rasschaert et al., 1990) and in the region between the s and m protein genes of mhv strain s (yokomori and lai, 1991 ) of a small plaque variant of tgev (wesley et al., 1990) and of prcv (rasschaert et a/., 1990) . among the feline coronaviruses another deletion was found recently in fecv 79-1683 as compared to fipv in the region between the s and m protein genes (e. lewis and h. vennema, unpublished data) . alignment of nucleotide and amino acid sequences of tgev, ccv, fipv, and fecv revealed a close relationship among the strains of this cluster (table 1) . the four strains could be divided into two pairs on the basis of their homologies, tgev and ccv on the one hand and fipv and fecv on the other. both in ccv-and in fecv-infected cells 6b proteins are produced. their characterization showed that the fecv 6b protein is glycosylated, like the fipv 6b protein, while the ccv 6b protein is not. the observation that fipv, ccv, and fecv induce the synthesis of 6b proteins appears to compromise their antigenic distinction. however, the differences between the 6b proteins may allow discrimination using, e.g., monoclonal antibodies. the deletion of 238 nucleotides in fecv 79-l 683 was revealed bycdna-pcr, allowing discrimination from ccv (fig. 4) and fipv 79-l 146 (data not shown). it remains to be determined whether this is a universal distinguishing property of fipv and fecv. the feline coronaviruses used in this study are almost the same with respect to growth in tissue culture, protein composition, and antigenicity (boyle et a/., 1984; fiscus and teramoto, 1987) . small differences were also reported; the n protein of fecv is slightly smaller than that of fipv (tupper et al., 1987) . recently, a monoclonal antibody specific for the s protein of strain 79-1 146 was characterized (hohdatsu et al., 1991) . the close resemblance and the low incidence of fip despite the high proportion of feline coronavirus (fcv) seropositive cats have led to the hypothesis that fecv carriers are the source of fipv which is generated de nova from fecv by minor mutations (pedersen et a/., 1984; pedersen 1989) . our sequence comparisons show that fipv 79-l 146 and fecv 79-l 683 are related more closely to each other than they are to ccv. therefore, fipv 79-l 146 did not originate from fecv 79-1683 by insertion of genetic information; it would be too much of a coincidence that an insertion with 77% identity is present in exactly the same position in ccv. an alternative mechanism for de nova generation of fipv could be recombination, which is an established phenomenon for coronaviruses (lai, 1992) . avirulent fipv-strains and/or fecv-strains are and sds-page analysis of lysates from ccv-, fecv-, fipv-, and vtf7-3-infected cells, recombinant vaccinia virus vtf7-3-infected cells were transfected with the plasmid dnas ptcgb, ptegb, and ptfgb, as indicated above the lanes. one-half of each sample was treated with endo h, the other half was mock treated (indicated with + and -, respectively). structural proteins (s, n, and m) are indicated. in addition, the m protein bands are indicated with arrowheads. the region of the gel in which the 6b protein bands appear overlaps with that of m protein bands. recovery and characterization of a coronavirus from military dogs with diarrhoea. froc plaque assay, polypeptide composition and immunochemistry of feline infectious peritonitis virus and feline enteric coronavirus sequence of the nucleoprotein gene from a virulent british field isolate of transmissible gastroenteritis virus and its expression in saccharomyces cerevisiae recommendations of the coronavirus study group for the nomenclature of the structural proteins mrnas, and genes of coronaviruses single step method of rna isolation by acid guanidium thiocyanate-phenol-chloroform extraction sequence analysis of the b'end of the feline coronavirus fipv 79-1146 genome: comparison with the genome of porcine coronavirus tgev reveals large insertions a comprehensive set of sequence analysis programs for the vax antigenic comparison of feline coronavirus isolates: evidence for markedly different peplomer glycoproteins eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage t7 rna polymerase the polypeptide of m, 1400 of porcine transmissible gastroenteritis virus: gene assignment and intracellular location antigenic analysis of feline coronaviruses with monoclonal antibodies (mabs): preparation of mabs which discriminate between fipv strain 79-l 146 and fecv strain 79-1683 the nucleotide sequence of the peplomer gene of porcine transmissible gastroenteritis virus (tgev): comparison with the sequence of the peplomer protein of feline infectious peritonitis virus (fipv) sequence analysis of the porcine transmissible gastroenteritis corona virus nucleocapsid protein gene detection of gene expression. ln "pcr technology: principles and applications for dna amplification cleavage of structural proteins during assembly of the head of bacteriophage t4 rna recombination in animal and plant viruses localization of extensive deletions in the structural genes of two neurotropic variants of murine coronvirus jhm molecular cloning. a laboratory manual isolation of feline coronavirus from two cats with diverse disease manifestations carbohydrate-peptide linkages in glycoproteins and methods for their elucidation sequence analysis reveals extensive polymorphism and evidence of deletions within the e2 glycoprotein gene of several strains of murine hepatitis virus animal infections that defy vaccination: equine infectious anemia, caprine encephalitis, maedi-visna, and feline infectious peritonitis virus pathogenicity studies of feline coronavirus isolates 79-1146 and 79-1683 porcine respiratory coronavirus differs from transmissible gastroenteritis virus by a few genomic deletions enteric coronavirus tgev: partial sequence of the genomic rna, its organization and expression dna sequencing with chain termination inhibitors the biology of coronaviruses antigenic and biological diversity of feline coronaviruses: feline infectious peritonitis and feline enteritis virus a novel glycoprotein of feline infectious peritonitis coronavirus contains a kdel-like endoplasmic reticulum retention signal. f. viral intracellular transport of recombinant coronavirus spike proteins: implications for virus assembly. 1. viral primary structure of the membrane and nucleocapsid protein genes of feline infectious peritonitis virus and immunogenicity of recombinant vaccinia viruses in kittens genetic basis for the pathogenesis of transmissible gastroenteritis virus mouse hepatitis virus s rna sequence reveals that nonstructural proteins ns4 and ns5a are not essential for murine coronavirus replication. f. viral the authors thank raoul de groot and willy spaan for critical reading of the manuscript. j.w. was supported by a grant from solvay-duphar b.v., weesp, the netherlands. key: cord-254950-y6kayxie authors: morse, stephen s. title: mouse thymic virus (mtlv; murid herpesvirus 3) infection in athymic nude mice: evidence for a t lymphocyte requirement date: 1988-03-31 journal: virology doi: 10.1016/0042-6822(88)90262-0 sha: doc_id: 254950 cord_uid: y6kayxie abstract mouse thymic virus (mtlv; murid herpesvirus 3) is a lymphotropic herpesvirus that cytolytically infects developing t lineage lymphocytes in the thymus of neonatal mice. mtlv establishes a persistent infection and can be recovered indefinitely from infected mice, but nothing is known about requirements for this persistent infection. in order to determine whether t lineage lymphocytes are required for infection, young adult athymic nude (nulnu) mice and euthymic littermates were infected with mtlv and tested for virus shedding. although euthymic littermates regularly shed virus, in the nude mice only about 20% of isolation attempts up to 100 days postinfection were positive. blind passage yielded an additional three isolations out of 14 samples (21 %). in addition, unlike many other herpesviruses, the virus did not replicate in a number of epithelial and fibroblastic cell lines that were tested. these data confirm that the virus is preferentially t lymphotropic and suggest that infection may require t lineage lymphocytes. mouse iclymic virus (mtlv; murid herpesvirus 3) is a lymphotropic herpesvirus that infects and kills developing t lymphocytes in the thymus of neonatal mice (1) (2) (3) . the virus is also cytolytic for t lymphocytes in peripheral lymphoid tissues (4) . when adult mice are infected, the virus appears to bypass the thymus (2) but can be isolated indefinitely from salivary glands and on mouth swabs (1) (2) (3) . irrespective of virus dose and age at primary infection, regular lifetime virus shedding appears to be the rule (1, 2) . although thymus-derived lymphocytes appear to be the targets for primary infection in newborn mice, nothing is known about cells involved in persistent infection. the nude mouse is an ideal model for dissecting the effects of lymphotropic viruses. nude mice are athymic as a result of a genetic defect which prevents the development of the embryonic thymus (5) . because t lymphocytes mature in the thymus, this lymphocyte type is largely absent in nude mice (6). the nude gene is autosomal recessive; heterozygotes (+lnu) are phenotypically normal (5) and have essentially normal t lymphocyte function (6). in this study, experimentally infected nude mice and euthymic littermates were used to determine whether t lymphocytes are required for infection by mtlv. because there is no tissue culture system for assay of mtlv in vitro (2, 3) , the standard assay, and the most sensitive and reliable assay available at present is based on infectivity (thymic necrosis) in litters of newborn mice. the breeding stocks used to provide litters for our infectivity assay have been screened for thymic virus by infectivity testing of mouth swabs and salivary glands from retired breeders (i, 3) and are negative. in addition, the breeding stocks used were seronegative for all known murine pathogens. forvirus assay of mouth swabs, each swab was expressed into 1 ml dulbecco's modified eagle's medium supplemented with 10% fetal bovine serum and 50 pg gentamycin per milliliter (dme-1 0), and the liquid was inoculated intraperitoneally (0.05 ml per mouse; additional mice received up to 0.2 ml per newborn mouse) into litters of newborn (less than 24 hr old) balb/c mice (3). the pups were examined for thymic necrosis after 9-l 1 days (2, 3); any visible necrosis was considered positive. typically, these necrotic thymuses were ~20% of normal size. to determine whether mtlv infection requires thymus-derived lymphocytes, 4-week-old female icr swiss athymic nude (nulnu) and euthymic (+lnu) littermate controls (four each; memorial sloan-kettering cancer center nude mouse breeding colony) were inoculated intraperitoneally with either 40 or 200 ids0 of mtlv and virus shedding was tested by mouth swabs beginning 6 days after infection. all nude mice and littermate controls were negative for mtlv by virus isolation at the outset of the experiment (before inoculation). after inoculation, all the inoculated mice became infected with the virus. ability to recover virus was not correlated with input virus dose. virus could as readily be recovered from mice that had received 40 ids0 as from those that had received 200 idso. the infected mice remained healthy throughout the observation period (over 100 days), and no overt disease was attributable to mtlv infection in the nude mice. in the euthymic controls, all virus isolation attempts were positive through 100 days postinfection, with most of the inoculated pups developing macroscopic thymic necrosis (mean, 92%). in the athymic nude mice, by contrast, although virus was recovered at least once from each nude mouse (table l) , only 5 of 27 primary isolation attempts from day 6 through day 98 were positive, giving an isolation rate of 18.5%. in positive isolations, fewer inoculated pups showed thymic necrosis (mean, 14.2%) than pups that received swab fluids from controls (table 1) . for many viruses, virulence or tissue tropism can be altered by conditions of infection (7); introduction of mtlv into the nude mouse could therefore have altered virus infectivity. blind passage is a standard method for restoring virulence and increasing assay sensitivity (1, 2) . although limitations in the number of note. infected icr swiss athymic nude (nulnu) and euthymic (+/nu) littermate controls (total number of mice, eight) were tested forvirus shedding at the times shown. forvirus assay, mouth swabs were taken using cotton swabs moistened with dulbecco's modified eagle's medium + 10% fetal bovine serum and 50 pg gentamytin per milliliter (dme-1 0), and tested for mtlv by infectivity assay in newborn balb/c mice as described in the text. additional infectivity assays in normal newborn icr background swiss mice gave the same results. a values in parentheses represent number of nude mice yielding positive isolations on that day. infectivity assays from four nude mice through day 30, three on day 34, three on days 44/48 together, and two on day 98. litters available did not allow every negative sample to be tested, additional litters of normal newborn mice were inoculated with fresh homogenates (1 o-20%, w/v) of randomly selected negative thymuses and salivary glands, representing various test dates up to day 48, from 14 assay litters that had received swab fluids from nude mice. up to two blind passage cycles were performed. these passages yielded a total of three additional isolations (21%) ( table 2 ). in an additional biological passage, separate litters of normal neonatal mice were inoculated with swab fluids from each of the nude mice (day 50 postinfection) and these newborns were allowed to reach adulthood. homogenates of salivary glands and thymuses were prepared from these animals, and tested for mtlv by infectivity assay in fresh litters of newborn mice. one sample, from an animal that had received fluids from nude mouse 2, was positive on assay. this represented only the second isolation from this nude mouse, which was positive on primary isolation on day 6 and had been consistently negative thereafter. tissues from uninfected mice (as negative controls), or other negative samples, passaged at the same time as nude mouse samples were consistently negative. nude mice lack t lymphocyte function, but nonspecific immune functions, such as natural killer (nk) activity, are normal or even increased in the nude mouse (8). nk-deficient nude mice have recently been developed (9) . in order to determine whether reduced virus shedding in the nude mice could have been due to active suppression by nk cells or similar mechanisms, eight nk-deficient balb/c-c57 beige hybrid mice (life sciences, st. petersburg, fl) were infected with mtlv and tested at various times after infection. there were six positive or possibly positive isolations out of a total of 46 attempts (13%) from 1 to 14 weeks postinfection. virus was isolated consistently more than once from only one mouse, which was negative on day 6, positive on days 19, 26, and 41, positive with a lower level of virus shedding on day 33, and negative on day 48 and thereafter. one other nude mouse appeared to shed a low level of virus on day 19 and was possibly positive on day 34, but appeared negative on other isolation attempts including days 6, 41, and 48 postinfection. the other six nude mice were negative on all isolation attempts (e.g., days 6, 19, 26, 33-34, 41, 48 and thereafter through 14 weeks). thus, mtlv establishes a persistent infection in the nude mouse, but virus shedding may be reduced in comparison with euthymic mice. aside from thymusderived (t) lymphocytes, most other cell types appear to be normal in the nude mouse (5, 6) . therefore, if persistent mtlv infection were to involve mostly nonlymphoid cells, all isolations should have been positive after initial infection. that this did not appear to occur suggests that t lineage lymphocytes might well be the major target cells for persistent infection. this has been demonstrated in herpesvirus sylvilagus, a rabbit herpesvirus, which can be isolated from saliva and salivary glands of infected hosts, but the virus is associated with lymphocytes and not with epithelial cells (12) . mtlv similarly persists in salivary gland (1, 2), but electron microscopy of salivary gland epithelial cells of infected animals revealed no abnormalities (i), and no viral antigens could be detected in salivary gland epithelial cells (2) . there are two likely explanations for the apparently low level iof virus shedding in the nude mouse. although nude mice lack mature functional t lymphocytes, early t lineage precursors are present in the nude mouse (6, 8, 10); under certain conditions, these cells in the nude mouse can also differentiate extrathymically into mature t lymphocytes (7 1). in addition, several reports indicate that a small number of apparently matu"e (bearing the t lymphocyte marker thy 1) t lineage lymphocytes are present in nude mice throughout their lives (6, 10). a small population of this type could be persistently infected and responsible for the low level of virus shedding observed. this population reportedly increases with age or intercurrent infection (111. infection with the coronavirus mouse hepatitis virus (mhv), a common murine pathogen endemic in many nude mouse colonies, has also been reported to induce extrathymic t cell differentiation in nude mice (11) . some of our preliminary results (data not shown) suggest that nude mice older at primary infection, or nude mice that have been infected with mhv, appear more likely to shed mtlv. alternatively, there could be a secondary cell type (such as the macrophage) in which virus can persist at low levels even in the absence of t lymphocytes. macrophage infection by another t lymphocytolytic virus, human immunodeficiency virus (hiv-l), has been documented (13). our preliminary attempts to isolate mtlv from adherent peritoneal macrophages of persistently infected euthymic mice have been negative so far. however, low levels of infected cells might not be detected. specific molecular probes, such as genomic dna probes or monoclonal antibodies, are not presently available for this virus. we are in the process of producing specific high-titer antisera and other probes in order to test the presence of mtlv in various lymphoid and hematopoietic tissues and in nerve cells. as regards other susceptible cells, original attempts to grow the virus in mouse embryo cells or mouse embryo kidney cells, among others, were unsuccessful (1). our attempts to infect a variety of epithelial and fibroblastic cell cultures that support the replication of many other viruses have been uniformly negative. for infection, subconfluent monolayers of cells were incubated for 90 min with dme-10 containing 500-2000 ids0 mtlv and polybrene (30 pg/ml), then fed (dme supplemented with 5010 fetal bovine serum), incubated, and tested for mtlv by infectivity assay at 5-7 days and at 2 weeks. cells tested included ar42j rat glandular epithelium, balb cl.7 mouse fibroblast, balb 3t3 fibroblast, cl271 mouse mammary tumor, j774a.l mouse macrophage, nctc 1469 mouse liver, sc-l feral mouse embryo, tcmk-1 mouse kidney, 32 ill a rat hepatoma, a549 human lung carcinoma, hep-2 human epidermoid carcinoma, mdck canine kidney epithelium, vero african green monkey kidney, wish diploid human amniotic epithelium, 324k human kidney, and primary cultures of african green monkey kidney, cynomolgous monkey kidney, rabbit kidney, and rhesus monkey kidney. replicate cultures, observed up to a month for cytolysis or focus formation, remained negative throughout. additional cultures cocultivated with primary mtlv-infected thymocytes (5 x 105/ml, harvested day 5 postinfection) also remained uninfected. these results suggest that mtlv is unable to productively infect many cell lines that support the cytolytic replication of many viruses including other lymphotropic herpesviruses. taken together, these lines of evidence appear to implicate the t lineage lymphocyte as the primary target of both acute and persistent mtlv infection. co/d spring harbor conf. cell pro/if key: cord-269204-kajws5xo authors: nitschke, matthias; korte, thomas; tielesch, claudia; ter-avetisyan, gohar; tünnemann, gisela; cardoso, m. cristina; veit, michael; herrmann, andreas title: equine arteritis virus is delivered to an acidic compartment of host cells via clathrin-dependent endocytosis date: 2008-08-01 journal: virology doi: 10.1016/j.virol.2008.04.041 sha: doc_id: 269204 cord_uid: kajws5xo equine arteritis virus (eav) is an enveloped, positive-stranded rna virus belonging to the family arteriviridae. infection by eav requires the release of the viral genome by fusion with the respective target membrane of the host cell. we have investigated the entry pathway of eav into baby hamster kindey cells (bhk). infection of cells assessed by the plaque reduction assay was strongly inhibited by substances which interfere with clathrin-dependent endocytosis and by lysosomotropic compounds. furthermore, infection of bhk cells was suppressed when clathrin-dependent endocytosis was inhibited by expression of antisense rna of the clathrin-heavy chain before infection. these results strongly suggest that eav is taken up via clathrin-dependent endocytosis and is delivered to acidic endosomal compartments. equine arteritis virus (eav) is an enveloped positive-stranded rna virus (van dinten et al., 1997) of the family arteriviridae which belongs together with coronaviruses to the order nidovirales. horses are the typical target of eav. consequences of infection range from an asymptomatic, persistent carrier state to abortion or even to lethal haemorrhagic fever (snijder and meulenberg, 1998) . eav buds from the rough endoplasmic reticulum and the outer nuclear membrane, but not from the cell surface (wada et al., 1995) . eav contains seven structural proteins which are essential for virus infectivity and maturation. the nucleocapsid protein n forms an isometric core particle (de vries et al., 1992) , which is surrounded by the lipid envelope. n and the complex of the non-glycosylated membrane protein m and the major glycoprotein gp5 are required for virus assembly (wieringa et al., 2004) . the putative envelope heterotrimer of the glycosylated proteins gp2b, gp3, gp4 (ratio: 1:1:1) is a prime candidate for receptor-binding and for fusion of eav envelope with the cellular target membrane (wieringa et al., 2003 (wieringa et al., , 2004 . the function of the nonglycosylated envelope protein e is not yet known. two major cell entry pathways, the endosomal and the nonendosomal pathway, are known for enveloped virus to deliver the viral genome into the target cell. in both cases the release of the viral genome requires fusion of the viral envelope with the respective target membrane of the host cell, the endosomal or the plasma membrane. membrane fusion is mediated by a conformational change of viral proteins. for viruses utilizing the non-endosomal pathway (marsh and helenius, 2006) , like retroviruses (hiv-1) and paramyxoviruses (e.g. sendai virus) the conformational change of the viral glycoproteins is induced by interaction with the receptor of the host cell surface, thereby triggering fusion between the viral envelope and the plasma membrane. in the case of viruses which are taken up by cells via the endosomal pathway (marsh and helenius, 2006) , acidification of late endosomal lumen by the proton pump activity of v-type atpases (perez and carrasco, 1994) is essential for activating the fusion competence of viral glycoproteins. the low ph triggers the conformational change either directly as for example for the glycoprotein hemagglutinin of influenza virus (orthomyxoviruses) or indirectly by activation of endosomal proteases cleaving the ectodomain of the viral glycoprotein to enable the structural transition into a fusion competent state. the latter mechanism has been described for the fusion mediating s-protein of sars virus (coronaviruses) (simmons et al., virology 377 (2008) . recently, it has been shown for avian sarcoma/leucosis virus (aslv) that the conformational change of fusion mediating protein requires both receptor priming and low ph (mothes et al., 2000) . in this study we have investigated the entry pathway of eav using bhk cells as a target. the influence of substances interfering either with the endosomal pathways or with the acidification of late endosomal compartments, such as lysosomotropic agents and inhibitors of the vacuolar h + -atpase (v-type atpases) (perez and carrasco, 1994) , was studied by the plaque assay. to assess more specifically the role of clathrin-dependent endocytosis in eav infection we utilized a bhk-21 cell line with inducible expression of clathrin heavy chain (chc) antisense rna (iversen et al., 2001) . activation of the antisense rna causes a block of the clathrin-mediated endocytosis. our data provide evidence that the clathrin-dependent endocytic pathway is the major route of eav cell entry and that infection by eav requires a low ph trigger. to investigate whether eav cell entry proceeds via endocytic uptake we first studied the involvement of clathrin-coated pits in eav internalization using a bhk cell line that can be induced to express antisense rna of the clathrin heavy chain (chc) causing a selective block in clathrin-dependent endocytosis (iversen et al., 2001) . for expression of antisense chc (anti-chc) cells (tc−) were incubated in the absence of tetracycline (see materials and methods). to demonstrate the repression of chc-expression clathrin-dependent endocytosis of fluorescent transferrin was measured. while efficient transferrin uptake was observed for chc expressing cells (tc+), internalisation of transferrin was strongly inhibited for tc− cells (fig. 1a) . the results confirm previous studies that in tc− cells cathrin-dependent endocytosis is abolished (iversen et al., 2001) . bhkasc tc+ and tc− cells were infected with eav. infection was assessed by immunofluorescence detection of the nucleocapsid protein n (see materials and methods). for control cells (tc+), n was detected after 2h p.i. and much stronger after 24h p.i. (fig. 1b) . similar observations were made for bhk-21 cells (not shown). we surmise that n after 2h p.i. originated from uncoated, disassembled viruses in infected cells rather than from expression of new proteins, while n detected after 24h p.i. corresponded to newly expressed proteins. for cells expressing anti-chc (tc−) n protein was observed neither 2h p.i nor 24h p.i. (fig. 1b) showing that inhibition of clathrin-dependent endocytosis prevents cell infection. to confirm the result we performed an infectivity assay. the bhkasc cells were preincubated in the presence (tc+) and absence (tc−) of tetracycline. in the latter case antisense rna against clathrin heavy chain is expressed suppressing clathrin-dependent endocytosis. a. alexa fluor 633 transferrin uptake. chc expressing cells (tc+) display fluorescent transferrin (tf) with high intensities in intracellular vesicles, whereas cells (tc−) with suppressed chc expression show no or less intense intracellular internalisation of transferrin. uptake was measured one min after addition of transferrin (37°c). b.infection of bhkasc cells by eav. at 2h p.i. and 24h p.i. cells were fixed and examined, stained with a primary antibody against nucleocapsid protein and tritc labelled secondary antibodies, and examined by confocal microscopy. fluorescence images were recorded at identical gain settings. pcphase contrast microscopy. dicdifferential interference microscopy. supernatant of cell monolayers incubated for 20-22h after virus adsorption was harvested and probed for infectivity by the plaque assay (see materials and methods). while we observed strong infectivity of the supernatant from tc+ cells, we could not find any plaque formation when supernatants from tc− cells were used (data not shown). next we studied the influence of chlorpromazine known to abolish the formation of clathrin-coated endocytic vesicles by interfering with the interaction between the adapter protein ap-2 and the clathrincoated pit lattice (wang, rothberg, and anderson, 1993) . chlorpromazine as well as all other drugs used (see below) were present shortly before and during virus adsorption, but not during the subsequent plaque assay. as shown in fig. 2 a strong inhibition of infection of bhk-21 cells was observed upon treatment of cells with chlorpromazine. in agreement with this observation, we could detect the nucleocapsid protein n neither at 2h p.i nor at 24h p.i of chlorpromazine treated bhk-21 cells (60 µm chlorpromazine) by immunofluorescence (images not shown). the absence of any detectable intracellular n protein after 2h p.i. in chlorpromazine treated cells supports strongly that the early phase of virus infection is inhibited, and that it is unlikely, that suppression of virus production is due to inhibition of transport of viral proteins or assembly and budding of virions. in contrast to eav, chlorpromazine treatment of bhk-21 cells did not reduce infection by sv5 (fig. 2 ) belonging to parainfluenza viruses which are known to infect cells via a non-endocytic mechanism (lamb and kolakofsky, 2001) . this result confirms that treatment with chlorpromazine does not interfere with other cellular processes at least those which are important for virus maturation. taken together these results strongly argue for a clathrin-dependent endocytic uptake as the main cell entry route for eav. based on the observation that eav enters via endocytosis the host cell we asked whether eav infection requires the acidic environment of the endosomal compartment. to this end, we incubated cells with substances preventing endosomal acidification. we have used lysosomotropic agents as (i) ammonium chloride, a relatively weak base accumulating inside endosomal vesicles (brindley and maury, 2005; jin et al., 2005; ohkuma and poole, 1978) , (ii) bafilomycin a1 and concanamycin a, specific inhibitors of the vacuolar h + -atpase (v-atpase) in animal and other eukaryotic cells belonging to macrolide lactone ring antibiotics (drose and altendorf, 1997) , and (iii) monensin, an ionophore that disrupts the proton gradient across vesicular membranes (perez and carrasco, 1994) . preincubation of bhk-21 cells with ammonium chloride revealed an inhibitory effect on virus reproduction (fig. 3a) . incubation of cells with the vacuolar h + -atpase inhibitors bafilomycin a1 and concanamycin a caused also a suppression of eav infection (figs. 3b, c). even at 2nm of concanamycin a infection was completely abolished. a similar extent of infection suppression was observed for bafilomycin a1 at 20nm. it is known that concanamycin a has a higher inhibitory activity in comparison to bafilomycin a1 (drose and altendorf, 1997) . finally, eav infection was abolished in the presence of monensin (figs. 3d). at 100nm plaque reduction was diminished by about 50% with respect to the control. in agreement with this observation, we could detect the nucleocapsid protein n neither at 2h p.i nor at 24h p.i of monensin treated bhk-21 cells (400nm monensin) by immunofluorescence (images not shown). hence, all lysosomotropic agents used, showed a strong inhibitory effect on virus infection. in contrast to eav, neither concanamycin a nor bafilomycin a1 nor monensin treatment of bhk-21 cells did affect sv5 mediated plaque formation (fig. 3 ) consistent with the fact that infection by parainfluenza viruses does not require a low ph compartment (lamb and kolakofsky, 2001) . in conclusion, our results strongly argue that eav after clathrindependent endocytic uptake has to be exposed to the low ph environment of endosomes as presuppositions to release the viral genome into the cytoplasm. the low ph compartment might be required to expose eav to acid-dependent proteases, i.e. to cathepsins. it has recently been reported that cathepsins cleave the s-protein of sars (simmons et al., 2005) and gp1 of filoviruses (chandran et al., 2005; brindley et al., 2007) and that this cleavage is required to facilitate the acid-dependent conformational change required to activate the fusion capacity of those proteins. to explore whether proteases of the late acidic compartments may play a role in fusion activation of eav, we measured infection of bhk-21 cells upon incubation with leupeptin and e64d, which have been shown to inhibit acidic proteases in the endosomal compartment. leupeptin is a competitive inhibitor of serine and cysteine proteases, while e64d is an irreversible cysteine protease inhibitor. we did not observe any influence on eav infection up to concentrations of 100 µm leupeptin and 10 µm e64d appropriate concentrations formerly used for inhibition of those proteases (qiu et al., 2006; simmons et al., 2005) (data not shown). however, before conclusive results on this issue can be made further studies are warranted to demonstrate that inhibitors indeed are efficient to suppress endosomal proteases. since endosomal protease activity does not seem to play a role in infection by eav, it is more plausible that the low ph directly induces a conformational change of the (still unknown) glycoproteins of eav mediating membrane fusion. this is supported by our observation of a strong inhibition of infection of bhk cells when viruses were preincubated at ph b 6.0 (fig. 4) . the irreversible loss of infectivity upon low ph treatment resembles the behaviour of other enveloped viruses which requires an acidic ph for infection of host cells such as influenza viruses (earp et al., 2005) . the fusion mediating glycoprotein hemagglutinin of the influenza virus a undergoes a conformational change at acidic ph and elevated temperatures (korte et al., 1999; puri et al., 1990) . this conformational change is irreversible and abolishes the fusion activity and, thus, the infectivity of influenza viruses. infection of cells by sv5 was not affected by low ph preincubation of viruses (fig. 4) . it is known that the conformational change of the fusion mediating f protein of parainfluenza viruses is triggered by interaction with cellular receptors but not by acidic ph (lamb and kolakofsky, 2001) . to confirm the important role of acidic ph for infection of cells, we have performed 'low ph-bypass' experiments. those experiments show whether cells treated with inhibitors of virus entry can be infected by triggering fusion of viruses with the plasma membrane at low ph. for this purpose, cells were pretreated as described above either with chlorpromazine (60 µm), concanamycin a (4nm), or monensin (400nm) known to inhibit infection of bhk-21 cells (see figs. 2 and 3) . during virus adsorption, virus-cell complexes were incubated for 15 min at ph 5.0, 37°c, and subsequently re-neutralised (see materials and methods). while without low ph incubation infection was almost completely abolished (fig. 5) as expected (see above) we observed a very strong infection when virus-cell complexes were exposed to low ph. these experiments support that low ph triggers fusion of eav viruses. remarkably, infection was four to six folds higher in comparison to infection of cells not treated with inhibitors (control, without low ph incubation). obviously, the low ph incubation was much more efficient in activating the infection fig. 4 . influence of preincubation at acidic ph. eav (filled circles) or sv5 (open circles) were incubated at the indicated ph for 1h at 37°c. after ph neutralization of virus suspension, viruses were adsorbed to bhk cells and the plaque assay was performed (see materials and methods). data present mean ± standard error of estimate of three independent experiments. fig. 5 . low ph-bypass of inhibition of virus infection. bhk cells were pretreated as described above either with chlorpromazine (60 µm), concanamycin a (4nm), monensin (400nm), or mβcd. after the first 60 min of virus adsorption at 37°c, the ph was lowered to 5.0 for 15 min. after neutralization, virus-cell complexes were incubated for further 45 min and the plaque assay was performed as described in materials and methods. controlmock pretreatment, no low ph exposure. w/owithout low ph exposure. wwith low ph exposure. data present mean ± standard error of estimate of three independent experiments. potential of viruses in comparison to the natural entry pathway of viruses. infection of bhk-21 cells by sv5 was neither enhanced nor reduced by low ph exposure (data not shown) indicating also that cells were not affected by a temporary acidic environment. recent studies provide evidence that cell entry of enveloped viruses, for example murine coronavirus (choi et al., 2005; eifart et al., 2007) , could be associated with lipid domains enriched of cholesterol, so called rafts. to assess the role of cholesterol in eav entry, bhk-21 cells were depleted of cholesterol by preincubation with methyl-betacyclodextrin (mβcd, see materials and methods). at concentrations n5 mm mβcd eav infection was completely blocked (fig. 6a) . furthermore, treatment of cells with filipin iii which is known to form complexes with cholesterol in membranes abolished eav infection (fig. 6b) . as a control bhk-21 cells were infected with vsv, which enters cells independently of cholesterol and/or rafts (guyader et al., 2002) . indeed, cholesterol depletion of the bhk-21 cells by mβcd (fig. 6a) or treatment of cells with filipin iii (fig. 6b ) did not diminish infection by vsv. notably, we found a slightly enhanced infectivity of vsv upon filipin iii treatment. these findings strongly indicate an important role of cholesterol for eav infection. since the inhibitory effect of cholesterol depletion could be overcome by low ph-bypass (fig. 5) we conclude that cholesterol depletion inhibits virus uptake but not binding of viruses to cells. cholesterol is strongly engaged in the clathrin(rodal et al., 1999; subtil et al., 1999) as well as in the caveolae-dependent endocytic pathway (yumoto et al., 2006) . because inhibition of expression of the clathrin-heavy chain completely blocked infection (see above) the caveolae-dependent endocytic entry plays no role in eav uptake. hence, cholesterol depletion may interfere with the clathrin-dependent entry of eav. in summary, eav enters host cells via clathrin-dependent endcoytosis. after uptake endocytic vesicles are delivered to a late acidic endosomal compartment. low ph is essential for infection of the cell. since we have no indication that proteases of this late compartment are essential for infection, we surmise that low ph is required to trigger a conformational change of the structural virus protein mediating fusion. alternatively, it might be that eav in principle can fuse at neutral ph, but its cellular receptor is rapidly endocytosed and subsequent acidification in the endosome is required to release virus particles from the receptor. although the structural proteins of the eav envelope have been identified, it is not yet known which of the protein(s) is (are) responsible for mediating fusion. the gp2b/gp3/ gp4 complex is currently a prime candidate for the membrane-fusion activity of eav (wieringa et al., 2003 (wieringa et al., , 2004 . one may wonder whether the clathrin-dependent endocytosis is typical for infection of cells by arteriviruses. as shown recently mouse hepatitis viruses (mhv) belonging to coronavirus family may use different routes for cell entry. while mhv-a59 is taken up via clathrindependent endocytosis (eifart et al., 2007) , non-endocytic internalisation has been described for mhv-4 (jhm) and mhv-s4 ( (nash and buchmeier, 1997; kooi etal., 1991) . the entry pathway of eav shares similarities with that of another arteriviruses, the porcine reproductive and respiratory syndrome virus (prrsv). it has been shown that prrsv enters cells via receptor-mediated and low ph-dependent endocytic pathway through a ph-dependent mechanism like we have described here for eav (kreutz and ackermann, 1996; nauwynck et al., 1999) . kreutz and ackermann (1996) found that infection of a monkey kidney cell line by prrsv was strongly inhibited in the presence of 20 µm cytochalasin b pointing to an involvement of active transport of prrsv containing endocytic vesicles by microfilaments. lysosomotropic agents such as ammonium chloride and bafilomycin a1 suppressed prrsv infection of those cells at concentrations similar to that used in our study. taken together, these results suggest that viruses belonging to the family arteriviridae enter cells via a similar endocytic route. cell lines were propagated as adherent monolayer cultures. baby hamster kidney (bhk-21 c13) (american type culture collection) cells were grown in dulbecco's modified eagle medium (dmem) (cambrex, verviers, belgium) mixed one to one with leibovitz l-15 medium (cambrex, verviers, belgium) supplemented with 5% heat-inactivated fetal bovine serum (δfbs) (hyclone, logan, ut) called grow media (gm). these cells were maintained at 37°c in a humidified incubator at 5% co 2 . eav strain bucyrus (doll et al., 1957) were grown in roller cell culture flask in roller incubator at 5% co 2 , 37°c. the adherent, confluent bhk-21 monolayers were incubated with virus multiplicity (moi) of 1 in serumfree grow media at 37°c and 5% co 2 for 3h (0.25rpm). after adsorption, the inocula were washed and replaced with gm. the cell supernatant was harvested at 20-22h post infection (titer was determined by the plaque assay). after differential centrifugation to remove cellular debris (sequential spins of 15 min at 4°c at 4000×g, followed by 2 h at 4°c at 13000×g) the virus preparations were either used immediately or stored at −80°c in phosphate buffered saline (pbs, cambrex, verviers, belgium) . no influence of storage on virus infection was observed. the bhk-21-tta clathrin heavy chain/antisense (bhkasc) cell line, kindly provided by k. sandvig (institute for cancer research at the norwegian radium hospital, montebello), was grown in complete dmem (cambrex, verviers, belgium) supplemented with 10% heatinactivated fetal bovine serum (δfbs) (hyclone, logan, ut), 2 mm l-glutamine (cambrex, verviers, belgium), and 20µg/ml tetracycline in a cell culture flask at 5% co 2 , 37°c. for induction of chc antisense rna expression, tetracycline was removed from the medium. the viral titer was determined by the plaque assay using bhk-21 cells as indicator cells in emem (cambrex, verviers, belgium) with 1% δfbs, 1% l-glutamine (sigma, berlin, germany) and 0.9% v/v seaplaque agarose (cambrex, verviers, belgium). bhk-21 cells were plated on 6-well plates and grown in gm (2ml/well). the confluent bhk-21 monolayer was washed with pbs. sequential 10fold dilutions of virus were prepared and cells were inoculated with a volume of 1ml/well to determine the plaque forming units (pfu) which are plotted as the amount of plaques in%. a decrease of pfu by 10% corresponds to a reduction of plaques by one order of magnitude. after adsorption for 2h at 37°c and 5% co 2 the cells were washed again to remove also any lysosomotropic substance or inhibitor (see below). subsequently, the overlay, emem (cambrex, verviers, belgium) with 1% δfbs, 1% l-glutamine (sigma, hannover, germany) and 0.9% v/v seaplaque agarose (cambrex, verviers, belgium) was added. after 2days of incubation at 37°c and 5% co 2 the cells were stained with neutral red (biochrom, berlin, germany), fixed with 10% formalin (roth, karlsruhe, germany), and viral plaques were counted. for control experiments simian parainfluenza virus 5 (sv5) and vesicular stomatitis virus (vsv) (strain indiana) was used. in some cases, viruses (eav or sv5) were pretreated for 1 h at 37°c in serum-free growth medium adjusted to various acidic ph-values with 0.25 m citric acid. after neutralization of virus suspension, viruses were adsorbed to bhk cells and a plaque assay was performed. bafilomycin a1, chlorpromazine, concanamycin a, e64d, filipin iii, leupeptin, methyl-beta-cyclodextrin (mβcd), monensin were obtained from sigma (hannover, germany), and ammonium chloride from roth (karlsruhe, germany). substances were used within the following concentration range; bafilomycin a1 ranged from 5 to 40 nm, chlorpromazine from 5 to 100 µm, concanamycin a from 2 to 4 nm, e64d from 0.1 to 10 µm, filipin iii from 1 to 10 µm, leupeptin from 1 to 100 µm, methyl-beta-cyclodextrin from 1 to 10 mm, monensin from 100 to 600nm, and ammonium chloride from 5 to 30 mm. before addition of virus and performing the plaque assay, cells were treated with the substances for 30 min in gm without δfbs. subsequently, cells were infected in the presence of different drug concentrations and incubated for 2 h (see above). subsequently, lysosomotropic substances and inhibitors were washed out. only for mβcd and filipin iii cells were washed with pbs (with physiological conentrations of mg 2+ and ca 2+ ) before infection. to bypass any inhibitory effect of substances, virus was bound to bhk cells pretreated with substances for 2 h at 37°c and 5% co 2 as described above. after the first 60 min of virus adsorption, the ph was lowered to 5.0 for 15 min. after neutralization, virus-cell complexes were incubated for further 45 min and processed as described above. to account for strong infectivity and, hence, high amounts of plaques sequential 10-fold dilutions of virus were performed. the adherent, confluent bhkasc cell monolayers (tc− or tc+) were incubated with virus multiplicity (moi) of 1 in serum-free growth media at 37°c and 5% co 2 for 2h. after adsorption, the inocula were washed and replaced with growth medium. the cell supernatant was harvested at 20-22 h post infection and the titer was determined by plaque assay using bhk-21 cells. to induce the expression of antisense chc rna (anti-chc) in bhkasc cells, the cells were incubated for 18 h in the absence of tetracycline (tc−), for control cells tetracycline was present (tc+). the adherent, confluent bhkasc cell monolayers were incubated with virus multiplicity (moi) of approximately 10 in serum-free grow media at 37°c and 5% co 2 for 2h. after adsorption, the inocula were washed twice with pbs. cells were fixed at different time points post infection by pbs containing 3% formaldehyde, subsequently quenched with 100 mm glycin, permeabilized with 0.5% triton x-100, and then incubated with the primary mouse monoclonal antibody against eav nucleocapsid protein (vmrd inc., pullman usa) (deregt et al., 1994) for 30 min at 37°c. subsequently, cells were incubated with the appropriate tritc labelled secondary antibody against mouse igg developed in goat (sigma, hannover, germany). coverslips were obtained from marienfeld (germany). confocal fluorescence microscopy was done with an olympus fv-1000. bhkasc cells with and without removal of tetracycline from culture medium were seeded into 8-well µ-slide observation chambers (ibidi, martinsried, germany) and incubated for 24h at 37°c at 5% co 2 . prior to the experiment the culture medium was gently removed from the cells and exchanged against transferrin conjugated to alexa fluor 633 (invitrogen, paisley, uk) (10µg/ml final concentration) diluted medium (gm). immediately after adding of the marker dilution to the cells were incubated for 1h at 37°c and images were taken. confocal and phase contrast images were acquired with a zeiss laser scanning microscope lsm510 meta mounted on an axiovert 200 m inverted microscope using a 63× phase contrast oil immersion plan-apochromat objective na1.4. visualization of transferrin uptake was performed under identical conditions for the tc+ and tc− cells. endocytosis and a low-ph step are required for productive entry of equine infectious anemia virus ebola virus glycoprotein 1: identification of residues important for binding and postbinding events endosomal proteolysis of the ebola virus glycoprotein is necessary for infection murine coronavirus requires lipid rafts for virus entry and cell-cell fusion but not for virus release structural proteins of equine arteritis virus monoclonal antibodies to equine arteritis virus proteins identify the gl protein as a target for virus neutralization isolation of a filterable agent causing arteritis of horses and abortion by mares; its differentiation from the equine abortion, (influenza) virus bafilomycins and concanamycins as inhibitors of v-atpases and p-atpases the many mechanisms of viral membrane fusion proteins the role of endocytosis and low ph in cell entry of the murine hepatitis virus mhv-a59 role for human immunodeficiency virus type 1 membrane cholesterol in viral internalization endosome to golgi transport of ricin is independent of clathrin and of the rab9-and rab11-gtpases receptor-mediated entry by equine infectious anemia virus utilizes a ph-dependent endocytic pathway differentiation of acid-ph dependent andnondependent entry pathways for mouse hepatitis virus conformational intermediates and fusion activity of influenza virus hemagglutinin porcine reproductive and respiratory syndrome virus enters cells through a low ph-dependent endocytic pathway paramyxoviridae: the viruses and their replication, fields virology virus entry: open sesame retroviral entry mediated by receptor priming and low ph triggering of an envelope glycoprotein entry of mouse hepatitis virus into cells by endosomal and nonendosomal pathways entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages via receptor-mediated endocytosis fluorescence probe measurement of the intralysosomal ph in living cells and the perturbation of ph by various agents involvement of the vacuolar h(+)-atpase in animal virus entry conformational changes and fusion activity of influenza virus hemagglutinin of the h2 and h3 subtypes: effects of acid pretreatment endosomal proteolysis by cathepsins is necessary for murine coronavirus mouse hepatitis virus type 2 spike-mediated entry extraction of cholesterol with methyl-beta-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry the molecular biology of arteriviruses acute cholesterol depletion inhibits clathrin-coated pit budding an infectious arterivirus cdna clone: identification of a replicase point mutation that abolishes discontinuous mrna transcription ultrastructure and immunocytochemistry of bhk-21 cells infected with a modified bucyrus strain of equine arteritis virus mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation intra-and intermolecular disulfide bonds of the gp2b glycoprotein of equine arteritis virus: relevance for virus assembly and infectivity structural protein requirements in equine arteritis virus assembl clathrin-mediated endocytosis of fitc-albumin in alveolar type ii epithelial cell line rle-6tn we are indebted to peter j.m. rottier (university utrecht), kirsten sandvig (institute for cancer research at the norwegian radium hospital, montebello) for providing eav and bhkasc cells, respectively. we thank alexander kabatek (free university berlin) for helpful comments. this work was financially supported by grants to ah and mv from the dfg, germany. key: cord-262347-ejhz9rra authors: kappes, matthew a.; faaberg, kay s. title: prrsv structure, replication and recombination: origin of phenotype and genotype diversity date: 2015-03-07 journal: virology doi: 10.1016/j.virol.2015.02.012 sha: doc_id: 262347 cord_uid: ejhz9rra porcine reproductive and respiratory disease virus (prrsv) has the intrinsic ability to adapt and evolve. after 25 years of study, this persistent pathogen has continued to frustrate efforts to eliminate infection of herds through vaccination or other elimination strategies. the purpose of this review is to summarize the research on the virion structure, replication and recombination properties of prrsv that have led to the extraordinary phenotype and genotype diversity that exists worldwide. porcine reproductive and respiratory syndrome virus (prrsv) is the etiological agent of a worldwide epidemic designated porcine reproductive and respiratory syndrome (prrs). prrsv is highly host and tissue restricted to swine cells of the monocyte lineage, preferentially infecting the porcine alveolar macrophage (amφ) and is a persistent virus (duan et al., 1997; villarreal et al., 2000) . prrsv first emerged in the late 1980s as a "mystery" disease progressing through swine populations in both europe and north america collins et al., 1992; hopper et al., 1992; morin et al., 1991; wensvoort et al., 1991) . prevailing clinical symptoms were noted to be respiratory distress in young swine and widespread reproductive failure in pregnant sows including mummified, stillborn and aborted fetuses (goyal, 1993) . initial characterization of circulating european (type 1) and north american (type 2) genotype isolates was found to be surprisingly genetically divergent. although overall disease phenotype, gross clinical symptoms, genomic organization and temporal emergence were all similar, these strains differed by $ 40% at the nucleotide level collins et al., 1992; meulenberg et al., 1994; nelsen et al., 1999; wensvoort et al., 1991) . the degree of genetic heterogeneity suggests a protracted period of independent evolution on the two continents . molecular clock analysis predicts the divergence of the two genotypes from a common ancestor between a decade to over a century prior to clinical recognition (forsberg et al., 2001; yoon et al., 2013) , presumably from another host species (hanada et al., 2005; plagemann, 2003) . the origin of prrsv remains unknown and no secondary animal, human, or arthropod vectors have been identified to date (otake et al., 2003a (otake et al., , 2003b schurrer et al., 2005 schurrer et al., , 2004 zimmerman et al., 1997) . in the $ 25 years since the first emergence of prrsv, a global near worldwide epidemic has been sustained by a set of emerging and re-emerging strains supported by high frequency mutation and recombination (goldberg et al., 2003; meng, 2012; murtaugh et al., 2010) . prrsv remains the most economically devastating disease of swine and contributes to the deterioration of animal health through disease and the continual emergence of increasingly divergent and often virulent strains (gauger et al., 2012; han et al., 2006; holtkamp, 2011; tian et al., 2007) . other reviews have recently assessed the nidovirus family in broad terms, concentrating on what is similar between the family members and choosing to concentrate on cellular pathogenesis, but often leaving critical prrsv-specific details out of their discussions. therefore, the intent of this review is to examine what is known about prrsv virion structure and replication mechanisms that contribute to the highfrequency mutation and recombination observed, resulting in a vast array of phenotypically and genotypically divergrent strains. prrsv is a member of the arteriviridae family within the order nidovirales, which also includes the coronaviridae and roniviridae families. the nidovirus order constitutes a group of singlestranded positive-sense rna viruses which share a hallmark replication/transcription strategy, similar genomic organization, and a defining set of genetic elements, but differ in host species and range, disease phenotype, virion morphology, cellular tropism, genomic size and encoded content . the arteriviridae family is composed of five viruses which share similar genetic and biological characteristics such as genomic organization and content, morphology and a cellular tropism for the macrophage lineage . viral species include prrsv, simian hemorrhagic fever virus (shfv), lactate-dehydrogenase elevating virus (ldv), newly recognized wobbly possum disease virus (wpdv) in free-ranging australian brushtail possums (trichosurus vulpecula) and equine arteritis virus (eav; arterivirus prototype species) dunowska et al., 2012; plagemann and moennig, 1992) . the similar genomic organization, characteristic genetic elements and common functionality of orthologous proteins however have led to the acceptance of many putative functions for prrsv proteins, often derived from studies of the arterivirus prototype species eav and the more distantly related nidoviruses. each arterivirus infects only one animal species, in contrast to other nidoviruses such as some coronaviruses, which have been shown to transmit between species (hilgenfeld and peiris, 2013) . the prrsv genome varies from 14.9 kb to 15.5 kb in length and expresses a range of accessory and structural proteins through two distinct transcription mechanisms. the genomic organization and associated expression profiles are depicted in fig. 1 . the prrsv genome encodes a 5 0 proximal noncoding element (5 0 -untranslated region; 5 0 utr) of 217-222 nucleotides (nt; type 1) and 188-191 nt (type 2) in length yun and lee, 2013) . the 5 0 utr functions will be discussed below. directly downstream of the 5 0 utr has the large overlapping replicase open reading frames (orf). the orf1a/b share a single translational start site but are augmented by two ribosomal frameshift (rfs) sites at genomic positions 3889 nt (rfs1; nonstructural protein (nsp) 2) and 7695 nt (rfs2; nsp8/9) [vr-2332 (u87392) reference sequence] meulenberg et al., 1993b; nelsen et al., 1999; li et al., 2015) . two products are generated from the rfs1 site; a -1 rfs occurs approximately 7% of the time and results in an immediate termination of translation (nsp2n) , and a -2 rfs event occurs at $ 20% efficiency and yields a translational extinction in the -2 coding frame through the putative transmembrane domain of nonstructural protein (nsp2) . the large replicase polyproteins pp1a, pp1a-nsp2n, pp1a-nsp2tf, and pp1ab are generated from full-length genomic rna. using the type 2 prototype strain vr-2332 (u87392) for a reference, orf1a/b is encoded by a 5 0 proximal segment of approximately 12 kb [7512 nt orf1a, 4374 nt orf1b] yielding four distinct polyproteins including pp1a-nsp2n (1234 amino acids (aa); -1 rfs at rfs1), pp1a-nsp2tf (1,403aa; -2 rfs at rfs1), pp1a (2,503aa), and pp1ab (3,960aa; -1 rfs at rfs2) (fig. 1) . the replicase polyproteins are co-translationally and posttranslationally processed into at least 16 distinct nonstructural proteins (nsp) via the rfss and four virally encoded proteinases including papain-like cysteine proteinases 1α (plp1α; nsp1α), plp1β (nsp1β) and plp2 (nsp2), and the main serine proteinase (sp; nsp4) snijder et al., 2013) . plp1α and plpβ function to cleave the nsp1α ↓ nsp1β and the nsp1β ↓ nsp2 junction, respectively; plp2 is responsible for the cleavage of the nsp2 ↓ nsp3 junction and the main sp processes all remaining nsp products (nsp3-12) (han et al., 2009; snijder et al., 2013) . orf1a encodes pp1a encompassing nsps 1-8 and orf1ab encodes pp1ab composed of all known nsps (nsp1α/β, nsp2-6, nsp7α/β, nsp8-12) whereas nsp9 is a translational extension of nsp8 via a programmed -1 rfs at position vr-2332 7695 nt (rfs2) ( fig. 1 ) (meulenberg et al., 1993b; nelsen et al., 1999) . in contrast, the previously recognized structural proteins are encoded and individually expressed by a set of subgenomic rnas (sgrna) generated through a negative-strand intermediate (sgrna2-7; fig. 1 ) (van marle et al., 1999a) . sgrnas are genetically polycistronic (except rna7) but are assumed to be functionally monocistronic/bicistronic, where only the 5 0 terminal orf(s) is expressed (fig. 1) . sgrna 2 encodes orf2a/b which is translated to yield glycoprotein 2 (gp2) and a small unglycosylated envelope protein (e); orf3 is expressed from sgrna3 to yield gp3; and sgrna4 encodes orf4 yielding gp4. together gp2, gp3, and gp4 form a trimeric complex resulting in the minor glycoprotein complex which functions in viral entry and is heavily nglycosylated (das et al., 2010; wissink et al., 2005) . sgrna5 encodes orf5 and orf5a. orf5a codes for the orf5a protein, a small unglycosylated protein that is required for virus viability and orf5 codes for gp5, the major glycoprotein with a variable number of n-glycan residues surrounding the cell attachment domain (johnson et al., 2011; mardassi et al., 1996; robinson et al., 2013) . orf6 is expressed from sgrna6, resulting in the generation of the membrane protein (m). gp5 and m form a disulfide-linked heterodimer and together constitute the major glycoprotein complex on the virion, as was first shown for ldv (faaberg et al., 1995; mardassi et al., 1996) . finally, the nucleocapsid protein (n) is encoded by orf7 and is expressed from sgrna7. n is the major structural element within the prrsv virion which forms disulfide-linked homodimers, functions to package the viral genomic rna (grna), and is the only known structural protein which does not encode a transmembrane domain or to not have an ectodomain upon the prrsv virion (bautista et al., 1996; dea et al., 2000; doan and dokland, 2003; loemba et al., 1996; spilman et al., 2009; wissink et al., 2005; wootton and yoo, 2003) . recently, the nsp2 protein, coded for by the most variable region of the genome with insertions and deletions, was also shown to be incorporated into or onto ultrapurified virions of several prrsv strains as a set of differently sized protein isomers, presumably through its four to five membrane spanning regions near the c-terminal end (kappes et al., 2015) (han et al., 2010; kappes et al., 2013) . this surprising result increases the number of viral proteins, 10 or more (full-length nsp2 and its isomers, nsp2tf, gps 2-5, e, m, n, orf5a), that are exposed to the porcine immune system on entry of prrsv into swine alveolar macrophages veit et al., 2014) . the original work suggests that only 3 of these (gp5, m, and n) make up the majority of the protein content of prrsv (drew et al., 1995; mardassi et al., 1994a mardassi et al., , 1995 meulenberg et al., 1995; nelson et al., 1994) . however, the immense genetic and protein variation of all of these structural proteins, from the least conserved nsp2 region (han et al., 2006; tian et al., 2007) to the most conserved m protein (murtaugh et al., 1995; veit et al., 2014) , shows the complexity and the plasticity of the prrsv genome and virion structure. the 5 0 and 3 0 utrs flank the core protein coding regions of the prrsv genome (fig. 1 ). both the 5 0 and 3 0 utrs are implicated as essential components contributing to the viral strategies imparting replicative and translational functionality; however, the exact functions of the 5 0 and 3 0 utrs, and the associated mechanisms of interaction, are poorly understood. both encode conserved putative rna secondary structures important to replicative function. the 5 0 utr is encoded first within the prrsv genome and possesses a putative type i 5 0 cap structure (sagripanti et al., 1986) . the 5 0 utr is genetically variable, type 1 and 2 strains share approximately 50% genetic homology, and within each genotype, the pairwise identity is about 96% (lin et al., 2002; meulenberg et al., 1993a; nelsen et al., 1999; oleksiewicz et al., 1999; tan et al., 2001) . detailed studies of the distantly related coronavirus species, as well as arteriviruses, have shown the 5 0 utrs are regulators of genomic replication, transcription, and mrna translation, and are considered a necessary docking site for a variety of viral and host factors to complete these functions (choi et al., 2006; gao et al., 2012; liao and lai, 1994; lu et al., 2011; tahara et al., 1994; zhang et al., 1994) . the 3 0 utr is located directly downstream of orf7 and is encoded by approximately 150 nt excluding the polyadenylation site. the 3 0 utr [114 nt (type 1), 148 nt (type 2)] is also genetically diverse, sharing approximately 70% nucleotide identity between type 1 and type 2 sequenced isolates but about 96% pairwise nucleotide identity within each genotype choi et al., 2006; verheije et al., 2002; yin et al., 2013) . recent reviews extend upon the brief description presented here wang et al., 2014; yun and lee, 2013) . due to the unique attributes of nidovirus transcription and replication, including uncharacteristically large polycistronic rna genomes and the transcription of a nested set of 5 0 , 3 0 co-terminal sgrnas through a discontinuous transcription strategy, which is in itself a mechanism of recombination, nidoviral rna synthesis mechanisms have been suggested to be of unparalleled complexity among positive strand rna viruses van hemert et al., 2008) . prrsv replication closely ties three key features: rearrangement of host membranes to establish viral replication complexes (rc), synthesis and expression of grna, transcription of sgrna for the efficient expression of structural proteins, at the same time as the unique ability to produce aberrant prrsv sgrnas known as heteroclites (yuan et al., 2000 (yuan et al., , 2004 . genesis of grna (replication) and sgrna is inherently tied through the shared negative strand synthesis mechanism. modulation of negative-sense transcription through a non-stochastic mechanism yields either grna or one of six standard sgrnas (rna2-7) through an abortive disjoining/rejoining discontinuous transcription strategy ( fig. 1) (meng et al., 1996b; nelsen et al., 1999; pasternak et al., 2001; van marle et al., 1999a) . the characterization of the cellular entry mechanism that prrsv utilizes has been studied in detail, and will not be covered in this review (van breedam et al., 2010) . little is known about the establishment of prrsv infection, however, from the point postentry to the development of rc, including the formation of characteristic perinuclear double-membrane vesicles (dmvs) (knoops et al., 2012) . dmvs are believed to be derived from the endoplasmic reticulum (er) which are apparent sites of viral replication (pedersen et al., 1999) . it has been shown that the eav replicase proteins (orf1a/b) are sufficient to support viral replication (molenkamp et al., 2000b) , but that infectivity is dependent on the presence of the structural genes encoded at the 3 0 -end of the arterivirus genome molenkamp et al., 2000b; verheije et al., 2002) . upon entry, the grna serves as the mrna for immediate translation of the large replicase polyproteins. within orf1a, three proteins are recognized putative transmembrane proteins (nsp2, nsp3, and nsp5). the eav nsp2 and nsp3 were shown to be sufficient to modulate host cellular membranes into structures similar to those observed during viral infection . it is believed that membrane integration and possibly protein-protein interactions of these transmembrane proteins function to torque the existing membrane structures to form the dmvs; tethering the genesis and processing of the polyprotein (s) at the site of replication. additional viral or cellular interacting partners are not well defined. the mechanism of dmv formation is unknown but may include the modulation of autophagy and/or apoptosis pathways (breckenridge et al., 2003; chen et al., 2012; costers et al., 2008; cottam et al., 2014; huo et al., 2013; labarque et al., 2003; razi et al., 2009; sun et al., 2012; wang et al., 2014; yin et al., 2012; yu et al., 1999) . the core replicative machinery of prrsvthe rna dependent rna polymerase (rdrp; nsp9), the zinc-binding domain (zbd; or z; nsp10), the rna helicase (nsp10), and the conserved nidovirus uridylate-specific endoribonuclease, (nendou or u; nsp11)is encoded within orf1b (ulferts and ziebuhr, 2011) . the calculated rfs efficiency of the rfs1 (6-7% -1 rfs; 16-20% -2 rfs) and the rfs2 ( $ 20%) (den boon et al., 1991) demonstrates that orf1b (nsp9-12) is generated approximately once out of every six translational events (15%), suggesting the stoichiometric requirements for the core replicative machinery is low compared to the 5 0 encoded replicase proteins. rdrps form a characteristic right hand configuration (thumb, palm, finger(s)) with the thumb and fingers in contact to create a pocket for substrates (ferrer-orta et al., 2006) . comparison of single-and double-stranded rna virus rdrps show structural similarity even though there is low sequence homology between classes (ferrer-orta et al., 2006) . all polymerases share a core set of conserved motifs, suggesting a common ancestor (sabanadzovic et al., 2009) . the structure of the rdrp possesses an additional conserved motif (o'reilly and kao, 1998) . the rdrp of nidoviruses is phylogenetically clustered with the picorna-like virus superfamily koonin, 1991) but possesses a sdd (ser-asp-asp) signature, located within the active site on the palm side of the rdrp. the nidoviral sdd motif is a hallmark of the viral family that discriminates it from all other positive-sense rna virus groups that contain a gdd (gly-asp-asp) motif (den boon et al., 1991) . the sdd motif at this position was shown to be critical for eav replication (van dinten et al., 1999) ; surprisingly a s-g mutation within the prrsv rdrp was replication competent (grna) but displayed deficiencies in sgrna synthesis . another salient fact is that the arteriviral rdrp does not possess the 3 0 proofreading abilities that other nidoviruses display (lauber et al., 2013) . the rate of random mutation introduction is therefore elevated (forsberg, 2005; forsberg et al., 2001 forsberg et al., , 2002 , contributing to an abnormally high evolution rate estimated at between 4.71 â 10 2 and 9.8 â 10 2 /synonymous site/year (hanada et al., 2005) . nsp10 encodes the prrsv helicase protein (bautista et al., 2002) . the prrsv multi-domain helicase (hel) is composed of the core 1a and 2a canonical domains found in super-family 1 type helicases, a flexible accessory domain (1b), and a unique zinc-binding domain (zbd) (deng et al., 2014) . the prrsv hel functions to unwind dsrna in a 5 0 to 3 0 polarity (bautista et al., 2002) . both the flexible accessory domain and the zbd are critical to replicative function of eav including generation of grna and sgrna (van dinten et al., 2000; van marle et al., 1999b) . the helicase is predicted to function in concert with the rdrp to facilitate replication and transcription; however, it is not understood how the 5 0 to 3 0 directionality of the helicase and the 3 0 to 5 0 rdrp synthesis coordinate these activities (fang and snijder, 2010) . nsp11 harbors the nidoviral uridylate-specific endoribonuclease (nendou) domain (ulferts and ziebuhr, 2011) . originally described in coronaviruses, the nendou of eav was shown to be required for genome replication, and mutation of critical residues had varying deleterious effects on replication, and particularly on sgrna synthesis (posthuma et al., 2006) . in examining the core nuclease region of eav and prrsv, single-stranded rna was the preferred substrate, as was shown for the coronavirus sudden acute respiratory virus (sars). however, no dependence on the divalent cation mn 2 þ was seen. sars nsp15, the coronavirus orthologue, was previously shown to mn 2 þ dependent. eav nendou protein was also shown cleave 3 0 of pyrimidines, preferring uridine over cytidine, and releasing products with 2 0 ,3 0 -cyclic phosphate and 5 0 -oh ends. in addition, cleavage after unpaired over paired pyrimidines was preferred (nedialkova et al., 2009; ulferts and ziebuhr, 2011) . the function of nendou in nidovirus replication has not been established. how the prrsv rdrp initiates replication, either by a primer dependent mechanism or by de novo synthesis is also unknown. to assess the activity of the eav rdrp, a recombinant version of the polymerase was generated and assessed. eav rdrp activity was found in the absence of a primer with poly(u) or poly (c) templates but not with poly(a) templates, indicating a de novo initiation method in a template specific manner . introduction of primers to either the poly(u) or poly (c) templates reduced rdrp activity . incubation with non-complementary bases (i.e. for poly (u) template¼gtp and utp) did not result in isotopic labeling, which shows the rdrp did not function as a terminal transferase, and radioactively labeled primers were not incorporated into the synthetic non-viral templates . de novo polymerase activity could not be detected on viral templates however. this finding suggests the arterivirus rdrp is catalytically active without other viral factors and capable of de novo synthesis, but may require other viral or cellular co-factors to efficiently perform replication or transcription processes. similar research on prrsv has not been accomplished to date. it is generally believed that positive-sense rna viruses use conformational switches in their terminal noncoding regions in the form of higher order rna secondary structure to regulate translation, transcription of sgrnas, and genomic replication . to define the minimal cis-acting 3 0 genomic element required for efficient prrsv replication, progressive 3 0 deletions were introduced into self-limiting prrsv replicons encoding an internal ribosome entry site (ires)-driven luciferase (luc) reporter within the deleted region (choi et al., 2006) . only the smallest deletion, encoding the full m and n proteins, replicated to similar levels as the positive control. the next smallest deletion removing the m protein coding region but maintaining the complete n orf resulted in significant loss of genome replication (choi et al., 2006) . taken together, including the rdrp, hel, 5 0 utr, 3 0 utr, orf6, orf7, and other unknown viral proteins, these data show that key genetic elements or protein interacting partners are required for efficient prrsv replication and transcription, and are interspersed within multiple coding regions of the genome. viruses require the ability to selectively regulate transcription and translation processes both temporally and quantitatively in a highly ordered and balanced process (pasternak et al., 2000) . the expression mechanisms between the nonstructural replicase proteins and the structural proteins are fundamentally separated within the prrsv genome facilitating rapid expression of nsps from the grna and subsequent amplification of sgrna transcripts through a differential transcription cascade (pasternak et al., 2004) . sgrnas are synthesized by the viral rdrp through a highly ordered process encoded within the prrsv genome. the set of nested (nido latin: nested) sgrnas encode noncontiguous grna sequence including both the 5 0 utr and the polyadenylated 3 0 utr as well as one or more orfs from the 3 0 region of the genome (orf2-7) but lack the entire large $ 12 kb replicase coding region (orf1a/b) (fig. 1) pasternak et al., 2006; van berlo et al., 1982) . it was originally hypothesized that nidoviral sgrnas could be generated through a free 5 0 utr priming stage (baker and lai, 1990 ; baric et al., 1983 ) but was ultimately shown the 189 bp 5 0 untranslated of strain vr-2332 is shown as a bar encompassing two discrete regions, the 5 0 terminal sequence of 183 bases that differs approximately 5% between strains of the same genotype (gray bar) and the 100% conserved u/guaacc hexanucleotide on the distal end that serves as the transcription regulatory sequence (trs; black bar). the prrsv replicase complex is represented by a multi-point star. (a) production of conanical sgmrna. (step 1) sgrna synthesis initiates as ( à ) strand replication (blue bar) from the full-length ( þ ) sense (green bar) genome. rdrp interaction with a trs either results with a read-through and a continuation of ( à ) strand replication or, in the case of sgrna synthesis, ( step 2) disassociation of the replicating strain (body aauugg; white body) and ( step 3) re-joining at the 5 0 leader trs (leader aauugg; leader-body junction) through sequence complementarity annealing followed by completion of ( à ) strand sgrna synthesis. all sgrnas possess identical 3 0 and 5 0 termini (see fig. 1 ). (step 4) ( à ) sense sgrnas serve as template for generation of ( þ ) sense sgrna synthesis, required for structural protein translation. (b) production of heteroclite sgrna at unconventional leader-body junction sites to express aberrant proteins. that nidoviruses utilize a discontinuous sgrna transcription strategy (sawicki and sawicki, 1995; van marle et al., 1999a) . discontinuous replication proceeds through a replicative fusion of the viral genome 5 0 utr to one of many downstream 3 0 sites through base pairing interactions between sense and antisense stem-loop (sl) structures via long-range rna-rna interactions during negative strand synthesis (van marle et al., 1999a) . specifically, an antisense transcription-regulating sequence (trs) at or near the 5 0 end of each structural protein coding region (orf2-7) can each individually form a kissing-loop interaction with a conserved trs sequence (uuaacc), located at the 3 0 terminus of the 5 0 utr. sgrnas synthesis and grna replication utilize a similar initial synthesis mechanism, whereby negative strand transcriptional extension from the 3 0 termini is completed until a trs signal sequence within the body of the genome is encountered ( fig. 2a ) (den boon et al., 1996; pasternak et al., 2004) . the body trs is ordered into a sl structure encoding a conserved heptanucleotide primary sequence (body trs signal) (den boon et al., 1996) within the loop structure. this signal halts transcription of the negative strand and a "decision" is made between transcriptional readthrough and continuation of synthesis, or a disjoining of the transcriptional machinery and rejoining to the common leader trs (antisense to body-trs) by complementary base-pairing with a second sl structure within 5 0 utr (pasternak et al., 2003; van marle et al., 1999a) . the leader trs is located within the 5 0 utr directly upstream of the aug start codon of nsp1α and is part of a highly ordered and well conserved rna secondary structural motif between the 5 0 utr and the 5 0 nsp1α coding region. transcriptional read-through of all body trs sites will result in grna synthesis. decoupling from the genomic strand at the body trs and rejoining at the leader trs results in noncontiguous transcription that lacks a large central region of the genome, yielding one of at least six sgrna products (dependent on which body trs is utilized) ( fig. 2a) (pasternak et al., 2001) . the structural integrity of the eav nsp1 region, composed of two papain-like protease domains and a predicted n-terminal zinc finger, was also indispensible for transcription, and has been shown to interact with the cellular cofactor p100 (tijms et al., 2007 (tijms et al., , 2001 tijms and snijder, 2003) . again, all research completed on the arterivirus mechanism of subgenomic synthesis was through the use of the model eav strain adapted for growth in tissue culture. similar research on prrsv, with strain differences in replication rates and its immense depth of variation, may yield novel findings in this field. while the mechanism of sgrna generation is conserved, the discontinuous transcription process has been shown to be able to utilize both canonical and non-canonical body trs sites and can have strain specific derivations (den boon et al., 1996; meng et al., 1996b; nelsen et al., 1999) . alternative canonical and noncanonical body trs sites often precede the coding region of prrsv structural proteins, which function to drive sgrna synthesis to various degrees of efficiency, yielding major and minor sgrna species encoding the same structural protein. for instance, the na prrsv prototype strain (vr-2332) utilizes at least two different leader-body junction sites for the generation of sgrna4 and sgrna7 subspecies, and produces a separate species, sgrna5-1, for the expression of a truncated gp5 utilizing a downstream aug . other nuances for individual prrsv strains have been noted (lin et al., 2002; meng et al., 1996b) . examining mutational studies within the eav orf7 body trs (nucleocapsid) to abolish the generation of sgrna7, the most abundantly produced sgrna (van marle et al., 1999a) found that elimination of sgrna7 synthesis resulted in obvious increase in sgrna6, 5, 4, and 2 but production of sgrna3 remained unchanged. additionally, it was noted that a mutation within the leader trs, altering the fifth nucleotide of the conserved sequence (5 0 -ucaag-3 0 ) eliminated synthesis of all sgrnas except sgrna3 ( van marle et al., 1999a) . this effect is due to a unique non-canonical trs semi-independent generation of sgrna3.1, produced by both eav and prrsv (meng et al., 1996b; van marle et al., 1999a) . sgrna3.1 uses a non-trs body sequence of 5 0 -ucaauaccc-3 0 which lacks the 3 0 terminal c residue of the canonical trs sequence but possesses an additional five nucleotides (uaccc) that match the adjoining sequence downstream of the leader trs, allowing for sense/antisense base pairing. (van marle et al., 1999a) . data from single and double knockout mutagenesis studies of alternative (non-canonical) trs body sequences for eav showed that the expression of solely the minor sgrna species from alterative joining sites of the gp3, gp4, and gp5 structural proteins was sufficient for production of infectious progeny virus (pasternak et al., 2000) . it is not clear if the alternative trs body sites serve as a secondary mechanism to rescue deleterious mutations from the error-prone rdrp, or if they serve a dedicated purpose within the viral life cycle. knockout of the canonical eav trs body sequence of orf3, 4, or 5 resulted in infection rates at perceived wild-type levels (defined by ifa cellto-cell spread; 1-3 log pfu/ml reduction in titer) by utilizing secondary trs sequences within these coding regions (pasternak et al., 2000) . double knockout mutants of both the canonical and accessory body trs sequences surprisingly still resulted in generation of progeny virus, but at reduced levels (orf3 ¼2 log reduction, orf4¼ 3 log reduction, orf5¼5 log reduction; pfu/ ml). it is presumed that even if the use of alternative trs sequences results in inefficient sgrna synthesis, the two amplification cycles (genomic rna-( à ) sense sgrna synthesis-(þ ) sense sgrna) may result in sufficient sgrna copy numbers to allow a productive infection cycle to proceed. the authors noted that the reduction in pfu titers of the single or double trs mutants corresponded "very well" to the reduction in molar ratios of each respective rna subspecies (pasternak et al., 2000) . prrsv eu prototype strain lelystad virus (lv) sgrnas possess a conserved six nucleotide junction sequence of ucaacc (or similar sequence), but show heterogeneity at the junction site, suggesting the joining mechanism may be "imprecise" (meulenberg et al., 1993a) . studies on whole rna rt-pcr (cdna) of virally infected cells with na prrsv strains further identified a similar common leader-body junction sequence u(g)ua(g/c)acc (meng et al., 1996b; nelsen et al., 1999; oleksiewicz et al., 1999) . genetic heterogeneity was also noted at these junction sites, differing by a single base to a couple of nucleotides, showing there is slight flexibility within the disjoining and reattachment of the viral rdrp during this step (meng et al., 1996b; nelsen et al., 1999) . furthermore, the body trs motifs and the distance upstream from the starting aug for the expression of sgrnas differ between type 1 and type 2 prrsv, except for the predominant sgrna7 transcript (meulenberg et al., 1993a; nelsen et al., 1999) . on top of this, surveying northern analyses of different prrsv strains shows that each strain has different quantities of each sgrna transcript size, and often differ in their trs motifs (gauger et al., 2012; guo et al., 2013b; wang et al., 2008) . thus, the mechanism(s) regulating prrsv sgrna synthesis when comparing different viral strains appear to be more complex than appears when examining one viral strain in depth. defective interfering (di) rnas are a normally observed byproduct of positive-sense rna virus replication, particularly under high multiplicity of infection (m.o.i.) culturing conditions (masters, 2006; molenkamp et al., 2000a; pattnaik et al., 1992; xiao et al., 2011) . di rnas are generated through nonhomologous recombination between viral genomes resulting in random internal deletions but still encode the replication elements essential for generation of defective progeny virus including the genes encoding for the polymerase, essential replicase proteins, and capsid protein(s) (yuan et al., 2000) . unlike dis that have been described in other nidoviruses, a group of "heteroclite" sgrnas (heteroclite¼deviating from common forms or rules) were identified within prrsv replicative products of unusual structure but containing large internal deletions (yuan et al., 2000) . heteroclite sgrnas species were identified within infected cells, purified virions, porcine alveolar macrophages infected with field isolates, under natural infection conditions, within plaque-purified viral infections, and are assumed to result from homologous recombination at atypical nucleotide stretches (fig. 2b) (yuan et al., 2000 (yuan et al., , 2004 . the essential replicative products such as the viral rdrp were found to be absent within the heteroclite rnas, discriminating them from prototypical di genomes. in addition, heteroclites do not appear to interfere with ongoing genomic rna and sgrna transcription (yuan et al., 2000 (yuan et al., , 2004 . sequencing analysis showed a short site of two to seven conserved nucleotides between the 5 0 and 3 0 joining regions, but these aberrant trs motifs varied in sequence (yuan et al., 2004) . when rt-pcr products of culture supernatants of strain vr-23332 using a 5 0 and 3 0 primer pair were analyzed, at least 9 bands were discriminated (s1-s9). the result of this analysis and subsequent nucleotide sequencing showed that many similar-sized heteroclites were present in each band, but each band represented several individual heteroclites with different trs motifs. the 5 0 region of the heteroclites encoded terminal orf1a proteins including one or more of the papain-like proteases and joined within the downstream coding region either in-frame or within alternative reading frames, perhaps producing aberrant proteins (yuan et al., 2000 (yuan et al., , 2004 . these rna species persist in a range of experimental culturing conditions including low m.o.i. passage and plaque purification (yuan et al., 2000) , and were found to be packaged within the prrsv virion (yuan et al., 2004) . all identified heteroclites included at least the first 476 nt of the type 2 prototype, strain vr-2332, and later studies pinpointed 35 nucleotides in nsp1 that bound to the n protein and therefore was an important element in viral packaging (baig and zakhartchouk, 2011) . additional di rna that possesses many similar genetic features has been identified, but each contains a smaller deletion (nsp2-9) and encodes all structural proteins (xiao et al., 2011) . currently there is no known function for heteroclite sgrnas, but they have been proposed as a packaging vector to study the effect of viral factors, or the effect of exogenous elements on viral replication, translation, progeny phenotype, or immune response/modulation (yuan et al., 2000) . they may also allow for recombination events to occur in the background of an ongoing prrsv coinfection of two or more dissimilar strains. in this way, new viral sequences may be allowed to coexist with and interact with nascent viruses via recombination in the background, occasionally leading to new viral species with enhanced properties. hypothetical at this juncture, this concept should be evaluated by defined recombination studies. the mechanism of prrsv recombination is ill described (fig. 3) . homologous recombination was first described for nidoviruses using mouse hepatitis virus (mhv), a coronavirus, and the authors posited that less than full-length rna intermediates might be generated during viral rna replication. these early studies led to the proposal that mhv replication proceeded in a discontinuous and nonprocessive manner, perhaps at sites of secondary and tertiary structure thus generating free rna intermediates, which could be used in rna recombination via a copy-choice mechanism (lai, 1992; lai et al., 1985; makino et al., 1986) . in the absence of selection pressure, mhv rna recombination was found to be random, but that only certain recombinants are selected over passage in tissue culture, leading to the conclusion that there were "hotspots" for recombination (banner and lai, 1991) . recombination was detected during both negative and positive strand rna transcription, and took place not only between two different viral strains but also between one replicating viral rna and transfected non-replicating mhv rna fragments. furthermore, the recombinants could be detected after viral growth in cell culture and in animals, and successful recombination occurred more frequently within a hypervariable region which was also subject to deletion (liao and lai, 1992) . these results were shown to be representative of other coronaviruses, most notably infectious bronchitis virus (ibv), where numerous reports detail the ability of the virus to recombine in the field (kottier et al., 1995; toro et al., 2012; wang et al., 1993) . in the case of ibv, a pathogen of poultry, recombination has been shown to be robust, perhaps due to housing poultry in large flocks (cavanagh and davis, 1993) . most of these early conclusions mirror what has been shown to occur in prrsv. kapur et al. (1996) provided strong statistical evidence for intragenic recombination or gene conversion in orfs 2, 3, 4, 5 and 7, but not in orf 6. the first laboratory examination of prrsv recombination was done using two different prrsv strains to infect ma-104 cells. differential primer pairs were used in rt-pcr studies to examine cloned cell culture progeny for recombination events over an 1182 nucleotide span encompassing part of orf3 to orf5 of type 2 prrsv. five clones were selected for sequence analysis, which revealed that four clones each represented a single unique crossover, and one clone appeared to be a triple crossover recombinant. rnase treatment of the cell supernatant before rt-pcr analysis proved that the recombinant rnas were protected from degradation and therefore represented packaged viral rnas. finally, the investigators showed that the recombinants could be detected for up to three passages, but eventually were overtaken by one parental strain that had increased replication kinetics in the cultured cell line. recombination frequencies of up to 10% were estimated and recombinants could also be found in animals (murtaugh et al., 2001; yuan et al., 1999) . a similar experiment was completed with type 1 prrsv, reporting frequencies of only 0.1 and 2.5% rna recombination occurring within a 621 bp fragment, but also noting that recombination events are correlated with the size of the fragment analyzed (van vugt et al., 2001) . there are many reported algorithmically detected instances of recombination occurring between prrsv field strains of the same genotype, as well as defined coinfection studies in swine (li et al., 2009; liu et al., 2011; martin-valls et al., 2014; shi et al., 2013a) . recombination hotspots have been observed to take place within the 3 0 end structural genes, as well as in nsp2, and nsp9 (li et al., 2009; liu et al., 2011) . a more thorough analysis has shown that multiple breakpoints of recombination were detected by genetic algorithm recombination detection (gard) software all along the genome of both type 1 and type 2 isolates. gard analysis of 25 type 1 genomes produced 11 statistical breakpoints. similarly, 55 type 2 genomes led to the identification of 9 breakpoints (martin-valls et al., 2014) . rare evidence exists for nonhomologous recombination between the prrsv genome and other rna segments. a survey of 3188 field viruses (18 type 1) sequenced in the gp5 region (597-618 bp) by the university of minnesota veterinary diagnostic laboratory showed that a key segment coding for gp5 hypervariable region 1 was subject to insertions and deletions (faaberg, 2007) . this region contains 219 different patterns for an 8 amino acid stretch in hypervariable region 1, inducing non-neutralizing antibodies (ostrowski et al., 2002; plagemann et al., 2002) , but was also seen to have additions (up to 6 amino acids) or deletions (1 amino acid). two particular field isolates, encoding an extra 6 amino acids in the hypervariable region (nggmrtaansnsss), were found to be identical in nucleotide sequence (ggggggau-gaggaccgcc) in that 6 amino acid stretch to prrsv orf1 sequence, as well as many other swine host transcripts and other pathogens (unpublished data). although breakpoints and recombinants are valuable tools to understand viral evolution, there is a paucity of research directed toward understanding the viral and host machinery that prrsv utilizes to successfully carry out recombination. except for the illustration that mutations in the sdd motif of nsp9 of prrsv results in viral replication without sgrna transcription, and the hel and nendou domains being critical for both genomic and sgrna replication in eav and prrsv, no detailed mechanism for arterivirus homologous and nonhomologous recombination has been put forward. the molecular evolution of prrsv has been examined by many investigators in detail and will not be addressed in this review (frossard et al., 2013; shi et al., 2013a shi et al., , 2010a shi et al., , 2010b shi et al., , 2013b stadejek et al., 2013) . the cause of such rapid evolution may be primarily due to the lack of prrsv rdrp proofreading and tremendous viral recombination, resulting in an extraordinary diverse composition of isolates with varying pathogenicity. from the emergence of prrs in the united states in 1987 (keffaber, 1989) , it was apparent that there were several circulating viruses besides the usa prototype virus, vr-2332 . the disease was first recognized as mostly a reproductive disease, causing anorexia, late term abortions, and delayed return to estrus in sows. the infection of sows also led to increased preweaning mortality in young pigs that survived. histologically, interstitial pneumonitis, lymphomononuclear encephalitis, and lymphomononuclear myocarditis in piglets and focal vasculitis in the brain of the sow were seen. in nursing, growing, and finishing pigs, mild flu-like symptoms are evident, with pronounced hyperpnea, fever, and interstitial pneumonitis . in europe, similar disease phenotypes were observed, with the additional finding that sows sometimes had blue ears, but antigenic differences were seen between european virus isolates from different countries and these were radically dissimilar from united states and canadian isolates drew et al., 1995; hopper et al., 1992; paton et al., 1991; plana et al., 1992; wensvoort et al., 1992 wensvoort et al., , 1991 . similar findings based on herd clinical symptoms as well as seroconversion were also reported in the usa and canada (dea et al., 1992; morrison et al., 1992) . the first nucleotide sequencing efforts directed at the prrsv 3 0 -end of the genome confirmed these antigenic findings, revealing approximately 10% nucleotide differences between type 2 isolates and 40% between type 1 and type 2 isolates (drew et al., 1997; kapur et al., 1996; mardassi et al., 1994b; meng et al., 1994; meulenberg et al., 1994; pesch et al., 2005) . further evolution now places the divergence within both genotypes at 415% when comparing whole genomes (han et al., 2006; van doorsselaere et al., 2012) . investigators soon found that these differences were also reflected in the degree of pathogenicity caused by different viral isolates (halbur et al., 1995 (halbur et al., , 1996 . key events in prrsv diversity were the emergence of an "atypical" or "acute" variant that appeared in iowa, usa in the mid 1990s (meng et al., 1996a; mengeling et al., 1998) , the sudden appearance in 2001 of a novel class of prrsv named mn184 in minnesota, usa (han et al., 2006) , the notable type 2 highlypathogenic prrsv (hp-prrsv) in 2006 in china and subsequently most of asia (tian et al., 2007) , and the recent demonstration of enhanced pathogenicity of the type 1 lena strain (karniychuk et al., 2010) . some prrsv isolates have been shown to be neurovirulent (rossow et al., 1999; tian et al., 2007) , and most recently the ability of particular strains to depress the swine immune response has been shown to vary (brockmeier et al., 2012; guo et al., 2013a guo et al., , 2013b wang et al., 2013) . since the beginning, instances of increased virulence have emerged episodically in different regions of the world. in each case, however, the appearance of the novel isolates is sudden and the result of a significantly divergent prrsv genome sometimes accompanied by insertions or deletions in most often nsp2 (brockmeier et al., 2012) , and often containing detectable recombination breakpoints (shi et al., 2013a) . the remarkable phenotypic and genetic diversity is most likely the result of the innate attribute of prrsv persistence, replication, and recombination to yield an extraordinarily flexible viral genome, attempting to circumvent attempts to eradicate the pathogen from swine by the host response, vaccination or other means. mention of trade names or commercial products in this article is solely for the purpose of providing specific information and 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spatial dynamics the endoplasmic reticulum stressresponsive protein grp78 protects neurons against excitotoxicity and apoptosis: suppression of oxidative stress and stabilization of calcium homeostasis heteroclite subgenomic rnas are produced in porcine reproductive and respiratory syndrome virus infection characterization of heteroclite subgenomic rnas associated with prrsv infection recombination between north american strains of porcine reproductive and respiratory syndrome virus overview: replication of porcine reproductive and respiratory syndrome virus coronavirus leader rna regulates and initiates subgenomic mrna transcription both in trans and in cis mutational analysis of the sdd sequence motif of a prrsv rna-dependent rna polymerase studies of porcine reproductive and respiratory syndrome (prrs) virus infection in avian species this work builds upon the doctoral thesis of matthew kappes, department of veterinary microbiology and preventive medicine, iowa state university, ames, ia, 2014, entitled "identification and characterization of a novel structural protein of porcine reproductive and respiratory syndrome virus, the replicase nonstructural protein 2". project 3625-32000-108-00d of the usda agricultural research service provided support for dr. faaberg. dr. kappes was supported by bi vetmedica, inc., and by the usda agricultural research service. usda is an equal opportunity provider and employer. key: cord-252397-qlu7dilh authors: johnson, reed f.; via, laura e.; kumar, mia r.; cornish, joseph p.; yellayi, srikanth; huzella, louis; postnikova, elena; oberlander, nicholas; bartos, christopher; ork, britini l.; mazur, steven; allan, cindy; holbrook, michael r.; solomon, jeffrey; johnson, joshua c.; pickel, james; hensley, lisa e.; jahrling, peter b. title: intratracheal exposure of common marmosets to mers-cov jordan-n3/2012 or mers-cov emc/2012 isolates does not result in lethal disease date: 2015-11-01 journal: virology doi: 10.1016/j.virol.2015.07.013 sha: doc_id: 252397 cord_uid: qlu7dilh middle east respiratory syndrome coronavirus (mers-cov) continues to be a threat to human health in the middle east. development of countermeasures is ongoing; however, an animal model that faithfully recapitulates human disease has yet to be defined. a recent study indicated that inoculation of common marmosets resulted in inconsistent lethality. based on these data we sought to compare two isolates of mers-cov. we followed disease progression in common marmosets after intratracheal exposure with: mers-cov-emc/2012, mers-cov-jordan-n3/2012, media, or inactivated virus. our data suggest that common marmosets developed a mild to moderate non-lethal respiratory disease, which was quantifiable by computed tomography (ct), with limited other clinical signs. based on ct data, clinical data, and virological data, mers-cov inoculation of common marmosets results in mild to moderate clinical signs of disease that are likely due to manipulations of the marmoset rather than as a result of robust viral replication. infection with middle east respiratory syndrome coronavirus (mers-cov) has been associated with middle east respiratory syndrome commonly known as mers, a respiratory syndrome with acute severe hypoxic respiratory failure often accompanied by renal failure (arabi et al., 2014; assiri et al., 2013; zaki et al., 2012) . as of march, 2015 there have been approximately 1000 laboratory confirmed cases reported with a 36% case fatality rate (http://www.who.int/csr/disease/coronavirus_infections/). considering the geographical location of mers and the hajj pilgrimage which draws an estimated 2.5 million visitors, roughly 1.8 million of whom travel internationally (http://www.cdsi.gov.sa/english/ index.php?option=com_docman&itemid=173) mers-cov represents a global health risk. common signs and symptoms of mers include fever, cough, shortness of breath, and myalgia. gastrointestinal signs are also frequently observed which include vomiting, diarrhea, and abdominal pain (assiri et al., 2013) . often, mers patients also have underlying comorbidities such as diabetes, hypertension, and chronic cardiac or renal disease (assiri et al., 2013) . to guide development of mers countermeasures, an appropriate animal model must be identified and characterized. ideally, a laboratory animal model would demonstrate clinical signs consistent with all aspects of human disease. as with most infectious diseases, mice have been evaluated as a potential mers model for pathogenesis and countermeasure screening. balb/c and stat-1 knockout mice did not develop signs of disease, such as weight loss, nor could infectious virus be recovered from lung homogenates (coleman et al., 2014) . zhao et al. developed a murine model for mers by transduction of the respiratory tract with the putative mers-cov receptor, human dipeptidyl peptidase 4 (dpp4 or cd26), using an adenovirus construct (zhao et al., 2014) . infected mice developed limited clinical signs including a small degree of weight loss. histopathological analysis found peribronchial and perivascular lymphoid infiltrates which later progressed to an interstitial pneumonia. nearly 6 log 10 plaque forming units (pfu)/g of infectious virus could be detected in the lungs of infected dpp4 transduced mice (zhao et al., 2014) . the dpp4 transduced mouse model has been used to evaluate countermeasures and pathogenesis (channappanavar et al., 2014a (channappanavar et al., , 2014b . nonhuman primate (nhp) models are considered to be essential to understanding pathogenesis and evaluating countermeasures. results from several challenge studies of mers-cov in rhesus monkeys (macaca mulatta) have varied between laboratories. the first published nhp model used rhesus monkeys inoculated via multiple routes and evaluated for virological, immunological, and histopathological changes up to 6 days postinoculation . nhps demonstrated signs of pneumonia and virus could be detected in tissues and mucous membranes by quantitative reverse transcriptase polymerase chain reaction (qrt-pcr), but attempts to determine the load of infectious virus was not reported. a follow up study demonstrated that administration of interferon-alpha2b and ribavirin reduced viral burden and lessened disease . results from a natural history study of mers-cov-infected rhesus monkeys indicated that intratracheal inoculation induced a non-lethal disease with limited pathology observed in recovering animals at 28 days post-inoculation and infectious virus could be recovered from lung but not other tissues assayed (yao et al., 2014) . standard radiological examination revealed lung infiltrates at days 3 and 5 post-inoculation, suggesting virus-induced lung disease. more recently, falzarano et al. described multiple route inoculation of the common marmoset (callithrix jacchus) in which transcriptional changes indicating induction of immune, inflammatory, and repair pathways were cataloged and partial lethality was observed (falzarano et al., 2014) . here we characterize intratracheal (it) inoculation of mers-cov into common marmosets as a model for mers and to determine differences between two common isolates of mers-cov, mers-cov-jordan-n3/2012 virus and mers-cov-emc/2012 virus. we inoculated 4 groups of marmosets and followed disease progression by periodic physical exams that included computed tomography (ct). previously, positron emission tomography with computed tomography (pet)/ct has been used to evaluate tuberculosis therapies in common marmosets (via et al., 2013) , disease progression in monkeypox-virus-inoculated cynomolgus monkeys (dyall et al., 2011) , and influenza-a-virus-inoculated ferrets (jonsson et al., 2012) . ct has two distinct advantages when compared to standard x-ray radiography 1) ct provides three dimensional data, and 2) ct data can be quantified, which allows unbiased comparisons (elke et al., 2013; li et al., 2013; romanova et al., 2014) . an ex-vivo experiment with marmoset lung and kidney primary cultures found that these tissues could support mers-cov replication; this suggested that common marmosets might be developed as a suitable animal model for mers (fig. 1) . therefore, we sought to determine if intratracheal inoculation of marmosets would result in disease presentation similar to human disease. the experimental design is shown in fig. 2 . two pre-inoculation baseline ct's were performed to establish normal lung volumes and any pre-existing anomalies. mers-cov-jordan-n3/2012 virus (mers-jor, genbank kc776174) and mers-cov-emc/2012 virus (mers-emc, genbank jx869059) were obtained and propagated as described in materials and methods. to ensure that no gross cross-contamination of the mers-cov stocks used for the marmoset experiments occurred during preparation, the spike protein of each stock was sequenced and compared to reference sequences (cotten et al., 2013; frey et al., 2014) . the spike region was chosen for comparison due to high diversity associated with viral glycoproteins. strain-specific differences found in mers-jor and mers-emc reference sequences were maintained in our stocks (table 1) , indicating that no gross cross-contamination occurred. three single nucleotide polymorphisms (snps) were seen in our stocks, two in mers-emc and one in mers-jor. blastx alignments indicate that two of these snps lead to changes in the s2 protein sequence. the mers-jor stock had a t to c change at position 2636 which induced an i839t change in s2. two changes were observed in mers-emc c to t at position 2604 that did not alter amino acid sequence and a c to a change at 3044 which resulted in a n1016t change in s2. changes in the s2 region seen here are likely a result of serial passage in cell culture (frey et al., 2014) . body temperature, peripheral oxygen saturation, respiratory rate, and overall condition were evaluated at each physical exam. no increases in body temperatures above normal ranges were observed; subjects maintained peripheral oxygenation throughout the study, and respiratory rates increased above normal range sporadically throughout the study (fig. 3a) . tremors were noted on daily observations including and between days 3 and 9 postinoculation, but were not consistently observed (data not shown). subjects underwent blood withdrawal on days 0, 4 or 5, and at necropsy to determine if hematological parameters indicated changes consistent with disease. day 4 or 5 was chosen based on our data from mers-jor inoculated rhesus monkeys which demonstrated a peak in lung disease at day 5 post-inoculation by ct (manuscript in preparation). subjects did not develop clinically significant changes in total white blood cell count, lymphocyte number, monocyte number, or neutrophil number on day 4 or 5 post-inoculation and remained within the normal range, as indicated by shaded gray area (fig. 3b ). together, these data indicate that subjects did not develop systemic clinical disease. to determine if virus was disseminating or shedding, whole blood, oropharyngeal, rectal swabs, and fecal samples from mock and inactivated virus subjects, 2 subjects per group, were collected on days à 21, à8, 1, 3, 5, 7, 14 or 15, and 25. mers jordan subjects ex-vivo analysis of primary cells. cells were isolated from lung, kidney, and bronchoalveolar lavage (bal), and one-step growth kinetics were performed as described in materials and methods. lung, kidney and bal demonstrate that mers-cov is able to replicate to at least 4 log 10 pfu/ml. were swabbed on days à 21, à8, 1 (n ¼1), 2 (n ¼2), 3(n ¼1), 4 (n ¼2), 5(n¼ 1), 6 (n ¼2), 7(n¼ 1), 8(n ¼2) 14(n ¼3) and 32 and mers-emc exposed subjects were swabbed on days à 21(n¼ 3), -8 (n ¼3), 1(n ¼3), 3 (n ¼3), 5(n¼ 3), 7(n ¼3), 14(n ¼3), and 25 (n ¼3) as outlined in fig. 2 . these samples were assayed by qrt-pcr as described in materials and methods. all samples were negative for mers-cov, suggesting that virus did not disseminate nor was shed. furthermore, plaque assays were performed on necropsy tissues and no infectious virus could be detected. virus isolation was attempted using lung homogenates for two sequential passages on vero cells; no infectious virus could be detected. fluorescent reduction neutralizing assays were performed to determine if marmosets developed an antibody response to mers-cov (fig. 4 ). subjects inoculated with mers-jor developed a low antibody titer to mers-cov with an average titer of 1:100 across the group at study end, day 25-32 post-inoculation. animals inoculated with mers-emc developed an antibody titer of 1:80 for all 3 subjects. media only, and inactivated virus did not develop detectable neutralizing antibody responses to mers-cov. qualitative assessment of the imaging data indicates that mers-cov inoculated marmosets developed lung disease that mainly affected the medial and caudal regions of the lung. lung abnormalities could be observed in media-only and the inactivated virus inoculation groups, with the inactivated virus stimulating increased lung abnormalities when compared to the media only mock inoculated subjects (fig. 5a ). lung infiltrates presenting as air bronchograms did not completely clear by end of study (fig. 5b ). more importantly, quantitative assessment of abnormal lung volume data supported the qualitative assessment. the quantitative assessment suggests that mock inoculated subjects developed a short lived response that was observed one day post-inoculation but returned to near baseline by the day 7 ct and remained near that volume throughout the course of the study. subjects inoculated with inactivated virus demonstrated increased abnormal lung volumes when compared to the media only group, but not as elevated as the groups that received live virus. mers-cov inoculated groups demonstrated increasingly abnormal lung volumes beginning day 1 post-inoculation in an individual dependent manner that did not completely resolve by study end (fig. 5c and d). comparison of the mean peak values of diseased lung volume and percent relative change of abnormal lung volume for each group indicated no statistically significant difference (unpaired t-test) between the virus isolates ( fig. 5e and f). when the peak diseased lung volume of the infectious mers-emc receiving group is compared to the mock infected the pvalue ¼0.026 and when compared to the γ-irradiated virus receiving group it is p ¼0.548. comparison of the mers-jor group to the control groups indicates a similar pattern p ¼ 0.057 and p ¼0.1245, respectively. comparison of the % fold change in diseased lung volume between the mers-emc and mock group gave p ¼0.0049 and the γ-irradiated virus receiving group was 0.0195. comparison of the % fold change in diseased lung volume between the mers-jor and mock group gave p ¼0.2102 and p ¼0.1591. the data indicate that, in common marmosets, the genetic sequence differences between the isolates do not impact disease progression as indicated by changes in diseased lung volume and that the mers virion itself can induce an inflammatory response. however, a significant difference was observed between the mers-emc group and the mock exposed group when compared by fold change of diseased lung volume, which likely reflects the small group sizes. differences in temporal progression were observed between the individual subjects that did not segregate by virus isolate. one subject in the mers-emc inoculated group appeared to develop a secondary infection observed by ct that increased to study end, day 25 post-exposure. however, no infectious virus could be recovered, suggesting an opportunistic infection either due to repeated manipulations or effects of mers-cov. at necropsy, lung lesions included multifocal to coalescing interstitial pneumonia with consolidation of the dorsocaudal lung lobe in the mers-cov inoculated animals (fig. 6 ). gross pathology of mers-cov infected subjects revealed no to moderate changes. these changes include interstitial multifocal to coalescing moderate pneumonia as shown in fig. 6a . histopathological examination of select lymphoid tissues revealed mild lymphoid hyperplasia, with medullary histiocytosis of the mandibular and tracheobronchial lymph nodes in several of the mers-cov inoculated subjects, while the control animals showed minimal changes. examination of representative lung tissue samples showed interstitial lymphohistiocytic pneumonia with type ii pneumocyte and bronchial associated lymphoid tissue (balt) hyperplasia and a few syncytia were observed in the mers-cov inoculated animals ( fig. 6b and d) . in addition, the lung lesions observed in the mers-cov inoculated subjects are consistent with a chronic respiratory disease. in general, the gross and histopathological findings in the lung tissue did somewhat correspond with the ct findings. examination of representative lung tissue from a single subject that received mers-jor and demonstrated the greatest increase in diseased lung volume as seen by ct did not differ grossly or histologically from the other subjects. no signal was detected for the presence of mers-cov antigen by immunohistochemistry (ihc) performed on the lung tissue sections of the infected subjects. ideally, an animal model for mers will recapitulate the key features of the human disease in the severe form. therefore, an ideal mers model might result in lethal disease with severe bronchopneumonia and extrapulmonary complications such as renal failure. with the use of ct, we observed that it inoculation of common marmosets with mers-jor or mers-emc isolates resulted in a non-lethal disease characterized by limited clinical signs and moderate consolidative lung pathology that did not completely resolve by study end. no difference in clinical signs was observed between the two isolates or control subjects, nor could we detect mers-cov genome using a genome specific qrt-pcr assay with a sensitivity of 100 genome copies of mers-cov. detection of neutralizing antibody in the infectious virus receiving groups and not the γ-irradiated virus receiving group does suggest that an infectious process did take place which allowed for antigen processing for development of neutralizing antibodies. artifacts introduced by manipulation of the subjects should be accounted for by use of control subjects. control marmosets were inoculated with media or inactivated virus which also resulted in limited lung pathology. in the present study, examining the percent change in diseased lung volume determined a significant difference was observed between the mers-emc group and the mock group, but not between the mers-emc and γ-irradiated group, which suggests that some viral-induced disease process did occur. however, statistical significance was not found when the total diseased lung volumes were compared between any of the groups. across all groups, pathology findings were predominantly confined to the lungs showing chronic and resolving changes. the absence of viral antigen signal in the lung tissue sections by ihc might be a result of probing for the viral antigen only at a late stage of disease, when there was likely considerable clearance of the virus. falzarano et al. observed inconsistent lethality highlighted by lung pathology that shared some characteristics of mers following mers-emc exposure of common marmosets. three notable differences between the present study and falzarano et al. are 1) our use of control subjects receiving media or inactivated virus, 2) single it route vs. multiple inoculation routes (oral, intranasal, ocular, and it), and 3) the volume of lung inoculum (0.2 ml vs. 0.5 ml). similar to their rhesus experiment, their marmoset experiment was designed as a serial euthanasia study. six subjects were euthanized at early timepoints post-exposure, before the disease course could resolve. however, two subjects met moribund endpoint criteria on day 4, and 3 survived to day 20 post-inoculation. it is unknown what the true outcome of the subjects that were serially euthanized would have been and if the subjects that met endpoint criteria did so due to virus induced disease or subject manipulations, thus skewing the interpretation of disease severity. adding to the difficulty in data interpretation, qrt-pcr data is not supported by immunohistochemistry, em, or virus isolation. furthermore, the pattern of infectious virus isolation shown by falzarano et al. demonstrates that initially only the lungs contain infectious virus in 11/15 tissues (trachea and lung lobes) examined, by day 4 postinoculation virus can be isolated from fewer lung lobes (8/10 tissues), but virus can be isolated from the trachea. by day 6 postinoculation virus is detected in 1/8 of lung lobes and all tracheas examined. no quantification is provided, therefore it is difficult to determine if propagated infectious virus was recovered or merely the inoculum. based on our data and falzarano et al.'s data, it is possible that intratracheal inoculation results in mechanical damage to the respiratory epithelium, followed by inflammation and repair, which leads to restoration of mucociliary motion that clears the liquid (and virus) from the lung. in the absence of a lethal model, other quantitative measures become more valuable for demonstrating pathogenesis. in the present study, we demonstrated that ct is effective for evaluating disease progression and regression following mers cov exposure in common marmosets. ct data can be quantified, which provides an opportunity to effectively evaluate countermeasures in sub-optimal models and strengthen data gained in well-established models. in the case of mers, an optimal animal model must still be developed. staged euthanasia studies similar to the adenovirus transduced mouse mers model could be performed in nhps, but such studies are undesirable due to the ethical concerns of nonhuman primate use. vero cells (atcc catalog number ccl81) were maintained in dulbecco's modified eagle's medium (dmem) (hyclone, logan, ut) and supplemented with 10% fetal bovine serum (fbs) (sigma st. louis mo) at 37 1c with 5% co 2 . mers-cov isolates (jordan-n3/ 2012 and emc/2012) were propagated at a multiplicity of infection of 0.1 on vero cells in 5% fbs and 5% co 2 for 3-4 days postinoculation until cytopathic effect encompassed 75-80% of the vero cell monolayer. virus was concentrated by ultracentrifugation and pelleted through a 20% sucrose cushion at 221,000 â g for 2 h at 4 1c. the pellet was re-suspended in dmem 5% fbs, frozen and titered by limiting dilution plaque assay (see below). inactivated virus for the control groups was generated by treating virus with 60,000 gy of gamma irradiation from a 60 cobalt irradiator (jl shepherd 484-r2), followed by confirmation of inactivation. mers-cov-jordan-n3/2012 was a kind gift of armed forces health surveillance center, division of global emerging infections surveillance and response system and mers-cov-emc/2012 was kindly provided by vincent munster, laboratory of virology, national institute of allergy and infectious diseases (niaid). marmoset lung tissue was ground in a sterile petri dish using a plunger from a sterile syringe, digested with 100 u/ml of collagenase (life technologies, ny usa), and 10% fetal calf serum (fcs, hyclone) 37 1c for 30 min. the reaction was quenched, and the homogenate was passed through a 70-mm filter, washed, and centrifuged (500 â g, 5 min, 4 1c). after the final wash, cells were re-suspended in 5 ml of ack lysing buffer (life technologies) for 15 min and washed twice with 25 ml pbs/5 mm edta/0.5% bsa. washed and pelleted cells were re-suspended in rpmi (lonza, switzerland) supplemented with 10% fcs (hyclone, ut, usa) and 1% penicillin/streptomycin (ps) and plated. media was changed after 24 h. kidneys were processed similar to the lung but strained through 70 mm and 40 mm filters prior to the ack lysis step. bronchoalveolar lavage samples were centrifuged at 1000 â g for 10 min at 4 1c, re-suspended in rpmi 10% fcs, and 1% ps (life technologies) before plating. multi-step growth curves were performed on isolated primary cells infected with mers-jor at a multiplicity of infection of 0.1. supernatants were harvested at 0, 24, 48, and 72 h and stored at à80 1c until plaque assays were performed (described below). mers-cov isolates were sequenced using the primers in table 2 . spike genomic sequences were generated by aligning sanger to generate a comparison between study stocks and references, spike sequences obtained from sequencing and associated reference sequences were aligned using clustal omega with default settings (goujon et al., 2010; mcwilliam et al., 2013; sievers et al., 2011) . the resulting clustal alignment was filtered for alignment positions with at least one variant base using a custom python script. potential coding changes were assessed by blastx alignments (altschul et al., 1990) . ten common marmosets, ranging in weight from 250 g to 475 g and age from 3 to 9 years were divided into 4 groups. mock inoculated subjects (n ¼2) received dmem supplemented with 5% fbs by it inoculation. two subjects received inactivated virus by it inoculation; one subject received inactivated mers-jor virus isolate, and one subject received inactivated mers-emc virus isolate. three marmosets received 5 â 10 7 pfu of mers-jor and 3 others received 5 â 10 7 pfu of mers-emc by it inoculation. prior to handling, marmosets were anesthetized with isoflurane to effect. it inoculation was performed by placement of a 20 gauge â 1 in. catheter into the trachea followed by installation of the virus inoculum in a 0.2 ml volume followed by a 0.2 ml air flush of the syringe and catheter. all animal procedures were approved by the national institute of allergy and infectious diseases (niaid) animal care and use committee, and adhered to national institutes of health (nih) policies. the experiments were carried out at the niaid integrated research facility, an aaalac and aalas accredited facility. prior to and after inoculation, ct scans, physical exams, including temperature and weight measurements were performed, oropharyngeal and rectal swabs, and stool samples were collected (fig. 2) . nhps were monitored at least twice daily. a preestablished scale was used to evaluate subject health and disease progression. these criteria included: (1) overall clinical appearance, (2) labored breathing, (3) activity and behavior, (4), responsiveness, and (5) core body temperatures. no subjects met moribund clinical endpoint criteria by study end. eight of 10 marmosets were humanely euthanized for histopathological and virological analysis. blood withdrawal was performed pre-inoculation, either day 4 or 5 post-inoculation, and at necropsy. we acquired high resolution chest ct scans, without contrast, using a hybrid philips gemini 16 slice pet/ct scanner specifically designed to function in a biological safety level 4 environment. due to the rapid respiratory rate of marmosets, a breath hold could not be performed. the ct parameters were as follows: helical scan, 140 kvp, 300 ma s, 90 mm field of view, 0.5 mm/rotation table speed, high-resolution collimation of 16 â 0.75 mm 2 , 0.8 mm slice thickness, and 0.4 mm increment covering the whole lung. the images were acquired using a standard algorithm and reconstructed using a y-detail algorithm. images were analyzed as previously described (via et al., 2013) . unpaired t-test of peak abnormal lung volume was performed with graphpad prism 6.0 (graphpad software, ca, usa). complete blood cell differential count was determined from blood samples collected in edta-coated blood tubes and analyzed using a sysmex xt2000v ™ hematology analyzer (sysmex america). viral load in samples was determined by quantitative pcr using the following primer probe set: forward primer: 5ʹtggcc gtggtggttatcact 3ʹ, reverse primer: 5ʹctcaaaatcgtccatcca ctca3ʹ, and probe 5ʹ6-fam/caccccattccactatgagcgagacaac/ 36-tamsp/3ʹ. cycling conditions were 48 1c for 30 min and 95 1c for 10 min for the rt-step followed by 95 1c for 15 s and 60 1c for 60 s for 40 cycles. the taqman one-step rt-pcr master mix was used (life technologies, ny, usa). samples were extracted with trizol and screened for the presence of mers-cov using specific primers on an applied biosystems 7900ht fast real time pcr system (life technologies). the limit of detection was 100 gene copies. virus stock and tissue samples were excised at necropsy, flash frozen, and stored at à 80 1c. for tissues, a w/v homogenate between 10% and 30% was generated. serial 10-fold dilutions were made for virus stocks, growth curve samples, and tissues and incubated on confluent veroe6 cells using 0.8% tragacanth in emem 2% fbs, and 1% ps overlay and incubated for 5 days. following incubation, tragacanth overlays were removed, the monolayers were stained with crystal violet (0.1% crystal violet, 20% ethanol 10% formalin v/v), and plaques were enumerated. serum samples taken at necropsy or experiment end were heat inactivated at 56 1c for 1 h then serial diluted and mixed with 120 pfu (final moi¼1.0) of either mers-jor or mers-emc and incubated for 1 h. after incubation virus and serum mixture was added to veroe6 cells (atcc, manassas va) for 1 h at 37 1c, 5% co 2 on 96 well operetta (perkin-elmer) compatible plates. negative control (no virus and no serum) samples and positive control (virus þ rabbit polyclonal antibody to the mers spike protein (sino biological)) samples were also included. cells were washed and incubated for 24 h followed by fixation with 20% nbf. mers-cov infection was visualized by fluorescence using a mers-cov anti-spike antibody (sino biological) on an operetta high content imager (perkin-elmer). the dilution at which 50% inhibition of relative fluorescence intensity was observed was reported as the frna 50 . forty-one tissues from all major organ systems were collected and fixed in 10% neutral buffered formalin (nbf) for 30 days. following fixation in 10% nbf tissues were processed following standard procedures. hematoxylin and eosin (he) stain was applied using the leica automated staining system (leica, microsystems). stained slides were then examined via standard light microscopy. to detect mers-cov antigen, ihc was performed using a rabbit polyclonal antiserum against mers-cov (sino biological p.r. china) (1:1000). tissues from an uninfected control animal were used to validate all ihc procedures. he and ihc sections were examined by light microscopy by the veterinary pathologists (sy and lh). in this experiment, we sought to determine if there were virus specific differences in disease progression following intratracheal inoculation of common marmosets with middle eastern respiratory syndrome coronavirus, commonly known as mers-cov, with two common laboratory viral isolates (mers-emc and mers-jordan). in contrast to previous results, we observed a non-lethal disease and few, if any, signs of virus-specific pathology. in addition, we were unable to isolate infectious virus from tissues. computed tomography was used to evaluate disease progression and provide quantitative data for comparisons between mockexposed, inactivated virus-exposed, and virus-exposed subjects. the data indicate that marmosets do not faithfully replicate human mers pathogenesis and that alternate models must be developed to efficacy test medical countermeasures. basic local alignment search tool clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study virusspecific memory cd8 t cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection t cell-mediated immune response to respiratory coronaviruses wild-type and innate immune-deficient mice are not susceptible to the middle east respiratory syndrome coronavirus full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques evaluation of monkeypox disease progression by molecular imaging quantification of ventilation distribution in regional lung injury by electrical impedance tomography and xenon computed tomography infection with mers-cov causes lethal pneumonia in the common marmoset treatment with interferon-alpha2b and ribavirin improves outcome in mers-cov-infected rhesus macaques full-genome sequence of human betacoronavirus 2c jordan-n3/2012 after serial passage in mammalian cells a new bioinformatics analysis tools framework at embl-ebi molecular imaging reveals a progressive pulmonary inflammation in lower airways in ferrets infected with 2009 h1n1 pandemic influenza virus growth pattern analysis of murine lung neoplasms by advanced semiautomated quantification of micro-ct images analysis tool web services from the embl-ebi magnetic resonance imaging versus computed tomography for identification and quantification of intraventricular hemorrhage fast, scalable generation of high-quality protein multiple sequence alignments using clustal omega differential virulence and disease progression following mycobacterium tuberculosis complex infection of the common marmoset (callithrix jacchus) an animal model of mers produced by infection of rhesus macaques with mers coronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia rapid generation of a mouse model for middle east respiratory syndrome this work was supported by the national institute of allergy and infectious disease, division of intramural research. we are grateful to marisa st. claire, russell byrum, dan ragland, and the entire evps and irf team for their contributions to these studies. we key: cord-267718-t47i8hui authors: bao, jingyue; wang, qinghua; li, lin; liu, chunju; zhang, zhicheng; li, jinming; wang, shujuan; wu, xiaodong; wang, zhiliang title: evolutionary dynamics of recent peste des petits ruminants virus epidemic in china during 2013–2014 date: 2017-07-19 journal: virology doi: 10.1016/j.virol.2017.07.018 sha: doc_id: 267718 cord_uid: t47i8hui peste des petits ruminants virus (pprv) causes a highly contagious disease, peste des petits ruminants (ppr), in sheep and goats which has been considered as a serious threat to the local economy in africa and asia. however, the in-depth evolutionary dynamics of pprv during an epidemic is not well understood. we conducted phylogenetic analysis on genomic sequences of 25 pprv strains from china 2013–2014 outbreaks. all these strains clustered into a novel clade in lineage 4. an evolutionary rate of 2.61 × 10(−6) nucleotide substitutions per site per day was estimated, dating the most recent common ancestor of pprv china 2013–2014 strains to early august 2013. transmission network analysis revealed that all the virus sequences could be grouped into five clusters of infection, suggesting long-distance animal transmission play an important role in the spread of pprv in china. these results expanded our knowledge for pprv evolution to achieve effective control measures. peste des petits ruminants (ppr) is one of the most important diseases of sheep and goats, which is caused by a morbillivirus, peste des petits ruminants virus (pprv). alongside sheep and goats, pprv also affects wildlife species including captive, wild small ruminants and sub-clinically cattle and buffalo . ppr can cause a very high mortality reaching up to 100% in immunologically naïve populations. thus, ppr is regarded as a significant hurdle to the development of sustainable agriculture. in march 2015, office internationale des epizooties (oie) and food and agriculture organization (fao) officially launched a global program to eradicate ppr by 2030. the genome of pprv is 15,948 or 15,954 nucleotides (nt) in length and organized into six transcriptional units encoding six structural proteins, the nucleoprotein (n), the phosphoprotein (p), the matrix protein (m), the fusion protein (f), the hemagglutinin protein (h) and the polymerase or large protein (l) (bailey et al., 2005; bao et al., 2014) . the p transcription unit also encodes two non-structural proteins, c and v. after the first report of ppr in 1942 in côte d'ivoire, the disease has been reported in most parts of sub-saharan africa, north africa, the middle east, south asia, central asia, and east asia (banyard et al., 2014; dhar et al., 2002; kwiatek et al., 2011) . the first outbreak of ppr in sheep and goats in china was reported in tibet, china in 2007 (wang et al., 2009) . another outbreak of ppr in wild small ruminants in tibet, china was reported in 2008 (bao et al., 2011) . no further spread of the disease has been reported in china until late 2013. on november 30th, 2013, an outbreak of ppr was reported by the ministry of agriculture (moa) of china in a farm in huocheng county of yili region in xinjiang province. four more infected farms were identified in december 2013 in the same province. the disease has rapidly and widely spread to 21 provinces along with the movements of goats and sheep (banyard et al., 2014; wang et al., 2015; wu et al., 2015) . this epidemic of ppr in china continued to the end of june 2013, causing death of more than 16,000 sheep and goats. the spread of ppr in china has posed serious threat of this highly contagious disease on the large population of domestic small ruminants (more than 300 millions sheep and goats) and different species of wild small ruminants in china and neighboring countries. phylogenetic studies using partial n or partial f gene sequences have grouped pprv strains into four lineages (kwiatek et al., 2007; shaila et al., 1996) . the first three lineages were distributed in africa. and lineage iv included strains from asia and recently northern africa (kwiatek et al., 2011) . recent phylogenetic analysis revealed that the chinese tibetan strains and the chinese 2013-2014 strains were separately grouped into two clusters in lineage iv (wu et al., 2015) . historically, pprv molecular epidemiology has focused on the partial n (255 nt region; nucleotide site 1360-1614 of pprv genome) or partial f gene (322 nt region; nucleotide site 5779-6100 of pprv genome) sequences, preventing the in-depth evolutionary analysis among strains (cosseddu et al., 2013; munir et al., 2011; ozkul et al., 2002; padhi and ma, 2014; soltan and abd-eldaim, 2014) . genome sequences for pprv have recently been used for an evolutionary study on a global scale (muniraju et al., 2014c) . however, the evolutionary dynamics of pprv in an epidemic outbreak has not been studied. analysis on the evolution of pprv genome during the course of an epidemic may help the interpretation of field epidemiology data and the implementation of efficient control measures. such in-depth analysis has been conducted for several other rna viruses, such as human respiratory syncytial virus, hepatitis c virus, highly pathogenenic avian influenza virus and food-and-mouth disease virus (agoti et al., 2015; bataille et al., 2011; lu et al., 2015; otieno et al., 2016; valdazo-gonzalez et al., 2012) . the outbreak of ppr in china during 2013-2014 provided a unique opportunity to study the in-depth epidemiological and evolutionary dynamics of pprv during an epidemic. the diagnosis and the genetic characterization of the ppr viruses being involved in this epidemic have been reported (wu et al., 2015) . however, the molecular evolution of pprv between infected farms during the outbreak remains unclear. in this study, we present full-genome sequence data for the ppr epidemic in china from november 2013 to june 2014. phylogenetic analysis was used to study the evolutionary dynamics of pprv during an epidemic. full-length pprv genome sequences were achieved directly from clinical samples of infected animals taken from 25 infected farms in 21 provinces. the infected farms included in this study were identified from moa's animal disease surveillance ppr data archive (http:// www.syj.moa.gov.cn/dwyqdt/). clinical samples were screened with real-time rt-pcr with amplification targeting n gene for confirmation. although 3-5 animals were sampled for diagnosis in each infected farms, samples from one ppr-confirmed animal from each infected farm were used for genome amplification. we used clinical samples of nasal swabs, mesenteric lymph node and spleen. all clinical samples used for genome amplification were detailed in table 1 . viral rna was extracted and used directly for viral genome determination. fourteen pairs of oligonucleotide primers were used to amplify 14 overlapping fragments by reverse transcription-pcr as previously described (bao et al., 2014) . the pcr products were purified and sequenced with an abi 3730xl genome sequencer (applied biosystems, usa). an addition of 15 pprv genome sequences representing viral strains from different endemic country was obtained from genbank. the genbank accession number, the year and country of isolation were listed in table 2 . vaccine strains were not included in this study. all the genome sequences were aligned using megalign software in lasergene package. a phylogenetic tree was inferred by maximumlikelihood method from the nucleotide alignment of genome sequences using mega version 4 software, assuming a tn93 model of base substitution (equal substitution rates among sites and between transitional and transversional substitutions) (tamura et al., 2007) . the statistical significance of phylogenies constructed was estimated by bootstrap analysis with 1000 repetitions. temporal dynamics of pprv were analyzed with time-resolved phylogenies using the bayesian markov chain monte carlo (mcmc) method in the beast package 1.7.1 (drummond et al., 2002) . the jmodeltest program was used to select the best-fitting nucleotide substitution model (posada, 2009 ). the datasets were analyzed using the gtr+g substitution model under an uncorrelated exponential relaxed clock with an exponential growth model. the mcmc chains were run for 2 × 10 8 generations and sampled every 20,000 generation. convergence was assessed from the effective sample size (ess) with a 10% burn-in using tracer v1.6. ess value above 200 was accepted. the bayesian skyline plot, estimating the change in effective population size through time, was generated from the beast bayesian mcmc output files using tracer v1.6. the phylogenetic network was constructed using the median joining method in the program network (hajimorad et al., 2003) . this software uses a parsimony method to connect each sequence to its closest neighbor, and allows the creation of internal nodes, which could be interpreted as unsampled or extinct ancestral genotypes to link the existing genotypes in the most parsimonious way. the generated pprv sequences were deposited in genbank with the accession numbers given in table 1 . a total of 25 complete genomes were obtained for the ppr viruses circulating in china from november 2013 to june 2014. these pprv genomes were from 25 ppr infected farms in 21 provinces across china as shown in fig. 1 . we firstly investigated the sequence differences in the partial n gene (nucleotide site 1360-1614 of these genomes) and partial f gene sequence (nucleotide site 5785-6106 of these genomes). only one substitution site, c1457t, was found in the n partial region, showing two genotypes (t for strain chinagx2014 and c for other strains). one substitution site located in f partial region (c6090t), with a t in strain chinacq2014 and c for other strains. we further studied the sequence differences along the whole genome. these 25 china pprv genomes shared 99.86-99.99% nucleotide identities. a total of 96 substitution sites were found along the genome sequences of these 25 china pprv strains (table 3 ). the number of pair-wise nucleotide differences across the entire genome varied from 1 (between strains chinajs2014 and chinahb2014) to 23 (between strains chinanx2014 and chinajl2014; chinajs2014 and chinagd2014). this increased resolution suggested the advantage for tracking pprv short-term evolution using full-length genome sequence. a total of 32 substitution sites were found in the non-coding region. sixty four substitutes were identified in the coding region, among which 28 substitutes were found non-synonymous (ns). the highest ratio of ns substitutes were found in p gene (10 ns sites out of 12 sites), followed by f gene (4/6 sites), h gene (4/7 sites), l gene (8/30 sites), n gene (1/4 sites) and m gene (1/5 sites). we investigated the distribution of substitution sites at different codon positions (table 4) . a total of 17 nucleotide changes were found at the first codon position, among which 16 sites contributed to amino acid changes. at the second codon position, 16 nucleotide substitutions were found, with all of them contributing to amino acid changes. although 37 nucleotide changes were found at the third codon position, only 2 sites lead to amino acid changes. the finding suggested that the nucleotide substitutions at the first and second codon position contributed mainly to the amino acid changes. these 25 genomes obtained in this study were combined with the 15 genomes derived from genbank to generate a set of 40 genomes from 11 different epidemic countries in asia and africa: india, turkey, united arab emirates, oman, china, côte d'ivoire, nigeria, ethiopia, ghana, uganda and morocco. to identify the genome characteristics of the 25 china 2013-2014 pprv genomes, we conducted multiple sequence alignments between these 25 sequences and the other 15 pprv genomes. the genome length of all the china 2013-2014 pprv strains was 15,954 nucleotides (nt) with a 6-nt insert (tccctc) at nucleotide 5215 within the 5' utr of f gene, comparing to the 15,948-nt-long genome of all the other pprv strains. we identified a total of 150 nucleotide differences between the china 2013-2014 strains and all the other strains. only 45 substitution sites were found in the noncoding region. in total, 105 substitutes were identified in the coding region, among which 39 substitutes were found non-synonymous (ns). the highest ratio of ns substitutes were found in h gene (10 ns sites out of 14 mutation sites), followed by p gene (8/15 sites), n gene (5/11 sites), f gene (4/13 sites), l gene (12/43 sites) and m gene (0/9 sites). the distribution of substitution sites at different codon position was found as following: 21 nucleotide differences (causing 14 amino acid changes) at the first base, 12 nucleotide (12 amino acid) differences at the second base, 72 nucleotide (13 amino acid) changes at the third base. phylogenetic analysis using maximum likelihood revealed that all the pprv genomes from china between 2013 and 2014 could be grouped into a distinct clade in lineage iv. as shown in fig. 2 to analyze the evolutionary rate and the time to the most recent common ancestors (tmrca) of ppr viruses obtained in china, the 25 (fig. 3) . the latest divergence of pprv from xinjiang province to other provinces was dated between the end of october and the end of november in 2013. the earliest divergence of pprv among the other provinces except xinjiang was dated between the middle of november and the end of december in 2013. it is consistent with the one-month time difference between the outbreaks in xinjiang province in december 2013 and the first outbreak outside xinjiang province in the end of january 2014. time-resolved phylogenetic analysis was generated from all pprv genomes from china, 2013-2014. five clusters of sequences could be identified (cluster a-e, fig. 3 ). however, no distinct temporal and spatial distribution was found for samples in each cluster. as an example, cluster d included two samples from xinjiang province in december 2013 (china/xj2/2013 and china/xj4/2013), two samples from northeast area between march and april 2014 (chinahlj2014 and chinajl2014), and three samples from middle area in april 2014 (chinasax2014, chinahen2014 and chinahn2014). median joining phylogenetic analysis implemented by network also showed that all the 25 pprv sequences derived from china, 2013-2014 were mainly grouped in five transmission clusters. all clusters were connected at the base of the network through four internal nodes (mv1, mv2, mv3 and mv4 in fig. 4 ). mv1 was identified as a putative common internal node of all the 25 sequences by comparing these 25 sequences with the most closely related sequence, china/tibet/30/ 2007. china/xjyl/2013, which was sampled from the first reported outbreak during the 2013-2014 epidemic, was found to be most closely related to mv1 by 3 nt substitutions. all sequences in cluster d shared a same putative ancestor of mv3, which was derived from mv1 by 3 nt substitutions. all sequences in cluster e were originated from a putative ancestor of mv4, which was derived from mv3 by 1 nt mutation. it was noteworthy that in each cluster the sequence collected from xinjiang province was one of the most closely related to the putative ancestor. we identified two pairs of samples that were unambiguously connected in the phylogenetic network, which indicated the likely inter-farm transmission events. the first likely inter-farm transmission event involved a farm in hubei province in middle china (chinahb2014) and the other farm in jiangsu province along the east coast of china (chinajs2014), with a distance of 1000 km. the second likely inter-farm transmission event linked one farm in xinjiang province in western china (china/xj5/2013) and the other farm in shanxi province in the middle of china (chinasx2014), with a distance of 3000 km. we estimated the population dynamics of the genome of pprv using bayesian skyline plot (bsp) analysis. the plot depicted a consistent pattern of changes in the effective number of infections (n e t) through time. a sudden increase of viral population was found during january 2014, followed by a stable platform until june 2014 (fig. 5) . here we present the first intra-epidemic analysis of pprv genome by studying viruses from 25 farms infected during the 2013-2014 ppr outbreaks in china. this has demonstrated the potential of such studies to provide in-depth insights into the evolution of pprv in endemic country to achieve effective control measures. the 25 pprv genomes obtained during november 2013 and june 2014 in china showed considerable sequence variety. a total of 96 substitution sites were found along the genome sequences, among which only two sites were found in n gene or f gene partial region. five clusters were confirmed for the phylogeneitc analysis using genome sequences of these strains. however, only two genotypes were found for these strains in n gene or f gene partial region. it is suggested that although n gene or f gene partial sequence was widely used for pprv phylogenetic analysis, whole-genome sequences were more useful for detailed transmission study in a short period of time. sequence comparison showed that the distribution of nucleotide substitution along the pprv genome during this endemic was random. the genetic diversity of china 2013-2014 ppr viruses was mainly due to synonymous mutation. it is noteworthy that the highest ratio of ns substitutes was found in p gene. previous studies revealed that p protein of rinderpest virus played an important role in transcription and replication of the genome (kaushik and shaila, 2004; saikia et al., 2008) . the bottleneck effects occurring during virus transmission may result in the fixation of mutation in p gene. however, the limitation of data scale (only 25 sequences sampled during a short period of 7 months) deduced the failure of selection pressure analysis in this study (data not shown). further research should be conducted to cover longer time scale to reveal the selection pressure on the evolution of pprv in china. our estimation of the evolutionary rate of pprv genome in china during 2013-2014 was 2.61 × 10 −6 nucleotide substitutions per site per day (9.54 × 10 −4 nucleotide substitutions/site/year), which is consistent with previous prediction for the evolutionary rate of other paramyxoviruses (10 −3 -10 −4 nucleotide substitutions per site per year) (chong et al., 2010; furuse et al., 2010) . muniraju et al. estimated that the evolutionary rate of pprv genome was 9.09 × 10 −4 nucleotide substitutions/site/year according to the analysis based on 12 pprv genomes (muniraju et al., 2014c) . the tmrca of ppr viruses collected in china during 2013-2014 is estimated to be in early august 2013, which is three months earlier than the confirmation of the first outbreak in yili region of xinjiang province. it is possible that the virus was transmitted into xinjiang, china and kept undetected for several months. the presence of a single putative common ancestor around august 2013 for all pprv china 2013-2014 genome sequences provided clear evidence for a single introduction of the virus. the same conclusion was drawn by previous phylogenetic analysis on n gene or f gene partial sequence (wu et al., 2015) . phylogenetic analysis based on partial sequence of n gene revealed that china/xjyl/2013 was most closely related to ppr viruses circulating in middle-asian countries (kock et al., 2015; wu et al., 2015) . since no genome sequence of pprv central-asian strain was available, it is not possible to investigate the phylogenomic relationship between pprv china 2013-2014 strains and central-asia strains in this study. more genome sequences of pprv strains circulating in neighboring countries especially in central asia will help locate the origin of pprv china 2013-2014 epidemic. the virus caused two waves of outbreaks after introduction. the first wave of outbreaks was limited in xinjing province between november and december 2013, involving 5 sporadic outbreaks. the second wave of outbreaks started from the end of january 2013 to the beginning of june 2013. during this wave of outbreaks, more than 200 sporadic outbreaks were reported in 21 provinces. it is of question how the virus spread out of xinjiang province and then across most part of china. bsp analysis conducted in this study revealed that a sudden increase of viral population was found during january 2014, which was consistent with the occurrence of the second wave of outbreaks. the timing of this wave coincides with the spring festival (31st january 2014) which is associated with rising of lamb-consuming and therefore increased risk of long-distance animal transmission. field investigation revealed that the infected farms in xinjiang province had intended to fatten the purchased goats and sell to other provinces. in this study, we constructed the transmission pathway of the spread of virus basing on the full-length genome sequences. it revealed that viruses collected from the second wave of outbreaks shared three putative ancestor viruses with those from the first wave of outbreaks. it indicated that the second wave of ppr in china was caused by multi-origin transmission from xinjiang province. recently, much has been conducted to track the high-resolution transmission history of viruses by using full-genome sequencing. transmission network of the middle east respiratory syndrome coronavirus in saudi arabia was reconstructed using full genome sequences cotten et al., 2013) . emergence and evolution of norovirus in sydney was investigated by using genetic sequences (eden et al., 2014) . evolution of rift valley fever virus during an outbreak in kenya during 2006-2007 was described using genome sequences (bird et al., 2008) . in this study, we constructed the transmission pathway of the spread of ppr virus basing on the fulllength genome sequences and identified five clusters of infections. farms in each clusters showed dispersed geographical location, with farm-to-farm distance ranging from 360 km to 3400 km. it indicated that such transmission between farms was caused by long-distance animal movement. putative-ancestor-virus-centered radial shape of each cluster suggested that samples collected from different province might come from a same virus origin. previous epidemiologic analysis suggested that infected animals in different provinces could be traced back to some national livestock markets (fan et al., 2015) . these animals were found bought from the market and transferred thousands of kilometers away to the farms by trucks. the incubation period of ppr is typically 4-6 days, which may range between 3 and 14 days. according to the convenient highway network in china, it is suggested that the infected animals which were during incubation period were transferred from xinjiang province to some national livestock market and then mixed with sheep and goats in the market. it is of most possibility that the suspected animals in the market were infected with ppr and were transferred to the farms in other provinces by longdistance translocation during incubation period. we identified two pairs of samples that were unambiguously connected in the phylogenetic network, which indicated the likely inter-farm transmission events. the virus from chinajs2014 was one nucleotide different from the virus from chinahb2014. it indicated a single farm-to-farm transmission despite the long distance between these two infected farms. the network analysis revealed a number of long branches (more than 10 nt substitutions) between the putative ancestor virus and viruses collected from infected farms, which may indicate the presence of intermediate undetected infected farms. although more than 200 infected farms were reported during 20132014 pprv epidemic in china, only 25 of them were full-genome sequenced and analyzed in this study. another possibility is that long branches were a result of intra-farm evolution of the virus within an infected farm because we analyzed only one sample from each farm. in further research, analysis with samples collected from more infected farms and more samples from one infected farm will provide deeper information on transmission pathway. the possibility of further spread of ppr to neighboring countries is of great concern. among the 14 neighboring countries of china, ppr was endemic in five countries (india, bhutan, nepal, pakistan and afghanistan) in south asia and has been reported in two countries (kazakhstan and tajikistan) in central-asia (banyard et al., 2010; lundervold et al., 2004) . before 2013, no occurrence of ppr was reported in east asia and southeast asia. in september 2016, two outbreaks of ppr were reported to the office internationale des epizooties in western part of mongolia (http://www.oie.int). although no evidence was available to show that the disease in mongolia was transmitted from china, the widespread of ppr in china has put high risk of this disease to the neighboring ppr-free countries. this study showed that full genome sequence analysis can make an important contribution to understanding the intra-epidemic transmission of pprv. analysis of these data provided useful information for effectively control and finally eradication of the disease. this work was financially supported by the national key research and development program of china (2017yfd0502300), the animal diseases surveillance fund of the ministry of agriculture, china (08-52) and a scholarship from the china scholarship council to j.b (20143012). local evolutionary patterns of human respiratory syncytial virus derived from whole-genome sequencing hospital outbreak of middle east respiratory syndrome coronavirus full genome 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molecular evolution of peste des petits ruminants virus molecular evolutionary dynamics of respiratory syncytial virus group a in recurrent epidemics in coastal kenya prevalence, distribution, and host range of peste des petits ruminants virus genetic and epidemiological insights into the emergence of peste des petits ruminants virus (pprv) across peste des petits ruminants selection of models of dna evolution with jmodeltest phosphorylation status of the phosphoprotein p of rinderpest virus modulates transcription and replication of the genome geographic distribution and epidemiology of peste des petits ruminants virus emergence of peste des petits ruminants virus lineage iv in ismailia province mega4: molecular evolutionary genetics analysis (mega) software version 4.0 reconstruction of the transmission history of rna virus outbreaks using full genome sequences: foot-and-mouth disease virus in bulgaria in 2011 peste des petits ruminants virus in heilongjiang province peste des petits ruminants virus in tibet peste des petits ruminants viruses re-emerging in china the collection of the field samples involved in this study was conducted by the national ppr surveillance project. the diagnosis of china 2013-2014 samples was undertaken by the oie ppr reference laboratory located at the exotic diseases research center in china animal health and epidemiology center. key: cord-262441-slh52nxm authors: sakai, yusuke; kawachi, kengo; terada, yutaka; omori, hiroko; matsuura, yoshiharu; kamitani, wataru title: two-amino acids change in the nsp4 of sars coronavirus abolishes viral replication date: 2017-07-21 journal: virology doi: 10.1016/j.virol.2017.07.019 sha: doc_id: 262441 cord_uid: slh52nxm infection with coronavirus rearranges the host cell membrane to assemble a replication/transcription complex in which replication of the viral genome and transcription of viral mrna occur. although coexistence of nsp3 and nsp4 is known to cause membrane rearrangement, the mechanisms underlying the interaction of these two proteins remain unclear. we demonstrated that binding of nsp4 with nsp3 is essential for membrane rearrangement and identified amino acid residues in nsp4 responsible for the interaction with nsp3. in addition, we revealed that the nsp3-nsp4 interaction is not sufficient to induce membrane rearrangement, suggesting the participation of other factors such as host proteins. finally, we showed that loss of the nsp3-nsp4 interaction eliminated viral replication by using an infectious cdna clone and replicon system of sars-cov. these findings provide clues to the mechanism of the replication/transcription complex assembly of sars-cov and could reveal an antiviral target for the treatment of betacoronavirus infection. severe acute respiratory syndrome (sars) coronavirus (sars-cov) is the etiological agent of sars, an emerging life-threatening disease characterized by high fever, myalgia, nonproductive cough and dyspnea (booth et al., 2003; drosten et al., 2003; ksiazek et al., 2003; tsang et al., 2003) . sars was first recognized in february 2003, in the midst of a worldwide epidemic that resulted in 8096 patients and 776 deaths, with the last patient being reported in (de wit et al., 2016 . in 2012, nearly a decade after the sars epidemic, a novel coronavirus, the middle east respiratory syndrome (mers) coronavirus (mers-cov), emerged in saudi arabia (zaki et al., 2012) and has since spread to other countries in the middle east, north africa, europe and asia. sars-cov belongs to the genus betacoronavirus in the family coronaviridae, and is an enveloped virus with a positive-sense and single-stranded rna genome that has 5'capped and 3' poly (a)containing 14 open reading frames (orfs) (thiel et al., 2003) . the genomic size of sars-cov is extremely large among rna viruses and the 5' two-thirds of the genomic rna has two partially overlapping orfs, 1a and 1b. upon infection, the genomic rna is translated into two large polyproteins and then proteolytically cleaved into 16 mature viral proteins, nsp1 to nsp16 (prentice et al., 2004; thiel et al., 2003) . infection with most of the positive-stranded rna viruses induces rearrangement of host cell membranes that serve as the platform for a viral replication complex called the replication and transcription complex (rtc) (den boon and ahlquist, 2010; miller and krijnse-locker, 2008) . the membrane remodeling and consequent rtc formation induced by virus infection are critical for creating a site to support viral rna synthesis and for protecting newly synthesized rna from host innate immune systems (den boon and ahlquist, 2010) . like infection with other positive-stranded rna viruses, infection with coronaviruses, including sars-cov and murine hepatitis virus (mhv), also induces replication-associated membrane structures, which in the case of betacoronavirus infection consist of two interconnected membrane structures, double-membrane vesicles (dmvs) and convoluted membranes (cms) (goldsmith et al., 2004; gosert et al., 2002; knoops et al., 2008; snijder et al., 2006) . furthermore, infection with avian infectious bronchitis virus (ibv) induces a spherular structure derived from the endoplasmic reticulum (er), which has not been identified in cells infected with other coronavirus species (maier et al., 2013) . electron microscopy analysis of cells infected with coronavirus reveals that dmvs and cms localize to the proximity of the er and partially interconnect with the er, suggesting that the dmvs and cms are derived from host er membranes, despite the lack of canonical er membrane markers (gosert et al., 2002; hagemeijer et al., 2014; knoops et al., 2008; snijder et al., 2006; ulasli et al., 2010) . among the 16 nsps of coronavirus, nsp3, nsp4, and nsp6 have multiple transmembrane domains (harcourt et al., 2004; oostra et al., 2008; ratia et al., 2008) . although expression of nsp3, nsp4, and nsp6 is observed in rtc upon infection, it is localized in the er in cells individually expressed by the transfection with expression plasmids (kanjanahaluethai et al., 2007; oostra et al., 2008 oostra et al., , 2007 . in the case of mhv, interaction of nsp4 and the c-terminal one-third of nsp3 (nsp3c) was observed by immunoprecipitation analysis (hagemeijer et al., 2011) , and these proteins were colocalized in the perinuclear foci in cells upon co-transfection with expression plasmids, similar to those observed in infected cells (hagemeijer et al., 2014 (hagemeijer et al., , 2011 . these data suggest that coexpression of nsp3 and nsp4 results in a membrane rearrangement to induce dmvs and cms (hagemeijer et al., 2014) . mhv nsp4 is a glycosylated protein, and the glycosylated nsp4 is important for the virus-induced membrane rearrangement and the replication complex function and assembly of dmvs (clementz et al., 2008; gadlage et al., 2010) . analyses using confocal and electron microscopy have shown that the expression of nsp3, nsp4, and nsp6 of sars-cov induces formation of dmvs in the transfected cells (angelini et al., 2013) . however, limited information is available about the mechanism of interaction between nsp3 and nsp4 of sars-cov or the roles of such interaction on viral replication through the formation of membrane rearrangements. in this study, we demonstrated that the interaction of nsp3 with nsp4 plays a critical role in the replication of sars-cov through the rearrangements of host-derived membranes, suggesting that the membrane rearrangement induced by the nsp3-nsp4 interaction is crucial for optimal replication of sars-cov. 2.1. co-expression of nsp3c and nsp4 induces redistribution from the er to concentrated foci in the perinuclear region in previous reports, co-expression of sars-cov nsp4 with the cterminal one-third of nsp3 (nsp3c) in the cell was shown to redistribute these proteins from the er to concentrated foci in the perinuclear region (hagemeijer et al., 2011) . to confirm this redistribution of the proteins, we constructed the expression plasmids pcag nsp4-ha and pcag nsp3c-3xflag carrying an ha-tag and 3xflag tag in the c-termini of each protein, respectively (fig. 1a) . subcellular localization of nsp4 and nsp3c in 293t cells transfected with pcag nsp4-ha and/or pcag nsp3c-3xflag was determined by immunofluorescence assay (ifa) using anti-ha and anti-flag antibodies. although nsp3c and nsp4 were localized in the er when expressed individually, both nsp3c and nsp4 were detected in discrete foci in the perinuclear region in cells upon co-expression ( fig. 2a , b top panel), confirming that coexpression of nsp4 with nsp3c of sars-cov induces redistribution of the proteins from the er to concentrated foci in the perinuclear region. amino acid sequences of mhv and sars-cov predicted that the nsp4 of both viruses has four transmembrane domains. previous studies have demonstrated that an nsp4 deletion mutant of mhv lacking the first transmembrane domain did not re-localize upon co-expression with mhv nsp3c (hagemeijer et al., 2014) . to further determine the molecular mechanisms of the interaction between nsp4 and nsp3c of sars-cov to induce the perinuclear foci, expression plasmids encoding deletion mutants of nsp4, pcag nsp4 1-274-ha and pcag nsp4 1-313-ha (fig. 1b) were transfected together with pcag nsp3c-3xflag into 293t cells (fig. 2b 2nd and 3rd panels) . subcellular localization of nsp4 and nsp3c was examined by using anti-ha and anti-flag antibodies, respectively, at 30 h posttransfection. co-expression of nsp3c with nsp4 deletion mutants missing the third and fourth (nsp4 1-313-ha) or second to fourth transmembrane regions (nsp4 1-274-ha) exhibited re-localization to the concentrated foci ( fig. 2b 2nd and 3rd panels), suggesting that the large lumenal loop between the first and second transmembrane regions of nsp4 of sars-cov is responsible for induction of the concentrated foci upon co-expression with nsp3c. 2.3. two domains in the large lumenal loop of nsp4 are essential for induction of the concentrated foci upon co-expression with nsp3c to determine the region(s) in the large lumenal loop of sars-cov nsp4 responsible for re-localization upon co-expression with nsp3c, expression plasmids encoding deletion in the large lumenal loop of nsp4, pcag nsp4 δ112-164-ha, pcag nsp4 δ164-197-ha, and pcag 293t cells were transfected with either pcag nsp3c-3xflag or pcag nsp4-ha and then fixed with 4% pfa at 30 h posttransfection. nsp3c and nsp4 were stained with rabbit anti-ha antibody and mouse anti-flag antibody, followed by cf594-conjugated anti-mouse igg and cf488-conjugated anti-rabbit igg, respectively. cell nuclei were stained with hoechst 33342. (b) co-expression of wild type nsp4, nsp4 1-274, nsp4 1-313, nsp4 δ112-164, nsp4 δ164-197 or nsp4 δ220-234 together with nsp3c. at 30 h posttransfection, the cells were fixed with 4% pfa and stained as described above. white arrows represent the concentrated foci. y. sakai et al. virology 510 (2017) [165] [166] [167] [168] [169] [170] [171] [172] [173] [174] nsp4 δ220-234-ha (fig. 1b) were transfected together with pcag nsp3c-3xflag into 293t cells (fig. 2b 4th, 5th, and bottom panels). subcellular localization of nsp4 and nsp3c was examined at 30 h posttransfection. co-expression of nsp3c with an nsp4 deletion mutant lacking the region from 164 to 197aa but not with mutants lacking the regions from 112 to 164aa or from 220 to 234aa exhibited relocalization to the concentrated foci ( fig. 2b 4th, 5th, and bottom panels), suggesting that the large lumenal loop of sars-cov nsp4 has at least two functional domains responsible for induction of the concentrated foci upon co-expression with nsp3c. 2.4. amino acid residues from positions 112-164 in the large lumenal loop of nsp4 are responsible for binding to nsp3c to determine the binding region in sars-cov nsp4 responsible for the interaction with nsp3c in more detail, 293t cells transfected with either pcag nsp4 δ112-164-ha, pcag nsp4 δ164-197-ha, or pcag nsp4 δ220-234-ha together with pcag nsp3c-3xflag were lysed at 30 h posttransfection and subjected to immunoprecipitation analysis by using anti-ha antibody. the wild type nsp4, nsp4 δ164-197 and nsp4 δ220-234 but not nsp4 δ112-164 were co-immunoprecipitated with nsp3c ( fig. 3a) , suggesting that the amino acid residues spanning from 112 to 164 in nsp4 are responsible for binding to nsp3c. to further confirm the binding of nsp4 to nsp3c in situ, a proximity ligation assay (pla) based on antibodies tagged with a circular dna probe was performed by using a duolink® in situ pla kit (invitrogen). once the antibodies are in close proximity, the probes can be ligated together and then amplified with a polymerase. the pla signals were detected in the cytoplasm of cells co-expressing nsp3c with either wild type nsp4, nsp4 δ164-197 or nsp4 δ220-234 but not with nsp4 δ112-164 at 30 h posttransfection (fig. 3b ), supporting the notion that the amino acid residues from 112 to 164 of sars-cov nsp4 are responsible for the interaction with nsp3c. interestingly, the nsp4 δ220-234 failed to induce re-localization upon co-expression with nsp3c ( fig. 2b bottom panel), but interaction with nsp3c was detected by using immunoprecipitation and pla assays ( fig. 3a and b), indicating that the interaction between nsp4 and nsp3c was not sufficient to induce the re-location concentrated foci. these results suggest that another host or viral factor(s) is involved in the re-location concentrated foci induced by interaction between nsp4 and nsp3. 2.5. h120 and f121 in nsp4 are crucial to induce membrane rearrangements to determine the crucial amino acid residue(s) in sars-cov nsp4 required to induce membrane rearrangements through the interaction with nsp3c, we constructed additional expression plasmids encoding deletion mutants of sars-cov nsp4, pcag nsp4 δ112-126-ha and pcag nsp4 δ126-164-ha, as shown in fig. 1b . immunoprecipitation analysis showed that the amino acid residues from 112 to 126 in nsp4 are important for binding to nsp3c (fig. 4a) . moreover, the alignment of amino acid sequences corresponding to the amino acids from 112 to 126 in nsp4 of sars-cov revealed that two amino acid residues, h120 and f121, were conserved among betacoronaviruses that included bovine coronavirus (bcov), mhv, and mers-cov (fig. 4b ). next, to determine the role of h120 and f121 in nsp4 on the interaction with nsp3c, the expression plasmid pcag nsp4 h120n/f121l-ha, in which h120 and f121 were replaced with n and l, respectively, was co-transfected with pcag nsp3c-3xflag into 293t cells. immunoprecipitation analysis showed that nsp4 h120n/f121l abrogated binding to nsp3c (fig. 4a ) and ifa showed that nsp4 h120n/ f121l lost the ability to re-localize concentrated foci upon co-expression with nsp3c ( fig. 4c ). western blot analysis showed that the amounts of nsp4 h120n/f121l in cells co-transfected with nsp3c were comparable to those of nsp4-wt in co-transfected with nsp3c ( fig. 4d) . to determine the effect of the two amino acid residues, h120 and f121, in sars-cov nsp4 on the membrane rearrangements thorough interaction with nsp3c, 293t cells transfected with pcag nsp4-ha or pcag nsp4 h120n/f121l-ha together with pcag nsp3c-3xflag at 30 h posttransfection were subjected to transmission electron microscopy (tem) analysis. tem analysis revealed that cm-like structures were detected in cells co-expressing nsp3c with nsp4 but not with nsp4 h120n/f121l ( fig. 5a and b) . these results suggest that the amino acid residues h120 and f121l in sars-cov nsp4 play crucial roles in the membrane remodeling through their interaction with nsp3c. to determine the effect of the two amino acid residues, h120 and f121, in sars-cov nsp4 on the viral propagation, we generated a mutant virus possessing substitutions of h120 and f121 to n and l, respectively, by using a reverse genetics system based on the bacterial artificial chromosome (bac), as described previously (almazán et al., 2006) . briefly, cdna of sars-cov was assembled in the bac under the control of the cytomegalovirus immediate-early promoter and flanked at the 3'end by 25-bp of poly(a) followed by an hdv ribozyme and a bgh termination sequence (almazán et al., 2006) , and designated pbac-sars-wt. a mutant clone, pbac-sars-h120n/f121l, was generated by introducing an h120n/f121l substitution in nsp4 into the parental pbac-sars-wt (fig. 6a) . to determine the effect of h120n/f121l mutation in nsp4 on the infectious particle formation and viral rna replication of sars-cov, infectious titers in the culture supernatants and intracellular viral rna in huh7 cells transfected with either pbac-sars-wt or pbac-sars-h120n/f121l were determined at 72 h posttransfection (fig. 6b) . although the infectious titers in the supernatants reached 5.62e+04 tcid50/ml and the amounts of viral rna determined by real-time pcr were increased in a time-dependent manner in cells transfected with pbac-sars-wt, no infectious titer in the supernatants and no replication of intracellular viral rna was detected in those transfected with pbac-sars-h120n/f121l ( fig. 6c and d). these results suggest that the amino acid residues h120 and f121 in nsp4 play an important role in viral rna replication through the membrane remodeling. next, to confirm the importance of h120 and f121 in nsp4 on the replication of sars-cov, we inserted a reporter gene into pbac-sars-h120n/f121l. briefly, the accessory gene 7ab in pbac-sars-h120n/ f121l was replaced with renilla luciferase and firefly luciferase genes by using a red/et recombination system counter-selection bac modification kit, to obtain pbac-sars-h120n/f121l-rluc and pbac-sars-h120n/f121l-fluc, respectively (fig. 6e) . as a control, pbac-sars-wt-fluc, in which the 7ab gene was replaced with a firefly luciferase gene, was generated. pbac-sars-h120n/f121l-rluc was transfected into 293t cells together with either pbac-sars-wt-fluc or pbac-sars-h120n/f121l-fluc, and the luciferase activities in cell lysates were determined at 24 h posttransfection (fig. 6f) . the expressions of renilla luciferase and firefly luciferase were detected in cells transfected with pbac-sars-h120n/f121l-rluc together with pbac-sars-wt-fluc but not with pbac-sars-h120n/f121l-fluc (fig. 6g) . these results suggest that propagation of the replicationdeficient mutant sars-cov possessing a substitution of h120n/f121l in nsp4 was rescued by the co-infection with a wild type sars-cov. to determine the biological significance, with respect to viral rna replication, of the membrane remodeling induced by the interaction of nsp4 with nsp3c, we employed an rna replicon system in which sars-cov rna replicates but does not produce any infectious particles, as described previously (almazán et al., 2006; tanaka et al., 2012) . we introduced h120n/f121l mutations in nsp4 of the parental replicon, pbac-sars-rep-wt, to generate pbac-sars-rep-h120n/f121l (fig. 7a) . as a negative control, we generated pbac-sars-rep-sad carrying a mutation in nsp12 deficient in rnadependent rna polymerase activity by the replacement of d760 to a760 (subissi et al., 2014) (fig. 7a) . northern blot analysis of 293t cells at 24 h and 36 h posttransfection with pbac-sars-rep-wt, pbac-sars-rep-sad or pbac-sars-rep-h120n/f121l using an n gene-specific probe revealed that no mrna was detected in cells transfected with either pbac-sars-rep-h120n/f121l or pbac-sars-rep-sad, in contrast to those transfected with pbac-sars-rep-wt (fig. 7b) . to further confirm the impairment of replication of the replicon carrying mutations in h120 and f121, the levels of renilla luciferase in 293t cells transfected with pbac-sars-rep-wt, pbac-sars-rep-sad or pbac-sars-rep-h120n/f121l were determined at 24 h and 36 h posttransfection. as we expected, expression of renilla luciferase was detected in cells transfected with pbac-sars-rep-wt, but not in those with pbac-sars-rep-h120n/f121l or pbac-sarsrep-sad (fig. 7c) , suggesting that both h120 and f121 in sars-cov nsp4 play critical roles in the viral replication by remodeling the membrane through binding with nsp3. like other positive-strand rna viruses, coronaviruses induce a variety of membrane rearrangement structures (goldsmith et al., 2004; gosert et al., 2002; snijder et al., 2006) , including the dmvs and cms that serve as a scaffold for replication/transcription complexes (hagemeijer et al., 2014; maier et al., 2013) . upon sars-cov infection, viral rna is translated into 16 mature viral proteins, nsp1 to nsp16, by processing with two viral proteinases (prentice et al., 2004) , and some of them localize to dmvs and cms (knoops et al., 2008) . in sars-cov, nsp3, nsp4 and nsp6 contain multiple hydrophobic and membrane-spanning domains (hagemeijer et al., 2014) , and co-expression of nsp3, nsp4 and nsp6 was shown to result in the formation of dmvs and cms (angelini et al., 2013) . in addition, hagemeijer et al. showed that co-expression of nsp4 with the cterminal one-third of nsp3 re-translocates both proteins into concentrated foci that predominantly localize in the perinuclear region (hagemeijer et al., 2011) . further, the large lumenal loop between the first and second transmembrane domains of mhv nsp4 was shown to be essential for the induction of membrane rearrangement (hagemeijer et al., 2014) . although many studies have investigated the role of the co-expression of nsp3 and nsp4 in inducing host membrane rearrangement the molecular mechanisms underlying the interaction of nsp3 with nsp4 and the role of this interaction in viral replication are largely unknown. in the present study, we identified the crucial amino acid residues in nsp4 responsible for the interaction with nsp3 and demonstrated that the membrane rearrangement induced by the interaction of nsp3 with nsp4 is crucial for formation of viral replication complexes. we also showed by means of immunoprecipitation and pla assays that the amino acids residues from positions 112-164 and from 220 to 234 of nsp4 are critical for the induction of membrane rearrangement upon co-expression with nsp3, and the amino acids residues from positions 112-164 of nsp4 form the binding site with nsp3. sparks et al. demonstrated that both the first and second lumenal loops of nsp4 are critical for viral replication by using an mhv infectious clone (sparks et al., 2007) . although the second lumenal loop of nsp4 does not participate in the membrane rearrangement (hagemeijer et al., 2014) , the role of the lumenal loops in the viral replication is still unclear. the roles of the glycosylation in the first lumenal loop of nsp4 on the membrane rearrangement have been well studied. mhv has two glycosylation sites (n176 and n237) and sars-cov has one glycosylation site (n131) (clementz et al., 2008; gadlage et al., 2010; oostra et al., 2008) . interestingly, morphologically aberrant dmvs were observed in cells upon infection with a mutant mhv possessing nsp4 with no glycosylation site and rna replication was impaired (beachboard et al., 2015; gadlage et al., 2010) . although we did not examine the effect of mutation of n131 in nsp4 on the membrane rearrangement and viral propagation in this study, the h120n/f121l mutations in nsp4, which are distant from the n131 glycosylation site, induced a complete loss of membrane rearrangement and impairment of viral rna replication, suggesting that the specific interaction of nsp4 with nsp3 plays roles in viral replication. in addition, substitution of the conserved cysteine residues to serine affected the membrane rearrangement of mhv nsp4 (hagemeijer et al., 2014) . although we did not focus on the cysteine residues in sars-cov nsp4 in this study, we did note that three cysteines between 220 and 234 were well conserved in coronaviruses including mhv, sars-cov and coronaviruses in other genera. because nsp4 δ 220-234 was capable of binding to nsp3c, the nsp4-nsp3 interaction is likely independent of cysteine-mediated interaction. further studies are needed to understand the role of the cysteine residues in nsp4 of sars-cov on viral replication. many viruses utilize host factors that are involved in the intracellular transport of virus-induced vesicles (den boon and ahlquist, 2010; miller and krijnse-locker, 2008) . the vesicle coat proteins known as copii proteins transport proteins from the rough er to the golgi apparatus, and are involved in the production of vesicles in cells infected with poliovirus (pv) (rust et al., 2001) . adp-ribosylation factors, which play a role in the regulation of membrane dynamics and protein transport, are localized to the pv-induced membranes in cells infected with pv (belov et al., 2007) . vesicle-associated-membrane protein-associated proteins (vaps) remodel the er by interacting with nir2 to mediate formation of hcv-induced membranes (moriishi and matsuura, 2007) . although the precise interaction of the coronavirus rtc with host proteins is largely unknown, erad tuning pathway is suggested to be involved in the rtc formation of mhv (hagemeijer et al., 2014; reggiori et al., 2010) , since rtc proteins coexist with the erad regulator proteins edem1 and os9, and the erad machinery mediated by erad regulator proteins participates in the formation of lc3-i-positive rtc (noack et al., 2014; reggiori et al., 2010) . in this study, we found that the nsp4 mutant lacking the amino acid residues from positions 220-234 is capable of binding to nsp3c but lacks induction of re-localization ( fig. 3 and 4) and membrane rearrangement (fig. 5) , suggesting that other host or viral factor(s), such as edem1 and os9, participate in the membrane rearrangement induced by the interaction between nsp4 and nsp3. in addition, we identified two amino acids residues (h120 and f121) in nsp4 that are well conserved among betacoronaviruses, including mers-cov, but not among alphacoronaviruses or gammacoronaviruses. these residues were essential for the membrane rearrangements induced by the interaction with nsp3, suggesting that the mode of interaction of nsp4 with nsp3 differs among the different types of coronaviruses. in fact, cells infected with infectious bronchitis virus (ibv), which belongs to gammacoronavirus, induce structures called zippered er and spherules in addition to dmvs (maier et al., 2013) . such morphological difference of virus-induced membrane structures among coronaviruses may be due to functional and structural difference of nsp3 and nsp4. further studies will be needed to elucidate the mode of interaction of nsp4 with nsp3 differs among the different types of coronaviruses. rna replicon and infectious cdna carrying the replacement of h120n/f121l in nsp4 were lethal, suggesting that nsp3-nsp4 binding and induction of the membrane rearrangements correlates with viral propagation. moreover, we showed that co-infection with a wild type virus rescued the replication of the replication-deficient sars-cov carrying a mutation in nsp4, suggesting that the membrane rearrangements inducing by the nsp3-nsp4 interaction are necessary to provide a site for the viral genome replication. two cysteine proteases, nsp3 and nsp5 of sars-cov, participate in processing of the polyprotein to release other nsps, and chemical compounds that have been shown to suppress the proteinase activities (konno et al., 2016; ratia et al., 2008; thanigaimalai et al., 2013) . therefore, while further studies will be needed to elucidate the molecular mechanisms underlying nsp4-mediated membrane rearrangements, our data provide important information about a function of such rearrangements-namely, the nsp4 mutations h120n and f121l impair viral rna replication by impairing the membrane rearrangements. therefore, interventions to block the interaction of nsp4 with host or viral proteins might be a novel target for the development of antivirals for betacoronaviruses. cultures of 293t (human kidney), bhk-21 (hamster kidney), huh7 (human hepatocellular carcinoma) and veroe6 (monkey kidney) cells were maintained in dulbecco's modified minimum essential medium (dmem) (nacalai tesque, kyoto, japan) containing 10% heat-inactivated fetal bovine serum (fbs), 100 u/ml penicillin, and 100 mg/ml streptomycin. all cells were cultured in a humidified 5% co2 atmosphere at 37°c. the plasmids were transfected into 293t cells by use of polyethyleneimine (pei) (polysciences, warrington, pa). at 4 h posttransfection, the media were replaced with fresh dmem containing 10% fbs. the pcr products of sars-cov nsp3c with a c-terminal 3×flagtag sequence and sars-cov nsp4 with a c-terminal ha-tag sequence were cloned into pcaggs-mcs, yielding pcag nsp3c-3×flag and pcag nsp4-ha, respectively. an inverse pcr procedure using pcag nsp4-ha as the template was employed to generate pcag nsp4δ220-234-ha, pcag nsp4δ164-197-ha, pcag nsp4 δ112-164-ha, and pcag nsp4 h121n/f122l-ha by using a kod mutagenesis kit (toyobo, osaka, japan). the sequences of all of the constructs were confirmed with an abi prism 3100 genetic analyzer (applied biosystems, tokyo, japan). 293t cells cultured on poly-l-lysine (pll)-coated glass bottom dishs (matsunami glass ind. ltd., osaka, japan) were cotransfected with pcag nsp3c-3×flag together with the indicated expression plasmids of nsp4. at 30 h posttransfection, the cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (pbs) for 30 min at 4°c. after washing one time with pbs, the cells were permeabilized for 15 min at room temperature with pbs containing 0.1% tween-20. the cells were then incubated with a mixture of rabbit anti-ha antibody (1:300; mbl, aichi, japan) and mouse anti-dykddddk (flag) antibody (1:300; wako, osaka, japan) for 1 h at rt, washed three times with pbs, and incubated with a mixture of cf488-conjugated anti-rabbit igg (1:500; sigma, st. louis, mo), cf594-conjugated antimouse igg (1:500; sigma), and hoechst 33342 (dojindo, kumamoto, japan) for 1 h at rt. finally, the cells were washed three times with pbs and observed with a fluoview fv1000 laser scanning confocal microscope (olympus, tokyo, japan). 293t cells were cotransfected with pcag nsp3-3×flag together with the indicated expression plasmids of nsp4. at 32 h posttransfection, the cells were lysed with lysis buffer (10 mm hepes, ph 7.4, containing 150 mm nacl and 0.5% tritonx-100). the cell lysates were incubated with mouse anti-ha antibody (covance, richmond, ca) for 1 h at 4°c, and the immunoprecipitates were collected by centrifugation after incubation with protein a/g plus-agarose (santa cruz biotechnology, santa cruz, ca) for 1 h at 4°c. the immunoprecipitates were incubated for 15 min at 42°c in laemmli sample buffer and then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. the proteins were transferred to polyvinylidene difluoride membranes (millipore, bedford, ma) and then incubated with rabbit anti-ha antibody (1:2000; mbl) or rabbit anti-flag antibody (1:2000; mbl) for 16 h at 4°c. after washing three times, the membranes were incubated with horseradish peroxidase (hrp)-conjugated anti-rabbit igg (sigma) for 1 h at rt. the immunocomplexes were visualized with chemi-lumi one ultra (nacalai tesque) and detected by using las-4000epuv (fujifilm, tokyo, japan). 293t cells were cultured on pll-coated glass bottom dishs, cotransfected with pcag nsp3c-3×flag together with the indicated expression plasmids of nsp4, fixed with 4% pfa in pbs, permeabilized with pbs containing 0.1% tween-20, and then incubated with primary antibodies as described above. an in situ proximity ligation assay (pla) was performed using a duolink® in situ pla kit (invitrogen, carlsbad, ca) according to the manufacturer's instructions. briefly, after washing three times, the samples were incubated with anti-mouse plus (invitrogen) and anti-rabbit minus (invitrogen) for 1 h at rt. after washing two times, a ligase solution (invitrogen) was applied for 30 min at 37°c. the samples were washed and a mixture of amplifica-tion green solution (invitrogen) and polymerase solution (invitrogen) was applied for 1 h at 37°c. the samples were then washed a final time, mounted using mounting medium with dapi (invitrogen) and examined with a fluoview fv1000 laser scanning confocal microscope (olympus). 293t cells were cultured on a gridded glass bottom dish (mattek, ashland, ma) and transfected with pcag nsp4-ha and pcag nsp3c-3×flag as described above. at 30 h posttransfection, the cells were fixed with 2.5% glutaraldehyde in 0.1 m cacodylate buffer (ph 7.4) containing 7% sucrose. the cells were postfixed for 1 h with 1% osmium tetroxide and 0.5% potassium ferrocyanide in 0.1 m cacodylate buffer (ph 7.4), dehydrated in a graded series of ethanol, and embedded in epon812 (taab, reading, uk). ultrathin (80 nm) sections were stained with saturated uranyl acetate and lead citrate solution. electron micrographs were obtained with a jem-1011 transmission electron microscope (jeol, tokyo, japan). the bac clone carrying a full-length infectious genome of the urbani strain of sars-cov, pbac-sars-wt, was generated according to a previously report (almazán et al., 2006) . the bac dna of sars-cov-rep, which was kindly provided by luis enjuanes, was used as a backbone bac sequence to generate pbac-sars-wt. the bac infectious clone carrying two amino acid substitutions, h120n and f121l, in nsp4 was generated by modification of the pbac-sars-wt as a template using a red/et recombination system counter-selection bac modification kit (gene bridges, heidelberg, germany), yielding pbac-sars-h120n/f121n. the sequences of the two introduced mutations were confirmed as described above. the recombinant viruses were recovered according to established protocols, as described previously (almazán et al., 2006) . virus infectivity was determined by a 50% tissue culture infectious dose (tcid 50 ) tcid50 assay with vero cells. briefly, the viruses were serially diluted and inoculated onto monolayers of vero cells. after 1 h of absorption, the cells were washed with serum-free dmem and cultured in dmem containing 1% fbs. at 72 h postinfection, cells were fixed with 10% formaldehyde neutral buffer solution, and tcid 50 _ was calculated by the spearman and karber algorithm. total rna was prepared from cells by using a purelink rneasy mini kit (thermofisher scientific), and first-strand cdna was synthesized using a revertra ace qpcr rt kit (toyobo). the level of each cdna was determined by using thunderbird sybr qpcr mix (toyobo), and fluorescent signals were analyzed by using an abi prism 7000 system (applied biosystems). the sars-cov n and gapdh genes were amplified using the following primer pairs: 5'tgggtccaccaaatgtaatgc -3' and 5'-aagccaaccaacctcgatctc -3' for sars-cov n and 5'-gaaggtgaaggtcggagt-3' and 5'-gaagatggtgatgggatttc3' for gapdh. the value of sars-cov n mrna was normalized to that of gadh mrna. the construction of the sars-cov-derived replicon carrying a renilla luciferase reporter gene (rluc), pbac-sars-rep-wt (previously referred to as pbacwt-rluc), has been described elsewhere (almazán et al., 2006; tanaka et al., 2012) . the bac dna of sars-cov-rep, which was kindly provided by luis enjuanes, was used as a backbone bac sequence to generate pbac-wtrluc. a red/et recombination system counter-selection bac modification kit (gene bridges) was used to generate mutations (h120n and f121l) in nsp4 and a mutation (d760a) in nsp12 by using pbac-sars-rep-wt as a template, yielding pbac-sars-rep-h120n/f121l and pbac-sars-rep-sad. the sequences of the two introduced mutations were confirmed with an abi prism 3100 genetic analyzer, as described above. lysates of 293t cells transfected with the indicated bac dna were lysed with passive lysis buffer (promega). luciferase activity was determined by using a dual-luciferase assay system (promega, madison, wi) and an ab-2200 luminometer (atto, tokyo, japan). northern blot analysis was performed by using total intracellular rnas as described previously (tanaka et al., 2012) , and visualization was performed using a digoxigenin (dig) luminescence detection kit (roche). construction of a severe acute respiratory syndrome coronavirus infectious cdna clone and a replicon to study coronavirus rna synthesis severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce doublemembrane vesicles mutations across murine hepatitis virus nsp4 alter virus fitness and membrane modifications hijacking components of the cellular secretory pathway for replication of poliovirus rna clinical features and 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reticulovesicular network of modified endoplasmic reticulum design and synthesis of a series of serine derivatives as small molecule inhibitors of the sars coronavirus 3cl protease a novel coronavirus associated with severe acute respiratory syndrome infectious bronchitis virus generates spherules from zippered endoplasmic reticulum membranes modification of intracellular membrane structures for virus replication host factors involved in the replication of hepatitis c virus how viruses hijack the erad tuning machinery topology and membrane anchoring of the coronavirus replication complex: not all hydrophobic domains of nsp3 and nsp6 are membrane spanning localization and membrane topology of coronavirus nonstructural protein 4: involvement of the early secretory pathway in replication identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins a noncovalent class of papain-like protease/deubiquitinase inhibitors blocks sars virus replication coronaviruses hijack the lc3-i-positive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication cellular copii proteins are involved in production of the vesicles that form the poliovirus replication complex ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex genetic analysis of murine hepatitis virus nsp4 in virus replication one severe acute respiratory syndrome coronavirus protein complex integrates processive rna polymerase and exonuclease activities severe acute respiratory syndrome coronavirus nsp1 facilitates efficient propagation in cells through a specific translational shutoff of host mrna development of potent dipeptide-type sars-cov 3cl protease inhibitors with novel p3 scaffolds: design, synthesis, biological evaluation, and docking studies mechanisms and enzymes involved in sars coronavirus genome expression a cluster of cases of severe acute respiratory syndrome in hong kong qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia we thank ms. kaede yukawa for secretarial assistance, ms. kanako yoshizawa for technical assistance, dr. shinji makino (university of texas medical branch) for helpful insights, drs. makoto sugiyama and naoto ito (gifu university) for kindly providing research reagents, and drs. luis enjuanes and marta l. dediego for kindly providing a sars-cov-rep. wk was supported by a grant-in-aid for scientific research (16k08811) and by funds from the takeda science foundation. key: cord-265173-70wyecwj authors: trujillo-uscanga, adrian; gutiérrez-escolano, ana lorena title: host cell p53 associates with the feline calicivirus major viral capsid protein vp1, the protease-polymerase ns6/7, and the double-stranded rna playing a role in virus replication date: 2020-08-27 journal: virology doi: 10.1016/j.virol.2020.08.008 sha: doc_id: 265173 cord_uid: 70wyecwj p53 is implicated in several cellular pathways such as induction of cell-cycle arrest, differentiation, senescence, and apoptosis. p53 is activated by a broad range of stress signals, including viral infections. while some viruses activate p53, others induce its inactivation, and occasionally p53 is differentially modulated during the replicative cycle. during calicivirus infections, apoptosis is required for virus exit and spread into the host; yet, the role of p53 during infection is unknown. by confocal microscopy, we found that p53 associates with fcv vp1, the protease-polymerase ns6/7, and the dsrna. this interaction was further confirmed by proximity ligation assays, suggesting that p53 participates in the fcv replication. knocked-down of p53 expression in crfk cells before infection, resulted in a strong reduction of the non-structural protein levels and a decrease of the viral progeny production. these results indicate that p53 is associated with the viral replication complex and is required for an efficient fcv replication. feline calicivirus (fcv) belongs to the vesivirus genus of the caliciviridae family, which currently comprises eleven genera containing viruses that infect a wide range of vertebrates (vinjé et al., 2019) . caliciviruses have a single-stranded, positive-sense rna genome (grna) of 6.4-8.5 kilobases (kb) in length, which is covalently attached to the viral protein genome-linked (vpg) in its 5' end and is polyadenylated. fcv genomic rna (grna) is composed of 3 open reading frames (orfs) . the expression of the orf1 results in a polyprotein that is processed by the viral protease-polymerase, the non-structural protein (ns) 6/7, to generate the six non-structural viral proteins (ns1-ns6/7). both orf 2 and 3 encode for the structural proteins; the orf2 gives rise to the precursor of the major capsid protein vp1, which is further cleaved by the ns6/7 protease-polymerase to generate the leader of the capsid (lc) protein and the mature vp1, whereas the orf3 encodes for the minor capsid protein vp2 necessary to produce infectious viral particles (herbert, brierley, & brown, 1996) (s. v. sosnovtsev, belliot, chang, onwudiwe, & green, 2005) ; both vp1 and vp2 are translated from a subgenomic rna (sgrna). fcv uses the junctional adhesion molecule 1 (f-jam1) as its functional receptor (makino et al., 2006) , enters its target cells through clathrin-mediated endocytosis (stuart & brown, 2006) followed by the release of the genomic rna into the cytosol where it is immediately translated to produce the ns proteins, essential for the replication complexes (rcs) formation (bailey et al., 2010) . rcs are membranous-derived compartments where the synthesis of both the grna and the sgrna occurs. once synthesized, the sgrna is promptly translated to produce the structural proteins; therefore, these membranous components contain high j o u r n a l p r e -p r o o f amounts of viral rna and proteins, being vp1 the most abundant viral protein (green et al., 2002) . fcv viral capsid is composed of 90 dimers of vp1 and a few copies of vp2. both structural proteins are assembled concomitantly with the grna to form the viral progeny, which exits the infected cells by apoptosis (stanislav v. sosnovtsev, prikhod'ko, belliot, cohen, & green, 2003) (barrera-vázquez et al., 2019) . apoptosis is a highly regulated process essential for the establishment of the calicivirus immunopathogenic phenotype, particularly to facilitate virus dissemination in the host [reviewed in: (peñaflor-téllez, trujillo-uscanga, escobaralmazán, & gutiérrez-escolano, 2019) ]. moreover, recent evidence indicates that apoptosis is a mechanism to suppress the translation of induced interferonstimulated genes (isg) to impair the host innate immune response to norovirus infection (emmott et al., 2017) . protein p53 (p53), a 393 amino acid protein expressed from the tp53 gene (saha, kar, & sa, 2015) , also known as the "guardian of the genome" that functions as a dna sequence-specific transcriptional regulator. p53 controls various cellular pathways such as cell cycle arrest at the g1/s regulation point on dna damage recognition, differentiation, and senescence by activating dna repair proteins when the dna has sustained damage, and apoptosis when dna damage is irreparable [reviewed in: (aloni-grinstein et al., 2018) (mishra & laboratories, 2015) ]. one of the most important proteins activated by p53 is the cyclindependent kinase (cdk) inhibitor1 (p21), implicated in the progression of the cell cycle, transcription regulation, and apoptosis (karimian, ahmadi, & yousefi, 2016) . j o u r n a l p r e -p r o o f p21 is considered as an indicator of p53 activity (georgakilas, martin, & bonner, 2017) . p53 exists in an inactive state in normal cells due to its interaction with its negative regulator, the mouse double-minute-2 homolog (mdm2) protein (momand, zambetti, olson, george, & levine, 1992) ; however, dna damage produced by cell stress conditions induces its dissociation and the further activation of p53 by its accumulation, posttranslational modifications, and conformational changes [reviewed in: (kastenhuber & lowe, 2017) ]. on activation, p53 may induce cellcycle arrest for dna repair, or may induce apoptosis to eliminate the altered cells; however, what determines p53 to stimulate one pathway or the other is still a matter of investigation [reviewed in: (kastenhuber & lowe, 2017) ]. as p53 is activated by several and varied stress signals, it is not surprising that virus-infected cells harbor altered p53 functions to achieve successful replication and spread throughout the host [reviewed in: (sato & tsurumi, 2013 ) (aloni-grinstein et al., viruses modulate p53 functions in different manners: single-stranded rna viruses such as the human coronavirus nl63 (hcov-nl63) induces its degradation of p53 (yuan et al., 2015) to ensure viral growth in infected cells (ma-lauer et al., 2016) , zika (zikv) and west nile (wnv) activate p53 to facilitate their replication (el ghouzzi et al., 2016) (teng et al., 2017) (yang et al., 2008) while adenovirus even though p53 is implicated in the induction of apoptosis and in many viral infections, its role during calicivirus infection has not been studied. here we found that p53 interacts with fcv dsrna, the protease-polymerase ns6/7, and vp1 in the rc; moreover, knockdown of p53 resulted in a significant reduction of the ns viral protein synthesis and virus production, indicating its role for efficient viral replication. cells and virus infection. the crandell feline kidney (crfk) cells (crandell, fabricant, & nelson-rees, 1973) obtained from the american type culture collection (atcc) (rockville, md) were grown in eagle's minimal essential medium with earle's balanced salt solution and 2mm l-glutamine that was modified by the atcc to contain 1.0mm sodium pyruvate, 0.1mm nonessential amino acids, 1.5 g/l sodium bicarbonate. the medium was supplemented with 10% bovine fetal serum, 5000 u of penicillin, and 5 μg/ml of streptomycin. cells were grown in a 5% co 2 incubator at 37°c (santos-valencia et al., 2019) . the fcv urbana strain was obtained by the reverse genetic system using the pq14 infectious clone (stanislav sosnovtsev & green, 1995) . virus titers were determined by plaque assay as previously described (escobar-herrera, medina-ramírez, & gutiérrez-escolano, 2007) . western blot assays. mock and fcv infected cells with fcv at an multiplicity of j o u r n a l p r e -p r o o f infection (moi) of 5 were washed with phosphate buffer saline (pbs), lysed in np40 lysis buffer (150 mm nacl; 1% np40, tris ph 8) and boiled for 10 min. forty µg of total protein cell extracts were analyzed by sds-page and transferred to a 0.22 µm nitrocellulose membranes. the membranes were blocked with 5% skimmed milk in tris-buffered saline (500 mm tris, 50 mm nacl) in 0.05% tween (tbs-t) for 30 minutes at room temperature (rt) and incubated overnight at 4°c with the following antibodies: mouse anti-p53 (sc-374087, santa cruz biotechnology) in a 1:500 dilution in tbs-t-5% skimmed milk; rabbit anti-p21 (sc-397, santa cruz biotechnology); rabbit anti-fcv ns6/7 (kindly donated by ian goodfellow) diluted in a 1:10 000 pbs-5% skimmed milk; and mouse anti-annexin a2 (anxa2) used as previously reported (santos-valencia et al., 2019) . the membranes were washed with 0.05% tbs-t and incubated 2 hrs with the appropriate anti-rabbit-hrp or anti-mouse-hrp secondary antibodies diluted in 1:10 000 pbs-5% skimmed milk at rt and developed using chemiluminescence (piercse). quantification of protein levels was achieved by measuring band intensities in the scanned images using imagej software (http://rsb.info.nih.gov/ij) and expressed as relative expression units. the escherichia coli bl-21 strain was transformed with 1 µg of the fcv vp1-gst expression vector and grown to od 600 of 0.6, and protein expression was induced by 0.5 mm of isopropyl β-d-1-thiogalactopyranoside (iptg) for 4 hrs at 37c gently shaken. the recombinant protein was purified by electroelution for 1 hr at 120 v. mock-infected crfk protein cell lysates (600 µg) were incubated with 50 µl of glutathione-agarose beads for 2 hrs at 4c with gentle shaking. the mixture was centrifuged at 14,000 rpm for 5 minutes at 4c, and the supernatant was recovered, mixed with 50 µl of glutathione-agarose beads and 10µg of the recombinant vp1-gst protein and incubated 2 hrs at 4c with gentle shaking. the mixture was centrifuged at 14,000 rpm at 4°c, and the beads were washed three times with cold pbs and eluted with 20 mm reduced glutathione-50 mm tris-hcl (ph 8), and centrifuged at 14,000 rpm at 4c. the eluted products were analyzed by western blotting using the corresponding antibodies. forty µg of gst protein (corresponding to an equimolar amount of vp1-gst protein) was used as a negative control. j o u r n a l p r e -p r o o f overlay assays. to determine the direct interaction between p53 and vp1, 5 µg of vp1-gst or gst were subjected by sds-page and transferred to a nitrocellulose membrane. the membrane was blocked as was mentioned before and incubated with 10 µg of p53-his in pbs-5% skimmed milk for 2 hrs at rt. after three washes with pbs, the anti-his antibody was incubated (on) at 4ºc in gentle shaking. the secondary anti-mouse antibody was diluted (1:10,000) in pbs-5% milk. the interaction was analyzed by chemiluminescence. sirna-mediated knockdown of p53. for the sirna-mediated knockdown of p53 expression, transfections were carried out as previously described (cancio-lonches et al., 2011) . briefly, crfk cells were plated in a 24-well plate to reach 80% confluence. after 24 hrs, 200 nm of the non-targeting-sirna (qiagen) or p53-sirnas (qiagen) and 2.5µl lipofectamine were mixed separately with 100 µl opti-mem, respectively for 10 min at rt. the two mixtures were combined, incubated at rt for 10 min, and then diluted in 100 µl opti-mem. the mixture was added directly to the cells, and transfection with the sirnas was carried out at 37°c for 8 hrs, followed by the addition of 200 µl growth medium and additional incubation for 24 hrs. after transfection, cells were infected with fcv at an multiplicity of infection (moi) of 5, and at 5 and 7 hpi were lysed with laemmli 1x buffer. the levels of the p53, viral protease-polymerase ns6/7, and it is widely known that viruses can modulate p53 steady-state by changing its expression, activity, localization, and by translational modifications [reviewed in (sato & tsurumi, 2013 ) (aloni-grinstein et al., 2018 ]. to determine if the levels or the subcellular localization of p53 were modified during fcv infection, the expression of p53 in mock-infected and infected crfk cells were determined by western blotting and immunofluorescence assays ( fig. 1 and 2) . total protein j o u r n a l p r e -p r o o f extracts from mock-infected and infected cells at 0, 1, 3, 5, 7, 9, and 11 hrs were submitted to sds-page, and p53 relative expression was determined by western blotting. similar levels of p53 were observed in both mock-infected and infected cells in all hpi analyzed ( fig. a1 and b1) . however, the levels of p21, a gene downstream of p53, were found downregulated from 7 and up to 11 hpi, suggesting a modified transcriptional activity of p53 at late hpi ( figure a1 and c1). the finding that p53 levels were similar during infection correlates with the immunofluorescence assays results where the fluorescence intensity of p53 was similar in both mock-infected and infected cells and in all the conditions tested (3, 5, and 7 hpi) (fig. 2) . likewise, the localization of p53 was observed mainly in the cytoplasm in both mock-infected and infected cells (fig. 2) . in contrast, increasing amounts of ns6/7 and vp1 protein were observed from 3 hpi in both western blotting ( interaction between fcv vp1 and p53 was also predicted by in silico analysis. a p53 constructed three-dimensional model was obtained using the raptorx server (http://raptorx.uchicago.edu)(10.1093/bioinformatics/btt211), and a molecular docking predicted between p53 and vp1 using the cluspro protein-protein docking (cluspro.org) was performed. a similar energy binding ∆g° (cal/mol) average calculated for vp1-p53 (∆g°=-1802) interaction and p53-mdm2 ∆g°=-1806), a well know interaction used as a positive control was obtained (momand et al., 1992) (chen, marechal, & levine, 1993) , in contrast to the low energy binding of p53-gst (∆g°=-1256) used as a negative control (fig. 3) , supporting the notion that p53 interacts with fcv vp1. moreover, the in vitro interaction of vp1 and p53 was determined by an overlay assay. vp1-gst, gst, and p53-his (5 µg) were subjected to sds-page and transferred to a nitrocellulose membrane, and incubated with the recombinant p53-his followed by the anti-his antibody ( supplementary fig. 4) . a band of 95 kda, corresponding to the molecular weight of vp1-gst, was observed (line 1), suggesting the interaction between these two proteins. the negative and positive controls are shown ( supplementary fig. 4 , lines 2, and 3). (green et al., 2002) . since we have seen that much of the colocalization between p53 and vp1 is observed in the perinuclear area, we speculate if p53 could be recruited to the rc. therefore, the colocalization of p53 and the viral dsrna was determined by immunofluorescence assays using the anti-p53 (red) and anti-dsrna (green) antibodies (fig. 4) . while the p53 signal was observed mainly in the cytoplasm of both mock-infected and infected cells at 3, 5, and 7 hrs, the dsrna signal was only observed in the perinuclear area from the infected cells at 5 and 7hpi (supplementary fig. 5 and fig. 4a) . a colocalization between p53 and the dsrna was observed in the perinuclear area, with a pcc 0.2 ± 0.03 and 0.50 ± 0.09 at 3 and 5 hpi respectively ( supplementary fig. 5 ) and 0.59 ± 0.11 at 7 hpi (fig. 4a) , indicating that as the infection progresses p53 is recruited to the fcv rc and associates with the dsrna. to confirm the direct interaction between p53 and the dsrna, a pla analysis was performed in fcv infected crfk cells. a positive signal (red dots) in the cytoplasm was detected in fcv infected cells at 7 hpi stained with both anti-p53 and anti-dsrna antibodies, strongly suggesting that p53 interacts with the dsrna in the fcv rc at this time post-infection (fig. 4b) . moreover, the interaction of p53 with other components of the rc, the ns6/7 protein was also detected (fig. 4b) , confirming that p53 is recruited in the rc. no signal was detected in mock-infected cells incubated with both primary antibodies or in infected cells incubated with the anti-p53 antibody alone, demonstrating the specificity of the signal (fig. 4b and supplementary fig. 6 ). the reduction of p53 expression resulted in a delay of the cytopathic effect, and a reduction in the viral protein production and fcv replication. to evaluate if p53 plays a role on fcv replication, crfk cell monolayers were transfected with nt-sirna or p53-sirna for 24 hrs and infected with fcv at an moi of 5. after 5 and 7 hrs, the cytopathic effect (cpe), the levels of viral protein and virus progeny production were analyzed (fig. 5) . a delay in the cpe of cells transfected with the p53-sirna at 5 and more evident at 7hpi, was observed when compared to the cpe from the non-targeting-sirna treated infected cells (fig. 5b ). this observation correlates with a 95 and 91% reduction in the ns6/7 viral protein levels at 5 and 7 hpi compared to the levels of ns6/7 in cells transfected with the non-targeting sirna (fig. 5c-5e ). this reduction in viral protein observed in the p53 sirna-transfected cells was not the consequence of a reduction of cell viability or cell proliferation ( supplementary fig. 7a and 7b) or any gross effects on the host protein synthesis since the levels of glyceraldehyde-3-phosphate dehydrogenase (gapdh) or anxa2 were unaffected ( fig. 5c and 5d) . a 60% decrease in p53 levels in transfected crfk cells with specific sirnas in comparison to the levels from cells transfected with the non-targeting (nt) sirna was obtained by band quantification using the imagej software (fig. 5a ). due to the reduction of viral proteins as a result of p53 knockdown, virus production was evaluated by plaque assays. p53 knockdown resulted in a 90.2 and 95% reduction in the fcv progeny production at 5 and 7hpi, respectively (fig. 5f ), indicating that p53 is required for fcv replication. therefore, its expression is an indicator of the p53 transcription activity (georgakilas et al., 2017) . notwithstanding, p53 can also control other pathways such as autophagy, metabolism, cell pluripotency, and plasticity, and facilitates an iron-dependent form of cell death known as ferroptosis (kastenhuber & lowe, 2017) . to regulate its steady-state, p53 is labeled by the mdm2 ubiquitin ligase for its proteasomal ubiquitin-dependent pathway degradation in unstressed cells, where p53 levels are to low to be detected (levine & oren, 2009 ). upon cell stress as dna damage, p53 is phosphorylated and evade the proteasomal degradation; then it is stabilized and activated as a transcription factor, leading to cell cycle arrest or apoptosis induction by p53-mediated gene expression cascades. therefore, the cell pathways linked to p53 dynamics are mediated by its expression levels and its post-translational modifications [reviewed in: (sato & tsurumi, 2013) . upon viral infections, the p53 steady-state can be modified as a result of cell stress and favors an efficient replicative cycle. as widely reported, some viruses can j o u r n a l p r e -p r o o f activate p53; some others induce its inhibition and/or degradation, and some more can modulate both its activation and degradation at specific stages of the infection [reviewed in: (aloni-grinstein et al., 2018) ]. even though it is well known that all the members of the caliciviridae family induce apoptosis, the role of p53 in this pathway or in other stages of viral replication has not been studied; therefore, the aim of this work was to determine if p53 host cell protein has a role in the replicative cycle of fcv, one of the best models to study the replication of this family. during fcv infection, p53 expression levels remained unchanged, and its subcellular localization was mostly in the cytoplasm in both mock-infected and infected cells. however, its transcription activity was modified during infection; as the relative expression of the cyclin-dependent kinase inhibitor p21 one of the targets of p53 activity was reduced from 7 and up to 11 hpi with fcv. this result correlates with the fact that inhibition of apoptosis is a function of p21 [reviewed in: (gartel & tyner, 2002) ]. the colocalization between p53 and the fcv vp1 observed form 3hpi, and that increased at later times post-infection was observed, suggest that both proteins interact from the early stages of infection. to this regard, during influenza a virus (iav) infection, p53 is activated in a biphasic pattern: in the early stages and later, at the middle-late phase of infection, during the apoptotic onset (shen et al., 2009) (turpin et al., 2005) (xue wang et al., 2014) . while iav protein ns1 a nonessential virulence factor, interacts with p53 inhibiting its transcriptional activity and apoptosis (xiaodu wang et al., 2010) ; the iav nucleoprotein (np), with multiple roles such as the viral rna transcription, replication, and packaging (portela & j o u r n a l p r e -p r o o f digard, 2002) , interacts with p53, increasing its transcriptional activity (xiaodu wang et al., 2012) , and modulating the immune response (b. wang et al., 2018) . therefore, we first wanted to determine if p53 interacted with fcv vp1. the in vitro association of both p53 and vp1 was suggested by molecular docking in silico analysis as well as by far-western blotting and further corroborated by pull-down and overlay assays. moreover, the interaction between p53 and vp1 was confirmed in infected cells by pla assays. regulation of p53 is also the result of the interaction of viral capsid proteins with mdm2, its main regulatory protein. the c-terminus of the of the zikv capsid protein interacts with mdm2, activating the death of infected neural cells (teng et al., 2017) ; while the capsid protein of the wnv interacts with the human double minute-2 (hdm2), which is sequestered into the nucleolus with the resulting stabilization of p53 and apoptosis induction (yang et al., 2008) . if mdm2 homolog in crfk cells interacts with any fcv protein or if it is modified during infection remains to be determined. taking into account that vp1 is the most abundant protein in the rcs (green et al., 2002) , it was likely that p53 was recruited in these membranous structures. the colocalization of p53 with the dsrna in the perinuclear region showed by immunofluorescence assays, and the interaction of both molecules demonstrated by pla assays strongly suggests that p53 is present in the rcs; therefore, the association between vp1 and p53 could be taking place in these cellular compartments. furthermore, the association of p53 with the protease-polymerase ns6/7 protein by pla corroborates the presence of p53 in the rcs. considering that p53 is associated with three fcv components, we hypothesized that it might have a role for efficient viral replication. the role of p53 in the fcv replication was demonstrated by knocking down its expression with specific p53-sirnas. even though p53 is an abundant protein, the cells treated with the specific p53-sirnas showed at least a 60% reduction of these protein levels, which caused a delay in the cytopathic effect of the fcv-infected cells, when compared to the infected cells treated with the non-targeting sirna. this delay in the cytopathic effect correlates with a strong reduction of the ns6/7 viral protein expression at 5 and 7hpi and a 1log reduction in the viral progeny production, indicating that p53 participates for an efficient fcv replication, a role that is for the first time reported for a member of the caliciviridae family. taken into account that p53 is recruited in the rc, where replication of both grna and sgrnas, as well as vp1 translation, occur and that p53 is associated with vp1, ns6/7, and with the dsrna, it could be possible that p53 participates in these viral processes. on the one hand, rna-linked p53 has been reported to be a major biological active form of p53 (samad & carroll, 1991) . p53 in the cytoplasm exerts high levels of 3´ to 5' exoribonuclease activity on ss-dna, ds-dna, ss-rna, ds-rna, and rna/dna substrates (derech-haim, friedman, hizi, & bakhanashvili, 2020) (grinberg, teiblum, rahav, & bakhanashvili, 2010) . some rna viruses such as coronaviruses, toroviruses, and roniviruses encode for 3' to 5´exoribonucleases that are critically involved in the synthesis of multiple rnas from its large genomic rna templates and performs a proofreading function required for high-fidelity replication (minskaia et al., 2006) . moreover, the human immunodeficiency virus (hiv)-1, which has a small rna genome, the cytoplasmic p53 increases the accuracy of dna synthesis by the reverse transcriptase (rt) (bakhanashvili, novitsky, lilling, & rahav, 2004 ). thus, it is possible that p53 may play a role for an efficient fcv rna replication. on the other hand, the low levels of ns proteins when p53 was knocked down could be the result of an inefficient translation or reduced levels of the grna. in this regard, since vp1 is also present in the rc and associated with p53, and the rdrp ns6/7 is associated with p53, it is possible that p53 through its interaction with vp1 contributes to the rdrp activity, as during norovirus replication (subba-reddy, goodfellow, & kao, 2011) . interestingly, p53 can direct either the complete degradation of and decrease in the level of cellular dsrna, or incomplete dsrna degradation that results in the generation of short dsrna products (grinberg et al., 2010) . it would be interesting to determine if p53 could participate in the generation of the fcv grna or sgrna. p53 can be extensively posttranslationally modified in response to various types of cellular stress that are implicated in the regulation of its levels, as well as its dna binding and transcriptional activities. since modifications in p53 are implicated in the induction or repression of a variety of its target genes, it is possible that this protein can have other functions during infection. here we have shown that p53 is associated with fcv elements present in the rc, such as the dsrna; however, the specific role of this interaction is not yet known. p53 acts as a transcription factor by binding to some response elements (p53re) within the genomic dna (b. wang, xiao, ko, & ren, 2010) . moreover, p53 also interacts with the genomes of double-stranded dna viruses. p53 binds to a consensus p53 binding sequence within the non-coding region of the human papillomavirus 77 (hpv77) genome and activates its activity in response to uv j o u r n a l p r e -p r o o f radiation (purdie et al., 1999) . more recently, two novel functional p53 responsive elements (re) have been identified in the hsv-1 genome, modulating the expression of viral proteins that may determine the progression of the lytic phase or the establishment of latency (hsieh, kuta, armour, & boehmer, 2014) . hepatitis b virus (hbv) transcription and replication are repressed by p53 sequence-specific binding to an enhancer element within its genome. this transcriptional effect of p53, when bound to this viral dna region, can be modulated by adjacent enhancer elements as well as with interactions with other dna-proteins (ori et al., 1998). p53 can also bind to the long terminal repeat of the hiv-1 that mediates mutant p53 transactivation (gualberto et al., 1995) . an interesting finding was that different conformations of p53 recognize different dna binding sites and mediate distinct biological functions. although p53 has been typically considered a sequence-specific dna-binding transcription factor, its interaction with different cellular rnas has been described in a variety of contexts [reviewed in: (riley & maher, 2007) ]. p53 binds to the 5'-utr of its own mrna (mosner et al., 1995) ; to the 5.8s ribosomal rna (rrna), and to the cdk4 and the fibroblast growth factor 2 (fgf-2) mrna [reviewed in: (riley & maher, 2007) ]. although p53-rna interactions are mediated by its nucleicacid-binding domain responsible for dna recognition, they are most probably sequence nonspecific [reviewed in: (riley & maher, 2007) ]. however, no reports regarding the interaction of p53 with a viral rna have been previously described. here we found that p53 interacts with three components of the fcv rc: vp1, ns6/7, and the dsrna. the knockdown of p53 caused a significant reduction of viral protein synthesis and virus yield, indicating its role for an efficient viral j o u r n a l p r e -p r o o f replication. future studies will provide further insights into the specific mechanism of action of p53 in fcv replication. were calculated by pearson's coefficient correlation using the icy software (http://icy.bioimageanalysis.org). b) recombinant fcv vp1-gst or gst proteins coupled to glutathione-agarose beads were incubated with total mock-infected protein extracts. after several washes, the eluted proteins were analyzed for the presence of p53 by western blotting using anti-p53 and anti-gst antibodies. c) proximity ligation assay (pla-duolink) between vp1 and p53 in fcv infected cells at 7 hrs. pla signals (red) represent dual-recognition against p53 and vp1. dapi was used for nuclear (blue) staining. the cells were examined in a zeiss lsm 700 confocal microscope. monolayers of mock-infected or fcv crfk cells were infected at an moi of 5 at 3, and 5 hrs, and immunostained with anti-p53 (red), and anti-dsrna (green) antibodies, followed by alexa fluor 594 (red) and alexa fluor 488 (green) respectively. dapi was used for nuclear (blue) staining. the cells were examined in a zeiss lsm 700 confocal microscope. images correspond to a z-stack of 15 slices and are representative of three independent experiments. merged and zoom images are indicated. colocalization rates of p53 and dsrna at 3hpi, (0.2 ± 0.03) j o u r n a l p r e -p r o o f and 5hpi (0.50 ± 0.09) were calculated by pearson's coefficient correlation using the icy software (http://icy.bioimageanalysis.org). p53 and the viral connection: back into the future ‡ feline calicivirus p32, p39 and p30 proteins localize to the endoplasmic reticulum to initiate replication complex formation p53 in cytoplasm may enhance the accuracy of dna synthesis by human immunodeficiency virus type 1 reverse transcriptase the feline calicivirus leader of the capsid protein causes survivin and xiap downregulation and apoptosis nucleolin interacts with the feline calicivirus 3' untranslated region and the protease-polymerase ns6 and ns7 proteins, playing a role in virus replication mapping of the p53 and mdm-2 interaction domains development, characterization, and viral susceptibility of a feline (felis catus) renal cell line (crfk) p53 regulates its own expression by an intrinsic exoribonuclease activity through au-rich elements zika virus elicits p53 activation and genotoxic stress in human neural progenitors similar to mutations involved in severe forms of genetic microcephaly and p53 norovirus-mediated modification of the translational landscape via virus and host-induced cleavage of translation initiation factors a carboxymethyl-cellulose plaque assay for feline calicivirus the role of the cyclin-dependent kinase inhibitor p21 in apoptosis p21: a two-faced genome guardian isolation of enzymatically active replication complexes from feline calicivirus-infected cells p53 in cytoplasm exerts 3′→5′ exonuclease activity with dsrna expanding the p53 regulatory network: lncrnas take up the challenge a proliferative p53-responsive element mediates tumor necrosis factor alpha induction of the human immunodeficiency virus type 1 long terminal repeat detection of the orf3 polypeptide of feline calicivirus in infected cells and evidence for its expression from a single, functionally bicistronic, subgenomic mrna identification of two novel functional p53 responsive elements in the herpes simplex virus-1 genome multiple functions of p21 in cell cycle, apoptosis and transcriptional regulation after dna damage putting p53 in context. cell the first 30 years of p53: growing ever more complex p53 down-regulates sars coronavirus replication and is targeted by the sars-unique domain and plpro via e3 ubiquitin ligase rchy1 junctional adhesion molecule 1 is a functional receptor for feline calicivirus discovery of an rna virus 3′→5′ exoribonuclease that is critically involved in coronavirus rna synthesis p53: an overview the mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation negative feedback regulation of wild-type p53 biosynthesis p53 binds and represses the hbv enhancer: an adjacent enhancer element can reverse the transcription effect of p53 immune response modulation by caliciviruses the influenza virus nucleoprotein: a multifunctional rna-binding protein pivotal to virus replication the promoter of a novel human papillomavirus (hpv77) associated with skin cancer displays uv responsiveness, which is mediated through a consensus p53 binding sequence p53-rna interactions: new clues in an old mystery structural and sequential context of p53: a review of experimental and theoretical evidence the tumor suppressor p53 is bound to rna by a stable covalent linkage annexin a2 associates to feline calicivirus rna in the replication complexes from infected cells and participates in an efficient viral replication genome guardian p53 and viral infections influenza a virus induces p53 accumulation in a biphasic pattern rna transcripts derived from a cloned full-length copy of the feline calicivirus genome do not require vpg for infectivity feline calicivirus vp2 is essential for the production of infectious virions feline calicivirus replication induces apoptosis in cultured cells denv up-regulates the hmg-coa reductase activity through the impairment of ampk phosphorylation: a potential antiviral target entry of feline calicivirus is dependent on clathrin-mediated endocytosis and acidification in endosomes vpg-primed rna synthesis of norovirus rna-dependent rna polymerases by using a novel cell-based assay an integrative analysis reveals a central role of p53 activation via mdm2 in zika virus infection induced cell death influenza virus infection increases p53 activity: role of p53 in cell death and viral replication ictv virus taxonomy profile: caliciviridae influenza a virus facilitates its infectivity by activating p53 to inhibit the expression of interferon-induced transmembrane proteins the p53 response element and transcriptional repression stabilization of p53 in influenza a virus-infected cells is associated with compromised mdm2-mediated ubiquitination of p53 the non-structural (ns1) protein of influenza a virus associates with p53 and inhibits p53-mediated transcriptional activity and apoptosis novel pandemic influenza a (h1n1) virus infection modulates apoptotic pathways that impact its replication in a549 cells west nile virus capsid protein induces p53-mediated apoptosis via the sequestration of hdm2 to the nucleolus p53 degradation by a coronavirus papain-like protease suppresses type i interferon signaling we thank romel rosales, josé humberto pérez-olais, rosa m. del ángel, and juan ludert for many helpful suggestions and critical comments on the manuscript.we also thank clotilde cancio-lonches and yoloxochitl paredes-morales for technical assistance. key: cord-266585-jfjrk9gy authors: fang, shouguo; chen, bo; tay, felicia p.l.; ng, beng sern; liu, ding xing title: an arginine-to-proline mutation in a domain with undefined functions within the helicase protein (nsp13) is lethal to the coronavirus infectious bronchitis virus in cultured cells date: 2007-02-05 journal: virology doi: 10.1016/j.virol.2006.08.020 sha: doc_id: 266585 cord_uid: jfjrk9gy genetic manipulation of the rna genomes by reverse genetics is a powerful tool to study the molecular biology and pathogenesis of rna viruses. during construction of an infectious clone from a vero cell-adapted coronavirus infectious bronchitis virus (ibv), we found that a g–c point mutation at nucleotide position 15526, causing arg-to-pro mutation at amino acid position 132 of the helicase protein, is lethal to the infectivity of ibv on vero cells. when the in vitro-synthesized full-length transcripts containing this mutation were introduced into vero cells, no infectious virus was rescued. upon correction of the mutation, infectious virus was recovered. further characterization of the in vitro-synthesized full-length transcripts containing the g15526c mutation demonstrated that this mutation may block the transcription of subgenomic rnas. substitution mutation of the arg132 residue to a positively charged amino acid lys affected neither the infectivity of the in vitro-synthesized transcripts nor the growth properties of the rescued virus. however, mutation of the arg132 residue to leu, a conserved residue in other coronaviruses at the same position, reduced the recovery rate of the in vitro-synthesized transcripts. the recovered mutant virus showed much smaller-sized plaques. on the contrary, a g–c and a g–a point mutations at nucleotide positions 4330 and 9230, respectively, causing glu–gln and gly–glu mutations in or near the catalytic centers of the papain-like (nsp3) and 3c-like (nsp5) proteinases, did not show detectable detrimental effect on the rescue of infectious viruses and the infectivity of the rescued viruses. coronaviruses cause severe diseases affecting human and other animal species. in 2003, a novel coronavirus (sars-cov) was identified as the causative agent of severe acute respiratory syndrome (sars) (marra et al., 2003; rota et al., 2003) . the potential risk to public health posed by sars-cov and other coronaviruses and the lack of specific antiviral agents and vaccines have triggered a global research effort to characterize this family of viruses at the molecular and cellular levels. major advances in studies of the biological functions of individual viral proteins and replication mechanisms are currently being made by genetic manipulation of coronaviral genomes using reverse genetics and targeted recombination approaches (almazan et al., 2000; casais et al., 2001 casais et al., , 2003 casais et al., , 2005 coley et al., 2005; hodgson et al., 2006; koetzner et al., 1992; masters and rottier, 2005; sanchez et al., 1999; youn et al., 2005a youn et al., , 2005b yount et al., 2000 yount et al., , 2002 yount et al., , 2003 yount et al., , 2005 . avian infectious bronchitis virus (ibv), a group 3 coronavirus, causes an acute and contagious disease in chickens with a significant impact on the poultry industry worldwide. ibv contains a 27.6 kb single-stranded, positive-sense rna genome. in the virus-infected cells, six mrna species, including the genome-length mrna 1 and five subgenomic mrnas (mrna 2-6), are produced by a discontinuous rna transcription mechanism. each mrna species possesses a 64 nucleotides leader sequence derived from the 5′ end of the virology 358 (2007) 136 -147 www.elsevier.com/locate/yviro genome (boursnell et al., 1987) . subgenomic mrnas 2, 3, 4, and 6 encode the four structural proteins, i.e., spike glycoprotein (s), envelope protein (e), membrane protein (m), and nucleocapsid protein (n). the 5′ two-third region of mrna 1 comprises two large orfs, 1a and 1b, and encodes two polyproteins. the two polyproteins are proteolytically cleaved by two virus-encoded proteinases, the papain-like and 3c-like proteinases, into 15 functional proteins (nsp2-nsp16) liu, 1998a, 1998b; lim et al., 2000; liu et al., 1995 liu et al., , 1997 liu et al., , 1998 ng and liu, 1998 xu et al., 2001) . compared to other coronaviruses, nsp1 is absent in ibv but nsp2 is considerably larger liu, 1998a, 1998b; liu et al., 1995) . in general, ibv shares close similarities in the genome organization, gene expression, and rna replication with other coronaviruses but is non-infectious to human. these properties make ibv an attractive model system for studying the biology and pathogenesis of coronavirus. in construction of an infectious ibv clone by in vitro assembly of five cloned rt-pcr fragments from a vero celladapted ibv beaudette strain, a g-c (g15526c) point mutation at nucleotide position 15,526 was found to be lethal to the infectivity of ibv on vero cells. no infectious virus could be rescued from vero cells electroporated with in vitro-synthesized full-length transcripts containing this mutation. as this mutation causes arg132-pro mutation in a domain with unknown function within the helicase protein (nsp13), it implies that this region might play certain roles in the functionality of the helicase protein. on the contrary, a g-c (g4330c) and a g-a (g9230) point mutations at nucleotide positions 4330 and 9230, respectively, causing glu-gln and gly-glu mutations in or near the catalytic centers of the papain-like (nsp3) and 3c-like (nsp5) proteinases, did not impair the infectivity of the in vitrosynthesized transcripts containing these mutations. further characterization of the in vitro-synthesized full-length transcripts containing the g15526c mutation demonstrated that this mutation blocks the transcription of subgenomic rnas. substitution mutation of the arg132 residue to a positively charged amino acid (lys) affected neither the infectivity of the in vitro-synthesized transcripts nor the growth properties of the rescued virus. however, mutation of the arg132 residue to a leu, which is conserved in most of other coronaviruses at the same position, reduced the viral recovery rate of the in vitrofig. 1 . in vitro assembly of full-length cdna clone derived from a vero cell-adapted ibv beaudette strain. (a) diagram of the genome organization of ibv. the regions coding for the replicase polyproteins, the structural proteins s, e, m, and n, the accessory proteins 3a, 3b, 5a, and 5b, and the 5′-and 3′-utr are shown. also shown are the regions of the five rt-pcr fragments, the t7 promoter at the 5′-end of fragment a, and the 30 as at the 3′-end of fragment e. (b) preparation of the five cdna fragments, assembly of the five fragments into a full-length cdna clone, and in vitro transcription of the full-length transcripts. the five cdna fragments covering ibv sequences from nucleotides 1-5751 (lane 2), 5752-8693 (lane 3), 8694-15,520 (lane 4), 15,521-20,900 (lane 5) , and 20,901-27,608 (lane 6), respectively, were obtained by digestion of corresponding plasmid dna with either bsmbi or bsai, purified from agarose gel, and analyzed on a 0.8% agarose gel (lanes 2-6). equal amounts of the purified fragments were ligated using t4 dna ligase (lane 7) and analyzed on a 0.4% agarose gel. the in vitro assembled full-length cdna was used as templates for generation of the full-length in vitro transcripts, which were analyzed on a 0.8% agarose gel (lane 10). lanes 1, 7, and 9 show dna markers, and numbers on the left indicate nucleotides in kilobases. synthesized full-length transcripts. the mutant virus showed much smaller-sized plaques. this study reveals the essential role of a domain with previously unassigned functions within the helicase protein in coronavirus replication. construction of a full-length cdna clone derived from a vero cell-adapted ibv beaudette strain by in vitro assembly of five rt-pcr fragments to construct a full-length ibv clone, five fragments (a to e) spanning the entire ibv genome were obtained by rt-pcr of total rna extracted from vero cells infected with a vero cell-adapted ibv beaudette strain (p65) (shen et al., 2003 (shen et al., , 2004 fang et al., 2005) . to facilitate the assembly of the full-length cdna in vitro, restriction sites for either bsmbi or bsai were introduced into both the 5′ and 3′ ends of the fragments (fig. 1a) . in fragment a, a 19-nucleotide sequence corresponding to the t7 rna promoter (table 1) was inserted into the 5′ end of the ibv genome to facilitate in vitro transcription using the t7 polymerase (fig. 1a) . the primers used to amplify these fragments are listed in table 1 . the pcr products were purified from agarose gel and cloned into either pcr-xl-topo (invitrogen) or pgem-t easy (promage) vectors. for the convenience of digestion using the restriction enzyme bsmbi, the nhei-and ecori-digested fragment a was subcloned into pkt0 which contains a bsmbi site 400 bp upstream of the t7 promoter sequence (fig. 1a) . two to three independent clones for each construct were selected for sequencing. the complete sequences of the five fragments, determined by automated nucleotide sequencing, are summarized in table 2 . the five fragments were then prepared by digestion of the corresponding constructs with either bsmbi or bsai and purified (fig. 1b, lanes 2-6) . the full-length clone was made by ligation of the purified fragments in vitro (fig. 1b, lane 8) and used as the template for in vitro transcription. the fulllength in vitro-synthesized transcripts were generated using the mmessage mmachine t7 kit (ambion, austin, tex) (fig. 1b , lane 10). as coronavirus n gene transcripts were shown to enhance the recovery of the rescued virus from the in vitrosynthesized full-length transcripts (casais et al., 2001; youn et al., 2005a youn et al., , 2005b yount et al., 2000 yount et al., , 2002 , the n transcripts were generated from a linearized pkt0-ibvn construct containing ibv n gene and the 3′-utr region. the full-length transcripts together with the n transcripts were introduced into vero cells by electroporation. however, it was consistently observed that no infectious virus could be recovered from cells transfected with the full-length transcripts together with the n transcripts. sequencing comparison of the five fragments with the vero cell-adapted ibv strain (p65, accession no. dq001339) shows nucleotide changes at 16 positions (table 2 ). among them, 11 a unique mutations found only in the cloned fragments. b same as the published sequences but different from p65. caused unique amino acid changes (table 2) . to assess the deleterious effects of these mutations on the infectivity of the full-length clone, correction of some of the mutations was carried out by site-directed mutagenesis. three point mutations, g4330c, g9230a, and g15526c, were chosen based on the fact that they are located either in or near the catalytic centers of the two viral proteinases or in a region of the rna helicase protein with undefined functions. four full-length cdna clones either with correction of the mutations at all three positions (ribv) or combination of two positions (e.g., g4330c containing g-c mutation at nucleotide position 4330 but without mutations at the other two positions) were constructed. rna transcripts generated from the four full-length cdna clones were introduced into vero cells together with the n transcripts by electroporation. at 2 days post-electroporation, a typical cpe of the vero cell-adapted ibv, the formation of giant syncytial cells (fang et al., 2005) , was observed in cells transfected with transcripts generated from cdna clones ribv, g4330c, and g9230a. cpe was extended to almost the whole monolayers at 3 days post-electroporation ( fig. 2a) . no cpe was observed in cells transfected with transcripts generated from clone g15526c (fig. 2a) . rt-pcr analysis of the subgenomic mrna 5 was performed to confirm if cpe observed is caused by the replication of ibv. sequencing of the rt-pcr fragment generated from cells transfected with ribv showed correct sequence in the leader/body junction region of the subgenomic mrna (fig. 2b ). further sequencing of the rt-pcr fragments covering regions with unique amino acid mutations confirmed the recovery of ibv (ribv) from the in vitro-synthesized fulllength transcripts. compared to the parent ibv strains, ribv contains 8 amino acid mutations (table 2 ). to test if these mutations may affect the growth properties and genetic stability of the rescued virus, ribv was propagated on vero cells for 5 passages, and the plaque sizes and growth kinetics were determined and compared with wild type ibv p65. in cells infected with ribv, the average plague size is 0.56 ± 0.028 mm, which is slightly smaller than the average plaque size of 0.68 ± 0.034 mm in cells infected with wild type ibv (fig. 3a) . analysis of the growth curves demonstrated that ribv exhibited very similar growth properties as the wild type virus (fig. 3a) . further characterization of ribv was subsequently carried out by analysis of viral rnas and structural proteins. northern blot and western blot analyses showed the detection of very similar amounts of viral rnas (fig. 3b ), and s, n, and m proteins ( fig. 3c ) in cells infected with wild type and ribv, respectively, at 24 h post-infection. when probing with anti-n protein antibodies, other species migrating faster than the fulllength products on sds-page were also observed ( fig. 3c ). they may represent premature termination and cleavage products of n protein (unpublished observation). it was also noted that variable amounts of these species were detected in cells infected with wild type and ribv (fig. 3c ). the significance of these variations is unclear at the moment. taken together, these results confirm that ribv is stable and possesses very similar growth properties as wild type ibv. as no infectious virus was recovered from cells transfected with g15526c mutant transcripts, rt-pcr amplification of the negative strand rna was performed to check if rna replication occurred in these transfected cells. total rna was extracted from vero cells transfected with wild type and g15526c mutant transcripts at 24 and 48 h post-electroporation, respectively. reverse transcription was performed by using equal amount of rna and the sense-primer ibv14931-f (5′-14,931 gct-tatccactagtacatc 14,949 -3′), and pcr was carried out by using the sense-primer ibv14931-f and the antisense-primer ibv15600-r (5′-15,600 cttctcgcacttctgcacta-gca 15,578 -3′). if replication of viral rna occurred, a 670 bp pcr fragment would be expected. as shown in fig. 4a , rt-pcr fragments amplified from negative strand rna templates were obtained from cells transfected with both wild type (lanes 2 and 4) and the mutant transcripts (lanes 3 and 5). sequencing of the pcr fragments confirmed that they represent the correct sequences. as a negative control, the in vitro-synthesized transcripts were mixed with total rna extracted from normal vero cells and were used as a template for rt-pcr. no rt-pcr fragment was detected (fig. 4a, lane 6) , confirming that the detection of negative strand rna from cells transfected with mutant transcripts is due to the replication of viral rna. further quantitation of the negative strand rna transcription in cells transfected with wild type and g15526c mutant transcripts was carried out by real-time pcr. at 24 and 48 h post-electroporation, transcription of the negative rna in cells electroporated with wild type transcripts was 24.76-and 945.54-fold, respectively, higher than that in cells electroporated with the g15526c mutant transcripts. these results confirm that transcription of the negative strand rna has taken place in cells transfected with the mutant transcripts, but with much lower efficiency. fig. 3 . analysis of the growth properties of wild type (p65) and ribv. (a) plague sizes and one-step growth curves of wild type and ribv. monolayers of vero cells on a 6-well plate were infected with 100 μl of 1000-fold diluted virus stock and cultured in the presence of 0.5% carboxymethy cellulose at 37°c for 3 days. the cells were fixed and stained with 0.1% toluidine. to determine the one-step growth curves of wild type and ribv, vero cells were infected with the viruses and harvested at 0, 4, 8, 12, 16, 24, and 36 h post-inoculation, respectively. viral stocks were prepared by freezing/thawing of the cells three times, and tcid50 of each viral stock was determined by infecting five wells of vero cells on 96-well plates in triplicate with 10-fold serial dilution of each viral stock. error bar shows standard error of the mean. (b) northern blot analysis of the genomic and subgenomic rnas in cells infected with wild type and ribv. ten micrograms of total rna extracted from vero cells infected with wild type and ribv, respectively, was separated on 1% agarose gel and transferred to a hybond n+ membrane. viral rnas were probed with a dig-labeled dna probe corresponding to the 3-end 680 nucleotides of the ibv genome. total rna extracted from mock-infected cells was included as negative control. numbers on the left indicate nucleotides in kilobase, and numbers on the right indicate the genomic and subgenomic rna species of ibv. (c) western blot analysis of viral protein expression in cells infected with wild type and ribv. vero cells infected with wild type (lane 1) and ribv (lane 2) were harvested, lysates prepared and separated on sds-10% polyacrylamide gel. the expression of s, n, and m proteins was analyzed by western blot with polyclonal anti-s, anti-n, and anti-m antibodies, respectively. the same membrane was also probed with antiactin antibody as a loading control. numbers on the left indicate molecular masses in kilodaltons. rt-pcr amplification of subgenomic mrnas was carried out to check if a low level of subgenomic mrna synthesis could occur in cells transfected with the mutant transcripts. total rna prepared from the transfected cells 2 days postelectroporation was used in the rt reaction with the sense-primer ibv-leader (5′-26 ctattacactagccttgcgct 46 -3′) for the detection of negative-stranded sgrna, and the antisenseprimer ibv24803-r (5′-24,803 ctctggatccaataacc-tac 24,784 -3′) for the detection of positive-stranded sgrna. the two primers were then used for pcr. if transcription of subgenomic mrnas did occur, a 415 bp pcr product corresponding to the 5′-terminal region of the subgenomic mrna 4 and a 1010 bp fragment corresponding to the 5′terminal region of the subgenomic mrna 3 would be expected. as shown in fig. 4b , a dominant 415 bp band and a weak 1010 bp band were observed in cells electroporated with wild type full-length transcripts at 2 days post-electroporation (lanes 2 and 5). sequencing of the pcr fragments confirmed that they represent the correct sequences of the corresponding regions of the subgenomic mrnas 3 and 4, respectively. however, the same pcr products were not detected in cells electroporated with the mutant transcripts (fig. 4b, lanes 3 and 6) . as a negative control, the amplified fragments were not detected in cells without electroporation (fig. 4b, lane 4) . the failure to detect both negative-and positive-stranded sgrnas in cells transfected with the mutant transcripts show that the g15526c mutation leads to the disruption of subgenomic rna transcription. to further demonstrate that the failure to rescue infectious virus from the g15526c mutant transcripts is due to a defect in subgenomic rna transcription, the full-length clones with and without the g15526c mutation were used to generate recombinant ibv expressing the enhanced green fluorescent protein (egfp) by replacing the 5a gene with egfp. full-length transcripts containing egfp were synthesized in vitro and introduced into vero cells together with the n transcripts by electroporation. at 2 days post-electroporation, single cpe with the expression of egfp was observed in cells transfected with the full-length transcripts without the g15526c mutation vero cells electroporated with in vitro-synthesized transcripts derived from the in vitro assembled full-length clones containing egfp either with or without g15526c mutation. phase-contrast and fluorescent images were taken 2, 3, and 5 days post-electroporation, respectively. (fig. 4c) . gradually increased cpe and fluorescent cells were observed from 3 to 5 days post-electroporation (fig. 4c) . at 5 days post-electroporation, cpe and fluorescent cells were extended to almost the whole monolayer (fig. 4c) . however, it was consistently observed that much less infectious virus was recovered from cells transfected with this construct. furthermore, the recombinant virus rapidly lost infectivity when passaged on vero cells; the recovered virus maintains minor infectivity only for one passage. in cells transfected with the fulllength transcripts containing the g15526c mutation, neither cpe nor cells expressing egfp were observed (fig. 4c) , demonstrating that g15526c mutation led to total demolition of the egfp expression. as egfp could be expressed only if subgenomic rnas were synthesized but can be observed even if a single cell was transfected and expressed the protein to a certain level, these results reinforce the conclusion that the g15526c mutation blocks subgenomic rna transcription. mutational analysis of the r132 residue of the helicase protein g15526c mutation resulted in the substitution mutation of the r132 with a pro (r132p) of the helicase protein. sequence comparison of the ibv helicase protein with other known coronaviruses showed that r132 residue is located adjacent to a conserved motif (fig. 5a) . in all sequenced coronaviruses, only ibv has a charged amino acid (r132) at this position (fig. 5a) . to assess if a positive charge amino acid at this position is essential for the function of the protein, mutation of r132 to a lys (r132k) was carried out. meanwhile, a conserved leu residue (ile in the case of tgev) was found at this position in all other coronaviruses, mutation of r132 to a leu (r132l) was also included. in vitro full-length transcripts containing the r132k and r132l mutations were electroporated into vero cells. as shown in fig. 5b , transcripts generated from wild type (r132) and r132k mutant constructs showed very similar infectivity after introduction into vero cells. typical cpe was observed in large areas of the monolayers at 3 days post-electroporation (fig. 5b) , and recombinant viruses were recovered. however, r132l transcripts were found to be less infectious. in cells electroporated with transcripts generated from this mutant, typical cpe was observed in much restricted areas of the monolayer at 3 days post-electroporation (fig. 5b) . the growth properties of the r132k and r132l mutant viruses on vero cells were tested by analysis of plaque sizes and growth curves of passage 5 mutant viruses. compared to cells infected with wild type recombinant virus (average plaques size is 0.56 ± 0.028), plaques with similar size were observed in cells infected with the r132k mutant virus (fig. 6a) . the average plaque size in cells infected with r132k mutant virus is 0.59 ± 0.029 mm. in cells infected with the r132l mutant virus, much smaller-sized plaques were observed (fig. 6a) . the average plaque size in cells infected with this mutant virus is 0.24 ± 0.017 mm. however, analysis of the growth curves of wild type and mutant viruses demonstrated that the mutant viruses exhibited very similar, or even better, growth properties as the wild type recombinant virus (fig. 6a) . the r132k and r132l mutant viruses were subsequently characterized by analysis of viral rnas and structural proteins. northern blot analysis showed the detection of very similar amounts of genomic and subgenomic rnas in cells infected with ribv and the two mutant viruses at 24 h post-infection (fig. 6b) . similarly, western blot analysis of cells infected with ribv, r132k, and r132l mutant viruses showed that similar amounts of s, n, and m proteins were detected at 24 h post-infection (fig. 6c, lanes 1-3) . when probing with anti-n protein antibodies, other species migrating faster than the full-length products on sds-page were also observed (fig. 6c) . they may represent premature termination and cleavage products of n protein (unpublished observation). it was also noted that variable amounts of these species were detected in cells infected with wild type and different mutants (fig. 6c) . the genetic stability of r132k and r132l mutant viruses was tested by propagation of the viruses on vero cells for 5 passages. sequencing analysis of the fifth passages of the two mutant viruses showed that the mutations are stable. no reversion to the original sequences or mutation to other nucleotides was found in the position. in vitro assembly of full-length coronavirus clones, generation of full-length transcripts in vitro using a bacteriophage dna-dependent rna polymerase, and recovery of infectious viruses by introduction of the in vitro-synthesized transcripts into cells, first used by yount et al. (2000) , are a rapid and reliable approach to construct infectious clones from large rna viruses. it has been successfully used to construct infectious fig. 6 . analysis of the growth properties of ribv, r132k, and r132l mutant viruses. (a) plague sizes and one-step growth curves of ribv, r132k, and r132 mutant viruses. monolayers of vero cells on a 6-well plate were infected with 100 μl of 100-fold diluted virus stock and cultured in the presence of 0.5% carboxymethy cellulose at 37°c for 3 days. the cells were fixed and stained with 0.1% toluidine. to determine the one-step growth curves of ribv, r132k, and r132l mutant viruses, vero cells were infected with the viruses and harvested at 0, 4, 8, 12, 16, 24 , and 36 h postinoculation, respectively. viral stocks were prepared by freezing/thawing of the cells three times, and tcid50 of each viral stock was determined by infecting five wells of vero cells on 96-well plates in triplicate with 10-fold serial dilution of each viral stock. error bar shows standard error of the mean. (b) northern blot analysis of the genomic and subgenomic rnas in cells infected with ribv, r132k, and r132l mutant viruses. ten micrograms of total rna extracted from vero cells infected with ribv, r132k, and r132l mutant viruses, respectively, was separated on 1% agarose gel and transferred to a hybond n+ membrane. viral rnas were probed with a dig-labeled dna probe corresponding to the 3-end 680 nucleotides of the ibv genome. numbers on the left indicate nucleotides in kilobase, and numbers on the right indicate the genomic and subgenomic rna species of ibv. (c) western blot analysis of viral protein expression in cells infected with wild type and r132 mutant viruses. vero cells infected with wild type recombinant ibv (lane 1), r132k (lane 2), and r132l (lane 3) were harvested, lysates prepared, and separated on sds-10% polyacrylamide gel. the expression of s, n, and m proteins was analyzed by western blot with polyclonal anti-s, anti-n, and anti-m antibodies, respectively. the same membrane was also probed with anti-actin antibody as a loading control. numbers on the left indicate molecular masses in kilodaltons. clones for several coronaviruses, including transmissible gastroenteritis virus (tgev), mouse hepatitis virus (mhv), sars-cov, and ibv (youn et al., 2005a (youn et al., , 2005b yount et al., 2000 yount et al., , 2003 . in the process of developing an infectious ibv clone from a vero cell-adapted ibv beaudette strain using this approach, a g-c point mutation at nucleotide position 15,526 was found to be lethal to the infectivity of the in vitrosynthesized full-length transcripts on vero cells. no infectious virus could be rescued from vero cells electroporated with transcripts containing this mutation. this mutation causes arg132-pro substitution in a domain within the helicase protein (nsp13) with undefined functions, indicating that this domain may be essential for the functionality of the helicase protein. multiple enzymatic activities have been assigned to the nsp13 helicase protein. these include rna and dna duplex-unwinding activities, ntpase and dntpase activities, and an rna 5′-triphosphatase activity that might be involved in the formation of the 5′-cap structure of viral rnas (ivanov et al., 2004) . the protein is comprised of two domains: a putative n-terminal zinc binding domain, which spans the nterminal region of the protein from approximately amino acids 1 to 77, and a c-terminal helicase domain covering the c-terminal part of the protein from amino acids 279 to the cterminal end (ivanov et al., 2004) . a ser-pro substitution located immediately downstream of the putative zinc binding domain of nsp10, the equivalent rna helicase protein in equine arteritis virus (eav), caused defect in subgenomic mrna transcription (van dinten et al., 1997 (van dinten et al., , 2000 . more detailed analysis of the zinc binding domain of nsp10 from eav and nsp13 from human coronavirus 229e by mutagenesis studies showed that this domain could modulate the enzymatic activities of the helicase domain (seybert et al., 2005) . through this regulatory role and some yet to be discovered mechanisms, the zinc binding domain is shown to be critically involved in the replication and transcription of coronavirus rna. in this study, introduction of the in vitro-synthesized fulllength transcripts containing the g15526c (r132p) mutation was shown to be totally defective in subgenomic rna transcription, a phenotype similar to, but appears to be much more severe than, the ser-pro mutation in eav (van dinten et al., 1997 (van dinten et al., , 2000 . mutation of the arg132 residue to a positively charged amino acid (lys) does not affect the infectivity of the in vitro-synthesized transcripts as well as the growth properties of the rescued virus. however, mutation of the arg132 residue to leu, a conserved residue at the same position in most of other known coronaviruses, impaired the recovery rate of the in vitrosynthesized transcripts. the recovered mutant virus showed much smaller-sized plaques. the r132 residue is located 57 amino acids downstream of the last his (h75) residue in the putative zinc binding domain (fig. 5a) and 147 amino acids upstream of the helicase motif 1 (seybert et al., 2005) . so far, no functional domain has been found in this region of the helicase protein. why the r132p substitution, located outside of the two functional domains of the helicase protein, shows severe phenotypic defect in subgenomic rna transcription and the infectivity of ibv is not clear at the moment. three possibilities were considered. first, r132 may be part of the n-terminal zinc binding domain. as no studies have been attempted to define the boundary of the two domains, it is not even certain that an independent domain with unique function may exist in this region. r132p mutant virus shares certain phenotypic similarity to the ser-pro mutant eav, such as the absence of subgenomic mrna transcription (van dinten et al., 1997 (van dinten et al., , 2000 , suggesting that the two mutations might disrupt a similar function of the helicase protein. however, as biochemical characterization of the effect of r132p mutation on the enzymatic activities of ibv nsp13 helicase protein is currently lacking, it would be difficult to draw a conclusion that the two mutants share mechanistically similar characteristics. second, r132 is located in a region with a high degree of amino acid conservation in all known coronaviruses. since the main defect of r132p mutant virus is in the subgenomic rna transcription, one possibility is that this region may be involved in the subgenomic rna synthesis by interacting with host or other viral functional proteins. alternatively, as a positively charged amino acid is required to maintain the full function of the protein, it would be possible that this region may be involved in binding of the helicase protein to viral rna during rna replication and transcription. finally, mutation to a pro would lead to the disruption of the three dimensional structures of the two domains. biochemical characterization of the involvement of this region in the enzymatic activities of the protein and determination of its three dimensional structures are underway to address these possibilities. the g433c (e-q) point mutation near the catalytic center of the plp domain in nsp3 affects neither the recovery of infectious virus from the full-length synthesized in vitro transcripts nor the infectivity of the rescued virus. similarly, efficient recovery of infectious virus from the in vitro transcripts containing the g9230a (g-e) point mutation in the 3clp was obtained. the two mutant viruses are genetically stable and show similar growth properties with the wild type recombinant virus. even though the two mutations are located in or near the catalytic centers of the two viral proteinases, the mutations did not alter their enzymatic activities. this relatively high degree of tolerance to mutations in non-essential regions of important functional proteins would minimize the occurrence of lethal mutations during the replication cycles of rna viruses and increase the adaptability of these viruses to a changed environment. vero cells were cultured at 37°c in minimal essential medium (mem) supplemented with 10% fetal bovine serum (fbs), penicillin (100 units/ml), and streptomycin (100 μg/ml). a vero cell-adapted ibv beaudette strain (65 passages on vero cells (p65)) (shen et al., 2003 (shen et al., , 2004 fang et al., 2005) was propagated in vero cells in fbs-free mem. five fragments spanning the entire ibv genome were obtained by rt-pcr from vero cells infected with the vero cell-adapted ibv p65 at a multiplicity of approximately 1. briefly, total cellular rna was extracted from the infected vero cells with tri reagent (molecular research center, inc.), according to the manufacturer's instructions. reverse transcription was performed with expand reverse transcriptase (roche) using reverse primers ibv-5753r, ibv-8694r, ibv-15532r, ibv-20930r, and ibv-27608r (table 1) . each cdna fragment was amplified from rt products by pcr using kod hot start dna polymerase according to the manufacturer's instructions (novagen). the pcr products were purified from agarose gels and cloned into pcr-xl-topo (invitrogen) or pgem-t easy (promage) vectors. subsequently, fragment a was removed from pcr-xl-topo by digestion with nhei and ecori and subcloned into pkt0 vector. two to three independent clones of each fragment were selected and sequenced by automated sequencing using specific primers and the abi dye termination sequencing method. sequence comparison, assembly, and analysis were performed by using blast and dna star software. mutations were introduced into the corresponding fragments by using quickchange site-directed mutagenesis kit (stratagene) and confirmed by sequencing of the whole fragments. in vitro assembly of full-length cdna clones plasmids were digested with either bsmbi (fragment a) or bsai (fragments b, c, d, and e). the digested plasmids were separated on 0.8% agarose gels containing crystal violet. bands corresponding to each of the fragments were cut from the gels and purified with qiaquick gel extraction kit (qiagen inc.). fragments a and b, and fragments c, d, and e were first ligated with t4 dna ligase at 4°c overnight. the two reaction mixtures were then mixed and further ligated at 4°c overnight. the final ligation products were extracted with phenol/chloroform/isoamyl alcohol (25:24:1), precipitated with ethanol, and detected by electrophoresis on 0.4% agarose gels. full-length transcripts were generated in vitro using the mmessage mmachine t7 kit (ambion, austin, tx) according to the manufacturer's instructions with certain modifications. briefly, 30 μl of transcription reaction with a 1:1 ratio of gtp to cap analog was sequentially incubated at 40.5°c for 25 min, 37.5°c for 50 min, 40.5°c 25 min and 37.5°c for 20 min. the n transcripts were generated by using a linearized pkto-ibvn containing ibv n gene and the 3′-utr region as templates. a 1:2 ratio of gtp to cap analog was used for the transcription of ibv n gene. the in vitro-synthesized full-length and n transcripts were treated with dnasei and purified with phenol/chloroform. vero cells were grown to 90% confluence, trypsinized, washed twice with cold pbs, and resuspended in pbs. rna transcripts were added to 400 μl of vero cell suspension in an electroporation cuvette and electroporated with one pulse at 450 v, 50 μf with a bio-rad gene pulser ii electroporator. the transfected vero cells were cultured overnight in 1% fbs-containing mem in a 60 mm dish or a six-well plate and further cultured in mem without fbs. analysis of the negative strand and subgenomic rnas by rt-pcr, real-time pcr total rna was extracted from vero cells electroporated in in vitro-synthesized transcripts after treatment with dnasei, using tri reagent (molecular research center, inc.) at 24 or 48 h post-electroporation. reverse transcription was performed with expand reverse transcriptase (roche) using equal amount of rna. after optimization, real-time pcr was performed using lightcycler faststart dna master sybr green i kit according to the manufacturer's instructions (roche). vero cells were infected with wild type and mutant viruses at a multiplicity of approximately 1, and total rna was extracted from the infected cells. ten micrograms of rna was added to a mixture of 1× mops, 37% formaldehyde, and formamide and incubated at 65°c for 20 min before subjected to gel electrophoresis. the segregated rna bands were transferred onto a hybond n+ membrane (amersham biosciences) via capillary action overnight and fixed by uv crosslinking (stratalinker). hybridization of dig-labeled dna probes was carried out at 50°c in hybridization oven overnight. membranes were washed 3 times for 15 min each with the probe buffer, before proceeding to detection with cdp-star (roche) according to the manufacturer's instructions. vero cells were infected with wild type and mutant viruses at a multiplicity of approximately 1. total proteins extracted from the infected vero cells were lysed with 2× sds loading buffer in the presence of 200 mm dtt plus 10 mm of iodoacetamide and subjected to sds-page. proteins were transferred to pvdf membrane (stratagene) and blocked overnight at 4°c in blocking buffer (5% fat free milk powder in pbst buffer). the membrane was incubated with 1:2000 diluted primary antibodies in blocking buffer for 2 h at room temperature. after washing three times with pbst, the membrane was incubated with 1:2000 diluted anti-mouse or anti-rabbit igg antibodies conjugated with horseradish peroxidase (dako) in blocking buffer for 1 h at room temperature. after washing three times with pbst, the polypeptides were detected with a chemiluminescence detection kit (ecl, amersham biosciences) according to the manufacturer's instructions. confluent monolayers of vero cells on six-well plates were infected with 100 μl of 100-fold diluted virus stock. after 1 h of incubation at 37°c, cells were washed twice with pbs and cultured in 3 ml of mem containing 0.5% carboxymethy cellulose for 3 days. the cells were fixed and stained with 0.1% toluidine. vero cells were infected with wild type and recombinant ibv and harvested at different times post-infection. viral stocks were prepared by freezing/thawing of the cells three times. the 50% tissue culture infection dose (tcid50) of each sample was determined by infecting five wells of vero cells on 96-well plates in duplicate with 10-fold serial dilution of each viral stock. engineering the largest rna virus genome as an infectious bacterial artificial chromosome completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus reverse genetics system for the avian coronavirus infectious bronchitis virus recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism gene 5 of the avian coronavirus infectious bronchitis virus is not essential for replication recombinant mouse hepatitis virus strain a59 from cloned, full-length cdna replicates to high titers in vitro and is fully pathogenic in vitro selection of and recombination between minor variants lead to the adaptation of an avian coronavirus to primate cells neither the rna nor the proteins of open reading frames 3a and 3b of the coronavirus infectious bronchitis virus are essential for replication multiple enzymatic activities associated with severe acute respiratory syndrome coronavirus helicase repair and mutagenesis of the genome of a deletion mutant of the coronavirus mouse hepatitis virus by targeted rna recombination characterization of a papain-like proteinase domain encoded by orf1a of the coronavirus ibv and determination of the cterminal cleavage site of an 87 kda protein characterization of the two overlapping papainlike proteinase domains encoded in gene 1 of the coronavirus infectious bronchitis virus and determination of the c-terminal cleavage site of an 87 kda protein identification of a novel cleavage activity of the first papain-like proteinase domain encoded by open reading frame 1a of the coronavirus avian infectious bronchitis virus and characterization of the cleavage products identification, expression and processing of an 87k polypeptide encoded by orf1a of the coronavirus infectious bronchitis virus proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein: identification of a 10 kda polypeptide and determination of its cleavage sites proteolytic mapping of the coronavirus infectious bronchitis virus 1b polyprotein: evidence for the presence of four cleavage sites of the 3c-like proteinase and identification of two novel cleavage products the genome sequence of the sars-associated coronavirus coronavirus reverse genetics by targeted rna recombination identification of a 24 kda polypeptide processed from the coronavirus infectious bronchitis virus 1a polyprotein by the 3c-like proteinase and determination of its cleavage sites further characterization of the coronavirus infectious bronchitis virus 3c-like proteinase and determination of a new cleavage membrane association and dimerization of a cysteinerich, 16-kda polypeptide released from the c-terminal region of the coronavirus infectious bronchitis virus 1a polyprotein characterization of a novel coronavirus associated with severe acute respiratory syndrome targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence a complex zinc finger controls the enzymatic activities of nidovirus helicases emergence of an avian coronavirus infectious bronchitis virus (ibv) mutant with a truncated 3b gene: functional characterization of the 3b gene in pathogenesis and replication a single amino acid mutation in the spike protein of coronavirus infectious bronchitis virus hampers its maturation and incorporation into virions at the nonpermissive temperature infectious rna transcribed in vitro from a cdna copy of the human coronavirus genome cloned in vaccinia virus an infectious arterivirus cdna clone: identification of a replicase point mutation that abolishes discontinuous mrna transcription the predicted metal-binding region of the arterivirus helicase protein is involved in subgenomic mrna synthesis, genome replication, and virion biogenesis further identification and characterization of novel intermediate and mature cleavage products released from the orf 1b region of the avian coronavirus infectious bronchitis virus 1a/1b polyprotein contribution of trafficking signals in the cytoplasmic tail of the infectious bronchitis virus spike protein to virus infection in vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a59 reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice this work was supported by the agency for science technology and research, singapore, and by a grant from the biomedical research council (bmrc 03/1/22/17/220), agency for science, technology and research, singapore. key: cord-260376-29ih5c9v authors: guo, jian-ping; petric, martin; campbell, william; mcgeer, patrick l title: sars corona virus peptides recognized by antibodies in the sera of convalescent cases date: 2004-07-01 journal: virology doi: 10.1016/j.virol.2004.04.017 sha: doc_id: 260376 cord_uid: 29ih5c9v we synthesized on cellulose membranes 4942 ten-amino-acid peptides which included all of the sequences predicted for the severe acute respiratory syndrome (sars) corona virus. we probed these membranes with four pairs of acute and convalescent sera from recovered sars cases. we correlated positively reacting peptides with the in vitro sars-cov neutralizing activity of the samples. we found that convalescent sera with high neutralizing activity recognized exclusively only a limited number of peptides on the membranes. this suggests that antibodies against the epitopes represented by these peptides could be responsible for much of the sars-cov neutralizing activity. the findings have implications for monitoring humoral responses to sars-cov as well as for developing a successful sars vaccine. a newly identified virus named sars-cov has been established as the etiological agent of severe acute respiratory syndrome (sars). a total of 8422 probable cases of this highly infectious disease had been reported to who by august 2003 including 916 deaths (http:// www.who.int/csr/sars/en/). the viral genome has been sequenced (marra et al., 2003; rota et al., 2003) . it consists of 29 751 nucleotides which contain 15 identifiable open reading frames (ay274119). peptides incorporating all of the sequences predicted in the open reading frames of the sars-cov genome were prepared on derivatized cellulose membranes using a robotic peptide synthesizer (autospot asp 222, intavis bioanalytical instruments, lagenfeld, germany). the peptides were 10 amino acids long and overlapped by eight residues. each peptide on a membrane was therefore shifted from the one previous by two amino acids towards the c-terminal end. the arrangement of the 4942 peptides on sets of four membranes covering the 15 open reading frames (orfs) is illustrated in fig. 1 . membranes were probed with pairs of acute and convalescent sera from four cases who recovered from a sars infection. as controls, serum from one case that failed to survive, and one from a healthy, nonexposed volunteer was utilized. samples were diluted 100-fold in buffer and then applied to a membrane for 1 h at 37 jc. the membranes were developed with horseradish peroxidase-conjugated goat antibodies against human igg, igm, or iga. positive spots were then identified following treatment with ecl chemiluminescence reagents. sera were tested for the presence of antibodies against sars-cov in a standard virus neutralizing test. serial 2fold dilutions of each serum from 1/8 to 1/1024 were incubated with 100 plaque-forming units of sars-cov (tor-2 isolate, marra et al., 2003) for 2 h and then added to monolayers of vero e6 cells. the cultures were examined after 72 h for the presence of characteristic cytopathic effects. the dilution before the one at which cytopathic effects were first noted was recorded as the antivirus titer. all sera, which showed evidence of antivirus activity, were retested following heat treatment at 56 jc for 30 min. this process confirmed the presence of neutralizing antibodies and eliminated the possibility of nonspecific, heat-sensitive factors contributing to the neutralizing activity. the results of neutralization assays for the various sera are shown in table 1 , along with the age and sex of the respective patients. acute sera were collected on presentation, and convalescent sera collected 1 month later in case 1, and at least 2 weeks later in cases 2, 3, and 4. the acute serum of case 3 had a neutralizing antibody titer of 1/8, which indicates the beginning of an immune response. other acute sera as well as the acute serum of the deceased case taken 7 days after illness onset, and the healthy control, had no detectable neutralizing activity. these data indicate that the four recovered cases developed antibodies with viral neutralizing potency between the time of acute and convalescent serum sampling. therefore, those peptides strongly recognized on membranes probed with convalescent sera, but not with acute or control sera, should be the most immunodominant and may include sars-cov epitopes that are vulnerable to neutralization by antibody. this analysis is limited to linear epitopes since peptides on the membranes do not replicate conformational epitopes or those that develop from posttranslational modifications such as glycosylation. three examples of membranes reacted with acute and convalescent sera and developed with anti-iga antibodies are shown in fig. 1. fig. 1b compares the second panel of membrane 1 probed with serum from the unexposed control, the deceased case, convalescent serum from case 1, and acute and convalescent serum from case 2. the panel has a total of 408 peptides encoded from the orf 1a open reading frame (see fig. 1a ). no spots were strongly immunostained except for a single triad in the two convalescent sera. the peptides in the triad have the following sequences: sddyiklngp, dyiklngplt, and iklngpvg. the sequence iklngp is common to all three. fig. 1c compares the third panel of membrane 2 probed with the acute and convalescent serum of case 2, and the deceased case. the panel has 408 peptides encoded from the s-protein open reading frame. one triad of spots was immunopositive, in the convalescent, but not the acute serum. the triad has the following sequences: fqpfqqfgrd, pfqqfgrdvs, and qqfgrdvsdf. the common sequence is qqfgrd. fig. 1d compares the fourth panel of membrane 3 probed with acute and convalescent serum of case 2, and the convalescent serum of case 1. the bottom half of the membrane has peptides encoded for the complete nucleocapsid (n) protein open reading frame. one triad was immunopositive in the two convalescent sera but was not positive in the corresponding acute serum of case 2. the triad has the following sequences: qlpqgttlpk, pqgttlpkgf, and gttlpkgfya with the common sequence being gttlpk. shown in table 2 are the 24 overlapping membrane peptides that were recognized exclusively, or much more strongly, in multiple pairs of convalescent compared with the respective acute sera. none of these peptides was recognized by the serum from the deceased case or the healthy control. there were two overlapping sequences from orf 1a, one from orf 1b, six from n-protein, eleven from s-protein, two from m-protein, one from e protein and one from orf 10. the remaining open reading frames were not reactive in more than one serum sample. in the table, those peptides listed with a sequence of 14 amino acids were identified as a combination of three adjacent spots on the membranes, whereas those listed with 12 were identified as a combination of two adjacent spots. common sequences therefore include residues 5 -10 of the 14-amino-acid peptides and 3-10 of the 12-aminoacid peptides. the note to the right of m4p1 shows the key to the orfs with peptide totals in brackets. this membrane was spotted with dye instead of amino acids. in actual membranes, each spot is a 10-amino-acid peptide with adjacent spots being shifted by two amino acids. characterization of the immune response against these single case epitopes promises to provide important insights into their role in the resolution of infection. however, epitopes recognized by multiple convalescent sera may be the most important targets of neutralizing antibodies. (b -d) examples of membrane panels probed with various serum samples and developed with peroxidase-labeled goat antihuman iga; (b) m1p2 probed with control serum, acute as well as convalescent serum from case 2, convalescent serum from case 1, and serum from the deceased case. notice the triad of spots recognized only in the serum of the two convalescent cases. the peptide sequences from orf 1a are sddyiklngp, dyiklngplt, and iklngpltvg. (c) p3m2 probed with acute and chronic serum from case 2 and serum from the deceased case. panel 3 has peptides from s-protein. notice the triad of spots recognized only by the convalescent serum. the peptide sequences from s-protein are fqpfqqfgrd, pfqqfgrdvs, and qqfgrdvsdf. (d) p4m3 probed with acute and convalescent serum from case 2 and convalescent serum from case 1. notice the triad of spots recognized in the two convalescent sera. the peptide sequences from n-protein are qlpqgttlpk, pqgttlpkgf, and gttlpkgfya. additional peptides to those shown in table 2 were recognized only by convalescent serum from one of the four cases. among these single case examples were peptides from orf 3, orf 4, orf 9, orf 13, and orf 14, indicating that the virus expresses these proteins and that antibodies against certain of their epitopes are being produced. characterization of the immune response against these single case epitopes promises to provide important insights into their role in the resolution of infection. however, epitopes recognized by multiple convalescent sera may be the most important for developing antibodies that will protect against the virus. our knowledge of the functions of the sars-cov proteins is mostly derived from analogies with those of other well-characterized corona viruses. over 21 kb of the total genome is taken up by orf 1a and orf 1b, which are presumed to encode for proteins such as proteases and polymerases that are associated with virus replication (marra et al., 2003; ruan et al., 2003) . the genome is sufficiently unique to be proposed as belonging to a separate group designated as group iv (marra et al., 2003; ruan et al., 2003) , but this view is not consensual and it has been noted that sars-cov is most closely related to group 2 coronaviruses (snijder et al., 2003) . nevertheless, the structural proteins s, m, n, and e have been clearly identified in virus grown in infected cells (marra et al., 2003; ruan et al., 2003) . the spike, or s-protein, protrudes from the outer surface and is a strong candidate for facilitating viral entry into host cells. li et al. (2003) have reported that the sars-cov sprotein efficiently binds to angiotensin converting enzyme 2 of sars-cov permissive vero e6 cells, suggesting that this binding is a key step in sars-cov infectivity. interaction between the s-protein of a corona virus and specific mammalian receptors can determine species sensitivity. for example, when the s-protein ectodomain of mouse corona hepatitis virus was replaced with that of the feline infectious peritonitis virus, the viral particles were no longer infective for mouse cells, but became infective for feline cells (kuo et al., 2000) . sars-cov has been reported to infect domestic cats and ferrets (martina et al., 2003) so these relationships may have relevance to this broader species susceptibility. a further example of interaction between the s-protein and mammalian receptors involves cd13 or aminopeptidase n. it is the main mammalian receptor for the s-protein of many coronaviruses. substitution on human cells of the human cd13 n-glycosylation site with the porcine n-glycosylation site resulted in a failure to bind human corona virus-229e (wentworth and holmes, 2001) . antibodies directed against the 11 epitopes of s-protein identified in table 2 are therefore potential candidates for neutralizing activity. antibodies against m-protein and eprotein epitopes could also be important although they are smaller and are believed to be less exposed. the protein encoded by orf 10 may be displayed on the viral surface and an immune response to it may contribute to the resolution of disease. antibodies against orf 1a, orf 1b, and n-protein probably are unlikely to have contributed to the in vitro neutralization results. n-protein is internal and is probably inaccessible in the brief exposure time of the neutralization test, and the products of orf 1a and orf 1b are not believed to be structural proteins. nevertheless, they may be very important to in vivo protection. given the complexity of antibodies produced in each convalescent serum, it would be difficult to predict what the relative contribution of each to the overall neutralizing activity might be, particularly when antibodies against conformational or carbohydrate-dependent epitopes might be present. nevertheless, high titers of those consistently represented in the various convalescent sera might be sufficient to confer immunity. numerous strategies have been suggested for producing a sars vaccine (degroot, 2003) . one possibility is to use a combination of peptides, such as those listed in table 2 , as a sequences with 14 amino acids represent three adjacent peptides with a common sequence from residues 5 to 10. sequences with 12 amino acids represent two adjacent peptides with a common sequence from residues 3 to 10. the amino acid positions (aa) in sars-cov open reading frames are taken from blast at ncbi: http://www.ncbi.nlm.nih.gov/blast/blast.cgi. polyvalent antigen. peptide-based vaccines have heretofore had only limited success but this can be attributed to a lack of knowledge as to which peptides to use. such uncertainty is reduced by analyzing the antibody pattern in sera from cases that have successfully resolved the infection. peptides making up the antigen can be screened in advance for homologies to mammalian proteins, reducing the possibility of self-attack. they can also be easily modified to cope with viral mutations that could escape traditional vaccines. significant mutations have already been identified in sars-cov (ruan et al., 2003; martina et al., 2003) , indicating the necessity of developing adaptable vaccines. polypeptide vaccines should be safe, highly stable, and easy to administer. antibodies cloned against particularly sensitive peptides could be used as therapeutic agents. the type of analysis described here may have general utility in designing peptide vaccines and therapeutic antibodies against other infectious agents such as hiv. cellulose-bound overlapping peptides (10 mers) derived from all proteins encoded by the 15 orfs of the sars genome were synthesized using an autospot robot provided by intavis bioanalytical instruments. the software used for designing the arrays was from digen, jerini biotools gmbh, berlin, germany. membranes derivatized with a polyethylene (peg) linker and a free amino terminal, as well as 9-fluorenylmethyloxycarbonyl (fmoc) amino acids were obtained from intavis bioanalytical instruments. the fmoc amino acids (0.25 m) were dissolved in dimethylformamide (dmf, sigma, oakville, ont. canada) and then activated with 1-hydroxybenzotriazole (hobt, sigma) and n, n v -disopropylcarbodiimide (dipc, sigma) for at least 15 min. they were then delivered to the membrane in 60-nl aliquots per spot by the robotic synthesizer. fifteen minutes after completion of each cycle, the membranes were removed from the apparatus and treated with 2% acetic anhydride in dmf to acetylate any free remaining amino groups. they were then washed and further treated with 20% piperidine in dmf to remove the fmoc protecting group. this was followed by washing with dmf and methanol, and finally by air drying. the membrane was then precisely repositioned on the robotic apparatus to initiate the next coupling cycle. after the final cycle, the side chain protecting groups were removed by treatment with a solution of the following composition: 5 ml of 50% trifluoracetic acid; 5 ml dichloromethane (dcm); 300 al triisopropyl silane (sigma); and 200 al water. membranes were then washed twice with dcm, twice with dmf, and twice with methanol. after air drying, membranes were stored in a sealed bag at 4 jc until used. membranes were rehydrated by treating first with methanol, then 50% methanol followed by washing three times in 50 mm tris-buffered saline/0.2% tween 20 (tbs-t). they were then blocked with 5% skim milk in tbs-t overnight at 4 jc. after washing three times with tbs-t, the membranes were incubated at 37 jc for 1 h with 1/100 dilutions of serum samples of patients and controls. after washing, membranes were treated with 1/5000 dilutions (0.2 ag/ml) of goat antihuman iga, igg and igm antibodies conjugated to horseradish peroxidase (sigma). after washing in tbs-t, membranes were developed with an ecl chemiluminescence kit provided by amersham pharmacia biotech (buckinghamshire, england). four milliliters of the two ecl western blotting detection reagents were mixed before being applied on the membranes. the membranes were scanned using a bio-rad fluorescent imager (hercules, ca) and saved for later analysis. the membranes were regenerated by incubation in 8 m urea/1% sds overnight followed by 60 min in 50% ethanol/10% acetic acid. after this step, membranes were washed three times in methanol, and after air drying stored at 4 jc in a sealed bag until reuse. in initial development of the sars-cov serum neutralization test, comparisons were made between the microneutralization test and a 50% plaque reduction assay. testing of several sera consistently produced assay results where the 50% plaque reduction titer was 2-fold greater than in the microneutralization test. following this validation, the microneutralization test system was adopted for greater convenience despite the 2-fold greater sensitivity of the plaque reduction assay. typical results of this assay are illustrated in fig. 2. how the sars vaccine effort can learn from hivspeeding towards the future, learning from the past retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus sars virus infection of cats and ferrets characterization of a novel coronavirus associated with severe acute respiratory syndrome comparative full-length genome sequence analysis of 14 sars coronavirus isolates and common mutations associated with putative origins of infection unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage molecular determinants of species specificity in the coronavirus receptor aminopeptidase n (cd13): influence of n-linked glycosylation the authors are grateful to drs. a mcgeer and d. skowronski for help in obtaining specimens and to dr. k karunakarum for initial assistance in processing the membranes. we are also grateful to dr. mitoshi kunimatsu of nagoya city university for help in programming the robotic synthesizer. this research was supported by a cihr grant on which m.p. is a co-investigator, a grant to p.l.m. from the province of british columbia via the sars accelerated vaccine initiative (savi), and support from individual british columbians to the kinsmen laboratory of neurological research. key: cord-266617-z8uecyl6 authors: pavesi, angelo title: asymmetric evolution in viral overlapping genes is a source of selective protein adaptation date: 2019-04-03 journal: virology doi: 10.1016/j.virol.2019.03.017 sha: doc_id: 266617 cord_uid: z8uecyl6 overlapping genes represent an intriguing puzzle, as they encode two proteins whose ability to evolve is constrained by each other. overlapping genes can undergo “symmetric evolution” (similar selection pressures on the two proteins) or “asymmetric evolution” (significantly different selection pressures on the two proteins). by sequence analysis of 75 pairs of homologous viral overlapping genes, i evaluated their accordance with one or the other model. analysis of nucleotide and amino acid sequences revealed that half of overlaps undergo asymmetric evolution, as the protein from one frame shows a number of substitutions significantly higher than that of the protein from the other frame. interestingly, the most variable protein (often known to interact with the host proteins) appeared to be encoded by the de novo frame in all cases examined. these findings suggest that overlapping genes, besides to increase the coding ability of viruses, are also a source of selective protein adaptation. many viruses produce novel genes inside pre-existing genes by overprinting of a de novo frame onto an ancestral frame (atkins et al., 1979; keese and gibbs, 1992; rancurel et al., 2009; sabath et al., 2012) . the high prevalence of overlapping genes in viruses has been attributed to the advantage of maximizing the gene information content of small viral genomes (miyata and yasunaga, 1978; lamb and orvath, 1991; pavesi et al., 1997) . in detail, the gene-compression hypothesis states that the size of the viral capsid imposes a biophysical limit on the size of the viral genome, thus making overprinting the most adequate strategy to gain new function (chirico et al., 2010) . in alternative, the gene novelty hypothesis argues that the birth of overlapping genes is driven by selection pressures favoring evolutionary innovation (brandes and linial, 2016) . this hypothesis is supported by the finding that overlaps, thought for a long time to be restricted to viruses, also occur in the large genomes of prokaryotic (delaye et al., 2008; fellner et al., 2015) and eukaryotic organisms (szklarczyk et al., 2007; bergeron et al., 2013; vanderperre et al., 2013) . a particularly interesting feature of overlapping genes is that they represent an intriguing example of adaptive conflict. indeed, they simultaneously encode two proteins whose freedom to change is constrained by each other (sander and schulz, 1979; krakauer, 2000; peleg et al., 2004; allison et al., 2016) , which would be expected to reduce the adaptive ability of the virus (simon-loriere et al., 2013) . we would expect, in principle, that overlapping genes are subjected to strong evolutionary constraints, as a single nucleotide substitution can impair two proteins (see the codon position "21" in fig. 1) . a typical example of "constrained evolution" is that occurring in hepatitis b virus (hbv), whose short genome (3.2 kb) contains a high percentage (50%) of overlapping coding regions (mizokami et al., 1997; zhang et al., 2010) . however, overlapping genes can also show a less conservative pattern of change, because of a high rate of non-synonymous substitutions in one frame (positive adaptive selection) with concurrent dominance of synonymous substitutions in the other (negative purifying selection). examples of positive selection concern the overlapping genes that encode the tat and vpr proteins of simian immunodeficiency virus (hughes et al., 2001) , the p19 and p22 proteins of the tombusvirus family of plant viruses (allison et al., 2016) , and the orf2 and orf5 proteins of trichodysplasia spinulosa-associated polyomavirus (kazem et al., 2016) . we can hypothesize for overlapping genes a first evolutionary model in which the two proteins they encode are subjected to similar selection pressures. when selection is strong both proteins (or protein regions) are highly conserved (e.g. the rnase domain of polymerase and the amino-terminal half of the x protein in hbv; see fig. 4 in mizokami et al., 1997) . when selection is not too strong both proteins can vary considerably (e.g. the spacer domain of polymerase and the pres1/s2 domain of the surface protein in hbv; see fig. 4 in mizokami et al., 1997) . this model is named "symmetric evolution", because the number of amino acid substitutions of one protein is expected to be not significantly different from that of the other. it corresponds to the "shared model" by fernandes et al. (2016) . in alternative, we can hypothesize for overlapping genes an evolutionary model in which the two proteins they encode are subjected to significantly different selection pressures. support for this model, which implies adaptive selection on one frame and purifying selection on the other, was provided both by viral (hughes et al., 2001; fujii et al., 2001; guyader and ducray, 2002; stamenković et al., 2016) and mammalian overlapping genes (szklarczyk et al., 2007) . this model is named "asymmetric evolution", because the number of amino acid substitutions of one protein is expected to be significantly different from that of the other. it corresponds to the "segregated model" by fernandes et al. (2016) . we recently assembled a dataset of 80 viral overlapping genes whose expression is experimentally proven (pavesi et al., 2018) , with the aim to provide a useful benchmark for systematic studies. a first analysis of the dataset revealed that overlapping genes differ significantly from non-overlapping genes in their nucleotide and amino acid composition (pavesi et al., 2018) . we also found that the vast majority of the 80 overlaps of the dataset have one or more homologs, suggesting further comparative studies. in the present study, i investigated the evolution of viral overlapping genes by sequence analysis of 75 pairs of homologs. the first aim of the study was to determine which of the two evolutionary models described above is the prevailing one. the second aim was to identify the type of nucleotide substitution that significantly affects the pattern of symmetric/asymmetric evolution. finally, the third aim was to assess whether the most variable protein (in the case of asymmetric evolution) is that encoded by the ancestral or the de novo frame. 2.1. selection criteria for homologous overlapping genes i first extracted from the dataset of 80 overlapping genes experimentally proven (s1 dataset from pavesi et al., 2018) the amino acid sequence of the two proteins encoded by each overlap. for each protein, i searched for homologs against the non-redundant protein sequences ncbi database using blastp (altschul et al., 1997) . when blastp did not detect any homolog i used tblastn, which compared the protein query sequence against the nucleotide collection ncbi database translated in all reading frames. i used tblastn because the amino acid sequence of the protein encoded by one of the two overlapping frames (usually that discovered more recently) may not be reported in many viral genomes present in the ncbi database (pavesi et al., 2018) . the selection of homologous overlapping genes was based on three criteria. the first was an equal length of the homolog. it was met in the great majority of cases (72 out of 80). in the remaining cases, the homolog was only slightly shorter than the query sequence. the exception was the overlap capsid protein/assembly activating protein (aap) of adeno-associated virus-2, whose homolog encodes an aap 9 amino acids shorter in the amino-terminal region and 26 amino acids shorter in the carboxy-terminal region. the second criterion was a homolog yielding, for both the encoded proteins, an alignment with no insertion/deletion (indel) or with a minimal number of indels. in the latter case, i imposed the rule that indel(s) must be located at the same amino acid position in the alignments of the two pairs of proteins (see for example the overlap polymerase/2b protein of spinach latent virus, which is the first overlap in supplementary file s1). by imposing this rule, i could align the two homologous nucleotide sequences in full accordance with the corresponding protein sequences. the alignment of protein sequences was carried out with clustal omega (sievers and higgins, 2014) . the third criterion concerned the cases in which i found multiple homologs meeting the two criteria described above. in these cases, i selected the most distantly related homolog, with the aim to cover the largest evolutionary space. the choice to select only one homolog for each overlapping gene was due to the fact that collection of a larger sample of homologs is limited to a few overlaps, mainly those occurring in virus species that are human pathogens (e.g. influenza and hepatitis viruses or sars and ebola viruses). the search for homologs yielded a dataset of 80 pairs of homologous overlapping genes (supplementary file s1). thirty-seven homologs came from a different virus species, in accordance with the ictv taxonomy (king et al., 2018) (https://talk.ictvonline.org/taxonomy/). the mean nucleotide identity between overlaps and homologs was 70.7%, with a standard deviation (sd) of 9.4%. the remaining 43 homologs came from isolates belonging to the same virus species. in this case, the mean nucleotide identity between overlaps and homologs was 89.6% (sd = 7.1%). for each pair of homologous overlapping genes, the supplementary file s1 contains the following information: i) the nucleotide sequence of the upstream frame and that of the homolog; ii) the amino acid sequence of the protein encoded by the upstream frame (up1) and that of the protein encoded by the homolog (up2); iii) the nucleotide sequence of the downstream frame (shifted of one nucleotide 3' with respect to the upstream frame) and that of the homolog; iv) the amino acid sequence of the protein encoded by the downstream frame (down1) and that of the protein encoded by the homolog (down2); v) the alignment of up1 with up2 and the percent amino acid identity; vi) the alignment of down1 with down2 and the percent amino acid identity; vii) the chisquare analysis, which compared by a 2 x 2 contingency-table the number of the amino acid identities and differences in the up1-up2 alignment with that in the down1-down2 alignment (cut-off of significance = 3.84; 1 degree of freedom; p < 0.05). 3.2. half of overlapping genes evolve in accordance with the asymmetric model i carried out a preliminary analysis using the t-student test for paired data. for each pairs of homologous overlaps, i counted the number of amino acid identities between up1 and up2 and that between down1 and down2. i then calculated the absolute value of the difference between them. the null hypothesis was a mean difference orientation of overlapping genes, with the downstream frame having a shift of one nucleotide 3′ with respect to the upstream frame. there are 3 types of codon position (cp): cp13 (bold character), in which the first position of the upstream frame overlaps the third position of the downstream frame; cp21 (underlined character), in which the second position of the upstream frame overlaps the first position of the downstream frame; cp32 (italic character), in which the third position of the upstream frame overlaps the second position of the downstream frame. based on the genetic code, a nucleotide substitution at first codon position causes an amino acid change in 95.4% of cases, at second codon position in 100% of cases, and at third codon position in 28.4% of cases. thus, nucleotide substitutions at the codon positions "13" and "32" are usually non-synonymous in one frame and synonymous in the other. nucleotide substitutions at the codon position "21" are almost all non-synonymous in both frames. virology 532 (2019) [39] [40] [41] [42] [43] [44] [45] [46] [47] between paired observations close to zero, indicating that overlapping genes evolve in accordance with the symmetric model. the null hypothesis was rejected (t-student = 5.91; 79 degrees of freedom; p = 10 −5 ), indicating that overlapping genes can also evolve in accordance with the alternative asymmetric model. in order to identify which and how many overlapping genes undergo symmetric or asymmetric evolution, i then compared the amino acid diversity between up1 and up2 to that between down1 and down2. i used the contingency-table chi-square test (snedecor and cochran, 1967) with a cut-off value of 3.84 for 1 degree of freedom (p < 0.05). i classified a pair of homologous overlaps as a case of symmetric evolution, if the number of amino acid substitutions in the up1-up2 alignment did not significantly differ from that in the down1-down2 alignment (chi-square < 3.84). an example is given by the overlap ns1 protein/ns2 protein from dendrolimus punctatus densovirus. for the ns1 protein, i found 73 identities and 86 differences when compared to the homolog from hordeum marinum itera-like densovirus. for the ns2 protein, i found 71 identities and 88 differences, yielding a chi-square value (0.01) largely below the cut-off of significance. in alternative, i classified a pair of homologous overlaps as a case of asymmetric evolution, if the number of amino acid substitutions in the up1-up2 alignment was significantly different from that in the down1-down2 alignment (chi-square > 3.84). an example is given by the overlap movement protein/replicase from turnip yellow mosaic virus. for the movement protein, i found 302 identities and 323 differences when compared to the homolog from watercress white vein virus. for replicase, i found 454 identities and 171 differences, yielding a chisquare value (76.3) largely above the cut-off of significance. the chi-square test was highly sensitive. for example, i found that the overlap capsid protein/p31 protein from maize chlorotic mottle virus undergoes asymmetric evolution, in spite of a nucleotide identity with the homolog extremely high (96.7%). indeed, the number of amino acid differences between p31 and homolog (12 out of 149 sites) was significantly higher than that between capsid and homolog (2 out of 149 sites) (chi-square = 6.07; p = 0.01). based on this finding, i set the upper limit of sensitivity of the chi-square test to a nucleotide identity between overlap and homolog of 97%. this filter limited the analysis to 75 (out of 80) pairs of homologous overlaps. overall, i found that 38 overlapping genes evolve in accordance with the asymmetric model (significantly different selection pressures on the two proteins). the highest chi-square value (113.8) concerned the overlap from apple stem grooving virus, which encodes the 36kd movement protein and the polyprotein linker-domain. indeed, the amino acid diversity between linker-domain and homolog (39%; 125 differences and 195 identities) was ten-fold higher than that between movement protein and homolog (4%; 13 differences and 307 identities). i found that the remaining 37 overlapping genes evolve in accordance with the symmetric model (similar selection pressures on the two proteins). the occurrence of similar selection pressures can yield two highly conserved proteins. for example, analysis of the overlap 3a protein/3b protein from human sars coronavirus revealed that the amino acid diversity between 3a and homolog is remarkably low (5.3%; 6 differences and 108 identities), as well as that between 3b and homolog (8.8%; 10 differences and 104 identities). however, the occurrence of similar selection pressure can also yield two proteins with a remarkably less conserved pattern of change. this is the case of the overlap from spinach latent virus, which encodes the zincfinger domain of polymerase and the 2b protein. sequence analysis revealed that the amino acid diversity between zinc-finger domain and homolog is considerably high (47%; 47 differences and 54 identities), as well as that between 2b and homolog (44%; 44 differences and 57 identities). the analysis of amino acid diversity in the 75 pairs of homologous overlapping genes is summarized in fig. 2 . it shows, for each overlap, the percent amino acid (aa) identity of the two encoded proteins with those encoded by the homolog. the subset of the 37 overlapping genes under symmetric evolution ( fig. 2a) contains 31 overlaps in which both proteins have high conservation (aa identity > 50%), 5 overlaps in which both proteins have poor conservation (aa identity < 50%) and 1 overlap with a protein having an aa identity above 50% and the other below 50%. the subset of the 38 overlapping genes under asymmetric evolution (fig. 2b ) contains 24 overlaps in which both proteins have high conservation (aa identity > 50%), 1 overlap in which both proteins have poor conservation (aa identity < 50%) and 13 overlaps with a protein having an aa identity above 50% and the other below 50%. finally, a list of the 75 overlapping genes, classified in accordance with the symmetric or asymmetric model (37 and 38 cases, respectively), is given in supplementary table s1. 3.3. validation of the model of symmetric/asymmetric evolution by analysis of the pattern of nucleotide substitutions in homologous overlapping genes in accordance with wei and zhang (2014) , i first classified the nucleotide sites of each overlapping gene into four categories depending on the impact of potential mutations on the two encoded proteins. the four categories are referred as nn, sn, ns, and ss sites, respectively, where n stands for non-synonymous change and s stands for synonymous change. that is, if all potential mutations at a site cause nonsynonymous change in both proteins, it is a nn site, and so on. i then classified the nucleotide substitutions occurring in the homolog into four categories: nn, sn, ns, and ss. using the contingency-table chisquare test, i compared the number of sn and ns sites in each overlapping gene with the number of sn and ns substitutions in the homolog. under symmetric evolution, i would expect a chi-square value below the cut-off of significance (3.84; 1 degree of freedom), that is a full concordance between the number of sn and ns sites and that of sn and ns substitutions. for example, in the overlap orf4/orf5 from barley yellow striate mosaic virus i counted 49 sn sites and 51 ns sites. in the homolog from maize yellow striate virus, i classified 23 nucleotide substitutions into the sn category and 28 substitutions into the ns category. the chi-square test yielded a value (0.08) largely below the cut-off of significance. under asymmetric evolution, i would expect a chi-square above the cut-off of significance, that is a significant discordance between the number of sn and ns sites and that of sn and ns substitutions. for example, the overlap capsid protein/ns4 protein from bluetongue virus (serotype 10) has 56 sn sites and 46 ns sites. the homolog from bluetongue virus (serotype 16) has 5 nucleotide substitutions belonging to the sn category and 29 substitutions to the ns category. the chisquare test yielded a value (15.07) largely above the cut-off of significance. the analysis of the pattern of nucleotide substitutions in the 75 pairs of homologous overlaps revealed 39 and 36 cases of symmetric and asymmetric evolution, respectively (supplementary table s2 ). this result was in accordance with that obtained previously (from analysis of the amino acid diversity, see supplementary table s1) in the 87% of cases (65 out of 75). overall, i found a total of 33 overlaps under symmetric evolution (they are marked with a single asterisk in supplementary tables s2a) and a total of 32 overlaps under asymmetric evolution (they are marked with a double asterisk in supplementary table s2b ). a list of the 32 overlapping genes under asymmetric evolution is given in table 1 . these findings were not affected by the fact that some homologs came from a different virus species, while others from an isolate within the same virus species. under symmetric evolution, i found 14 and 19 overlaps with the homolog within and between species, respectively. under asymmetric evolution, i found 18 and 14 overlaps with the homolog within and between species, respectively. finally, a further validation of the model of symmetric/asymmetric a. pavesi virology 532 (2019) 39-47 evolution was provided by a correlation test between the chi-square value from analysis of amino acid substitutions and the distribution of nucleotide substitutions at the codon positions "32" and "13" (fig. 1) . given the orientation of overlapping genes in our dataset (fig. 1) , a substitution at the codon position "32" (cp32) is usually synonymous in the upstream frame and always non-synonymous in the downstream frame, while a substitution at the codon position "13" is almost always non-synonymous in the upstream frame and usually synonymous in the downstream frame. under symmetric evolution, the number of substitutions at the codon position "32" is expected to be close to that at the codon position "13", yielding a similar distribution of the amino acid substitutions in the two pairs of homologous proteins. under asymmetric evolution, the number of substitutions at the codon position "32" is expected to be significantly higher (or lower) than that at the codon position "13", yielding a different distribution of the amino acid substitutions in the two pairs of homologous proteins. by comparing the upstream frame of each overlap with that of the homolog, i calculated the absolute value (abs) of the difference between the percent frequency (%f) of substitutions at the codon position "32" (%f.cp32) and that at the codon position "13" (%f.cp13). i then carried out a correlation test between abs (%f.cp32 -%f.cp13) and the chi-square value from analysis of amino acid substitutions. as the chisquare test depends on the extent of the sample (here the length of the protein encoded by the overlap), i normalized the chi-square value in accordance with the cohen's rule (cohen, 1988) . normalization was the square root of the ratio between the chi-square value and the overall length of the two proteins encoded by the overlap (e.g. the highest chisquare value, 113.83, was converted into the highest normalized chi-square value, 0.42). i found a significantly positive correlation between abs (%f.cp32 -%f.cp13) and the normalized chi-square value (r = 0.88; t-student = 14.36; one tailed p < 0.00001; 63 degrees of freedom) (fig. 3) . as expected, this result indicates that asymmetric evolution is significantly affected by an unbalanced distribution of the nucleotide substitutions at the codon positions "32" and "13". to answer the question, i investigated the genealogy of the 32 overlapping genes under asymmetric evolution. identifying which gene is ancestral and which one is de novo (the genealogy of the overlap) can be done by examining their phylogenetic distribution, under the assumption that the gene with the most restricted distribution is the de novo one (rancurel et al., 2009) . this approach yielded a set of 34 overlapping genes with a reliably predicted genealogy (see table 1 in sabath et al., 2012 and table 1 in pavesi et al., 2013) . this set included 16 out of the 32 overlaps under asymmetric evolution. another approach to infer the genealogy of overlapping genes is the codon-usage method. it is based on the assumption that the ancestral gene, which has co-evolved over a long period of time with the other viral genes, has a distribution of synonymous codons significantly closer to that of the viral genome than the de novo gene (keese and gibbs, 1992; sabath et al., 2012; pavesi et al., 2013; willis and masel, 2018) . due to the shortness of most overlapping genes, the method has been improved, with the aim to evaluate the correlation between the codon-usage patterns of overlapping and non-overlapping genes with a fig. 2 . analysis of the amino acid diversity in the 75 pairs of homologous overlapping genes. each pair of columns shows: i) the percent amino acid identity between the protein encoded by the upstream frame of the overlap and that encoded by the homolog (dark column); ii) the percent amino acid identity between the protein encoded by the downstream frame of the overlap (shifted of one nucleotide 3′ with respect to the upstream frame) and that encoded by the homolog (gray column). the horizontal line separates well-conserved homologous pairs (aa identity > 50%) from not well-conserved homologous pairs (aa identity < 50%). (a) subset of the 37 overlapping genes under symmetric evolution. (b) subset of the 38 overlapping genes under asymmetric evolution. the numbering of overlapping genes is in accordance with that given in supplementary table s1 . the underlined numbers indicate the overlaps in which the pattern of symmetric evolution (4 cases out of 37) or that of asymmetric evolution (6 cases out of 38) was not confirmed by chi-square analysis of the nucleotide diversity. virology 532 (2019) 39-47 table 1 list of the 32 overlapping genes evolving in accordance with the asymmetric model. minimal loss of information (pavesi, 2015) . using the improved version of the codon-usage method (pavesi, 2015) , i could predict the genealogy of 18 out of the 32 overlapping genes under asymmetric evolution. in 11 cases, the prediction by codon-usage was concordant with that established by the phylogenetic method. in the remaining 7 cases, the prediction was provided only by the codon-usage method (supplementary table s3 ). the overlap p130/p104 of providence virus is notable, as the ancestral frame p104 was acquired from another viral genome by distant horizontal gene transfer (pavesi et al., 2013) , which makes the codon usage an unreliable predictor of the genealogy. the prediction yielded by phylogenetics is supported by the finding that p104, unlike p130, has a wide phylogenetic distribution (pavesi et al., 2013) . overall, i collected a set of 23 overlapping genes, all under asymmetric evolution and with known genealogy (15 overlaps with a shift of the de novo frame of one nucleotide 3′ with respect to the ancestral frame and 8 overlaps with a shift of two nucleotides 3'). interestingly, i found that in all cases the most variable protein is that encoded by the de novo gene (table 2) . 3.5. symmetric and asymmetric evolution in the same overlap: the case of the overlap polymerase/large envelope protein of hepatitis b virus (hbv) chi-square analysis indicated that the overlap polymerase/large envelope protein of hbv evolves in accordance with the symmetric model (supplementary tables s1 and s2 ). on the other hand, theoretical and experimental studies (pavesi, 2015; lauber et al., 2017) demonstrated that this long overlap (1167 nt) is subjected to modular evolution, as the spacer domain of polymerase and the s domain of the large envelope protein originated de novo by overprinting. thus, the overlap can be subdivided into two regions: a 5′ region (480 nt), in which the spacer domain of polymerase (de novo gene product) overlaps the pre-s domain of envelope (ancestral gene product), and a 3' region (687 nt), in which the reverse transcriptase domain of polymerase (ancestral gene product) overlaps the s domain of envelope (de novo gene product). i carried out a chi-square analysis of the 2 regions of the overlap independently, under the hypothesis that they may have been subject to different evolutionary pressures. this analysis revealed that the 5' region of the overlap undergoes asymmetric evolution, because the amino acid diversity of the spacer domain (33.7%; 54 differences and 106 identities) is significantly higher than that of the pre-s domain (19.4%; 31 differences and 129 identities) (chi-square = 7.75; p = 0.005). asymmetric evolution was confirmed by analysis of the pattern of nucleotide substitutions (chi-square = 10.13; p = 0.001). in addition, chi-square analysis revealed that the 3' region of the overlap undergoes symmetric evolution, as the amino acid diversity of the reverse transcriptase domain (7.4%; 17 differences and 212 identities) does not significantly differ from that of the s domain (11.8%; 27 differences and 202 identities) (chi-square = 2.04; p = 0.15). symmetric evolution was confirmed by analysis of the pattern of nucleotide substitutions (chi-square = 1.61; p = 0.20). with the aim to further validate these findings, i carried out a further analysis using, as homolog, the most distantly related overlap of woolly monkey hbv (79.9% of nucleotide identity). again, chi-square analysis of the amino acid and nucleotide diversity revealed asymmetric evolution in the 5′ region and symmetric evolution in the 3' region. details of both analyses are reported in the supplementary file s2. finally, the finding that the spacer domain of polymerase (de novo gene product) is significantly more variable than the pre-s domain (ancestral gene product) confirms that the most variable protein, under asymmetric evolution, is usually that encoded by the de novo gene. several researchers have developed methods for estimating the strength of selection pressure on overlapping genes (pedersen and jensen, 2001; sabath et al., 2008; de groot et al., 2008; mir and schober, 2014; wei and zhang, 2014) . all methods evaluate, in both overlapping frames, the ratio of non-synonymous nucleotide substitutions to synonymous nucleotide substitutions (dn/ds) by correctly taking into account the problem of the interdependence between sequences imposed by the overlap. the aim is to assess if there is neutral evolution or positive selection in one frame (dn/ds higher than 1) and purifying selection (strong constraints) in the other frame (dn/ds lower than 1). however, the only method having an accessible implementation is that by sabath et al. (2008) . yet, the method has some limitations, as it restricts the analysis to the homologous overlaps in which the two encoded proteins have both an amino acid diversity smaller than 50% or greater than 5%. in the dataset examined here (see the first 75 pairs of homologous overlaps in supplementary file s1), these limitations would have considerably reduced the size of the sample from 75 to 43 pairs of homologous overlaps. i thus chose an approach focused, at first instance, on the evaluation of the amino acid diversity of homologous overlapping proteins, which is the final result of the complex pattern of the interdependent nucleotide substitutions that occur in dual-coding regions. unlike previous studies, limited to a few virus species (sabath et al., 2012; zaaijer et al., 2007; liang et al., 2010; shukla and hilgenfeld, 2015; brayne et al., 2017) , i examined a large dataset of 75 overlaps from 59 virus species. a possible limitation of the study concerns the selection criteria for homologous overlapping genes. in particular, the first two stringent criteria (an equal length of the homolog and an alignment with a minimal number of indels) led to exclusion, for some overlaps, of highly divergent homologs. an example is given by the overlap p3n-pipo/ polyprotein of turnip mosaic virus, in which the length of the p3n-pipo protein is quite variable among the different potyvirus species, ranging from 60 to 115 amino acids (hillung et al., 2013) . thus, the dataset used in this study likely underestimates the sequence diversity of overlapping genes, as it was created mainly to ensure a high quality in the homologous relationship. the finding that 32 out of 65 overlapping genes (table 1 ) undergo asymmetric evolution is striking, as well as that the most variable protein is encoded by the de novo gene in all cases examined (table 3 ). in particular, i would point out the overlap orf3/orf4 from tobacco bushy top virus, which encodes two proteins entirely nested within each other. this peculiar arrangement is similar to that of the overlap p19/ p22 from tomato bushy stunt virus, in which the de novo p19 protein fig. 3 . correlation between the normalized chi-square value (from analysis of amino acid substitutions) and the absolute value (abs) of the difference between the percent frequency (%f) of nucleotide substitutions at the codon position "32" (%f.cp32) and that at the codon position "13" (%f.cp13). empty circles indicate the 33 overlapping genes under symmetric evolution. black circles indicate the 32 overlapping genes under asymmetric evolution. virology 532 (2019) 39-47 table 2 list of the 23 overlapping genes with known genealogy and evolving in accordance with the asymmetric model. shows a previously unknown structural fold an a previously unknown mechanism of binding to small interfering rnas (vargason et al., 2003; baulcombe and molnár, 2004; scholthof, 2006) . i believe that structural or functional studies on the de novo orf3 protein from tobacco bushy top virus could reveal new interesting features. in addition, i would point out the overlap polymerase (pb1 subunit)/pb1-f2 protein of human influenza a virus. it shows, when compared to the homolog from duck, a sixteen-fold increase of substitutions at the codon position "32" (89.2%) with respect to the codon position "13" (5.4%). this yields only 3 amino acid differences between the two pb1 subunits and as many as 35 differences between the two pb1-f2 proteins. interestingly, the de novo pb1-f2 protein has been shown to largely contribute to viral pathogenicity by a pleiotropic effect (chen et al., 2001; varga et al., 2011; yoshizumi et al., 2014) . several other de novo proteins under asymmetric evolution are known to play a role in viral pathogenicity. eight de novo proteins (arfp, vp5, l*, x, vf1, pb1-f2, p6, and nss) act as suppressor or antagonist of the interferon response by the host (park et al., 2016; lauksund et al., 2015; sorgeelos et al., 2013; wensman et al., 2013; mcfadden et al., 2011; varga et al., 2011; garcía-rosado et al., 2008; jääskeläinen et al., 2007) . four de novo proteins (p19, p69, ac4, and movement protein) act as suppressor of rna silencing (silhavy et al., 2002; chen et al., 2004; chellappan et al., 2005; yaegashi et al., 2008) . two de novo proteins (apoptin and pb1-f2) act as apoptosis factor (noteborn et al., 1994; chen et al., 2001) . finally, the de novo protein pa-x has the ability to selectively degrade the host rna-polymerase ii transcripts (khaperskyy et al., 2016) . however, another possible limitation of the study depends on the fact that the subset of overlapping genes evolving asymmetrically and with known genealogy (23 overlaps) is too small to conclude that the de novo protein is always the preferred target of selection. furthermore, overlapping genes are subjected to a variety of selection pressures that are independent of the orientation of the overlapping frames relative to one another. 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simple method for estimating the strength of natural selection on overlapping genes the x proteins of bornaviruses interfere with type i interferon signalling gene birth contributes to structural disorder encoded by overlapping genes inhibition of long-distance movement of rna silencing signals in nicotiana benthamiana by apple chlorotic leaf spot virus 50 kda movement protein influenza a virus protein pb1-f2 translocates into mitochondria via tom40 channels and impairs innate immunity independent evolution of overlapping polymerase and surface protein genes of hepatitis b virus evolutionary selection associated with the multi-function of overlapping genes in the hepatitis b virus the author is grateful to alessio peracchi (university of parma) and alberto vianelli (university of insubria) for helpful suggestions. special thanks to xinzhu wei (university of michigan) for valuable comments and suggestions and to gianmarco del vecchio for preparing the figures. the author thanks the anonymous referees and the editor alexander e. gorbalenya for their helpful feedback and suggestions. the study was financed by the miur (ministero dell'università e della ricerca). supplementary data to this article can be found online at https:// doi.org/10.1016/j.virol.2019.03.017. key: cord-262226-7kwkla73 authors: fang, shouguo; xu, linghui; huang, mei; qisheng li, frank; liu, d.x. title: identification of two atr-dependent phosphorylation sites on coronavirus nucleocapsid protein with nonessential functions in viral replication and infectivity in cultured cells date: 2013-07-09 journal: virology doi: 10.1016/j.virol.2013.06.014 sha: doc_id: 262226 cord_uid: 7kwkla73 coronavirus encodes an extensively phosphorylated and highly basic nucleocapsid (n) protein. previous studies have identified ser190, ser192, thr378 and ser379 as the phosphorylation sites for coronavirus infectious bronchitis virus (ibv) n protein. in this study, we show that phosphorylation at thr378 and ser379 sites is dependent on the ataxia-telangiectasia mutated (atm) and rad3-related (atr), a kinase activated during ibv replication. introduction of ala substitutions at these two sites individually, in combination of the two and together with other two sites (ser190 and ser192) into an infectious ibv clone did not affect recovery of the recombinant viruses containing the mutations. a mutant virus (ribv-nm4) carrying the four ala substitutions grew at a similar, if not better, growth rate as wild type virus. this study reveals a cellular kinase responsible for phosphorylation of a coronavirus n protein at two positions and the functional consequence of this modification on coronavirus replication. coronavirus is an enveloped virus with a single strand, positive-sense rna genome of 27-30 kb length. in cells infected with coronavirus, a 3′-coterminal nested set of 6-9 mrnas species, including the genome-length mrna (mrna1) and 5-8 subgenomic mrna species (mrna2-9), is expressed. the genomelength mrna1 encodes two overlapping replicase proteins in the form of polyproteins 1a and 1ab, which are processed by virusencoded proteinases into at least 15 putative nonstructural proteins (nsp1-nsp16) (fang et al., 2008 . the four structural proteins, spike (s), envelope (e), membrane (m) and nucleocapsid (n), are encoded by subgenomic mrnas. in addition, several putative nonstructural proteins, such as 3a, 3b, 6, 7a, 7b, 8a, 8b, 9b, are also encoded by subgenomic mrnas thiel et al., 2003) . coronavirus n protein contains multiple functional domains. sequence comparisons and structural studies have identified three main structural domains, although their primary sequence conservation is low (lai and cavanagh, 1997; li et al., 2005) . of this the middle domain is an rna-binding domain, capable of binding both coronavirus-and non-coronavirus-derived rna sequences in vitro tan et al., 2012; zhou et al., 1996) . the motifs for ribosome binding and nucleolar localization signals have been assigned to domain iii . more recently, chang et al. (2009) proposed that all coronavirus n proteins may share a similar modular organization. in cells expressing the n protein, it localizes either to the cytoplasm alone or to the cytoplasm and nucleolus . this nucleolar localization has been shown to be a common feature of the coronavirus family . the prime function of the protein is to associate with the genomic rna to form a ribonucleoprotein complex (rnp) and viral core (davies et al., 1981; escors et al., 2001; narayana et al., 2000; riso et al., 1996) . the protein may also play an important role in the replication of the genomic rna (chang and brian, 1996) , and in the transcription and translation of subgenomic rnas (sgrna) (almazán et al., 2004; baric et al., 1988; stohlman et al., 1988; tahara et al., 1994; zúñiga et al., 2010) . in addition, n protein might inhibit host cell proliferation or delay cell growth, possibly by disrupting cytokinesis (chen et al., 2002; wurm et al., 2001) . it can also stimulate strong humoral and cellular immune response, making it a potential vaccine candidate (kim et al., 2004) . coronaviruse n protein is an extensively phosphorylated and highly basic protein. it varies from 377 to 455 amino acids in length and has high serine content (7-11%) as potential targets for phosphorylation. this protein contains several basic amino acid-rich regions functioning as nucleolar localization signals and rna-binding motifs, and a serine/arginine (s/r) rich motif (li et al., 2005 , fan et al., 2005 tan et al., 2006) . biochemical characterization and mutagenesis studies demonstrated that coronavirus n protein was also posttranslationally modified by covalent attachment to the small ubiquitin-like modifier (sumo). sumoylation may play a role in the self-association and homo-oligomerization of the n protein, and its interference of host cell division (li et al., 2005) . infectious bronchitis virus (ibv), a prototype coronavirus, is the etiological agent of infectious bronchitis, which impairs the respiratory and urogenital tracts of chickens (cavanagh, 2007) . similar to other coronaviruses, ibv n protein is heavily phosphorylated. by using mass spectroscopic analysis, two conserved amino acid clusters, ser190ser192 and thr378ser379, in ibv n protein were identified as the two regions for phosphorylation (chen et al., 2005) . subsequently, a total of six residues (ser162, ser170, thr177, ser389, ser424 and thr428) were identified as phosphorylation sites for mouse hepatitis virus (mhv) a59 n protein (white et al., 2007) , and four phosphoserines at positions 9, 156, 254 and 256 were identified in transmissible gastroenteritis virus (tgev) n protein (calvo et al., 2005) . in addition, the host cell kinase (s) responsible for phosphorylation of coronavirus n protein is beginning to emerge. one example is glycogen synthase kinase-3, which was shown to be involved in regulation of the phosphorylation of severe acute respiratory syndrome coronavirus (sars-cov) n protein . current evidence suggests that phosphorylation may play important roles in regulation of the functions of coronavirus n protein. these included regulation of its subcellular location, viral rna transcription, particle assembly, immunoreactivity and specificity (calvo et al., 2005; jayaram et al., 2005; shin et al., 2007; surjit et al., 2005; spencer et al., 2008) . however, the functional relevance of n protein phosphorylation has not been rigorously tested in the context of virusinfected cells using an infectious clone system, and host cell kinases responsible for phosphorylation of other coronavirus n proteins are not reported. ibv infection perturbs cell cycle progression and arrests cell at the s and g2/m phases (li et al., 2007; wilson and rangasamy, 2001; wurm et al., 2001) . more recently, ibv infection was shown to induce dna damage response and activation of the ataxiatelangiectasia mutated (atm) and rad3-related (atr) kinase/ checkpoint kinase-1 (chk1) pathway partly through interaction between coronavirus nsp13 and dna polymerase delta (pol δ) (xu et al., 2011) . in this study, we show that phosphorylation at the thr378 and ser379 sites is dependent on atr, a kinase activated during ibv replication. introduction of ala substitutions at these two sites individually, in combination of the two, and together with the other two sites (ser190 and ser192), into an infectious ibv clone did not affect the recovery of recombinant viruses containing the mutations. a mutant virus (ribv-nm4) carrying ala substitutions at all the four previously identified sites grew at a similar, if not better, growth rate as wild type virus. this study reveals a cellular kinase responsible for phosphorylation of a coronavirus n protein at two sites and the functional consequence of this modification on coronavirus replication in virusinfected cells. in a previous study, we showed the phosphorylation of a number of atr-specific substrates, including chk1, the best studied atr substrate on ser317 and ser345, and replication protein a2 (rpa2), a component of the heterotrimeric rpa complex, on ser4/8 in ibv-infected cells (xu et al., 2011) . the specific residues for the atm kinase activity including chk2 on thr68 and atm on ser1981, however, were not detected in the same infected cells, demonstrating the activation of the atr-chk1 pathway during ibv infection (xu et al., 2011) . during this study, we noted that, when cells were infected with ibv at high multiplicity of infection and an excess amount of samples was loaded, a batch of pchk1-specific antibodies was also able to detect clearly a band migrating slightly more rapidly than pchk1 in ibv-infected cells, but not in cells infected with the ultraviolet (uv)-inactivated ibv at 8 and 12 h post-infection (fig. 1) . it should be pointed out that this observation was not consistently made when pchk1-specific antibodies from different sources were used. it was also noted that apparently more pchk1 was detected in cells treated with uv-inactivated ibv (fig. 1) . this increased detection of pchk1 may represent cellular contamination from the inoculums, as the virus stocks used for uv irradiation were prepared by freezing/thawing the infected cells and were not purified. re-probing of the same membrane with antibodies against ibv n protein detected the n protein at the exact position ( fig. 1 ). this result suggests that the protein band detected by anti-pchk1 may be a phosphorylated form of the ibv n protein. a series of experiments was then carried out to confirm if this indeed represents a phosphorylated form of ibv n protein and if atr is responsible for its phosphorylation. first, cells infected with ibv were analyzed by western blot with antibodies against ibv n protein and atm/atr substrates. as shown in fig. 2a , the fulllength 48-kda ibv n protein and several smaller bands, representing either premature or cleavage products derived from n protein (li et al., 2005) , were detected by western blot with anti-ibv n antibodies. the 48-kda band was also detected efficiently by the anti-atm/atr substrate antibodies (fig. 2a) . in addition, a background band with unknown identity was also detected from both mock-and ibv-infected cells by this antibody (fig. 2a) . the involvement of atr in the phosphorylation of ibv n protein was then analyzed by addition of 10 and 15 μm of schisandrin b (schb), a specific atr inhibitor functioning in ibv-infected cells as demonstrated in a previous study (xu et al., 2011) cells at 4 h post-infection. to more specifically analyze the full-length ibv n with an unrelated antibody, a recombinant ibv (ribv-fn) with a flag tag added to the n-terminus of the ibv n protein was constructed and used in this study. the recombinant virus showed very similar growth kinetics as wild type ibv (fig. 2b ). the cells were harvested at 24 h post-infection and analyzed by western blot. as observed in our previous study (xu et al., 2011 ), schb exhibited a certain level of inhibitory effect on ibv replication. ibv n protein was reduced to 78% and 55% when 10 and 15 μm, respectively, of schb were added, as revealed by western blot using anti-flag antibodies and densitometry analyses (fig. 2c) . probing of the same membrane with antibodies against atm/atr substrates detected much less phosphorylated n protein in the presence of schb (fig. 2c) . quantification of the band intensities by densitometry after normalizing to the total n protein showed that the atr-dependent phosphorylation of n protein was reduced to 47% and 2% after addition of 10 and 15 μm of schb, respectively (fig. 2c ). the phosphorylation of ibv n protein by atr was then analyzed in cells overexpressing the n protein. cells transfected with ibv n construct were subjected to uv irradiation at 22 h posttransfection in the presence or absence of 10 μm of schb. western blot analysis with antibodies against ibn n and atm/atr substrates showed that significantly less phosphorylated ibv n protein was detected in the uv-irradiated, transfected cells in the presence of 10 μm of schb (fig. 2d ). in the absence of 10 μm of schb, a 2.8-fold increase of the atr-dependent phosphorylation of n protein was detected in the transfected cells after uv-irradiation, compared to that in the cells without uv-irradiation ( fig. 2d ). in the presence of 10 μm of schb, a 1.5-fold increase of the atr-dependent phosphorylation of n protein was detected by the same comparison (fig. 2d ). these results would lend more support to the conclusion that atr is one of the kinases responsible for phosphorylation of ibv n protein. to systematically map the atr-dependent phosphorylation of ibv n protein, ala substitutions at the four previously identified sites (ser190, ser192, thr378 and ser379) as well as two potential new sites (ser65 and thr109) predicted to be atr substrates (fig. 3a) were carried out. the ala substitutions were then introduced into an ibv infectious clone either individually, or in combination of two, four, five or six, and nine recombinant ibvs were recovered from these constructs (fig. 3a) . all the nine mutant viruses were passaged in cells up to eight passages and their genetic stability was determined by nucleotide sequencing of the entire n protein-coding region. the sequencing results confirmed that the ala substitutions were stably maintained in all the mutant viruses, and no additional mutations were introduced into the n protein during passaging. the growth properties of the recombinant viruses (passage 8) were then determined. as shown in fig. 3b , very similar growth kinetics was found between wild type ibv and the mutant viruses. one notable difference was that all mutant viruses reached their peak titers at 32 h post-infection, while wild type virus reached the peak titer at 24 h post-infection (fig. 3b) . wild type and seven mutant viruses (nm1-7, passage 3) were first used to infect h1299 cells. total cell lysates were prepared and analyzed by western blot with antibodies against atm/atr substrates. the results showed that the atr-dependent phosphorylation of n protein was detected only in cells infected with wild type, nm1 and nm2 mutant viruses (fig. 4a) . the same band was not detected in cells infected with other five mutant viruses (nm3-nm7) (fig. 4a) . as all these five mutant viruses contain ala substitutions at thr378 and ser379 positions, it suggests that phosphorylation of ibv n protein at these two sites is atrdependent. re-probing of the same membrane with pchk1specific antibodies detected a much reduced amount of the phosphorylated n protein in cells infected with nm4 mutant virus, and moderately reduced amounts of the phosphorylated n protein in cells infected with nm3, nm5, nm6 and nm7 mutant viruses, respectively (fig. 4a) . the additional two mutant viruses, nm8 and nm9 containing the ala substitutions at thr378 and ser379, respectively (fig. 3a) , were then used to assess the relative contribution of the two residues to the atr-dependent phosphorylation of ibv n protein. h1299 cells were infected with wild type, nm3, nm8 and nm9 mutant viruses, and total cell lysates were prepared. western blot analysis of total cell lysates with antibodies against atm/atr substrates showed, once again, detection of the atr-dependent phosphorylation of ibv n protein in cells infected with the virus (fig. 4b) . no obvious phosphorylation of ibv n protein was detected by the same antiserum in cells infected with the three mutant viruses (fig. 4b) . these results indicate that both amino acid residues are phosphorylated by atr and the neighboring sequence may affect this atr-dependent phosphorylation of ibv n protein. it was also noted that pchk1 was not readily detected by western blot using both atm/atr substrate and pchk1-specific antibodies under the conditions shown in fig. 4a and b. the reason may be due to the relatively low abundance of pchk1, compared to the phosphorylated n protein in virus-infected cells. by immunofluorescence with antibodies against ibv n protein. as shown in fig. 4a , massive syncytium formation was observed in cells infected with both wild type and mutant viruses. no distinguishable difference in syncytium formation and subcellular distribution of n protein between wild type and the mutant viruses was observed (fig. 5a) . western blot analysis of another set of the infected cells with anti-ibv n antibodies showed the presence of both full-length and other processed forms of the n protein (li et al., 2005) (fig. 5b) . interestingly, slightly more n protein was detected from cells infected with the mutant virus at this time point (fig. 5b) . the growth properties of the mutant virus were then studied by plaque formation and growth kinetics studies. as shown in fig. 5c , plaques with similar sizes were found in cells infected with both wild type and ibvnm4 mutant viruses at 24 h post-infection. analysis of the growth kinetics of wild type and the mutant viruses also showed very similar growth properties for both viruses, although the mutant virus reached its peak titers at 24 h post-infection, which was 8 h later than did wild type virus (fig. 5c) . interestingly, the mutant virus remained at peak titers until the end of the time course at 32 h postinfection (fig. 5c) . it is not clear if this may represent an altered cellular response to the infection caused by this mutant virus. the buoyant density of ibvnm4 mutant virus was then analyzed by ultracentrifugation using 10-50% sucrose gradients, after collection of the virus from culture supernatants by spinning down through a 20% sucrose cushion. eleven fractions were collected and analyzed by western blot with antibodies against ibv n protein, which was used as a marker for the presence of virus particles. both wild type and mutant viral particles were found to be present mainly in fractions 7 and 8 (50.4% for wild type and 58.4% for ibvnm4) (fig. 5d) . it was noted that more viral particles (32.0%) were found in fraction 7 for wild type ibv, but more virus particles (39.2%) were found in fraction 8 for the mutant virus (fig. 5d) . these results demonstrate that the mutant virus may have a slightly lower buoyant density than wild type virus. further study would be required to confirm if this difference may reflect the difference in the phosphorylation of the n protein. coronavirus n protein plays multiple functions during the viral replication cycle. available evidence suggests that most of its at 24 h post-infection, the cultured media were collected and cell debris was removed by centrifugation at 10,000 rpm for 10 min. the virus particles were pelleted through a 20% sucrose cushion. the pellets were resuspended in 1 ml medium and subjected to ultracentrifugation in a 10-50% linear sucrose gradient. eleven fractions were collected and the presence of virus particles in each fraction was analyzed by western blot with antibodies against ibv n protein. the percentage of virus in each fraction was calculated after determination of the amount of n protein in each fraction by densitometry. functions may be regulated by the phosphorylation of the protein (calvo et al., 2005; jayaram et al., 2005; shin et al., 2007; surjit et al., 2005; spencer et al., 2008) . in this study, two previously identified phosphorylation sites in ibv n protein, thr378 and ser379, are shown to be phosphorylated by atr, which is activated by ibv-induced dna replication stress during ibv infection of cells (xu et al., 2011) . ala substitutions of these two residues together with the other two previously identified phosphorylation sites (ser190 and ser192) as well as two predicted sites for atr individually or in combination did not affect the recovery of infectious viruses containing the mutations. in this study, we provided evidence suggesting that phosphorylation of ibv n protein at thr378 and ser379 by atr appears to play a limited, if any, regulatory function in ibv replication and infectivity in cultured cells. as ibvnm4 also covers ala substitutions at other two previously identified phosphorylation sites (ser190ser192), it would point to the possibility that phosphorylation of coronavirus n protein at these four sites may represent a by-effect of host response to virus infection. coronavirus infection may activate a diversity of host cell signal transduction pathways as well as kinases, which would in turn lead to the phosphorylation of viral n protein. this may explain why more phosphorylated n protein is incorporated into ibv virions (jayaram et al., 2005) , as extensive phosphorylation of n protein would occur at a relatively late stage of the viral infection cycle. it is currently unclear if this is specific to ibv, as some other coronaviruses, such as mhv, tend to incorporate hypophosphorylated n protein into virions (white et al., 2007) on the other hand, we cannot rule out the possibility that phosphorylation of ibv n protein may occur at other positions in cells infected with nm4 and other related mutant viruses, and phosphorylation at these alternate sites would functionally compensate for the loss of phosphorylation at these four positions. this possibility would be supported by the observation that significantly more phosphorylated n protein was detected in cells infected with nm5, nm6 and nm7 mutant viruses, than in cells infected with nm4. it appears that additional ala substitutions at ser65 and thr109 in these three mutant viruses might induce more phosphorylation of ibv n protein at a site(s) recognized by the pchk1-specific antibodies used. in this context, it would be interesting to determine which phosphorylation site(s) of ibv n protein may be recognized by this pchk1-speific antiserum. data present in this study are inconclusive, but appear to indicate that both thr378 and ser379 may be recognized specifically or nonspecifically by the pchk1 antibodies used, as significantly reduced amounts of the phosphorylated n protein were detected in cells infected with mutant viruses containing mutations at these two positions, compared to that in cells infected with wild type and other mutants. however, as certain amounts of the phosphorylated n protein were still detected by this antiserum in cells infected with these mutant viruses, other phosphorylation site (s) must exist. multiple cellular kinases are involved in phosphorylation of coronavirus n protein. this may constitute another layer of regulatory mechanisms controlling the phosphorylation status of coronavirus n protein in virus-infected cells. for example, both thr378 and ser379 residues could be phosphorylated by casein kinase ii (ckii), as predicted by computer-aided programs. it was reported that ckii can be stabilized by atr-dependent phosphorylation of chk1 at ser317 in response to stalled dna replication (martin and ouchi, 2008) . this in turn would lead to phosphorylation of phosphatase and tensin homolog protein (pten) at thr383. as ibv infection activates the atr/chk1 pathway, phosphorylation of ibv n protein at multiple sites may be also regulated by a similar mechanism. in addition, phosphorylation of n protein is most likely a dynamic process, timely regulated during the infection cycle. in this study, we showed that the phosphorylated n protein was detected by a pchk1-specific antibody at as early as 8 h postinfection, as well as at 24 h post-infection. analysis of the effects of ala substitutions on the phosphorylation status of ibv n protein in virus-infected cells was carried out at 24 h post-infection in most of the studies shown here. this time point was chosen based on the consideration that slightly delayed growth kinetics was observed in most of the mutant viruses. it was noted that more phosphorylated n protein was detected in cells infected with some mutant viruses (mainly nm1 and nm2 mutant viruses) than that in cells infected with wild type virus. this may reflect this dynamic regulatory process at this time point. the successful recovery of viruses from all the mutant constructs confirms that phosphorylation at these positions is not essential for the viability of ibv in cultured cells. in fact, western blot analysis of viral protein expression and viral growth kinetics analysis showed that most of the mutant viruses grew similarly well as wild type virus. this is confirmed by detailed analysis of the growth properties and kinetics of ibvnm4, showing that the mutant virus grew equally well, if not better, as wild type virus, although a slightly delayed growth kinetics was observed. interestingly, after reaching peak titer at 24 h post-infection, the mutant virus remains at its peak titer until the end of the time course at 32 h post-infection. this is in contrast to wild type virus, which reached the peak titer at 16-24 h post-infection. the titers were gradually declined afterward. the underlying mechanisms for this difference are currently unknown, but it may reflect the difference in virus-host interactions between wild type and the mutant virus. one possibility is that the mutant virus may have an altered ability to induce cell death or cell cycle arrest. a more thorough study would be required to understand this issue more. in conclusion, this study has provided strong evidence that activation of atr/chk1 pathway during ibv infection is responsible for phosphorylation of ibv n protein at two previously identified sites. phosphorylation of n protein at these two positions, however, may play limited, if any, regulatory functions in ibv replication and infectivity in cultured cells. this would meanwhile reinforce our previous conclusion that ibv infection leads to efficient activation of the atr pathway (xu et al., 2011) . vero (atcc ccl-81) cells were maintained in dmem supplemented with 10% fetal bovine serum (fbs), penicillin (100 units/ ml) and streptomycin (100 μg/ml) at 37 1c in a 5% co 2 environment. h1299 (atcc crl-5803) cells were maintained in complete rpmi 1640 medium (jrh) supplemented with 10% newborn calf serum. both vero and h1299 cells were routinely used to culture vero-adapted ibv (liu et al., 1995 (liu et al., , 1998 tay et al., 2012) the passage 65 (p65, designated wild type virus (wtibv) of vero-adapted ibv beaudette was described before (fang et al., , 2007 . the ibv strains were propagated in vero cells and the tissue culture infective dose (tcid 50 ) of viral stocks was calculated by the reed-muench method (yamada and liu, 2009 ). viral stocks were kept at −80 1c until use. schisandrin b (schb, shanghai tauto biotech co. ltd., china) was dissolved in dmso and stored at −80 1c, as previously described (xu et al., 2011) . antibodies against chk1ser317 and phospho-(ser/thr) atm/ atr substrates were purchased from cell signaling technology (beverely, ma, usa). antibody against flag was from sigma-aldrich, and antibodies against actin and tubulin were from santa cruz (santa cruz, ca). polyclonal antibodies against ibv n and s were raised in rabbits (xu et al., 2010; yamada and liu, 2009 ). horseradish peroxidase (hrp)-linked goat anti-rabbit secondary antibodies, hrp-linked goat anti-mouse secondary antibodies were purchased from dako (glostrup, denmark). inhibitors schb was added to cells 1 h prior to infection or 8 h post-transfection, and kept in the media during the infection. cells of 90-95% confluency grown on 12-well plates were infected with ibv at a multiplicity of infectivity of approximately 1, and were harvested at the indicated time points post-infection. plasmid pkt-0 which has t7 promoter for transcription was used for transient transfection using the vaccinia/t7 recombinant virus liu, 2000, 2002) . the pkt-n construct that encoded full-length n protein was previously reported (li et al., 2005) . epitope-tagged constructs were made by overlap pcr and cloned into the pkt-0. point mutations were made by site-directed mutagenesis using the quikchange™ kit (stratagene). cells at about 90% confluency were infected with the recombinant vaccinia/t7 virus for 1 h followed by transfection of plasmid dna using the effectene transfection reagent (qiagen). each sample was lysed with 1 â sds sample loading buffer without bromophenol blue and the protein concentration was determined by the bio-rad protein assay kit. equal amounts of total protein were separated by sds-page and transferred to pvdf membranes. membranes were incubated with a primary antibody, subsequently with hrp-conjugated secondary antibody, and detected using the ecl advance western blotting detection kit (amersham). vero cells were cultivated in 4-well chamber slides (iwaki). ibv-infected cells were washed with phosphate buffered saline (pbs) supplemented with 10% normal goat serum, fixed with 4% paraformaldehyde in pbs for 15 min, and permeabilized with 0.2% triton x-100 for 10 min. if staining was performed by incubating cells with polyclonal antibodies against ibv n protein and subsequently with the fitc-conjugated anti-rabbit igg. cells were examined by fluorescent microscopy. the detailed procedure to create a full-length ibv cdna clone was previously reported (tan et al., 2006; fang et al., 2007) . the region of the ibv cdna covering the n gene was replaced with mutant n genes, and subsequently ligated into full-length ibv cdna. full-length transcripts were generated in vitro using the mmessage mmachine t7 kit (ambion, austin, tex). wild type ibv n gene transcripts were also generated to enhance the recovery of virus. vero cells at about 90% confluency were trypsinized, washed twice with ice-cold pbs, and resuspended in pbs. rna transcripts were introduced into vero cell by using the bio-rad gene pulser ii electroporator. cells were cultured overnight in 1% fbs-containing dmem in a 6-well plate and further incubated in dmem without fbs. at 48 h post-electroporation, viral rna replication was investigated by 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replication and transcription mechanisms and enzymes involved in sars coronavirus genome expression characterization of cellular furin content as a potential factor determining the susceptibility of cultured human and animal cells to coronavirus infectious bronchitis virus infection identification of mouse hepatitis coronavirus a59 nucleocapsid protein phosphorylation sites viral interaction with the host cell sumoylation system glycogen synthase kinase-3 regulates the phosphorylation of severe acute respiratory syndrome coronavirus nucleocapsid protein and viral replication localization to the nucleolus is a common feature of coronavirus nucleoproteins and the protein may disrupt host cell division coronavirus infection induces dna replication stress partly through interaction of its nonstructural protein 13 with the p125 subunit of dna polymerase delta the cellular rna helicase ddx1 interacts with coronavirus nonstructural protein 14 and enhances viral replication proteolytic activation of the spike protein at a novel rrrr/s motif is implicated in furin-dependent entry, syncytium formation and infectivity of coronavirus infectious bronchitis virus in cultured cells the infectious bronchitis virus nucleocapsid protein binds rna sequences in the 3′ terminus of the genome coronavirus nucleocapsid protein facilitates template switching and is required for efficient transcription this work was partially supported by a competitive research programme (crp) grant (r-154-000-529-281), the national research foundation, singapore. key: cord-269986-jdcw59r2 authors: regan, andrew d.; cohen, rebecca d.; whittaker, gary r. title: activation of p38 mapk by feline infectious peritonitis virus regulates pro-inflammatory cytokine production in primary blood-derived feline mononuclear cells date: 2009-02-05 journal: virology doi: 10.1016/j.virol.2008.11.006 sha: doc_id: 269986 cord_uid: jdcw59r2 feline infectious peritonitis (fip) is an invariably fatal disease of cats caused by systemic infection with a feline coronavirus (fcov) termed feline infectious peritonitis virus (fipv). the lethal pathology associated with fip (granulomatous inflammation and t-cell lymphopenia) is thought to be mediated by aberrant modulation of the immune system due to infection of cells such as monocytes and macrophages. overproduction of pro-inflammatory cytokines occurs in cats with fip, and has been suggested to play a significant role in the disease process. however, the mechanism underlying this process remains unknown. here we show that infection of primary blood-derived feline mononuclear cells by fipv wsu 79-1146 and fipv-df2 leads to rapid activation of the p38 mapk pathway and that this activation regulates production of the pro-inflammatory cytokine tumor necrosis factor alpha (tnf-alpha) and interleukin-1 beta (il-1 beta). fipv-induced p38 mapk activation and pro-inflammatory cytokine production was inhibited by the pyridinyl imidazole inhibitors sb 203580 and sc 409 in a dose-dependent manner. fipv-induced p38 mapk activation was observed in primary feline blood-derived mononuclear cells individually purified from multiple spf cats, as was the inhibition of tnf-alpha production by pyridinyl imidazole inhibitors. coronaviruses are a diverse family of enveloped positive-stranded rna viruses that infect a wide range of species including humans. coronaviruses are divided into three groups in which group 1 and 2 infect mammals and group 3 infects birds (perlman et al., 2008) . feline coronaviruses (fcovs) belongs to group 1 and are classified as either serotype i or ii depending on the sequence of their spike (s) protein (rottier, 1999) . in addition, each serotype is divided into two biotypes designated as either feline enteric coronavirus (fecv) or feline infectious peritonitis virus (fipv) based on their pathological outcome in cats (vennema et al., 1998) . fecv is ubiquitous amongst felines and causes mild to often unapparent enteritis, while fipv leads to a lethal systemic infection marked by severe granulomatous inflammation (pedersen et al., 1984a (pedersen et al., , 1984b weiss and scott, 1981) . the mechanism underlying this drastic difference in disease between the two biotypes remains elusive, namely because fecv and fipv isolates from the same serotype are virtually indistinguishable on the genetic and antigenic level. however it has been shown that the two biotypes possess markedly different abilities to infect cells of the immune system, with fipv isolates possessing an extended tropism that allows for the infection of macrophages and monocytes (stoddart and scott, 1989) . recent studies have suggested that this alteration in tropism may be due to mutations in the s protein that affect protein cleavage and fusion activation during entry (regan et al., 2008; rottier et al., 2005) . viral pathogens that infect immune cells (e.g. human immunodeficiency virus (hiv) and dengue virus) are known to induce aberrant cytokine production, a process which is proposed to play a role in the pathological outcome of their respective diseases (fantuzzi et al., 2003; kedzierska and crowe, 2002; leong et al., 2007) . studies of cats with fip have shown that cytokine expressions are altered as compared to healthy animals (dean et al., 2003; kiss et al., 2004) . specifically it has been noted that expression of the pro-inflammatory cytokine tumor necrosis factor alpha (tnf-alpha), interleukin-1 beta (il-1 beta) and interleukin-6 (il-6) are significantly increased in cats with fip, and are likely produced by infected macrophages and monocytes (kiss et al., 2004; takano et al., 2007a takano et al., , 2007b . it has been shown that tnf-alpha is able to induce feline t-cell apoptosis, making it the most likely causative agent of t-cell lymphopenia in fipvinfected cats (dean et al., 2003; takano et al., 2007a) . in addition tnfalpha has been shown to increase expression of the fcov receptor aminopeptidase n (apn) causing target cells to be more susceptible to viral infection and further exacerbate the disease (takano et al., 2007b) . however despite their critical role in the pathological outcome of fip, the mechanism regulating fipv-induced upregulation of pro-inflammatory cytokines remains undescribed. mitogen-activated protein kinases (mapks) are a family of proteins that serve as components of signaling pathways within cells in order to process and respond to extracellular stimuli (raman et al., 2007) . typically, receptors on the cell surface initiate signaling cascades, which lead to phosphorylation and translocation of mapks to the nucleus where they regulate transcriptional activators (whitmarsh, 2007) . in recent years, it has become clear that mapks also regulate processes outside of the nucleus such as mrna translation and cytoskeletal remodeling (frevel et al., 2003; huang et al., 2004) . three major mapk pathways have been identified which are conserved in all eukaryotic cells ranging from yeast to mammals. these pathways are designated as extracellular signal-regulated kinases 1 and 2 (erk1/2), c-jun n-terminal kinases (jnk1) and p38 mapk (pearson et al., 2001) . in general the erk pathway is activated by proliferative stimuli, while the jnk and p38 mapk pathways are activated by extracellular stresses such as ultraviolet light, heat and osmotic shock (pearson et al., 2001) . p38 mapk was originally identified as the target of pyridinyl imidazole compounds that were shown to inhibit the production of il-1 and tnf-alpha in lipopolysaccharide (lps)-stimulated human monocytes (lee et al., 1994) . subsequent studies have shown that the p38 mapk pathway is responsible for the phosphorylation of a large group of transcriptional and translational response elements which directly regulate the expression of a wide variety of proinflammatory cytokines (kumar et al., 2003) . due to its involvement in cytokine regulation, we reasoned that the p38 mapk pathway might play a role in the increased production of pro-inflammatory cytokines observed in cats with fip. in this study we examined the activation of the p38 mapk pathway in response to infection by fipv in primary feline blood-derived mononuclear cells. we also investigated the role of p38 mapk in tnf-alpha, il-1 beta and il-6 production, and the effect of p38 mapk inhibitors on these processes. the p38 mapk pathway has been shown to be activated by multiple viral pathogens during infection (adamson et al., 2000; banerjee et al., 2002; dumitru et al., 2006; erhardt et al., 2002; holloway and coulson, 2006; zachos et al., 1999) . to determine whether the p38 mapk pathway is activated during the infectious lifecycle of fipv, primary feline blood-derived mononuclear (pfbm) cells were inoculated with either fipv-1146 or fipv-df2 at an moi of 100. untreated cells and infected cells ranging from 15 min to 12 h p.i. were lysed and analyzed by western blot with the anti-phospho-p38 mapk mab (3d7). untreated cells showed a minimal level of p38 mapk phosphorylation, however addition of either virus isolate caused rapid phosphorylation of p38 mapk (n600% increase) within 15 min p.i. (figs. 1a and c) . p38 mapk underwent dephosphorylation by 60 min and then showed a second phase of phosphorylation later in infection between 6 and 12 h p.i., which was less pronounced (fig. 1a) . to determine whether viral replication was required for fipv-induced p38 mapk activation, uv-inactivated virus was added to pfbm cells and analyzed by western blot as described above (fig. 1b) . uv-treated fipv also induced p38 mapk phosphorylation, however the activation was not biphasic, and instead remain sustained throughout the 12 h time-course (fig. 1b) . membranes were re-probed with anti-p38 mapk (n-20) pab to show that an equal amount of p38 mapk was present in each sample (figs. 1a and b) . to further confirm that the p38 mapk pathway is activated by fipv, pfbm cells were inoculated with fipv-1146 at an moi of 100 before fixing the cells for immunofluorescent microscopy. p38 mapk again showed a rapid phosphorylation by 15 min p.i. while the total amount of p38 mapk remained unchanged (fig. 2) . in addition the p38 mapk in infected cells showed increased nuclear localization as compared to untreated cells, a phenomenon highly associated with the regulation of transcriptional activators (fig. 2) . these data indicate that the p38 mapk pathway is activated during infection of pfbm cells by fipv, and that viral replication is dispensable for this activation to occur. the p38 mapk pathway was first discovered by investigating the target of pyridinyl imidazole compounds which blocked lps-induced cytokine induction in human monocytes (lee et al., 1994) . to test the effect of pyridinyl imidazole inhibitors on fipv-induced p38 mapk phosphorylation, pfbm cells were treated with 10 μm of either sb 203580 or sc 409 (or 0.1% dmso as a control) for 2 h before inoculating with fipv-1146 or fipv-df2 at an moi of 100. 15 min p.i. cells were lysed and analyzed by western blot with the anti-phospho-p38 mapk mab (3d7). cells which were pretreated with dmso alone showed rapid fipv-induced phosphorylation of p38 mapk, however those which were pretreated with either sb 203580 or sc 409 showed no activation as compared to uninfected cells (fig. 3) . these data demonstrate that fipv-induced p38 mapk activation is blocked by pyridinyl imidazole inhibitors. activation of the p38 mapk pathway has been shown to be required for replication of some viruses including the murine coronavirus mouse hepatitis virus (mhv) (banerjee et al., 2002) . to investigate whether activation of the p38 mapk pathway is required for replication of fipv, pfbm cells were treated with 10 μm of either sb 203580 or sc 409 (or 0.1% dmso as a control) for 2 h before inoculating with fipv-1146 or fipv-df2. 12 h p.i. cells were fixed and stained for with the anti-fipv n protein mab (17b7.1). as shown in fig. 4 , pretreatment with p38 mapk inhibitors has no significant effect on fipv replication in pfbm cells. activation of p38 mapk by viral pathogens has been shown to induce the production of pro-inflammatory cytokines such as tnfalpha, il-1 beta and il-6 (banerjee et al., 2002; griego et al., 2000; lee et al., 2005a lee et al., , 2005b sloan and jerome, 2007; wang et al., 2004; yurochko and huang, 1999) . to investigate whether tnfalpha production in fipv-infected pfbm cells is regulated by p38 mapk activation, pfbm cells were treated with 10 μm of either sb 203580, sc 409 or 0.1% dmso for 2 h before inoculating with fipv-1146 or fipv-df2 at an moi of 100. 24 h p.i. supernatants were collected, concentrated and analyzed by western blot with the anti-tnf-alpha (n-19) pab. cells which were pretreated with dmso alone showed significant production of tnf-alpha, however those which were pretreated with either sb 203580 or sc 409 showed no detectable production of tnf-alpha as compared to uninfected cells (fig. 5a ). to quantify the production of tnf-alpha, infections were performed as described above, except at 24 h p.i. supernatants were collected and analyzed by anti-tnf-alpha capture elisa. infected cells which were pretreated with dmso alone showed significant production of tnf-alpha (n750 pg/ml) however pretreatment with 10 μm sb 203580 and 10 μm sc 409 resulted in a 8-fold and 4-fold reduction in tnf-alpha production respectively (fig. 5b) . uninfected cells produced no tnf-alpha, or were below the detection level of the assay (data not shown). overall these data indicate that production of the pro-inflammatory cytokine tnf-alpha in fipvinfected pfbm cells is regulated by activation of the p38 mapk pathway. to show that the reduction of fipv-induced tnf-alpha production by sb 203580 and sc 409 was specific to the pyridinyl imidazole inhibitors, pfbm cells were treated with either sb 203580 or sc 409 at a range of concentrations (10 μm, 1 μm or 0.1 μm) or 0.1% dmso for 2 h before inoculating with fipv-1146 or fipv-df2 at an moi of 100. as seen in our previous data, pfbm cells which were pretreated with dmso alone produced significant amounts of tnfalpha however pretreatment with sb 203580 and sc 409 resulted in a significant reduction in tnf-alpha production in a dose-dependent manner (fig. 6) . it is known that individual animals can vary in their reaction to infection by fipv (kiss et al., 2004) . to determine whether or not fipvinduced p38 mapk activation was specific to a single animal, pfbm cells were individually prepared from six additional spf cats (07pgp2, 07pgv4, 07pgv5, 07fgr2, 07fgv6, 07fjm5). pfbm cells individually purified from each animal were inoculated with fipv-1146 at an moi of 100. untreated cells and infected cells (15 min p.i.) were lysed and . 24 h p.i. supernatants were collected and tnfalpha production was quantified by anti-tnf-alpha capture elisa (b). tnf-alpha produced from untreated cells was below the detection limit of the assay (b10 pg/ml). fig. 6 . fipv-induced tnf-alpha production is inhibited in a dose-dependent manner by sb 203580 and sc 409. pfbm cells were pretreated with either sb 203580 or sc 409 at a range of concentrations (10 μm, 1 μm or 0.1 μm) or 0.1% dmso for 2 h before inoculating with fipv-1146 at an moi of 100. 24 h p.i. supernatants were collected and tnf-alpha production was quantified by anti-tnf-alpha capture elisa. tnf-alpha from untreated cells was below the detection limit of the assay (b10 pg/ml). analyzed by western blot with the anti-phospho-p38 mapk mab (3d7). consistent with our previous data, untreated cells showed a minimal level of p38 mapk phosphorylation while addition of fipv caused a rapid phosphorylation of p38 mapk in pfbm cells from all six cats (fig. 7) . membranes were re-probed with anti-p38 mapk (n-20) pab to show that an equal amount of p38 mapk was present in each sample (fig. 7) . in addition, the regulation of tnf-alpha production by p38 mapk was analyzed in pfbm cells from all six spf cats. pfbm cells from each animal were treated with 10 μm sc 409 or 0.1% dmso for 2 h before inoculating with fipv-1146 at an moi of 100. 24 h p.i. supernatants were collected and analyzed by anti-tnf-alpha capture elisa. while the baseline level of tnf-alpha production differed slightly amongst all of the cats tested, treatment with the p38 inhibitor sc 409 resulted in the same trend observed in our previous experiments: a significant reduction in tnf-alpha levels (between 3fold to 8-fold) (fig. 8) . these data taken together suggest that fipvinduced activation of the p38 mapk pathway in pfbm cells represents a common mechanism by which this virus promotes tnf-alpha production in cats. cats with fip have also been reported to show increased levels of the pro-inflammatory cytokines il-1 beta and il-6. to investigate whether il-1 beta and il-6 production in fipv-infected pfbm cells is regulated by p38 mapk activation, pfbm cells were treated with 10 μm of either sb 203580, sc 409 or 0.1% dmso for 2 h before inoculating with fipv-1146 at an moi of 100. 24 h p.i. supernatants were collected and analyzed by anti-il-1 beta and anti-il-6 capture elisa. infected cells which were pretreated with dmso alone showed significant production of il-1 beta (n200 pg/ml) however pretreatment with 10 μm sb 203580 and 10 μm sc 409 resulted in a 7-fold and 4-fold reduction in il-1 beta production respectively (fig. 9) . neither infected nor uninfected pfbm cells produced significant levels of il-6 ( fig. 9) . overall, these data indicate that both tnf-alpha and il-1 beta production in fipv-infected pfbm cells is regulated by p38 mapk activation, a situation that does not apply to il-6. modulation of signaling pathways by viruses is becoming recognized as a key pathogenic determinant in viral diseases mediated by aberrant host immunological responses. in the case of fip, cytokine production is markedly altered between animals with disease as compared to healthy animals, with overproduction of the proinflammatory cytokine tnf-alpha in particular being indicative of a poor outcome (kiss et al., 2004) . feline tnf-alpha causes apoptosis in feline t-cells (implicating it as the causative agent of t-cell lymphopenia), and upregulates the fipv receptor apn making target cells more susceptible to infection in vitro (dean et al., 2003; kiss et al., 2004; takano et al., 2007a takano et al., , 2007b . it has been shown previously that fipv-infected monocytes upregulate the expression of tnf-alpha, however the mechanism regulating this process remains undescribed. in this study we show that infection by fipv causes a rapid activation the p38 mapk pathway in pfbm cells, and that this process directly regulates production of the pro-inflammatory cytokines tnf-alpha and il-1 beta. as shown in fig. 1 , fipv-induced p38 mapk activation in pfbm cells occurs in a biphasic temporal pattern which mimics that observed with other viral pathogens that activate mapk pathways during infection such as influenza virus (pleschka et al., 2001) . at present we are unable to define the mechanism by which fipv particles are able to activate the p38 mapk pathway, however the rapid nature of the initial activation suggests that it occurs early during entry; likely due to interactions between the s protein and its receptor. this model is further supported by the observation that uv-inactivated virus also induce rapid activation of the p38 mapk pathway. this activation is markedly different than that reported in mhv infected cells, where activation did not occur until 6-12 h p.i., and uv-treated viral particles did not induce phosphorylation of p38 mapk (banerjee et al., 2002) . it notable that the fipv receptor apn localizes to lipid rafts (navarrete santos et al., 2000; nomura et al., 2004) which are known to be a signaling portal for the p38 mapk pathway (calzolari et al., 2006; head et al., 2006; olsson and sundler, 2006; sugawara et al., 2007; wang et al., 2006; zeidan et al., 2008) . in fact it has recently been shown that rhinovirus activates the p38 mapk pathway through the actions of lipid rafts and rhoa (dumitru et al., 2006) . further investigation will be necessary to determine the role of apn and lipid rafts in the initial phase of fipv-induced p38 mapk activation and tnf-alpha/il-1 beta production. interestingly, uv-inactivated fipv induced prolonged p38 mapk activation, rather than the biphasic activation induced by untreated viral particles. this suggests that fipv may activate p38 mapk during entry, but then suppresses p38 mapk during the early phase of replication. the second phase of fipv-induced p38 mapk activation induced by untreated viral particles (6 h p.i.) may be caused by the production of pro-inflammatory cytokines. it has been shown that tnf-alpha can itself activate the p38 mapk through signaling associated with the cytoplasmic domain of its receptors tnf receptor 1 (tnfr1)-associated death domain protein (tradd) and tnf receptor-associated factor 2 (traf2) (carpentier et al., 1998; hsu et al., 1995 hsu et al., , 1996 . therefore tnf-alpha produced during the initial phase of fipv-induced p38 mapk activation, may be the cause of the latter phase of activation. pretreatment with the pyridinyl imidazole inhibitors sb 203580 and sc 409 blocked production of tnf-alpha and il-1 beta suggesting that p38 mapk directly regulates production of the cytokines in fipvinfected pfbm cells. the upregulation of il-6 production was not fig. 8 . fipv-induced tnf-alpha production by pfbm cells from six spf cats is inhibited by sc 409. pfbm cells were individually prepared from three male (07pgp2, 07pgv4, 07pgv5) and three female (07fgr2, 07fgv6, 07fjm5) spf cats. cells from each animal were inoculated with fipv-1146 at an moi of 100. 24 h p.i. supernatants were collected and tnf-alpha production was quantified by anti-tnf-alpha capture elisa. tnf-alpha from untreated cells was below the detection limit of the assay (b 10 pg/ml). observed in fipv-infected pfbm cells, suggesting that another cell type may be responsible for its production in cats with fip. at this time the mechanism by which p38 mapk regulates pro-inflammatory cytokine production in fipv-infected pfbm cells is unknown, however regulation of cytokines by mapks in analogous systems occurs by affecting either transcriptional regulation, translational regulation, or both (kumar et al., 2003) . for example the recently emerged severe acute respiratory syndrome coronavirus (sars-cov) is also known to infiltrate immune cells such as monocytes and macrophages and activate the p38 mapk pathway (belyavsky et al., 1998; franks et al., 2003; gu et al., 2005; nicholls et al., 2003) . sars-cov infection causes a p38 mapk-dependent phosphorylation of downstream transcriptional regulators such as activating transcription factor 1 (atf-1) and signal transducer and activator of transcription 3 (stat-3), as well as translational regulators such as mapk activate protein kinase 2 (mapkapk2) and the eukaryotic initiation factor 4e (eif4e) (mizutani, 2007; mizutani et al., 2004a mizutani et al., , 2004b . as seen in fig. 2 it appears that fipv causes increased p38 mapk nuclear localization suggesting that the activation of transcription factors likely play a role in proinflammatory production in pfbm cells, however this also does not exclude a role for translational regulation. future studies examining the role of downstream transcriptional and translational regulators in fipv-infected pfbm cells should clarify the mechanism regulating this process. another aspect complicating the treatment of fip is the diverse reactions to infection displayed by cats with the disease (kiss et al., 2004) . our results suggest that activation of the p38 mapk pathway and its regulation of tnf-alpha production is common to pfbm cells of all cats, however further sampling of animals throughout different geographic regions will be required to confirm this conclusion. pyridinyl imidazole compounds have been shown to be efficacious therapeutic agents for blocking the mediators of chronic inflammatory diseases such as rheumatoid arthritis (kumar et al., 2003) . in fact, several p38 mapk inhibitors have shown promise in animal models of inflammatory diseases and some have even reached human clinical trials (kumar et al., 2003) . our results show a clear activation of p38 mapk by fipv during infection, and that this activation is responsible for pro-inflammatory cytokine production which is a key contributor to the pathological changes observed in cats with fip. this raises this possibility that p38 mapk inhibitors, alone or in conjunction with other therapies, may possess therapeutic benefits in the treatment of cats with fip. primary feline blood-derived mononuclear (pfbm) cells were individually purified from four male spf cats (animal id# 07pjo7, 07pgp2, 07pgv4, 07pgv5) and three female spf cats (animal id# 07fgr2, 07fgv6, 07fjm5) (liberty research, waverly, ny) using a standard ficoll-paque gradient (ge healthcare) as specified by the manufacturer. cells were seeded in 24-well plates with tissue culturetreated glass coverslips and allowed to attach overnight. after washing, cells were incubated in the presence of 5% co 2 at 37°c in rpmi-1640 media ph7.4 supplemented with 10% fetal bovine serum (fbs), 2 mm glutamine, 100 u/ml penicillin and 10 μg/ml streptomycin. the purity of pfbm preparations were routinely checked by immunofluorescence microscopy using the marker dh59b (veterinary medical research & development inc., pullman, wa). crandell-reese feline kidney cells were obtained from the american type culture collection (atcc) and cultured and maintained according to atcc guidelines. fipv wsu 79-1146 (fipv-1146) was obtained from the atcc. fipv-df2 was provided by dr. ed dubovi (animal health diagnostic center, new york state college of veterinary medicine, cornell university). both viruses were grown by inoculating crfk cells at a moi of 0.01 and collecting supernatant after cpe was observed in 80% of cells which typically occurred between 48 and 72 h. supernatant was fig. 9 . production of il-l beta and il-6 by fipv-infected pfbm cells. pfbm cells were pretreated with 10 μm of either sb 203580 or sc 409 (or 0.1% dmso as a control) for 2 h before inoculating with fipv-1146 at an moi of 100. 24 h p.i. supernatants were collected and il-1 beta and il-6 production was quantified by anti-il-1 beta or anti-il-6 capture elisa. clarified by a low speed centrifugation step (1250× g for 10 min) and viral particles were then pelleted by centrifugation at 28,000 rpm in a sw28 rotor (sorvall) for 60 min. pellets were resuspended in phosphate-buffered saline (pbs). virus titers were determined by plaque assays on crfk cells using standard techniques. for uvinactivation, a thin layer of viral suspension was exposed to uv light (30 w) at a distance of 10 cm for 5 min. inactivation was verified by performing infection assays in crfk and pfbm cells as described. the anti-phospho-p38 mapk (thr180/tyr182) (3d7) rabbit monoclonal antibody (mab) was obtained from cell signaling technologies (danvers, ma). the anti-p38 mapk (n-20) goat polyclonal antibody (pab) and anti-tnf-alpha (n-19) goat pab were obtained from santa cruz biotechnology (santa cruz, ca). the anti-fipv nucleocapsid (n) protein mab (17b7.1) was provided by dr. ed dubovi (animal health diagnostic center, new york state college of veterinary medicine, cornell university). anti-cd127a mab dh59b was obtained from veterinary medical research and development, inc. (pullman, wa). the feline tnf-alpha elisa kit (tnf-alpha/tnfsf1a), feline il-1 beta elisa kit (il-1 beta/il-1f2), feline il-6 elisa kit, and associated antibodies and detection reagents were obtained from r&d systems (minneapolis, mn). the p38 mapk inhibitors 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1h-imidazole (sb 203580) and 4-(3-(4-chlorophenyl)-5-(1-methylpiperidin-4-yl)-1h-pyrazol-4-yl) pyrimidine (sc 409) were obtained from calbiochem (san diego, ca). pfbm cells were incubated in low-serum media (1% fbs) for 12 h before inoculation with the specified virus at an moi of 100, or pretreatment with the specified inhibitor for 2 h followed by infection. for p38 mapk activation experiments, cells were lysed at the specified time-points in lysis buffer (1% triton x-100, 50 mm tris-hcl, 150 mm nacl, 1 mm edta, 1 mm dtt, 50 mm beta-glycerophosphate, 100 mm sodium vanadate, ph 7.4) supplemented with 1× complete protease inhibitor cocktail (roche). lysates were clarified by centrifugation at 13,000 rpm in a table-top centrifuge at 4°c for 15 min before freezing at −80°c for later analysis. for immunofluorescence assays, cells were fixed at the specified time-points with 3% paraformaldehyde. for analysis of cytokine production, supernatant was collected 24 h postinoculation (p.i.) before freezing at −80°c for later analysis. fixed cells were labeled with the specified antibodies as described previously (chu et al., 2006) . cells were viewed on a nikon eclipse e600 fluorescence microscope, and images were captured with a sensicam em camera and analyzed with iplab software. sds sample buffer was added to lysates and the reaction was heated at 95°c for 10 min before separation using a 4-20% sds-page gel at 200 v for 2 h. gels were electroblotted to pvdf membrane at 200 a for 2 h, blocked with 5% bovine serum albumin and probed with the specified antibody at 4°c for 12 h. membranes were developed using either anti-rabbit antibody (southern biotech, 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fatal severe acute respiratory syndrome human coronavirus 229e binds to cd13 in rafts and enters the cell through caveolae the role of lipid rafts in lps-induced signaling in a macrophage cell line mitogen-activated protein (map) kinase pathways: regulation and physiological functions pathogenic differences between various feline coronavirus isolates pathogenicity studies of feline coronavirus isolates 79-1146 and 79-1683 influenza virus propagation is impaired by inhibition of the raf/mek/erk signalling cascade differential regulation and properties of mapks differential role for low ph and cathepsin-mediated cleavage of the viral spike protein during entry of serotype ii feline coronaviruses the molecular dynamics of feline coronaviruses acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein herpes simplex virus remodels t-cell receptor signaling, resulting in p38-dependent selective synthesis of interleukin-10 intrinsic resistance of feline peritoneal macrophages to coronavirus infection correlates with in vivo virulence the lipid raft proteins flotillins/reggies interact with galphaq and are involved in gq-mediated p38 mitogen-activated protein kinase activation through tyrosine kinase a "possible" involvement of tnf-alpha in apoptosis induction in peripheral blood lymphocytes of cats with feline infectious peritonitis tnf-alpha, produced by feline infectious peritonitis virus (fipv)-infected macrophages, upregulates expression of type ii fipv receptor feline aminopeptidase n in feline macrophages feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses sustained activation of p38 mitogen-activated protein kinase and c-jun n-terminal kinase pathways by hepatitis b virus x protein mediates apoptosis via induction of fas/fasl and tumor necrosis factor (tnf) receptor 1/tnf-alpha expression hsp70 enhances macrophage phagocytosis by interaction with lipid raft-associated tlr-7 and upregulating p38 mapk and pi3k pathways pathogenesis of feline infectious peritonitis: pathologic changes and immunofluorescence regulation of gene transcription by mitogen-activated protein kinase signaling pathways human cytomegalovirus binding to human monocytes induces immunoregulatory gene expression herpes simplex virus type 1 infection stimulates p38/c-jun n-terminal mitogen-activated protein kinase pathways and activates transcription factor ap-1 leptin-induced cardiomyocyte hypertrophy involves selective caveolae and rhoa/rock-dependent p38 mapk translocation to nuclei we thank marc antoniak for helpful advice and discussions during the course of this work, and ed dubovi for kind provision of reagents. we also thank a damon ferguson for technical assistance. adr was supported grant t32ai007618 (training in molecular virology and pathogenesis) from the national institutes of health. work in the author's lab was supported by the winn feline foundation and the george sydney and phyllis redmond miller trust. key: cord-268467-btfz6ye8 authors: schreiber, steven s.; kamahora, toshio; lai, michael m.c. title: sequence analysis of the nucleocapsid protein gene of human coronavirus 229e date: 1989-03-31 journal: virology doi: 10.1016/0042-6822(89)90050-0 sha: doc_id: 268467 cord_uid: btfz6ye8 abstract human coronaviruses are important human pathogens and have also been implicated in multiple sclerosis. to further understand the molecular biology of human coronavirus 229e (hcv-229e), molecular cloning and sequence analysis of the viral rna have been initiated. following established protocols, the 3′-terminal 1732 nucleotides of the genome were sequenced. a large open reading frame encodes a 389 amino acid protein of 43,366 da, which is presumably the nucleocapsid protein. the predicted protein is similar in size, chemical properties, and amino acid sequence to the nucleocapsid proteins of other coronaviruses. this is especially evident when the sequence is compared with that of the antigenically related porcine transmissible gastroenteritis virus (tgev), with which a region of 46% amino acid sequence homology was found. hydropathy profiles revealed the existence of several conserved domains which could have functional significance. an intergenic consensus sequence precedes the 5′-end of the proposed nucleocapsid protein gene. the consensus sequence is present in other coronaviruses and has been proposed as the site of binding of the leader sequence for mrna transcriptional start. this region was also examined by primer extension analysis of mrnas, which identified a 60-nucleotide leader sequence. the 3′-noncoding region of the genome contains an 11-nucleotide sequence, which is relatively conserved throughout the coronavirus family and lends support to the theory that this region is important for the replication of negative-strand rna. human coronavirus 229e (hcv-229e) belongs to one of two major antigenic groups of human coronaviruses (macnaughton, 1981) . it shares antigenic relationships with other coronaviruses, such as porcine transmissible gastroenteritis virus (tgev), feline infectious peritonitis virus (fipv), and canine coronavirus (ccv). the other well-characterized human coronavirus, hcv-oc43, is in a separate antigenic group which includes mouse hepatitis virus (mhv) and bovine coronavirus (bcv). both human coronaviruses are mainly respiratory pathogens and have been estimated to cause up to 25% of common colds (mcintosh et a/., 1974; wege et a/., 1982) . they have also been implicated in gastrointestinal diseases (resta et a/., 1985) . furthermore, the isolation of coronaviruses bearing an antigenic relationship to hcv-oc43 from the central nervous system of two patients with multiple sclerosis has suggested a possible etiologic relationship between human coronaviruses and multiple sclerosis (burks et a/., 1980) . this possibility is supported by the observation that neurotropic strains of mhv cause demyelination in the central nervous system of rodents (weiner and stohlman, 1978) . thus, human coronaviruses are important human pathogens. the structural and biochemical properties of several coronaviruses, particularly mhv and avian infectious sequence data from this article have been deposited with the embugenbank data libraries under accession no. jo441 9. ' to whom requests for reprints should be addressed. 2 present address: department of virology, tottori university, school of medicine, yonago 683, japan. peritonitis virus (ibv), have been well characterized (lai et a/., 1987; boursnell et a/., 1987) . the virion contains a single-stranded, positive-sense rna molecule (molecular weight 6-8 x 1 o6 da) (lai and stohlman, 1978) associated in a helical conformation with nucleocapsid proteins (n) . the viral nucleocapsid is enclosed by an envelope, in which are embedded at least two types of viral proteins, the peplomer (e2) and matrix (el) glycoproteins. coronavirus rna replication occurs in the cytoplasm of infected cells and is mediated by a virusencoded rna-dependent rna polymerase (brayton et a/., 1982) . the virus-specific mrna in infected cells comprises a genomic-sized rna plus six subgenomic mrna species. these mrnas are arranged in a nested-set structure, which is characterized by rnas having common 3'-termini but extending for varying lengths in the 5'direction (lai et al., 1981) . only the 5'proximal regions of each mrna are translated (rottier et a/., 1981) . a unique feature of the structure of coronavirus is the existence, at the 5'-end of each mrna, of an identical leader sequence. this sequence is derived from the 5'-end of the genomic rna and is of approximately 70 nucleotides in length (lai eta/., 1983 (lai eta/., , 1984 . recent evidence has supported a role for the leader sequence in mediating a novel type of discontinuous transcription of genomic rna (baric et a/., 1985; makino et al., 1986; . in contrast to other coronaviruses, the molecular biology of human coronaviruses is relatively poorly understood. the genomic rna of both hcv-229e and hcv-oc43 has a molecular weight of approximately 6 x 1 o6 da (hierholzer et al., 1981) . the six subgenomic rna species appear to have lower molecular weights than those of the corresponding mhv rnas (weiss and leibowitz, 198 1) . the structure of these mrnas is not yet known. analysis of purified hcv-229e virions has revealed three major polypeptides: a glycosylated protein with a molecular weight of 180 kda, a phosphorylated nucleocapsid protein of 50 kda, and a family of polypeptides with molecular weights of 25, 23, and 21 kda (kemp et al., 1984) . in addition, several minor nonstructural polypeptides of 107, 92, and 39 kda have been identified (kemp et al., 1984) . the functions of these proteins have not yet been characterized. to further understand the molecular biology of hcv-229e, we have initiated molecular cloning and sequence analysis of hcv-229e rna. in this paper we report the sequence analysis of the gene encoding the nucleocapsid protein of hcv-229e. in addition, the mrna leader sequence was also identified. the results are compared with sequences of other coronaviruses including mhv, bcv, ibv, and tgev. hcv-229e (obtained from dr. j. fleming, university of southern california) was propagated at low multiplicities of infection in human fetal lung cells l132 (kennedy and johnson-lussenberg, 197511976 ) using dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal calf serum. virus purification and preparation of virion rna following a virus adsorption period of 1 hr at 37", hcv-229e-infected l132 monolayers were incubated at 37" for 24 to 48 hr, at which time the cell culture fluid was harvested. viruses were precipitated from 2 liters of culture fluid with 50% ammonium sulfate and centrifuged at 8000 rpm for 30 min. the pellet was resuspended in nte buffer (0.1 m naci, 0.01 m tris-hydrochloride (ph 7.2), 1 mm edta) and then placed on a discontinuous sucrose gradient consisting of 60, 50, 30, and 20% (w/w) sucrose in nte buffer and centrifuged at 26,000 rpm for 13 hr at 4" in a beckman sw28.1 rotor. the virus band at the interface between 50 and 30% sucrose was collected and diluted threefold with nte buffer. the diluted virus suspension was centrifuged on a linear sucrose gradient at 26,000 rpm in an sw28.1 rotor for 4 hr at 4". the virus band was collected and treated with proteinase k (0.2 mg/ml) for 20 min at 37", followed by 1% sds for 30 min at 37". genomic rnawas extracted with phenol and then with phenol/chloroform, and precipitated with ethanol. monolayers of l132 cells grown in 100 x 20-mm culture dishes were infected with hcv-229e. cells were incubated in phosphate-free dmem containing 1% dialyzed fetal calf serum 4 hr prior to rna extraction. actinomycin d (1 pg/ml) (sigma) and [3zp] or-thophosphate (70 &i/ml) (icn radiochemicals) were added at 3 and 2 hr, respectively, prior to rna extraction at 15 hr postinfection (p.i.). cells were collected in cold phosphatebuffered saline and centrifuged at 5000 rpm for 3 min at 4". the pellet was mixed with cold 0.5% nonidet-p40 in nte buffer, incubated for 10 min at 4', and then centrifuged at 5000 rpm for 3 min. the supernatant was transferred to a fresh tube containing l/10 vol of 10% sds at room temperature and vortexed briefly. intracellular rna was extracted with phenol and phenol/ chloroform and precipitated with ethanol. poly(a)-containing rna was selected by oligo(dt)-cellulose chromatography as previously described (makino et al., 1984) . to examine the kinetics of viral mrna synthesis, intracellular rna was extracted from virus-infected l132 monolayers in 60 x 15-mm culture dishes at 7, 21, 29, 46, and 58 hr postinfection. cdna cloning cdna cloning was performed using a modified method of gubler and hoffman (1983) . the poly(a)containing rna extracted from 229e-infected l132 monolayers was precipitated, dried, and resuspended in 6.72 ~1 of autoclaved water. the rna was incubated with 10 mm methylmercuric hydroxide in an 8 ~1 total volume for 10 min at room temperature. first-strand cdna synthesis was carried out in a 50-~1 reaction mixture containing 60 units rnasin (promega biotec), 10 mm mgci,, 100 mm kci, 50 mm tris-hci (ph 8.3 at 42") 10 mm dtt, 1.25 mm dntps, 40 &i [a-32p]datp (3000 ci/mmol), 28 mm ,&mercaptoethanol, and 10 ng oligo(dt),2-,s primer. after 5 min at room temperature, 40 units of amv reverse transcriptase (life science) was added and the mixture was incubated for 1 hr at 42". the reaction was stopped by adding 4.4 ~1 of 250 mm edta. the products were extracted with phenol/ chloroform and precipitated with ethanol containing 0.3 m ammonium acetate. for second-strand synthesis, the loo-~1 reaction mixture contained 5 mm mgci,, 100 mm kci, 20 mm tris-hci (ph 7.5) 50 pgl ml bovine serum albumin (bsa), 10 mm ammonium sulfate, 0.15 mm p-nad, 100 pm dntps, 25 units of escherichia co/i dna polymerase i, 2 units of e. co/i dna ligase, and 0.8 units of rnase h. sequential incubations were for 1 hr at 12" and 1 hr at 22". the reaction was stopped by the addition of 8.7 ~1 of 250 mm edta and the products were extracted with phenol/ chloroform and precipitated with ethanol in the presence of 0.3 mammonium acetate. homopolymeric tailing of double-stranded cdna with poly(c) was carried out in a 1 ~-pi reaction mixture containing 10 units of terminal transferase, 200 mm potassium cacodylate, 0.5 mm co&, 25 mm tris-hci (ph 6.9), 2 rnn/l dlt, 250 pg/ml bsa, and 50 pm dctp at 37" for 4 min. the dc-tailed double-stranded dna was annealed to 200 pg of dg-tailed pstl-cut pbr322 plasmid in 20 ~1 of a buffer containing 10 mni tris-hci (ph 7.4), 100 mm naci, and 0.25 mh/l edta. the mixture was incubated for 5 min at 68" and then cooled slowly overnight. the annealed molecules were used to transform e. co/i mci061 as described (dagert and erhlich, 1979) . colonies grown on lb/tetracycline plates were incubated at 37" for 12 hr and transferred to colony/plaque screen disks (new england nuclear). bacterial lysis and dna fixation were carried out according to the methods previously described (grunstein and hogness, 1975) . the disks were prehybridized in a solution containing 0.2% polyvinylpyrrolidone (mw 40,000), 0.2% ficoll (mw 400,000), 0.2% bsa, 0.05 mtris-hci (ph 7.5) 1% sds, 1 l\/i naci, 10% dextran sulfate, and 100 pg/ml denatured salmon sperm dna at 65" for 6hr. fragments derived from either the 5'-or 3'-ends of gene 7 were labeled with 32p by nick-translation and added to the solution. hybridization was carried out for 20 hr at 65". the disks were then washed twice in 2~ ssc (0.3 lvi naci, 30 mlvi sodium citrate) at room temperature, twice in 2x ssc containing 1% sds for 30 min at 65", and twice in 0.1 x ssc at room temperature for 30 min. the disks were air-dried and exposed to xray film at -70". intracellular rna from virus-infected cells was denatured by glyoxal treatment and separated by electrophoresis on a 1% agarose gel containing 10 mll/l sodium phosphate (ph 7.0) as described previously (mc-master and carmichael, 1977) . rna transfer to biodyne nylon filters (icn radiochemicals) and subsequent hybridization were performed according to the method described by thomas (1980) . a synthetic oligodeoxyribonucleotide was 5'-end-labeled with [y-~'p]atp by polynucleotide kinase (pedersen and haseltine, 1980) . the total amount of poly(a)-containing rna extracted from 229e-infected cell monolayers in three 150 x 20-mm culture dishes was incubated in 8 pi of distilled water containing 10 mm methylmercuric hydroxide for 10 min at room tem-perature. a further incubation was carried out in a 50-~1 reaction volume containing 60 units of rnasin (promega), 10 mm mgc12, 100 mm kci, 50 ml\/l tris-hci (ph 8.3 at 42") 10 mm dlt, 1.25 mn/ldntps, 28 mm ,&mercaptoethanol, 5'-end-labeled synthetic oligodeoxyribonucleotides, and 20 units of amv reverse transcriptase (life science) for 1 hr at 42". reaction products were extracted with phenol/chloroform, precipitated with ethanol, and then analyzed by electrophoresis on a 6% polyacrylamide gel containing 8.3 m urea. the primer-extended product was identified by autoradiography and eluted from the gel according to the published procedure (maxam and gilbert, 1977) . sequencing was carried out by the dideoxyribonucleotide chain termination method (sanger et al., 1977) as well as the chemical modification procedure (maxam and gilbert, 1977) . in the first method, fragments of cdna inserts generated by various restriction endonucleases were cloned into the ml 3 vectors mp18 and mp19 (messing and vierira, 1982) . [(u-~~s]-datp was used as a label. sequence data were also obtained by chemical modification (maxam and gilbert, 1977) of various cdna fragments subcloned into the pt7-3 vector (tabor and richardson, 1985) . in the second method, cdna fragments were 3'-end-labeled with klenow fragment at internal restriction sites or, alternatively, at the polylinker cloning site of pt7-3. end-labeled cdna restriction fragments were separated by electrophoresis on preparative polyacrylamide gels (maxam and gilbert, 1980) and purified as described previously (hansen et a/., 1980; hansen, 1981) . sequencing of the primer-extended product of mrna7 was performed by the chemical modification procedure (maxam and gilbert, 1977) . sequence analysis was performed by the lntelligenetics and seqaid programs. hydropathy profiles were constructed using the pepplot program of the university of wisconsin computer genetics group, which employs both the kyle-doolittle (kd) and goldman, engelman, steitz (ges) algorithms. to determine the optimum time for extracting 229especific mrnas, we first studied the kinetics of virusspecific mrna synthesis. intracellular rna was extracted from infected l132 monolayers at specified times p.i. the rna was separated by agarose gel electrophoresis (fig. 1) . as can be seen, viral mrna synthesis could be detected as early as 7 hr p.i. and reached maximum at 29 hr p.i. thereafter, total rna synthesis gradually declined. by 46 hr p.i. onlythe most abundant mrna species were evident. the number and size of these mrna species are comparable to those of mhv mrnas and are in agreement with previously published results (weiss and leibowitz, 1981) . significantly, mrna 2a, which was previously found only in bcv-infected cells and proposed to encode hemagglutinins (king et a/., 1985; keck et a/., 1988) was not present. this is consistent with the finding that hcv-229e does not have hemagglutinating activity (hierholzer, 1976) . the relative amounts of the mrna species were the same throughout the replication cycle. therefore, in all of our subsequent experiments, the virus-specific intracellular rnas were extracted at 15 hr p.i. molecular cloning of hcv-229e genomic rna and intracellular virus-specific mrnas cdna cloning was initially performed using virion genomic rna as a template. the sizes of inserts in the resultant cdna clones ranged from 0.2 to 0.5 kb in length. one clone, a34, contained a 0.45-kb insert, which was subsequently characterized by restriction mapping and northern blot analysis. the 0.45kb fragment was labeled with 32p by nick-translation and hybridized with intracellular rna from 229e-infected cells. the result, shown in fig. 2 , revealed that the fragment hybridized to each of the mrna species. this result suggested that the hcv229e subgenomic mrnas possess a nested-set structure similar to other coronaviruses and that a34 represented a cdna clone of either the 3'-end of the genomic rna or the leader sequence. cloning was subsequently carried out using intracellular rna from 229e-infected cells as a template. the resulting cdna clones were screened by colony hybridization using the 0.45-kb fragment from clone a34 as a nick-translated probe (fig. 3) . several positive colonies were identified and characterized further. clone l8 contained a 3.6-kb insert but lacked a 3'-poly(a) tail. clone l37, which contained an insert of 1.7 kb, overlapped l8 but was 0.1 kb shorter at the 3'-end. this clone also lacked a poly(a) sequence (see below). therefore, additional cdna clones were isolated using a 0.24-kb bal i-ecori fragment of l8 (fig. 3a) as a probe. these latter clones were further characterized by southern blot analysis. clone slo contained an insert of 0.8 kb which overlapped the 3'-ends of the two previous clones and extended another 0.4 kb in that direction. figure 3b shows the orientation and sizes of clones l8, l37, sl 0, and a34 with reference to theviral genome. restriction enzyme sites used for sequencing are also shown. to determine the sequence of the 3'-end of hcv-229e genome, various restriction fragments of l8, l37, and slo were subcloned into ml 3 vectors. for l8, only the 1.2-kb fragment extending from an internal pstl site toward the 3'-end was sequenced. clone l37 was also sequenced in part. figure 3c shows the cdna fragments and strategy used in sequencing. each region a primer extension study was carried out using a synthetic oligodeoxyribonucleotide complementary to an 18.mer sequence underlined near the 5'.end of the gene. the 3'noncoding region contains a conserved sequence which is shown by the double line. the intergenic conserved sequence, tctaaact, is also shown (dotted line) was verified by dideoxy chain termination sequencing of both strands or by the chemical modification method. clone sl 0 was found to have a poly(a) stretch of 34 bases. figure 4 shows the complete dna sequence with a translation of the main open reading frame (orf) in one-letter amino acid code. this orf extends from base 147 to base 13 13 and predicts a 389 amino acid protein with a molecular weight of 43,366 da. this predicted molecular weight is slightly smaller than the measured molecular weight of the nucleocapsid protein of hcv-229e, which is 50 kda as determined by sds-polyacrylamide gel electrophoresis (macnaughton, 1980) . the difference is probably due to phosphorylation or other modification of the protein. the predicted protein shares features with the nucleocapsid proteins of tgev, mhv, bcv, hcv-oc43, and ibv (kapke and brian, 1986; skinner and siddell, 1984; armstrong er a/., 1983; lapps et a/., 1987; kamahora et al., 1988; boursnell et a/., 1985) . namely, the protein is highly basic and rich in serine residues. sixty percent of the amino acid residues are basic and 12% are acidic. there are 39 serine residues (10% of total), which are presumed to be sites of phosphorylation (stohlman and lai, 1979) . when compared to tgev, with which hcv-229e shares antigenic properties, both n proteins have identical amounts of basic and acidic amino acids and serine residues and similar molecular weights (kapke and brian, 1986) . figure 5 shows a schematic diagram of the possible orfs obtained by translating the nucleotide sequence. the orf in frame 3 is likely the one which encodes the nucleocapsid protein. in frame 2, the 5'-flanking region probably contains part of the sequence of the matrix protein encoded by gene 6. this possibility is sup--i1 i ii i iii -----__ 1 iii1 -i -llil i ii ii i i i iii i ii 2-j i i 111111 ii i1111111 lllll iii i ii, 1111 i ill1 iiii i 3 i i i i ill i i i11 i i i i i ported by the finding that reading frame 2 remains open at the extreme 5'-end. furthermore, the sequence tctaaact, which is found in the intergenic regions of several other coronaviruses (kapke and brian, 1986; skinner and siddell, 1984; armstrong et a/., 1983; lapps et al., 1987; kamahora et a/., 1988; budzilowicz eta/., 1985) is also present between the presumed initiation codon of the main orf and the 3'-end of gene 6. this sequence is the proposed site of fusion of the leader sequence with the mrna coding region makino et al., 1986; budzilowicz et al., 1985) . the 3'-noncoding region contains the sequence tggaagagcca, 75 nucleotides from the 3'-end (fig. 4) which is relatively conserved among coronaviruses and is found at approximately the same location in all of these viral genomes (kapke and brian, 1986; skinner and siddell, 1984; armstrong et a/., 1983; lapps et al., 1987; kamahora et a/., 1988; boursnell et al., 1985) ( table 1) . there is only one nucleotide difference in this conserved sequence when it is compared with that of tgev, bcv, and hcv-oc43. two and three nucleotide differences are found in ibv and mhv, respectively. this conservation of sequence and location suggests that it may be important for viral rna replication. in frame 1, there are several additional orfs of at least 30 amino acids. some of these, including one found in the 3'-noncoding region, lack appropriate translation start sites. another long internal orf is found from base 322 through 693. this contains an appropriate initiation sequence and encodes a hypothetical protein of 13,974 da, which is rich in leucine residues (17%). the significance of this orf remains to be defined. the mrnas of coronaviruses contain a stretch of leader sequence which is derived from the 5'-end of the viral genome and exhibits homologywith the intergenic consensus sequence budzilowicz et al., 1985) . since our cdna clones did not appear to contain leader sequences, we used primer extension studies to determine the sequence of the hcv-229e leader rna. a synthetic oligodeoxyribonucleotide which was complementary to an 18-mer sequence located near the 5'-end of the gene (fig. 4) was end-labeled and used in a primer extension study with poly(a)-selected intracellular mrna as a template. the reaction products, separated by agarose gel electrophoresis, revealed six bands (data not shown). since these bands were most likely to represent the primerextended products of the individual mrna species, the smallest and most abundant band, corresponding to the primer-extended product of mrna7, was eluted and sequenced by the chemical modification method (maxam and gilbert, 1977) . the sequence of the 3'-end of the primer-extended product was identical to the l8 sequence from nucleotides 129 to 17 1. at nucleotide 128, immediately 5' to the proposed leader mrna fusion site, the sequence diverged from the l8 sequence and revealed a putative 60-base leader sequence which is shown in fig. 6 . the figure also shows a degree of homology with the leader sequence of ibv. considerably less homology exists between the leader sequence of hcv-229e and those of hcv-oc43 and mhv-jhm (data not shown). this report presented the primary sequence of the nucleocapsid gene and leader sequence of hcv-229e. when compared to the known sequences of other coronaviruses (kapke and brian, 1986; skinner and siddell, 1984; armstrong et a/., 1983; lapps et al., 1987; kamahora et al., 1988; boursnell et al., 1985) , common features of coronavirus nucleocapsid proteins emerged; namely, they are highly basic and have a high proportion of serine residues, which have been shown 30 40 50 60 i i i i i i hcv-22 9e 5'-cttaag*taccttat*ctatcta*caaatagaaaag **ttgctttttagactttgtgtc*ta*cttc . . . . . . . . . . . . :: : : ::: :: : : :: : :: :::: :::. . ::: : :: : ibv 5'-acttaagatagatattaatatatatctattacactagccttgc**gctagatttttaa*cttaacaaa..... fig. 6. hcv-229e mrna leader sequence compared to the leader sequence of ibv. the ibv leader extends for at least 16 nucleotides in the 3' direction. to be sites of phosphorylation (stohlman and lai, region of 46% homology within the amino-terminal 1979). the relationship between the nucleocapsid one-third of the protein which extends from residues genes of wv-229e and tgev is particularly interest-29 to 134 in hcv-229e, and 41 to 146 in tgev. furing since the viruses are antigenically related (mac-thermore, approximately 10 amino acids downstream naughton, 1981). the predicted molecular weights of from the homologous region in both proteins lies an the n protein and the number of potential phosphoryla-area which is abundant in serine residues, suggesting tion sites of both viruses are almost identical. although that this may be an important functional domain of the these two viruses have little nucleotide sequence ho-molecule. to further examine such functional homolmology between their nucleocapsid genes, the amino ogy between the two proteins, hydropathy profiles acid sequences are homologous within a limited re-were constructed (fig. 7) . the contour of these plots gion. amino acid sequence analysis revealed several suggests that a certain degree of functional homology structural features common to both viruses, which may exists within the first and last one-third of each molehave functional significance. for instance, there is a cule, with an additional region around position 200. the peak around position 200 occurs just after the serine-rich region of the molecule. the relative conservation of these regions suggests a possible role in the interaction of the n protein with the viral genome. similar structural features exist among the n proteins of hcv-229e, ibv, mhv, hcv-oc43, and bcv (skinner and siddell, 1984; lapps et a/., 1987; kamahora et a/., 1988; boursnell et a/., 1985) . this is demonstrated by the hydropathy profiles of these proteins, which are also shown in fig. 7 . further studies are required to reveal the functional significance of the conserved domains. another interesting finding is the open reading frame internal to the main coding region of the hcv-229e n gene. thus far, two other coronaviruses, bcv and mhv-jhm, have been found to contain internal orfs in gene 7 (skinner and siddell, 1984; lapps eta/., 1987) which are preceded by optimum translation initiation signals according to kozak's consensus sequence (kozak, 1983) . the predicted amino acid sequences could encode hypothetical proteins of molecular weights 13,973; 14,842; and 23,057 for hcv-229e, mhv-jhm, and bcv, respectively. interestingly, all three sequences are abundant in leucine residues (17 to 19%). hcv-oc43 also has two smaller internal orfs encoding potential leucine-rich proteins of 8830 and 16,297 molecular weights (kamahora et a/., 1988) . further studies to determine whether this hypothetical protein can be detected in 229e-infected cells or by in vitro translation of a full-length cdna clone (i.e., l8) are in progress. finally, the 3'-noncoding conserved sequence of gene 7 lends additional support to a common ancestry for coronaviruses, regardless of antigenic subgroup. this sequence has been proposed as a recognition site for the virus-encoded rna-dependent rna polymerase prior to negative-strand synthesis (kapke and brian, 1986) . certainly future studies must focus on examining the role of this conserved region in the viral replication cycle. sequence of the nucleocapsid gene from murine coronavirus mhv-a59 characterization of leader-related small rnas in coronavirus-infected cells: further evidence for leader-primed mechanism of transcription sequences of the nucleocapsid genes from two strains of avian infectious bronchitis virus completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus characterization of two rna polymerase activities induced by mouse hepatitis virus. 1. viral three intergenic regions of coronavirus mouse hepatitis virus strain a59 genome rna contain a common nucleotide sequence that is homologous to the 3'end of the viral mrna leader sequence two coronaviruses isolated from central nervous system tissue of two multiple sclerosis patients prolonged incubation in calcium chloride improves the competence of escherichia coli cells colony hybridization: a method for the isolation of cloned dnas that contain a specific gene simple and very efficient method for generating cdna libraries use of solubilizable acrylamide disulfide gels for isolation of dna fragments suitable for sequence analysis chemical and electrophoretic properties of solubilizable disulfide gels purification and biophysical properties of human coronavirus 229e the rna and proteins of human coronaviruses sequence analysis of nucleocapsid gene and leader rna of human coronavirus oc43 sequence analysis of the porcine transmissable gastroenteritis coronavirus nucleocapsid protein gene temporal regulation of bovine coronavirus rna synthesis characterization of viral proteins synthesized in 229e-infected cells and effect(s) of inhibition of glycosylation and glycoprotein transport /76). isolation and morphology of the internal component of human coronavirus, strain 229e bovine coronavirus hemagglutinin protein comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles replication of coronavirus rna. /n "rnagenetits characterization of leader rna sequences on the virion and mrnas of mouse hepatitis virus, a cytoplasmic virus mouse hepatitis virus a59: messenger rna structure and genetic localization of the sequence divergence from the hepatotropic strain mhv-3 coronavirus: a jumping rna transcription presence of leader sequences in the mrna of mouse hepatitis virus the rnaof mouse hepatitis virus sequence analysis of the bovine coronavirus nucleocapsid and matrix protein genes the polypeptides of human and mouse coronaviruses structural and antigenic relationships between human, murine and avian coronaviruses leader sequences of murine coronavirus rna can be freely reassorted: evidence for the role of free leader rna in transcription analysis of genomic and intracellular viral rnas of small plaque mutants of mouse hepatitis virus a new method for sequencing dna sequencing end-labeled dna with base-specific chemical cleavages coronavirus infection in acute lower respiratory tract disease of infants analysis of singleand double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange a new pair of ml3 vectors for selecting either dna strand of double-digest restriction fragments a micromethod for detailed characterization of high molecular weight rna antigenic relationship of the feline infectious peritonitis virus to coronaviruses of other species isolation and propagation of a human enteric coronavirus translation of three mouse hepatitis virus strain a59 subgenomic rnas in xenopuslaevisoocytes dna sequencing with chain-terminating inhibitors the 5'-end sequence of the murine coronavirus genome: implications for multiple fusion sites in leaderprimed transcription nucleotide sequencing of mouse hepatitis virus strain jhm messenger rna 7 phosphoproteins of murine hepatitis virus a bacteriophage t7 rna polymerase/promoter system for controlled exclusive expression of specific genes hybridization of denatured rna and small dna fragments transferred to nitrocellulose the biology and pathogenesis of coronaviruses viral models of demyelination comparison of the rnas of murine and human coronaviruses we thank carol flores for assistance in preparation of the manuscript. this work was supported by public health service research grants nsl8146 and all 9244 from the national institutes of health and grant 1449 from the national multiple sclerosis society. s.s.s. is supported by a postdoctoral training fellowship from the national institutes of health grant ns07149. key: cord-264359-m9j3pcj1 authors: vrijsen, r.; wouters, m.; boeye, a. title: resolution of the major poliovirus capsid polypeptides into doublets date: 1978-05-15 journal: virology doi: 10.1016/0042-6822(78)90093-4 sha: doc_id: 264359 cord_uid: m9j3pcj1 abstract using the ph gradient electrophoretic technique of vrijsen and boeyé, the coat protein of poliovirus types 1 and 2 was resolved into six components, cl to c6, instead of the classical vp1-2-3 (the much smaller polypeptide vp4 was excluded from this study). the multiple components ran true upon reelectrophoresis, and there were no technical artifacts. their resolution did not depend on a particular method for the preparation or disruption of the virion. the c1–c6 components of ph gradient electrophoresis and the classical vp1–vp3 polypeptides were examined with regard to (i) their migration in normal and ph gradient reelectrophoresis and (ii) their kinetics of leucine incorporation in emetine-stopped pulses. it is concluded that c1 and c2 were both derived from vpl, c3 and c4 from vp2, and c5 and c6 from vp3. the vpl-vp3 group of poliovirion polypeptides can be resolved into multiple components by normal polyacrylamide gel electrophoresis (page) in the presence of sodium dodecyl sulfate (sds) (vanden berghe and boeye, 1972) ; however, resolution into six components is achieved more easily and reproducibly in the presence of a ph gradient. in this paper, "normal electrophoresis" will refer to sds-page in a system entirely buffered at ph 7.2; "ph gradient electrophoresis" means that the catholyte was maintained at ph 11.0 and the anolyte was maintained at ph 6.5. under the latter conditions, the gel (initially at ph 7.2) is rapidly invaded by an alkaline zone (vrijsen and boeye, 1978) . the converging results of reelectrophoresis and emetine-stopped pulse-labeling experiments are presented which show that the "classical" vpl, vp2, and vp3 each yield a doublet in ph gradient electrophoresis. the two polypeptides in each doublet appear to be nonidentical, though encoded by similar regions of the viral genome. a preliminary account of these findings appeared elsewhere (boeye, 1976) . ' author to whom requests for reprints should be addressed. virus. poliovirus of strains la/s3 (type 1) and mef-1 (type 2) was grown in suspended hela cells, labeled, and purified as described (boeye, 1965; vanden berghe and boeye, 1973) . extraction of capsidpolypeptides. a suspension of poliovirus in 0.01 m sodium phosphate buffer (ph 7.2) supplemented with 1% sds and 1% mercaptoethanol was incubated for 1 hr at 60". after cooling, sucrose was added to a final concentration of 10% and the mixture was electrophoresed immediately. gels and electrophoresis. all procedures were as described by vrijsen and boeye (1978) . the length of the gels was 9 cm and the potential was 25 v. radioactivity profiles. gels were cut into l-mm slices, and these were either counted after adding soluene and a toluene-based scintillation mixture or eluted for 24 hr at room temperature in 0.3 to 0.5 ml of 0.1% sds. eluate samples of 20 ~1 were counted in bray's mixture. normal counting time was 10 min, but this was increased to 200 min as needed. when required, eluate pools were concentrated by ultrafiltration in a minicon b-15 (amicon) apparatus. for each series of radioactive profiles, one gel loaded with cold la/s3 protein was incorporation studies using enietine (modified from rekosh, 1972) . hela cells were collected 3 hr after high-multiplicity infection and brought to a density of 4 x 10" cells/ml at 37" in eagle's medium devoid of amino acids. r3h]leucine was added 15 min later, and its incorporation was completely stopped after a short time (to be specified) by adding 200 pg/ml of emetine (sigma) and a looo-fold excess of unlabeled leucine. after 6 hr of incubation, the cells were collected by centrifugation and frozen to -70". the supernatant was again centrifuged for 90 min at 40,000 rpm. the pellet, which contained the extracellular virus, was resuspended in phosphate buffer. to prepare cytoplasmic extracts, the thawed cells were resuspended for 15 min in rsb supplemented with excess leucine, disrupted in a dounce homogenizer, and centrifuged to remove the nuclei. the extracellular virus and 120 pg of carrier virus were added to the cytoplasmic extract. the mixture was dialyzed against 0.01 m phosphate buffer (ph 7.2) containing 0.5% sds and layered onto a 15 to 30% sucrose gradient. the sucrose solutions contained 0.01 m phosphate (ph 7.2) and 0.5% sds. the virus was collected as the 160 s peak after centrifugation for 75 min at 29,000 rpm. the "c-virus used for co-electrophoresis in these experiments was separately labeled with ['4c]leucine, purified as above, and added immediately before protein extraction. in ph gradient electrophoresis figure 1 shows the six-band pattern (excluding vp4) into which the protein of la/s3 poliovirions was resolved by ph gradient electrophoresis and the proposed designation of the bands. normal sds-page of the same virion protein ( fig. 2a) yielded only the classical vpl-2-3 pattern and a trace of vpo. samples of uniformly 14c-labeled la/s3 protein were submitted to either normal or ph gradient electrophoresis. the resulting radioactivity profiles (not shown) confirmed the results illustrated in figs. 1 and 2a and were in excellent agreement with frc. 1. ph gradient sds-page of virion polypeptides (strain la/s3). duration of electrophoresis, 24 hr; catholyte at ph 11.0 and anolyte at ph 6.5. scale, distance in millimeters from cathodic end. cl-c6, designation of bands. laemmli (1970) (3 ma/gel, 5.5 hr); the acrylamide and bisacrylamide concentrations in the separation gel were increased to 12.5 and 0.3331, respectively. scale, distance in millimeters from cathodic end. wouters. and boeye the densitometric scans of duplicate gels stained with coomassie blue. as illustrated in fig. 2b , discontinuous electrophoresis according to laemmli (1970) failed to yield the fine resolution which was achieved by ph gradient electrophoresis. our usual purification procedure of la/s3 virus involves the collection of extracellular virus at about 28 hr postinfection, concentration by flash evaporation, two rounds of centrifugation, a butanol extraction, and ecteola -cellulose chromatography (boeye, 1965) . this procedure was modified in three different ways in separate experiments: (i) the virus was centrifuged in a 15 to 30% sucrose gradient and collected from the 160 s peak as an additional purification step; or (ii) intracellular (instead of extracellular) virus was harvested by freezing and thawing of the cells, the other steps of the procedure remaining unchanged; or (iii) the virus was harvested prematurely (9 hr p.i.) by homogenizing the cells and purified by sucrose gradient centrifugation in the presence of 0.5% sds (for a detailed description of the procedure, see under materials and methods, incorporation studies using emetine). none of the modifications to the usual virus purification procedure altered the results of normal or ph gradient electrophoresis of the viral capsid polypeptides. the protein extraction procedure was also modified in two different ways. instead of the procedure described under materials and methods, the virus suspension was either (i) heated for 5 min at 100' in the presence of 1% sds and 1% mercaptoethanol or (ii) dialyzed twice for 24 hr at 37" against 100 vol of 0.01 m phosphate buffer (ph 7.2) containing 0.1% sds and 5 m urea in the first and 0.1% sds and 0.1% mercaptoethanol in the second dialysis (after vanden berghe and boeye, 1972) . again, the results of both normal and ph gradient electrophoresis of the viral protein were unchanged. in the last step of our usual purification procedure, the virus is eluted from ec-teola-cellulose using 0.02 m phosphate buffer of ph 7.2. reelution of the column using the same buffer supplemented with 0.5 m nacl yielded a mixture of virions and empty particles from which the latter were purified as the 80 s peak in a 15 to 30% sucrose gradient. these empty particles were found by the normal electrophoresis to contain vpo, vpl, and vp3 but no vp2 or vp4, which is in agreement with the known composition of procapsids (maize1 et al., 1967; phillips and fennell, 1973) . the components resolved by ph gradient electrophoresis were vpo, cl, c2, and a c5-c6 group (fig. 3) . thus, the protein of procapsids could be further resolved by ph gradient electrophoresis, just like that of mature virions. tritiated mef-1 (type 2) virion protein was co-electrophoresed in the ph gradient technique with "c-labeled la/s3 (type 1) protein, and the results are shown in fig. 4 . both virus strains yielded essentially the same six-peak pattern with minor differences: (i) all mef-1 components migrated slightly slower than their la/s3 homologs, and (ii) c2 was more abundant in mef-1 than in la/s3 protein. ations had been carried out at ph 7.2; (ii) after a 24-h ph gradient electrophoresis, the gel segment containing components cl to c6 was eluted. the eluate was concentrated, resubmitted to the original protein extraction procedure, and reelectrophoresed in the normal system; the results were similar to those shown in fig. 2a and identical to those of controls in which the first electrophoresis was omitted. thus, pretreatment of the sds complexes of poliovirion polypeptides at ph 11, either in free solution or in an acrylamide gel, failed to produce detectable alterations in their migration patterns. the experiments to be presented below, in which single components were resolved by ph gradient electrophoresis and rerun in the same system, allow the same conclusion. in addition, (i) incipient resolution of vp2 and vp3 into doublets was also achieved by electrophoresis at a lower ph, including the normal (ph 7.2) technique, and (ii) nonviral test proteins failed to yield multiple components in ph gradient electrophoresis (vrijsen and boeye, 1978) . it is concluded that ph gradient eiectrophoresis does not cause the observed multiplicity of the poliovirus capsid polypeptides but merely resolves preexisting components. in summary, fine resolution of the poliovirus capsid polypeptides was achieved by ph gradient electrophoresis and the results were independent of (i) the virus purification or (ii) protein extraction procedures, (iii) the state of maturation (procapsid or mature virion), and (iv) the serotype (type 1 or 2). origin of the multiple origin of the components resolved in ph components in ph gradient electrophoresis since the migration of the poliovirus gradient ebctrophoresis polypeptides in ph gradient electrophoresis takes place at about ph 10.5 to 11 (vrijsen and boeye, 1978) , one may well wonder whether this alkaline ph does not cause partial degradation of the polypeptides. the following experiments were performed to detect possible alkaline degradation: (i) poliovirion protein was dialyzed for 24 hr at room temperature against a mixture of 1% mercaptoethanol and 1% sds in 0.01 m phosphate buffer at ph 11.0; the ph was then lowered to 7.2, the extraction procedure was repeated, and the polypeptides were electrophoresed using the normal technique. the polypeptide pattern obtained after this alkaline pretreatment was similar to that of fig. 2a and indistinguishable from that of a control where all oper-two kinds of reelectrophoresis experiments were performed. first, tritiated poliovirion protein was electrophoresed using the normal technique, the gel being sliced and eluted. aliquots of the slice eluates were used to determine the radioactivity profile (fig. 50 ) and the remainder was combined into pools corresponding to peaks vpl, vp2, and vp3 and to the regions in between these peaks. 14c-labeled virus was added to each pool, and the mixture was extracted and reelectrophoresed using both the normal and ph gradient technique. as shown in fig. 5a -5c, the pools corresponding to vpl, vp2, and vp3 each yielded a single peak under normal reelectrophoresis; the intermediate fractions, as expected, yielded two peaks (results not shown). the vp1 material in ph gradient reelectrophoresis yielded mainly cl (fig. 5d) fig. 5. normal and ph gradient reelectrophoresis of vpl, vp2, and vp3 (strain la/s3). (0) primary electrophoresis of "h-labeled la/s3 virion protein (normal technique, 24 hr). slices were eluted in 0.5 ml of 0.1% sds, of which 20 1.11 was used to determine the radioactivity profile. full circles show fractions that were subsequently joined into three pools (representing vpl, vp2, and vp3) for reelectrophoresis; 40-to 60-1.11 samples of these pools were mixed with "c-virus, submitted to the protein extraction procedure, and reelectrophoresed simultaneously in the normal and ph gradient system. material yielded a broad peak covering the ity, no conclusive results were obtained whole c3-c4 area (fig. 5e) , and the vp3 with c2 or c4). material yielded both c5 and c6 (fig. 5f ). taken together, the reelectrophoresis ex-the reverse kind of reelectrophoresis was periments establish the electrophoretic also performed. in this case, the protein equivalence of cl and vpl, which means was first submitted to ph gradient electrothat the same molecules which migrate as phoresis. the excised peaks were rerun in cl in ph gradient electrophoresis migrate the ph gradient system for further purifias vp1 using normal electrophoresis. simcation and finally reextracted and reelectroilarly, c3 appears to be derived from vp2, phoresed in the normal system. the results and both c5 and c6 appear to be derived (not shown) can be summarized as follows: from vp3. the results concerning c2 and cl on normal reelectrophoresis comigrated c4 remained inconclusive. it may be rewith vpl, c3 with vp2, and both c5 and c6 with vp3 (due to insufficient radioactiv-called, however, that procapsids having vp1 and lacking vp2 yielded c2 but neither c3 nor c4 (fig. 3) , and this suggests that c2 is derived from vp1 and c4 from vp2. the combined evidence yields three tentative doublets: vp1 = cl + c2, vp2 = c3 + c4, and vp3 = c5 + c6. genetic relations between doublet polypeptides rekosh (1972) used the labeling kinetics of the poliovirus capsid polypeptides in emetine-stopped pulses of r3h]leucine incorporation to determine the order of the rna regions encoding the polypeptides. as the length of the pulses was increased, vpl, vp3, and vp2 became labeled in turn. the rekosh experiment was easily reproduced using our la/s3 strain and the normal electrophoretic technique to separate vpl, vp2, and vp3 (not shown). the ex-periment was repeated using the ph gradient technique, and the most relevant results are shown in fig. 6 . cl was the first component into which [3h] leucine was incorporated (fig. 6a) , and the next was c6 (this component was exceptionally represented by twin peaks). after 3 min, there was no detectable "h radioactivity in the c3 to c4 area (fig. 6a ), but these components became progressively labeled as pulse length increased (figs. 6b-6d) . the data did not allow any conclusion about c2, which was present only in trace amounts, nor about c5, which appeared as a shoulder in some of the 3h-profiles (figs. 6a and 6b). to determine the 3h/'4c ratio for c5, this component was further purified by reelectrophoresis. to this end, the relevant fractions (figs. 6b-6d) were pooled, con:',-centrated, reextracted, and reelectrophoresed as before (ph gradient technique, 38 hr). for purposes of comparison, the same procedure was also applied to pools of fractions representing cl, the c3-c4 area, and c6. the 3h/'4c ratios were computed from the radioactivity profiles (not shown), normalized, and plotted against the duration of the pulse (fig. 7) . the data show the existence of three different labeling kinetics represented by cl, and c5-c6 group, and the order of labeling was vpl-vp3-vp2 or (cl, c2)-(c5, c6)-(c3, c4), according to whether the polypeptides were analyzed by normal or by ph gradient electrophoresis. thus, the six electrophoretie components, cl to c6, resolved in ph gradient electrophoresis could be attributed to three doublets according to their location on the viral rna, and these doublets were the same that were independently proposed above as the tentative conclusion of reelectrophoresis experiments. in this experiment, la/s3 virus of sufhcient radioactivity was used to allow reelectrophoresis of single-slice eluates (fig. 8) . the polypeptide pattern after ph gradient electrophoresis is shown in fig. 80 . the eluates of fifteen different slices were each mixed with 14c-labeled virus and submitted again to an exact repetition of the original protein extraction procedure and to ph gradient electrophoresis. the most relevant radioactivity profiles are shown in figs. 8a-bg, and the results can be summarized as follows: (i) material collected at or near the center of peaks cl, c5, or c6 upon reelectrophoresis yielded a single peak (figs. 8a, be, and sc). this shows the stability of the protein-sds complexes throughout the extraction procedure and the absence of conversion to the doublet partner; (ii) material taken from the left, center, or right of peak cl migrated identically upon reelectrophoresis (results partly shown in figs. ba-bb), thus showing the original cl peak to contain a single molecular species. this also held true for peaks c5 (figs. bd-8f) and c6 (figs. 8f-8g); (iii) material collected between two of the original peaks again yielded two peaks (figs. bb, 8d, and 8f); (iv) c3 and c4 were not resolved, but the presence of two kinds of molecules was demonstrated by the progressive shift to the right upon reelectrophoresis of successive fractions (figs. bc-8d ). this single-slice reelectrophoresis experiment allows two conclusions: (i) no interconversion among doublet partners could be demonstrated, and (ii) at least those components which could be clearly resolved in the primary radioactivity profile (i.e., cl, c5, and cs) ran true upon reelectrophoresis. discussion the improved resolution of poliovirus capsid polypeptides by ph gradient electrophoresis reported by vrijsen and boeye (1978) was confirmed and shown to be independent of the serotype and of the methods used for virus purification and protein extraction. it is therefore unlikely that the multiple components observed were preparative artifacts, as have been observed with proteins of coronaviruses (sturman, 1977) . the possibility that they were formed by partial degradation of the protein during electrophoresis was also examined and rejected. whereas normal sds-page or discontinuous electrophoresis according to laemmli (1970) resolved only vpl, vp2, and vp3, ph gradient electrophoresis usually resolves six components (cl-cs). the pattern was not entirely constant, however, as neighboring components sometimes failed to separate (e.g., c3-c4 in fig. 80 ; c5-c6 in fig. 4) ; on the other hand, the results occasionally suggested further resolution of a single component (e.g., c3 in figs. 4 and 5d-5f). due to these and other technical difficulties, no single experiment yielded all of the information being sought, and a clear picture emerged only by juxtaposition of the results. according to this picture, cl and c2 formed a doublet corresponding to vpl, c3 and c4 a second doublet corresponding to vp2, and c5 and c6 a third doublet corresponding to vp3. with regard to the cl-c2 doublet, for example, this correspondence means (i) that the molecules which migrate as either cl or c2 in ph gradient electrophoresis will migrate as vp1 in normal sds-page, and (ii) that cl and c2 in emetine-stopped pulses are labeled equally and according to the same kinetics as vpl. thus, although we demonstrated a higher number of electrophoretie components, our findings are still basically in agreement with the accepted vpl-2-3-4 scheme for the composition of the poliovirus capsid. several sds complexes of the same pro-vrijsen, wouters, and boeye tein may coexist, at least when a limited amount of sds is made available for binding; however, saturation is reached at a sds/protein weight ratio of about 4.5:1 (stoklosa and latz, 1975) ? and this ratio was largely exceeded in our extraction procedure (sds/protein ratio ca 3o:l) and during electrophoresis (unlimited supply of sds). even so, the question may be asked whether the two electrophoretic components in each doublet could not be different dodecyl sulfate complexes of the same polypeptide. therefore, we looked for possible interconversion to the doublet partner upon isolation of single components and exact recreation of the conditions under which the protein-detergent complexes were originally formed (extraction of capsid protein). since no interconversion was found and each component ran true upon reelectrophoresis, it may be tentatively concluded that the two polypeptides in each doublet were not identical. if so, they must differ slightly in charge, molecular weight, or both. such differences may conceivably originate in the accumulation of mutants as a result of serial passages (phillips and fennell, 1973) . to prevent any such accumulation, however, each virus population examined in this work was grown from a single plaque by only four passages, and a mutational origin of the observed multiplicity can therefore be excluded. the observed microheterogeneity probably originates in the posttranslational processing of the proteins. since the capsid proteins are carved out of a common precursor by successive cleavages (summers and maizel, 1968; jacobson and baltimore, 1968) , microheterogeneity may result from multiplicity of the cleavage sites. ambiguity of cleavage was already invoked to explain differences in the migration velocity of homologous polypeptides of different poliovirus strains (cooper et al., 1970) or the appearance of doublets (beckman et al., 1976) . however, no direct proof of ambiguity was offered and the case rests on the unproven assumption that differences in migration velocity in sds-page necessarily reflect differences in molecular weight. ambiguity of cleavage would be expected to reveal itself in multiple n-terminal or cterminal sequences. the terminal sequences of the major capsid polypeptides of mengo virus were determined and no mention was made of ambiguities (ziola and scraba, 1976) . this is also true for footand-mouth disease virus (adam and strohmaier, 1974; matheka and bachrach, 1975) , except that in an earlier paper (bachrach et al., 1973) an ambiguity in the c-terminal sequence of vp2 was reported. until and unless ambiguity of cleavage is demonstrated by means of sequence analysis, other possible causes of microheterogeneity, such as phosphorylation, deamidation, and glycosylation cannot safely be ruled out. isolation of the coat proteins of foot-and-mouth disease virus and analysis of the composition and n-terminal end groups isolation of the structural polypeptides of foot-and-mouth disease virus and analysis of their c-terminal sequences cleavage site alterations in poliovirus-specific precursor proteins molecular weight of a poliovirus protein picornaviral structure and protein synthesis evidence for ambiguity in the posttranslational cleavage of poliovirus proteins polypeptide cleavages in the formation of poliovirus proteins cleavage of structural proteins during the assembly of the head of bacteriophage t4 composition of artificially produced and naturally occurring empty capsids of poliovirus type 1 nterminal amino acids of foot-and-mouth disease virus peptides polypeptide composition of poliovirions, naturally occurring empty capsids, and 14 s precursor particles gene order of the poliovirus capsid proteins electrophoretie behavior of protein dodecyl sulfate complexes in the presence of various amounts of sodium dodecyl sulfate characterization of a coronavirus: i, structural proteins: effects of preparative conditions on the migration of protein in polyacrylamide gels evidence for large precursor proteins in poliovirus synthesis new polypeptides in poliovirus stepwise degradation of poliovirus and top component by concentrated urea gel electrophoresis of protein-dodecyl sulfate complexes in a ph gradient and improved resolution of poliovirus polypeptides structure of the mengo virion: iv, amino-and carboxyl-terminal analysis of the major capsid polypeptides we are grateful for the excellent technical assistance of a. de rees. this research was supported by the fonds voor kollektief fundamenteel onderzoek (belgium). key: cord-255841-3laov764 authors: duquerroy, stéphane; vigouroux, armelle; rottier, peter j.m.; rey, félix a.; jan bosch, berend title: central ions and lateral asparagine/glutamine zippers stabilize the post-fusion hairpin conformation of the sars coronavirus spike glycoprotein() date: 2005-05-10 journal: virology doi: 10.1016/j.virol.2005.02.022 sha: doc_id: 255841 cord_uid: 3laov764 the coronavirus spike glycoprotein is a class i membrane fusion protein with two characteristic heptad repeat regions (hr1 and hr2) in its ectodomain. here, we report the x-ray structure of a previously characterized hr1/hr2 complex of the severe acute respiratory syndrome coronavirus spike protein. as expected, the hr1 and hr2 segments are organized in antiparallel orientations within a rod-like molecule. the hr1 helices form an exceptionally long (120 å) internal coiled coil stabilized by hydrophobic and polar interactions. a striking arrangement of conserved asparagine and glutamine residues of hr1 propagates from two central chloride ions, providing hydrogen-bonding “zippers” that strongly constrain the path of the hr2 main chain, forcing it to adopt an extended conformation at either end of a short hr2 α-helix. to initiate a productive infection, all viruses must translocate their genome across the cell membrane (reviewed by smith and helenius, 2004) . enveloped viruses achieve this step by membrane fusion, a process mediated by specialized envelope proteins present at the virus surface. for coronaviruses such as the recently emerged sars coronavirus (sars-cov), the spike (s) glycoprotein is responsible for both cell attachment and entry by triggering fusion of the viral and cellular membrane. this type i membrane protein can be divided into two domains of similar size, s1 and s2 (fig. 1a) . s1 forms the bulbous globular head and is responsible for cell attachment; its amino acid sequence is less conserved than s2, which forms the membrane-anchored stalk region and is responsible for membrane fusion. the coronavirus spike protein has all the essential features of a bona fide class i viral fusion protein (smith and helenius, 2004) , including the occurrence of two heptad repeat regions in its s2 domain (de groot et al., 1987) . most class i viral fusion proteins, with influenza virus hemagglutinin (ha) as a prototype (skehel and wiley, 2000) , are expressed as precursor proteins, which are endoproteolytically cleaved by cellular proteases, giving rise to a metastable complex of a receptor-binding subunit and a membrane fusion subunit. upon receptor binding at the cell membrane or as a result of protonation after endocytosis, the fusion proteins undergo a dramatic conformational transition that leads to the exposure of a hydrophobic fusion peptide and insertion into the target membrane. the free energy released upon subsequent refolding of the fusion protein to its most stable conformation 0042-6822/$ -see front matter d 2005 elsevier inc. all rights reserved. doi:10.1016/j.virol.2005.02.022 through association of the two hr regions is believed to facilitate the close apposition of viral and cellular membranes and the subsequent lipid merger. the coronavirus spike protein has, however, some characteristics that set it apart from class i fusion proteins, such as the lack of a cleavage requirement and the presence of an internal fusion peptide. we and others have previously shown that, analogous to other class i fusion proteins, peptides corresponding to the hr regions of the mouse hepatitis coronavirus (mhv, bosch et al., 2003) and sars-cov (bosch et al., 2004; ingallinella et al., 2004; xu et al., 2004a; tripet et al., 2004) can fold into a stable rod-like structure, consisting of three hr1 helices in association with three hr2 peptides in antiparallel orientation. this complex, supposedly representing the post-fusion conformation with the predicted fusion peptide upstream of hr1 and the transmembrane segment downstream of hr2 positioned at the same end, juxtaposes the cellular and viral membrane thereby facilitating membrane fusion and consequently virus entry. here, we report our analysis of the structure of this hr1/hr2 complex of the sars-cov as determined by x-ray crystallography to 2.2 2 resolution. this analysis complements two recent reports (supekar et al., the s1 and s2 regions are labeled. fp: putative fusion peptide region. tm: transmembrane region. sp: signal peptide. (b) sequence alignments of the hr1 and hr2 regions of coronavirus spike proteins of the three different groups (g1 to g3) and the unclassified sars coronavirus (blue arrow) using clustalw (thompson et al., 1994) . see the supplementary material for the complete names of the viruses used. the numbers at the top line correspond to the sars amino acid sequence, the structure of which is described in the text. the alignment is color-coded according to sequence conservation: red, strictly conserved; green, highly conserved; blue, conserved; black, variable. the alignment in the hr2n region was manually modified to match the structural superposition with the corresponding region of the mhv protein (pdb accession numbers 1wdf and 1wdg, xu et al., 2004c) . residues with ordered electron density have a grey background in the sars line. the cloned fragment contained all residues between the boxed columns (further highlighted with a vertical empty arrow below the sequence). two additional lines at the bottom summarize the following: the bregisterq line provides the abcdefg heptad repeat assignment with letters in black for the residues actually observed in a helical conformation in the structure, with the two hr1 stutters in grey background. note the insertion of exactly two heptad repeats, both in hr1 and hr2, in the s protein of group 1 coronaviruses. the binteractionq line shows the two salt bridges (1 and 2, see fig. 2b ) in a yellow background. in the case of hr1, this line provides also the residues participating in the asparagine/glutamine zipper shown in fig. 3 (labeled bnq), the knob-into-hole interactions with either partner within the trimer (labeled b or c), and the residues lining the central cavity shown in fig. 2b (labeled with a star). residues forming salt bridges 1 and 2 connecting hr1 with hr2 have a yellow background, with a number below to indicate the partner in each chain. note that salt bridge 1 is conserved, but is sometimes made with hr1 residue 900 (from the previous helical turn) instead of 903. the structure shows that the side chain of residue 900 can contact the hr2 1188 side chain equally well. blue background columns indicate hr1 residues interacting with the two central chloride ions. vertical grey background columns identify hr2 residues in the extended segments (hr2n and hr2c) that pack their side chains into hydrophobic pockets in the hr1 interhelical grooves. 2004; xu et al., 2004b) describing the structures of three related constructs of the core fragment of this protein. indeed, the structure described here provides important additional insights for understanding the determinants of the stabilization of the hr1 central core and the constraints imposed to the hr2 main chain by the internal coiled coil. the membrane fusion core fragment of the sars-cov s glycoprotein was crystallized in a rhombohedral space group and the crystals diffracted just beyond 2.2 2 resolution. the details of the construct used, the production and purification procedure, the crystallization and the x-ray structure determination are provided in materials and methods, given as supplementary material. the crystal asymmetric unit contains two hr1/hr2 heterodimers lying about two different crystallographic 3-fold axes. the packing environment of the two independent molecules is however very similar. the hr1/hr2 complex is an a-helical trimeric bundle (figs. 2a and b) containing 378 amino acids in total, its maximal length and diameter being 123 2 and about 30 2, respectively. the total molecular surface buried from solvent upon trimer formation is 3000 2 2 per hr1/hr2 heterodimeric subunit. in the intact protein, an intervening polypeptide segment of 173 amino acids -from residues 974 to 1146 -connects the two segments, hr1 and hr2, as diagrammed in fig. 1a . the hr1 segment folds as a 22-turns-long a-helix that is involved in parallel interactions with its 3-fold related symmetry mates throughout its whole length, forming a 120-2-long central trimeric bundle which buries 2350 2 2 of accessible surface for each helix. the interhelical grooves of this bundle accommodate the hr2 segment running antiparallel to the hr1 helix in a pattern typical of the post-fusion conformation of class i fusion proteins. the accessible surface buried by each hr2 peptide is 1460 2 2 . the overall fold and quaternary structure yields a very stable protein rod having the hr1 n-termini and the hr2 ctermini clustered together at one end. this organization of the post-fusion form implies that both of the membraneinteracting segments, the fusion peptide and the tm region, are brought into close proximity by the fusogenic conformational change of the s protein, similar to all the other membrane fusion proteins of known structure. as expected, there is essentially one side chain per turn of the hr1 a-helix participating to the central hydrophobic core of the molecule, resulting in 23 amino acids from each chain (labeled to the left of fig. 2a ) interacting with their symmetry mates at the central 3-fold axis. positions baq and bdq alternate every other turn according to the helical wheel diagram of fig. 2d . the long hr1 a-helix does not display the typical bheptad repeatq parameters, in which the average periodicity is 3.5 residues per turn -that is, 7 residues every two turns (crick, 1953) -but rather displays a mean periodicity of 3.64 residues per turn, which is very close to the canonical a-helix periodicity (pauling et al., 1951) . the baq and bdq positions therefore drift away from the hydrophobic core (fig. s1 , supplementary materials) and become out of register after several turns, effectively shifting the face of the helix that faces the hydrophobic core at the 3-fold axis. this results in the presence of bstuttersq in which the 3-4-3-4 periodicity of the heptad repeat becomes 3-4-4-3-4 at two positions (indicated in fig. 2a ). the helical wheel of fig. 2d has the residues labeled along their helical positions for the 22 turns, after correction for the stutters. this assignment is also indicated in the sequence alignment of fig. 1b , which shows the baq through bgq positions of the heptad repeats, and the babcdq (first stutter) or bdefgq (second stutter) insertions indicated under the hr1 sequence. two strong peaks along the 3-fold molecular axis, with heights between 8 and 10 j, are evident in the electron density maps calculated with the final phases, from a model for the hr1 and hr2 polypeptides, respectively. pink spheres on the central 3-fold axis indicate the chloride ions. in a, the axes of the hr1 helices in the trimer are drawn as green tubes, highlighting the two stutters in red. the coiled coil axis is dark red (vertical at the trimer center). the helical turns are numbered from n-to c-terminus for one of the subunits (black and white numbers are used for hr1 and hr2, respectively). the n-and c-terminal ends of the model are indicated for one hr1/hr2 heterodimer. the 2 columns between the vertical scale bar on the left and the ribbon diagram indicate the residues and the 3-and 4-residue repeat pattern of the side chains facing the 3-fold axis of the coiled coil. black and red fonts indicate polar and non-polar side chains, respectively. residues within green boxes are strictly conserved. red boxes in the second column highlight the stutters. in b, the side chains of polar residues within the hydrophobic core are drawn in green and labeled. water molecules are indicated as small red spheres. inter chain salt bridges (1 and 2, labeled in blue, corresponding to lys 903 to glu 1188 and lys 929 to glu 1163, respectively) are also indicated (basic side chains are in blue, acidic in red). the central cavity is displayed as a gold surface. (c) slab of the model viewed down the 3-fold axis to show the chloride ions. pink arrows indicate the center of the slab in panel b: top and bottom panels display chloride ions 1 and 2, respectively. the hydrogen bonding network propagating from the central ions toward the outside-which highly constrains the hr2 main chain, is indicated. several of the asn and gln residues labeled are part of the asn/gln zipper (see text). as a guide for orientation, the axes of the 3 hr1 a-helices are drawn and labeled in green. the top panel is a view from below the atom, and the bottom panel from above it, relative to panel 2b. (d) helical wheel after correction for the stutters (as in the register line of fig. 1b ). hr1 left, hr2 middle: as in panel a, polar and non-polar side chains are black and red, respectively (notice the strong amphipathic character of the two helices). positions a and d are highlighted within a circle with a red background. the right panel shows a diagram of the interactions in the 6-helix bundle. refined in the absence of any atom at those sites (i.e., bomit mapsq). both of the independent molecules in the crystal display this feature. the oxygen atom of a water molecule does not have enough electrons to account for this extra density, and we have interpreted each of the peaks as corresponding to a bound chloride ion, which is the most common ion in all the buffers used to produce and purify the protein. indeed, introducing a chloride ion in the model at these sites leads to refined thermal parameters (bb factorsq) for the ion that are roughly the same as those of the surrounding amino acid side chains (about 20 2 2 ), whereas introducing a water molecule leads to abnormally low b factors. the two chloride ions are labeled 1 and 2 according to their distance to the hr1 n-terminus, and are respectively chelated by gln 902 and asn 937 and their 3-fold symmetric counterparts. as shown in figs. 2a and d, these are among the few polar buried residues of hr1. the gln/ asn nature of residues at these two positions is strictly conserved among coronaviruses (see the alignment of fig. 1b ). chloride ion 1 is directly liganded by the ne atoms of the three equivalent gln 902 side chains, with tetrahedral coordination geometry (fig. 2c , top panel). the fourth ligand for such a sphere of coordination would have been on the 3-fold axis below the ion, but in this case, it is absent, the ion being held in place by van der waals contacts with hydrophobic side chains from the next helical turn (ile 905 in the 4th turn) interacting at the 3-fold axis. chloride ion 2 is liganded via water molecules because the asn 937 side chain is also involved in interactions with the hr2 main chain (see below). this site contains 6 ordered water molecules in total, 2 per subunit, directly surrounding the 3-fold axis (fig. 2c, bottom panel) . only one water molecule is in direct contact with the ion, the second is hydrogen bonded to the first one, to its own symmetry mate in the trimer and to the main chain carbonyl of asn 937. the coordination geometry of chloride ion 2 is the same as that of the first ion but the tetrahedral bpyramidq is inverted, in this case, the fourth ligand would be on the 3-fold axis above the ion, which is held in place by the ring of hydrophobic side chains of val 934 above it (with the molecule in the orientation of fig. 2a) . the side chains of both gln 902 (which is strictly conserved) and asn 937 are engaged in a network of hydrogen bonds, both with main chain amide and carbonyl groups and with the side chains of adjacent residues (including asn 901 which is also strictly conserved, see fig. 1 ) and with the main chain of hr2 (at gly 1182 for gln 902, and ser 1156 for asn 937). thus, a stabilizing hydrogen bonding network propagates from the central cl à ion all the way to the periphery of the molecule, playing a role in constraining the hr2 main chain conformation (see below). in addition to gln 902 and asn 937, which chelate the central ions, the other polar side chains directed toward the 3fold axis are gln 895 (which is at the very first turn and so is not in the hydrophobic bcoreq of the molecule) and thr 923, at the 9th turn ( fig. 2a) . this region is where the hr2 helix inserts in the lateral groove, between turns 6 and 10 of hr1, resulting in the packing of 6 helices along 5 helical turns. both ser 919 (position bgq, 8th turn) and thr 923 are found pointing toward the interior of the molecule here, where usually hydrophobic residues are present, leaving an internal hydrophilic cavity of about 21 2 3 at the 3-fold axis (depicted in gold in fig. 2b ). the sequence alignment (fig. 1b) shows that position 923 is semi-conserved: it is threonine in about half of the sequences examined and valine in the others. valine has a non-polar side chain of about the same volume as threonine and so is also unable to fill the cavity. furthermore, the other residue lining the cavity, ser 919, is often glycine or alanine in most coronaviruses, which have smaller side chains and so the corresponding proteins will have an even bigger cavity. in several group i coronaviruses, both valine and alanine are present at positions 923 and 919, in which case, the cavity will have only hydrophobic boundaries. the presence of this cavity will undoubtedly have a negative impact on the stability of the molecule, suggesting that the helical portion of hr2, which packs against the hr1 helix precisely in this region, strengthens the molecule by providing more rigidity in this weak point. the hr2 polypeptide contains a central helix of 5 complete turns flanked by two extended regions, hr2n and hr2c, at the amino-and carboxy-terminal ends of the central helix, respectively. in fig. 1b , the heptad repeat positions are labeled such that the first hydrophobic position bdq is right before the first helical turn (turn 0), and the last is at the end of the 5th turn, amounting to a total of 6 hydrophobic side chains from positions baq and bdq -mostly leucines and isoleucines -which interact with the hydrophobic core of hr1. the corresponding helical wheel is shown in fig. 2d , with the helix oriented such that the baq and bdq positions of the helix face the hr1 interhelical groove. the extended segments of hr2 also contribute hydrophobic side chains to the central hydrophobic cluster, 10 in total (6 from hr2n and 4 from hr2c, shown in fig. 3) . thus, in total, 16 side chains from hr2 contribute to the stability of the core. the n-terminus of hr2 lies in between turns 16 and 17 of the hr1 helix, and the c-terminus reaches turn 1, so that hr2 contacts most of the hr1 helix, except for its 5 cterminal turns, as illustrated in figs. 2a, b and 3 . the main-chain amide groups of a-helices are -by definition -engaged in hydrogen bonding with the main chain carbonyl of a residue located four residues downstream (pauling et al., 1951) in an n to n + 4 patternwhere n denotes the number of an amino acid -in contrast to 3/10 helices in which the pattern is n to n + 3. the ends of the helix are special because the amide groups at the nterminus and the carbonyl groups at the c-terminus do not have a hydrogen bonding partner from within the helical main-chain; in general these interactions have to be satisfied by alternative hydrogen bond acceptors (constituting an ncap) or donors (c-cap), except when the ends of the helix are directly exposed to solvent (presta and rose, 1988; richardson and richardson, 1988 ). in the hr1/hr2 complex, the long hr1 a-helix has both its termini exposed to solvent, with a few non-helical residues at its n-terminus in which the sequence 890-gly-ile-gly-892 breaks the helix. in contrast, the short hr2 helix is strongly capped at both ends. the three exposed amide groups at the n-terminus of hr2 are residues 1161 to 1163. they are capped by asn 1159-a position in which there is either asn or asp in the sequence alignment of fig. 1 . the oy atom of asn 1159 accepts a hydrogen bond from the amide group of residue 1161, and at the same time interacts with the amide group 1162 via ordered water molecules. these waters are also stabilized by lateral hydrogen bonding to the salt bridge glu-1163 to lys-929 (from hr2, 10th turnfig. 2b ). thus, all three residues (1159, 1163 and 929-two from hr2 and one from hr1, and which are all nearly strictly conserved, see fig. 1 ) participate indirectly in the capping of this amide group. finally, the amide 1163 donates a hydrogen bond to the main chain carbonyl of residue 1160, in a 3/10 helix interaction. the latter carbonyl also accepts a hydrogen bond from the amide 1164, within a standard n to n + 4 a-helix pattern. thus, the n-cap is provided almost entirely from within the hr2 sequence. the hr2 helix makes four a-helical turns followed by a 3/ 10 helical turn (labeled in fig. 2b ), that is, there is a disruption of the normal helical pattern, which is obvious in the ribbon diagrams. the exposed carbonyls of the 4th turn belong to residues 1171 to 1173. the first two carbonyls are capped by asn 1175 (strictly conserved) and gln 917, respectively, the side chains of which also hydrogen bond to each other. the asn 1175 side chain, a residue within the hr2 helix, thus perturbs the geometry of the helix by introducing a side chain to main chain hydrogen bond. however, asn 1175 is part of a strictly conserved n-glycosylation site, and it is very likely that, once glycosylated, its ny atom would not be available for hydrogen bonding to the main chain. thus, the observed perturbation of the hr2 helix, in which the chain switches from an a to a 3/10 helix at the 4th turn, may simply be an artifact resulting from producing the protein in escherichia coli. the third carbonyl, 1173, makes a 3/10 interaction with the amide group of residue 1176. the 3/10 5th helical turn of hr2 also exposes its carbonyl groups (1175 and 1176), the second one being capped by gln 908 and lys 911. therefore, the hr2 c-cap is set in place, to an important extent, by interactions with hr1. the polypeptide chain at either end of the hr2 helix is maintained in an extended conformation by a string of figs. 2a and b , in which all the main chain atoms of the hr2n and hr2c extended segments of one of the subunits are represented as ball and stick, as well as the side chains of the asn and gln residues participating in the zipper. the atoms are colored according to atom type: grey are carbon atoms (light and dark for atoms of hr1 and hr2, respectively), red and blue indicate oxygen and nitrogen atoms, respectively. hydrogen bonds between gln and asn side chains and main chain atoms are displayed as hatched cyan tubes. at the top, the arrows indicate the segments that connect to the fusion peptide (fp) and trans-membrane (tm) region. the boxes indicate the regions blown up in the right panels. the a-helical 5 turns of hr2 are numbered. right panels. the top and bottom panel zoom into the asn/gln zippers that constrain the hr2c and hr2n segments, respectively. the model was slightly rotated in each of the two panels, with respect to the view in the left panel, for clarity. hydrophobic hr2 side chains fitting into pockets in the hr1 grooves are shown in green. asn and gln side chains are colored as in the left panel. all residues indicated are labeled, red boxes highlighting highly conserved residues. the interactions displayed in this figure, together with the interactions that form an n-and c-cap to the hr2 helix as described in the text, highly constrain the hr2n and hr2c main chain. asparagine and glutamine side chains from the hr1 coiled coil, which hydrogen bond to the hr2 main chain as illustrated in fig. 3 . the asparagine ny atoms (or the glutamine ne atoms) donate a hydrogen bond to the main chain carbonyls, and the oy (or oe) accept one from the main-chain amide groups, resulting in a pattern that partially mimics the one seen by a h-strand. this extensive hydrogen-bonding network zips the hr2 main chain along the hr1 interhelical grooves. two important peripheral ionic hr1/hr2 interactions are also observed at either end of the hr2 helix. the first one is between e1163 and k929 and the second one between k903 and e1188, as illustrated in fig. 2b . the residues participating in these salt bridges -especially the first one -are also highly conserved among coronaviruses. the most important discovery revealed by the structure of this particular construct of the fusion core of the sars cov s glycoprotein is the extent of the constraints imposed on the hr2 main chain by the central hr1 coiled coil. the striking arrangement of conserved asn and gln residues, which provides a hydrogen bonding network propagating from two central ions at the 3-fold axis of the coiled coil, stretches the main chain of hr2 and results in confinement of its helical portion to only 5 turns in total at the center, capped at either side. from the amino acid sequences of many coronaviruses s proteins, it had been predicted that hr2 by itself would display at least four to five heptad repeats, which would lead to a helical region of at least 8 turns (see fig. s2 in the supplementary material). this prediction has been further substantiated experimentally by the high degree of a-helicity (81% for sars-cov and 89% for mhv) observed by circular dichroism with the hr2 peptide in the absence of hr1 (bosch et al., 2003 (bosch et al., , 2004 . one of the structures recently reported by supekar et al. (2004) , displayed in fig. 4 (third panel, pdb code 1beq) shows that hr2 segments not interacting with hr1 do adopt an a-helical conformation. this led the authors to propose that the c-terminal segment of hr2 would continue as a helix as it approaches the n-terminus of hr1. fig. 4 (compare the 1st and the 3rd panels) shows that this is not the case, as the internal hr1 coiled coil actually maintains this hr2 segment in an extended conformation all the way up to the hr1 first helical turn. the structure drawn in the second panel (1bez) lacked the first turns of hr1 and the c-terminal end of hr2 to provide this information, while the resolution -and thus the quality of the resulting atomic model -of the 1wnc structure (xu et al., 2004b) was not enough for a detailed analysis of the hydrogen-bonding interactions. given the different lengths of the structures reported (fig. 4) , a question arises concerning the actual n-terminus of the hr1 coiled-coil in the post-fusion conformation of the intact s protein. the hydrophobic cluster analysis (hca) provided in fig. s2 (supplementary material) suggests that the two glycines at positions 890 and 892 are indeed likely to break the helix also in the intact molecule, as they do in the structure of the fragment reported here. accordingly, the ahelical hca pattern switches at this position to one of alternating polar/non-polar side-chains along the sequence, which is more typical of h-strands or extended conformations. this extended segment of the hr1 chain, of about 10 amino acids, would connect to a glycine/alanine-rich region (residues 855 to 880) that has all the characteristics of typical viral fusion peptides. this is reminiscent of the influenza virus ha2 protein in its post-fusion conformation, in which the fusion peptide -which is comprised between ha2 residues 1 to 22 -is connected to the n-terminus of the central coiled coil (at residue 38) by a segment of polypeptide for which there is visible electron density in the crystals between amino acids 33 and 38. the visible portion of the connector is in an extended conformation and provides an n-cap to the neighboring a-helix in the trimer (chen et al., 1999) . in the present case, we see ordered electron density for residues 890 to 892 in an extended conformation (although in this case, they do not provide an n-cap to the hr1 helix). the 5 n-terminal amino acids of the construct (885 to 889) are disordered. in contrast, at the c-terminus of hr2, there is clear density in the crystals all the way to the last amino acid of the construct, tyr 1188, with the chain ending with a single turn of a-helix between residues 1184 and 1188. interestingly, the structure 1beq shows that these residues are indeed part of a longer helix (these amino acids are actually in turn 7 of the 1beq-hr2 helix, labeled in fig. 4 , third panel, top) ending after turn 8 at residue 1193. it is clear from fig. s2 that the tm region begins around amino acid 1194. the presence of one ahelical turn in our structure suggests that the hr2 chain may connect in a-helical conformation to the lipid bilayer, bringing the fused membrane to about the location drawn in fig. 4 . it is likely that in the 1beq structure, the presence of the shorter hr1 segment causes the observed disruption of the hr2 helix at turns 4 and 5 (see fig. 4 , 3rd panel, bottom) because all the elements for capping the helix at that position are present, but the downstream zipper is missing. indeed, the residues corresponding to turn 6 in 1beq are seen in extended conformation in our structure, owing to the strong constrains imposed to the hr2 main chain by the central coiled coil. in the absence of hr1, hr2 could well adopt a straight a-helical conformation since there are no glycine nor proline residues in this region, which are known to be a-helix breakers. our separate observations that in the absence of hr1, the hr2 peptide has a very high a-helical content as observed by em and circular dichroism (bosch et al., 2003 (bosch et al., , 2004 argue in favor of this interpretation. fig. 4 . recapitulation of the current structural data on the fusion core of the sars cov glycoprotein s. the left panel (framed in red) displays the structure described in this report in a ribbons representation in which the three hr1 segments are in primary colors and hr2 in grey. the other three panels are labeled with the corresponding pdb accession code of the structure depicted (pdb codes 1beq and 1bez from supekar et al., 2004, and 1wnc from xu et al., 2004b) . in all panels, both the trimeric molecule (top) and one subunit (bottom) are displayed. all panels are colored identically, with segments containing amino acids that are not present in our current model (in the left panel) colored white. the images are all at the same scale, with the horizontal bars providing a means to align them so that the n-terminus of the hr2 helix is at the same height in each panel. the bottom panel indicates the number of the n-and cterminal ends of the constructs represented. at the top, a roughly-to-scale diagram of a putative bfused membraneq, with its aliphatic portion in blue and the hydrophilic lipid heads in orange, is drawn at about the expected distance from the structures, as deduced from the amino acids that are missing between the nand c-termini and the membrane interacting segments of the protein, the n-terminal fusion peptide and the c-terminal trans-membrane region. the hr1 coiled coil -or at least its n-terminal portion -is believed to form only when the fusogenic conformational change of the s protein takes place, triggered by receptor binding at the target cell surface, so that it is not present in the fusion-active conformation of the molecule at the surface of infectious virions. in the absence of the n-terminal portion of the hr1 coiled coil, the hr2 regions are likely to be part of the stalk of the s protein pre-fusion conformation, since they are connecting to the tm regions and have a strong propensity to adopt an a-helical conformation as discussed above. the pre-fusion conformation of s is trimeric (delmas and laude, 1990) , and it is thus likely that the a-helical hr2 segments are also part of its trimerization interface, stabilized by additional segments of this very large glycoprotein. indeed, several (iso)leucine to alanine mutations in hr2 were shown to strongly impair oligomerization of the mhv s protein (luo et al., 1999) . the results from the structural studies on the fusion core would therefore suggest that the prefusion arrangement of this region of the s protein is strongly disturbed and refolds after formation of the hr1 coiled coil. the rearrangement is such that presumably the pre-fusion trimer has to dissociate and then reassociate around the hr1 coiled coil. a putative transient dissociation of the pre-fusion trimer may help explain the topological problems encountered when all three subunits of a trimer are simultaneously connected to two separate membranes to then fuse them into a single lipid bilayer. it is, therefore, possible that trimer dissociation and re-association during the fusogenic conformational transition is indeed part of a more general membrane fusion mechanism, which would be valid for all class i fusion proteins. an additional unnoticed feature in the previously reported structures is the presence of ions at the 3-fold axis, liganded by polar residues, a feature that has been observed in the structures of similar coiled coils from class i fusion proteins of other viruses (baker et al., 1999; fass et al., 1996; malashkevich et al., 1999; weissenhorn et al., 1998) . the presence of these polar residues within the hydrophobic cores has been proposed to provide a register to the interactions (akey et al., 2001) , resulting in a single pattern of hydrophobic contacts, and so avoid possible miss-folding by packing of helices shifted by one or two turns along the axis of interaction. in the particular case reported here, it is obvious that the presence of the two ions provides clear anchors. one interesting observation is the likely conservation of the central cavity with its destabilizing effect on the molecule. this suggests that the residues lining the cavity in the postfusion form are important for an alternative conformation of the protein, before the fusogenic conformational change. it thus appears from the structure that the protein has been forced to evolve alternative ways to compensate for the destabilizing effect of the cavity, strengthening the molecule from the outside with the presence of the hr2 helical segment and further stabilizing the arrangement by the salt bridges and the zipping of the asparagines at either end. taken together, all of these features of the structure account for the observed relatively high stability of the (hr1/hr2) 3 complex, as indicated by its resistance to proteolytic degradation and the fact that the thermal dissociation in sds gels is 60 8c and 80 8c for sars-cov and mhv, respectively (bosch et al., 2004) . these values suggest that the melting point in the absence of sds would be much higher, although calorimetry measurements with the proteins resulting from these constructs have not been made. in contrast to most of the class i fusion proteins that have been studied structurally until now, the coronavirus s protein is special because is does not undergo an activating cleavage near the n terminus of the fusion peptide. in influenza virus, the structural studies have shown that the conformational transition of the hemagglutinin projects the fusion peptide by a distance of 100 2 away from its original location in the metastable form of the protein. this is possible because this peptide is located at the very n-terminus of ha2, and therefore it does not carry along any upstream polypeptide segment. the situation is completely different in the case of the coronaviruses, and it is likely that the polypeptide segment preceding the fusion peptide will have to act like a rope that can follow the projection of the fusion peptide. this would imply that the segment preceding the fusion peptide does not have a very rigid and stable structure in the pre-fusion form so that it can be unwound during the conformational change. however, such flexibility is not obvious from the amino acid sequence (see the hca pattern in fig. s2 ), and only a structure of the pre-fusion form of the s-protein can clarify this issue. these features of the s protein suggest, however, that in order for the fusogenic conformational transition to take place, a relatively high energy barrier has to be overcome, and this process could be facilitated also by having a very low energy minimum for the final stable conformation. finally, the structure described in this manuscript can provide a rational basis for developing potent inhibitors of entry of the sars-cov, by blocking the formation of the membrane-fusion core of the molecule. as shown in fig. 3 , there are indeed a number of pockets identified along the interhelical grooves of hr1 into which putative inhibitors can be designed to bind and disturb the correct association of hr2. far acknowledges support from the cnrs and inra, the sesame program of the brégion ile-de-franceq, the cnrs programs pcv and bdynamique et réactivité des assemblages biologiquesq buried polar residues in coiled-coil interfaces structural basis for paramyxovirus-mediated membrane fusion the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex severe acute respiratory syndrome coronavirus (sars-cov) infection inhibition using spike protein heptad repeat-derived peptides n-and c-terminal residues combine in the fusion-ph influenza hemagglutinin ha2 subunit to form an n cap that terminates the triple-stranded coiled coil the fourier transform of a coiled-coil evidence for a coiled-coil structure in the spike proteins of coronaviruses assembly of coronavirus spike protein into trimers and its role in epitope expression retrovirus envelope domain at 1.7 angstrom resolution structural characterization of the fusion-active complex of severe acute respiratory syndrome (sars) coronavirus amino acid substitutions within the leucine zipper domain of the murine coronavirus spike protein cause defects in oligomerization and the ability to induce cell-tocell fusion core structure of the envelope glycoprotein gp2 from ebola virus at 1.9-a resolution the structure of proteins: two hydrogen-bonded helical configurations of the polypeptide chain helix signals in proteins amino acid preferences for specific locations at the ends of a helices receptor binding and membrane fusion in virus entry: the influenza hemagglutinin how viruses enter animal cells analysis of alpha-helical coiled coils with the program twister reveals a structural mechanism for stutter compensation structure of a proteolytically resistant core from the severe acute respiratory syndrome coronavirus s2 fusion protein clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice structural characterization of the sars-coronavirus spike s fusion protein core crystal structure of the ebola virus membrane fusion subunit, gp2, from the envelope glycoprotein ectodomain characterization of the heptad repeat regions, hr1 and hr2, and design of a fusion core structure model of the spike protein from severe acute respiratory syndrome (sars) coronavirus crystal structure of severe acute respiratory syndrome coronavirus spike protein fusion core structural basis for coronavirus-mediated membrane fusion. crystal structure of mouse hepatitis virus spike protein fusion core we thank stephane bressanelli and enrico stura for their participation in parts of this project. we gratefully acknowledge matthijs raaben for his technical assistance in the construction of the hr1-hr2 linker plasmid and the expression and purification of the hr1-hr2 protein complex; c. schulze-briese and t. tomikazi for help during diffraction data collection; j. navaza and j. lepault for discussions; g. aumont and c. branlant for support. diffraction data were collected at synchrotrons: sls, paul supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.virol.2005.02. 022. key: cord-255738-r8zfdsix authors: ge, feng; luo, yonghu; liew, pei xiong; hung, eugene title: derivation of a novel sars–coronavirus replicon cell line and its application for anti-sars drug screening date: 2007-03-30 journal: virology doi: 10.1016/j.virol.2006.10.016 sha: doc_id: 255738 cord_uid: r8zfdsix the severe acute respiratory syndrome (sars) outbreak in 2002, which had a high morbidity rate and caused worldwide alarm, remains untreated today even though sars was eventually isolated and controlled. development and high-throughput screening of efficacious drugs is therefore critical. however, currently there remains a lack of such a safe system. here, the generation and characterization of the first selectable, sars–coronavirus (sars–cov)-based replicon cell line which can be used for screening is described. partial sars–cov cdnas and antibiotic resistance/reporter gene dna were generated and assembled in vitro to produce the replicon transcription template, which was then transcribed in vitro to generate the replicon rna. the latter was introduced into a mammalian cell line and the transfected cells were selected for by antibiotic application. for the antibiotic-resistant cell lines thus generated, the expression of reporter gene was ensured by continued monitoring using fluorescent microscopy and flow cytometry. the suitability of this replicon cell line in drug screening was demonstrated by testing the inhibitory effect of several existing drugs and the results demonstrate that the sars–cov replicon cell lines provide a safe tool for the identification of sars–cov replicase inhibitors. the replicon cell lines thus developed can be applied to high-throughput screening for anti-sars drugs without the need to grow infectious sars–cov. severe acute respiratory syndrome (sars) is a potentially fatal atypical pneumonia that arose in guangdong province of the people's republic of china in november 2002, quickly spreading to 26 countries on five different continents and causing large-scale outbreaks in hong kong, singapore and toronto in early 2003 (peiris et al., 2003b) . sars was recognized in late 2002 and by the end of the viral outbreak in july 2003, there have been more than 8000 sars cases reported worldwide and 774 sars-attributed deaths . this outbreak had a severe and profound impact on public health and economies worldwide, reminding us of the danger emerging infectious diseases bring to densely populated societies. coronaviruses (order nidovirales, family coronaviridae, genus coronavirus) are a group of viruses with large, enveloped crown-like virions and positive-sense single-stranded rna genomes (siddell et al., 1983) . the genomes of coronaviruses range from 27 to 32 kb, the largest of any of the known rna viruses. all coronaviruses share the characteristic 3′ co-terminal and nested-set structure of sub-genomic rnas in addition to unique rna synthesis strategies, genome organization, nucleotide sequence homology and structural proteins particular only to coronaviruses (cavanagh et al., 1995) . the etiologic agent of sars was identified as a novel coronavirus (sars-cov) (peiris et al., 2003a; drosten et al., 2003; ksiazek et al., 2003; poutanen et al., 2003) although the genomic sequence of sars-cov does not resemble any of the three recognized groups of coronaviruses. soon after the disease was recognized, sars-cov was confirmed as the causative agent of sars; by demonstrating that the coronavirus could be used to experimentally infect and induce interstitial pneumonitis in cynomolgus macaques, thus fulfilling koch's postulates kuiken et al., 2003) . although the 2002/2003 sars epidemic was eventually controlled by patient isolation, there is neither an effective treatment for sars presently nor an efficacious vaccine to prevent infection (peiris et al., 2003b) . the significant morbidity and mortality, including its potential for reemergence, make it necessary to develop effective methods to treat and prevent the disease. therefore, in the fight against sars, it is important to develop antiviral agents that can specifically inhibit the rna synthesis of sars-cov. to maximize the chance of finding efficacious anti-sars drugs, high-throughput screening of large chemical libraries for compounds that can block sars-cov replication must be carried out. however, the high infectivity and virulence of sars-cov render this kind of research very dangerous. therefore, there is a need for an anti-viral agent identification system that does not involve the use of live virus. to this aim, partial viral rna genomes have been constructed such that they replicate and persist in dividing cells without producing viral particles (kaplan and racaniello, 1988; liljestrom and garoff, 1991; khromykh and westaway, 1997; behrens et al., 1998; lohmann et al., 1999; pang et al., 2001; shi et al., 2002; thumfart and meyers, 2002; hertzig et al., 2004) . these viral replicons were derived from viral genomes through the deletion of all or some structural genes. due to the absence of viral structural genes, virion proteins are not synthesized in the cells and therefore no infectious viral particle could be produced by the cells. however, since all transand cis-acting components required for viral rna synthesis are retained, these partial viral rnas could replicate autonomously in the cells. this report describes the construction of the first sars-cov-derived replicon cell line. this sars-associated replicon cell line is based on the use of sars replicon cdnas generated by reverse genetic techniques. the results demonstrate that these sars-cov-derived replicon cell lines can be used to test candidate sars-cov replicase inhibitors without the need to grow infectious sars-cov. our method of developing replicon constructs was based on the strategy previously used to assemble the full-length cdna construct sars-cov (yount et al., 2003) . the strategy for the construction of the replicon is illustrated in fig. 1 . viral envelope-protein coding genes s, e and m were excluded from the replicon to disable virion synthesis, production and secretion. the nucleocapsid gene, n, was retained because the nucleocapsid protein has been shown to be required for viral rna synthesis (almazan et al., 2004; hertzig et al., 2004) and that the sequence involved in the regulation of the expression of a coronavirus 3′-proximal gene is more than ∼ 50 nt upstream of the gene (alonso et al., 2002; jeong et al., 1996) . therefore, to achieve relatively native expression of n gene from the replicon, a region ∼ 60 nt upstream of n orf was included in the replicon. the green fluorescent protein-blasticidin deaminase fusion (gfp-blar) gene was also included into the replicon to enable easy selection and detection of repliconcontaining cells. gfp-blar gene was inserted between orf 1 and n, not at the 5′ or 3′ end of the replicon, in order to minimize any possible deleterious effect in the synthesis of replicon rna. the expression of gfp-blar was driven by the transcription regulatory sequence of orf s, which was included in the replicon and occurring at a position right upstream of the gfp-blar gene. following this strategy, six cdna subclones that span the sars-cov genome were used in the assembly of recombinant sars-cov replicon cdna. the green fluorescent protein-blasticidin deaminase fusion (gb) gene was amplified from the plasmid ptracer™-cmv/ bsd/lacz using pcr and included into the replicon constructs. in addition, the cdnas constructed were flanked by restriction sites that leave unique interconnecting junctions of 3 or 4 nts in length (sapi or bsai). as these sticky ends are not complementary to the majority of other sticky ends, which are generated by the same enzyme at other sites in the dna, replicon cdnas can be systematically assembled by in vitro ligation. the total dna from the above ligation reaction, after an extraction using phenol-chloroform, was used directly as the template for the in vitro transcription and synthesis of the sars-cov replicon rna. for a rough quality inspection, an aliquot of the post-transcription mixture was analyzed on a denaturing agarose gel and the transcripts seen were of the appropriate lengths (data not shown). the purified sars-cov replicon rna was used to transfect bhk-21 cells and the transfected cells were subjected to blasticidin selection to enrich for the replicon-carrying cells. as fluorescence indicates successful transfection and selection of sars-cov replicon rna, transfected cells were observed for fluorescence microscopically after 5 days of blasticidin treatment. clusters of green-fluorescent cells were observed and a typical green-fluorescent cell cluster is shown in fig. 2a . as observed, there was no sign of any cytotoxicity due to the sars-cov replicon and the morphology of the greenfluorescent cells was typical of bhk-21 cells. the transfected cultures were further maintained under blasticidin selection to obtain cells that carried the sars-cov replicon consistently. autonomous replication of viral rna was also successfully maintained, northern blot analysis of total cellular rnas prepared from scr-1 revealed the presence of full-length replicon rna and replicon rna derived transcripts encoding gb and n genes. the n gene probe detected replicon rna and replicon rna-derived gb-n and n rnas. the gb probe detected only those rnas containing the gb gene; replicon rna and replicon rna-derived gb-n mrna (fig. 2b ). gfp expression of scr-1 has been studied by fluorescence microscopy and flow cytometry for a period of 3 months (over 40 passages under blasticidin selection). as shown in fig. 2c , the average green fluorescence intensity value of src-1 culture remained at a constant level and was in excess of that of the parent bhk-21 culture. these results were consistent when compared to previous findings (hertzig et al., 2004) . hertzig et al. (2004) analyzed the gfp expression of hcov 229e replicon cells by flow cytometry for a period of 4 months (over 50 passages under g418 selection) and they showed that the percentage of green fluorescent cells remained at a constant level of 40-60% throughout this period. thus, these data indicate that although replicon cells may express sufficient gfp-blar to survive blasticidin selection, the amount of gfp blar protein was insufficient to be detected by flow cytometry. a possible cause for differential gfp blar protein expression in replicon cells is the efficiency of functional replicon rna uptake during transfection. thus, this analysis shows that the sars-cov replicon persists efficiently and grows consistently in the cells under selection for substantial periods of time and would be suitable to use for anti-sars drug screening purposes. furthermore, scr-1 cells that have been stored for 1 month in liquid nitrogen and re-cultured still displayed green fluorescence indistinguishable from cells that have been passaged continuously (data not shown). subsequent passage of cell culture supernatants onto bhk-21 cells also did not result in either blasticidin resistance or gfp expression, thus demonstrating that the replicon particles are not released by passaging supernatants into fresh cultures. sequence analysis of the replicon rna purified from scr-1 cells soon after selection in blasticidin (passage number 6) found no sequence differences compared with the published sequence of sars-cov strain sin2774 (genbank accession number ay283798). at passage number 40, the rna from scr-1 cells was purified and examined again by sequence analysis. at this later passage, three nucleotide changes were detected that resulted in three amino acids changes: two in orf 1 and one in n gene (table 1) . since these nucleotide changes were not encoded by the cloned cdna and no adaptive mutations had occurred in the early passage, these three mutations, most likely, have been acquired during replication in the later passage. it remains important to elucidate whether the mutations facilitate efficient replication of replicon rna in scr-1. to develop a fast and convenient antiviral screening protocol for the detection of sars-cov replicase inhibitors, three previously described antiviral drugs were tested, all of which have been evaluated in the context of sars-cov inhibition in tissue culture (yount et al., 2003; cinatl et al., 2003; so et al., 2003) . the compounds were applied in different concentrations on scr-1 cells and gfp expression was monitored by flow cytometry analysis, fluorescent microscopy and quantitative real-time pcr 3 days later. untreated cells served as controls and the cytotoxic effect of each compound was assessed in parallel on parental bhk-21 cells. as shown in fig. 3 , incubation of scr-1 cells with e64-d at 0.4 mg/ml reduced not only the level of reporter gene expression and the copy number of replicon rna but also the overall percentage of green fluorescent cells. importantly, no cytotoxicity of e64-d was observed at or near inhibitory concentrations. ribavirin showed low inhibitory activity at the concentration of 0.4 mg/ml and significant cytotoxic effects were observed at higher concentration, 0.5-5 mg/ml. as such, ribavirin was considered to be inactive against sars-cov replication. glycyrrhizin showed low inhibitory effect at the concentration of 2 mg/ml. these results indicate that the cysteine proteinase inhibitor e64-d appears to represent a promising candidate for the inhibition of sars-cov replicase function. on the other hand, the therapeutic efficacy of the other drugs may be limited because of their low specific inhibitory effect and significant cytotoxicity. taken together, these results show that the replicon cell lines thus developed would be useful for anti-sars drug screening and will provide a tool to study candidate anti-sars agents. in the past 15 years, the concept of autonomously replicating rnas (replicon rnas) has been applied to a number of positivestrand rna virus systems and has led to the establishment of novel antiviral screening assays and vectors (bartenschlager, 2002; lo et al., 2003) . baric's group constructed a transmissible gastroenteritis virus (tgev) replicon for the expression of heterologous gfp gene (curtis et al., 2002) and thiel's group generated a non-cytopathic, selectable replicon rna (based on hcov 229e) for the identification of coronavirus replicase inhibitors (hertzig et al., 2004) . nevertheless, the genomic sequence data of sars-cov reveal that this novel agent did not belong to any of the known groups of coronaviruses, including two human coronaviruses, hcov oc43 and hcov 229e (peiris et al., 2003a; drosten et al., 2003) . therefore, despite the presence of functional replicon rna assays (hertzig et al., 2004) , there is a need for the development of sars-cov replicon cell lines, which would allow a rapid and safe identification of inhibitors that are specific for sars-cov. in this study, the first selectable sars-cov based replicon cell line was successfully established and characterized for the purpose of screening anti-sars drugs. the sars-cov replicon cell line we described here will be a valuable tool for the development of anti-sars therapeutics. our results with the following replicase inhibitors clearly demonstrate that our sars-cov replicon system facilitates the identification of these inhibitors in tissue culture. replicase inhibitor ribavirin, although active against a wide range of viruses and used as sars therapy (so et al., 2003) , was shown in this study to be inactive against sars-cov in vitro at noncytotoxic concentration, which is in line with previous reports (tan et al., 2004; cinatl et al., 2003; wu et al., 2004) . next, the finding that e64-d inhibitor had selective activity against sars-cov is in line with a study by yount et al. (2003) , who showed that e64-d effectively inhibited sars-cov replication in tissue culture. for glycyrrhizin, the variation in results obtained in this study as compared to previously published results may be due to assay conditions including virus strain, detection method and compound concentration and handling (cinatl et al., 2003) . notably, other independent groups also observed the lack of anti-sars-cov activity of glycyrrhizin (hertzig et al., 2004) . this indicates that glycyrrhizin may not inhibit coronavirus replication but instead exert antiviral effects during virus adsorption or release. with an easily detectable gfp expression and the absence of virion production, our viral replicon cell line represents a simpler and safer system for anti-viral agent identification than a live virus infection system. thus, the efficacy of candidate inhibitors can be evaluated by measuring the fluorescence intensity of the replicon cells before and after the addition of drugs. that means our cell-based system can be easily automated and used in large-scale screening of anti-sars-cov agents. furthermore, our viral replicon cell-based system can be used to test individual antiviral agents designed based on certain biochemical principles or new drugs targeting at multiple regions of the sars-cov orf 1 or n gene. since no infectious virus is formed, the assay represents a safe protocol that can be performed in biosafety level 2 laboratories. compared to anti-viral agent identification systems based on purified proteins or nucleic acids, our sars-cov replicon cell line has two advantages: first, if a candidate inhibitor can inhibit replication of our replicon rna, which occurs intracellularly, it thus demonstrates that this agent can permeate the cell. secondly, the cytotoxicity of the candidate inhibitor can also be observed simultaneously by noting its cell morphology. two critical indexes of a candidate inhibitor-its inhibitory effect and cytotoxicity, can therefore be met using our sars-cov replicon cell line. for an anti-viral agent identified using a purified-bimolecular-based system, further tests on cell delivery and cytotoxicity of the agent has to be done separately. a disadvantage of our replicon cell-based system is that the structural genes s, e and m are not included in the replicon rna. therefore, it cannot be used to screen for drugs that act on cellular and viral targets involved in receptor binding, virus entry, genome encapsulation and virus release. however, structural genes of coronaviruses change rapidly and anti-viral agents that target these genes have less consistent efficacy. thus, anti-viral agents should target regions highly conserved in the coronavirus genome such as orf 1, which is the most conserved region found. in conclusion, our replicon system provides a convenient and safe screening system for the identification of drug candidates selectively active against sars-cov. the protocols and reagents developed in this study will be useful for gaining insights into the mechanisms of rna synthesis of this pathogen. potential additional applications include the development of rna vaccines against sars-cov and rna vectors for long-term gene expression. the baby hamster kidney (bhk)-21 cell line was purchased from american typical culture center (atcc). bhk-21 cell line was maintained in dmem medium (gibco) supplemented with 10% foetal calf serum (gibco) (d10). sars-cov strain sin2774 virion rna (a gift of prof. t. k. chow, department of microbiology, national university of singapore) was used as the template for cdna synthesis. the reverse genetic strategy for constructing the desired sars-cov replicon is illustrated in fig. 1 . the reverse transcription reaction was performed using the superscript iii first strand kit (invitrogen) as described in the manufacturer's manual with some modifications (nathan et al., 1995) . the reverse transcription was primed using oligonucleotide 9r (5′-gtcattctccta agaagctattaaaatcacatgg-3′) and 09r (5′-gattcaggtctc attgtcctccac ttgc taggtaatcc-3′). the cdnas were denatured for 30 s at 94°c and amplified by pcr with elongase amplification system (invitrogen) with 35 cycles at 94°c for 30 s, 55°c for 30 s, and 68°c for 1.5 to 6 min depending on the size of the amplicon. the amplicons were isolated from agarose gels and cloned into topo ii ta vectors (invitrogen) according to the manufacturer's directions. the following primers were used in the isolation of the sars a1 subclone (froward, 5′-cacgctcttcagcatac taatacgactcactata-gatattaggtttttacctacccaggaaaag-3′, reverse, 5′-gaatgagctcttcatggtaatggttgagttgg-tacaagg-3′); a2 subclone (forward, 5′-gaatgagct-cttcaccaaatgcgagttttgataatttcb-3′, reverse, 5′-caaccatccatgatatgaacatagc-3′); b subclone (froward, 5′-ccgtttctgcaa tggttaggatg-3′, reverse, 5′-ggctgctgtagtcaatggtatgatg-3′); c subclone (froward, 5′-gcagatcaggctatgacccaaatgtac-3′, reverse, 5′-tgggaggctt atgtgacttgc-3′); d subclone (froward, 5′-gtgcctgtattaggagaccattcc-3′, reverse, 5′-gtatcaggtctcaatgttcgtttagttgt-taacaagaatatcac-3′) and n subclone (froward, 5′-cattcaggtctcattggtaccttcatgaaggtcacc-3′, reverse, 5′-gtcattctcctaagaagctattaaaatca-catgg-3′). the gb dna was amplified as described above, except that the template was ptracer™-cmv/bsd/lacz vector dna (1 ng) and the primers were forward, 5′-cgtggatc-cggtctctacatggcctcca aaggagaagaac-3′ and reverse, 5′-ccagaattcggtctcaccaattagccctc-cca cacataaccag-3′. two to five independent clones of each sars amplicon were isolated and sequenced using a panel of primers located about 0.5 kb from each other on the sars insert and an abi model automated sequencer. the sequencing data thus obtained were compared with the published sequence of sars-cov strain sin2774 (genbank accession number ay283798) using the megalign module of the sequence analysis software lasergene (dnastar). a consensus sequence for each of the cloned fragments were determined, and when necessary (i.e., a1, b and d subclones), a consensus clone was assembled using restriction enzymes and standard recombinant dna techniques to remove unwanted amino acid changes except silent mutations. each of the plasmids was grown to high concentration, isolated and digested or double-digested with sapi or bsai according to the manufacturer's direction (neb). the appropriately sized cdna inserts were isolated from 0.7 ∼ 1.2% agarose gels and extracted using the qiaex ii dna purification kit (qiagen). the a1 + a2 + b, c + d, and gb + n fragments were ligated overnight and isolated. the a1a2b + cd + gbn cdnas were ligated overnight at 4°c. the resulting dna was extracted first by phenol-chloroform-isoamyl alcohol (25:24:1), then by chloroform, and precipitated in the presence of 67% ethanol and 0.1 m sodium acetate (ph 5.2). shortly before the performance of in vitro transcription (see below), the extracted dna was pelleted by centrifugation, washed with 70% ethanol, air-dried and finally dissolved in 10 μl of rnasefree water. the detailed cloning strategy, plasmid maps and sequences are available from the author upon request. the t7 in vitro transcription system mmessage mmachine kit (ambion), which also includes the rna 5′-capping function, was used to generate the sars-cov replicon rna. for 2 h at 37°c, a 30-μl reaction mix were performed with 4.5 μl of a 30 mm gtp stock, resulting in a 1:1 ratio of gtp to cap analog. to remove the dna template, 1 μl of dnase i (2 u/μl) was then added and the reaction mix was incubated at 37°c for 15 min. to polyadenylate the rna synthesized, the reaction mixture was treated further by the reagents from the poly (a) tailing kit (ambion). the final product was purified by adding 30 μl of licl precipitation solution (ambion) to pellet the rna. the rna was washed once with 1 ml of 70% ethanol, air-dried, and finally dissolved in 20 μl of rnase-free water. bhk-21 cells grown to 50% confluence in a 6-well plate were transfected with 10 μg of sars-cov replicon rna and 10 μl of the transfection agent lipofectamine 2000 (invitrogen) in opti-mem medium (invitrogen) according to the manufacturer's instructions. one day after transfection, blasticidin (invitrogen) was added to a final concentration of 10 μg/ml to the culture medium to select for the replicon-carrying cells. two weeks later individual cell clones were isolated and expanded until analysis or storage in liquid nitrogen. the blasticidin-resistant cell line generated was designated as scr-1 and subjected to analyses pertinent to the sars-cov replicon that they carried. the parent cell line bhk-21 was used as the negative control in these analyses. fluorescence microscopy and flow cytometry were used to observe the green fluorescence of gfp blar protein expressed from the sars-cov replicon. the cells were observed under an olympus ix70 inverted fluorescence microscope and the images were recorded using image-pro plus (media cybernetics). the cells were then scanned for green fluorescence and light scattering using a beckman coulter epics altra flow cytometer. the data collected were analyzed using the win-mdi 2.7 data analysis program (the scripps research institute). to analyze whether the introduced replicon rna can mediate coronavirus-specific discontinuous transcription of l-gb-n and l-n mrnas (fig. 4) , northern blot analysis was performed. for northern blot analysis, total cellular rnas from bhk-21 or scr-1 cells were isolated using the rneasy mini kit (qiagen) according to the animal cell protocol supplied by the manufacturer. samples (10 μg) of total cellular rnas were denatured, electroporated and transferred to a hybond-n + positively charged nylon membrane (ambion) as per the manufacturer's instructions and hybridized with fluoresceinlabelled probes specific for the gb gene or the sars n gene. the gb and n probes were prepared from the gb and n gene using the gene images random prime labeling module (amersham). the hybridization of probes to the immobilized rnas on the nylon membrane and subsequent signal generation were done using the reagents from the gene images cdp-star detection module (amersham). chemiluminescent signals fig. 4 . generation of sub-replicon rnas through discontinuous transcription of sars-cov replicon rna in the replicon-carrying cells. the black box represents the 72-nt leader rna sequence, derived from the 5′ end of the replicon, located at the 5′ end of each sub-replicon rna. the size of each rna shown is exclusive of the poly-a tail. emitted from the blot were detected by exposing a hyperfilm-mp x-ray film (amersham) to the blot for durations between 1 to 10 min, followed by the photographic development of the exposed film. to obtain the complete sequence of the sars-cov replicon persisting in the scr-1 cells, the total cellular rnas isolated from src-1 cells at passage number 6 and 40 were used as the templates. the rna was amplified by rt-pcr and the pcr products were gel-purified and sequenced directly. primers used in the rt-pcr and sequence analysis are available upon request. to prove that the replicon particles are not released by passaging supernatants into fresh cultures, supernatants obtained from scr-1 cell cultures were passaged onto fresh bhk-21 cells and examined for blasticidin resistance and gfp expression. to identify potential agents active against sars-cov replication, three drugs approved for clinical use in the treatment of viral infections were tested. e64-d and glycyrrhizin (sigma) were included in this test due to their previously reported activities against sars-cov replication (yount et al., 2003; cinatl et al., 2003) . ribavirin (icn pharma) was also used as it represents a widely used class of nucleoside analogs, which inhibits viral polymerases (so et al., 2003) . scr-1 cells were cultured in a 96-well plate in dmem containing 10% fbs and 10 μg/ml blasticidin. the culture media was removed after a 1-day incubation when the cells reached 80-90% confluence and replaced by fresh medium without blasticidin. graded doses of drugs were added on monolayers of scr-1 cells and an untreated well was served as control. three days later, the cells were harvested and analyzed by fluorescence microscopy and flow cytometry. four duplicate tests were performed. inhibition of reporter gene expression was calculated as reduction of the fluorescence intensity of gfp-positive cells by setting the fluorescence intensity of untreated src-1 cells at 100%. in parallel, the cytotoxicity of drugs was assayed on bhk-21 cells using celltiter 96 aqueous non-radioactive cell proliferation assay kits (promega). real-time rt-pcr analyses were performed to quantify the copy number of replicon rna in scr-1 cells before and after drug treatment. total cellular rnas from bhk-21 or scr-1 cells were isolated using the rneasy mini kit and one-step artus sars lc rt-pcr kit (qiagen, hamburg, germany) was used for real-time quantitative amplification of sars-cov replicon rna. real time rt-pcrs were performed according to the instructions of the manufacturer. the amplicon and primers were as described . briefly, 5 μl of total rna extract was added to a capillary tube containing 15 μl of rt-pcr reagents and loaded into the lightcycler (roche diagnostics gmbh, mannheim, germany). the thermal cycling conditions were as follows: rt was performed at 50°c for 10 min; and amplification was performed for 1 cycle of 95°c for 10 s, followed by 50 cycles of 2 s of denaturing at 95°c, 12 s of annealing at 55°c, and 10 s of extension at 72°c. finally, cooling was performed at 40°c for 30 s. total rna extracted from bhk-21 cells was used as the negative control. internal sars-cov standards, which allow the determination of the copy number of replicon rna, were supplied with the assay kit. real-time pcr signals were analyzed using the lightcycler software (roche; version 5.32), and the sizes and uniqueness of pcr products were verified by performing both melting curves and agarose gel electrophoresis. the copy number of replicon rna was determined by direct comparison with the internal standards. replicon rna expression levels are expressed as number of copies/μg total rna. all samples were run in triplicate and the average value of the copy number was employed to quantify replicon rna. the nucleoprotein is required for efficient coronavirus genome replication transcription regulatory sequences and mrna expression levels in the coronavirus transmissible gastroenteritis virus hepatitis c virus replicons: potential role for drug development characterization of an autonomous subgenomic pestivirus rna replicon virus taxonomy. sixth report of the international committee on taxonomy of viruses glycyrrhizin, an active component of liquorice roots, and replication of sars-associated coronavirus heterologous gene expression from transmissible gastroenteritis virus replicon particles identification of a novel coronavirus in patients with severe 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syndrome in canada construction and characterization of subgenomic replicons of new york strain of west nile virus the biology of coronaviruses development of a standard treatment protocol for severe acute respiratory syndrome inhibition of sars coronavirus infection in vitro with clinically approved antiviral drugs feline calicivirus: recovery of wild-type and recombinant viruses after transfection of crna or cdna constructs small molecules targeting severe acute respiratory syndrome human coronavirus reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus ge feng gratefully acknowledges the continuous support by dr. zhang xian-en over many years. we thank dr. yu hongxiang and professor mary ng for their excellent assistance, and the roche company that provided compounds for the assay experiment. this study was funded by a grant from the biomedical research council, singapore. key: cord-253894-4u5yt7b7 authors: senkevich, tatiana g.; koonin, eugene v.; moss, bernard title: vaccinia virus f16 protein, a predicted catalytically inactive member of the prokaryotic serine recombinase superfamily, is targeted to nucleoli date: 2011-09-01 journal: virology doi: 10.1016/j.virol.2011.06.017 sha: doc_id: 253894 cord_uid: 4u5yt7b7 the f16l gene of vaccinia virus (vacv) is conserved in all chordopoxviruses except avipoxviruses. the crocodile poxvirus f16 protein ortholog has highly significant similarity to prokaryotic serine recombinases and contains all amino acids that comprise the catalytic site. in contrast, f16 orthologs encoded by other poxviruses show only marginally significant similarity to serine recombinases, lack essential amino acids of the active site and are most likely inactive derivatives of serine recombinases. nevertheless, the conservation of f16l in non-avian poxviruses suggested an important function. however, a vacv mutant with the f16l gene knocked out replicated normally in dividing and quiescent cells. the f16 protein was synthesized early after infection and detected in virus cores. when expressed in infected or uninfected cells, f16 accumulated in nucleoli depending on the level of expression and confluency of cells. evidence was obtained that f16 forms multimers, which might regulate concentration-dependent intracellular localization. the poxviridae comprise a family of large, complex viruses with 130 to n300 kbp double-stranded dna genomes that infect numerous vertebrate and invertebrate animals (moss, 2007) . vaccinia virus (vacv) was used as the vaccine for smallpox eradication and is currently employed as a live recombinant vector for expression studies and development of vaccines against other pathogens and cancer (moss, 1996) . vacv, by far the most intensively studied poxvirus, replicates entirely in the cytoplasm of infected cells and encodes many of the proteins required for its growth as well as numerous proteins modulating virus-host interactions. the genome of vacv comprises a subset of genes that are common to all or most poxviruses and essential for replication in cell culture and another subset of genes that are specific for the orthopoxvirus genus, mostly nonessential in cell culture and involved in host interactions (upton et al., 2003) . those genes that are conserved throughout the poxviridae have been functionally characterized to varying degree of detail. however, many genes that are present in most but not all chordopoxviruses remain to be analyzed. one example is the uncharacterized vacv f16l gene, which has been retained in all chordopoxviruses except for avipoxviruses. our interest in this gene, apart from its considerable evolutionary conservation, was stimulated by evidence that the f16 protein ortholog of nile crocodile poxvirus (crv) is homologous to site-specific serine recombinases of prokary-otic transposons and bacteriophages (afonso et al., 2006) . here we provide a detailed computational analysis of the f16 protein indicating that it is unlikely to have serine recombinase activity, and experimental data showing that the f16l gene is not required for virus growth in cell culture. remarkably, the f16 protein specifically localized to the nucleoli of infected and uninfected cells. our specific interest in the f16 protein of vacv was aroused by the report of afonso and co-workers (afonso et al., 2006) that the putative protein encoded by the 051 gene of crv shares similarity with both bacterial transposon resolvases and homologs of vacv f16, which were detected in all chordopoxviruses except avipoxviruses. starting with the vacv f16 sequence, an iterative database search using the psi-blast program (altschul et al., 1997 ) revealed a distant relationship to crv051 and the lack of any detectable homolog in avipoxviruses. whereas the crv protein showed statistically highly significant similarity to numerous proteins of the ser recombinase superfamily from bacterial and archaeal transposons and bacteriophages (grindley et al., 2006; olorunniji and stark, 2010; smith and thorpe, 2002) , the vacv f16 and the other poxvirus orthologs showed only marginally significant similarity (expect value of~0.03 (altschul et al., 1997) ). excluding crv, the detection of homology of the f16 proteins with ser recombinases required at least two psi-blast iterations, accounting for the absence of such annotation in the virology 417 (2011) 334-342 literature. nevertheless, the validity of this relationship was strongly supported by additional search results using the hhsearch method (soding, 2005 ) that detects ser recombinases as proteins with the greatest similarity to a hidden markov model generated from the alignment of poxvirus f16 proteins with a 97% confidence. the region of sequence conservation between f16 proteins and ser recombinases encompasses the entire catalytic domain of the latter that consists of approximately 140 amino acids (fig. 1) . all structural elements of the resolvase/recombinase domain appear to be conserved in f16 as indicated by the comparison of the predicted secondary structure of f16 and the crystal structure of resolvases of the ser recombinase superfamily (fig. 1) . strikingly, however, most of the individual conserved amino acid residues of the resolvases/ recombinases have matches only in the crv protein but not in the other poxvirus f16 sequences. specifically, all 8 amino acid residues that comprise the catalytic site of the ser recombinases, namely tyr6, arg8, ser10, gln14, gln19, asp36, arg68, and arg71 (using the amino acid numbering of tn3 resolvase which is nearly identical to res_1gdt included in the alignment in fig. 1 ) are conserved in the crv protein (with the substitution of glu for asp36 that is found in many ser recombinases), whereas only two, ser10 and gln19, are represented in the rest of the f16 proteins ( fig. 1) . although ser10 is the main catalytic residue of ser recombinases that is directly responsible for the nucleophilic attack on the phosphodiester bonds in the target dna, the other residues in the catalytic site, in particular the three conserved arg, are also essential for catalysis stark, 2009, 2010) . given the concerted loss of 6 of the 8 catalytic residues, and in particular the three essential arg residues, it can be confidently predicted that f16 proteins are inactivated derivatives of ser recombinases that possess no enzymatic activity. thus, whatever the function of these proteins in poxvirus reproduction might be, it does not involve site-specific recombination. most of the ser recombinases contain a dna-binding helix-turn-helix domain to the c-terminus (or less commonly to the n-terminus) of the catalytic domain. the approximately 100 amino acid region following the recombinase homology domain of f16 does not show detectable similarity to any helix-turn-helix domains, although it is predicted to adopt a primarily alpha-helical fold (not shown). this unique domain might mediate functionally important protein-dna and/or protein interactions. the conservation of f16 among chordopoxviruses suggested that it had an important function. yet the likelihood that the protein lacks ser recombinase activity left us with no clue as to what that function might be. our first question was whether f16 was essential for vacv replication. we succeeded in knocking out the f16l gene by replacing it with the enhanced green fluorescent protein (egfp) gene under the control of the synthetic early/late vacv promoter (chakrabarti et al., 1997) . green fluorescent plaques formed and the knockout virus vδf16 was plaque purified, suggesting that the function of f16 is nonessential at least in bs-c-1 cells used for its isolation. plaques produced by vδf16 were compared with wild type (wt) virus on several conventionally used cell lines (bs-c-1, rk13, hela, and a543) as well as on two primary cell cultures, chick embryo fibroblasts and human foreskin fibroblasts (hffs). no difference was detected in the plaque size or appearance of the mutant and the wt virus on any of the tested cells ( fig. 2 and data not shown). furthermore, no differences in the yields of vδf16 and wt virus were found in a single round of infection of bs-c-1, hela, and rk13 cells ( fig. 3a and data not shown). based on our recent studies with the vacv dna ligase (paran et al., 2009) , we considered that the f16 protein might have a more critical role in resting cells. replicate cultures of human foreskin fibroblasts cells were cultivated for 5 days in 0.2% serum to achieve quiescence, which was confirmed by a decrease in the amount of the cell cycledependent dna ligase i transcript, and infected with a low multiplicity of each virus. virus yields were determined over a 48 h period. however, no difference was found in the rate of formation or amount of wt and mutant viruses (fig. 3b ). the viral protein selection consists of one representative of 9 of the 10 chordopoxvirus genera that encode this protein (with the exception of avipoxviruses). the ser recombinase selection consists of four sequences of putative resolvases and recombinases with the greatest similarity to f16 and three sequences for representative crystal structures. the secondary structure lines show the structural elements for f16 proteins that were predicted using the psipred program (buchan et al., 2010) and the structural elements extracted from the 1gdt structure; e indicates extended conformation (beta-strand) and h indicates alpha-helix. asterisks above and beneath the alignment indicate invariant amino acid residues in the f16 sequences and in the selected ser recombinase sequences, respectively. the catalytic residues of ser recombinases and the matching residues in the poxvirus sequences are shaded gray. the catalytic serine is shown by white type on black background. the alignment was constructed using the mofft program (katoh et al., 2005) and slightly manually adjusted to improve the alignment of secondary structure elements. the poxvirus sequences are denoted by an abbreviated virus name followed by the gene name; the ser recombinase sequences are denoted by the abbreviated species name followed by abbreviated protein annotation; for the sequences with resolved crystal structures, the pdb accession numbers are indicated. for each protein, the number of the first and last amino acid residues in the aligned region is indicated before and after the sequence, respectively. the orthologs of f16 are 231 amino acids long in all orthopoxviruses and 209 to 230 amino acids in other genera. abbreviations (genbank accession number is given in parentheses): vac_wr, vacv, strain wr (yp_232937.1); mcv, molluscum contagiosum virus (np_043980.1); sqpox, squirrel poxvirus (abd51455.1); dpv, deerpox virus (yp_002302379.1); lsdv, lumpy skin disease virus (np_150464.1); myx, rabbit myxoma virus (acb28648.1); yaba, yaba monkey tumor virus (np_938286.1); orf, orf virus (np_957793.1); crpv, crocodilepox virus (yp_784241.1); ostri, oscillochloris trichoides (zp_07686770.1); spthe, sphaerobacter thermophilus (yp_003320473.1); syaci, syntrophus aciditrophicus (yp_462099.1); niham, nitrobacter hamburgensis (yp_579027.1); tp901-1, lactococcus phage tp901-1; res_1gdp, gamma-delta resolvase (from e. coli transposon); sulso, sulfolobus solfataricus (for the last three sequences, the pdb accession numbers are indicated instead of the genbank accession numbers); res, resolvase; rec, recombinase. inspection of the sequence immediately upstream of the f16 gene suggested the presence of an early promoter and the absence of a late promoter moss, 1989a, 1989b) . indeed, the f16 gene was expressed early in infection as shown by the accumulation of the f16 protein containing a 3xflag tag on the c-terminus (fig. 4 ). f16-3xflag was detected by western blotting at 2 h after infection, only slightly increased in amount between 4 and 24 h, and accumulated in the presence of cytosine arabinoside (arac), an inhibitor of dna synthesis that prevents vacv intermediate and late gene expression. as a control, the blot was probed with antibody to the wellcharacterized late 4b core protein and its precursor p4b. these proteins were detected between 6 and 8 h after infection and were not seen in the presence of arac (fig. 4) . f16-3xflag was detected in virions purified by sucrose gradient centrifugation following two sucrose cushions. as shown in fig. 4 , the ratio between f16 in virus particles and the 24 h cell extract was only slightly lower than the ratio for the abundant virion protein p4b/4b, suggesting that f16 is a bona fide virion protein. vac proteins are generally considered to be present in the core or membrane fraction of virus particles depending on their ability to be extracted from purified virions by non-ionic detergents in the presence or absence of reducing agent. we compared the extraction of f16-3xflag with well-characterized core (4b) and membrane (l1) proteins. f16-3xflag was mainly in the insoluble fraction, like 4b and unlike l1, during different treatments and was thus classified as a core protein (fig. 5 ). most vacv virion proteins are expressed after viral dna replication and are present predominantly in cytoplasmic factories, which are the sites of viral dna, rna and protein synthesis as well as fig. 2 . comparison of plaques produced by wt vacv and vδf16. monolayers of bs-c-1 african monkey kidney cells, primary hff cells and primary chick embryo fibroblasts (cef) were infected with wt vacv or vδf16. after 48 h, the cells were stained with crystal violet (bs-c-1 cells) or antibody to vacv followed by a secondary antibody conjugated to horseradish peroxidase. at each time point, aliquots were lysed with sodium dodecyl sulfate and reducing agent. in addition, virus particles were purified from dounce homogenates by sedimentation through two successive sucrose cushions and one sucrose gradient. the proteins from the lysates and purified virions were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a membrane. the blot was probed with antibody to the flag epitope tag of f16 and to the late core protein 4b and its precursor p4b. antibodies were detected by chemiluminescence. fig. 5 . fractionation of proteins from purified vf16-3xflag virions. virus particles were purified as described in the legend to fig. 4 and treated with np40 detergent with or without dithothreitol (dtt), nacl, and deoxycholate (doc) as indicated by plus and minus signs. the suspensions were centrifuged and the supernatant (s) and pellet (p) fractions were analyzed by western blotting with antibodies to the flag epitope of f16, the 4b core protein and its precursor p4b, and to the l1 membrane protein. virion assembly (katsafanas and moss, 2007) . f16, however, was expressed starting early in infection, before viral dna synthesis and factory formation. unexpectedly, in non-confluent cells before 8 h of infection f16 was detected in the nuclei, where it was concentrated in the nucleoli and to some extent on the nuclear membrane in addition to the cytoplasm (fig. 6a) . f16 was detected in virus factory regions, presumably accounting for its incorporation into virions, but was not concentrated there (figs. 6a and c). localization in the nucleoli also occurred when cells were infected in the presence of the dna replication inhibitor arac (fig. 6b ). in confluent cells and at later times after infection, f16 was distributed throughout the cell but there was still some nucleoplasmic staining ( fig. 6c and data not shown) . a similar inverse correlation between localization in nucleoli and density of cell monolayer has been observed for the nucleoprotein np of influenza virus (bui et al., 2002) . furthermore, when a human codon optimized copy of the f16 gene under the control of the cytomegalovirus (cmv) immediate early promoter was expressed at relatively low levels in nonconfluent, uninfected cells, nucleoli targeting also occurred (figs. 6d and e). two other vac proteins, i3 and b1, containing a c-terminal 3xflag tag and expressed from a transfected plasmid under the control of the cmv promoter were analyzed in parallel with f16-3xflag, and no nucleolar or nuclear membrane staining was detected (not shown). the association of f16 with nucleoli was confirmed by fig. 6 . intracellular localization of f16-3xflag in infected and uninfected cells. (a-c) hela cells infected with vf16-3xflag for 7 h. a, infected non-confluent hela cells; b, same in the presence of arac; c, infection as in panel a but cells were confluent. in panels a, b and c, f16 was stained with anti-flag monoclonal mouse antibody followed by a secondary antibody conjugated with alexafluor 647 and appears red; dna stained with dapi appears blue. white arrowheads point to cytoplasmic viral dna factories. (d-g) uninfected hela cells transfected with plasmid encoding f16-3xflag. d and e, relatively low level expression in non-confluent cells; f and g, high level of expression in non-confluent and confluent cells, respectively. in d, e, f and g, f16 was stained with an anti-flag polyclonal rabbit antibody followed by a secondary antibody conjugated with alexafluor 488 and appears green; in d, nucleolin was stained with an anti-nucleolin polyclonal rabbit antibody followed by a secondary antibody conjugated with alexafluor 647 and appears red; in e, nucleophosmin was stained with an anti-nucleophosmin monoclonal mouse antibody followed by a secondary antibody conjugated with alexafluor 647 and appears red; in f and g, dapi staining appears blue. colocalization with nucleolin/c23 (fig. 6d ) and nucleophosmin/b23 (fig. 6e) . different anti-flag antibodies were used in the latter doublestaining experiments, namely an anti-flag mouse monoclonal antibody with an anti-nucleolin rabbit polyclonal antibody and a rabbit polyclonal anti-flag with mouse monoclonal anti-nucleophosmin antibody. thus, two independent anti-flag antibodies detected the same staining pattern for f16, corroborating the specificity. as in infected cells, the localization of f16 depended to a great extent on the level of expression in an individual cell. in transfected cells with high f16 expression level, either the entire cell or only the cytoplasm was stained with flag antibody (figs. 6f and g) . the same pattern of f16 accumulation was detected in hela cells, bs-c-1 and rk13 cells infected with vf16-3xflag or transiently transfected with a plasmid expressing f16-3xflag from the cmv promoter (not shown). invariant ser8 and 11 are not required for targeting f16 to nucleoli all poxvirus orthologs of f16 share the invariant ser11 that aligns with the catalytic ser10 of the ser recombinases (fig. 1) . the striking conservation of this ser in all poxvirus orthologs in spite of the absence of other catalytic residues could suggest an important function, e.g., phosphorylation required for nucleolar targeting. similarly, ser8 is conserved in all poxvirus f16 orthologs except for crv. to assess the potential importance of ser11 and ser8 for the targeting of f16 to the nucleoli, two mutants were constructed in which ser11 or ser8 was replaced by ala. however, no difference in nucleoli targeting was observed after transfection of the corresponding plasmids into cells (not shown). thus, these conserved serine residues are not required for the targeting of f16 to the nucleoli. the f16 protein does not contain known nuclear and/or nucleolar targeting signals (emmott and hiscox, 2009) . in order to determine which part of the protein might be responsible for targeting to nuclei and nucleoli, we investigated the localization of a truncated mutant of f16 as well as the n-terminal and c-terminal domains of f16 expressed separately. in the truncated mutant of f16 (t), the 21 cterminal amino acids were removed (fig. 7a) . this region includes a predicted alpha-helix that is conserved in the chordopoxvirus f16 homologs (not shown) and potentially could be involved in proteinprotein interactions. the t mutant was still targeted to the nucleoli, but showed much less cytoplasmic staining than the full-length protein (fig. 7b ). in addition, when highly expressed, the t mutant exhibited a characteristic halo that lined the inner side of the nuclear membrane (fig. 7b) . the n-terminal domain, which is homologous to ser recombinases, and the unique c-terminal domain of f16 were not targeted to the nucleoli under any conditions. the n-terminal domain was distributed throughout the cell but was specifically excluded from the nucleoli, whereas the c-terminal domain was localized exclusively to the cytoplasm (fig. 7b) . these results suggest that for the nucleolar targeting of f16 interaction between structural elements located in the n-terminal and c-terminal domains might be required. as nucleoli are the sites of rrna synthesis, we investigated whether expression and nucleolar localization of f16 reduced r rna synthesis. cells were transfected with f16-3xflag expressed from the cmv promoter, incubated for 48 h during the last 3 h of which the uridine analog 5-ethynyluridine (eu) was present (jao and salic, 2008) . following fixation, f16-3xflag was detected by antibody and the labeled rna was discerned by "click" chemistry, a coppercatalyzed covalent reaction between azide and alkyne. a comparison of f16 transfected and non-transfected cells in the same field did not suggest decreased rrna synthesis (fig. 8) . the absence of nuclear/nucleolar targeting signals suggested to us that interaction of f16 with a cellular protein might modulate its intracellular localization. attempts to show an interaction of f16 with cellular nucleolin or nucleophosmin by coimmunopurification, however, were unsuccessful (tgs, unpublished). however, we did find that f16 forms multimers by cotransfecting cells with plasmids expressing f16-3xflag and f16-v5. both tagged versions of f16 coimmunopurified with each other following incubation with antiflag or anti-v5 antibody as shown by western blotting (fig. 9) . these results indicate that f16 exists as dimers or higher order multimers. the homology between f16 and ser recombinases has interesting evolutionary implications. the crv homolog of f16 is the first likely ser recombinase to have been detected in any eukaryote or eukaryotic fig. 8 . rna synthesis in hela cells transfected with f16-3xflag. cells were transfected for 48 h and incubated with the uridine derivative eu for the last 3 h. f16 was stained with an anti-flag polyclonal rabbit antibody followed by a secondary antibody conjugated with alexafluor 488 and appears green; dapi staining appears blue; rna containing 5-ethynyl uridine (eu) was stained with alexafluor 647 azide and appears red. a merge is shown in the lower right box. fig. 9 . formation of f16 multimers. hela cells were cotransfected with plasmids expressing f16-3xflag and f16-v5 tag or with f16-3xflag and a control plasmid or f16-v5 and a control plasmid. cytoplasmic extracts were immunopurified on agarose beads with anti-flag or anti-v5 antibodies. agarose-bound proteins were analyzed by sdspolyacrylamide gel electrophoresis followed by western blotting with anti-flag or anti-v5 antibodies. virus (afonso et al., 2006) . thus, the most parsimonious evolutionary scenario involves acquisition of a ser recombinase gene by the ancestor of chordopoxviruses from a transposon or a bacteriophage. this scenario is reminiscent of other probable bacterial or bacteriophage contributions to the evolution of poxviruses, in particular, the holiday junction resolvase a22 (garcia et al., 2000) and the predicted deubiquitinase g6 (senkevich et al., 2008) as well as the primasehelicase d5 and the dna-packaging atpase a32 that are shared with other nucleocytoplasmic large dna viruses (koonin and yutin, 2010) . the subsequent evolution of chordopoxviruses apparently involved the abrogation of the recombinase activity of f16, as evidenced by the substitution of key amino acids in the active site region. this loss of activity occurred after the divergence of crv [the deepest chordopoxvirus branch (afonso et al., 2006; koonin and yutin, 2010) ] from the common ancestor with the other chordopoxviruses but prior to the radiation of the latter, and the complete loss of this gene in the avipoxvirus branch. the loss of recombinase activity was apparently accompanied by an overall acceleration of evolution and apparently a change of function. assuming the latter scenario, what is the acquired new function of the poxvirus f16 homologs? given its conservation and early expression shown here, a role in viral dna replication or recombination seemed one possibility. however, knockout of the f16l gene had no measurable effect on virus growth in any of the many tested cell cultures including resting primary hffs. another gene with similar biological features is the g6r gene that is conserved in all poxviruses and encodes a putative deubiquitinating enzyme (senkevich et al., 2008) . knockout of the g6r gene has no effect on virus growth in all tested cell cultures and leads only to a slight attenuation in intranasal mouse infection. conservation of these genes in most poxvirus genera suggests that their functions are not directly involved in the virus growth cycle but may be involved in generic aspects of virus-host interaction as opposed to interactions between viruses and their specific hosts. the targeting of the f16 protein to the nucleoli, whether expressed by vacv or by transfection in uninfected cells, was unexpected. however, the myxoma virus ankyrin repeat protein m148r, which also lacks a defined nucleolar localization motif, has been reported to localize in the nucleolus (blanie et al., 2009) . deletion of m148r does not perturb replication in vitro but reduces virulence in a rabbit model. another vacv protein, e3l, has been detected in the nuclei of infected cells but its nuclear function (if any) has not been characterized (yuwen et al., 1993) . although f16 had a 3xflag tag, the latter is unlikely to cause nuclear or nucleolar targeting as the vacv proteins i3 and b1, containing a c-terminal 3xflag tag and expressed from a transfected plasmid under the control of the cmv promoter, were analyzed in parallel with f16-3xflag and no nucleolar or nuclear membrane staining was detected (tgs, unpublished). moreover, the 3xflag has been used in numerous studies of subcellular localization of proteins, and no specific effect on nuclear and/or nucleolar targeting has been reported. in particular, the 3xflag tag was used in a genome wide screen of herpesviruses for protein subcellular localization, with no specific effects on protein targeting (salsman et al., 2008) . the amino acid sequence of f16 does not contain known nuclear or nucleoli targeting signals (emmott and hiscox, 2009 ) and our studies indicated that both the n-and c-terminal regions of f16 are required for localization to nucleoli. a likely possibility is that f16 targets the nucleus and nucleoli by interacting with a cellular protein. thus far, we have been unable to detect an interaction of f16 with cellular nucleolin or nucleophosmin by coimmunopurification (tgs, unpublished) but interactions with other cellular proteins remain a possibility. we did find that f16 interacts with itself to form homodimers or higher order multimers. if there is competition between f16 binding to itself and to a cell protein, this could explain the inverse correlation of nucleolar targeting with the level of expression and confluency of cells. conditions in which the concentration of f16 is increased (high expression) or the cell protein is decreased (confluency), might favor multimer formation and cytoplasmic localization. targeting of viral proteins to nucleoli is a frequent theme in the study of virus-host interactions. for example, a genome-wide screen of three distinct herpesviruses has shown that at least 12 herpesvirusencoded proteins specifically localize to the nucleolus (salsman et al., 2008) . targeting of viral proteins to nucleoli has been described not only for dna viruses such as herpesviruses, adenoviruses, and papovaviruses, and rna viruses, such as retroviruses and influenza, that possess a defined nuclear stage of reproduction, but also for proteins of rna viruses without any nuclear stage such as coronaviruses, arteriviruses and filoviruses as well as several groups of rna viruses of plants (hiscox, 2007; hiscox et al., 2010; taliansky et al., 2010) . concomitantly, it has become evident than nucleoli are not only the factories of ribosome biosynthesis, as previously thought, but multifunctional and dynamic structures that contain hundreds of cellular proteins and are involved in a variety of signaling pathways, including cell cycle control, regulation of cell division, apoptosis, antiviral defense, cancer, and more (boisvert et al., 2007; sirri et al., 2008) . for many viruses, trafficking of viral proteins through nucleoli is an important step in their growth cycle, and accordingly, the viral proteins directed to nucleolus are essential for the viral replication in cell culture. for example, herpesvirus and human immunodeficiency virus proteins that are targeted to the nucleolus have been implicated in viral mrna processing (hiscox et al., 2010) . several proteins of adenoviruses and herpesviruses cause redistribution of nucleolar components, reorganization of the nucleolus and interference with nucleolar function, including disruption of rrna synthesis, processing and trafficking (hiscox et al., 2010) . vacv affects various host cell processes, in particular translation is shut down at least partly through decapping of host mrnas (parrish et al., 2007) and by localization of the translation apparatus within viral factories (katsafanas and moss, 2007) . host transcription is impaired by mechanisms that have not been well characterized (puckett and moss, 1983 ). jefferts and holowczak (jefferts and holowczak, 1971) reported decreased nucleolar rna synthesis in vacv-infected l cells and detected peptide from 14 c-labeled virions in nucleoli. in our studies, the f16 protein was specifically targeted to the nucleoli of transfected hela cells but it had no obvious effect on rrna synthesis as determined by eu incorporation and click chemistry. in a preliminary experiment, we did not see greater eu labeling of hela cell nucleoli in cells infected with the f16 deletion mutant compared to wt virus (tgs, unpublished) . thus, f16 may affect another, perhaps signaling functions of the nucleoli. further studies of this phenomenon could reveal an additional layer of manipulation of host cell functions by poxviruses. standard procedures for preparation and maintenance of hela and bs-c-1 cells originally obtained from the american type culture collection (manassas, va) and propagation, titration and purification of vacv were used (earl et al., 1998a (earl et al., , 1998b . primary hffs were provided by a. mcbride (national institute of allergy and infectious diseases, bethesda, md), primary chick embryo fibroblasts were prepared from 10-day-old embryos and used in the first passage. on monolayers of bs-c-1 cells, vacv produced clear plaques that were visible after staining with crystal violet; on other cell monolayers, where plaques were less easily discerned, infected cell foci were detected by immunostaining with anti-vacv antibody, followed by protein a conjugated to horseradish peroxidase (carroll and moss, 1997) . recombinant viruses were derived from the wr strain (atcc vr-1354). vδf16 and vf16-3xflag were constructed by inserting a universal dna fragment containing the egfp gene preceded by vacv early/late synthetic promoter ctaaaaattgaaattttattttttttttttggaata-taa via homologous recombination into the desired place in the vacv genome. the universal fragment (e/l-gfp) was surrounded on both sides by 500 nt flanks to allow homologous recombination into the specific site. all dna pieces were assembled by overlapping pcr and transfected into bs-c-1 cells infected with 1-2 pfu/cell of vacv. after 24 h, lysates were prepared, new bs-c-1 cell monolayers were infected and green fluorescent plaques of recombinant viruses were isolated and clonally purified. in vδf16, the coding sequence of the f16 gene excluding 19 nucleotides from the 3′ end was replaced by the e/l-gfp cassette. replacement of f16 by gfp was confirmed using pcr primers that flanked f16; the size of the product was that predicted. in vf16-3xflag the sequence encoding the 3xflag gactacaaagaccat-gacggtgattataaagatcatgacatcgattacaaggatgacgatga-caagtga was linked to the last codon of f16, followed by the e/l-gfp cassette and then by the last 7 nucleotides of the f16 orf including its stop codon. the 3′-end nucleotides of f16 orf were kept to prevent disruption of the downstream f15l gene promoter. duplicate wells containing cells in 24 well plates were infected with virus for 1 h, washed 3 times with medium, incubated for different times at 37°c and harvested. infected cells were lysed by 3 freezethaw cycles, sonicated, and virus titers were determined by plaque assay on bs-c-1 cells in semi-liquid 0.5% methylcellulose medium. total cell extracts in sds-page loading buffer were sonicated to reduce viscosity and proteins were analyzed on nupage gels (invitrogen, carlsbad, ca) and transferred to nitrocellulose membrane. membrane was incubated with antibodies in 5% nonfat milk in phosphate buffered saline (pbs)-0.1% tween-20 for different times at room temperature or 4°c, then secondary horseradish peroxidaseconjugated antibodies, and the blot was developed with supersignal west pico, dura or femto chemiluminescent substrates (pierce, rockford, il) depending on hemiluminescence intensity. a copy of f16-3xflag gene optimized for expression in human cells was synthesized by geneart (regensburg, germany) and cloned into pcdna3.1(+) vector (invitrogen), which contains the cmv immediate early promoter. mutations and deletions were constructed by overlapping pcr and cloned into the same vector. hela cells were transfected with plasmid dna using lipofectamine 2000 (invitrogen) according to the protocol of the manufacturer and analyzed after 48 h. as controls, plasmids containing two additional vac proteins, i3 and b1, containing a c-terminal 3xflag tag under the control of the cmv promoter were constructed antibodies all commercially available primary antibodies were from sigma (st. louis, mo). for confocal microscopy, anti-flag m2 mouse monoclonal, anti-flag rabbit polyclonal, anti-nucleolin rabbit polyclonal and anti-b23 mouse monoclonal antibodies were used followed by alexafluor 488 or 647 goat anti-mouse or goat anti-rabbit antibodies (invitrogen). for western blotting horseradish peroxidase conjugated m2 mouse monoclonal antibody and polyclonal rabbit antibodies against vacv proteins l1 (r180), provided by g. cohen and r. eisenberg (university of pennsylvania) and p4b/4b (r. doms and b. moss, unpublished) were used followed by secondary horseradish peroxidase conjugated antibodies. immunoprecipitations bs-c-1 cells, in 12-well dishes, were cotransfected with plasmids expressing f16-3xflag and f16-v5 or cotransfected with each plasmid and a vector plasmid. after 48 h of incubation at 37°c, cells were disrupted in pbs-0.5% np40. cytoplasmic extracts were preincubated with igg beads for 1 h at +4°c and one-half of each extract was loaded on agarose beads with covalently bound anti-flag antibody (sigma) or anti-v5 antibody (sigma) and incubated overnight at +4°c with constant rotation. agarose beads were washed 4 times with pbs-0.5% np40, proteins were eluted in sds-polyacrylamide gel loading buffer and resolved in a sds 10% polyacrylamide gel, transferred to a nitrocellulose membrane and incubated with horse radish peroxidase-conjugated anti-flag antibodies (sigma), then the membrane was stripped and incubated with horse radish-conjugated anti-v5 antibody (invitrogen). for detection of cellular rna by confocal microscopy, the click-it kit rna imaging kit with alexa floor 594 azide (invitrogen) was used. hela cells were transfected and incubated with uridine derivative eu for the last 3 h of a 48 h incubation and then fixed and stained with alexa fluor 594 azide according to the manufacturer's protocol. hela cells on coverslips were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% triton x-100 in pbs for 10 min, incubated with primary antibodies in pbs-5% fetal calf serum for 1-18 h at room temperature or 4°c, stained with secondary alexa fluor antibodies for 1 h followed by dapi staining for 10 min and mounted on slides with prolong gold antifade reagent (invitrogen). a leica sp2 inverted four-channel microscope was used for imaging. the search of the non-redundant protein sequence database (ncbi, nih, bethesda) was performed using the psi-blast program with the conditional compositional score matrix adjustment (altschul et al., 1997 (altschul et al., , 2005 . additional search of protein sequence databases was performed using the profile hidden markov model approach implemented in the hhsearch program (soding, 2005) . protein secondary structure was predicted using multiple alignments as implemented in the psipred program (mcguffin et al., 2000) . genome of crocodilepox virus gapped blast and psi-blast: a new generation of protein database search programs protein database searches using compositionally adjusted substitution matrices m148r and m149r are two virulence factors for myxoma virus pathogenesis in the european rabbit the multifunctional nucleolus protein annotation and modelling servers at university college london nucleo-cytoplasmic localization of influenza virus nucleoprotein depends on cell density and phosphorylation host range and cytopathogenicity of the highly attenuated mva strain of vaccinia virus: propagation and generation of recombinant viruses in a nonhuman mammalian cell line compact, synthetic, vaccinia virus early/late promoter for protein expression the structure of vaccinia virus early promoters the structure of vaccinia virus late promoters preparation of cell cultures and vaccinia virus stocks generation of recombinant vaccinia viruses nucleolar targeting: the hub of the matter bacterial-type dna holliday junction resolvases in eukaryotic viruses mechanisms of site-specific recombination rna viruses: hijacking the dynamic nucleolus nucleolar proteomics and viral infection exploring rna transcription and turnover in vivo by using click chemistry rna synthesis in vaccinia-infected l cells: inhibition of ribosome formation and maturation mafft version 5: improvement in accuracy of multiple sequence alignment colocalization of transcription and translation within cytoplasmic poxvirus factories coordinates viral expression and subjugates host functions origin and evolution of eukaryotic large nucleocytoplasmic dna viruses the psipred protein structure prediction server genetically engineered poxviruses for recombinant gene expression, vaccination, and safety poxviridae: the viruses and their replication the catalytic residues of tn3 resolvase catalysis of site-specific recombination by tn3 resolvase cellular dna ligase i is recruited to cytoplasmic vaccinia virus factories and masks the role of the vaccinia ligase in viral dna replication vaccinia virus d10 protein has mrna decapping activity, providing a mechanism for control of host and viral gene expression selective transcription of vaccinia virus genes in template dependent soluble extracts of infected cells genome-wide screen of three herpesviruses for protein subcellular localization and alteration of pml nuclear bodies a conserved poxvirus nlpc/p60 superfamily protein contributes to vaccinia virus virulence in mice but not to replication in cell culture nucleolus: the fascinating nuclear body diversity in the serine recombinases protein homology detection by hmm-hmm comparison involvement of the plant nucleolus in virus and viroid infections: parallels with animal pathosystems poxvirus orthologous clusters: toward defining the minimum essential poxvirus genome nuclear localization of a double-stranded rna-binding protein encoded by the vaccinia virus e3l gene we thank catherine cotter and george katsafanas of our laboratory for cell culture and helpful discussions on confocal imaging, respectively. dr. sundar ganesan of the biological imaging facility of the research technologies branch, niaid provided help with confocal microscopy. the study was supported by the division of intramural research, national institute of allergy and infectious diseases, national institutes of health. key: cord-254747-vox5xsgd authors: deng, xufang; baker, susan c title: an “old” protein with a new story: coronavirus endoribonuclease is important for evading host antiviral defenses date: 2018-04-01 journal: virology doi: 10.1016/j.virol.2017.12.024 sha: doc_id: 254747 cord_uid: vox5xsgd here we review the evolving story of the coronavirus endoribonuclease (endou). coronavirus endou is encoded within the sequence of nonstructural protein (nsp) 15, which was initially identified as a component of the viral replication complex. biochemical and structural studies revealed the enzymatic nature of nsp15/endou, which was postulated to be essential for the unique replication cycle of viruses in the order nidovirales. however, the role of nsp15 in coronavirus replication was enigmatic as endou-deficient coronaviruses were viable and replicated to near wild-type virus levels in fibroblast cells. a breakthrough in our understanding of the role of endou was revealed in recent studies, which showed that endou mediates the evasion of viral double-stranded rna recognition by host sensors in macrophages. this new discovery of nsp15/endou function leads to new opportunities for investigating how a viral endou contributes to pathogenesis and exploiting this enzyme for therapeutics and vaccine design against pathogenic coronaviruses. we provide a brief outline of the major research findings related to coronavirus (cov) endoribonucleases (endou) in table 1 . in the text below, we describe the experimental approaches that led to these findings and compare the activity of cov endou with reports of other viral and host ribonucleases. initial studies focused on identifying the products of the cov replicase polyprotein, pp1ab (depicted in fig. 1a ). heusipp et al. used a murine monoclonal antibody (10g11) that recognizes amino acid residues 6158-6164 of human cov 229e pp1ab (heusipp et al., 1997) . they identified a 41-kda polypeptide in virus-infected cells and found that this polypeptide was a product of the viral 3c-like proteasemediated cleavage of pp1ab. this protein localizes to the perinuclear region, as detected by immunofluorescence assay, similar to other pp1ab-derived polypeptides (heusipp et al., 1997) . shi and coworkers obtained similar findings while studying mouse hepatitis virus (mhv) (shi et al., 1999) . they generated a rabbit antiserum against amino acids 6679-6821 of mhv pp1ab and found that this antiserum detected a 35-kda product in infected cells. they also found that this protein colocalized with de novo synthesized viral rna, and therefore postulated that this viral protein associated with the viral rna replication/ transcription machinery (shi et al., 1999) . later, the corresponding polypeptides that these antibodies recognized were defined as the 15th cleavage product of pp1ab (called nsp15), counting from the aminoterminus to the carboxyl terminus of pp1ab (ziebuhr et al., 2000; snijder et al., 2003) . after the outbreak of severe acute respiratory syndrome (sars) in [2002] [2003] , and once a cov had been confirmed as the etiological agent of sars, researchers intensively scrutinized cov genomic sequences to better understand this novel human pathogen. by comparative genomic characterization of cov replicases, snijder et al. reported that the c-terminus of nsp15 has high sequence similarity to the xenopus laevis poly(u)-specific endoribonuclease and therefore predicted that nsp15 possesses endou activity (snijder et al., 2003) . based on the available sequence information of viruses from the order nidovirales at that time, the endou was considered a nidovirus-specific marker (called nendou) (fig. 1b) (snijder et al., 2003) . the members of the family arteriviridae, including equine arteritis virus (eav) and porcine respiratory and reproductive syndrome virus (prrsv), also have endou domains within nsp11. however, it was later discovered that the presence of the endou domain is not universal in all nidoviruses. nam dinh virus, the first insect nidovirus belonging to the family mesoniviridae, and roniviruses that infect invertebrates, do not encode an endou domain (nga et al., 2011; lauber et al., 2012) . these findings suggest that the endou domain may only serve as a signature for vertebrate nidoviruses, including covs and arteriviruses (fig. 2 ). previous reviews have comprehensively summarized the biochemical and structural features of nidovirus ribonucleases (ulferts and ziebuhr, 2011; snijder et al., 2016) . here, we highlight the key experiments with respect to nidovirus endou and provide recent updates. studies performed by ivanov et al. (2004) and bhardwaj et al. (2004) first demonstrated the endou activity of sars-cov nsp15 in vitro (ivanov et al., 2004; bhardwaj et al., 2004) . the wild-type (wt) nsp15 and its mutants with alanine (ala) substitutions of the putative catalytic residues were expressed in e. coli. the recombinant nsp15-wt, but not the mutants, could efficiently cleave single-stranded (ss) and doublestranded (ds) rnas. in contrast, neither ssdna nor dsdna molecules could be processed by nsp15, demonstrating its predicted ribonuclease-as opposed to deoxyribonuclease-activity. blocking either the 5′ or the 3′ terminus of the rna substrates by covalent modifications with fluorescein or puromycin, respectively, had no effect on the rna cleavage (bhardwaj et al., 2004) , confirming that nsp15 is an endorather than an exoribonuclease. similar endou activity was detected in other cov nsp15s, including human cov 229e, mhv, infectious bronchitis virus (ibv), and turkey cov (ivanov et al., 2004; bhardwaj et al., 2004; cao et al., 2008) , and in the nsp11s of arteriviruses eav and prrsv (nedialkova et al., 2009) . hexamerization of nsp15 is critical for its endou activity. guarino and coworkers found that cov nsp15 could be present in solution as either monomers or hexamers in a protein concentration-dependent manner. the hexamer is the fully active form of endou that binds rna and executes optimal endou activity (guarino et al., 2005) . the residues in the n-terminal domain of nsp15 are critical for hexamer formation (guarino et al., 2005) . in addition, nsp15 requires divalent metal ions as a co-factor for rna cleavage and prefers mn 2+ over mg 2+ or other divalent cations (ivanov et al., 2004; bhardwaj et al., 2004) . addition of mn 2+ significantly affects the protein conformation, enhances rna binding, and increases endou activity (ivanov et al., 2004; bhardwaj et al., 2004) . in contrast to cov nsp15, arterivirus nsp11 forms dimers in solution and does not require divalent cations as a cofactor for activity in vitro (nedialkova et al., 2009; shi et al., 2016) . instead, a concentration of mn 2+ that greatly stimulated the activity of cov nsp15 inhibited the endou activity of eav and prrsv nsp11s. cov endou was revealed biochemically to hydrolyze the 3′ end of pyrimidines, with a preference for uridylates, and release products with 2′, 3′-cyclic phosphate and 5′-hydroxyl ends (ivanov et al., 2004; bhardwaj et al., 2004) . this finding seems to implicate a broad range of targets; however, the endou activity of cov nsp15 can be affected by secondary structure and modification of the rna substrate. bhardwaj et al. found that nsp15 preferentially cleaved unpaired uridylates in hairpin-structured rnas and that the neighboring nucleotides of targeted sites also influenced hydrolysis (bhardwaj et al., 2006) . on the other hand, ivanov et al. found that 2′-o-ribose methyl groups present on the substrate rna blocked endou-mediated cleavage (ivanov et al., 2004) . these data suggest that multiple factors might limit the range of endou targets. this is reasonable because the endou activity of nsp15 is likely to be tightly regulated during infection in cells to avoid unwanted cleavage on viral and/or cellular targets. for example, cov nsp16 is a 2′-o-ribose methyltransferase, whose function could theoretically block the endou-mediated cleavage of viral rnas. similar to cov nsp15, arterivirus nsp11 also prefers 3′ of uridylates for cleavage and yields products with 2′, 3′-cyclic phosphate ends (nedialkova et al., 2009) . further studies are needed to address whether the endou activity of arterivirus nsp11 is restricted by rna modifications or secondary structures. guarino and coworkers visualized single particles of sars-cov nsp15 using electron microscopy (guarino et al., 2005) and the same group later reported an 8.3 å structure of nsp15 by cryoelectron microscopy (bhardwaj et al., 2006) . they reported that the nsp15 hexamer comprises a dimer of trimers and proposed that the rna substrate binds to the inter-trimer interface. x-ray crystal structures of sars-cov and mhv nsp15s were first solved by ricagno et al. and xu et al., respectively (ricagno et al., 2006; xu et al., 2006) . these high-resolution structures define cov nsp15 as a separate endou family with unique folds that differ from cellular endoribonucleases (fig. 3a) . the monomer of sars-cov nsp15 consists of three domains: a small nterminal domain (ntd) (residues 1-62, cyan), a middle domain (residues 63-191, magenta), and a large c-terminal domain (ctd) (re-sidues192-345, green). the endou is located in the ctd. the monomeric structure of mhv nsp15 can be superimposed onto that of sars-cov nsp15, except for a flexible loop structure in the middle domain of mhv nsp15 (fig. 3b ). this flexible loop is encoded by a viral rna packaging signal sequence, which is present in mhv nsp15 but not in sars-cov nsp15 (kuo and masters, 2013) . these structural studies demonstrated that the presence of the flexible loop did not alter the overall folding of mhv nsp15 relative to sars-cov nsp15. in addition, through these and other structural studies (ricagno et al., 2006; xu et al., 2006; joseph et al., 2007; bhardwaj et al., 2008) , the nsp15 hexamer was again confirmed to be a dimer of trimers. as shown in fig. 3c , three monomers form a trimer and two trimers interact headto-head to form a hexamer. the ntds line up in the center of the hexamer, while the ctds face outward. this architecture allows the nsp15 hexamer to possess six active sites. the extensive contact between subunits through the ntd and middle domain is critical for hexamerization. the crystal structure of arterivirus nsp11 was also recently reported (shi et al., 2016; zhang et al., 2017) . these structural studies revealed that the monomer of nsp11 contains only two domains: ntd and ctd (no middle domain). the monomeric structures of cov nsp15 and arterivirus nsp11 could not be superimposed except in the ctd (catalytic domain). distinct from the hexameric structure of cov nsp15, nsp11 monomers assembled into an asymmetric dimer (shi et al., 2016) . virus-encoded endoribonuclease is a genetic signature of nidoviruses that infect vertebrates. a phylogenic tree of 32 representative nidoviruses was generated based on a conserved region of rdrp (sequnces and genbank assession numbers are available upon request). multiple sequence alignment and phylogeny analyses were conducted with the programs muscle and phyml, respectively (available at http:// www.phylogeny.fr/). the phylogenic tree was generated using dendroscope software version 3 with default parameters. virology 517 (2018) 157-163 since the crystallographic studies of cov nsp15 revealed that the active site of endou is structurally similar to that of rnase a, researchers evaluated small molecule inhibitors of rnase a for their ability to inhibit nsp15 activity. ortiz-alcantara et al. tested several commercially available rnase a inhibitors (benzopurpurin b, congo red, and others) and reported that their 50% inhibitory concentration (ic 50 ) values for inhibiting the endou activity of sars-cov nsp15 ranged from 0.2 μm to 40 μm. benzopurpurin b was shown to have a broad-spectrum activity and could inhibit nsp15 orthologs from mhv and ibv with ic 50 values of 0.4 μm and 0.2 μm, respectively (ortiz-alcantara et al., 2010). these rnase a inhibitors had variable effects on cov replication in cell cultures. in plaque formation assays, treatment with congo red resulted in 27-fold and 4-fold reductions in mhv and sars-cov titers, respectively, while benzopurpurin b led to marginal inhibition of both covs (ortiz-alcantara et al., 2010) . although the impact of these rnase a inhibitors on cov replication requires more comprehensive investigation, these early results suggest that the similarity between nsp15/endou and rnase a may provide a basis for exploiting small molecule inhibitors to modulate viral endou activity. due to its unique enzymatic activity and co-localization with the replicating viral rna, nsp15/endou was initially thought to play an important role in virus replication. however, mhv encoding catalyticdefective endou exhibited only a subtle defect in rna synthesis and a slight reduction in viral titers (~1 log) compared to wt virus when evaluated in fibroblasts (kang et al., 2007) . similar results were obtained for sars-cov and hcov-229e nsp15 mutants generated using either reverse genetics or replicon systems (ulferts and ziebuhr, 2011) . these data indicate that the endou activity of nsp15 is not essential for cov replication, as was initially proposed (snijder et al., 2003; ivanov et al., 2004; bhardwaj et al., 2004) . intriguingly, nsp15 may indirectly affect virus replication through other mechanisms. ivanov et al. demonstrated that when the conserved aspartic acid (asp)-298 of hcov-229e nsp15 was replaced with an ala, its endou activity was eliminated, viral rna synthesis was completely abolished, and no viable virus was recovered (ivanov et al., 2004) . similar phenotypes were observed for an mhv nsp15 mutant with an ala substitution of asp-324 (equivalent to asp-298 of hcov-229e) (kang et al., 2007) . it is unclear how the asp residues affect viral rna synthesis. kang et al. predicted that asp-324 is critical for an ionic-bond network and observed that the ala substitution resulted in an insoluble protein when mhv nsp15 was expressed in e. coli (kang et al., 2007) . these data suggest that the ala substitution may prevent the nsp15 protein from folding correctly. since the proteins adjacent to nsp15 are critical replicative components, any protein-folding issue with nsp15 may have an effect on the proteolytic processing of the neighboring components, thereby leading to a nonviable phenotype. overall, current evidence indicates that the endou activity of cov nsp15 is dispensable for viral rna synthesis and virus replication in cell culture. further work is required to address any non-endou-mediated role of nsp15 protein in virus replication. mutagenesis of arterivirus nsp11 revealed pleiotropic effects of endou on the viral life cycle (posthuma et al., 2006; sun et al., 2016) . similar to cov nsp15, mutations in the asp-3014 and asp-3038 residues (corresponding to mhv nsp15 asp-324 and asp-351, respectively) in eav nsp11 resulted in a nonviable phenotype. compared with the mild effect of mutating the catalytic histidine (his) residues (hi-s262ala and his277ala) of mhv nsp15, eav infectious clones with catalytic residue mutations (his2963ala/gln and his2978ala/gln) exhibited smaller plaque sizes, reduced rna synthesis, and dramatic titer reductions up to 5 log units. other substitutions at conserved, noncatalytic residues of eav nsp11 resulted in an intermediate phenotype: intermediate plaque sizes and~1-2 log reduction in titers. similar results were obtained with the prrsv nsp11 mutant viruses (sun et al., 2016) . these data indicate that arterivirus nsp11 may be involved in virology 517 (2018) 157-163 viral rna synthesis. however, similar to the asp324ala mutation of mhv nsp15, both asp3014ala and asp3038ala mutations in eav nsp11 rendered the protein insoluble, again raising the question of whether or not these mutations influence viral rna synthesis through interfering with proteolytic processing of the orf1b polyprotein. the absence of an endou domain in insect nidoviruses and invertebrate roniviruses further indicates that endou activity is not required for the unique rna synthesis strategy of nidoviruses (nga et al., 2011; lauber et al., 2012) . the aforementioned studies highlight the extensive efforts of the field to investigate the characteristics of nsp15/endou and its role in virus life cycle. the endou activity of cov nsp15 was found to play a non-essential role in viral rna synthesis and replication in immortalized fibroblast cells. recent work with different cell types and in vivo experiments revealed a novel function of nsp15/endou in virus replication and pathogenesis and provided a new direction of study with respect to this "old" protein. cov nsp15 was first suggested to possess interferon (ifn) antagonism capabilities through ectopic expression experiments. frieman et al. used an alphavirus replication-defective vector (vrp) to screen sars-cov proteins that suppress vrp-induced ifn responses. one of the identified ifn antagonists of sars-cov was nsp15 (frieman et al., 2009) . later, arterivirus nsp11 was also identified as an ifn antagonist and its endou activity was found to mediate inhibition of ifn-beta induction (beura et al., 2010; shi et al., 2011) . several other studies also reported that cov nsp15 and arterivirus nsp11 inhibit cellular innate responses in ectopic expression experiments (lei et al., 2009; wang et al., 2015) . these data seem to indicate that these two proteins function as ifn antagonists; however, the endou activity of overexpressed nsp15/nsp11 may unexpectedly affect the activities of the reporters used in these assays. we found that transfected mhv nsp15 reduced the signal of both ifn-reporter firefly luciferase and the internal control renilla luciferase (hackbart m, deng x, and baker s, unpublished data). a similar result was obtained with the overexpressed prrsv nsp11 (shi et al., 2016) . these observations imply that nsp15/nsp11 may execute non-specific cleavage when ectopically expressed. since cov nsp15 is part of the viral replicase/transcriptase complex (rtc), it is reasonable to predict that its endou activity is tightly regulated during viral infection to avoid unwanted cleavage. in line with this prediction, we and others reported a specific, punctate, perinuclear localization of cov nsp15 during viral infection (heusipp et al., 1997; shi et al., 1999; deng et al., 2017; athmer et al., 2017) , while ectopically expressed nsp15 was distributed throughout the cytoplasm (cao and zhang, 2012) . hence, we advise caution when interpreting the results of overexpression studies, as the nature of the endou activity was only revealed after studying nsp15/nsp11 in the context of viral infection. it was first discovered that the endou activity of nsp15 mediates the evasion of host recognition of viral dsrna by infecting primary macrophages with endou-deficient covs (deng et al., 2017; kindler et al., 2017) . infection with mhv endou-deficient mutants stimulated mouse bone-marrow derived macrophages (bmdms) to produce a remarkably high level of type i ifns during the early phase of infection compared to wt infection. this ifn response is mda5-dependent, as both the ifn mrna and protein levels were not elevated in mda5-deficient bmdms. moreover, the replication of endou-deficient covs was severely impaired in primary macrophages. interestingly, the ifn-induced antiviral response is not the only player responsible for this replication defect, as the titers of the endou-deficient covs were not completely restored in bmdms that lack critical genes (e.g. mda5, mavs, and irf3/5/7) involved in the ifn response (deng et al., 2017; kindler et al., 2017) . these data suggest that other antiviral pathways may also contribute to the observed replication defect. in support of this, it was found that the infection of endou-deficient covs also activate the pkr and the oas-rnase l pathways (deng et al., 2017; kindler et al., 2017) , which both execute potent antiviral functions, discussed further below. indeed, the replication of endou-deficient covs could be partially restored in pkr/ rnase l-double knockout cells (kindler et al., 2017) . replication was only fully restored in type i ifn receptor-knockout macrophages, as these cells not only have a defect in ifn signaling but also express very low basal levels of pkr and oas relative to wt cells (deng et al., 2017; birdwell et al., 2016) . taken together, these studies using live viruses in primary cells effectively illustrated the ifn antagonistic properties of cov endou. as mentioned above, infection with endou-deficient covs also activates the pkr and oas-rnase l pathways in macrophages (deng et al., 2017; kindler et al., 2017) . pkr is a dsrna-activated protein kinase and serves as a dsrna sensor. activated pkr phosphorylates eukaryotic initiation factor 2α (eif2α), resulting in inhibition of host and viral mrna translation. thus, pkr-mediated translation shutoff plays an important role in the host antiviral defense (barber, 2005) . the endou-deficient cov-infected macrophages exhibited increased levels of phosphorylated eif2α and decreased levels of translation (deng et al., 2017; kindler et al., 2017) , indicating that pkr was activated during infection. another piece of evidence of pkr activation was that endou-deficient covs induced rapid apoptotic cell death in infectedmacrophages (deng et al., 2017) . it has been shown that pkr-mediated translation shutoff leads to apoptosis in macrophages (hsu et al., 2004) . when the endou-deficient cov-infected macrophages were treated with a pkr-specific inhibitor (c16), the level of apoptosis was significantly reduced (deng et al., 2017) . this result further supports the hypothesis that endou-deficient covs activate pkr. interestingly, loss of pkr expression or inhibition of its activity only partially restored the replication of endou-deficient covs in macrophages (kindler et al., 2017) . moreover, treatment with the pkr inhibitor did not affect ifn induction or rnase l-mediated ribosomal rna degradation in the endou-deficient cov infected-macrophages (deng et al., unpublished data) . these results imply that the infection of endou-deficient cov activates multiple host dsrna sensors independently, including mda5, pkr, and oas. oas is a protein family of 2′, 5′-oligoadenylate (2-5a) synthetases. upon activation, oas can synthesize 2-5a, which binds to and activates rnase l. rnase l is a host ribonuclease that executes global degradation of host and viral rnas. thus, oas and rnase l constitute a potent host antiviral system. macrophages infected by the endou-deficient covs exhibited an early, rnase l-mediated degradation of ribosomal rna, demonstrating that the oas-rnase l system was activated (deng et al., 2017; kindler et al., 2017) . lack of mda5 expression or treatment with the pkr inhibitor did not affect virus-induced rna degradation (deng et al., 2017; kindler et al., 2017) , suggesting that the nsp15-mediated blockage of oas-rnase l activation is independent of the mda5-ifn and pkr pathways. loss of rnase l expression does not restore the replication of endou-deficient covs in macrophages. taken together, these results suggest again that multiple antiviral pathways, including mda5-ifn, pkr, and the oas-rnase l system, were activated during infection with endou-deficient covs. the antiviral defense executed by these pathways contribute together to the replication defect of endou-deficient covs in macrophages. interestingly, some covs encode a 2′, 5′-phosphodiesterase (pde) (e.g. mers-cov orf4b and mhv ns2) (thornbrough et al., 2016; zhao et al., 2012) . this cov pde also prevents rnase l-mediated rna degradation through digesting the 2-5a produced by oas. thus, the presence of two antagonists of the oas-rnase l system in some cov genomes represents a functional redundancy or tissue-specific roles for these antagonists. in fact, not all covs encode a pde, and it has been reported that loss of the pde activity mitigated mhv pathogenicity in the mouse liver but not in the brain, suggesting a liver-specific effect of cov pde activity in vivo (roth-cross et al., 2009; zhao et al., 2011) . several endou-deficient covs have been evaluated in fibroblast cell lines and no marked phenotypes were obtained (mild reduction of viral rna synthesis and replication) (discussed above). we speculate that the activation of an ifn response and apoptosis in macrophages is due to high levels of basal expression of host sensors, such as mda5, pkr, and oas (deng et al., 2017; birdwell et al., 2016) . indeed, when the expression of oas was induced by pre-treatment with ifn, the immortalized fibroblast cells also exhibited rna degradation upon infection with endou-deficient cov (kindler et al., 2017) . in line with this, without the stimulation of ifn, constitutively expressed oas and pkr in the mda5-deficient macrophages is capable of sensing the endou-deficient cov infection and implementing antiviral processes (deng et al., 2017) . although the production of ifn is dispensable for the activation of the oas-rnase l system and pkr, type i ifn receptors or a direct signal is required for maintaining the high basal expression of these ifn-inducible genes (deng et al., 2017; birdwell et al., 2016) . this is biologically relevant because macrophages and other myeloid cells are quick responders to early virus infection, responding even before ifn is highly induced. it is unclear whether other cell types, such as epithelial cells, behave similar to macrophages, but at least mouse embryonic fibroblasts have been shown to display the nsp15/endoumediated effects (kindler et al., 2017) . due to robust activation of antiviral responses, mhv endou-deficient mutants also exhibited a marked attenuation in vivo relative to mhv-wt. depending on the inoculation route, mhv infection can result in hepatitis or encephalitis in c57/bl6 mice. strikingly, regardless of which infection route (intraperitoneal or intracranial injection) was used, we found that mhv endou-deficient mutants were highly attenuated (deng et al., 2017) . when mice were inoculated intraperitoneally using a high dose of the mutant virus, there was no detectable viral titer in the liver or spleen at day 3 post-infection and only a minimal detection of viral mrna in mesenteric lymph nodes at day 1 post-infection. when a sensitive encephalitis model was used, mice infected with mhv endou-deficient mutants exhibited only a transient loss of body weight and recovered completely. this significant attenuation is attributed to the loss of endou-mediated evasion of host antiviral defenses. we found that endou-deficient mutants maintained wt-level virulence in type i ifn receptor knockout (ifnar -/-) mice (deng et al., 2017) . similar results obtained by kindler et al. showed that the mutant virus was only detected in the organs from ifnar -/mice but not wild-type or other gene-deficient (mda5 -/-, tlr7 -/-, and mda5 -/-/tlr7 -/-) mice at day 2 post-infection (kindler et al., 2017) . interestingly, even though mhv endou-deficient mutants were highly attenuated and exhibited limited replication in vivo, the pre-infected mice were protected from subsequent lethal challenges of wild-type mhv in both disease models (deng et al., 2017) . these results demonstrate that nsp15 plays an important role in virus pathogenesis and illustrate the potential use of endou-deficient covs as vaccine candidates. 1.11. how does nsp15 mediate the evasion of host sensors? as an endou, it is plausible that nsp15 may degrade viral dsrna to prevent host recognition. previous studies of pestivirus envelope glycoprotein (e rns ) and lassa virus nucleoprotein linked viral ribonuclease activity to type i ifn antagonism through degrading viral dsrna (python et al., 2013; qi et al., 2010; hastie et al., 2011) , although no direct evidence for this linkage has been obtained. for cov nsp15, kindler et al. detected an increased level of dsrna in endou mutantinfected cells by flow cytometry using a dsrna-specific monoclonal antibody (mab) (kindler et al., 2017) . this mab recognizes dsrna molecules longer than 40 bp in length, regardless of the sequence, meaning that a long dsrna could potentially bind multiple mab molecules. to saturate the mab-dsrna binding, we tested serial concentrations of antibody but did not detect any significant change of dsrna levels by either flow cytometry or immunofluorescence analysis (deng et al., 2017) . the discrepancy between these two studies may be ascribed to the experimental settings in respective experiments. however, it is also possible that the methods used in these studies may not be sensitive enough to detect changes in dsrna levels if the wt-nsp15 produces dsrna cleavage products that are > 40 bp, such that the uncleaved and cleaved dsrnas are indistinguishable for binding by the anti-dsrna antibody. importantly, it has been demonstrated that the endou activity of cov nsp15 is influenced by secondary structures and modifications of dsrna (ivanov et al., 2004; bhardwaj et al., 2006) . because these factors may limit the number of cleavage events and/or the targets of endou, nsp15-mediated cleavage may produce few overall cleavage products that are < 40 bp. consequently, nsp15mediated cleavage may not reduce the level of total dsrna in the cell, but rather may hydrolyze long dsrnas into shorter cleavage products that are sufficiently short to evade recognition by host sensors (e.g. mda5) but not by the anti-dsrna antibody. it has been documented that cov dsrnas form cytoplasmic aggregated foci during replicating in cells (becares et al., 2016; hagemeijer et al., 2012) . these dsrna foci co-localize with the viral rtcs early during infection. interestingly, we noted that during the early stages of infection, dsrna foci were not co-localized with the rtcs in the endou-deficient cov-infected cells. this decrease in colocalization relative to wt infection resulted in a dispersed pattern of dsrna foci, such that some dsrnas did not appear to associate with the rtcs. it is not known whether these "free" dsrnas trigger host sensors, but this dispersed distribution of dsrna does notably coincide with early activation of the ifn response and other dsrna sensors (e.g. pkr and oas). overall, additional studies with new methods are needed to characterize the intracellular localizations and the fates of dsrna in cov-infected cells. the detailed strategy used by cov endou to evade host recognition remains enigmatic. more studies are needed to address several key questions: (i) what is the natural target of endou? (ii) how does the endou activity of nsp15 alter the fate of dsrna? (iii) how is this endou activity regulated to avoid unwanted cleavage events? (iv) do any interaction partners (viral or cellular) of nsp15 participate in regulating its endou activity (bhardwaj et al., 2012; athmer et al., 2017) ? answers to these questions will be essential for understanding the mechanism of the endou-mediated evasion of host dsrna sensors. additionally, it is possible that endou serves as a conserved antagonist in vertebrate nidoviruses. a similar phenotype has been observed in human blood-derived macrophages infected with the hcov-229e nsp15 mutant virus (kindler et al., 2017) , which is a representative alphacoronavirus. whether arterivirus nsp11 also functions as an ifn antagonist during infection is still unknown. one obstacle to studying arterivirus nsp11 mutants in cell culture is that mutations of catalytic residues have been reported to severely affect viral replication even in ifn-deficient cells (posthuma et al., 2006; sun et al., 2016) . nonetheless, given the striking roles of cov endou in macrophages and in vivo, it will be 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endoribonuclease activities of porcine reproductive and respiratory syndrome virus nsp11 was essential for nsp11 to inhibit ifn-β induction a dimerization-dependent mechanism drives the endoribonuclease function of porcine reproductive and respiratory syndrome virus nsp11 unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage the nonstructural proteins directing coronavirus rna synthesis and processing nonstructural protein 11 of porcine reproductive and respiratory syndrome virus suppresses both mavs and rig-i expression as one of the mechanisms to antagonize type i interferon production nidovirus ribonucleases: structures and functions in viral replication the nonstructural protein 11 of porcine reproductive and respiratory syndrome virus inhibits nf-κb signaling by means of its deubiquitinating activity new antiviral target revealed by the hexameric structure of mouse hepatitis virus nonstructural protein nsp15 structural biology of the arterivirus nsp11 endoribonucleases antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns2 protein is required for virus replication and liver pathology cell-type-specific type i interferon antagonism influences organ tropism of murine coronavirus virus-encoded proteinases and proteolytic processing in the nidovirales we thank robert c. mettelman and aaron volk for assistance with editing the manuscript. our studies are supported by national institutes of health r01 ai085089 (to scb). key: cord-264331-uvi8ucz4 authors: singh, shailbala; briles, worthie e.; lupiani, blanca; collisson, ellen w. title: avian influenza viral nucleocapsid and hemagglutinin proteins induce chicken cd8(+) memory t lymphocytes date: 2010-04-10 journal: virology doi: 10.1016/j.virol.2009.12.029 sha: doc_id: 264331 cord_uid: uvi8ucz4 the avian influenza viruses (aivs) can be highly contagious to poultry and a zoonotic threat to humans. since the memory cd8(+) t lymphocyte responses in chickens to aiv proteins have not been defined, these responses to h5n9 aiv hemagglutinin (ha) and nucleocapsid (np) proteins were evaluated by ex vivo stimulation with virus infected non-professional antigen presenting cells. secretion of ifnγ by activated t lymphocytes was evaluated through macrophage induction of nitric oxide. aiv specific, mhc-i restricted memory cd8(+) t lymphocyte responses to np and ha were observed 3 to 9 weeks post-inoculation (p.i.). the responses specific to np were greater than those to ha with maximum responses being observed at 5 weeks p.i. followed by a decline to weakly detectable levels by 9 weeks p.i. the cross-reaction of t lymphocytes to a heterologous h7n2 aiv strain demonstrated their ability to respond to a broader range of aiv. avian influenza viruses (aiv) within the orthomyxoviridae family have segmented, negative sense rna genomes. these viruses, natural infectious agents of waterfowl and shorebirds, are classified according to their transmembrane hemagglutinin (ha) and neuraminidase (na) glycoproteins (alexander, 2000; krauss et al., 2007; olsen et al., 2006; webby and webster, 2001) . due to their incredibly broad avian host range, aiv strains have been isolated from many different species of birds including ducks, gulls, geese, psittacines and poultry (alexander, 2000; olsen et al., 2006) . aiv strains with all 16 hemagglutinin (ha) and 9 neuraminidase (na) types have been isolated from waterfowl or shore birds (fouchier et al., 2005; krauss et al., 2007) . depending on the virulence of the virus in poultry, isolates from poultry are classified as either low pathogenic (lp) or highly pathogenic (hp) (alexander, 2000; collisson et al., 2008) . lpaiv strains cause asymptomatic to mild respiratory and enteric tract infections while the highly pathogenic strains cause clinical illness and systemic infections. infections of poultry, especially with the highly pathogenic strains, result in severe economic losses (capua and marangon, 2003; tollis and di trani, 2002) . human influenza viruses, including those causing high morbidity and significant mortality, such as the h1n1 from 1918, h2n2 from 1957 and h3n2 from 1968 have been shown to have avian origins (capua and alexander, 2002; taubenberger et al., 2001) . even the currently circulating swine origin h1n1 human influenza virus encodes two genes of aiv origin (babakir-mina et al., 2009; garten et al., 2009) . since 1996, highly pathogenic h5n1 aiv strains isolated in hong kong have been infecting and subsequently causing deaths in humans, although person-to-person transmission is apparently rare (capua and alexander, 2002; perdue and swayne, 2005; ungchusak et al., 2005) . poultry is a logical intermediate host for adaptation of viral strains from wild birds to humans and other mammals, such as swine (webby and webster, 2001; webster, 1997) . indeed, human adapted strains have been shown to consist of genome segments of avian, swine and human origin (webby and webster, 2001; webster, 1997) . vaccination efficacy is traditionally determined by the demonstration of protective humoral immunity, especially targeting aiv ha and by putative neutralization of viruses (collisson et al., 2008; suarez et al., 2006; swayne and kapczynski, 2008) . while sterile immunity may depend on humoral responses to homologous ha, effector and memory cd8 + t cell immunity in mice has been shown to diminish disease preventing mortality, and even morbidity swain et al., 2004) . humoral immunity of chickens to aiv is well characterized but little information is available regarding the more difficult to evaluate, viral specific t cell immune responses (kwon et al., 2008; seo and webster, 2001; swayne and kapczynski, 2008) . because mice are not natural hosts of aiv, all the immunological characterization in mice is based only on mouse adapted viruses. it is relevant to define the t lymphocyte mediated immune responses in chickens since aivs are established pathogens of poultry and can be transmitted directly from chickens to humans. avian t lymphocytes have been stimulated ex vivo with mhc matched chicken kidney cells (ckc) serving as non-professional antigen presenting cells (apcs) and by the adoptive transfer of mhc matched t lymphocytes to naïve chicks prior to viral challenge (pei et al., 2003; seo et al., 2000) . the availability of a number of poultry lines with defined mhc (located within the chicken b locus) greatly facilitates the evaluation of the adaptive t lymphocyte responses in chickens (miller et al., 2004) . studies targeting acute infections with a strain of infectious bronchitis virus (ibv), an avian coronavirus, have identified specific cd8 + t cell responses . adoptive transfer of either effector t cells prepared from birds 10 days post-infection (p.i.) or of memory t lymphocytes prepared from birds 3 weeks after infection with ibv, provided protection against acute disease after viral challenge (pei et al., 2003; seo et al., 2000) . following infection with h9n2 aiv, seo et al. (2002) described cd8 + t cell responses that correlated with crossprotection to an h5n1 strain. protection by effector cd8 + t lymphocytes prepared at 7 to 10 days p.i. with aiv was demonstrated following adoptive transfer 1 day prior to aiv challenge (seo and webster, 2001) . however, none of these studies with aiv identified the individual aiv proteins housing t lymphocytes epitopes or described the kinetics of the t lymphocyte mediated memory responses to aiv. this current study describes the memory responses of peripheral blood t lymphocytes from chicks to aiv ha and/or np protein. dna plasmid vectors expressing aiv proteins were used to delineate the responses induced individually by either ha or np proteins. responses were evaluated following ex vivo stimulation with mhc-i matched or mismatched apcs. both np and ha induced aiv specific memory t lymphocyte responses between 3 and 9 weeks p.i. although the t lymphocyte response induced by np was greater than the response induced by ha until 7 weeks p.i., no differences between the responses to the two proteins were detected by 9 weeks p.i. the maximum response of memory t lymphocytes against either aiv np or ha protein was observed at 5 weeks p.i. in order to determine the t lymphocyte responses to individual aiv proteins, the np and ha genes of the low pathogenic h5n9 (turkey/wis/68) strain were cloned into the pcdna3.1/v5-his-topo ta vector (invitrogen, carlsbad, ca). the eukaryotic expression of the proteins encoded by the plasmids was determined by ifa in cho-k1 cells 48-h post-transfection with plasmids expressing either np or ha (fig. 1) . no fluorescence was observed in cells transfected with plasmid encoding for lacz. the in vivo expression of aiv ha and np and the antibody response to these proteins in the chickens was confirmed by detecting the presence of antibodies specific for ha and np at 3 weeks p.i. with the plasmids. hi titers of sera from 6 chickens inoculated with the ha expression plasmid using the homologous strain ranged from 5 to 6.5 log 2 (gmt). sera from chickens inoculated with ha plasmid failed to inhibit hemagglutination by a heterologous h7n2 aiv. no hi activity was detected in sera of the 4 chickens inoculated with pbs. elisa titers for antibodies against np in 6 np plasmid inoculated chickens ranged between 2.5 and 3.0 log 10 (gmt) at 3 weeks p.i. (fig. 2) . memory t cell responses were detected from weeks 3 to 9 p.i. with ha and np plasmids because the t cell specificity for individual aiv proteins have not previously been reported in chickens, t lymphocyte responses to ha, np or a combination of both ha and np (hn) were determined following inoculation of b19/b19 chickens with plasmids expressing the ha or np orf. in the absence of wellestablished elisa or intracellular cytokine staining methods to examine avian t lymphocyte mediated responses an indirect ifnγ assay (no production from hd11 cells, a chicken macrophage cell line) was precisely standardized for evaluating the ex vivo activation of t lymphocytes by apcs. ckc infected with aiv were used as non-professional apcs for the stimulation of t lymphocytes. as demonstrated in other studies, the primary effector t lymphocyte response observed at 10 days p.i. had declined to basal level by 16th day p.i. (data not shown). considering previously conducted adoptive transfer studies, which identified specific memory t cells to ibv by 3 weeks p.i. with maximal responses occurring between weeks 5 and 6 p.i. (pei et al., 2003) , the memory aiv response of peripheral blood memory t lymphocytes were also evaluated between 3 and 9 weeks p.i. with ha, np, or hn plasmids. the kinetics of the t lymphocyte mediated responses was determined on the same birds during the course of the study. all data represent the response from individual birds. following ex vivo co-culture with mhc matched b19/b19 apcs infected with h5n9 virus, memory responses were detected in t cell preparations obtained from all chickens receiving plasmids expressing either aiv protein by 3 weeks p.i. since neither supernatants from the t cells cultured with uninfected apcs nor t cells from pbs inoculated birds cultured with infected mhc matched b19/b19 birds produced ifnγ (data not shown), the memory t lymphocyte activity was considered aiv specific. the ex vivo stimulation of the t cells from plasmid inoculated birds with mhc mismatched b2/b2 apcs could only induce basal levels of no (fig. 3) , indicating the memory t lymphocyte responses from each group of chickens receiving aiv plasmids was highly mhc restricted. the maximum memory t cell responses to np were detected at 5 weeks p.i. however, while still detectable, memory t cells responses at 9 weeks p.i, were dramatically diminished for all birds receiving the aiv expression plasmids (ha, np or hn). during weeks 3 through 7, the activity of t cells from the ha plasmid inoculated birds was significantly less than that of t cells isolated from birds receiving either np or np plus ha plasmids (fig. 3 ). in addition to weaker apc induced stimulation, the levels of ha specific memory t lymphocyte responses were similar at weeks 3, 5 and 7 p.i. these results were observed for two independent experiments. the phenotype of activated subpopulations of t lymphocytes following co-culture with apcs expressing aiv antigens was determined using flow cytometric analyses (table 1 ). in order to evaluate the type of t lymphocyte population expanding in response to infected apcs, the proliferation of the lymphocytes from chickens inoculated with both np and ha (hn) plasmids was determined at 5 weeks p.i. lymphocyte populations were gated using a chicken pan lymphocyte cd44 specific mab and mabs specific for either cd4 or cd8 t cell antigens . the relative increases in the populations of cd8 + t lymphocytes harvested from each ha and np (hn) plasmid inoculated chicken were between 62% and 91% following ex vivo stimulation with aiv expressing apcs, in contrast to the increase of 31% to 37% in the cd4 + t lymphocyte populations. the increase in the population of t lymphocytes harvested from pbs inoculated birds was 7% and 1% for cd8 + and cd4 + t lymphocyte subpopulations, respectively, following co-culture with aiv infected apcs. the preferential increase in cd8 + cells correlates with their anticipated expansion following exposure to non-professional apcs endogenously expressing aiv and expressing surface mhc-i. a rationale for targeting cellular immunity is the potential for cross-reactivity between vaccine and heterologous virus. since the availability of the peripheral t lymphocytes was limited and the same birds were being used throughout the study, all aspects of the memory t lymphocyte mediated responses could not be evaluated at same time points. at 8 weeks p.i. the capacity for memory t lymphocytes specific for the np and ha proteins of the h5n9 strain to be stimulated with an h7n2 (a/turkey/virgina/158512/02) strain of aiv was determined following co-culture with mhc matched apcs infected with either aiv strain (fig. 4) . both heterologous h7n2 and homologous h5n9 aiv infected apcs significantly stimulated ifnγ production from memory t lymphocytes isolated from either ha or np inoculated chickens (p ≤ 0.01) compared with pbs inoculated chickens. regardless of the strain used to infect apcs, the observed memory responses generated by t cells obtained from chickens receiving the np plasmid were again statistically greater (p ≤ 0.008) than that generated by t cells harvested from ha plasmid inoculated chickens. memory t cells could be readily detected between 3 and 7 weeks p.i. with plasmids expressing either the np or the ha proteins. in order to evaluate the memory t lymphocyte responses of chickens inoculated with infectious aiv, chicks with the b19/b19 haplotype were inoculated with the low pathogenic h5n9/turkey/wis/68 strain and blood was collected at 5 weeks p.i. the ex vivo activation of t lymphocytes by aiv infected apcs was determined by the indirect ifnγ assay (fig. 5) . the mean average no production induced by the ex vivo stimulation of t lymphocytes from infected birds with b19/ b19 apcs was specific compared with the uninfected, mhc matched apcs (p ≤ 0.005). the responses to aiv infected apcs were mhc restricted as demonstrated by only basal level activation by b2/b2 apcs. flow cytometric analysis was used to determine the phenotype of the t lymphocyte subpopulations from the infected chickens responding to ex vivo stimulation. the relative increase in the population of cd8 + t lymphocytes from h5n9 infected chickens was 46% to 95% while the increase in the cd4 + t lymphocyte population ranged from 6 to 28% following co-culture with mhc-i matched, h5n9 aiv infected apcs ( table 2 ). the increase in the population of the lymphocytes from uninfected chickens was only 1 to 14% and 10 to 19% for cd8 + and cd4 + t lymphocytes, respectively. although increases were observed in the cd4 + lymphocytes, the greater increased proliferation of cd8 + lymphocytes from birds infected with the low pathogenic virus was consistent with detection of a preferential mhc-i restricted aiv specific, cd8 + memory t cell response. this is the first study delineating the response of chicken memory cd8 + t lymphocytes to specific aiv proteins. studies evaluating the cd8 + t lymphocyte response to influenza virus in mice have identified np as housing the dominant cd8 + t cell epitopes (flynn et al., 1998; kreijtz et al., 2007; o'neill et al., 2000; townsend et al., 1984; yewdell et al., 1985) . in contrast, human cytotoxic t lymphocytes (ctl) have a broader repertoire and the response is directed to multiple influenza viral proteins, including ha (gianfrani et al., 2000; gotch et al., 1987; jameson et al., 1998; kedzierska et al., 2006; kreijtz et al., 2008) . this study has shown that similar to the responses in humans, memory cd8 + t lymphocytes in chickens are directed against both aiv ha and np proteins (gianfrani et al., 2000; jameson et al., 1998; kreijtz et al., 2008; mcmichael et al., 1986b) . significantly greater responses were induced by np than by ha at 3 and 5 weeks p.i. the aiv specific t cell responses were primarily mhc-i restricted as non-professional apcs of b19 and b2 haplotypes were used for ex vivo stimulation of t cells and the apcs of b2 haplotype chickens either failed to stimulate or could only weakly stimulate the t lymphocytes derived from the b19 line. therefore, responding t cells were primarily of the cd8 + phenotype, which also showed significantly greater proliferation than cd4 + t lymphocytes in response to ex vivo apc mediated stimulation. similar to the mhc-i restricted t lymphocyte responses demonstrated following infection with ibv, cd8 + t cell memory responses to aiv ha and/or np were detected by 3 weeks p.i. (pei et al., 2003) . preliminary studies in our lab have also demonstrated an elevation in the levels of expression of cd44 molecule on the surface of the responding t lymphocytes. greater cd44 expression has been described to be a characteristic of mouse and human memory t lymphocytes (curtsinger et al., 1998) . the current studies quantified the protein specific t lymphocyte responses of the same birds until 9 weeks p.i. the responses increased from 3 weeks p.i. until 5 weeks p.i. however, by 9 weeks p.i. with plasmids expressing either np or ha aiv protein, the memory t cell activity had declined to significantly lower levels. the decline in the more vigorous cd8 + t lymphocyte response to np was more rapid after 5 weeks than the cd8 + t cell response stimulated by ha, such that by 9 weeks p.i. the responses to both proteins individually or in combination were similar. a decline in the cd8 + t lymphocyte mediated protection at 10 weeks after challenge of h9n2-infected chicks with h5n1 had been observed by seo and webster (2001) . similarly, a decline in the memory t lymphocyte response specific to influenza virus infection has also been reported in humans (mcmichael et al., 1983) . furthermore, our study also proved the efficacy of the plasmid delivery approach in providing a mechanism to evaluate the t cell response to aiv ha and np proteins, either individually or in combination. protection studies were not included following inoculation of the aiv plasmids because of increased biosafety requirements for aiv. however, hemagglutinating antibodies which can be correlated with protection were demonstrated (toro et al., 2007) . the antibodies specific for the ha cloned from a h5n9 virus failed to prevent h7n2 virus-mediated hemagglutination. since each ex vivo stimulation assay required specific numbers of necessary controls for the validity of the results of the assay and availability of limited numbers of t lymphocytes for each time, all assays could not be conducted at the same time point. although at 8 weeks p.i. the response of the memory t lymphocytes was significantly lower than the response at 5 weeks p.i., it remained detectable and the apcs infected with h7n2 aiv were able to effectively ex vivo stimulate the t lymphocytes from h5 inoculated chickens. these observations indicate that despite the absence of shared hi antibody epitopes, ha does have at least one cd8 + epitope that is shared between both strains of the virus. adoptive transfer studies of cd8 + t lymphocytes specific for ibv and aiv have demonstrated their importance in protection against heterologous viruses (pei et al., 2003; seo et al., 2000; seo and webster, 2001) . in mice, adoptive transfer of memory cd8 + t lymphocytes specific for influenza virus np has also been shown to be protective against viral challenge (lukacher et al., 1984; taylor and askonas, 1986) . in vivo inoculation of chicks with a dna plasmid expressing the ibv nucleocapsid protein was shown to provide ctl mediated protection against acute respiratory disease . consistent with the greater response to np from the homologous virus, t lymphocytes of chicks inoculated with np exhibited greater cross reactivity with the heterologous h7n2 virus than t cells from the birds inoculated with ha. the amino acid sequences in ha from the two aiv strains were 41% identical while the amino acid sequences of np were 97% identical. the more conserved nature of np, in comparison to ha could be responsible for the greater cross reactive response (portela and digard, 2002) . although the variation could also be attributed to the differences in the amount of antigen generated and presented by the infected apcs (busch and pamer, 1998; crowe et al., 2003; deng et al., 1997; doherty et al., 1994) , both np and ha responses were similar by 9 weeks p.i. using the same standardized cell assay. the differences in the repertoire of t cell epitopes along with the variations in the binding affinity of t cell epitopes with mhc-i can also contribute to the differences in the cd8 + t cell mediated response to influenza viral proteins (cao et al., 1996; mcmichael et al., 1986a) . chickens have 27 distinct defined mhc haplotypes (miller et al., 2004) . further studies aimed at determining the response to aiv proteins in different mhc lines of chickens is important for identification of immunodominant proteins which can elicit conserved cd8 + t lymphocyte responses amongst various mhc haplotypes. although the cross reactivity of memory cd8 + t lymphocytes may not prevent the infection of chickens with a heterotypic strain of aiv, it could contribute to the more rapid clearance of the virally infected cells and augment the protection against clinical illness. this study establishes that chickens cd8 + t lymphocytes respond to aiv np and ha proteins. the ability of the other aiv proteins to stimulate cd8 + t 3 . chicken memory t lymphocyte responses to aiv ha and np proteins between 3 and 9 weeks p.i. with np and/or ha expression plasmids. chickens of the b19/b19 mhc haplotype were inoculated with dna plasmids expressing aiv ha, np or both ha and np (hn). memory t lymphocytes were stimulated ex vivo with h5n9 aiv infected mhc matched b19/b19 and mismatched b2/b2 apcs. production of no by hd11 macrophage cells induced by the secretion of ifnγ from stimulated t lymphocytes was used to quantify lymphocyte activation. results represent the average (± s.e.) of two separate experiments. each ex vivo stimulation assay is denoted by the source of t lymphocytes and mhc haplotype of the apcs infected with the virus. the differences in stimulation by matched and mismatched apcs were significant (p ≤ 0.003 to 0.02) for each inoculated antigen and time point. the responses to hn (p ≤ 0.03) at 3 weeks and np (p ≤ 0.03) and hn (p ≤ 0.007) at 5 weeks p.i. were significantly greater than the responses to ha at the same time points. the p value for the difference between responses to np and ha at 3 weeks p.i. was 0.07. lymphocytes of chickens needs to be evaluated. future experiments would also analyze the expression of various cytokines by the activated t lymphocytes. inclusion of aiv protein targets that can induce cross reactive cd8 + t lymphocyte responses in addition to humoral immunity in chickens is critical for the development of efficacious vaccines which can provide protective immunity against a broader range of aiv types. low pathogenic aivs, h5n9 (a/turkey/wis/68) and h7n2 (a/ turkey/virginia/158512/02), were propagated in the allantoic sacs of 10-day-old embryonated chicken eggs (ece). the allantoic fluid was harvested 48 h p.i. and the presence of virus was verified by an hemagglutination activity (ha) test according to the oie guidelines (http://www.oie.int/eng/normes/mmanual/2008/pdf/2.03.04_ai. pdf). viruses were quantified by titrating in ece and expressed as embryo infectious dose 50 (eid 50 ) (beard, 1989) . embryonated eggs of mhc defined b19/b19 and b2/b2 lines of chickens were obtained from dr. briles' laboratory at northern illinois university (dekalb, il). after hatching, chicks were housed in a specific pathogen-free environment at the vivarium facility, western university of health sciences, pomona, ca. viral infection studies in chickens were conducted at the biosafety level 2 lab animal research resource animal facility, texas a&m university, college station, tx. all procedures involving the use of chickens were approved by and conducted according to guidelines established by the institutional animal care and use committees of western university of health sciences and/or texas a&m university. cloning of np and ha into a eukaryotic expression plasmid rna from h5n9 (a/turkey/wis/68) was extracted from allantoic fluid using the rneasy mini kit (qiagen, valencia, ca) according to the manufacturer's protocol. first strand cdna was synthesized with improm-ii™ reverse transcriptase enzyme (promega, madison, wi) using aiv specific universal 12 primer (5′agca/gaaagcagg 3′) (urabe et al., 1993) . pfu polymerase (stratagene, la jolla, ca) was used to amplify the open reading frames (orf) of ha and np using specific primer pairs ha-forward 5′-accatggaaagaatagtgatt-3′and ha-reverse 5′-gatgcaaattctgca-3′ and np-forward 5′-accat-ggcgtctcaaggcacc-3′ and np-reverse 5′-attgtcatactcctctgc-3′, respectively. taq dna polymerase (new england biolabs, ipswich, ma) was then used to add ta overhangs on the pfu amplified pcr product. the amplified cdna products were cloned into the eukaryotic expression vector pcdna3.1/v5-his-topo ta (invitrogen, carlsbad, ca). big dye terminator v1.1cycle sequencing kit (applied biosystems, foster city, ca) was used to sequence the cloned gene segments and the sequence analysis was performed at genoseq, ucla, los angeles, ca. following confirmation of the sequence of the orfs, in vitro expression of np and ha proteins were determined using an indirect immunofluorescence assay (ifa) on cho-k1 cells transfected with the plasmids expressing aiv proteins. known chicken serum, positive for aiv (nvsl, ames, ia) was used at a dilution of 1:100 as primary antibody. mouse anti-chicken igg fitc at a dilution of 1:500 (southern biotech, birmingham, al) was used as the secondary antibody. primary ckc lines were established from 10-day-old chicks of b19/b19 and b2/b2 mhc haplotype lines as described previously . ckc of the tenth passage were infected with aiv and used as non-professional apcs for the stimulation of the cd8 + t lymphocytes. the presence of mhc-i on ckc lines and the absence of mhc-ii was confirmed by flow cytometric analysis, using anti-chicken mhc-i or anti-chicken mhc-ii r-phycoerythrin conjugated monoclonal antibodies (mabs) (southern biotech, birmingham, al) (data not shown). three-week-old specific, pathogen-free chickens of the b19/b19 mhc haplotype were inoculated, intramuscularly (i.m.), with 500 μg of cdna expressing ha alone or np alone, or of 500 μg of each ha and np (hn) combined. control birds were inoculated with either pcdna 3.1 vector expressing lacz (lacz) or pbs. for viral inoculations, b19/ b19 chicks were inoculated at 3 weeks of age, intranasally, with 10 8 eld 50 of the low pathogenic h5n9/tur/wis/68 aiv strain. serum samples were prepared from blood collected from the jugular vein of chickens at 3 weeks p.i. to evaluate the humoral responses against aiv ha and np. hemagglutination inhibition (hi) assays, according to oie guideline (http://www.oie.int/eng/normes/mmanual/ 2008/pdf/2.03.04_ai.pdf), were used to identify antibodies specific to h5n9 virus (a/turkey/wis/68) ha. hi mediated by the anti-h5 antibodies against h7n2 aiv was also evaluated. titers were expressed as geometric mean titers (gmt). titers of ≤ 1 log 2 were assigned a titer of 1 log 2 . np specific antibodies were determined using the aiv plus elisa kit (synbiotics, kansas city, mo) as described by the manufacturer. effector t lymphocytes used for ex vivo stimulation were prepared from peripheral blood mononuclear cells (pbmc) from 2 to 4 individual chickens per group (pei et al., 2003) . briefly, blood was collected from the jugular vein at 3, 5, 7 and 9 weeks p.i. and diluted 1:2 in alsever's solution (sigma-aldrich, st. louis, mo). pbmc were prepared by ficollhistopaque (histopaque-1077, sigma-aldrich, st. louis, mo) density gradient centrifugation . viable cells were collected from the interface and washed twice with phosphate buffered saline (pbs, ph 7.4). cells were resuspended in 3 ml of rpmi 1640 (invitrogen, la jolla, ca) supplemented with 10% fetal bovine serum (gemini bio-products, west sacramento, ca), 2 mm l-glutamine, and 0.1 mm mem non-essential amino acids. b lymphocytes were removed by passing the cell suspension through complete rpmi equilibrated nylon wool column and adherent cells were removed by incubating the cell preparation in 25 cm 2 tissue culture flasks as described previously . flow cytometric analysis of cells labeled with anti-chicken cd3 fitc mabs (southern biotech, birmingham, al) as described previously determined that the lymphocyte population recovered after nylon wool separation and removal of adherent cells was 97% positive for the expression of the t lymphocyte marker, cd3 (bohls et al., 2006) . t lymphocytes from pbmc were stimulated ex vivo with mhc b19/ b19 (matched) and b2/b2 (mismatched) apcs. apcs at a concentration of 1 × 10 5 cells/ml in 96-well tissue culture plates were incubated for 8 h at 39°c, 5% co 2 . apcs were infected with 1x10 5 eld 50 of h5n9 (a/turkey/wis/68) virus for 1 h followed by removal of the virus and cells before washing 3 times with dmem supplemented with 10% fbs. one × 10 6 t lymphocytes in complete rpmi were added to each well. cells were co-cultured for 24 h, before the media was collected and centrifuged. the clarified supernatants were used to quantify ifnγ production by activated t lymphocytes using a nitric oxide detection assay. at 5 weeks p.i. the pelleted t lymphocytes were collected for facs analysis to measure the lymphocyte proliferation. each ex vivo stimulation assay was conducted in duplicate. a modified indirect ifnγ assay based on no production (crippen et al., 2003; karaca et al., 1996; pei et al., 2003) from hd11 cells (a chicken macrophage cell line) was used to quantify the ifnγ secreted from ex vivo activated t lymphocytes by apcs. briefly, cells were incubated in individual wells of 96-well plates at a concentration of 1 × 10 5 cells/well in complete rpmi media for 2 h at 39°c, 5% co 2 prior to the addition of 150 μl supernatants from t lymphocyte-apcs cultures. after 24 h of incubation, the accumulation of nitrite in supernatant from stimulated hd11 cells was measured using the griess reagent assay according to the manufacturer's protocol (sigma-aldrich, st. louis, mo). to ensure that the data represent the nitric oxide produced specifically by the ifnγ mediated stimulation of hd11 cells and not due to any other soluble inducing factors, nitrite concentration in each sample was normalized by subtracting the nitrite concentration of supernatants from control apcs cultured without t lymphocytes. the concentration of nitrite produced was determined using sodium nitrite solutions with a concentration of 1 to 20 μmol as standards. after ex vivo stimulation with aiv infected apcs, t lymphocytes were collected and dual labeled with phycoerythrin-conjugated mabs specific for cd44 and fluorescein labeled mabs specific for either cd8 or cd4 (southern biotech birmingham, al) as previously described . flow cytometric analysis was used to determine the concentration of t lymphocyte subpopulations. a minimum of 10 4 events were collected for each sample. the percentage of cd44 + lymphocytes expressing either cd4 or cd8 surface antigen was determined using flowjo™ (treestar, inc., ashland, or). cell proliferation was calculated as the percent increase in the population of cd4 + or cd8 + t lymphocytes cultured with uninfected apcs after in vitro stimulation by virus-infected apcs for 24 h. the nitric oxide concentrations were expressed as the average of 4 to 6 birds per group. anova (analysis of variance) with significance of p ≤ 0.05 was used to determine statistical differences. a review of avian influenza in different bird species origin of the 2009 mexico influenza virus: a comparative phylogenetic analysis of the principal external antigens and matrix protein a laboratory manual for the isolation and identification of avian pathogens the use of flow cytometry to discriminate avian lymphocytes from contaminating thrombocytes mhc class i/peptide stability: implications for immunodominance, in vitro proliferation, and diversity of responding ctl the weak cd8 + ctl response to an influenza hemagglutinin epitope reflects limited t cell availability avian influenza and human health the use of vaccination as an option for the control of avian influenza evolving vaccine 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cytotoxic t lymphocytes and protects chickens from acute infection adoptive transfer of infectious bronchitis virus primed alpha beta t cells bearing cd8 antigen protects chicks from acute infection protective cross-reactive cellular immunity to lethal a/goose/guangdong/1/96-like h5n1 influenza virus is correlated with the proportion of pulmonary cd8(+) t cells expressing gamma interferon avian influenza vaccination in north america: strategies and difficulties t cell responses to influenza virus infection: effector and memory cells strategies and challenges for eliciting immunity against avian influenza virus in birds integrating historical, clinical and molecular genetic data in order to explain the origin and virulence of the 1918 spanish influenza virus influenza nucleoprotein-specific cytotoxic t-cell clones are protective in vivo recent developments in avian influenza research: epidemiology and immunoprophylaxis protective avian influenza in ovo vaccination with non-replicating human adenovirus vector cytotoxic t cell recognition of the influenza nucleoprotein and hemagglutinin expressed in transfected mouse l cells probable person-to-person transmission of avian influenza a (h5n1) mdbk cells which survived infection with a mutant of influenza virus a/wsn and subsequently received many passages contained viral m and ns genes in full length in the absence of virus production emergence of influenza a viruses influenza virus: transmission between species and relevance to emergence of the next human pandemic influenza a virus nucleoprotein is a major target antigen for cross-reactive anti-influenza a virus cytotoxic t lymphocytes this work was supported by usda-csrees-nri grant 2006-35204-16560 and usda-aicap grant 2007-35203-18070. we thank victoria hampton, lisa griggs, omar alvarado and vinayak brahmakshatriya for technical assistance. special thanks to dr. twani crippen for her generous gift of hd11 cells and advice with the assays. we are grateful to dr. roger smith for help with the flow cytometry. we acknowledge dr. maisie dawes, dr. ghida banat, dr. miguel saggese, and dr. yvonne drechsler for their help and advice with the manuscript. key: cord-256737-ptjng78b authors: mcbride, corrin e.; machamer, carolyn e. title: palmitoylation of sars-cov s protein is necessary for partitioning into detergent-resistant membranes and cell-cell fusion but not interaction with m protein date: 2010-09-01 journal: virology doi: 10.1016/j.virol.2010.05.031 sha: doc_id: 256737 cord_uid: ptjng78b coronaviruses are enveloped rna viruses that generally cause mild disease in humans. however, the recently emerged coronavirus that caused severe acute respiratory syndrome (sars-cov) is the most pathogenic human coronavirus discovered to date. the sars-cov spike (s) protein mediates virus entry by binding cellular receptors and inducing fusion between the viral envelope and the host cell membrane. coronavirus s proteins are palmitoylated, which may affect function. here, we created a non-palmitoylated sars-cov s protein by mutating all nine cytoplasmic cysteine residues. palmitoylation of sars-cov s was required for partitioning into detergent-resistant membranes and for cell-cell fusion. surprisingly, however, palmitoylation of s was not required for interaction with sars-cov m protein. this contrasts with the requirement for palmitoylation of mouse hepatitis virus s protein for interaction with m protein, and may point to important differences in assembly and infectivity of these two coronaviruses. coronaviruses are enveloped positive strand rna viruses that infect many avian and mammalian species, including humans. these viruses target a variety of tissues and generally cause mild disease. in humans, coronaviruses are responsible for approximately 20% of common cold cases (larson et al., 1980) . however, in 2002, a novel human coronavirus, severe acute respiratory syndrome coronavirus (sars-cov), emerged in the guangdong province of china (kuiken et al., 2003; rota et al., 2003) . sars-cov is unlike any other human coronavirus to date, causing severe respiratory disease and death in 10% of infected patients (who, 2003) . while many enveloped viruses assemble at the plasma membrane, coronaviruses assemble intracellularly and bud into the lumen of the endoplasmic reticulum golgi intermediate compartment (ergic) (klumperman et al., 1994) . to produce infectious virus, the envelope proteins must be targeted to the ergic for virus assembly. many coronavirus envelope proteins localize near the assembly site when exogenously expressed alone (corse and machamer, 2000; klumperman et al., 1994) ; however, others rely on lateral interactions with other envelope proteins to localize to the virus assembly site (mcbride et al., 2007; nguyen and hogue, 1997; opstelten et al., 1995) . like other coronaviruses, sars-cov encodes 3 envelope proteins, spike (s), envelope (e) and membrane (m) (marra et al., 2003; rota et al., 2003) . the s protein is the second most abundant protein in the virion envelope. it is a type-i membrane protein that determines host cell tropism and is responsible for virus-cell as well as cell-cell fusion (cavanagh, 1995; gallagher and buchmeier, 2001) . the sars-cov s glycoprotein is large (approximately 180 kda) and heavily glycosylated with 23 potential n-linked glycosylation sites (marra et al., 2003; rota et al., 2003) . the cytoplasmic tail of the sars-cov s is palmitoylated (petit et al., 2007) and contains a weak endoplasmic reticulum retrieval signal that helps it localize to the virus assembly site when co-expressed with sars-cov m (mcbride et al., 2007) . the m protein is the most abundant protein in the virion envelope. m can form homo-oligomers and acts as a scaffold for virus assembly, interacting with s, e and the viral nucleocapsid hogue and machamer, 2008) . sars-cov m has an n-linked glycosylation site, three transmembrane domains and a long cytoplasmic tail (voss et al., 2006) . the e protein is the least abundant protein in the virion envelope although it has an important role in virion budding and release (dediego et al., 2007; fischer et al., 1998; kuo and masters, 2003; machamer and youn, 2006; ortego et al., 2007 ortego et al., , 2002 . although the topology and glycosylation of sars-cov e is controversial, it has a single hydrophobic domain and is palmitoylated on its cytoplasmic tail (reviewed in (liu et al., 2007) . virology 405 (2010) [139] [140] [141] [142] [143] [144] [145] [146] [147] [148] protein palmitoylation is a common post-translational modification that occurs on cytoplasmic cysteine residues. protein palmitoylation occurs by the addition of the fatty acid palmitate to a protein via a thioester linkage (resh, 2006a) . protein palmitoylation can greatly increase the hydrophobicity of a protein, which can in turn affect protein activity. transmembrane proteins are commonly palmitoylated on cysteine residues that are at or near the lipid bilayer (resh, 2006a) . cytoplasmic proteins can also be palmitoylated, inducing membrane association (greaves and chamberlain, 2007) . palmitoylation is generally reversible and can be highly dynamic (linder and deschenes, 2007) . addition of palmitate to proteins can greatly influence a protein's trafficking, stability, localization and interaction with other proteins (delandre et al., 2009; mccormick et al., 2008; van itallie et al., 2005) . because of the great versatility of protein palmitoylation, it can be used to dynamically regulate protein function (baker et al., 2003; iwanaga et al., 2009) . palmitoylation of viral proteins can play an important role in virus assembly and infection (grantham et al., 2009; majeau et al., 2009; rousso et al., 2000) . the s and e proteins of several coronaviruses have been shown to be palmitoylated (boscarino et al., 2008; corse and machamer, 2002; liao et al., 2006; lopez et al., 2008; petit et al., 2007; thorp et al., 2006) ; however, not much is known about the role of this modification. coronavirus s protein palmitoylation appears to be important for cell-cell fusion (petit et al., 2007) , infectivity and virus assembly (thorp et al., 2006) . in addition, for mouse hepatitis virus (mhv), it has been shown that s protein palmitoylation is important for interaction with the m protein (thorp et al., 2006) . coronavirus e protein palmitoylation is important for e protein stability, virus assembly and production (boscarino et al., 2008; lopez et al., 2008) . although many important roles for palmitoylation of coronavirus envelope proteins have been suggested, few studies have analyzed all of the possible palmitoylated cysteine residues with regard to function (petit et al., 2007) . pharmacological inhibition of palmitoylation is commonly used (thorp et al., 2006) , although this has deleterious effects on general cellular functions since palmitoylation is a common modification (mikic et al., 2006; resh, 2006b) . in this study, we addressed the role of sars-cov s protein palmitoylation without the use of general palmitoylation inhibitors by mutating the 9 potentially palmitoylated cytoplasmic cysteine residues. we show that palmitoylation is not necessary for sars-cov s protein stability, localization and trafficking. we confirm previous reports that suggest a role for sars-cov s protein palmitoylation in cell-cell fusion and also provide evidence for the importance of palmitoylation in sars-cov s partitioning into detergent-resistant membranes (drms). importantly, we show that sars-cov s palmitoylation is not necessary for efficient interaction with sars-cov m, which differs from published experiments for mhv (thorp et al., 2006) and suggests a significant difference between the two viruses that may have important implications for virus assembly and infectivity. results from other labs have shown that coronavirus spike proteins are palmitoylated (bos et al., 1995; petit et al., 2007) ; however, there is no information about the compartment in which this post-translational modification occurs. since the protein acyltransferases responsible for protein palmitoylation are localized throughout the entire secretory pathway (tsutsumi et al., 2008) , it is possible that coronavirus spike proteins could be palmitoylated at the er, golgi or the plasma membrane. after glycoproteins are synthesized, their sugars become modified as the protein traffics through the secretory pathway. in the medial golgi, glycoproteins become resistant to digestion with endoglycosidase h (endo h) (herscovics, 1999) . to determine if sars-cov s becomes palmitoylated in a pre-medial golgi compartment, hek293t cells exogenously expressing sars-cov s were labeled for 30 min with 35 s-methionine/ cysteine to measure total protein expression or 3 h-palmitic acid to measure palmitoylated protein. while 35 s-methionine/cysteine labels newly translated proteins, 3 h-palmitic acid labels both newly made and pre-existing proteins. after radiolabeled cells were lysed, s protein was immunoprecipitated, denatured and digested with endo h. as expected, upon endo h treatment, there was a population of newly made 35 s-labeled s protein that had not yet trafficked past the medial golgi and was thus sensitive to endo h digestion (fig. 1a, left) . there was also a population of 3 h-palmitic acid labeled s protein that was sensitive to digestion with endo h (fig. 1a, right) , indicating that s can be palmitoylated before it traffics through the golgi en route to the plasma membrane. this result suggests that at least a portion of sars-cov s is palmitoylated in a pre-medial golgi compartment but does not rule out the possibility of additional palmitoylation in a postmedial golgi compartment. palmitoylation is reversible and proteins can be palmitoylated and de-palmitoylated rapidly (linder and deschenes, 2007) . generally, viral proteins are stably palmitoylated, but there are examples of proteins containing palmitate chains that are rapidly turned over (rocks et al., 2005) . to determine if sars-cov s palmitoylation was a stable or dynamic modification, we performed a pulse-chase assay. hek293t cells expressing sars-cov s were pulse labeled with 35 smethionine/cysteine or 3 h-palmitic acid and chased for various times. the 3 h-palmitate label (fig. 1b , right) was similar in stability to the total 35 s-methionine/cysteine labeled population (fig. 2, left) . this suggests that palmitoylation of sars-cov s is a stable posttranslational modification. like other coronaviruses spike proteins, sars-cov s has multiple cysteine residues in its cytoplasmic tail that could be palmitoylated. sars-cov s, like mhv s, has 9 cytoplasmic cysteines that are putative palmitoylation sites ( fig. 2a ) (petit et al., 2007) . to determine the role of sars-cov s palmitoylation, we mutated all 9 of the cytoplasmic cysteine residues to alanines. to ensure that the mutant sars-cov s was not palmitoylated, we radiolabeled hek293t cells expressing wild-type or mutant s with 35 s-methionine/cysteine or 3 h-palmitic acid. while wild-type and mutant sars-cov s proteins were expressed at similar levels, only wild-type sars-cov s was protein palmitoylation can have dramatic effects on protein turnover. palmitoylation often increases protein stability (ochsenenbauer-jambor et al., 2001) ; in support of this, mhv e palmitoylation was reported to increase its half-life (lopez et al., 2008) . to determine if palmitoylation affects the stability of s, we calculated the half-lives of sars-cov s and s pn . hek293t cells expressing sars-cov s or s pn were pulse labeled and chased for various times. wild-type and mutant s proteins were immunoprecipitated and analyzed. the percentage of protein remaining relative to the zero chase time was calculated for each time of chase. both sars-cov s and s pn had similar half-lives of approximately 2.75 h (fig. 3) . this result suggests that palmitoylation of sars-cov s does not affect turnover or stability when s protein is exogenously expressed. sars-cov s localizes to the plasma membrane when exogenously expressed alone in cells (bisht et al., 2004; hofmann et al., 2004; schwegmann-wessels et al., 2004; simmons et al., 2004) . transmembrane protein trafficking as well as soluble protein translocation to membranes can both be regulated by protein palmitoylation (resh, 2006a) . in fact, palmitoylation of different cysteines on the same protein can lead to dramatic differences in subcellular localization (roy et al., 2005) . to determine if palmitoylation alters the subcellular localization of sars-cov s, we performed indirect immunofluorescence microscopy on hek293t cells expressing sars-cov s or s pn . hek293t cells expressing s or s pn were fixed, permeabilized and costained with antibodies to sars-cov s and golgin-160 (a golgi marker). sars-cov s and s pn were both present throughout the secretory pathway, with staining at the cell surface and some internal concentration at the golgi complex co-localizing with golgin-160 ( fig. 4a ). to further assess the localization of sars-cov s and s pn at the plasma membrane, we performed surface staining on nonpermeabilized cells with mouse anti-sars-cov s. subsequently, cells were washed, fixed, permeabilized and then stained for total s protein with rabbit anti-sars-cov s. surface staining of intact cells revealed that sars-cov s and sars-cov s pn were both present at the plasma membrane (fig. 4b ). these results suggest that blocking palmitoylation does not affect the subcellular localization of sars-cov s when exogenously expressed. although both sars-cov s and s pn were present at the cell surface, it is possible that there could be a difference in the amount of protein at the plasma membrane at steady state if palmitoylation affects a post-golgi trafficking step. to determine if palmitoylation affects the steady-state levels of s protein at the plasma membrane, we performed a cell surface biotinylation assay. hek293t exogenously expressing sars-cov s or s pn were surface biotinylated at 0°c with a membrane-impermeable biotinylating reagent. biotinylated s proteins were captured with streptavidin agarose resin and analyzed by sds-page followed by western blotting. sars-cov s and s pn were both present in similar amounts at the plasma membrane, with 8-9% of the total protein expressed surface biotinylated at steady state ( fig. 4c ). previous data from our lab and others have shown that coronavirus s and m proteins can interact directly (godeke et al., 2000; hsieh et al., 2008; huang et al., 2004; mcbride et al., 2007; nguyen and hogue, 1997; youn et al., 2005) . palmitoylation has been shown to regulate protein-protein interactions (shmueli et al., 2010) . using the palmitoylation inhibitor 2-bromopalmitate (2-bp) (thorp et al., 2006) or truncation analysis (bosch et al., 2005) , it has been indirectly shown that palmitoylation of mhv s protein is necessary for interaction with mhv m. when the sars-cov s protein is exogenously expressed alone, it localizes to the cell surface. however, when co-expressed with sars-cov m, sars-cov s localizes to the golgi complex, co-localizing with m (mcbride et al., 2007) . to determine if sars-cov s palmitoylation is necessary for interaction with m, we performed indirect immunofluorescence microscopy. hek293t cells exogenously co-expressing sars-cov m and sars-cov s or s pn were fixed, permeabilized and co-stained with antibodies to sars-cov s and sars-cov m. upon co-expression with sars-cov m, s was retained intracellularly at the golgi complex and co-localized completely with m (fig. 5a) . surprisingly, sars-cov s pn was also retained intracellularly at the golgi when co-expressed with sars-cov m (fig. 5a) . palmitoylation did not affect the steady-state localization of sars-cov s when co-expressed with m by indirect immunofluorescence microscopy but might influence the trafficking of s in the absence or presence of sars-cov m. to determine if palmitoylation affects the trafficking of sars-cov s, we performed a pulse-chase assay and measured acquisition of endo h resistance. hek293t cells expressing sars-cov s or s pn in the absence or presence of sars-cov m were pulse labeled with 35 s-methionine/cysteine and chased for various times. s protein was immunoprecipitated, denatured and digested with endo h. s and s pn had similar trafficking kinetics through the secretory pathway. after 40 min of chase, 70-80% of both sars-cov s and s pn were resistant to digestion with endo h, implying that 70-80% of the pulse-labeled protein had moved past the medial golgi, presumably en route to the plasma membrane (fig. 5b) . interestingly, when co-expressed with sars-cov m, carbohydrate processing of both s and s pn was dramatically reduced and only 10-20% of the labeled protein was resistant to endo h digestion after 40 min of chase (fig. 5b) . thus, both sars-cov s and s pn interacted with m and were retained in a pre-medial golgi compartment, which is consistent with the previously published cis-golgi localization of some coronavirus m proteins (swift and machamer, 1991) . taken together, these results and previously published in vitro binding data (mcbride et al., 2007) suggest that palmitoylation of sars-cov s is not essential for interaction with sars-cov m. this reveals a potentially important difference between assembly of sars-cov and mhv. one of the best known functions of protein palmitoylation is directing incorporation into specific membrane domains that are resistant to extraction with cold triton x-100. detergent-resistant membranes (drms) are sometimes referred to as lipid rafts and are regions of membrane that are enriched in cholesterol and glycosphingolipids (brown, 2006) . drms may represent important signaling centers at the plasma membrane although they can also be found earlier in the secretory pathway at the golgi (simons and van meer, 1988) . mhv s is enriched in drms and enrichment was reduced by treating cells with 2-bp prior to isolation (thorp et al., 2006) . to determine if sars-cov s partitions into drms and if that partitioning is dependent on s protein palmitoylation, we isolated drms by floatation on a sucrose step gradient after cold triton x-100 extraction. fractions were collected and 10% of each fraction was analyzed by sds-page and western blotting with antibodies to sars-cov s. to identify which fractions contained drms, a small portion of each fraction was spotted onto nitrocellulose and incubated with hrp-cholera toxin b which binds to gm1, a glycolipid enriched in drms (brown, 2006) . gm1 was primarily present in fraction 4 for both samples (fig. 6, bottom panel) . sars-cov s was also present in fraction 4 (fig. 6 top panel) while s pn was completely absent from fractions that contained drms (fig. 6 middle panel) . amido black staining of the membrane ensured that similar amounts of total protein were present in each lane for both s and s pn (data not shown). it is important to note that the upper fully glycosylated mature form of sars-cov s was enriched in drms while the immature partially processed form of sars-cov s was present in the more dense fractions. these results demonstrate that sars-cov s palmitoylation is necessary for s partitioning into drms. approximately 8% of total s protein is at the plasma membrane at steady state by biotinylation (fig. 4c) , and an average of 15% of total s is present in detergentresistant membranes at steady state. thus, it is likely that all s at the plasma membrane is present in drms. a role for coronavirus s protein palmitoylation in membrane fusion has been suggested (bos et al., 1995; chang et al., 2000; petit et al., 2007; shulla and gallagher, 2009) . however, these studies have either used global palmitoylation inhibitors like 2-bp, which can be toxic to cells (mikic et al., 2006) , or only partial mutagenesis of palmitoylated cysteines. to determine a role for sars-cov s cytoplasmic cysteine residues in cell-cell fusion, we compared the fusion activities of sars-cov s and s pn in vero cells, which express the functional sars-cov receptor, ace2. vero cells expressing sars-cov s or s pn were briefly trypsinized to activate the protein for fusion (simmons et al., 2004) . the number of nuclei per syncytia (≥3 nuclei) was counted 24 h after trypsinization. vero cells expressing sars-cov s had extensive syncytium formation with approximately 5 nuclei per syncytia, but syncytium formation was absent in cells expressing sars-cov s pn (fig. 7) . these results suggest a critical role for s protein palmitoylation in cell-cell fusion. in conclusion, we have shown that palmitoylation is dispensable for the stability, subcellular localization and trafficking of sars-cov s. however, sars-cov s palmitoylation is important for partitioning into drms and for cell-cell fusion. most importantly, sars-cov s palmitoylation is not important for interaction with sars-cov m. sars-cov m retained non-palmitoylated s in the golgi and reduced the extent of non-palmitoylated s trafficking through the secretory pathway as well as wild-type sars-cov s. these results demonstrate a significant difference between sars-cov and mhv that may have significant implications for virus assembly and infection. protein palmitoylation is a common post-translational modification where a 16 carbon fatty acid chain is added to cysteine residues on proteins. palmitoylation can be dynamic and plays an important role in regulating protein function and activity. in fact, alterations in protein palmitoylation have been shown to affect protein stability, trafficking and subcellular localization as well as protein-protein interactions (linder and deschenes, 2007; lopez et al., 2008; resh, 2006a; roy et al., 2005; thorp et al., 2006; van itallie et al., 2005) . transmembrane proteins can be palmitoylated throughout the secretory pathway and cytoplasmic proteins can be palmitoylated at different secretory pathway membranes. additionally, many important regulatory proteins, signaling molecules and trafficking components have been shown to be palmitoylated (linder and deschenes, 2007) . in line with the important role of palmitoylation in cellular processes, global inhibition of palmitoylation has deleterious effects on cellular function (mikic et al., 2006; resh, 2006b) . in addition to the palmitoylation of endogenous cellular proteins, many viral proteins are palmitoylated. perhaps the most well-known examples of palmitoylated viral proteins are the influenza virus ha and m2 proteins (holsinger et al., 1995; sugrue et al., 1990; veit et al., 1991) . however, there are many more examples of viral protein palmitoylation including those encoded by hepatitis c virus (majeau et al., 2009 ), human immunodeficiency virus (rousso et al., 2000) , herpes simplex virus (nozawa et al., 2003) and some coronaviruses (bos et al., 1995; corse and machamer, 2002; petit et al., 2007) . inhibition or disruption of viral protein palmitoylation can have negative effects on important protein-protein interactions in the virion envelope (yu et al., 2006) , virus assembly (boscarino et al., 2008; lopez et al., 2008; majeau et al., 2009; thorp et al., 2006; ye et al., 2004) , infectivity (boscarino et al., 2008; lopez et al., 2008; rousso et al., 2000; yan et al., 2004) , subcellular protein localization (nozawa et al., 2003) and even protein stability (lopez et al., 2008) . two of the coronavirus envelope proteins, s and e, contain conserved palmitoylated cysteine residues in their cytoplasmic tails. palmitoylation of the coronavirus e protein does not seem to be important for golgi localization (boscarino et al., 2008; corse and machamer, 2002; lopez et al., 2008) or virus entry (lopez et al., 2008) but has been implicated in protein stability and efficient virus growth (boscarino et al., 2008; lopez et al., 2008) . e proteins have 2-3 potentially palmitoylated cytoplasmic cysteine residues. the work performed to determine the role of coronavirus e protein palmitoylation used mutagenesis of all potentially palmitoylated cysteine residues, which conclusively implicated or eliminated possible roles for e protein palmitoylation. coronavirus s proteins contain a cysteine-rich domain in their cytoplasmic tails with at least 7 cysteine residues (hogue and machamer, 2008) . coronavirus s protein palmitoylation has been implicated in cell-cell fusion, virus-cell fusion, virus assembly and infectivity; however, studies focusing on the role of s protein palmitoylation have so far mutated only some of the palmitoylated cysteines (petit et al., 2007; shulla and gallagher, 2009) or used pharmacological inhibitors of protein palmitoylation (thorp et al., 2006) . these methods and results led to only a partial disruption of protein palmitoylation and partial phenotypes. here, we mutated all 9 cytoplasmic cysteine residues to conclusively determine the role of sars-cov s palmitoylation. we constructed a palmitoylation-null mutant protein, sars-cov s pn , with all 9 cytoplasmic cysteine residues mutated to alanines, which was not palmitoylated (fig. 2) . the compartment in which envelope proteins become palmitoylated has not been determined for any coronavirus. when sars-cov s was exogenously expressed in cells, we identified a population of palmitoylated, endo h sensitive sars-cov s (fig. 1a) . this suggests that at least some sars-cov s protein is palmitoylated in a pre-medial golgi compartment. mhv s can also be palmitoylated in an early compartment (van berlo et al., 1987) . however, it will be important to determine if s protein palmitoylation occurs similarly during an infection when other viral proteins are present. also, we determined that palmitoylation of sars-cov s is a relatively stable post-translational modification (fig. 1b) , which appears to be common among palmitoylated viral proteins (linder and deschenes, 2007) . this does not rule out the possibility that different cysteines may have differential rates of palmitate turnover; however, it does suggest that there is always a population of s protein that is palmitoylated to some extent. unlike the mhv e protein, disruption of sars-cov s palmitoylation did not affect the stability of the protein since sars-cov s and s pn had similar half-lives when expressed exogenously (fig. 3) . however, like the e protein, sars-cov s palmitoylation does not appear to be important for subcellular localization (fig. 4) (boscarino et al., 2008; corse and machamer, 2002; linder and deschenes, 2007; lopez et al., 2008) . we confirmed previous results published for mhv and sars-cov that s palmitoylation is important for cell-cell fusion. however, we show a complete inhibition of cell-cell fusion (not a partial reduction) when cells expressed s pn (fig. 7) . this abolition of cell-cell fusion was not due to a reduction in the amount of sars-cov s pn at the plasma membrane ( fig. 4c) but possibly due to an exclusion of sars-cov s pn from drms (fig. 6) . in fact, drms have been implicated in cell-cell fusion events at the plasma membrane (mukai et al., 2009; teissier and pecheur, 2007) . although it is possible that mutating all 9 cysteine residues to alanines dramatically disrupted the conformation of the sars-cov s protein, this seems unlikely since s pn was stable, trafficked through the secretory pathway properly (fig. 5b) , had the correct subcellular localization and could still interact with the m protein (fig. 5a) . most importantly, we show that palmitoylation of sars-cov s is not necessary for interaction with the m protein. previous results from our lab suggested sars-cov s and m could interact in vitro using a recombinant sars-cov s cytoplasmic tail purified from bacteria. this recombinant s protein was not palmitoylated, yet it was able to interact with in vitro transcribed and translated sars-cov m (mcbride et al., 2007) . in that in vitro experiment, only the tail of the sars-cov s protein was used; here we confirm those results using different in vivo assays using the full length protein. we showed that sars-cov m was able to retain s pn at the golgi complex similarly to wild-type s (fig. 5a) . also, sars-cov m was able to reduce the amount of s pn carbohydrate processing (fig. 5b ) and the amount of s pn at the plasma membrane (data not shown) similar to wild-type sars-cov s. these results suggest a significant difference between mhv and sars-cov. for mhv, very low concentrations of 2-bp, which only slightly reduced the amount of mhv s palmitoylation, had dramatic results on the ability of mhv s to form a complex with m. this resulted in reduced mhv s incorporation into virions, which led to a reduction in infectious virus (thorp et al., 2006) . mhv s appears to be extremely sensitive to changes in palmitoylation levels, where as sars-cov s can better tolerate disruptions in palmitoylation. an interesting possibility is that wild-type fully palmitoylated s and palmitoylation-null s both interact equally well with m, but only partially palmitoylated s protein does not interact efficiently with m protein. it is also possible that an endogenous protein fig. 6 . sars-cov s palmitoylation is necessary for partitioning into detergent-resistant membranes. at 24 h post-transfection, detergent-resistant membranes (drms) were extracted from hek293t cells expressing sars-cov s or s pn using cold triton. drms were isolated using discontinuous density ultracentrifugation, and fractions were collected from the top. s protein was identified by western blotting (upper and middle panels) and drms were identified using hrp-cholera toxin b, which binds ganglioside gm1 (bottom panel). a representative image of 3 independent experiments is shown. was sensitive to low concentrations of 2-bp in the studies using mhv; this could have subsequently affected mhv s trafficking, localization and/or stability since none of these variables were tested after 2-bp treatment. we attempted to use virus-like particle (vlp) production to determine if sars-cov s palmitoylation was important for virus assembly, but the ability of s and s pn to be released from membranous vesicles when expressed alone in 293t cells precluded our ability to determine the contribution of palmitoylation to assembly using this assay. our results suggest that there would be no difference between s and s pn assembly into virions since both can interact equally well with m; however, it is possible that spike incorporation into virions could depend on more than interaction with m protein. even if sars-cov s pn is well incorporated into assembling virions, overall infection may be limited by the cell-cell fusion defect seen with s pn . also, recently published work suggests an unexpected role for mhv s cytoplasmic cysteine residues in virus-cell fusion (shulla and gallagher, 2009 ). here we examined the ability of palmitoylation-null sars-cov s to interact with m without complications from virus infection; however, it will also be important to determine the role of s protein palmitoylation in infected cells by inserting the mutations into an infectious clone. there is no clear consensus sequence for protein palmitoylation. palmitate adducts at different cysteines can have different dynamics (linder and deschenes, 2007) and with the large number of potentially palmitoylated cysteines in coronavirus s proteins, there could be many possibilities for differential s protein regulation based on which cysteines are palmitoylated and when. identifying palmitoylated sars-cov s cysteine residues will prove to be difficult due to the 9 potentially palmitoylated cysteines; a large number of combinatorial cysteine mutations would have to be made to fully uncover the role of each. also, mutagenesis of a palmitoylated residue could induce palmitoylation of residues that are not usually modified. although s and s pn are both present at the plasma membrane in similar amounts, s pn is excluded from drms. while this is interesting, a more important observation revolves around the amount of sars-cov s at the cell surface. based on our calculations, only 7-8% of sars-cov s protein expressed in cells is present at the plasma membrane at steady state; however, 15% of total s is present in drms. this suggests that all s present at the plasma membrane is in drms. since drms form in the late golgi, it is likely that s is enriched in these domains before trafficking to the plasma membrane. it seems that coronaviruses have evolved multiple mechanisms to control the amount and distribution of s at the plasma membrane. these mechanisms include er retrieval (lontok et al., 2004; mcbride et al., 2007) , endocytosis (lontok et al., 2004; petit et al., 2005; youn et al., 2005) , lateral protein-protein interactions (mcbride et al., 2007; opstelten et al., 1995) and sequestration in drms (thorp et al., 2006) . it is possible that too much s at the cell surface may compromise a productive infection. in support of this idea, previous work in our lab where the er retrieval and endocytosis signals of the infectious bronchitis virus (ibv) s protein were mutated in an infectious clone showed massive syncytia formation early after transfection but failed to generate any infectious virus (youn et al., 2005) . this suggests that too much s at the plasma membrane is detrimental to infection, possibly due to premature cell death. anchoring in drms insures that any s that is present at the plasma membrane is functional and fully fusion competent. thus, only small amounts of s protein at the cell surface are required to ensure efficient cell-cell fusion. it is also possible that mhv relies heavily on s protein palmitoylation for interaction with m because mhv s does not contain an er retrieval signal. the sars-cov s er retrieval signal presumably increases the possibility of s-m interaction by promoting cycling of s through the budding compartment. without this mechanism, mhv may rely on palmitoylation to present the s protein cytoplasmic tail properly for interaction with m. it will be interesting to determine the role of ibv s palmitoylation in s-m interaction since ibv s contains a canonical er retrieval signal. in conclusion, our results suggest that sars-cov s palmitoylation is important for s partitioning into drms and cell-cell fusion. however, sars-cov s palmitoylation was not necessary for s protein stability, trafficking or subcellular localization. additionally, we conclude that sars-cov s palmitoylation is not necessary for efficient interaction with m protein, which is different from previously published results for mhv (thorp et al., 2006) . this suggests there are differences in the requirements for coronavirus assembly that could translate into important differences in virus infection and spread. hek293t and vero cells were maintained in dulbecco's modified eagle's medium (dmem) (invitrogen/gibco, grand island, ny) supplemented with 10% fetal bovine serum (atlanta biologicals, lawrenceville, ga) and 0.1 mg/ml normocin (invivogen, san diego, ca) at 37°c and 5% co 2 . expression plasmids pcaggs/sars-cov s and pcaggs/sars-cov m were previously described (mcbride et al., 2007) . pcaggs/sars-cov s pn was generated using quikchange (stratgene, la jolla, ca) site-directed mutagenesis to introduce the following mutations sequentially into pcdna 3.1/sars-cov s: c1217a, c1218a, c1222a, c1223a, c1225a, c1230a, c1232a, c1235a and c1236a. the mutated region of sars-cov s pn was excised from pcdna3.1 and subcloned into pcaggs-mcs expression vector (niwa et al., 1991) using ecorv and xhoi. transient transfections were performed using fugene6 transfection reagent (roche, indianapolis, in) as per the manufacturer's instructions. when co-expressed with sars-cov m, there was a decrease in the expression level of sars-cov s and s pn . to counteract this decrease, we co-transfected s and m at a 3:1 ratio. briefly, 1 day after plating cells, 50% confluent 35 mm dishes of hek293t or vero cells were transfected with a total of 1.5 μg of dna per dish when expressing sars-cov s or s pn alone. when sars-cov s or s pn was coexpressed with sars-cov m, 2 μg of dna was used per dish: 1.5 μg pcaggs/sars-cov s or s pn and 0.5 μg sars-cov m. for detergentresistant membrane isolation, a 60% confluent 10 cm dish of hek293t cells was transfected with 12 μg of dna. rabbit anti-sars-cov s (mcbride et al., 2007) , rabbit antisars-cov m (mcbride et al., 2007) and rabbit anti-golgin 160 (hicks and machamer, 2002) polyclonal antibodies were previously described. mouse anti-sars-cov s monoclonal antibodies were from biodefense and emerging infections (bei) research resources (manassas, va). alexa 488-conjugated donkey anti-mouse igg was from invitrogen/ molecular probes (eugene, or), and texas red-conjugated donkey anti-rabbit igg was from jackson immunoresearch (westgrove, pa). horseradish peroxidase-conjugated (hrp) anti-rabbit igg was from amersham/ge healthcare (piscataway, nj). at 24 h post-transfection, hek293t cells were pulse labeled and chased as previously described (mcbride et al., 2007) . briefly, cells were starved in methionine/cysteine-free dmem, labeled for 20 min with 50 μci of 35 s methionine/cysteine, expre 35 s 35 s (perkin elmer, waltham, ma) in methionine/cysteine-free dmem per 35 mm dish and then chased for the times indicated. cells were lysed in detergent solution (50 mm tris-hcl [ph 8.0], 1% np-40, 0.4% deoxycholate, 62.5 mm edta) containing protease inhibitor cocktail (sigma, st. louis, mo). lysates were clarified for 10 min at 4°c at 16,000 ×g and sds was added to a final concentration of 0.2%. sars-cov s and s pn were immunoprecipitated with rabbit anti-sars-cov s polyclonal antibodies (mcbride et al., 2007) sds, 20% glycerol, 0.025% bromophenol blue and 5% 2-mercaptoethanol) and samples were subjected to 8% sds-page. to reduce variation in the sars-cov s and s pn half-life experiment, cells were first seeded on a 10-cm dish. the following day, cells were transfected with 12 μg of pcaggs/sars-cov s or s pn . 20 h post-transfection, cells were trypsinized and seeded onto 35 mm dishes. cells were allowed to reattach for 4 h and were then labeled as described above. labeled proteins were visualized by molecular imager fx phosphoimager (biorad) and quantified using quantity one software. at 24 h post-transfection, hek293t cells were labeled with 3 hpalmitic acid as previously described (corse and machamer, 2002) . briefly, hek293t cells were washed and incubated for 20 min in serum-free dmem. cells were labeled for 30 min at 37°c with 250 μci of 3 h-palmitic acid ([9,10-3 h(n)]-) dried under n 2 and resuspended in dmem supplemented with 10% fbs, 50 mm hepes [ph 7.2] and 1x non-essential amino acids (invitrogen/gibco, grand island, ny). a parallel dish was labeled for 30 min with 50 μci 35 s-methionine/ cysteine as described above to detect total s protein. cells were chased for various times and lysed and immunoprecipitated as described above. for endo h assays, samples were eluted and digested as described above. for all other assays, samples were eluted in 1x laemmli sample buffer. samples were subjected to 8% sds-page, gels were impregnated with 2,5 diphenyloxazole (ppo) and processed by fluorography at −80°c. hek293t cells were prepared for indirect immunofluorescence microscopy as previously described (mcbride et al., 2007) . cells were seeded onto glass coverslips treated with 1 mg/ml poly-l-lysine, mol wt n300,000 (sigma, st. louis, mo) to improve cell adherence during processing. briefly, at 24 h post-transfection hek293t cells were washed in pbs and fixed for 10 min in 3% paraformaldehyde in pbs. fixative was quenched in 10 mm glycine in pbs (pbs/gly) and cells were permeabilized for 3 min in 0.5% tx-100 in pbs/gly. cells were washed in pbs/gly and co-stained with primary antibodies diluted in 1% bovine serum albumin (bsa) in pbs/gly as follows: mouse anti-sars-cov s (1:100) and rabbit anti-golgin 160 (1:500), mouse anti-sars-cov s (1:100) and rabbit anti-sars-cov s (1:400), or mouse anti-sars-cov s (1:100) and rabbit anti-sars-cov m (1:400). cells were washed in pbs/gly and co-stained for 15 min with secondary antibodies as follows: alexa 488 donkey anti-mouse (1:500) and texas red donkey anti-rabbit (1:400). cells were washed in pbs/gly and mounted in 0.1 m n-propylgallate in glycerol. images were obtained with an axioscop microscope (zeiss, thornwood nj) equipped for epifluorescence using a sensys charge-coupled device camera (photometric, tucson, az) and ip lab software (scanalytics, vienna, va). cells were seeded onto dishes treated with 1 mg/ml poly-l-lysine mol wt n300,000 (sigma, st. louis, mo) to improve cell adherence during processing. at 24 h post-transfection, hek293t cells were washed with pbs and biotinylated in 1 mg/ml sulfo-nhs-lc-biotin (pierce/thermoscientific, rockford, il) in pbs for 30 min at 0°c. after washing in pbs, the biotinylation reaction was quenched for 3 min with pbs containing 50 mm glycine. cells were lysed in lysis buffer (10 mm hepes [ph 7.2], 0.2% np-40, 150 mm nacl) containing protease inhibitor cocktail at 0°c for 10 min. lysates were clarified for 10 min at 16,000 × g at 4°c. 10% of the sample was reserved for quantification of total s protein. biotinylated proteins were isolated overnight at 4°c using washed streptavidin agarose resin (pierce/ thermoscientific, rockford, il). streptavidin beads were washed in lysis buffer and biotinylated proteins were eluted in 1x laemmli sample buffer for 3 min at 100°c. samples were subjected to 8% sds-page then transferred to polyvinylidene di-flouride membrane (pvdf), (millipore, bedford,ma) for western blotting. pvdf membranes were blocked for 30 min in 5% non-fat dry milk in tris buffered saline with tween (tbst, 150 mm nacl, 10 mm tris-hcl [ph 7.4], 0.05% tween-20). membranes were incubated overnight at 4°c with rabbit anti-sars-cov s polyclonal antibody diluted 1:5,000 in 5% non-fat dry milk made in tbst. membranes were washed in tbst and then incubated at room temperature for 1 h with hrp-conjugated anti-rabbit igg diluted 1:10,000 in 5% non-fat dry milk made in tbst. membranes were washed in tbst then treated with hyglo chemiluminescence reagent (denville scientific, metuchen, nj) as per the manufacturer's instructions. membranes were analyzed using a versa doc imaging station (biorad, hercules, ca) and quantified using quantity one software (biorad). detergent-resistant membranes (drms) were isolated by using discontinuous density ultracentrifugation. at 24 h post-transfection, hek293t cells were washed with cold pbs and lysed in 1 ml cold 1% triton x-100 in tne (10 mm tris-hcl [ph 7.4], 150 mm nacl, 5 mm edta) for 1 h at 0°c, with occasional mixing. ten percent of the lysate was reserved for quantification of total s protein. all of the following steps were performed at 0°c: 1 ml of the lysate was mixed with 1 ml of 85% sucrose in tne and placed in a pre-cooled sw41 ultracentrifuge tube (beckman, palo alto, ca), 4 ml of 35% sucrose in tne was overlaid on the lysate containing 85% sucrose, 1 ml of 5% sucrose in tne was overlaid on the 35% sucrose and 5 ml of tne was overlaid on the 5% sucrose. samples were centrifuged for 18 h at 285,000×g at 4°c. after ultracentrifugation, drms were visible floating near the 5%/35% sucrose interface. after removal of 3.5 ml of tne from the top of the gradient, 600-μl fractions were collected. to identify fractions containing drms, 3 μl of each fraction was dot blotted onto nitrocellulose membrane and dried. after blocking for 30 min in 5% non-fat dry milk in tbst, the membrane was washed in tbst and incubated for 1 h with hrpcholera toxin b (1:25,000) (molecular probes/invitrogen) in 3% 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virion assembly and infectivity discovery of protein-palmitoylating enzymes fatty acid acylation of viral proteins in murine hepatitis virus-infected cells palmitoylation of claudins is required for efficient tight-junction localization the m2 protein of influenza a virus is acylated characterization of severe acute respiratory syndrome coronavirus membrane protein summary of probable sars cases with onset of illness from 1 sars coronavirus induces apoptosis in vero e6 cells genetic analysis of determinants for spike glycoprotein assembly into murine coronavirus virions: distinct roles for charge-rich and cysteine-rich regions of the endodomain contribution of trafficking signals in the cytoplasmic tail of the infectious bronchitis virus spike protein to virus infection palmitoylation and polymerization of hepatitis c virus ns4b protein this work was supported by national institutes of health grant r21 a1072312 (to c.e. machamer) and a ford foundation pre-doctoral diversity fellowship (to c.e. mcbride).monoclonal anti-sars-cov s protein (similar to 341c), nr-617 was obtained through the nih biodefense and emerging infections research resources repository, niaid, nih.we also thank the machamer lab for helpful discussion and comments on the manuscript. key: cord-268341-103xf3dw authors: parra, beatriz; hinton, david r.; lin, mark t.; cua, daniel j.; stohlman, stephen a. title: kinetics of cytokine mrna expression in the central nervous system following lethal and nonlethal coronavirus-induced acute encephalomyelitis date: 1997-07-07 journal: virology doi: 10.1006/viro.1997.8613 sha: doc_id: 268341 cord_uid: 103xf3dw abstract the potential role(s) of cytokines in the reduction of infectious virus and persistent viral infection in the central nervous system was examined by determining the kinetics of cytokine mrna expression following infection with the neurotropic jhm strain of mouse hepatitis virus. mice were infected with an antibody escape variant which produces a nonlethal encephalomyelitis and compared to a clonal virus population which produces a fulminant fatal encephalomyelitis. infection with both viruses induced the accumulation of mrnas associated with th1and th2-type cytokines, including ifn-γ, il-4, and il-10. peak mrna accumulations were coincident with the clearance of virus and there was no obvious differences between lethally and nonlethally infected mice. tnf-α mrna was induced more rapidly in lethally infected mice compared to mice undergoing a nonfatal encephalomyelitis. rapid transient increases in the mrnas encoding il-12, inos, il-1α, il-1β, and il-6 occurred following infection. nonlethal infections were associated with increased il-12, il-1β, and earlier expression of il-6, while lethal infections were associated with increased inos and il-1α mrna. these data suggest a rapid but differential response within the central nervous system cells to infection by different jhmv variants. however, neither the accumulation nor kinetics of induction provide evidence to distinguish lethal infections from nonlethal infections leading to a persistent infection. accumulation of both th1 and th2 cytokines in the central nervous system of jhmv-infected mice is consistent with the participation of both cytokines and cell immune effectors during resolution of acute viral-induced encephalomyelitis. introduction (wesselingh et al., 1994) . variations between viral infections resulting in cns the goal of the immune response during viral infection inflammation prompted an examination of the temporal is to limit replication via induction of both nonspecific and induction of cns cytokines during fatal and nonfatal cns specific antiviral effectors. acute viral infections of the ceninfections by variants of the jhm strain (jhmv) of mouse tral nervous system (cns) result in vigorous, but in some hepatitis virus (mhv). in immunocompromised hosts instances limited, host immune responses (sedgwick and jhmv replicates unchecked in the cns demonstrating dorries, 1991) . in contrast to responses in the periphery the importance of immune effectors in limiting cns virus where limiting virus replication can generally be carried replication ; houtman and out with minimal regard to tissue damage, within the cns fleming, 1996b; . effector checks and balances minimize inflammatory-mediated mechanisms implicated in protection and clearance of damage while limiting viral-induced cytopathology. al-jhmv from the cns include cell-mediated immunity and though a wide range of immune effectors are often induced, both neutralizing and nonneutralizing antibodies. jhmv predominant anti-viral mechanisms appear related to the provides an interesting paradigm of acute viral encephapathogenesis strategy of the individual agent. for example, litis not only because of its associated demyelination infection of mice with lymphocytic choriomeningitis virus (weiner, 1973; lampert et al., 1973) but also because induces a predominant cd8 / cytotoxic t lymphocyte (ctl) some immune effector mechanisms prevent death via response (lehmann et al., 1988) . by contrast, resolution of directly reducing cns virus replication while other immeasles virus-encephalitis in mice is mediated by cd4 / t mune effectors prevent death without significantly altercells and correlates with the local production of ifn-g ing virus replication hout-(finke et al., 1995) . finally, resolution of sindbis virus-inman and fleming, 1996b; . a duced encephalitis is related to induction of neutralizing common theme appears to be prevention of neuronal infection by reducing viral load or preventing neuronal infection, most likely via cytokines. tion and clearance of jhmv from the cns are not yet and rarely neurons (2.2v-1). these viruses contrast to the predominantly neuronotropic oblv-60 variant previously clear. the antiviral effects of cd8 / t cells appear to be due to direct lysis of infected cells; however, cd8 / and examined (pearce et al., 1994; . cd4 / t cells may also exert antiviral activity indirectly material and methods via cytokine secretion (biron, 1994) . neither infected neurons nor oligodendrocytes appear susceptible to major mice and viruses histocompatibility (mhc) class i-mediated killing in vivo, c57bl/6 mice were purchased from the jackson laboconsistent with the inability of jhmv-specific ctl to clear ratory (bar harbor, me) at 6 weeks and maintained in virus from infected oligodendroglia (stohlman et al., the university of southern california vivarium. all mice 1995b). furthermore, clearance of jhmv from the cns were used at 7 weeks of age. to produce a lethal infecis inhibited, but not abolished, in mice genetically defition, mice were infected by intracerebral inoculation (i.c.) cient in perforin-mediated cytolysis (lin et al., 1997) . with 100 pfu of the plaque-purified dm isolate of jhmv these data suggest the possibility that cytokines contrib(stohlman et al., 1982) in a volume of 32 ml. this virus has ute to either clearance or protection from jhmv infection. the plaque size and pathogenesis similar to the parental during jhmv infection of the cns there is an abrupt suckling mouse brain pool of jhmv originally described increase in mrna encoding interleukin-1 (a and b), ilby weiner (1973) and produces a lethal encephalomyeli-6, tumor necrosis factor (tnf)-a, and interferon (ifn)-g, tis with minimal demyelination apparent at the time of at the time of maximal decrease in virus replication and death. to produce a sublethal infection, mice were inmononuclear cell infiltration (pearce et al., 1994) . no ifnfected with 25 pfu of the 2.2v-1 monoclonal antibodyg mrna was detected in immunodeficient mice, sugderived neutralization-resistant variant of jhmv (fleming gesting this cytokine may be important during viral clearet al., 1986) . this variant replicates predominantly in oliance (pearce et al., 1994) . consistent with this concept, godendroglia producing a flaccid paralysis. although vimice treated with anti-ifn-g are more susceptible to ral antigen is cleared from survivors by 30 days postinfec-jhmv, while administration of ifn-g provides protection tion (p.i.), viral rna persists for at least 12 months (adami (smith et al., 1991) . il-6, tnf-a, and type 2 nitric oxide et al., 1995) . groups of at least 3 mice were sacrificed synthase (inos) have also been detected in the cns at various times p.i. immunosuppression was induced during acute jhmv infection (sun et al., 1995; by lethal irradiation (850r) 24 hr prior to infection. shamet al., 1995a; while il-1b, il-6, tnf-a, infected mice were injected i.c. with 32 ml of sterile endoand inos were detected in the cns of chronically intoxin-free phosphate-buffered saline (pbs). fected mice (sun et al., 1995) . the complex interactions of multiple immune effector mechanisms during jhmv virus titration infection may reflect both the relative immune privilege virus titers were determined by plaque assay using of the cns (sedgwick and dorries, 1991) as well as the monolayers of dbt cells as previously described (stohlspecific tropism of the virus for cns cell types. neuroman et al., 1982) . one-half of the brain was homogenized tropic mhv isolates differ in tropism and include viruses using tenbrock tissue homogenizers in 2.0 ml of dulbecwith predominant tropisms for astrocytes, microglia, and co's pbs, ph 7.4. the remaining half was taken for histooligodendroglia as well as neurons (fleming et al., 1986; pathology or rna extraction (see below). following cenperlman and reis, 1987; kyuwa and stohlman, 1990; trifugation at 1500 g for 7 min at 4њ, supernatants were pearce et al., 1994; stohlman et al., 1995a) . the balance assayed immediately or frozen at 070њ. data presented between limiting viral replication and preserving cns are the average titer of groups of three or more mice. function occasionally results in incomplete viral clearance and a persistent cns infection which may or may antibody titration not involve the continued presence of infectious virus jhmv-specific igm, igg1, and igg2a antibodies were quantitated by elisa as previously described (lin et al., 1996b) . persistence of infectious virus correlates with the 1997) using rabbit anti-mouse igm, igg1, or igg2a antipresence of ctl escape variants (pewe et al., 1996) . bodies (cappel, costa mesa, ca). concentrations of se-to understand the complex interrelationships between rum antibodies were expressed as the highest dilution encephalitis, protection, and viral clearance leading to a with o.d. values three times above background level. persistent infection of the cns, the expression of pro-neutralizing antibodies were tested in serum as preand anti-inflammatory cytokine mrnas in the cns of viously described (lin et al., 1997) . mice undergoing either lethal or sublethal jhmv infection were compared. the two jhmv chosen for study infect histology either primarily microglia and astrocytes, less frequently oligodendroglia and neurons (dm) or primarily oligoden-histopathologic analysis was performed as previously described (stohlman et al., 1995a) . briefly, tissues were droglia, much less frequently microglia and astrocytes, fixed for 3 hr in clark's solution (75% ethanol, 25% glacial with 32 p-atp-labeled internal oligonucleotide probes. membranes were washed (three times; 21 ssc, 0.1% acidic acid) and embedded in paraffin. sections were stained with hematoxylin and eosin or luxol fast blue. distri-sds; room temperature), exposed to storage phosphor screens (molecular dynamics, sunnyvale, ca), and bution of jhmv antigen was examined by immunoperoxidase staining (vectastain-abc kit; vector laboratory, burlin-scanned using a phosphorimaging scanner (molecular dynamics). game, ca) using the anti-jhmv mab j3.3 specific for the viral nucleocapsid protein (fleming et al., 1983) . radioactive signals of cytokine cdna were quantified and normalized to the house-keeping enzyme hypoxanthine phosphoriboxyltransferase (hprt) values to adjust cytokine mrna expression for differences in cdna as previously described (cua et al., 1995 (cua et al., , 1996 . the sample with the highest specific brains were processed individually to prevent contamination. rna was isolated from half brains by homogeni-activity was designated the 100% maximal response and values for the remainder were derived as percentage of zation at room temperature in guanidinium isothiocyanate using tenbrock tissue homogenizers as previously the highest value. data shown are mean values for 3-4 mice at each time point { 1 standard deviation. described (cua et al., 1995) . samples were sheared prior to centrifugation through 5.4 m cesium chloride at 100,000 g for 18 hr to isolate rna. the cdna were pre-results pared using avian myeloblastosis reverse transcriptase acute and subacute jhmv-induced encephalitis (promega, madison, wi) and oligo dt primers (promega) for 60 min at 42њ. expression of cytokine mrna was fatal encephalomyelitis induced by jhmv is associated with minimal demyelination (kyuwa and stohlman, determined by semiquantitative pcr analysis, following procedures previously described (cua et al., 1995 (cua et al., , 1996 (cua et al., ). 1990 houtman and fleming, 1996b) . this contrasts with infection by 2.2v-1 which produces an acute nonfatal en-pcr was performed using amplitaq dna polymerase (perkin-elmer, branchburg, nj) and specific cytokine cephalomyelitis with extensive demyelination (fleming et al., 1986; wang et al., 1990) . although both viruses primers for ifn-g, il-1a, il-1b, il-4, il-6, il-10, tnf-a (murphy et al., 1993; cua et al., 1996) , and il-12p40. the replicated rapidly to high titer in the cns (fig. 1a) , jhmvinfected mice succumbed within 8 days while 2.2v-1-sequences of the il-12p40 oligonucleotides primers and probe used are as follows: 5 primer, gac cct gcc infected mice underwent a subacute disease with little or no mortality (fig. 1b) . peak 2.2v-1 replication was at cat tga act ggc; 3 primer, caa cgt tgc atc cta gga tcg; oligoprobe, tgt ctg cgt gca agc tca day 3 while the peak of jhmv replication was delayed until day 5. 2.2v-1 clearance began at day 5 p.i. and by gga. amplification was carried out using 35 cycles of one denaturation step at 94њ (45 sec), primer annealing day 7 virus was nearly undetectable. by contrast, titers in jhmv-infected mice initially decreased at day 7 p.i. at 59њ (45 sec), extension step at 72њ (1.5 min), followed by a final extension step for 7 min. for il-4 and inos a and detectable virus was still present in the cns of moribound mice at day 8 p.i. (fig. 1a ). during lethal jhmv nested pcr was performed by using internal primers in a second round of pcr (25 cycles) under the conditions infection, virus replication within the cns is not reduced as rapidly as in mice which survive infection ( fig. 1a ) described above. the oligonucleotide primers used in the second pcr for il-4 were the corresponding se-consistent with the notion that rapid clearance correlates positively with protection. consistent with these findings, quences for the 5-primer and the probe described by cua et al. (1996) . the nucleotide sequence for the il-4 immunohistologic examination of the brains of jhmvinfected mice at day 7 showed abundant viral antigen oligonucleotide probe was ttg aag gag gtc aca gga gaa ggga (sideras et al., 1987) . the 5 and 3 outer in regions of encephalitis while only focal residual viral antigen was found in 2.2v-1-infected animals (fig. 2) . en-primer sequences for inos were gcc ttc cgc agc tgg gct gt and atg tgg tag cca cat ccc gag cephalitis was prominent in mice infected with either 2.2v-1 or jhmv and no differences in the amount or distri-cc, respectively (lyons et al., 1992) . internal 5 and 3 inos primers were agc tac tgg gtc aaa gac aag bution of mononuclear cell infiltrates were found at day 7 (fig. 2) . no serum neutralizing antibodies were de-agg ct and the 3 outer primer, respectively. the oligonucleotide probe consisted of the sequence ctc cct tected in either group by day 9 postinfection, even though the virus titer in the cns had declined over 3 log 10 (lin tcc gaa gtt tct ggc agc a. for quantification, pcr products were diluted in dena-et al., 1997; data not shown). in contrast to neutralizing antibodies, igm was first detected at day 5 post-2.2v-1 turing solution (0.4 n naoh, 25 mm edta), neutralized with tris-hci (1.0 m; ph 8.0), and transferred to 0.45 infection (data not shown) and both igg1 and igg2a were detected as early as 7 days p.i. (fig. 1c) . the igg1 and mm nytran membranes (schleicher & schuell, keene, nh) using a minifold i dot blot apparatus (schleicher & igg2a response suggest the absence of a shift toward either a th1-or th2-type response reported to be in-schuell). membranes were hybridized (60њ; overnight) volved in the response to sindbis virus-induced encepha-deficient mice, which showed no evidence of ifn-g mrna, suggests tnf-a may also contribute to inos litis (wesselingh et al., 1994) . mrna induction (colasanti et al., 1995; gazzinelli et al., 1993) . consistent with this notion, tnf-a mrna was first proinflammatory cytokines detected at day 3 in mice undergoing a lethal infection the mrna encoding ifn-g increased in both groups and at day 5 in mice sublethally infected (fig. 3c ). similar of mice through day 5 postinfection, consistent with the to the kinetics of ifn-g, tnf-a mrna increased until rapid accumulation of both nk and t cells in the cns of death of lethally infected mice. in mice undergoing a infected mice (williamson et al., 1991; williamson, 1992) sublethal infection, tnf-a mrna declined following the (fig. 3a) . no ifn-g mrna was detected in either shampeak of virus replication and approached baseline levels infected mice or in infected immunodeficient mice. durby 14 days p.i. ing the lethal jhmv infection ifn-g mrna did not in-similar to both tnf-a and inos, il-12 is secreted from crease between day 5 and day 7. however, in mice macrophages during the induction of cell-mediated imundergoing a sublethal infection the level of ifn-g mrna munity and protects from a number of viral infections continued to increase to day 7 and remained elevated, via a ifn-g-dependent mechanism (ozmen et al., 1995; suggesting the possibility that ifn-g is important followorange and biron, 1996) . no il-12 mrna was found foling infection with a jhmv variant tropic for oligodendroglowing sham infection; however, il-12 mrna increased lia. even though ifn-g mrna increased during the early rapidly and peaked at 3 days following both infections phase of infection, a sharp transient increase in inos (fig. 4a ). increased il-12 mrna also occurred in immu-mrna was detected at day 5 p.i. in mice with a lethal nodeficient mice at 3 days p.i., suggesting a direct reencephalomyelitis (fig. 3b) . only a slight increase was sponse to infection which may be related to the recently detected in mice undergoing subacute encephalomyelidescribed ifn-g-independent induction of il-12 (heinzel tis. interestingly, infection of immunodeficient mice with et al., 1996) . il-12 mrna levels decreased after day 3 jhmv induced the accumulation of inos mrna to apand nearly approached base line levels found in uninproximately 50% the level found in infected immunocomfected mice by 14 days p.i. petent mice, suggesting a direct response to viral infec-the il-1b mrna level found at day 1 p.i. declined by 3 days p.i., consistent with induction of an early transient tion. the increase in inos and tnf-a mrna in immunoincrease in il-1b mrna in sham-infected mice (fig. 4b ). subacute infection (fig. 4c ) and then declined but never returned to baseline. in lethally infected mice the peak il-1b mrna peaked at day 5 following sublethal infection and subsequently declined as virus was cleared of il-1a mrna was delayed (day 5 p.i.) and then declined as the animals succumbed to infection (fig. 4c ). il-6 from the cns. following a lethal infection, the quantity of il-1b mrna increased from day 3 p.i. until death. il-mrna peaked at day 5 postinfection in lethally infected mice and declined by day 7 as virus was cleared from 1a mrna peaked at day 3 p.i. in the mice undergoing a the cns (fig. 4d) . in contrast to the lethal infection, the of il-4 mrna expression following acute and subacute infections showed that the levels increased in parallel levels of il-6 mrna increased rapidly and peaked at day 3 p.i. following subacute infection. the level then through day 7 p.i. (fig. 5a ). in 2.2v-1-infected mice, the level of il-4 mrna continued to increase until day 9 p.i. declined rapidly by day 5 and had reached baseline levels by day 9 p.i. no il-6 mrna was detected in sham-and then declined slightly by day 14. infected mice, suggesting a rapid response to virus infection. very low levels of il-6 mrna were detected in im-discussion munodeficient mice infected with either virus. jhmv produces an acute cns infection associated with several immune effector mechanisms, including th2-related cytokines both cd4 / and cd8 / t cells houtman and fleming, 1996b) . kinetic analysis of cellular igg1 and igg2a virus-specific antibodies were detected in survivors of jhmv infection; however, there appeared cns infiltrations during jhmv infection of mice shows that nk cells accumulate prior to cd8 / t cells, which in to be little relationship between induction of antibody and control of jhmv infection within the cns. induction of both turn precede accumulation of cd4 / t cells and macrophages (williamson et al., 1991; williamson, 1992) . there isotypes suggest that th1 and th2 cytokines are induced by jhmv infection. the kinetics of il-10 mrna induction is no direct evidence for a role of nk cells in suppressing jhmv replication (houtman and fleming, 1996a) ; how-was of interest due to the association of il-10 with reduced th1 activity in vitro and with remission during exper-ever, cd8 / ctl appear to be critical immune effectors (williamson and stohlman, 1990; stohlman et al., 1995b) . imental allergic encephalomyelitis (kennedy et al., 1992) . il-10 mrna was first detected at day 3 p.i. in lethally recent analysis of jhmv pathogenesis in mice deficient in perforin suggests that in addition to cytolytic effectors infected mice, but not until day 5 postinfection in the cns of the mice undergoing a subacute encephalitis. however, other immune components also contribute to sterilizing immunity (lin et al., 1997) . similarly, the adoptive transfer at the time most lethally infected mice were about to succumb to infection (day 7), there was no difference in the of virus-specific cd4 / t cells to jhmv-infected mice demonstrates that some clones protect via reducing viral peak levels of il-10 mrna between the two groups. the kinetics of il-10 mrna accumulation differed between the replication (yamaguchi et al., 1991) , while others protect without reducing virus replication , groups; il-10 mrna accumulation in mice undergoing a sublethal infection was slower and remained at peak lev-suggesting that cytokines may play an important role in providing sterile immunity. els until day 9 p.i., prior to declining to near basal levels by day 14. no il10 mrna was detected in the cns of in general the kinetics of cytokine mrna expression correlated with the temporal presence of cns infiltrating sham-infected or infected immunodeficient mice. no il-4 mrna was detected following a single amplification dur-mononuclear cells. many cytokine transcripts, with the exceptions of il-12, il-1a, and il-6, were maximally ex-ing lethal or sublethal jhmv infections. however, after a second amplification, low abundant mrnas were de-pressed by 7 day p.i., near the peak inflammatory cell infiltration and during the elimination of virus from the tected (fig. 5a) . no il-4 mrna was detected in either sham-infected or infected immunosuppressed mice fol-cns (williamson et al., 1991; williamson, 1992) . previous data using the oblv-60 jhmv variant which has a selec-lowing two amplifications (data not shown). the kinetics tive tropism for neurons suggested a correlation between increasing time following subacute infection, consistent with the resolution of encephalitis. it is interesting that ifn-g induction, t cell accumulation, and reduction of virus replication (pearce et al., 1994) . the semiquantita-the cns of mice with active macrophage-mediated demyelination (day 14 p.i.) showed little evidence of tnf-tive kinetic analysis of ifn-g mrna in the cns of mice undergoing both lethal and sublethal jhmv infections a mrna, consistent with the inability of anti-tnf-a to prevent jhmv-mediated demyelination (stohlman et al., supports the positive correlation between ifn-g and viral clearance. however, the oblv-60 jhmv variant is 1995a). a surprising number of mrnas peaked relatively early cleared from the cns of ifn-g-deficient mice , consistent with ifn-g exhibiting poor in vitro following jhmv infection. the mrnas encoding inos, il-12, il-1a, il-1b, and il-6 peaked either prior to or anti-jhmv activity (zhang et al., 1997) and inability of rifn-g to inhibit cns virus replication (smith et al., 1991) . coincident with initiation of viral clearance. in most cases (except inos mrna) the levels were either higher or these data contrast with other viral-induced encephalopathies in which ifn-g plays a significant role (kundig et increased more rapidly in the mice undergoing subacute infections. accumulation of inos mrna was first de-al., 1993; finke et al., 1995) , including some (yu et al., 1996) , but not all (wesselingh et al., 1994) , neuronotropic tected in mice undergoing a lethal infection coincident with the initial detection of ifn-g mrna. however, the viruses. the kinetics of ifn-g mrna induction suggests that it may play a more prominent role in the pathogene-mrna levels declined as virus replication declined, suggesting a direct effect of virus on inos induction. in sis of jhmv variants with predominant tropisms for microglia, astrocytes (jhmv), or oligodendroglia (2.2v-1). contrast to lethal infections, inos mrna lagged detection of ifn-g in mice undergoing subacute infections and the isotype diversity of the anti-jhmv antibody response suggests that both th1 and th2 subsets of cd4 / increased to less than 50% the level detected in mice undergoing a lethal infection. similar to the recent data t cells are activated during infection. il-4 mrna accumulation in the cns corresponds to infiltration of th2 cells demonstrating low levels of inos in the cns of both nude mice and mice deficient in ifn-g , (cua et al., 1995) and kinetic analysis suggests that t cells expressing th2 cytokine profiles are recruited into inos mrna in immunodeficient mice was approximately 50% the levels detected in the cns of intact mice at day the cns with nearly equal kinetics in both lethally and sublethally infected mice (fig. 5a) . il-4 increases the 3 p.i. although jhmv is susceptible to inhibition by inos in vitro, inos is not associated with in vivo protection severity of encephalitis (ikemoto et al., 1995) and could potentially play a role in jhmv persistence via inhibition . il-12, predominantly produced by cells of the myelo-of viral clearance (moran et al., 1996) . in support of the recruitment of th2 cells, il-10 mrna also increased with monocytic lineage, is associated with the induction of th1 cd4 / t cells (brunda, 1994) . il-12 mrna peaked kinetics similar to those of ifn-g and il-4. whether this difference in detection of th2 cytokines is due to differ-early (day 3) in mice undergoing both lethal and sublethal jhmv infections. however, no significant differences ences in mouse strains or the selective tropism of the virus is not known. it is interesting that although il-10 is were found comparing mrna levels in immunodeficient mice to intact mice. this may suggest that jhmv infection secreted by activated microglia in vitro (lodge and sriram, 1996) , no il-10 mrna was detected in sham-induces transcription of il-12 mrna in cns cells. in addition, 2.2v-1 infects predominantly, but not exclu-infected or immunodeficient mice. this contrasts with other cytokine mrna detected in either sham-infected sively, oligodendroglia, while jhmv infects predominantly microglia and astrocytes. the relatively higher or virus-infected immunodeficient hosts (see below). tnf-a mrna is induced following jhmv infection level of il-12 mrna in 2.2v-1-infected mice suggests the possibility that oligodendroglia transcribe il-12 mrna in (pearce et al., 1994; stohlman et al., 1995a; sun et al., 1995) and tnf-a is present during both the acute and response to jhmv infection, similar to the induction of il-12 mrna following measles virus infection of oligo-persistent jhmv infections. tnf-a mrna is not translated in jhmv-infected cells (stohlman et al., 1995a ), al-dendroglia (yamabe et al., 1994 . during both the lethal and sublethal infections the il-though it may be secreted by adjacent but not infected cells. in addition, inhibition of tnf-a, which prevents 1a mrna peaks appear to coincide with replication and not clearance, suggesting that infection induces a rapid experimental autoimmune encephalitis (ruddle et al., 1990) , has no effect on either jhmv-induced encephalitis induction of il-1a mrna. these data contrast to the association of il-1a mrna and the clearance of the oblv-or demyelination (stohlman et al., 1995a) . as anticipated, based on the relative tropism of the two viruses analyzed, 60 variant of jhmv (pearce et al., 1994) , suggesting an additional difference in cytokine responses depending tnf-a mrna accumulated initially in the cns of mice infected with jhmv. however, by day 5 p.i. there was on the tropism of the virus analyzed. il-1b mrna, previously detected in the cns of jhmv-infected mice little difference in the levels of tnf-a mrna in the two groups. finally, the level of tnf-a mrna decreased with (pearce et al., 1994) , increased directly after infection at day 1 p.i. however, the level was approximately the same suggests it may play a positive role in reducing the extent of cns inflammation thereby inadvertently contributing as the level detected in sham-infected mice, suggesting it was induced by trauma. in all mice the levels subse-to persistent infection. some aspects of our data, i.e., the rapid induction of il-12 mrna in mice infected with 2.2v-quently dropped by day 3 p.i. the levels of il-1b mrna peaked at day 5 following 2.2v-1 infection and at day 7 1, suggest that infection of specific cell types may influence the induction of cytokine mrna (yamabe et al., following jhmv infection, suggesting il-1b mrna was also induced by infection. analysis of the levels in immu-1994) . this supports the notion that the cytokine mrna patterns more closely reflect diversity of the immune re-nodeficient mice were consistent with the notion that infection, and not immune infiltrates, contributed the ma-sponse to an individual agent, although differential secretion of cytokines following infection of unique cns cell jority of the il-1b mrna levels. il-6, another pleiotropic cytokine with numerous ef-types cannot be ruled out (benveniste, 1992; sun et al., 1995) . while the kinetics of ifn-g, il-4, and il-10 showed fects on immune responses (van snick, 1990) , was also detected early following both lethal and sublethal infec-little difference between the groups undergoing lethal or sublethal infections, mrnas encoding il-6 and il-1b tions. by contrast, il-6 mrna was also only detected at 6 days p.i. with the neuronotropic oblv-60 jhmv variant either appeared more rapidly (il-6) or accumulated to higher levels (il-1b) following infection with 2.2v-1 virus. (pearce et al., 1994) . kinetic analysis shows that the levels of il-6 mrna peaked at day 3 post-2.2v-1 infection by contrast the induction of inos and il-1a mrnas were increased in mice undergoing a lethal infection. these and at day 5 post-jhmv infection. interestingly, analysis of the mrna levels in the immunodeficient mice showed data suggest that an early induction of il-6, and possibly il-1b, are associated with sublethal infection or the dif-virtually no induction of il-6 mrna, suggesting that in contrast to il-1a and il-1b an intact immune response ferent tropisms exhibited by these two jhmv variants. however, during both infections the mrna levels de-was required for il-6 mrna induction. rapid induction of il-6 mrna following jhmv infection is consistent with creased as virus was cleared. similarly, there appears to be an inverse correlation between a rapid induction other models of viral-induced encephalitis in which it also precedes ifn-g (moskophidis et al., 1991) . although of inos mrna and sublethal disease, consistent with the recent demonstration that although inos is protective in both il-6 and il-10 are cofactors for ctl induction (chen and zlotnik, 1991; takai et al., 1988) , kinetic analysis is vitro, inhibition of inos activity in vivo appears to have no effect on jhmv pathogenesis . taken consistent with the notion that il-6, and not il-10, may be involved in the induction or recruitment of jhmv-specific together, kinetic analyses of the induction of cytokine mrna during the lethal and sublethal jhmv infections ctl. jhmv infection induces il-6 secretion from both brain endothelial cells and astrocytes following in vitro are consistent with the accumulation of both th1-and th2-associated cytokines and support the interaction of infection with jhmv (joseph et al., 1993) , consistent with data showing that it is produced by resident cns cells multiple cellular and soluble effector mechanisms whose balance may be critical in providing protection and steri-following infection with lymphocytic choriomeningitis virus (frei et al., 1989) . it is interesting that il-6 mrna lizing immunity. peaks first in mice infected with the 2.2 v-1 variant compared to jhmv, which infects a significantly larger num-acknowledgments ber of astrocytes. the rapid induction of il-6 and il-we thank wen-qiang for excellent technical assistance. this work 1b following infection with 2.2v-1 is consistent with the was supported by grant ns18146 from the national institutes of health. induction of these mrna in oligodendroglia infected by escherichia coli lipopolysaccharide and tumor necrosis factor alpha a paradigm for virus-induced demyelinating disease. trends microbiol. 5, 9-14. stimulation recovery from acute virus infection. role of cytotoxic t lymphocytes in the eliminatiation factor self-antigen-intion of lymphocytic choriomeningitis virus from spleens of mice duced th2 responses in experimental allergic encephalomyelitis (eae)-resistant mice exposure to t helper 2 cytokines in vivo before encounter with antigen selects for perforin-mediated cytolysis regulation of microglial activation t helper subsets via alterations in antigen-presenting cell function gamma interferon is a major mediator of antiviral defense in experiing and functional expression of an inducible nitric oxide synthase from a murine macrophage cell line antigenic relationships of murine coronaviruses: analysis using monoclonal antibodies to jhm (mhv-4) rus-infected mice production of random classes of immunoglobulins in brain tissue during persistent viral infection paralleled by secretion of in-weiner on the cellular source and function of interleukin 6 produced in the central nervous system in viral disease. ase chain reaction method in schistosoma mansoni infected mice an absolute and restricted requirement for il-12 in natural killer cell ifn-g production and antiviral bral toxoplasmosis is induced by in vivo neutralization of tnf-a and correlates with the down-regulated expression of inducible nitric defense the in vivo oxide synthase and other markers of macrophage activation ifng independent production of il-12 during murine endotoxemia cytokine induction during t-cell mediated clearance of mouse hepa-immunol dissociation of demyelination titis virus from neurons in vivo the astrocyte is a target cell in mice and viral clearance in congenitally immunodeficient mice infected with murine coronavirus jhm pathogenesis of mouse hepatitis virus-induced demyelination cytotoxic t cell-resistant variants are selected in a virus-induced small amounts of exogenous il-4 increase the severity of demyelinating disease grunencephalitis induced in mice by the intranasal infection of herpes simplex virus type 1 interleukin-6 induction in vitro in mouse brain endothelial cells and astrocytes allergic encephalomyelitis the immune system response by exposure to mouse hepatitis virus (mhv-4, jhm) igg1 induction factor: a single molecular entity with multiple of mice with experimental autoimmune encephalomyelitis reveals that il-10 mrna expression correlates with recovery 89-100. dependent ifn-g exerts an antiviral effect in the central nervous system but not in peripheral solid organs pathogenesis of a neurotropic of two plaque morphology variants of the jhm neurotropic strain murine coronavirus strain jhm in the central nervous system. seminar virol mechanisms of demyelination in jhm virus encephalomyelitis. electron microscopic cells prevent a lethal infection but do not inhibit virus replication tumor necrosis between the in vitro and in vivo effects of nitric oxide on a neurotropic murine coronavirus mouse hepatitis virus-specific cytotoxic t lymphocytes protect from lethal infection without eliminating virus from the central williamson hepatitis virus strain jhm virus-specific t cells in the central nervous activation of astrocytes in the spinal cord of mice chronically infected with a neurotropic coronavirus b cell stimulatory factor-2 is involved in the differentiation of yamabe 8, gene expression in measles-infected adult human glial cells protection of mice from a lethal coronavirus infection in the central induced by murine hepatitis virus, jhm strain (mhv-4) is immunologically mediated pathogenesis of demyelination induced by a mouse hepatitis virus (jhm virus) role of interferon-g in immunity to herpes simplex virus expression of gamma interferon by a coronavirus defectivealphavirus encephalitis suggests a predominant type 2 t cell response effective clearance of key: cord-258286-lodjcj8c authors: zhang, xuming; hinton, david r.; cua, daniel j.; stohlman, stephen a.; lai, michael m.c. title: expression of interferon-γ by a coronavirus defective-interfering rna vector and its effect on viral replication, spread, and pathogenicity date: 1997-07-07 journal: virology doi: 10.1006/viro.1997.8598 sha: doc_id: 258286 cord_uid: lodjcj8c abstract a defective-interfering (di) rna of the murine coronavirus mouse hepatitis virus (mhv) was developed as a vector for expressing interferon-γ (ifn-γ). the murine ifn-γ gene was cloned into the di vector under the control of an mhv transcriptional promoter and transfected into mhv-infected cells. ifn-γ was secreted into culture medium as early as 6 hr posttransfection and reached a peak level (up to 180 u/ml) at 12 hr posttransfection. the di-expressed ifn-γ (de-ifn-γ) exhibited an antiviral activity comparable to that of recombinant ifn-γ and was blocked by a neutralizing monoclonal antibody against ifn-γ. treatment of macrophages with de-ifn-γ selectively induced the expression of the cellular inducible nitric oxide synthase and the ifn-γ-inducing factor (igif) but did not affect the amounts of the mhv receptor mrna. antiviral activity was detected only when cells were pretreated with ifn-γ for 24 hr prior to infection; no inhibition of virus replication was detected when cells were treated with ifn-γ during or after infection. furthermore, addition of ifn-γ together with mhv did not prevent infection, but appeared to prevent subsequent viral spread. mhv variants with different degrees of neurovirulence in mice had correspondingly different levels of sensitivities to ifn-γ treatmentin vitro,with the most virulent strain being most resistant to ifn-γ treatment. infection of susceptible mice with de-ifn-γ-containing virus caused significantly milder disease, accompanied by more pronounced mononuclear cell infiltrates into the cns and less virus replication, than that caused by virus containing a control di vector. this study thus demonstrates the feasibility and usefulness of this mhv di vector for expressing cytokines and may provide a model for studying the role of cytokines in mhv pathogenesis. 1992). resistance to ifn-g may lead to incomplete viral clearance and contribute to the establishment of persis-interferon-g (ifn-g) is a pleiotropic cytokine produced tent infection (moskophidis et al., 1994) . by contrast, ifnby activated cd4 / and cd8 / t cells and natural killer g is also involved in inflammatory processes. ifn-g incells (trinchieri and perussia, 1985; pestka and langer, duces the expression of many other inflammatory cyto-1987; ijzermans and marquet, 1989) , which exerts both kines, such as interleukin-1 (il-1) and tumor necrosis antiviral and immunomodulatory effects. these include factor (tnf), and acts synergistically with these cytokines the activation of mononuclear phagocytes, enhancement (wong and goeddel, 1986) . the multitude of immunoof the generation of oxygen-free radicals, modulation of modulatory effects of ifn-g makes it a particularly interclass i and ii major histocompatability complex (mhc) esting cytokine for studying viral pathogenesis. in the antigen expression, and promotion of differentiation of central nervous system (cns), no cells constitutively exboth t and b cells (for reviews, see references by pestka press ifn-g. during encephalomyelitis, for example as and langer, 1987; benveniste, 1992) . it plays an ima result of mouse hepatitis virus (mhv) infection, actiportant role in the early phase of many viral infections vated nk cells and t cells which pass through the blood(wheelock, 1965; wong and goeddel, 1986; leist et al., brain barrier into the cns express ifn-g (bukowski et 1989; klavinskis et al., 1989; feducchi and carrasco, al., 1983; pearce et al., 1994) . in addition to its effects on 1991; ramsey et al., 1993; heise and virgin iv, 1995; mononuclear cells, ifn-g acts upon cells of the cns, rodriguez et al., 1995) , inhibiting the replication of a varisuch as astrocytes, microglia, and macrophages (benety of viruses prior to activation of antiviral effector cytoveniste, 1992) . toxic t lymphocyte (ctl) or antibodies. because of its mhv, a murine coronavirus, causes a variety of disantiviral activity, ifn-g has been implicated in virus cleareases in rodents, such as hepatitis, enteritis, and neuroance and resolution of viral infection (ramshaw et al., logical diseases, depending on the viral strain (cheever et al., 1949; gledhill and niven, 1955; ishida et al., 1978) . lination (stohlman et al., 1982; lai and stohlman, 1992) . may allow studies of the interaction between mhv and the host's immune system by expressing immunoregula-the dl variant derived from the parental jhmv causes an acute, fulminant, necrotizing encephalomyelitis with tory proteins at the foci of viral infection. minimal or no demyelination. by contrast, the neuroattenuated variant 2.2-v-1 derived from dl produces a nonfa-materials and methods tal encephalomyelitis with extensive demyelination virus and cells (fleming et al., 1986 (fleming et al., , 1987 wang et al., 1992) . disease outcome also depends on the genetic background, the the following virus strains were used in this study: the developmental stage, and the immunological status of neuropathogenic mhv strain jhm isolate (dl), which is the host. previous studies have shown that immunocoma large plaque variant derived from the parental jhm petent mice infected with mhv exhibited increased exstrain (stohlman et al., 1982) ; the small plaque variant pression of a number of cytokines, including il-1, il-6, ds (stohlman et al., 1982) ; the neutralization-escape mu-tnf-a, and ifn-g, in the cns at the time of viral cleartant 2.2-v-1 (fleming et al., 1987; wang et al., 1992) , and ance (pearce et al., 1994) . however, the role of these strain a59, which is both neurotropic and hepatotropic. cytokines in mhv pathogenesis is not fully understood. the murine astrocytoma cell line (dbt) (hirano et al., for example, it has been suggested that ifn-g may not 1974) and j774.1 macrophage cell line (obtained from be necessary for induction of the mhc class i molecules the american type culture collection) were used for in on neural cells in vivo (pearce et al., 1994) , a prerequisite vitro experiments. dbt cells were also used for plaque to ctl-mediated clearance (stohlman et al., 1995) . howassay. ever, ifn-g treatment ameliorates mhv-induced disease (smith et al., 1991) , suggesting that either the antiviral plasmid construction role or the immunomodulatory role of ifn-g is a critical a previously constructed plasmid p25cat (liao and component of mhv infection. lai, 1994) , which contains the plasmid bluescript (pro-mhv contains a single-strand, positive-sense rna gemega) sequence with a cat gene inserted behind an ig nome of 31 kb (lee et al., 1991) . it undergoes rapid recombisequence in the disse cdna (makino et al., 1988a) , was nation, probably due to its large rna genome and the used as the basic di vector. for cloning the murine ifnspecial properties of its rna-dependent rna polymerase g gene into the di vector, a cdna fragment containing . similarly, defective interfering (di) rnas are the complete ifn-g gene (kindly provided by dr. j. a. frequently generated in mhv-infected cells. recently, re-frelinger, university of rochester) was generated by combinant di rnas have been developed which can replipolymerase chain reaction (pcr) using a pair of primers. cate in the presence of a helper mhv (makino et al., 1988a, the 5 sense primer (5-taactagtaatctaatctaa-1991; van der most et al., 1991) . we have modified an mhv actttaaggaatgaacgctacacact-3) contains a re-di rna and developed an expression vector. this di rna striction enzyme spei site (underlined), the coronavirus contains both the 5-and the 3-ends, an internal region of intergenic sequence (in boldface), and the first 16 nucleothe parental mhv genome (makino et al., 1988b) , and an tides of the ifn-g open reading frame (orf). the 3 intergenic (ig) sequence, which is a recognition signal for antisense primer (5-tcagaattcaatcagcagcgasubgenomic mrna transcription, followed by an exoge-ctcct-3) contains the last 15 nucleotides of the ifn-g nous gene. upon transfection of this di rna into mhv-orf and a restriction enzyme ecori site (underlined). infected cells, a subgenomic mrna is synthesized and the after restriction enzyme digestion of the pcr products inserted gene expressed. this system has been used to with spei and ecori, a 0.5-kb cdna fragment was puriexpress the chloramphenicol acetyltransferase (cat) profied by low-melting-point agarose gel electrophoresis tein and the coronavirus structural protein hemagglutinin/ and directionally cloned into the spei and ecori sites of esterase (he) in mhv-infected cells (liao and lai, 1994; p25cat, resulting in pde-ifn-g (fig. 1a) . the resulting liao et al., 1995) . these proteins are expressed only in construct contains the ifn-g gene placed behind the ig infected cells during virus replication, thus providing some sequence between genes 6 and 7 (ig7) of mhv. degree of targeted gene expression. furthermore, the expressed he protein can be incorporated into virus particles, rna transcription and transfection and the expression can be detected in serial virus passages (liao et al., 1995) . thus, this di rna expression plasmid dna (pde-ifn-g) was linearized with xbai, and rna was transcribed in vitro using t7 rna polymer-system provides an alternative to an infectious full-length cdna clone, which is still not available, for studying the ase according to the manufacturer's recommended procedure (promega). rna transfection was carried out molecular biology and pathogenesis of coronaviruses. in the present study, we have used this di rna system using the dotap method (boehringer-mannheim) as described previously (zhang et al., 1994) . briefly, mono-to express the murine ifn-g gene. the expressed ifng exhibited antiviral activity, prevented virus spread in layers of dbt cells grown at approximately 70% confluence in 60-mm petri dishes were infected with mhv at vitro, and altered viral pathogenesis in mice. this system de-ifn-g rna. following centrifugation at 4000 g for 30 min, supernatants were tested for ifn-g using a sand-cells were washed with phosphate-buffered saline (pbs) and covered with 2 ml of prewarmed eagle's minimum wich elisa as previously described (cua et al., 1995) . r4-6a2 (anti-ifn-g) (american type culture collection) essential medium (mem) containing 1% newborn calf serum (intragen). five to ten micrograms of in vitro tran-serum-free hybridoma supernatant was used to coat 96well plates. biotinylated xmg-1.2 (anti-ifn-g) was ob-scribed rnas were mixed slowly with 10 ml of dotap (boehringer-mannheim) in hbs buffer (20 mm hepes; tained from pharmingen. avidin-peroxidase and o-phenylenediamine (opd) were obtained from sigma chemical 150 mm nacl; ph 7.4), and incubated at room temperature for 10 min. the mixture was then added to the cell co. recombinant ifn-g (rifn-g) (zymogen) was used as elisa standard, and the concentration of ifn-g is re-culture. the final concentration of dotap was 5 mg/ml. ported in international units per milliliter (u/ml). enzyme-linked immunosorbent assay (elisa) for ifn-g mhv replication in the presence of ifn-g to quantitate expression of ifn-g, medium was collected at 4, 6, 8, 10, 12, and 24 hr posttransfection from dbt cells were seeded at a concentration of 5 1 10 5 cells per well into 24-well plates and incubated for 24 hr dbt cells infected with jhm or a59 and transfected with at 37њ in mem containing 5% newborn calf serum. j774.1 extension. pcr products were analyzed by agarose gel electrophoresis. cells were seeded at a concentration of 5 1 10 4 cells per well into 24-well plates and incubated for 24 hr at 37њ in dulbecco's modified mem (dmem) containing 10% dot blot analysis fetal calf serum. cells were treated with various concen-rt-pcr products were quantitated using the dot blot trations of the di-expressed ifn-g (de-ifn-g) or rifn-g method previously described (murphy et al., 1993 ; cua and infected with viruses at an m.o.i. of 1, 0.1, 0. 01, or et al., 1995) . briefly, pcr-amplified cdna (10 ml) was 0.001. after virus adsorption for 1 hr, the respective medenatured in 90 ml of denaturing solution (0.4 n naoh dium with or without ifn-g was added and the cells were and 25 mm edta) for 10 min and neutralized by the incubated for the indicated periods of time. addition of an equal volume of 1 m tris-hcl, ph 8.0. samples were transferred to a nylon membrane via a isolation and detection of intracellular mrnas minifold i dot blot apparatus (schleichel and schuell), to study the effects of ifn-g treatment on the expresand the wells were washed with 51 ssc (4.38% sodium sion of cellular genes [inducible nitric oxide synthase chloride, 2.2% sodium citrate). membranes were air-dried (inos), interferon-g-inducing factor (igif), and mhv reand the cdna was fixed using a stratalinker uv oven ceptor (mhvr)], macrophage cells (j774.1) were grown (stratagene). following prehybridization [6% 101 ssc, to 90% confluence in 60-mm petri dishes and then treated 0.5% sodium dodecyl sulfate (sds), 0.1 mg/ml salmon with medium from cells expressing de-ifn-g or de-cat, sperm dna] at room temperature for 30 min, 32 p-labeled both of which had been irradiated with uv to inactivate specific probes (table 1) were added. following hybridhelper virus. at 24 and 48 hr after treatment, cells were ization at 60њ, the membranes were washed three times collected and intracellular rna was isolated as dewith 21 ssc containing 0.1% sds for 10 min, air dried, scribed previously (zhang et al., 1994) . to determine the and scanned on an ambis radioanalytic imaging system effects of mhv infection on the expression of cellular (ambis systems). total counts of each duplicate sample genes, j774.1 cells were infected with mhv-jhm virus at for inos, igif, and mhvr at each time point were noran m.o.i. of 0.01 at 24 hr after ifn-g treatment. rna was malized to the control hprt. the blots were further autoisolated at 24 hr postinfection. the rna samples were radiographed. used for synthesis of cdnas by reverse transcription (rt) with random priming hexamers (boehringer-mannheim). mice to detect individual genes, cdna pools were subjected to pcr amplification using gene-specific primers (table c57bl /6 mice were purchased at 7 weeks of age from the jackson laboratory. mice were infected with 1 1 10 5 1). the gene encoding the housekeeping enzyme hypoxanthine phosphoribosyltransferase (hprt) was used as pfu of a59 expressing de-ifn-g or de-cat. preliminary experiments showed no difference in virus replication in an internal control. the pcr was performed for 20 cycles under the following condition: 95њ for 1 min for denatur-the cns comparing parental a59 and a59 virus containing the de-cat vector. ation, 56њ for 1 min for annealing, and 72њ for 2 min for virus titers in the cns were determined by homogenization of half of the brain in pbs followed by plaque assay on monolayers of dbt cells as previously described (stohlman et al., 1995) . the remaining half of the brains were fixed in clark's solution (75% ethanol, 25% glacial acetic acid), embedded in paraffin, and stained with hematoxylin and eosin to examine the extent of encephalitis or with the immunoperoxidase method (vectastain abc kit; vector laboratories, burlingame, ca) using the anti-nucleocapsid monoclonal antibody j.3.3. (fleming et al., 1983) to determine the percentage of cuture medium from dbt cells infected with jhm virus and transfected virus-infected cells. with either de-ifn-g or de-cat rna was harvested at various time points posttransfection, and virus titers were determined by plaque assays. expression of ifn-g using an mhv di rna vector cell metabolism prior to infection or it may be that interferon acts at an early stage of viral replication. the murine ifn-g gene was cloned into the mhv di to distinguish these possibilities, the culture medium rna vector (liao et al., 1995) under the control of the harvested from jhm-infected and de-ifn-g-transfected mhv ig7 sequence. the resulting rna, de-ifn-g rna, cells late in infection was used to infect dbt cells. this was transfected into mhv-infected cells, and the producmedium contained not only jhm virus but also ifn-g tion of ifn-g in the culture medium was detected by (180 u/ml) (fig. 1) . therefore, ifn-g was present through-elisa. as shown in fig. 1b , when mhv-jhm was used out the infection, beginning with the initiation of viral as helper virus, ifn-g was secreted into the medium (20 infection. no significant differences in virus titer released u/ml) as early as 6 hr posttransfection and increased from the de-ifn-g-and de-cat-infected cells were dewith time. at 24 hr posttransfection, when cell monotected (both yielded approximately 10 6 pfu/ml) (data not layers were completely lysed, the amount of ifn-g shown). thus, ifn-g has little antiviral effect even when reached approximately 180 u/ml. when a59 was used present at the initiation of viral infection. as helper virus, the production of ifn-g was detected at in view of the known mechanisms of action of ifn-a 80 u/ml at 6 hr posttransfection and reached a maximum and -b, whose antiviral activities require preadsorption (approximately 180 u/ml) earlier (at 12 hr posttransfecto cells prior to viral infection (bianzani and autonelli, tion) (fig. 1c) , consistent with the observation that a59 1989), we examined the effects of pretreatment of cells replicates faster than jhm. these results indicated that with ifn-g prior to infection. for this study, the culture mhv di vector can be used for the production of a semedium from jhm-infected and de-ifn-g-transfected creted cytokine during mhv infection in vitro. cells was uv-irradiated to inactivate infectious virus and then used as a source of ifn-g to pretreat dbt cells. twenty-four hours later, cells were infected with jhm or replication in vitro a59 virus at m.o.i.'s ranging from 0.1 to 0.001 in the continual presence of de-ifn-g. virus titers were deter-ifn-g exerts multiple biological functions both in vitro and in vivo (trinchieri and perussia, 1985; pestka and mined at 24 hr postinfection. as shown in fig. 3a , de-ifng exhibited a slight inhibitory effect on jhm replication langer, 1987), but its effects on coronavirus infections have not been extensively examined. we first determined (approximately 1 log 10 reducation in virus titer), when an m.o.i. of 0.001 was used; similar results were obtained whether di-expressed ifn-g had antiviral effects on helper viral replication. virus titers in the medium of dbt with a59 virus (fig. 3a) , suggesting that pretreatment of cells with ifn-g prior to viral infection induces an antiviral cells infected with jhm and transfected with de-ifn-g rna were determined at various time points after infec-state. this inhibitory effect was less pronounced when higher m.o.i.'s were used (data not shown), suggesting tion and compared to de-cat rna-transfected cells. figure 2 shows that the virus titers in the presence of de-that the observed antiviral activity was weak and could be overcome by a higher virus titer. ifn-g were lower by approximately half a log 10 compared to cultures transfected with the de-cat rna. this differ-to further establish that the antiviral effect was due to the specific effects of ifn-g, the uv-inactivated de-ifn-ence was small but reproducible, suggesting that ifn-g exerts at most a weak antiviral effect. the absence of g preparation was preincubated for 2 hr with a hamster neutralizing monoclonal antibody specific for rifn-g. significant anti-viral effect of ifn-g in this system could be due to the requirement for interferon to modify host antiviral effects were completely blocked by this treatthe uv-irradiated supernatants were used either as a source of ifn-g or as a control (cat) to pretreat cells for 24 hr, and the cells were then infected with either jhm or a59 at an m.o.i. of 0.001. after virus adsorption, cells were incubated with the same supernatants for 24 hr, and the virus titers in culture medium at 24 hr postinfection were determined by a standard plaque assay. (b) neutralization assay of ifn-g. both uv-irradiated supernatants (ifn-g and cat) were incubated with 1 mg/ml of a hamster anti-ifn-g neutralizing monoclonal antibody for 2 hr at room temperature prior to being used for pretreatment of cells. subsequent procedures were the same as in (a). ment (fig. 3b ), demonstrating that ifn-g, but not the repli-log 10 , similar to the data obtained with dbt cells. thus, the absence of strong antiviral effects of ifn-g is not cation of the di vector itself, was responsible for the antiviral activity. these combined results suggest that due to nonresponsiveness of cells to ifn-g. ifn-g has a weak antiviral effect, which was evident only di rna-expressed ifn-g prevents virus spread when cells were pretreated with ifn-g prior to infection. the relatively weak antiviral effects of ifn-g also could the results described above indicated that antiviral be due to the possibility that dbt cells do not respond effects of ifn-g could be demonstrated only when cells well to ifn-g. since it is known that macrophages are were pretreated with ifn-g before viral infection and particularly sensitive to ifn-g treatment (ijzermans and when a low m.o.i. was used. they suggested the possibil-marquet, 1989), we further determined the inhibitory efity that ifn-g could prevent virus spread, if virus initially fects of ifn-g on mhv replication in an mhv-susceptible infects only a small number of cells. to establish an in macrophage cell line (j774.1). j774.1 cells were previtro model for studying the potential effects of ifn-g in treated with various concentrations of rifn-g for 24 hr preventing virus spread, uv-irradiated culture medium before and throughout virus infection. as shown in fig. from de-ifn-g-transfected cells, which contained ifn-g 4, both a59 and jhm were inhibited by rifn-g by 1 to 2 at 180 u/ml, was mixed with a very low titer of jhm virus at approximately one infectious particle in each well of a 24-well plate. cells were observed for cytopathic effects daily for 4 days and the number of fusion plaques was counted. results of these experiments are presented in table 2 . the number of plaques increased more slowly when the de-ifn-g was present (for example, from 1 plaque on day 1 to 12 plaques on day 4), as compared to those in the control wells, in which diexpressed cat preparation was used (i.e., from 1 plaque on day 1 to 30 plaques on day 2 and too numerous to count by day 3) (table 2) . initially, the plaque sizes in the presence of ifn-g were indistinguishable from those of the control wells (data not shown); however, by day 3 or 4 postinfection, while all plaques in the ifn-g-treated cultures remained of uniform size, plaques in the absence of ifn-g became numerous and heterogeneous it has been suggested that ifn-g induces a number no. of plaques c on of cellular proteins and enzymes which either act as t cells (okamura et al., 1995) . mhvr is a member of virus. one milliliter of each culture medium was then mixed with jhm the biliary glycoprotein (bgp)/carcinoembryonic antigen virus and added to the cell monolayers, so that an average of 1 pfu per well was present. (cea) family and serves as a receptor for mhv infection b each sample was quadruplicated in 4 wells of a 24-well plate. (williams et al., 1991) . treatment of cells with di-exc plaques were counted in the liquid medium using a light micropressed ifn-g for 24 hr increased the expression of inos scope. and igif mrnas. mhv infection did not affect the expresd uc, uncountable due to extensive cytopathic effects and detachment of cells. due to the rapid spread of progeny virus before ifn-g exhibited its antiviral effect (data not shown). similar results were obtained when various concentrations of rifng (50, 100, and 150 u/ml) were used, suggesting that 50 u/ml rifn-g is sufficient to prevent virus spread in vitro (data not shown). sensitivity of different jhm variants to ifn-g treatment in vitro was assessed in an effort to determine whether the ifn-g sensitivity correlates with the pathogenicity of the virus in vivo. three jhm variants with different degrees of neurovirulence were used: dl (ld 50 1-5 pfu), ds (ld 50 100-200 pfu), and 2.2-v-1 (ld 50 2000-10,000 pfu) (stohlman et al., 1982 (stohlman et al., , 1995 fleming et al., 1986 fleming et al., , 1987 . dl causes little demyelination and infects predominantly neurons whereas variant 2.2-v-1 causes extensive demyelination and infects predominantly glial cells with a particular tropism for oligodendrocytes. variant ds causes less demyelination than variant 2.2-v-1. dbt cells pretreated with ifn-g (180 u/ml) for 24 hr were infected, and the same concentrations of ifn-g were maintained throughout the infection. at 24 hr postinfection, culture medium was collected and virus titer determined by plaque assay. as shown in fig. 6 , a reduction of approximately 2.5 log 10 in pathogenicity in vivo, groups of c57bl/6 mice were infected with 1 1 10 5 pfu of a59 virus containing either de-ifn-g small numbers of perivascular and subarachnoid mononuor de-cat. preliminary experiments showed no difference clear cells, the brains of the de-ifn-g-expressing group in virus replication in cns between mice infected with pashowed widespread meningomyeloencephalitis with promrental a59 virus and those infected with a59-de-cat (data inent perivascular cuffs, infiltration of mononuclear cells not shown). at 6 days postinfection, four mice in each group into the parenchyma, and subarachnoid infiltrates (fig. 8) . were sacrificed and the brains were examined for mhv this result supports the immunostimulatory effects of ifntiter and histological changes. the remaining mice in each g. although this experiment used only a small number of group were monitored daily for survival. table 3 shows that mice, the data suggest that expression of immunomodulathere was approximately 2.4 log 10 less virus in the cns of tory molecules from the di vector can alter the pathogenemice infected with a59 expressing de-ifn-g vector comsis of mhv-induced disease. pared to the mice infected with a59 expressing de-cat vector. correspondingly, all the mice infected with de-ifng-expressing a59 survived the entire 21-day observation the molecular basis for the relative ifn resistance of different mhv strains is not yet known. previous studies this study demonstrates that the mhv di rna system have shown that the neutralization-escape mutant 2.2-vcan be utilized as a vector to express the ifn-g gene 1 of jhm strain has a single nucleotide mutation at posiand that the ifn-g protein is translated and secreted tion 3340 of the s gene, which results in a leucine to from infected cells as a biologically active molecule. phenylalanine substitution (wang et al., 1992) . whether these data represent the first successful attempt to exthis single mutation affects the sensitivity of the virus to press a mammalian cellular gene product using a coro-ifn-g remains unclear. in lymphocytic choriomeningitis navirus di rna vector. thus far, we have demonstrated virus, resistance of various virus strains to ifn-a/b or the feasibility of this di rna system for expressing a ifn-g in vitro correlates with their ability to establish prokaryotic bacterial gene cat (liao and lai, 1994) , a persistent infections in adult immunocompetent mice viral structural protein gene he (liao et al., 1995) , and (moskophidis et al., 1994) . one possibility is that ifn the mammalian cellular gene ifn-g (this report). these resistance allows enhanced viral replication and spread, studies showed a broad range of usage of this di rna facilitating exhaustion of antiviral ctl, thereby resulting system for expressing various genes of interest. in virus persistence. whether mhv utilizes a similar currently, an infectious, full-length cdna clone of mhv mechanism to modulate its infection in mice is an inter-rna is not available; therefore, it is difficult to unequivoesting issue. correlation between ifn resistance and cally elucidate the mechanism of pathogenesis of mhv viral pathogenicity has also been documented for meaat the molecular level. the development of a di rna sles virus, adenovirus, and herpes simplex virus type i expression system thus provides an alternative ap(carrigan and kehl-knox, 1990; su et al., 1990 ; kalvakoproach, allowing the expression of both viral and cellular lanu et al., 1991) . genes to be manipulated. further, this system allows the in vitro experiments showed that the di-expressed expression of heterologous gene products at the site of ifn-g had inhibitory effects on virus spread from initially viral replication. this system has an advantage over the infected cells to neighboring uninfected cells. the inhibipassive administration of cytokines for studying viral tory effect was more pronouced at a lower m.o.i., which pathogenesis, since cytokines usually have a short halfapparently allowed sufficient time for ifn-g to activate life, making it difficult to maintain high local concentraan antiviral state in adjacent uninfected cells. pretreattions at the site of infection. one drawback of the di ment of cells (astrocytoma and macrophages) with ifnsystem, however, is its limited expression. the di rna g is required to induce an antiviral state (figs. 3 and 4) , cannot be packaged beyond the fourth passage in vitro consistent with previous findings from studies of primary (data not shown). we have attempted to increase retenmouse macrophages (lucchiari et al., 1991) and other tion of the di rna via incorporation of a packaging signal. target cells (lewis, 1982) . expression of both inos and however, the expression level of the gene product was igif mrna in macrophages was induced by ifn-g. howreduced; no significant retention was found (lin and lai, ever, whether these molecules mediate the antiviral ef-1993). nevertheless, our data indicated that, during the fects of ifn-g is not clear. recently, it was demonstrated first several passages, the expression level of ifn-g was that inos expression did not play a significant role in such that a sufficiently high level of ifn-g can be mainthe pathogenesis of the mhv oblv60 strain (lane et al., tained locally at the beginning of viral infection. 1997). nevertheless, we can conclude from our study the virulence of several mhv variants correlates with that the antiviral effects of ifn-g are not mediated by their resistance to ifn-g treatment, suggesting that ifndown-regulation of mhvr. the precise mechanism of the g may play a role in the pathogenesis of mhv. an earlier antiviral effects of ifn-g will require additional studies, study analyzed the effects of ifn-g during jhm infection as there appears to be discordance between the antiviral using passive transfer of an anti-ifn-g-antibody (smith effects of no in vivo and its effects in vitro (lane et al., et al., 1991) . this treatment significantly enhanced virus 1997). replication and resulted in a higher mortality with dethe alteration of a59 neuropathogenesis by de-ifn-g creased survival times. ifn-g treatment of macrophages provides further support for the significance of ifn-g from a/j mice rendered them partially resistant to mhv3 in mhv infection. inhibition of ifn-g action by passive infection, whereas the macrophages from susceptible transfer of antibody (smith et al., 1991) enhanced virus balb/c mice did not respond to ifn-g, suggesting that replication and increased mortality, suggesting that local the resistance of mice to mhv3 infection involves the production of ifn-g by infiltrating leukocytes is a critical sensitivity of macrophages to ifn-g (lucchiari et al., component of the host response to mhv infection. in 1991; vassao et al., 1994a,b) . ifn-g was also shown to our experiments, the production of ifn-g by de-ifn-g be more effective than ifn-a/b in inducing an antiviral resulted in an exaggeration of the host response with state in macrophages infected with mhv (vassao et al., more prominent encephalitis, improved viral clearance, 1994a). these reports support the notion that ifn-g may and decreased mortality. the increased encephalitis may, in turn, induce local cytokine production and ctl play a role in mhv infection. the complete 263, 1-16. sequence (22 kilobases) of murine coronavirus gene 1 encoding the 1606-sequence as an upstream cis-acting element for coronavirus sub-1615. genomic mrna transcription a murine virus (jhm) causing disseminated encephalomyelitive-interfering rna as an expression vector: the generation of a tis with extensive destruction of myelin. i. isolation and biological pseudorecombinant mouse hepatitis virus expressing hemagglutiproperties of the virus deletion mapping of a mouse hepatiduced th2 responses in experimental allergic encephalomyelitis tis virus defective interfering rna reveals the requirement of an (eae)-resistant mice: th2-mediated suppression of autoimmune disinternal and discontiguous sequence for replication acquired interferon and tumor necrosis factor exert a synergistic blockade on immunity of a/j mice to mouse hepatitis virus 3 infection: dependence the replication of herpes simplex virus defectiveruses: analysis using monoclonal antibodies to jhm (mhv-4) virus. interfering particles of murine coronavirus: mechanism of synthesis virology primary structure and translation of a defective-interfering navirus jhm selected with monoclonal antibodies experimental demyelination induced by coronavidefective-interfering rna results from intergenic site insertion isolation and characterization of two plaque morphology variants of the jhm neurotropic strain mouse hepatitis virus-specific cytotoxic t lymphocytes protect from lethal infection without eliminating virus from the central ingitis virus to alpha/beta interferon and to gamma interferon detection of in vivo expression of interleukin-10 using a semi-quantitative polymerherpes simplex virus type i strain is associated with heightened sensitivity to alpha/beta interferon immune interferon: a pleiotropic lymphokine with multiple effects cloning of a new cytokine that induces ifn-g production by dation of coronavirus defective interfering rnas a genetic analysis of macrophage activation and specific antibodies in relation cytokine induction during t-cell-mediated clearance of mouse hepatitis virus from neurons in vivo the astrocyte is a target cell in mice persistently infected with mouse hepatitis virus, strain interferons and their actions. annu. of genetic heterogeneous mouse populations to mouse hepatitis virus infection sequence analysis of the spike protein gene of murine coronavirus variants: study of as effector molecules in the resolution of virus infection expression of cytokines by recombinant vaccinia viruses: a model leukocytes by phytohemagglutinin hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins tumour necrosis factor a and b inhibit virus replication and synergize with interferons coronavirus leader the role of gamma interferon in infection of susceptible mice with murine coronavirus, mhv-jhm gledhill, a. w., and niven, j. s. f. (1955) . latent virus as exemplified by activity. altogether, these data demonstrated that ifn-g mouse hepatitis virus (mhv). vet. rev. annotat. 1, [82] [83] [84] [85] [86] [87] [88] [89] [90] plays a critical role at least early in a59 infection. the haller, o. (1981) . inborn resistance of mice to orthomyxoviruses. curr.longer-term consequences of ever, cannot be definitively determined from this study heise, m. t., and virgin, iv, h. w. (1995) . key: cord-256036-gd53s4dv authors: sandmann, lisa; ploss, alexander title: barriers of hepatitis c virus interspecies transmission date: 2013-01-01 journal: virology doi: 10.1016/j.virol.2012.09.044 sha: doc_id: 256036 cord_uid: gd53s4dv hepatitis c virus (hcv) is a major causative agent of severe liver disease including fibrosis, cirrhosis and liver cancer. therapy has improved over the years, but continues to be associated with adverse side effects and variable success rates. furthermore, a vaccine protecting against hcv infection remains elusive. development of more effective intervention measures has been delayed by the lack of a suitable animal model. naturally, hcv infects only humans and chimpanzees. the determinants of this limited host range are poorly understood in part due to difficulties of studying hcv in cell culture. some progress has been made elucidating the barriers for the hcv lifecycle in non-permissive species which will help in the future to construct animal models for hcv infection, immunity and pathogenesis. hepatitis c virus (hcv) is a positive-sense, single stranded rna virus classified within hepacivirus genus of the flaviviridae family. hcv has a high propensity for establishing lifelong persistent infections, which are associated with a significant risk of progressive liver fibrosis and hepatocellular carcinoma. worldwide at least 130 million people are chronically infected resulting in an estimated 366,000 deaths due to cirrhosis and cancer annually (perz et al., 2006) . epidemiological data is incomplete and in the united states alone the frequency of chronic carriers may be twice as high (edlin, 2011) . thus, hcv poses a major public health threat. treatment options have improved but are still limited. furthermore, the current standard of care, consisting of a combination of pegylated interferon (ifn) alpha, the nucleoside analog, ribavirin (rbv), and one of two hcv ns3-4a protease inhibitors, boceprevir or telaprevir, is not well tolerated. prior to the approval of these directly acting antivirals (daas) roughly 50% of genotype 1 infected patients -clinically, the hardest hcv substrain to treat -who underwent treatment, cleared the infection. inclusion of a hcv protease inhibitor to the peg-ifn/rbv regiment has increased sustained virological response (svr) rates in certain clinical trial cohorts to 60-70% kwo et al., 2010; mchutchison et al., 2009; poordad et al., 2011) . currently, numerous viral and host proteins are being pursued as antiviral drug targets. second-generation protease inhibitors and several compounds interfering with the functions of the ns5a phosphoprotein and the rna-dependent rna polymerase, ns5b, are already being tested in clinical trials (yang et al., 2011) . a combination of orally administered daas with distinct mechanisms of action holds promise to boost svr rates across all hcv patient cohorts. despite these successes unexpected toxicity in late-stage clinical trials has led to the discontinuation or delays in development of some very potent compounds, including the protease inhibitor biln2061 (lamarre et al., 2003) , the nucleotide polymerase inhibitor bms-986094 (vernachio et al., 2011) , and the cyclophilin a antagonist, alisporivir (coelmont et al., 2009) . these challenges highlight the need for thorough pre-clinical testing in predictive animal models. likewise, the development of pan-genotypic prophylactic or therapeutic vaccines instrumental in containing the hcv epidemic in resourcepoor communities would be greatly accelerated by a tractable animal model. chimpanzees are the only available immunocompetent in vivo experimental system, but their use is limited by ethical concerns, restricted availability and prohibitively high costs. undoubtedly, more tangible animal models are needed not only to prioritize clinical development of vaccine and drug candidates, but also to gain deeper insights into virus-host biology. the study of hcv in conventional cell culture systems, i.e. human hepatoma cells, may not accurately reflect host responses to infection. with the advent of more sensitive detection methods for viral infection and improved technical ability to culture primary hepatocytes it is now possible to dissect hcv infection in a physiological-relevant environment (jones et al., 2010a; ploss et al., 2010) . however, even in the most advanced tissue culture platforms, including micropatterned primary hepatocyte co-cultures (mpccs; jones et al., 2010a; khetani and bhatia, 2008; ploss et al., 2010) , cultures of human fetal hepatocytes marukian et al., 2011) , or hepatocyte-like cells derived from induced pluripotentstem cells (ipscs; roelandt et al., 2012; schwartz et al., 2012; wu et al., 2012) , important features of liver biology as yet cannot be adequately recapitulated. within the liver, numerous cell types, including hepatocytes and various non-parenchymal cell subsets (i.e. liver sinusoidal endothelial cells, stellate cells, and oval cells), are arranged in an intricate three-dimensional architecture. nutrient and oxygen gradients within the liver result in a compartmentalization of the liver, referred to as zonation, affecting metabolism, detoxification and response to injury. whether those specific microenvironments impact hcv infection is not understood and may only be adequately modeled within the three-dimensional context of the liver. furthermore, a tractable animal model may be suitable to decipher mechanisms of viral persistence and pathogenesis and assess disease states and comorbidities, such as hiv, alcohol or nutritionally exacerbated viral hepatitis, or extrahepatic manifestations associated with hcv infection. the only hosts known to be naturally permissive to hcv infection are humans and chimpanzees (table 1 ). the basis for this limited species tropism is incompletely understood and the topic of this review. the chimpanzee model played an instrumental role in early characterization of non-a, non-b hepatitis (houghton, 2009) which ultimately led to the discovery of hcv as the etiologic agent for the classically defined non-a non-b hepatitis by michael houghton and his team in 1989 (choo et al., 1989) . subsequently, many important discoveries were facilitated by their use; for example it was first demonstrated in chimpanzees that in vitro transcribed rna from a hcv cdna clone was infectious (kolykhalov et al., 1997) . furthermore, experimental infection of chimpanzees with hcv helped to define the nature of protective immunity (farci et al., 1992; prince et al., 1992) and to demonstrate the significance of cellular subsets, most stringently the role of cd4 and cd8 t cells in controlling chronic hcv infection shoukry et al., 2003) . chimpanzees remain the only fully immunocompetent animal model for hcv infection and consequently, have and continue to play an important role in evaluating the preclinical efficacy of vaccine candidates (reviewed in houghton, 2011) . precedence for the efficacy of new treatment modalities, such as the ifn-free control of hcv infection with daas (olsen et al., 2011) or interference with the liver specific microrna (mir) 122 (lanford et al., 2010) was first shown in chimpanzees. despite their utility, the use of large apes in biomedical research has raised ethical concerns, which culminated in the ban of experiments conducted in chimpanzees in many countries. an nih moratorium on 'non-essential' chimpanzee research (nih, 2011) is likely to constrain hcv research in the future and has made the need for alternative animal models even more pressing. the natural host reservoir of hcv remains poorly defined. while chimpanzees can be experimentally infected with hcv, the prevalence in chimpanzees or other great apes, gorillas and orangutans in the wild is not known. in search of alternative, more readily accessible experimental models for hcv numerous species have been tested for their susceptibility to hcv. woodchucks, old-and new-world monkeys, including cynomolgus, rhesus, japanese, green monkeys, doguera (abe et al., 1993) , chacma baboons (sithebe et al., 2002) , cottontop tamarins (garson et al., 1997) and marmosets appear to be mostly resistant to hcv infection. surprisingly, tree shrews (tupaia belangeri) which are distantly related to primates (schmitz et al., 2000; xu et al., 2012) appear to support hcv infection to some level (xie et al., 1998; xu et al., 2007) (table 1 ) and were recently shown to develop signs of severe liver disease, including steatosis, fibrosis and cirrhosis (amako et al., 2010) . while these data are encouraging additional studies with larger cohorts of this outbred and thus, genetically heterogeneous species, are needed to define more clearly the natural course of hcv infection in tree shrews. genetically related viruses, which replicate more readily in non-human species have been considered as potential surrogates for the study of hcv in vivo. most prominently, gb viruses (gbv), named after the surgeon george baber (gb) who presented with acute viral hepatitis in the 1960s (deinhardt et al., 1967) , specifically gbv-b, can be transmitted to new world monkeys (bukh et al., 2001; karayiannis et al., 1989; lanford et al., 2003; schaluder et al., 1995; simons et al., 1995) . gbv-b and hcv both belong to the flaviviridae family but share only approximately 28% amino acid identity within their polyprotein, which constrains the utility of this model for testing of drugs specific to hcv or vaccine candidates formulated with hcv specific antigens. another caveat is the different course of gbv-b infection in tamarins and marmoset, which results in acute hepatitis butin contrast to hcv -does not establish chronicity. more recently, other viruses that are genetically more related to hcv than gbv-b have been detected in other species. non-primate hepaci-virus (nphv), also known as canine hepacivirus (chv) has been identified in dogs (kapoor et al., 2011) and horses (burbelo et al., 2012) . intriguingly, chv/nphv appears to have a broader tissue tropism in these species but has yet to be shown to cause hepatitis. comparative analysis of chv/nphv and hcv may help to shed light on potentially common viral and host factors determining viral host and tissue tropism. since hcv does not readily replicate in non-human species conceptually the most straightforward strategy would be to introduce the relevant tissue compartment -a human liverinto mice. to this end various approaches have been taken including transplantation of human liver pieces under the kidney capsule (ilan et al., 2002) , engraftment of ''human ectopic artificial livers'' (heals) in the peritoneal cavity (chen et al., 2011) or actual expansion of human hepatocytes within the liver parenchyma (reviewed in de jong et al. (2010) and meuleman and leroux-roels (2008b) of immunocompromised mice. for the latter approach, suitable xenorecipients combine two features; immunodeficiency to prevent graft rejection and liver injury to provide a growth stimulus for the normally quiescent hepatocytes as well as yield the transplanted human cells a competitive growth advantage over endogenous murine cells. the best-characterized liver injury mouse strains are urokinase-type plasminogen activator (upa) transgenic mice (heckel et al., 1990) and mice deficient in fumaryl acetoacetate hydrolase (fah; grompe et al., 1993) . when crossed to an immunodeficient background the livers of either line can be highly repopulated with human hepatocytes, rendering the human liver chimeric mice susceptible to hcv infection (bissig et al., 2010; mercer et al., 2001; meuleman et al., 2005) . such humanized mice have been invaluable to study basic aspects of hcv biology within the three-dimensional context of the liver and assessing preclinically the efficacy of antiviral compounds and biologics (reviewed in meuleman and leroux-roels (2008a) ). however, presumably due to their immunocompromised status hcv infected human liver chimeric mice generally do not exhibit signs of progressive liver disease, a process that is thought to be immune-mediated in humans. to overcome this caveat, engrafting components of a human immune system alongside human hepatocytes in a single recipient has been proposed (legrand et al., 2009 ). proof-of-concept for this strategy was provided in a recent study showing that mice co-injected with a mixture of human hematopoietic stem cells, fetal hepatoblasts and other nonparenchymal cells could be infected with hcv and develop signs of liver fibrosis (washburn et al., 2011) . while intriguing, human immune responses are generally weak in these reconstituted mice and additional modifications will be needed to improve both cellular complexity and functionality of the engrafted human immune system (reviewed in shultz et al. (2007) and willinger et al. (2011) ). the utility of these xenotransplantation models is hampered by low through-put, intricate logistics, high costs and technical difficulties in their production but also the inherent donor-to-donor variability and differences in the human chimerism between animals. an inbred mouse model for hcv would overcome the technical difficulties of the xenotransplantation model. mice with inheritable susceptibility to hcv could be produced fairly easily in large quantities, would be amendable to genetic manipulations on defined genetic backgrounds and viral infection could be analyzed with a vast amount of existing tools and reagents. the challenge is to elucidate and overcome restrictions for hcv to complete its lifecycle in mouse cells and mice ( fig. 1) . in recent years progress has been made in explaining aspects of the restricted host range of hcv. in the following sections, we will highlight barriers in the hcv lifecycle in non-permissive species, focusing on mice, and discuss putative strategies to overcome them. a large number of cellular factors has been implicated in facilitating hcv entry into human cells. in a coordinated multistep process (table 2 , reviewed in ploss and evans (2012) ) hcv, complexed with host lipoproteins in so called lipo-viroparticles, is thought to initially attach to glycosaminoglycans, then binds to low-density lipoprotein receptor (ldlr), the scavenger receptor class b type i (scarb1; scarselli et al., 2002) and the tetraspanin cd81 (pileri et al., 1998) on the hepatocyte surface, before engaging the tight junction proteins, claudin-1 (cldn1; evans et al., 2007) and occludin (ocln; liu et al., 2009; ploss et al., 2009) , ultimately resulting in endocytotic uptake (fig. 1 ). more recently, the receptor tyrosine kinases epidermal growth factor receptor (egfr) and ephrin receptor a2 (epha2; lupberger et al., 2011) and the cholesterol uptake receptor niemann-pick c1 like 1 (npc1l1; sainz et al., 2012) have been implicated in the viral entry process by indirectly influencing the composition of membrane microdomains. the c-type lectins liver/lymph node-specific intercellular adhesion molecule-3-grabbing integrin (l-sign, cd209l; cormier et al., 2004) , which is expressed on liver sinusoidal endothelial cells (lsecs) and the dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (dc-sign, cd209), expressed by dendritic and kupffer cells have been shown to bind to hcv e2 (gardner et al., 2003; lozach et al., 2003; pohlmann et al., 2003) . c-type lectins on sinusoidal cells may be able to trap hcv in the liver, but it is not known whether this can lead to productive infection of hepatocytes in trans or whether sequestered hcv is subjected to lysosomal degradation. other molecules such as cldn6, cldn9 (meertens et al., 2008; zheng et al., 2007) , transferrin receptor (tfr; martin and uprichard, 2010) and protocadherin 5 (pcdhb5; wong-staal et al., 2008) have been proposed to be involved in influencing hcv entry. however, cldn6, cldn9 nor pcdhb5 are expressed at appreciable levels in the liver (table 2) . thus, strong experimental evidence supporting their suggested relevance for viral entry and independent confirmation of these results still remain pending. in contrast to human hepatocytes, murine cells do not support hcv entry thereby creating a first and important barrier for a broader host tropism of the virus. in an attempt to define the determinants restricting hcv entry into non-human cells a cdna complementation screen was conducted in mouse cells ectopically expressing human cd81, scarb1 and cldn1 . human ocln was identified as the missing factor needed to facilitate viral entry into human and mouse cells. while cd81, scarb1, cldn1 and ocln are all required, only cd81 and ocln need to be of human origin to promote viral entry into mouse lymph node, liver,placenta lymph node, spleen, pancreas ã¾ ã¾ 54.5 gardner et al. (2003) cells ). these observations are in line with previous reports demonstrating that in contrast to cd81 of various non-human primate species, mouse cd81 does not support hcv pseudoparticle entry into human hepg2 hepatoma cells, which lack endogenous expression of cd81 (flint et al., 2006) . similarly, in human cells lacking endogenous ocln expression, primate sequences function equivalently to human ocln, whereas canine, hamster, mouse and rat ocln had intermediate to low activities, and guinea pig ocln was completely nonfunctional (michta et al., 2010) . for both cd81 and ocln, differences in the second extracellular loops are in part responsible for this apparent functional activity. in contrast, the residues (i32 and e48) within the first extracellular loop of cldn1 required for hcv entry (evans et al., 2007) are conserved between mice and man. thus, it is not surprising that mouse cldn1 supports hcv uptake. originally, it was shown that human but not mouse scarb1 can bind to soluble hcv e2 (scarselli et al., 2002) . nonetheless, in both in vitro ) and in vivo (dorner et al., 2011) infection assays mouse and human scarb1 were equally functional. these apparently conflicting data are likely attributable to the read-out system and highlight that hcv e2 binding may not be the most reliable proxy for functional entry assays. to date any differences in the susceptibility of cells of non-human origin to hcv entry are largely accounted for by differences in critical positions within respective orthologues of cd81, scarb1, cldn1 and ocln or their limited or absent expression. the observation that cd81 and ocln comprise the minimal set of human factors required to render mouse cells permissive to hcv entry in vitro provided the blue print for engineering a mouse that at least supports viral uptake in vivo. genetically humanized mice have been successfully constructed by expression of receptors and/or coreceptors in mice for other human pathogens, such as poliovirus (hcd155; racaniello and ren, 1994) , measles virus (hcd46/cd150; sellin and horvat, 2009), human coronavirus (hcd13; lassnig et al., 2005) , human immunodeficiency virus (hcd4/ccr5/cxcr4; klotman and notkins, 1996) and listeria monocytogenes (lecuit et al., 2001) . earlier attempts to render mice susceptible to hcv infection by transgenic expression of cd81 were not successful (masciopinto et al., 2002) . expression of human cd81 in a wide variety of tissues increased the binding of recombinant e2 to liver, thymocytes and splenic lymphocytes in comparison to non-transgenic controls, but due to the lack of human ocln human cd81-transgenic mice were resistant to hcv infection. recently, it was demonstrated that adenoviral delivery of human cd81 and ocln is sufficient to allow hcv infection of fully-immunocompetent inbred mice (dorner et al., 2011) . using intergenotypic virus chimeras expressing cre recombinase to activate a cellular reporter, in vivo uptake of diverse hcv genotypes could be conveniently quantified by bioluminescent imaging. utilizing blocking antibodies specific to cd81 or the viral envelope protein e2, expression of entry factor mutants and mice with a targeted disruption of the scarb1 gene validated uptake of hcv into murine hepatocytes in an hcv glycoprotein-mediated fashion. furthermore, it established a precedent for applying mouse genetics to dissect the viral entry process, in addition to validating the role of scarb1 for hcv uptake in vivo for the first time. beyond studying hcv entry, this model can be employed to evaluated passive and active immunization strategies (dorner et al., 2011, in press; giang et al., 2012) . the transient adenoviral delivery approach is high-throughput and allows rapid evaluation of mutant genes. however, in order to accurately reproduce the complex process of hcv entry in vivo, it will be important to achieve native expression patterns of the human hcv entry factor orthologues by using transgenic and/or knock-in approaches. recently, transgenic mice expressing human cd81, scarb1, cldn1 and ocln were generated, but did not appear to be permissive to hcv infection in vivo (hikosaka et al., 2011) . this apparent discrepancy between transient adenoviral and stable transgenic expression approaches can be in part explained by the lower level of entry factor expression in the transgenic mice and the requirement for a very sensitive reporter system to quantify viral entry (dorner et al., 2011) . as an alternative to this host adaptation approach to overcome species barriers it may be possible to adapt hcv to usage of entry factor orthologs from other species. using an unbiased selection approach, a laboratory strain of hcv was adapted to utilize mouse cd81 (bitzegeio et al., 2010) . taking advantage of the high mutational plasticity of hcv, three adaptive mutations in the viral glycoproteins e1 and e2 were identified that allowed the virus to enter cells expressing human scarb1, cldn1, ocln and mouse cd81. interestingly, the resulting murine-tropic (mt) hcv was also capable of efficiently utilizing mouse ocln and had a lower dependency on scarb1. whether mthcv is capable of infecting mouse hepatocytes in vitro or in vivo has yet to be shown. clearly, this study provides an important proof-of-concept for the validity of the viral adaptation approach but also raises a few potential caveats. the adaptive mutations within the viral envelope appear to have resulted in conformational changes. consequently, it remains to be tested whether mthcv faithfully recapitulates viral entry and raises concerns of whether the neutralizing capacity of antibodies or human specific therapeutics to block hcv entry can be adequately tested. the contribution of other host factors implicated in viral entry to the species tropism of hcv is not well understood. most human and murine orthologues share a high degree of amino acid sequence similarity (table 2 ) but, as discussed above, differences in individual residues can drastically affect viral uptake efficiency. the exact roles of egf receptor and ephrin receptor a2, which are both expressed in mouse and human hepatocytes, in this process are incompletely defined. both receptor tyrosine kinases (rtks) appear to mediate entry by regulating cd81-cldn1 co-receptor associations and viral glycoprotein-dependent membrane fusion (lupberger et al., 2011) . recently, it was shown that egfr activation is dependent on interactions between hcv and cd81 but not cldn1 (diao et al., 2012) . although, it is conceivable that expression of human egfr and/or ephrin receptor a2 could increase hcv entry efficiency in vivo their murine orthologues appear to exert sufficient functionality in genetically humanized mice. interestingly, although npc1l1 has been shown to play a role in the viral entry in human cells in vitro and in vivo (sainz et al., 2012) it appears to be dispensable for entry into mouse cells. in contrast to humans, the murine ortholog of npc1l1 has approximately 85% sequence similarity to the human protein and is abundantly expressed in the gut but not in the liver (altmann et al., 2004) . nonetheless, hcv is still capable of entering into hepatocytes of mice modified to express a combination of human cd81 and ocln (dorner et al., 2011) suggesting some level of redundancy for npc1l1 in this process. it is conceivable that hcv entry would be affected, perhaps becoming more efficient, if npc1l1 along with human cd81 and ocln was ectopically co-expressed in the liver, which remains to be experimentally tested. it was recently demonstrated that hepatic overexpression of human npc1l1 in transgenic mice (temel et al., 2007) or via adenoviral delivery modulates cellular and plasma lipid metabolism (kurano et al., 2012) . altered cellular lipid profiles can arguably affect the composition of membrane microdomains and consequently, the trafficking of membrane proteins including hcv entry factors. following receptor mediated endocytosis the viral membrane has to fuse with the endosome to eventually release the hcv rna genome from the nucleocapsid into the cytoplasm. it has been known that viral rna translation occurs in vivo, when the rna is directly introduced into mouse hepatocytes, e.g. by hydrodynamic delivery (mccaffrey et al., 2002) . this suggests that the hcv internal ribosome entry site (ires) within the 5 0 untranslated region is functional in mouse cells. recently, a genetically humanized mouse model was constructed utilizing cell culture produced recombinant hepatitis c virus to activate a cellular encoded reporter (dorner et al., 2011, in press ). in this model hcv is taken up into murine hepatocytes in a viral glycoprotein-dependent fashion providing further evidence that fusion and uncoating work efficiently in this background. the advent of hcv replicons, i.e. subgenomic or full-length genomes containing a dominant selectable marker such as neomycin amino transferase (blight et al., 2000; lohmann et al., 1999) , allowed for assessing whether hcv is capable of replicating in non-human cells. indeed, when these replicons were introduced into mouse hepatoma cell lines and embryonic fibroblasts, cell clones harboring replicating hcv rna could be identified at very low frequency (uprichard et al., 2006; zhu et al., 2003) . these data suggest that all necessary host factors are present in mouse cells and sufficiently similar to support hcv rna replication. however, it is conceivable that the viral rna replication machinery cooperates more robustly with essential human host factors than their murine counterparts. targeted and genome wide loss of function screens led to the discovery of numerous cellular factors putatively promoting or restricting hcv replication in human cells borawski et al., 2009; coller et al., 2009; jones et al., 2010b; li et al., 2009; ng et al., 2007; randall et al., 2007; reiss et al., 2011; supekova et al., 2008; tai et al., 2009; trotard et al., 2009; vaillancourt et al., 2009) . unfortunately, independent studies are often minimally overlapping and the relevance of many of these interactions to hcv biology remains to be demonstrated. strong experimental evidence has been provided for the critical role of cyclophilin a (cypa) in rna replication and virion assembly (kaul et al., 2009; yang et al., 2008) . another essential co-factor for rna replication independently identified by several groups borawski et al., 2009; reiss et al., 2011; tai et al., 2009; trotard et al., 2009) , is phosphatidylinositol 4 kinase iii (pi4kiii). pi4kiii is recruited to the membranous web via interaction with ns5a. the human and murine orthologues of cypa and pi4kiii share a high degree of sequence similarity at 98.2 and 98.6%, respectively. it is yet to be shown whether ectopic expression of human cypa and pi4kiii augments hcv rna replication in mouse cells. likewise, there is little evidence for the accumulation of adaptive mutations boosting viral rna replication in mouse cells by fostering more efficient interactions with mouse orthologues of host factors. cellular micrornas can drastically influence virus replication and pathogenesis (gottwein and cullen, 2008) . a liver specific microrna, mir-122, was shown to be critical for efficient hcv rna replication (jopling et al., 2005) . mir-122 is derived from a liver-specific noncoding polyadenylated rna transcribed from the gene hcr and interacts with two sites in the viral 5 0 non-translated region (ntr) (jopling et al., 2008 (jopling et al., , 2005 . those interactions presumably protect the 5 0 terminal viral sequences from nucleolytic degradation or from inducing innate immune responses to the rna terminus (machlin et al., 2011) , resulting in greater viral rna abundance in both infected cultured cells and in the liver of infected chimpanzees (lanford et al., 2010) . the exact sequence of mir-122 is highly conserved in vertebrate species; comparatively, mouse mir-122 is sequence-wise identical and with 50,000 copies per hepatocytes similarly expressed as the human counterpart (chang et al., 2004) . consequently, mir-122 is not likely to contribute to the restricted host tropism in non-permissive species. it is possible that cell-intrinsic, species-specific restriction factors, similar to those that control retroviral infection, such as fv1, trim5 or apobec3 cytidine deaminases (reviewed in bieniasz (2004) ), interfere with hcv rna replication in murine cells. however, heterokaryons of mouse and human cells capable of supporting the hcv lifecycle suggest that dominant negative inhibitors of hcv replication do not exist (frentzen et al., 2011) . differences in the magnitude, nature and kinetics of an antiviral response between hosts are known to restrict tropism of certain viruses, such as myxoma virus, which is only permissive in mouse cells that have impaired ifn responses . the interferon system also plays a pivotal role in limiting hcv both under physiological conditions and when triggered during therapeutic intervention. hundreds of ifn stimulated genes (isgs) are induced in the liver during hcv infection (reviewed in ) and isg products can potently inhibit hcv infection fig. 2) . pattern recognition receptors (prrs) such as toll-like receptors (tlrs)-3 and 7 in endosomes as well as a family of rig-i like receptors (rlrs), including the retinoic-acid-inducible gene i (rig-i) and the melanoma-differentiation-associated gene 5 (mda5) sense the presence of viral rnas. subsequently, host antiviral responses are activated resulting in the production of cytokines, such as type i and iii interferons (yoneyama et al., 2004) . when ifn signaling is blunted, hcv replicates more efficiently in both human hepatoma cells (blight et al., 2002) and primary human hepatocytes marukian et al., 2011) . similarly, in mouse embryonic fibroblasts with targeted disruptions in protein kinase r (pkr; chang et al., 2006) or interferon regulatory factor 3 (irf3; lin et al., 2010) rna replication is enhanced. whether disruption of these or other innate signaling pathways are sufficient to render mice expressing human hcv entry factors permissive to hcv infections has yet to be shown. hcv has devised mechanisms to counteract innate defenses but those may not work equally efficient in cells from different species. for example, ns3-4a serine protease is not only essential for processing of the hcv polyprotein but has been shown to cleave the mitochondrial associated antiviral signaling protein (mavs; meylan et al., 2005) , the t-cell protein tyrosine phosphatase (tc-ptp; brenndorfer et al., 2009 ) and tir domain-containing adapter inducing ifn (trif; li et al., 2005) thereby modulating antiviral signaling. the cleavage motifs of mavs and trif are partially conserved between mice and man/chimpanzees (fig. 2) and it remains to be demonstrated whether these antiviral evasion strategies are functional in murine cells. contrarily, mavs from certain new world monkey species, including rhesus macaques and baboons, both of which are resistant to hcv infection, is functional for interferon signaling even in the presence of heterologously expressed ns3/4a protease in human epithelial cells. thus, escape from hcv antagonism of innate immune sensing may explain at least part of hcv species restriction (patel et al., 2012) . furthermore, other aspects of hcvs antiviral evasion strategies may work less efficiently in mouse cells. for example, ns4b was shown to inhibit rlr-mediated ifn signaling through association with the stimulator of interferon genes (sting; nitta et al., in press) . similarly, overexpression data suggests that the hcv core protein interferes with ifn signaling downstream of the ifn receptor, presumably through direct interaction with stat1, leading to reduced phospho-stat1. the protein sequences of mouse and human stat1 are highly conserved ( 490% sequence identity), mouse and human sting are only ca. 60% identical (ca. 80% similar) which may result in a weaker interaction of ns4b with the murine orthologue and consequently, more exacerbated rlr signaling. the development of the infectious cell culture system, which recapitulates all parts of the viral lifecycle paved the way to study hcv assembly, egress and release. increasing evidence points to a connection between the viral lifecycle and the biogenesis of host very low-density lipoproteins (vldl) (huang et al., 2007; merz et al., 2011; ye, 2007) . very low-density viral particles can be detected in sera from hcvã¾ patients using antibodies targeting lipoproteinassociated proteins, notably apolipoproteins (apo) b and e (nielsen et al., 2006) . the role of specific apolipoproteins in virion assembly is incompletely understood. loss of function experiments have pointed towards apoa1 (mancone et al., 2011) , apob (huang et al., 2007) , and apoe (reviewed in bartenschlager et al. (2011) ) as potential players in this process. while human cells with a functional vldl pathway are capable of producing infectious hcv it was unclear whether other species would also support late stages of the hcv lifecycle. to investigate this process mouse hep56.1d cells harboring a subgenomic hcv replicon were transcomplemented with hcv core, e1, e2, p7 and ns2 proteins but only marginal amounts of infectious hcv were released . a comparative transcriptome analysis between naã¯ve mouse cells and those containing hcv replicons hinted that low levels of apoe in the replicon-containing cells might limit hcv assembly. indeed, complementation with human or mouse apoe was sufficient to rescue infectious hcv production from these cells, yielding infectious titers similar to those observed in the widely used human hepatoma cell line, huh-7.5. these data confirm the critical importance of apoe in hcv assembly and provide evidence that mouse cells can support the late stages of hcv lifecycle, if critical components of the vldl pathway are present. however, it remains unclear whether hcv can assemble in more physiologically relevant systems and importantly if mice are capable of producing infectious particles in vivo. substantial progress has been made in our understanding of the hcv lifecycle which is being translated into improved therapies. experimental systems devised to study hcv in cell culture have enabled comparative studies to elucidate the barriers that restrict hcv infection to humans and chimpanzees. while murine cells can be engineered to express the human orthologs of the entry molecules cd81 and occludin to allow entry, viral rna replication is still low in these cells. this is at least in part due to varying antiviral defenses. murine cells with an intact vldl pathway can produce infectious virions. taken together these observations raise the hope that an inbred mouse model for hcv infection can be achieved. recapitulating the entire viral lifecycle in a mouse would certainly be of great importance but it remains to be seen whether such a model would also mimic clinical relevant disease aspects. in humans pathogenesis in the context of chronic hcv infection is thought to be driven by persistent liver inflammation ultimately resulting in fibrosis, cirrhosis and liver cancer. thus, establishing hcv innate immune response to hcv infection. ch; chimpanzee, dsrna, double-stranded rna; er, endoplasmatic reticulum; hu, human; ifn, interferon; ifnar, interferon-alpha receptor; il, interleukin; irf, interferon regulatory factor; jak, janus kinase; ms, mouse; mavs, mitochondrial associated antiviral signaling protein; mda5, melanoma-differentiation-associated gene 5; pkr, protein kinase r; rh, rhesus macaque; rig-i, retinoic-acid-inducible gene i; ssrna, single-stranded rna; stat1, signal transducer and activator of transcription protein-1; stat2, signal transducer and activator of transcription protein-2; sting, stimulator of interferon genes; tlr, toll-like receptor; trif, tir-domain-containing adapter-inducing interferon; tyk, tyrosine kinase. inlet: sequence alignment of the ns3-4a cleavage motif within mavs from different permissive and non-permissive species. persistence in the presence of a functional immune system is likely necessary to model adequately hcv pathogenesis. studies characterizing chronicity in humanized mice engrafted with both human hepatocytes and components of a human immune system (washburn et al., 2011) as well as long-term follow up studies in tree shrews (amako et al., 2010 ) hint that it may become possible to dissect liver disease progression in small animal models. the course of hcv infection has been studied in great detail in chimpanzees and by and large mirrors adequately acute and chronic hcv infection in humans. whether hcv infection in other species such as inbred mice will follow a similar clinical course is unclear. however, even if hcv infections were to cause e.g. mostly acute hepatitis in mice inheritable traits rendering animals permissive to hcv could be combined with genetic backgrounds that are more prone for chronicity and liver disease progression. different approaches, including both viral and host adaptations are likely needed to construct a selection of hcv permissive animal models, which in combination may be suitable to replace the chimpanzee. a cost-effective animal model that is amendable to genetic manipulations and can be reproducibly produced at higher throughput would undoubtedly lend itself to address currently intractable problems in hcv research. lack of susceptibility of various primates and woodchucks to hepatitis c virus niemann-pick c1 like 1 protein is critical for intestinal cholesterol absorption pathogenesis of hepatitis c virus infection in tupaia belangeri expression of paramyxovirus v proteins promotes replication and spread of hepatitis c virus in cultures of primary human fetal liver cells assembly of infectious 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effectors of the type i interferon antiviral response modeling hepatitis c virus infection using human induced pluripotent stem cells current animal models: transgenic animal models for the study of measles pathogenesis memory cd8 ã¾ t cells are required for protection from persistent hepatitis c virus infection humanized mice in translational biomedical research isolation of novel virus-like sequences associated with human hepatitis lack of susceptibility of chacma baboons (papio ursinus orientalis) to hepatitis c virus infection identification of human kinases involved in hepatitis c virus replication by small interference rna library screening a functional genomic screen identifies cellular cofactors of hepatitis c virus replication hepatic niemann-pick c1-like 1 regulates biliary cholesterol concentration and is a target of ezetimibe kinases required in hepatitis c virus entry and replication highlighted by small interference rna screening replication of a hepatitis c virus replicon clone in mouse cells identification of a lipid kinase as a host factor involved in hepatitis c virus rna replication inx-08189, a phosphoramidate prodrug of 6-o-methyl-2 0 -c-methyl guanosine, is a potent inhibitor of hepatitis c virus replication with excellent pharmacokinetic and pharmacodynamic properties disruption of erk-dependent type i interferon induction breaks the myxoma virus species barrier a humanized mouse model to study hepatitis c virus infection, immune response, and liver disease improving human hemato-lymphoid-system mice by cytokine knock-in gene replacement characterization of the role of the protocadherin b famility in hcv entry productive hepatitis c virus infection of stem cell-derived hepatocytes reveals a critical transition to viral permissiveness during differentiation transmission of hepatitis c virus infection to tree shrews evaluating the phylogenetic position of chinese tree shrew (tupaia belangeri chinensis) based on complete mitochondrial genome: implication for using tree shrew as an alternative experimental animal to primates in biomedical research efficient infection of tree shrew (tupaia belangeri) with hepatitis c virus grown in cell culture or from patient plasma cyclophilin a is an essential cofactor for hepatitis c virus infection and the principal mediator of cyclosporine resistance in vitro anti-hcv drugs in the pipeline reliance of host cholesterol metabolic pathways for the life cycle of hepatitis c virus the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses claudin-6 and claudin-9 function as additional coreceptors for hepatitis c virus replication of hepatitis c virus subgenomes in nonhepatic epithelial and mouse hepatoma cells the authors thank dr. cynthia de la fuente for critical discussions and editing. l.s. is a fellow of the german national merit foundation and is supported by a stipend from the organization of supporters of the hannover medical school (gesellschaft der freunde der medizinischen hochschule hannover e.v.). a.p. is a recipient of the astellas young investigator award of the infectious disease society of america and a liver scholar award from the american liver foundation. work in the laboratory is supported in part by the starr foundation, the greenberg medical institute, the national institutes of health and the bill and melinda gates foundation. the funding sources were not involved in the writing of the report. key: cord-267377-wyhsxj6g authors: edwards, michael c.; weiland, john j. title: coat protein expression strategy of oat blue dwarf virus() date: 2014-01-14 journal: virology doi: 10.1016/j.virol.2013.12.018 sha: doc_id: 267377 cord_uid: wyhsxj6g oat blue dwarf virus (obdv) is a member of the genus marafivirus whose genome encodes a 227 kda polyprotein (p227) ostensibly processed post-translationally into its functional components. encoded near the 3' terminus and coterminal with the p227 orf are orfs specifying major and minor capsid proteins (cp). since the cp expression strategy of marafiviruses has not been thoroughly investigated, we produced a series of point mutants in the obdv cp encoding gene and examined expression in protoplasts. results support a model in which the 21 kda major cp is the product of direct translation of a sgrna, while the 24 kda minor cp is a cleavage product derived from both the polyprotein and a larger ~26 kda precursor translated directly from the sgrna. cleavage occurs at an lxg[g/a] motif conserved in many viruses that use papain-like proteases for polyprotein processing and protection against degradation via the ubiquitin-proteasome system. members of the genus marafivirus are alpha-like plant viruses (goldbach et al., 1991; rozanov et al., 1992) belonging to the family tymoviridae in the order tymovirales. these small, isometric, positive stranded rna viruses have approximately 6.3-6.8 kb genomes that are similar in organization to those of the tymoviruses and encode large polyproteins with methyltransferase, helicase, and polymerase motifs (reviewed in dreher et al., 2011) . while the tymovirus coat protein (cp) is encoded in a separate open reading frame (orf) just downstream of the polyprotein orf, marafivirus cp-encoding sequences are nested within, and are 3 0 co-terminal with, the orf encoding the polyprotein. the tymovirus genome expression strategy has been shown to employ a papain-like protease to process the large precursor polyprotein, while the cp is translated directly from a subgenomic rna (dreher et al., 2011) . a highly conserved core sequence known as the tymobox serves as a promoter for transcription of this sgrna (ding et al., 1990) . precise details of marafivirus genome expression have not been demonstrated, although edwards et al. (1997) proposed a model for expression of the oat blue dwarf virus (obdv) genome based in part on its genomic similarities with tymoviruses. marafiviruses possess a sequence analogous to the highly conserved tymobox subgenomic promoter sequence, known as the marafibox (izadpanah et al., 2002) , which is presumed to have a similar function. virions of marafiviruses, however, contain a major cp of 21 kda and a minor cp of 24 kda that differ only by an amino terminal extension present in the minor cp, whereas tymoviral particles contain a single cp. because these cps likely have a role in transmissibility of leafhopper-borne marafiviruses and the fact that there are structural differences between marafivirus and tymovirus genomes, the cp expression strategy of marafiviruses is an important feature to characterize. we now report the use of mutation analysis with an infectious obdv clone to dissect and analyze cp expression of obdv as a first step toward a better understanding of marafivirus gene expression. we also map the initial nucleotide at the 5 0 -terminus of the sgrna encoding the cps and show that it is consistent in position relative to the tymobox/ marafibox with that of sgrna termini of the tymoviruses. the obdv genomic region targeted to investigate cp expression and the mutants used in this investigation are shown in fig. 1 . our strategy employed mutation to create premature stop codons, disrupt proposed initiation codons and a potential protease cleavage site, as well as to disrupt the putative sgrna promoter sequence. inoculation of oat leaf protoplasts with obdv wild type transcripts (obdv-2r, genbank #gu396990) resulted in the accumulation after 24 h of readily detectable amounts of viral cp and grna þsgrna on western and northern blots, respectively. infection of protoplasts by obdv was confirmed using fluorescence microscopy to visualize a variant in which the entire major cp was replaced by the enhanced green fluorescent protein gene (not shown). initiation point for sgrna transcription and role of sgrna in cp production alignment of various tymo/marafibox sequences (a hallmark of the tymoviridae and putative promoter for sgrna synthesis of tymoand marafiviruses) reveals conservation of an adenine nucleotide $10 nt downstream from the 3 0 -edge of the core sequence (fig. 2) . this is the known 5 0 -end nucleotide for the sgrnas of turnip yellow mosaic virus (tymv), ononis yellow mosaic virus (oymv), and kennedya yellow mosaic virus (kymv) (ding et al., 1990; guilley and briand, 1978) . to determine whether the conserved a in that position in obdv (a 5573 ) is the 5 0 -terminal nucleotide of the obdv sgrna, 5 0 -race was performed on size-fractionated rna extracted from infected protoplasts and the resulting amplicons were sequenced (not shown). sequence data showed that a 5573 represents the 5 0 -end of the obdv sgrna encoding the viral cps and is the likely start site of transcription for this 939 nt sgrna (excluding the poly a tail). obdv is the first marafivirus for which this has been determined, thus providing the first experimental evidence that this highly conserved adenine nucleotide likely represents the 5 0 -terminus of other marafivirus sgrnas as well. these results are consistent with the evolutionary homology of the tymobox/marafibox sequence and its putative role as promoter of sgrna synthesis. to confirm this role, silent mutations (in relation to the polyprotein) were made in the marafibox in positions analogous the genomic rna possesses a 5 0 cap structure (star) and a 3 0 -poly a tail (a n ). the single, large orf encodes a polyprotein with domains specifying methyltransferase (mtr), protease (pro), helicase/ntpase (hel), and polymerase (pol) activities fused to the sequence encoding the cps. the major and minor coat proteins map to the same cp sequence and size differences between them are potentially determined by translation initiation at two different start codons (minor, aug 5581 and major, aug 5710 ). whereas the major cp is postulated to be translated primarily from sgrna, the minor cp could be produced by translation directly from the sgrna, by cleavage after gly-gly(▼) of either or both the large polyprotein or a protein initiated at aug 5581 , or by both direct translation and cleavage. the putative promoter for sgrna synthesis, the marafibox, is underlined and the start site for sgrna synthesis (this work) is indicated by the bent arrow. sites where premature termination codons were introduced are indicated by asterisks. mutant names are shown above the corresponding sequence locations, and amino acid changes in potential initiation and protease cleavage site codons are shown under the sequence. (b) mutant description and nomenclature. the mutant name is followed by indication of nucleotide/codon substitutions and the cognate amino acid change. mutant ijmn-9 represents a double mutant altering both the putative minor cp initiation codon and the potential cleavage site. to those known to severely reduce production of sgrna in tymv (schirawski et al., 2000) . mutation of two key nucleotides (mutant ab15-25) reduced transcription of the sgrna to an undetectable level even after 40 h of incubation, with a concomitant large reduction in accumulation of both forms of cp (fig. 2) . production of neither cp was completely eliminated, suggesting that either sgrna production was not completely shut down or that a low level of translation from grna occurred. an extraneous band migrating slightly faster than the minor cp appeared in some instances on western blots, perhaps representing a conformational variant (edwards et al., 1997) . interestingly, grna accumulation was also significantly reduced relative to that of wild type. this reduction appeared to be due to the loss of the major cp and not to the marafibox mutations, a view supported by similar reductions in grna accumulation for two other mutants whose ability to produce major cp was severely compromised (gh1-7 premature termination mutant and kl1-3 initiation codon mutant; fig. 2 ). the predicted initiation codons for the major and minor cps are functional in vivo as we noted previously (edwards et al., 1997) , two initiation codons (aug 5581 and aug 5710 ) exist with the potential to encode the minor and major cps, respectively. the possible roles of these codons in the production of the obdv cps were investigated using a combination of site-directed mutants designed to prevent translation either through introduction of premature stop codons or elimination of initiation codons aug 5581 and aug 5710 . results with point mutants generating premature stop codons (cd3-1, ef2-2, and gh1-7) confirmed and expanded our previous model for the origins of the major and minor cps of obdv. when a stop codon (uaa 5731 ) was placed downstream of the major cp initiation codon (mutant gh1-7), neither cp was detectable after 24 h (fig. 3 ) and grna accumulation was greatly reduced (figs. 2 and 3). however, a trace amount of major cp was detectable after 40 h (fig. 2) , possibly due to uaa suppression from this highly translated orf (lao et al., 2009) . with the stop codon (uag 5599 ) placed upstream of the major cp initiation codon (mutant ef2-2), abundant major cp was produced without detectable minor cp (fig. 3) . that the accumulation of grna and major cp was restored to near wild type levels in mutant ef2-2 suggests that the reduction of grna accumulation in mutant gh1-7 was linked to the absence of major cp expression. mutant cd3-1 (uaa 5578 ) positions the stop codon upstream of both the candidate start codon for the minor cp as well as the start codon for the major cp and thus precludes translation of either cp as a part of the polyprotein; both a minor and major cp are produced by this mutant (not shown). thus, complete translation of the entire polyprotein orf is not an absolute requirement for the production of either cp species, and aug 5581 is the probable initiation codon for the production of at least some fraction of the minor cp pool despite its close proximity to the 5 0 terminus of the sgrna. although such a short leader (8 nt for obdv, as close as 1 nt for other marafiviruses) is unusual, initiation of translation at aug codons positioned very close to the 5 0 end has been observed in fig. 2 . effects of marafibox and major coat protein (cp) gene mutations on accumulation of viral cps, and genomic and sgrnas. (a) nucleotide sequences from known (obdv, mrfv, belv, csdav) and proposed (swmv, blvs, gamav, grvfv, gsyv-1) marafiviruses aligned with those of pnmv (unassigned) and tymv (type member of the genus tymovirus). silent substitutions (u4 c, g 4a) introduced to produce mutant obdv-ab15-25 are shown and the adenine position representing the proposed start site of sgrna synthesis is indicated (bent arrow); underlined adenine residues below the horizontal arrow have been experimentally confirmed as sgrna 5 0 -ends. (b) accumulation of marafibox and major cp mutants in oat protoplasts. oat protoplasts were inoculated with capped transcripts of wild type clone pobdv, major cp mutants gh1-7 (premature termination mutant) and kl1-3 (initiation codon mutant), and ab15-25 (marafibox mutant) and incubated for 40 h. extracted viral rna and protein were detected on northern and western blots using a 3 0 -end dsdna probe and anti-obdv antiserum, respectively. the locations of major cp (maj. cp), minor cp (min. cp), genomic (grna), and subgenomic rna (sgrna), on the blots are shown. fig. 3 . accumulation of viral coat proteins (cps) and rna in oat protoplasts inoculated with premature termination and initiation codon mutants of the cp gene. oat protoplasts were inoculated with capped transcripts of wild type clone pobdv, premature termination mutants gh1-7 and ef2-2, and initiation codon mutants ij4-7 (minor cp), and kl1-3 and kl2-5 (major cp) and incubated for 24 h. methods for detection of viral protein and rna were as described in the legend for other biological systems (li and wang, 2004; elfakess and dikstein, 2008) . additional mutants were made to confirm the use of aug 5581 and aug 5710 as initiation codons in the production of minor cp and major cp, respectively. mutation of aug 5710 to uua (kl1-3) or uug (kl2-5) abolished accumulation of major cp in protoplasts, as expected, but also greatly reduced accumulation of viral rna (fig. 3) . however, near wild-type levels of viral rna and the major cp, along with reduced levels of the minor cp, accumulated in protoplasts inoculated with mutants ij4-7 and ij2-1, in which aug 5581 was replaced by gua or gug, respectively (fig. 3, ij2-1 not shown) . this supports the notion that the minor cp of obdv can be produced via proteolytic cleavage from the large polyprotein. proteolytic cleavage plays a role in formation of the minor cp species previous peptide sequence evidence indicated the presence of a potential papain-like protease cleavage site located immediately following gly 1834 of the obdv polyprotein (edwards et al., 1997) . taken together with the experimental evidence above, it is thus conceivable that functional minor cp might be produced via proteolytic cleavage from the large polyprotein precursor. to further investigate this possibility, the candidate cleavage site was changed from gly-gly 1834 to val-val 1834 (mutant mn6-1; fig. 4 ). typical accumulation of major cp was observed for this mutant, but the apparent size of the minor cp increased slightly, suggesting production exclusively from translation initiating at aug 5581 (fig. 4) . a similar result was obtained when gly-gly 1834 was changed to gly-val 1834 in mutant mn2-1 (not shown). when both the putative cleavage and initiation sites were knocked out by changing aug 5581 to gua in the mn6-1 background (double mutants ijmn9a and b), only major cp was detected in infected protoplasts. when combined with the results from premature stop codon mutant cd3-1 and initiation codon mutant ij4-7, it is clear the minor cp can be produced by cleavage of either or both the polyprotein and the minor cp originating from sgrna translation. the empirical evidence offered here, taken together with previous sequence analysis by edwards et al. (1997) , supports a model for obdv cp expression in which the major cp is translated directly from the 939 nt sgrna (excluding the polya tail), while the minor cp is cleaved from both the polyprotein and a minor cp precursor translated from the sgrna. the underlying reason for the existence of multiple means of expression of the minor cp is not evident. while cleavage from the replicase polyprotein provides a source of minor cp, it may be that production of minor cp exclusively through this mechanism doesn 0 t provide stoichiometric amounts necessary for virion assembly. it is also possible that viruses such as obdv are in an evolutionary transition toward cp production solely via sgrnas and that readthrough of the larger replicase polyprotein is vestigial. in any case, production of the minor cp through a cleavage mechanism provides a regulatory feature with probable functional significance for both replication and encapsidation. ultimately, the complexity of cp expression relative to that of tymoviruses may relate in some way to the infection by obdv of both plant and insect hosts. to consider this model in the larger context of cp expression in the marafiviruses as a group, amino acid sequences of known and proposed marafiviruses were aligned and compared (fig. 5) . in all cases, putative initiator methionine residues for both minor and major cps are evident. the putative initiator met for the minor cp is 5-15 amino acids downstream of the highly conserved gell motif that is encoded by the core sequence of the marafibox. with the exception of maize rayado fino virus (mrfv), switchgrass mosaic virus (swmv), and bermudagrass etched-line virus (belv), a conserved motif comprised of lxgg is present 7-8 amino acids downstream of the initiator met. since this incorporates the demonstrated cleavage site that creates the amino terminus of the obdv minor cp, it is reasonable to speculate that production of these other marafivirus minor cps also involves cleavage at this site. a similar motif is present immediately upstream of the putative initiator met in the polyproteins of mrfv, swmv, and belv. the swmv sequence encodes an alanine at the putative cleavage site, but that is consistent with the previous observations of kadaré et al. (1995) and sulea et al. (2006) , who noted the presence of g or a residues in the p1 and p2 positions of confirmed and predicted cleavage site sequences of tymo-and coronaviruses, respectively. thus, we propose the consensus sequence at the predicted cleavage site associated with minor cp expression in marafiviruses is lxg[g/a]. intriguingly, to counter cellular antiviral strategies that utilize the ubiquitin-proteasome system, the protease of the closely-related tymv cleaves ubiquitin conjugates from its rdrp at the same consensus sequence in addition to its role in polyprotein processing (chenon et al., 2012) . moreover, this consensus sequence is targeted by other viral cysteine proteases with both deubiquitinating and polyprotein processing activities (barretto et al., 2005; clementz et al., 2010; karpe and lole, 2011; wang et al., 2011) . the model for expression of obdv cps appears directly applicable to the majority of accepted and proposed marafiviruses, while an interesting variation of this strategy likely occurs with swmv, mrfv, and belv. for the latter, the predicted cleavage sites lie upstream of the predicted minor cp initiation codons, necessitating further studies to determine the precise cp expression strategies of these viruses. the location of the predicted cleavage site downstream from the initiator methionine of the minor cp would provide a consistent amino terminus for minor cp produced by cleavage of both the replicase polyprotein and a minor cp precursor translated directly from sgrna. irrespective of the role of protein cleavage in the formation of the minor cp n-terminus, an important function of the cleavage site may be to liberate the polymerase from the cp. the location of a putative protease cleavage site upstream of the initiator methionine for the minor cps of swmv, mrfv, and belv is consistent with this view. furthermore, the single cp of the closely-related but unclassified poinsettia mosaic virus (pnmv) is fused to the pnmv replicase polyprotein, and the amino acid sequence between the marafibox and the cp also harbors a predicted lxga cleavage site sequence (fig. 5) . thus, the lxg[g/a] motif is well conserved among viruses of the tymoviridae that possess a replicase-cp fusion, regardless of the precise location of the motif relative to the cp translation initiation codon. this is the first investigation of a marafivirus cp expression strategy and a step toward a better understanding of overall gene expression in marafiviruses. the sequence conservation and similarity with other viruses that utilize papain-like proteases as a means to process polyproteins and defend against protein degradation via the ubiquitin-proteasome system suggests that similar mechanisms are used by marafiviruses. with the existence of tymoviral and marafiviral infectious clones and techniques for their transmission, the tools are now available to investigate the roles of cps and other factors in the infection process within whole plants and their potential roles in marafivirus transmission by leafhoppers (edwards and weiland, 2010; spetz et al., 2008; weiland and dreher, 1989; weiland and edwards, 2011) . a full-length cdna clone of obdv from which infectious transcripts can be derived was generated previously (edwards and weiland, 2010) . this clone (pobdv-2r; genbank #gu396990) served as the parent plasmid in the generation of mutants of obdv with alterations in the cp gene(s). general amplification primers obd5140fwd and obd5901rev were used in conjunction with mutagenic primers (fig. 1, table 1 ) to produce overlapping amplicons. pcr reactions included 1 ng of linearized plasmid as template and conditions were as described in higuchi (1990) . typical reactions contained 1x platinum taq buffer, 0.1 mm each d(gatc)tp, 30 ng of each oligonucleotide primer, and 0.5 units of platinum taq high-fidelity dna polymerase (life technologies, grand island, ny usa) in a volume of 30 ml. thermocycling parameters were 95 1c, 30 s; 55 1c, 30 s; and 72 1c, 2 min per cycle over 30 cycles total. paired amplicons were mixed and subjected to a second round of amplification using only primers obd5140fwd and obd5901rev (higuchi, 1990) . digestion of the resulting amplicon with sal 1 (located at nts 5213 and 5831) released a 618 bp fragment that was used to replace the homologous region in pobdv-13b2 (a derivative of pobdv-2r in which the mcs was altered to remove the sal 1 site). capped and polyadenylated transcripts were synthesized as previously described (edwards and weiland, 2010) . the wild type pobdv-13b2 is designated pobdv herein. protoplasts were produced from oat (avena sativa l. cv 'rodney') as previously described (weiland and edwards, 1994) . the procedure routinely produced 10 6 protoplasts per gram fresh weight tissue from 7 day-old oat seedlings. a modification of the procedure by matsuda and dreher (2005) was used to transfect protoplasts with capped transcript rna. isolated protoplasts prepared from 3 g fresh weight of oat leaves were collected from the surface of a sucrose pad after centrifugation and diluted in 0.55 m mannitol/0.1% mes ph 5.6 (mm) containing 5 mm cacl 2 and 40 mm kcl (mmck). protoplasts were pelleted by centrifugation at 115 â g for 8 min. and resuspended in 4 ml mmck. approx. 5 â 10 5 protoplasts (per inoculation) were transferred to a microfuge tube, spun to collect the protoplasts, and the cells resuspended in 0.7 ml mmck. tubes containing protoplasts were incubated on ice for 30 min prior to inoculation. transcript rna was adjusted to a concentration of 0.2 mg/ml, and 5 ml was transferred to a 1.5 ml microfuge tube and placed on ice. a 0.8 ml aliquot of incubation medium (im) consisting of mm supplemented with micronutrients as previously described (weiland and edwards, 1994) was chilled on ice in a microfuge tube along with a 0.4 cm gap electroporation cuvette. chilled protoplasts were transferred to the tube containing transcript rna and the mixture was rapidly pipetted into the pre-chilled cuvette. contents of the cuvette were subjected to an exponential decay pulse of 100 v, 2 mf, and 480 ω, yielding a pulse duration of $110 ms (delivered by a btx ecm-600 electroporation system, harvard apparatus, holliston, ma, usa). following transfer of the contents of the cuvette to 0.8 ml of chilled im, samples were incubated on ice for 10 min. protoplasts were collected by centrifugation, resuspended in 1 ml fresh im containing 100 mg/ml cefotaxime, and incubated for 24 h or 40 h at 22 1c under continuous fluorescent light. each mutant was tested in protoplast inoculations at least 4 times and included parallel mock and pobdv inoculations within each experimental replication. samples were harvested and processed for the analysis of viral rna and protein as previously described (weiland and edwards, 1994) . total protein (for western blots) and nucleic acids (for northern blots) from 10 5 protoplasts were separated on 12% sodium dodecylsulfate polyacrylamide gels and 1% denaturing agarose gels, respectively, as indicated (edwards and weiland, 2010) . proteins were electroblotted from polyacrylamide gels to nitrocellulose membranes (schleicher and schuell [keene, n.h. usa], ba-85, 0.2 m), which subsequently were incubated in a 1:1000 dilution of rabbit anti-obdv, followed by incubation in a 1:2000 dilution of alkaline phosphatase-conjugated goat antirabbit igg (product #a0418, sigma-aldrich, st. louis, mo usa). protein complexes were detected on a kodak image station 2000 mm following treatment of the blot with lumiphos wb (thermo scientific, lafayette, co usa). nucleic acids transferred to positively-charged nylon membranes by capillary blotting (roche applied science, indianapolis, in usa) were probed with a digoxigenin-labeled, denatured dsdna probe representing nucleotides 5929-6508 of obdv-2r. after incubating blots with alkaline phosphatase-conjugated anti-digoxigenin igg, cdp-star (roche applied science, indianapolis, in usa) was added and viral rnas were detected by chemiluminescence. mapping of the 5 0 end of the sgrna total nucleic acids were prepared from obdv-infected oat protoplasts as previously described (edwards and weiland, 2010) . due to substrate competition with the genomic rna in the cdna generation step of the 5 0 -race procedure, gel-purified rna was used as template in the mapping of the 5 0 -end of the sgrna. thus, rnas were size-fractionated by agarose gel electrophoresis (1% gtg agarose [cambrex bioscience, rockland, me, usa], 1x tris-borate-edta buffer), and rna migrating a distance consistent with the estimated size of obdv sgrna ( $ 1.0 kb) was extracted and purified using the zymoclean™ gel rna recovery kit (zymoresearch, irvine, ca usa). following the manufacturer 0 s recommendations, primer obd5901rev (table 1 ) was used to prime cdna synthesis in the presence of smartscribe reverse transcriptase and anchor primers contained in the smartrace kit (clontech, mountain view, ca usa). final amplification employed virus-specific primer term2-221 k( à ) and a smartrace nested universal anchor primer and the products were cloned and sequenced (mwg operon, huntsville, al, usa). clustal-w (larkin et al., 2007) was used to align the resulting nucleotide sequences and the sgrna 5 0 -end determined; initial protein sequence alignments used to illustrate conservation of amino acid residues around the cp amino termini employed the same software. virus names, acronyms, and sequences used a designation of ( þ ) and ( à ) denotes complementary pair of primers where ( þ) is of "sense" orientation with respect to the genomic rna. b changes to ablate putative transcriptional promoter, translational initiation, and protease cleavage sites or produce premature termination of translation. c standard nucleotide designation in 5 0 -43 0 polarity with degeneracies where k ¼t þ g, m ¼a þ c, y¼ t þc, and r¼ a þg. the last nucleotide in each primer is numbered relative to its position in the sequence of obdv-2r (genbank accession #gu396990). nucleotides changed from the obdv-2r sequence are underlined. virus (swmv, nc_015522), turnip yellow mosaic virus (tymv, x16378). the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity a viral deubiquitylating enzyme targets viral rna-dependent rna polymerase and affects viral infectivity deubiquitinating and interferon antagonism activities of coronavirus papainlike proteases the tymobox, a sequence shared by most tymoviruses: its use in molecular studies of tymoviruses virus taxonomy. ninth report of the international committee on taxonomy of viruses first infectious clone of the propagatively transmitted oat blue dwarf virus oat blue dwarf marafivirus resembles the tymoviruses in sequence, genome organization, and expression strategy a translation initiation element specific to mrnas with very short 5 0 utr that also regulates transcription alpha-like viruses in plants nucleotide sequence of turnip yellow mosaic virus coat protein mrna recombinant pcr sequence of the coat protein gene of bermuda grass etched-line virus, and of the adjacent 'marafibox' motif expression of the turnip yellow mosaic virus proteinase in escherichia coli and determination of the cleavage site within the 206 kda protein deubiquitination activity associated with hepatitis e virus putative papain-like cysteine protease versatile dual reporter gene systems for investigating stop codon readthrough in plants clustal w and clustal x version 2.0 capped mrna with a single nucleotide leader is optimally translated in a primitive eukaryote, giardia lamblia in vivo translation studies of plant viral rnas using reporter genes conservation of the putative methyltransferase domain: a hallmark of the 'sindbis-like' supergroup of positive-strand rna viruses identification and functional analysis of the turnip yellow mosaic tymovirus subgenomic promoter symptomless infectious cdna clone of a norwegian isolate of poinsettia mosaic virus binding site-based classification of coronaviral papain-like proteases the leader proteinase of foot-and-mouth disease virus negatively regulates the type i interferon pathway by acting as a viral deubiquitinase infectious tymv rna from cloned cdna: effects in vitro and in vivo of point substitutions in the initiation codons of two extensively overlapping orfs evidence that the αa gene of barley stripe mosaic virus encodes determinants of pathogenicity to oat (avena sativa) linear-motion tattoo machine and prefabricated needle sets for the delivery of plant viruses by vascular puncture inoculation funding for this work was provided by usda-ars cris project number 5442-22000-048-00d. key: cord-008407-jbp8bxjz authors: derdeyn, cynthia a.; frey, teryl k. title: characterization of defective-interfering rnas of rubella virusgenerated during serial undiluted passage date: 1995-01-10 journal: virology doi: 10.1016/s0042-6822(95)80036-0 sha: doc_id: 8407 cord_uid: jbp8bxjz during serial undiluted passage of rubella virus (rub) in vero cells, two species of defective-interfering (di) rnas of approximately 7000 and 800 nucleotides (nts) in length were generated (frey, t. k., and hemphill, m. l., virology 164, 22–29, 1988). in this study, these di rnas were characterized by molecular cloning, hybridization with probes of defined sequence, and primer extension. the 7000-nt di rna species were found to be authentic di rnas which contain a single 2500to 2700-nt deletion in the structural protein open reading frame (orf) region of the genome. the 800-nt rnas were found to be subgenomic di rnas synthesized from the large di rna templates. analysis of the extent of the deletions using a reverse-transcription-pcr protocol revealed that the 3′ end of the deletions did not extend beyond the 3′ terminal 244 nts of the genome. the 5′ end of the deletions did not extend into the nonstructural protein orf; however, di rnas in which the subgenomic start site was deleted were present. following serial undiluted passage of seven independent stocks of rub, this was the only pattern of di rnas generated. di rnas of 2000 to 3000 nt in length were the majority di rna species in a persistently infected line of vero cells, showing that other types of rub di rnas can be generated and selected. however, when supernatant from the persistently infected cells was passaged, the only di rnas present after two passages were 7000 nts in length, indicating that this species has a selective advantage over other types of di rnas during serial passage. rubella virus (rub) is a member of the togavirus family and is the sole member of the rubivirus genus (francki et al, 1991) . the rub genome is a singlestranded, positive-polarity rna molecule of 9757 nucleotides (nts)in length (reviewed by frey, 1994) . the genome rna contains two long open reading frames (orfs). the 5' proximal orf encodes the nonstructural proteins (nsp-orf) and the 3' proximal orf encodes the structural proteins (sp-orf) in the order nh2-c-e2-e1-cooh. both nonstructural and structural proteins are proteolytically processed from a polyprotein precursor into the individual proteins. replication of the virus rna is carried out through a negative-polarity, genome-length intermediate rna species= initiation at the 3' end of the negative-polarity template leads to production of the genomic rna while initiation at an internal site on the negative-polarity rna template leads to production of a subgenomic rna (sg rna). the sg rna is translated to produce the structural proteins. the genome organization and replication strategy of rub is similar to that of the alphaviruses, the other togavirus genus whose members have been well-characterized (reviewed in strauss and strauss, 1986) . rub generates defective-interfering (di) rnas during both serial undiluted passage and persistent infection in cell culture (norval, 1979; bohn and van aistyne, 1981; terry etal, 1985; frey and hemphill, 1988; abernathy et al., 1990) . in a previous report from this lab (frey and hemphill, 1988) , di rnas were detected after four serial passages in vero cells. two di rna species were generated and maintained through 20 passages, a large species of approximately 7000 nts in length and a smaller species ranging from 700 to 1000 nts in length. concomitant with the appearance of di rnas, a decrease in the amount of standard genome rna and a drop in virus titer were observed (frey and hemphill, 1988) , indicating that these di rnas interfered with the replication of standard virus. in persistently infected vero cells, up to seven di rnas were detected and the number of these species varied as the persistently infected culture was propagated (abernathy et al, 1990) . the majority of the di rnas in persistently infected cells is less than 3000 nts in length (norval, 1979; frey and hemphill, 1988; abernathy et al., 1990) . this paper describes the molecular characterization of di rnas generated during serial passage of rub in vero cells. becco's minimal essential medium (dmem) (gibco-brl) supplemented with 5% fetal bovine serum, 10% tryptose phosphate, and gentamicin (20 #g/ml). vero cells were routinely subcultured at a 1,10 dilution every 7 days by wpsinization. the w-therien strain of rub was provided byj. wolinsky and the f-therien and m33 strains of rub were provided by j. k. chantler. some of these strains were plaque-purified in vero cells before use. supernatants from synovial cell explant cultures persistently infected with the f-therien and m33 strains of rub were provided byj. k. chantler. to amplify di-containing stock from the previous study (frey and hemphill, 1988) , passage 10 and passage 18 were passed twice in vero cells and f{na was extracted from cells infected with the second passage (p12 and p20). all other serial passages were done by initially infecting 1 x 106 vero cells with virus stock or supernatant from persistently infected cells. in each passage the supernatant was harvested 48 to 72 hr postinfection and one-fourth of the supernatant was used to infect 1 x 106 vero cells. vere cells were infected with standard rub (plaquepurified w-therien) at an m.o.i, of 0.1 pfu/cell or serial passage stock for 1 hr at 35 °, after which time the inoculum was removed and replaced with growth medium. intracellular rna was extracted between 48 and 72 hr p0stinfection (at the time that cytopathic effects were initially observable) bythe method of sawicki eta/. (1981) . northern gel analysis of intracellular rna was as previously described (hemphill eta/., 1988) with the exception that nylon membranes (micron separations, inc.) were used instead of nitrocellulose. synthesis of and hybridization with 32p-labeled positive-and negative-polarity rna probes containing the 3' 587 nts of the rub genome were as described previously (hemphill eta/., 1988) . northern transfers to be hybridized with radiolabeled oligonucieotide probes were prehybridized in 5x 8s0, 1% sds, 1 mm edta, containing 200 #g/ml of denatured salmon sperm dna for 2 hr or rapid hybridization buffer (amersham) for 30 min at 42 ° followed by addition 0fthe probe. oligonucleotide probes, synthesized using an abi model 381a synthesizer, were 5' end-labeled with [32p]atp using polynucleotide kinase as described previously (frey et al., 1989) . hybridizations in rapid hybridization buffer were done at 42 ° . for hybridizations in 5x ssc, the hybridization temperature for oligonucleotide probes was calculated using the following formula= th(in°)= 2 x(a+t) +4 x (g + c)-5, wherea, t, 0, and g correspond to the number of times each nucleotide occurs in the oligonucleotide probe. after hybridization, membranes were washed at the hybridization temperature twice in 2× ssc, 0.1% sds for 15 min and once inlx ssc, 1% sds for 15 min. washed membranes were wrapped in plastic wrap and exposed to kodak x-omat ar film at -80 ° between two cronex lightning plus intensifying screens (dupont). p12 intracellular rna was fractionated on a 15 to 30% sucrose gradient in 1x ten buffer [10 mmtris-hci (ph 8.0), 1 mm edta, 0.5 m naol] by high-speed centrifugation in a beckman sw41 rotor at 40,000 rpm for 4 hr at 25 °. rna fractions identified by northern hybridization to contain di rnas were used as a template for oligo-dtprimed cdna synthesis. first-and second-strand cdna synthesis was done using a promega riboclone cdna synthesis kit. double-stranded cdna was size-selected by spun column chromatography through a 1-ml sephacryl s-400 (pharmacia) column. the size-selected cdna was dctp-tailed using terminal deoxynucleotidyl transferase as'previously described (frey et a/., 1986) , annealed with pgem-3z(f-) (promega) tailed with dgtp, and transformed into maximum efficiency escherichia col~ dh5~ cells (bethesda research laboratories). colonies containing plasmids with rub-specific inserts were identified by colony blot hybridization using as a probe the ecori-hindlll fragment of robe12 [a plasmid containing a complete cdna copy of the rub genome (wang et al., 1994) ] labeled with [e32p]datp by random priming. unfractionated intracellular rna samples (1 #g) were used as templates for reverse transcription primed with oligo-dt (200 ng, promega) or oligonucleotide 51 (5'acg-tgcatgcctgcagt20-3') in 25-#1 reaction mixtures containing 0.05 m tris-hci (ph 8.3), 0.075 m kci, 3 mm mgci2, 1 mm datp, dctp, dgtp, and d-ci-p (pharmacia), 5 mm d-i-f, 40 units of rnasin ribonuclease inhibitor (promega), and 200 units of superscript rnase h-reverse transcriptase (bethesda research laboratories). the reaction was incubated at 45 ° for 1 hr followed by incubation at 97 ° for 5 min to inactivate the reverse transcriptase. ten microliters of the reverse transcription reaction was used as a template for amplification by the polymerase chain reaction (pcr) using one of three sets of oligonucleotide primers= 102 (5'-ccggtctagact-agtgcgggtttcgcgc-3', complementary to nts 9708 to 9725 of the rub genome) and 170 (5'-ccgaaq-i-cg-cgccaatctccacgac-3', colinear with nts 6360 to 6377 of the rub genome) (underlined sequences indicate flanking restriction sites incorporated to facilitate cloning); 102 and 173 (5'-ccggaa-iq-ccgactacag-cgcggagc-3', colinear with nts 6241 to 6258 of the rub genome); and 58 (5'-acgtgcatgcctgcagq-i--3', anchor sequences of oligonucleotide 51 used to prime reverse transcription) and 177 (5'-ccggaattoggtgot-ttgccgccgtt-3', colinear with nts 6120 to 6146 of the rub genome). the locations of the primer sets with respect to the rub genome are shown at the top of fig. 2. pcr was performed in a 100-or 200-#1 reaction mixture containing 10 mm kci, 20 mmtris-hci (ph 8.8), 10 mm ammonium sulfate, 2 mm magnesium sulfate, 0.1% triton x-100, 10% dmso (sigma), 80 ffg/ml bovine serum albumin (new england biolabs), 0.03% fl-mercaptoethanol (sigma), 200 ffm datp, dctp, dgtp, and d-!-i-p (pharmacia), 200 ng of each synthetic oligonucleotide primer, and 1.5 units of hot tub dna polymerase (amersham). the pcr reaction consisted of 30 cycles of 1 min ascending to 94 °, 1 min at 94 ° , 3 min descending to 50 ° , 1 min at 50 ° , 2 min ascending to 72 ° , and 2 min at 72 ° followed by 1 cycle of 30 min at 72 °. pcr products were purified from primers by spun column chromatography through a sephacryl s-400 column. pcr products were digested with ecori and xbal or ecori and pstl, size-selected by agarose gel electrophoresis or sephacryl s-400 spun column chromatography, and ligated into appropriately restricted pgem-3z(f-) (promega). one-half of the ligation reaction was used to transform competent e. coil strain sure cells (stratagene). colonies containing rub-specific recombinant plasmids were identified by colony blot hybridization using radiolabeled oligonucleotide 86 (5'-tggtgtgtg-tgccatac-3', complementary to nts 9540 to 9524 of the rub genome) as a probe. alternatively, following restriction some of the pcr amplification reactions were ligated with a bacteriophage x vector, xgem-2 (promega). duplicate plaque lifts were made of plates containing phage plaques. one lift was hybridized to radiolabeled oligonucleotide probe 10 (5'-ggcgaaacaacggtgggtga-3', complementary to nts 6453 to 6434 of the rub genome) and the other to 86 (5'-tggtgtgtgtgccatac-3', complementary to nts 9540 to 9524 of the rub genome). sequence determination dna sequencing of double-stranded plasmid templates was performed using the dideoxynucleotide chain termination method of sanger et al (1977) employing bacteriophage t7 dna polymerase (sequenase version 2.0, united states biochemical) with [e-aas]datp (1000-1500 ci/mmol, new england nuclear) or [~-32p]datp (3000 ci/mmol, new england nuclear or amersham), 7deaza gtp instead of gtp to reduce compression artifacts and synthetic oligonucleotide primers. the plasmid template was denatured prior to primer annealing by alkaline denaturation (haltiner et al, 1985) or heat denaturation (andersen et al, 1992) . the sequencing reactions were electrophoresed on 8% polyacrylamide-urea sequencing gels run at 2000 v. sequencing gels were fixed in 10% methanol, 10% glacial acetic acid, dried onto whatman 3mm filter paper under vacuum, and exposed to kodak x-omat ar film at -80 °. primer extension was performed using nonfractionated intracellular rna samples as templates. approxi. the extension reaction was incubated at 45 ° for 30 min. following an incubation at 90 ° for 5 rain, primer extension products were analyzed by electrophoresis on an 8% polyacrylamide sequencing gel. to amplify di rnas for sequence analysis, passage 10 and passage 18 stocks from the previous study (frey and hemphill, 1988) were used to infect vero cells and two subsequent passages were done, expanding the number of cells infected with each passage. rna was extracted from the ceils infected with the second passages of both stocks (p12 and p20). shown in fig. 1 is a northern blot of p12 rna hybridized with 3' terminal rna probes of both polarities. figure 1a shows the presence of two di rna populations of 7000 to 7500 and 700 to 850 nts in length. the sizes of the di rnas in p12 are similar to those which were previously reported. figure 1b shows the detection of a negative-polarity complement for only the larger di rna species. to clone the smaller di rna species, intracellular rna from p12-infected cells was fractionated by sedimentation through a continuous 15 to 30% sucrose gradient and fractions which contained this rna species were identified by northern hybridization. one gradient fraction which contained an abundance of the small di rna species and a small amount of the large di rna species was used as a template for cdna synthesis using oligo-dt as a primer. a cdna library was constructed using a plasmid vector (pgem-3z) and from the total of approximately 3000 recombinant colonies that were recovered, 3 colonies containing rub-specific sequences were identified. the sequences of the rub-specific inserts present in each of these 3 clones were determined (fig. 2 , bottom). all three clones contained a poly-a tail and the exact 3' terminus 0fthe genome, including several hundred nts into the e1 coding region, all three clones contained a single large deletion in the sp-orf extending from the e1 protein coding region to the capsid protein coding region, with the e2 protein coding region completely deleted. the deletions in the three clones were dissimilar and extended from nts 6795 to 9452 in 181, 6798 to 9370 in 182, and 6833 to 9443 in 183. the size of the deletion in all three clones was approximately 26% of the size of the rub genome. upstream from the deletion, two of the clones, 182 and 183, terminated in the capsid protein coding region while the largest clone, 181, extended to nt 740 of the genome. except for a large deletion in the sp-qrf, for the extent of the genome which each clone covered, each was a faithful copy of the rub genome (occasional point mutations did occur in each clone). within its boundaries, clone 181 contained a faithful copy of the nsp-orf and sg start site. since the template for this clone was presumably the large di rna species, it was likely that this species contained a complete copy of the nonstructural orf and the sg start site. the large deletion in the sp-orf would account for the reduction in the size of the large di rna species as compared to the genome. thus, the large di rna species could be capable of self-replication and the small di rna could be a sg rna synthesized from the large di rna template (accounting for the lack of a negative~polarity equivalent). these possibilities were investigated by northern hybridization to oligonucleotide probes representing different regions of the rub genome (locations of probes with respect to the genome rna are shown in the top of fig. 2 ). northern blots of p12 rna were first hybridized with radiolabeled oligonucleotide probes which were complementary to nts 1 to 45 (oligonucleotide 49, fig. 3a ) 100 to 117 (oligonucleotide 36, fig. 3b ), and 230t0 253 (oligonucleotide 88, data not shown) of the rub genome. all three of these 5' terminal oligonucleotide probes hybridized to the large di rna species but not to the small di rna species, indicating that the small di rna does not contain 5' terminal sequences. next, hybridization to northern blots of p12 rna was done with two radiolabeled oligonucleotide probes which are complementary to sequences immediately upstream (nt 6410 to 6430, oligonucleotide 11, fig. 30 ) and immediately downstream (nt 6430 to 6450, oligonucleotide 10, fig, 3d ) from the sg start site (nt 6430). the large di rna species hybridized to both probes while the small di rna hybridized to only the downstream probe, indicating that the small di rna contains sequences downstream but not upstream from the sg start site. finally, to determine whether the majority of di rnas present in p12 rna contained a deletion in the sp-orf, an oligonucleotide probe which is complementary to a sequence located within the e1 protein coding region (nts 8472 to 8488, eligonucleotide 83, fig. 3e ) that was deleted in all three cdna clones was hybridized to p12 rna. neither of the p12 di rna species hybridized to this probe, indicating that a deletion in the sp-orf was present in both the large and small populations of di rnas. in total, these data from oligonucleotide probe hybridization analysis are completely consistent with the hypothesis that the large di rna species contain a single large deletion in the sp-orf and that the small di rna species are sg rnas synthesized from the large di rna templates. analysis of the 5' terminus of di rna generated during serial passage as shown in figure 3a , oligonucleotide probes complementary to the exact 5' terminal sequences of the rub genome hybridized to the large di rnas. to confirm that these di rnas contained the authentic 5' end of the genome, primer extension was performed on intraceilular rna from standard rub-infected cells and p12 rna. approximately 1 #g of each rna was used as a template. figure 4 shows the results of primer extension using radiolabeled oligonucleotide primer 36 which is complementary to sequences located 100 nts from the 5' end of the genome. two primer extension products were produced from both rub-infected cell rna and p12 rna. the lower of these is caused by termination at a stable secondary structure formed by 5' terminal sequences while the upper product is due to termination at the 5' end of the genome (dominguez eta/., 1990) . even though p12 rna contained some genomic rna, which can serve as a template for primer extension, the 7000-nt di rna was clearly the most abundant template (refer to fig. 1) . theoretically, di rnas which served as a template for primer extension (the majority of 7000 nt di rnas hybridized to primer 36, fig. 3b ) and contained 5' termini that differed from the rubella virus genome would produce a primer extension product which migrated differently from that produced from the genome rna. only one primer extension product representing the 5' termini of the rna template was produced from both rub-infected cell rna and p12 rna, indicating that the majority of the large di rnas contain a 5' terminus similar to the standard genome. to determine whether the di rna species present in p12 rna were representative of di rnas routinely generated during undiluted serial passage of rub in vero cells, serial passages in vero cells of seven other rub stocks were made and the di rnas produced were analyzed by northern hybridization to the 3' terminal negative-polarity rna probe. these stocks were plaque-purified w-therien [wth(pp); this is a plaque-purified independent stock from that used to initiate the passage series that includes p12 and p20], f-therien received from another lab and passaged either before ( the di rna species present in p20 rna were also analyzed. as shown in figs. 5a and 5c, after 5 to 10 serial passages of each of these stocks, except for the synovial explant persistent infection supernatants (fig. 5c, lanes 4 and 5) , a di rna species similar in size to the large p12 di rna was generated. in most of these passages, a short di rna species was also generated, although the size and relative amount varied. hybridization of northern blots of p20, piv(ps), wthp10, and fthp10 rna with the 3' terminal positive-polarity rna probe (fig. 5b ) and the oligonucleotide probes used earlier (data not shown) confirmed that similar to the p12 di rnas, only the large di rnas present in each of these passage series contained 5' terminal sequences and produced a complementary negative-polarity species (fig. 5b) , that the small di rnas lacked sequences upstream from and contained sequences downstream from the sg start site, and that both di rna populations contained a deletion in the sp-orf. it was of interest that serial passage of supernatant from persistently infected vero ceils [piv(p5)] gave rise to di rnas similar to p12 since when the di rnas pres-re. 4. analysis of the 5' termini present in p12 rna by primer extension. primer extension was performed on unfractionated intracelluiar rna (rub, standard rub-infected rna; p12, p12 rna; mi, mockinfected cells rna) by reverse transcription using primer 36, which is complementary to nts 100 to 117 of the rub genome. primer extension products were analyzed by eleetrophoresis on an 8% polyacrylamide gel. the position of migration of the primer extension band correspond-jng to the 5' end of the genome is indicated on the left margin. the other band is a strong stop caused by a stem-loop structure formed by 5' terminal sequences (dominguez et al, 1990) . ent in that persistent infection were characterized it was found that although a large di rna species was present at certain passages, the majority di rna species was less than 3000 nts in length (abernathy et al., 1990) . subculture (sc) 133 of the persistently infected cell line was rescued from liquid nitrogen and after seven subcultures (sc140) supernatant fluid was harvested and passaged twice in vero cells. rna was extracted from the persistently infected cell line and cells infected with the first and second passage. as shown in fig. 6 , the primary fina species present in persistently infected cells was a species of 2000 to 3000 nts in length, although a large di rna and two shoaer di rnas were present. hybridization with the 3' terminal positive-polarity rna probe and oligonucleotide 49 showed that the 2000-to 3000nt di rnas contained 5' and 3' terminal sequences and synthesized a negative-polarity complement (data not shown). with exception of one of the short di rna species these species were present following one serial passage, although the relative amount of the 2000-to 3000rot di rna species was greatly reduced. by the second passage, only the large and short di rna species were present. the 3 cdna clones obtained from p12 rna contained nonidentical deletions, indicating that the di rna population was heterogeneous with respect to the deletions which they contained. the extent of the deletions in the p12 di rna population was analyzed by a protocol in which intracellular rna was a template for reverse transcription and pcr amplification (rt-pcr) using primer pairs which flank the deleted region. the strategy is diagrammed in the top of fig. 7 . amplification products were cloned into pgem-3z and 18 clones were isolated and sequenced. the extent of the deletion in each clone is given in table 1 . the deletions were heterogeneous, ranging in size from 2504 to 3047 nts. all of the deletions were different although some deletions shared one breakpoint. unexpectedly, 17 of the 18 rt-pcr clones had deletions which included the sg start site [the only p12 rt-pcr clone (120) which retained the sg start site was the result of a mispriming event during pcr amplification in which the 5' primer annealed downstream from the sg start site and thus selected for an amplification product that retained sequences downstream from the sg start site]. this rt-pcr technique was used to obtain information about the deletions present in di rnas from two other passages, p20 and piv(p5). of the 6 clones generated from p20 rna, all 6 retained the sg start site. of 3 rt-pcr clones generated from piv(p5) rna, all 3 contained a deletion of the sg start site. that the preponderance of rt-pcr clones from p12 rna lacked the sg start site was unexpected since it was in contrast to the results from the cdna library. to confirm that this was not due to selective cloning of rt-pcr products lacking the sg start site into the plasmid vector used (i,e., that sequences around the sg start site are not stable in this vector), the original p12 and p20 rt-pcr reactions were ligated into the x bacteriophage vector, xgem2, and recombinant plaques were screened by plaque hybridization to determine the proportion of p12 and p20 recombinants which retained the sg start site. plaques were transferred to nylon membranes in duplicate and hybridized against two oligonucleotide probes, 86 which is complementary to sequences near the 3' end of the rub genome and 10 which is complementary to sequences immediately downstream from the sg start site. all plaques containing rub-specific inserts will hybridize to oligo 86 while only those containing the sg start site will hybridize to oligo 10. ninety-seven of 100 (97%) of the p12 plaques with rub-specific inserts and 12 of 47 (25%) of the p20 plaques contained inserts ~s upstream from the sg start site. this sequence is the end of a 20-nt region which shares homology with the alphavirus sg promoter (frey, 1994) . of the 10 clones which retained the sg start site, all retained at least 200 nts downstream from the start site, and 9 retained over 360 nts downstream. the 3' breakpoints of the deletions were distributed over a 300nt region and all of the clones retained at least 244 nts from the 3' terminus of the genome. two regions of clustering of 3' breakpoints were observed. the first cluster occurred between nts 9228 and 9234 (uucaaga) and contained the breakpoints of 6 clones. the second cluster occurred between nts 9443 and 9455 (ccaagucag-cugc) and contained the breakpoints of 11 clones. of the 10 rt-pcr clones which retained the sg start site and extended into the sp-©rf, 6 maintained the reading frame of the s p -o r f across the deletion. no consensus sequence or sequence homology was identified on either side of any single deletion which could account for its generation. analysis of computer-generated secondary structures of both sides of the deletion did not reveal any significant secondary structures which could be responsible for generation of the deletions (data not shown). in this study, it was found that of the two species of novel virus-specific rnas generated by serial undiluted passage of rub in vero cells, the larger species was an :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: ® p~v(' rh)~ fie. 7. strategy of rt-pcr amplification of the sp-orf and locations of breakpoints of deletions. (a) diagrams of the rub genome rna and the large di rna, the relative locations of the three pcr primer sets used in amplification. primer numbers and restriction enzyme sites included in each primer to facilitate cloning are indicated. (b) the locations of the 5' breakpoints of the deletions in each clone plotted along the rub nucleotide sequence. open circles are clones derived from p12 rna, black circles are clones derived from p20 rna, and hatched circles are clones derived from piv(p5) rna. the boxed region indicates the enlarged area. nucleotide numbers correspond to the rub genome. the boxed termination codon uaa signals the end of the nsp-qrf. the 20-nt region which shares homology with the alphavirus subgenomic promoter region is underlined. the uuuaa sequence in which nine clones have breakpoints is underlined twice. (c) the locations of the 3' breakpoints of the deletion in each clone which were plotted along the rub nucleotide sequence. nucleotide numbers correspond to the rub genome. a predicted stem-loop structure shown to bind host cell proteins is indicated near the end of the e1 coding region. authentic di rna that contained a single deletion in the sp-orf and the smaller species was a sg rna synthesized from the large di rna. evidence supportive of the conclusion that the smaller species was a sg rna was as follows. first, no negative-polarity complement of the small di rna was detected, indicating that it was not replicated directly through an intermediate negative-polarity species. a negative-polarity complement of the small di rna species was detected previously by frey and hemphill (1988) but was most likely a result of the method of intracellular rna extraction that was used, which also resulted in the detection of a negative-polarity complement for the sg rna. these results were most likely due to nonspecific rnase digestion of single-stranded regions of double-stranded full-length replicative intermediates which contain both positive-and negative-polarity rnas. second, cloning of di rnas from p12 intracellular rna resulted in recovery of one clone, 181, that contained the majority of the nsp-orf and sg start site. the di rna from which this clone was derived was thus theoretically capable of synthesizing (or serving as a template for the synthesis of) a sg rna. third, both the large and small di rna species hybridized to a probe complementary to the 5' end of the sg rna. however, only the large di rna species hybridized to a probe complementary to sequences immediately upstream, indicating that the small di rna species does not contain sequences upstream from the sg stag site. finally, the frey and hemphill (1988) that the large di rnas, but not the small di rna, were encapsidated. several pieces of evidence suggested that the large di rnas conserve the entire nsp-orf and contain a single deletion in the structural orf. the largest clone recovered from p12 rna, clone 181, extended from the poly-a tail to nt 740 of the genome and contained a faithful copy of the nonstructural orf from nt 740 to the end of the orf. although the 5' end of the genome and nsp-orf was not present in any of the cdna clones generated, northern hybridization with probes complementary to sequences upstream from nt 740 demonstrated that the large di rnas contained the 5' end of the genome and the 5' end of the nsp-orf. a primer extension experiment indicated that the large di rna contained the authentic 5' end of the genome rna. the only deletion observed in clone 181 was located within the sp-orf and the shorter clones recovered from p12 rna (182 and 183) also contained a large deletion of the same region of the sp-orf that was deleted in clone 181. northern hybridization with a probe within the deleted region showed that both the large and small di rna population uniformly contained this deletion. the sizes of di rnas that contained the complete nsp-orf and deletions of the structural orf present in clones 181, 182, and 183 would be 7099, 7147, and 7185 nts, respectively, consistent with the size of the large di rnas. an'alysis of the extent of the sp-orf deletion by rt-pcr revealed that the 5' most deletion site preserved the termination codon of the nsp-orf and no deletions were detected that extended into the nsp-orf. rt-pcr analysis of the extent of the sp-orf deletions in the di rna population showed that the deletions were completely heterogeneous and ranged in size from 2504 to 3047 bases. the 5' most breakpoint was at the exact 3' end of the nsp-orf while at least 244 nts of 3' terminal sequence were maintained in all of the clones. this includes the predicted stem-loop structure located 58 nts from the 3' terminus that has been shown by nakhasi eta/. (1990) to interact with host cell proteins. analysis of the breakpoints of the deletions present in these clones did not reveal any consensus sequence or significant secondary structure that could be responsible for their generation. the breakpoints were distributed over regions of several hundred nts in the capsid protein and the e1 glycoprotein coding regions. however, cluste[s of breakpoints were observed on either side of the deletion. an unexpected result of the rt-pcr analysis was that in the majority of the p12 clones (17 of 18) the sg start site was deleted. the abundance of sg di fina detected in p12 rna by northern hybridization suggested that the large di rnas commonly retained the sg start site. additionally, oligonucleotide probes complementary to sequences immediately upstream and downstream from the sg start site hybridized to the large di rna in p12 rna. this result was also in contrast with the clones generated by cdna synthesis, in all 3 of which the 5' breakpoint of the deletion was downstream from the sg start. this result was not due to the inability of template containing the sg start site to be amplified by pcr since the majority of the p20 rt-pcr clones contained the sg start site. itwas also not due to instability of sequences in the vicinity of the sg start site in the plasmid vector pg em-3z since when p12 rt-pcr products were cloned into a bacteriophage x vector, 97% of the rub-specific recombinant plaques contained deletions of the sg start site. the preponderance of p12 rt-pcr clones that lack the sg start site possibly was due to selective amplifica-tion of smaller templates during pcr (i.e., those that lacked the sg start site). in contrast to rt-pcr clones from p12 rna, most of the rt-pcr clones from p20 rna retained the sg start site. thus, di rnas that both retain and delete the sg start site were maintained in the di rna population during serial passaging and the relative abundance of each varied. this is in distinct contrast to alphavirus di rnas which have been characterized to date, all of which delete the sg promoter and start site (schlesinger, 1988) . it is thought that deletion of the sg promoter provides a selective advantage for replication of the di rna over standard genomes because alphavirus di rna negativepolarity templates are uniformly available for replication and not used for sg rna synthesis. it is not clear why rub di rnas that retain the sg start site and produce a sg message are not only retained in the di rna population but are also the majority species at certain passages. in addition to maintenance of the sg start site, the overall structure of rub di rnas also contrasts with the structure of alphavirus di rnas, which usually contain a complex array of multiple internal deletions, duplications, rearrangements, and occasionally cell-derived sequences (schlesinger, 1988) . unlike rub di rnas, alphavirus di rnas have usually deleted most of the genomic sequences and are thus relatively small [often a fraction (i.e. one-fourth) of the total genome size]. interestingly, all of the clones in which the sg start site is retained also contained 200 to 360 nts downstream from the start site. the presence of functional fusion orfs created by the junction of noncontiguous regions of mouse hepatitis virus (mhv) gene 1 in two mhv di rnas was reported by makino et al. (1988 makino et al. ( , 1990 . novel proteins translated from the fusion orfs of both di rnas were detected in infected cells. thus, translation of fusion orfs may be a characteristic of mhv di rnas. supporting evidence that mhv di rna fusion orfs are translated was provided by de groot et al. (1992) who constructed a di cdna clone derived from a mhv di rnawhich contained a fusion orf and then generated nonsense and frameshift mutants that did not preserve the fusion orf. it was found that translation of the orf was required for efficient amplification of the mhv di rna transcripts. of the 10 p12 and p20 clones which retain the sg start site, only 6 maintained the reading frame of the sp-orf. thus translation of a fusion protein does not appear to be necessary for amplification of rub di rnas. similar species of rub di rnas were generated in five of seven independent serial passages and no other patterns of di rna generation were observed. in two other reports on rub di rna generation during serial passage, the species detected were also 7000 to 9000 nts in length (bohn and van alstyne, 1981; terry et al., 1985) . as observed in analysis of di rnas present in persistent infection, other types of rub di rnas can be generated. the 2000-3000 nt di rnathat was the major-ity intracellular species contained both 5' and 3' terminal sequences. thus, the prevalence of the large di rna species during serial passage is probably due to its having a selective advantage over other di rna species. one possibility is efficiency of encapsidation, a feature more critical for maintenance during serial passage than persistent infection. both the long di rna and the 2000to 3000-nt species from persistent infection were encapsidated as evidenced by their ability to be passaged, but the long di rna outcompeted the 2000-to 3000-nt species within two serial passages. however, the rub encapsidation sequences have not been defined and the disappearance of the 2000-to 3000-nt species within two passages could have been due to factors other than encapsidation. another possible selective advantage of the large di rna is that di rnas which contain the entire nsp-orf can theoretically synthesize their own nonstructural proteins and thus self-replicate. considering that the m.o.i, of standard virus in serially passaged stock is 0.1 to 0.01 pfu/cell (frey and hemphill, 1988) and that the eclipse phase during rub infection approaches 12 hr , a di rna which can self-replicate in a cell while waiting for infection of the cell by a standard helpervirus would have a selective advantage over di rnas which do not contain the nsp-orf and thus cannot self-replicate. effect of antiviral antibody on maintenance of long-term rubella virus persistent infection in vero cells a fast and simple technique for sequencing plasmid dna with sequenase using heat denaturation the generation of defectiveinterfering rubella virus particles sequence of the genome bna of rubella virus, evidence for genetic rearrangement during tegavirus evolution classification and nomenclature of viruses~ fifth report of the international committee on taxonomy of viruses molecular cloning and sequencing of the region of the rubella virus genome coding for glyceprotein el generation of defective-interfering 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rnas structure and replication of the alphavirus genome rubella virus rna, effect of high multiplicity passage construction of rubella virus genome-length cdna clones and synthesis of infectious rna transcripts we gratefully acknowledge robert b. simmons for expert photography and lee marr for critical reading of the manuscript. we thank hira nakhasi for providing the persistently infected culture from liquid nitrogen storage, geraldina dominguez for assistance with computer secondary structure analysis, and kostia pugachev for ~ cloning. this work was supported by public health service grant af21389 from the national institute of allergy and infectious disease. t.k.f. is the recipient of a public health service research career development award (ai-00923) from niaid. key: cord-272050-0u62j7nj authors: okamoto, kimiyuki; nagano, hideaki; iwakawa, hirooki; mizumoto, hiroyuki; takeda, atsushi; kaido, masanori; mise, kazuyuki; okuno, tetsuro title: cis-preferential requirement of a − 1 frameshift product p88 for the replication of red clover necrotic mosaic virus rna1 date: 2008-05-25 journal: virology doi: 10.1016/j.virol.2008.02.004 sha: doc_id: 272050 cord_uid: 0u62j7nj the genome of red clover necrotic mosaic virus (rcnmv) consists of rna1 and rna2. rna1 encodes n-terminally overlapping replication proteins, p27 and p88, which are translated in a cap-independent manner. the 3′ untranslated region of rna1 contains rna elements essential for cap-independent translation and negative-strand rna synthesis. in this study, we investigated how p27 and p88 were engaged in the replication of rcnmv genomic rnas by using dna vectors or in vitro transcribed rnas in protoplasts and in a cell-free extract of evacuolated by-2 protoplasts. our results show a cis-preferential requirement of p88, but not of p27, for the replication of rna1. this mechanism might help to facilitate a switch in the role of rna1 from mrna to a replication template. the viral replicase complex performs the replication of positive-strand rna viruses (ahlquist, 2002; buck, 1996) . the replicase complex consists of multiple proteins, including viral proteins, rna-dependent rna polymerase (rdrp) and auxiliary proteins, and host factors (ahlquist et al., 2003; van der heijden and bol, 2002) . cis-acting rna elements also play essential roles in viral rna replication. these rna elements are often distributed throughout the genomic rnas, including protein-coding regions. generally, the 3′ proximal nucleotide sequences of viral genomic rnas are essential to initiate negative-strand rna synthesis. these rna elements alone are often sufficient to function in recruiting the viral replicase supplied in trans for initiating rna synthesis (buck, 1996; kao et al., 2001) . also, the viral replicase in trans is involved in the replication of rnas that do not encode replicase component proteins in viruses with a multipartite genome. likewise, defective-rnas occurring naturally or created artificially as constructed-deletion variants can be efficiently amplified by a helper virus that supplies replicase component proteins in trans. in contrast, cis-preferential function of the viral encoded proteins or a coupling between translation and replication has been reported for several viruses, including alfalfa mosaic virus (amv) (neeleman and bol, 1999; van rossum et al., 1996) , clover yellow mosaic virus (cymv) (white et al., 1992) , bovine coronavirus (chang et al., 1994) , cowpea mosaic virus (van bokhoven et al., 1993) , poliovirus (hagino-yamagishi and nomoto, 1989; johnson and sarnow, 1991; novak and kirkegaard, 1994) , turnip crinkle virus (tcv) (white et al., 1995) , tobacco etch virus (mahajan et al., 1996; schaad et al., 1996) , tobacco mosaic virus (tmv) (lewandowski and dawson, 2000) , tomato bushy stunt virus (tbsv) (oster et al., 1998) , turnip yellow mosaic virus (tymv) (weiland and dreher, 1993) , and rubella virus (liang and gillam, 2001) . the coupling between translation and rna replication appears to play important roles in virus infection. however, the roles are poorly understood. red clover necrotic mosaic virus (rcnmv) is a positivestrand rna virus in the dianthovirus genus, tombusviridae family (van regenmortel et al., 2000) . its genome is divided into rna1 and rna2. rna1 encodes two n-terminal overlapping non-structural proteins of 27 kda (p27) and 88 kda (p88). p88 has an rdrp motif (koonin, 1991) and is produced by programmed − 1 ribosomal frameshifting lommel, 1994 and xiong et al., 1993b) . both p27 and p88 are required for viral rna replication. rna2 encodes a 35 kda movement protein (mp) required for viral cell-to-cell movement in plants (lommel et al., 1988; xiong et al., 1993a) . rna2 is not required for the replication of rna1 in protoplasts (mizumoto et al., 2003; osman and buck, 1987; paje-manalo and lommel, 1989) , but a stem-loop structure on the proteincoding region of rna2 is necessary for the synthesis of subgenomic rna from rna1 in an rna-mediated transactivation mechanism, which leads to the expression of coat protein (cp) (sit et al., 1998; tatsuta et al., 2005) . in addition to 5′-and 3′-untranslated regions (utrs) of rna2, the same stem-loop structure is essential for rna2 replication (tatsuta et al., 2005; turner and buck, 1999) . rna1 and rna2 have no cap structure at the 5′ end (mizumoto et al., 2003) and no poly-a tail at the 3′ end (mizumoto et al., 2002; xiong and lommel, 1989) . the 3′-utr of rcnmv rna1 contains cis-acting rna elements (te-3′ dr1) that can function as a cap-independent translational enhancer (mizumoto et al., 2003) . however, rna2 has no such cis-acting rna element. the strong link between the capindependent translation and replication of rna2 suggests that rna2 functions as mrna for translation only when generated de novo through the viral rna replication machinery (mizumoto et al., 2006) . the rna silencing suppression activity of rcnmv is also linked to viral rna replication (takeda et al., 2005) . thus, p27 and p88 play important roles not only in viral rna synthesis but also in the regulation of viral gene expression, and the suppression of host resistance during viral infection. we found previously that in vitro transcribed rna1 mutants encoding either p27 or p88 do not efficiently complement each other to accumulate detectable viral rnas in by2 protoplasts (takeda et al., 2005) . this suggests several possibilities including cis-preferential requirement of p27 or p88, or both for the replication of rna1. in this study, we investigated how p27 and p88 were engaged in the replication of rcnmv genomic rnas in protoplasts and in a cell-free extract of evacuolated by-2 protoplasts (byl) by using dna vectors or in vitro transcribed rnas expressing viral proteins and rnas. results indicated that rna1 mutants from which intact p88 was translated were efficiently replicated in the presence of p27 in protoplasts, whereas rna1 mutants from which p27 alone or no functional p88 was translated were replicated to very low levels in the presence of p88 and p27, even at levels sufficient to replicate rna2. together with the results obtained from in vitro experiments in byl, our results suggest a cis-preferential function of p88 in the replication of rna1. complementation in replication between p27-deficient and p88-deficient rna1 mutants dna vector plasmids used in this study are presented in fig. 1 . cowpea protoplasts were inoculated with pubrc1-p27, pubrc1-p88u, or their mixture. pubrc1-p27 expresses rna1 mutant that expresses p27 but not p88, and pubrc1-p88u expresses rna1 mutant that expresses p88 but not p27. virus rna accumulation was analyzed after 24 h of incubation by northern blotting using dig-labeled rna probe specific to rcnmv rna1. rna1 accumulated in inoculation with a mixture of pubrc1-p27 and pubrc1-p88u ( fig. 2a , lane 3), whereas no signal specific to rna1 was detected in inoculation with either pubrc1-p27 or pubrc1-p88u alone ( fig. 2a , fig. 1 . schematic representation of wild-type rcnmv rna 1 and 2 (pubrc1 and pubrc2, respectively) and mutants derived from rna 1 (referred to as the names of plasmids). these viral rnas had extra g residue at the 5′ end. the viral rna was initially synthesized in vivo from the 35s promoter of camv, and was cleaved by the ribozyme of satellite tobacco ringspot virus to generate the appropriate 3′ end. individual sites of the introduced mutations were explained and indicated by open triangles. lanes 1 and 2). similar results were obtained by using pubrc1-p88 whose transcripts were used in a previous study (takeda et al., 2005) . both pubrc1-p88u and pubrc1-p88 were expected to express wild-type p88 alone, but differed in silent mutations to eliminate a translational termination codon for p27 (fig. 1) . these results suggested that rna1 mutants expressing p27 or p88 alone, respectively, complemented each other for rna replication. replication of p27-deficient or p88-deficient rna1 mutants by supplying lacking replicase components in trans we next tested whether p27-deficient and p88-deficient rna1s could be replicated by supplying lacking replicase components from protein expression plasmids. cowpea protoplasts were inoculated with various combinations of p27, p88, rna1, and rna2-expressing plasmids, and viral rna accumulations were analyzed. rna1 accumulated efficiently in protoplasts inoculated with a mixture of pubrc1-p88u and pubp27 (fig. 2b , lane 2), although the accumulation level decreased in the presence of rna2 (fig. 2b, lane 4) . on the other hand, the accumulations of rna1 were extremely low or below detection limits in protoplasts inoculated with a mixture of pubrc1-p27 and pubp88 (fig. 2b , lane 1, data not shown), and the accumulation level of rna1 was consistently below detection limits in the presence of rna2 (fig. 2b, lane 3) . either combination of pubrc1-p88u and pubp27 or pubrc1-p27 and pubp88 supported the accumulation of co-inoculated rna2 efficiently (fig. 2b , lanes 3 and 4), indicating that p27 and p88 from these plasmids were sufficient to replicate rna2. thus, these results suggested that p88 mainly functioned in a cispreferential manner in rna1 replication, whereas p27 effectively functioned in trans to support the replication of rna1. if this idea is true, rna1 signals detected in the coinoculation experiments of pubrc1-p27 with pubrc1-p88u or pubrc1-p88 should reflect the preferential accumulation of p88-encoding rna1. to confirm this expectation, we used pubrc1-p27-dcp and pubrc1-p88u-dcp ( fig. 1 ), because these cp-deletion mutants allow one to distinguish rna1 mutants by size. in inoculations with a mixture of pubrc1-p27-dcp and pubrc1-p88u or a mixture of pubrc1-p27 and pucrc1-p88u-dcp, rna1 with a size expected from pubrc1-p88u or pubrc1-p88u-dcp preferentially accumulated ( fig. 2c , lanes 1 and 2). in addition, in vitro transcripts of rna1 mutants expressing p88 alone corresponding to pubrc1-p88u, pubrc1-p88, pubrc1-p88u-dcp, or pubrc1-p88-dcp were efficiently replicated by coinoculation with p27-expressing plasmids (data not shown). cis-acting rna elements are not involved in the failure of rescue of replication of rna1 mutants by p88 supplied in trans the above results do not rule out the possibility that failure in the accumulation of p88-deficient rna1 mutants by p88 supplied in trans might result from the disruption of cis-acting replication elements by the nucleotides inserted into or substituted in those rna1 mutants. pubrc1-p27 has six nucleotides inserted immediately downstream of the stop codon of the p27 orf (tagtaggctaa; inserted nucleotides in bold font) ( fig. 1 ; xiong et al., 1993b; takeda et al., 2005) . however, it is difficult to address the above question by gene manipulation in the region used to create pubrc1-p27, because any nucleotide changes in the region accompany changes in amino acid sequences in p88, which may affect rna replication. therefore, to avoid the effects of gene manipulation on amino acid sequences and cis-acting replication elements, we created other p88deficient rna1 mutants by substituting the u at position 871, thirty nucleotides downstream of the stop codon of p27, because base-substitutions in the region of 870-875 nt have been shown not to affect rna replication and systemic infection of rcnmv (kim and lommel, 1998) . pubrc1-871a and pubrc1-871g had a and g, respectively, at position 871, which create an additional stop codon to prevent p88 expression. pubrc1-871c had c at position 871 to create a synonymous codon for wild-type tyrosine (fig. 1) . these plasmids were inoculated into cowpea protoplasts alone or together with pubp88 or a mixture of pubp27 and pubp88. pubrc2 was also used to confirm the expression of p27 and p88. in inoculation with pubrc1-871a or pubrc1-871g alone, rna1 mutants did not accumulate to detectable levels, confirming the lack of expression of functional p88 from these rna1 mutants (fig. 3, lanes 3 and 4) . in contrast, rna1 from pubrc1-871c accumulated to a level similar to wild-type rna1 (fig. 3, lanes 5) . supplementation of p88, or both p88 and p27 in trans did not efficiently support the replication of rna1 mutants from pubrc1-871a and pubrc1-871g ( fig. 3. lanes 7, 8, 11 , and 12), whereas the trans-supplied p88 efficiently supported rna2 replication with co-inoculated pubrc1-871a or pubrc1-871g ( fig. 3. lanes 7 and 8) . the successful replication of rna1 from pubrc1-871c suggested that single-nucleotide substitutions at this position did not affect cis-acting rna elements required for replication. together, these results suggest that the impaired ability of rna1 mutants expressing p27 alone to be amplified by trans-supplied p88 is caused by a cis-preferential requirement of p88 for rna1 replication. effects of mutations in the gdd motif on rna1 replication in supply of wild-type p88 to investigate whether a complete translation process through p88 orf is involved in the cis-preferential requirement of p88 for rna1 replication, we used pubrc1-p88ugvd, in which the gly-asp-asp (gdd) motif was substituted to gly-val-asp (gvd) (fig. 1) . the gdd motif is conserved in rdrp (koonin and dolja, 1993) , and is required for rcnmv rna synthesis (bates et al., 1995) . cowpea protoplasts were inoculated with pubrc1-p88ugvd or pubrc1 together with or without pubp88p and pubrc2. in protoplasts inoculated with pubrc1-p88ugvd, the accumulation of rna1 was extremely low com-pared to that of wild-type rna1 from pubrc1 (fig. 4) . similar results were obtained in coinoculation with a mixture of pubp88 and pubrc2 (fig. 4) . these results indicated that wild-type p88 supplied in trans did not complement the impaired replication of rna1 mutant containing gvd motif. failure of the rescue in the replication of rna1 mutant with gvd motif by wild-type p88 suggests that the act of translation throughout the p88 orf is insufficient by itself to recruit replication factors including p88 to rna1, and supports the cis-preferential requirement of p88 for rna1 replication. the effect of the introduced mutation in the gvd mutant on cis-acting rna replication elements is discussed later. cis-preferential requirement of p88 for negative-strand rna synthesis in byl to further investigate the functions of p27 and p88 in rna1 replication, we analyzed negative-strand rna synthesis in a byl in vitro system using in vitro transcribed rna1 mutants. this system allows one to analyze negative-strand rna synthesis following translation from the input rcnmv rnas (iwakawa et al., 2007) . rna1 transcripts having mutations corresponding to pubrc1-871a, pubrc1-p88u, pubrc1-871a-dcp and pubrc1-p88u-dcp were incubated in various combinations in the byl in vitro system. immunoblot analysis using p27-antiserum confirmed the accumulation of p27 and p88 as expected from the nucleotide sequences of rna1 mutants (fig. 5, lanes 3 to 8) . in incubation of a mixture of rna1 mutants corresponding to pubrc1-p88u and pubrc1-871a-dcp and a mixture of those corresponding to pubrc1-p88u-dcp and pubrc1-871a, negative-strand rna1s with a size expected from the respective mutants expressing p88 were detected as a band with strong intensity (fig. 5, lanes 7 and 8) . rna bands with sizes corresponding to those expected for rna1 mutants expressing p27 were also detected in these lanes, but the intensity of the bands was similar to that of rna1 mutants incubated alone (fig. 5, lanes 3 to 6) . therefore, these rnas with weak signals were unlikely to be negative-strand rna products synthesized from the input rna1 mutants. together, the results obtained in the byl in vitro system indicated that negative-strand rna was synthesized only from rna1 mutants expressing p88 and in a cis-preferential manner. our study shows that only rcnmv rna1, from which p88 is translated, can be an effective template for viral rna replicase to initiate rna synthesis in the presence of p27. this suggests that p88 is required preferentially in cis for the replication of rna1, which contrasts with the efficient transacting activity of the protein for the replication of rna2. a cis-preferential function of the encoded protein in viral rna replication or coupling between translation and replication of viral rna has been reported in genomic, defective (d), or defectiveinterfering (di) rnas of several positive-strand rna viruses. in these phenomena, the process of translation, including the protein-coding capacity of the d or di rnas, may be essential for viral rna replication; alternatively the translated protein products included in the translation process may be essential for viral rna replication. for example, the replication of poliovirus di rna requires inframe deletion in the 2b non-structural protein (johnson and sarnow, 1991) and the replication of di rnas with amber mutations introduced into the 2a protein gene is not complemented in trans by the wild-type virus (novak and kirkegaard, 1994) . these observations suggest a cis-preferential requirement of either the encoded protein or the translation process itself through the coding region, or both, for rna replication. in the case of a prototype of cymv d rna, the process of translation rather than the encoded protein is considered essential for rna replication in the presence of helper cymv rna. the full-length d rnas of cymv containing prematurely terminated fusion orfs are not viable, whereas d rnas containing a variety of inframe deletions and insertions in the regions of the fusion orf of the d rna are viable, despite the observation that these fusion orfs are unlikely to encode functional proteins (white et al., 1992) . in the coupling between translation and replication of rcnmv rna1, a biologically active translation product rather than translation process appears important for rna1. this is because an rna1 mutant with the gdd motif replaced by gvd failed to replicate in the presence of p27 and p88 (fig. 4) . failure in the replication of this mutant suggests that the translation process through p88 orf, including − 1 frameshift, is not sufficient to recruit replication proteins and to initiate the replication of rna1. although the effects of the mutation introduced at position 1818 on cis-acting rna replication elements in the gvd mutant cannot be completely ruled out, no or little effect on rna1 replication of one nucleotide substitution (g to c) at position 1815 in the gdd motif (add in the mutant) (h. nagano and t. okuno, unpublished data) suggest that the impaired replication of the gvd mutant is mainly caused by amino acid change in p88, and not by nucleotide change in rna. no report for the occurrence of small rna replicons derived from rna1 in rcnmv infection supports the cis-preferential requirement of p88 for the replication of rna1. this is unlike rna2, which has cis-acting rna elements that can recruit replication proteins supplied in trans: the 3′ utr of rna2 or the trans-activator located in the mp orf are candidates for the cis-acting rna elements of rna2, because these elements are essential for the initiation of negative-strand rna synthesis (takeda et al., 2005; tatsuta et al., 2005; turner and buck, 1999; h. iwakawa and t. okuno, unpublished results) . in place of such rna elements in rna2, p88 may function as an element for recruiting replication factors including p27 in rna1 replication. binding of p88 to the proper region of rna1 through translation fig. 5 . analysis of translation products and negative-strand rna synthesis of rcnmv rna1 mutants in a cell-free byl system. in vitro transcribed uncapped rcnmv rnas, rc1, rc1-p88u, rc1-p88u-dcp, rc1-871a, and rc1-871a-dcp corresponding to dna vector plasmids, pubrc1, pubrc1-p88, pubrc1-p88-dcp, pubrc1-871a, and pubrc1-871a-dcp were incubated in byl reaction mixture at 17°c for 4 h. proteins and negativestrand rnas, respectively, were analyzed by western blotting with p27antiserum and northern blotting with specific probes for rna1 and rna2. may cause a structural change in rna1, which is required for the formation of rna replication complexes. the regions may include rna elements predicted in the 3′ proximal regions of rna1 that are essential for initiating rna synthesis (iwakawa et al., 2007) . a structural change in rna induced by viral protein and its translation has been reported in several rna viruses. in the rna bacteriophage ms2, translation of the cp gene is required for the expression of the replicase protein gene. the cp gene alone is accessible to ribosomes in the full-length genomic rna. the ribosomal binding site of the replicase gene is masked by a base pairing to an internal cp gene located at upstream of the viral genome, and the expression of the replicase protein gene is coupled to the expression of cp by the disruption of the base pairing and release of the replicase start site (berkhout and van duin, 1985) . cp-mediated rna structural change has also been reported in the 3′ utr of genomic rnas of amv, in which cp plays an important role in initiation of viral rna synthesis (olsthoorn et al., 1999) . in amv, non-structural replicase component proteins encoded by genomic rna 1 and rna2 are required in cis for the replication of rna1 and rna2 (neeleman and bol, 1999; van rossum et al., 1996) . what does cis-preferential requirement for p88 in rna1 replication means for rcnmv? cis-preferential requirement of p88 coupled with p88 translation will limit the number of rna1 molecules that can engage in rna replication, because a frameshifting event required to generate p88 occurs in less than 10% of translations, as assessed by the rabbit reticulocyte lysate in vitro translation assay or by transient expression assay of the chimeric rcnmv frameshift element-gus construct in nicotiana benthamiana protoplasts (kim and lommel, 1994; kim and lommel, 1998) . we have never detected p88 in rcnmvinfected cells and in incubation of wild-type rna1 in byl in our experimental conditions (unpublished data; fig. 5 ). in byl, p88 was detectable only in incubation of rna1 mutants from which p88 alone is translated. this implies that most wild-type rna1 can engage in translation to produce a large amount of p27 by functioning as a template for ribosomes without competing with rna replicase. in addition to frameshifting regulation, such regulation should lead to overproduction of p27 in excess to that of p88, which may be important for rcnmv rna replication and infection. on the other hand, cis-preferential requirement of p88 in rna1 replication may function to avoid or reduce access of translation factors to rna1 and to facilitate the replication of rna1. genomic rna of positive-strand rna viruses serves as a template for synthesis of negative-strand rna and as a template for synthesis of viral proteins. this results in a conflict between rna replication and translation pathways. the cispreferential requirement of viral proteins for rna replication may partly help to facilitate a switch in the role of genomic rna from mrna to a replication template. in polioviruses whose replication appears to be coupled with translation of encoded viral proteins (novak and kirkegaard, 1994) , the binding of viral protein 3cd represses translation and facilitates negative-strand rna synthesis (gamarnik and andino, 1998) . there remain several questions to be addressed for the roles of cis-preferential requirement of p88 in rna1 replication. for example, at what frequency is p88 used for cis-preferential replication of rna1? at least sufficient amounts of p88 should be available for rna2 replication. probably, p88 is released from rna1 at frequencies enough to support rna2 replication. it is also possible that rna2 may have the ability to deprive rna1 of translated p88. interestingly, the cis-preferential requirement of the frameshift product p88 in the replication of rcnmv rna1 is opposite to what was found in two other members of tombusviridae, tcv and tbsv. in these viruses, viral rnas expressing pre-readthrough products, p28 and p33 alone, respectively, are preferentially replicated over those expressing readthrough products alone in their coinoculation (white et al., 1995; oster et al., 1998; monkewich et al., 2005) . in tbsv, the preferential replication of viral rnas expressing p33 alone does not result from a cis-preferential function of p33, but rather results from translation-based inhibition of a cis-acting rna element (rii) that is positioned in the readthrough portion of the p92 orf (monkewich et al., 2005) . rii is considered to function for assisting the recruitment of viral rnas out of translation and into replication. thus, a cis-preferential function of viral replication proteins or the translation process appears to function in a different way in regulation between rna replication and translation in different viruses. plasmid clones pubrc1 (mizumoto et al., 2006) and pubrc2 (takeda et al., 2005) were previously described as plasmids from which wildtype rcnmv australian strain rna1 and rna2, respectively, were transcribed from the cauliflower mosaic virus 35s promoter in the introduced cells. pubrc1-p27 was created by insertion of the saci/smai fragment containing rcnmv rna1 mutant cdna from pbicrc1-p27 (takeda et al., 2005) into puc118 at the corresponding sites. pubrc1-p27dcp and pubrc1-p88u-dcp were derived from pubrc1-p27 and pubrc1-p88u, respectively, in which the xhoi/sacii region was replaced with that from pbicrc1-δcp (takeda et al., 2005) . other mutants, pubrc1-p88u, pubrc1-871a, pubrc1-871g, pubrc1-871c, and pubrc1-p88ugvd were created by pcr-based in vitro mutagenesis from pubrc1. in short, recombinant pcr products were digested by appropriate restriction enzymes that had unique recognition sites neighboring the mutation sites in pubrc1, and the fragments containing the mutation substituted the corresponding fragments in pubrc1. the absence of unexpected base change was confirmed by sequencing the resulting plasmids. these rna1 mutants transcribed from pubrc1 plasmids with harboring mutations are summarized in fig. 1 . the plasmids for protein expression, pubp27 and pubp88 were previously described (takeda et al., 2005) . all infectious dna plasmids were prepared by using qiagen plasmid midi kit (qiagen). the plasmids pucr1-871a, pucr1-871a-dcp, pucr1-p88u and pucr1-p88u-dcp were created by replacement of the aor51hi-bsiwi region in pucr1 (takeda et al., 2005) with the corresponding regions from pubrc1-871a, pubrc1-871-dcp, pubrc1-p88u and pubrc1-p88u-dcp, respectively. rna transcripts were synthesized from these plasmids by t7 rna polymerase after linearization by xmai. preparation and inoculation of cowpea (vigna unguiculata cv. california blackeye) protoplasts were previously described (mizumoto et al., 2003) . in brief, the inoculation was performed by polyethylene glycol-mediated inoculation method as described by sheen (http://genetics.mgh.harvard.edu/sheenweb/) using peg4000 (fluka). mixture of plasmids (10 µg each) or transcripts (1.5 µg each) were used as an inoculum. inoculated protoplasts (5 × 10 4 cells) were incubated for 24 h at 17°c (mizumoto et al., 2003) . total rna and protein were extracted from infected protoplasts using trizol reagent (invitrogen) and laemmli's sample buffer (laemmli, 1970) , respectively, and subjected to northern blot and immunoblot analysis. all inoculation experiments were repeated at least three times. preparation of cell extracts of evacuolated tobacco by-2 protoplasts (byl) and cell-free in vitro translation and replication were done as described (iwakawa et al., 2007) . uncapped in vitro transcripts (2 µg in each 1 µl) were added to 25 µl byl. the byl translation and replication mixture was incubated at 17°c for 4 h. aliquots (15 µl) of the reaction mixture were used for immunoblot analysis, and aliquots (10 µl) of the reaction mixture were used for northern blot analysis as described previously (iwakawa et al., 2007) . northern blot analysis was done as previously described (damayanti et al., 2002) . the digoxigenin (dig)-labeled rna probes specific to the 3′ utrs of rcnmv rna1 and rna2, and negative-strand rna1 and rna2, were synthesized in vitro and used for the hybridization as previously described (mizumoto et al., 2002) . the rna signals were detected with a luminescentimage analyzer (las 1000 plus; fuji photo film, japan). immunoblot analysis of p27 and p88 was performed as previously described (tatsuta et al., 2005) using ecl plus western blotting detection system (ge healthcare bio-science, usa). rna-dependent rna polymerases, viruses, and rna silencing host factors in positive-strand rna virus genome replication isolation and characterization of an rna-dependent rna polymerase from nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus mechanism of translational coupling between coat protein and replicase genes of rna bacteriophage ms2 comparison of the replication of positive-stranded rna viruses of plants and animals a cis-acting function for the coronavirus leader in defective interfering rna replication positional effect of deletions on viability, especially on encapsidation, of brome mosaic virus d-rna in barley protoplasts switch from translation to rna replication in a positive-stranded rna virus in vitro construction of poliovirus defective interfering particles cis-acting core rna elements required for negative-strand rna synthesis and cap-independent translation are separated in the 3¢-untranslated region of red clover necrotic mosaic virus rna1 three poliovirus 2b mutants exhibit noncomplementable defects in viral rna amplification and display dosagedependent dominance over wild-type poliovirus de novo initiation of viral rnadependent rna synthesis sequence element required for efficient − 1 ribosomal frameshifting in red clover necrotic mosaic dianthovirus identification and analysis of the site of − 1 ribosomal frameshifting in red clover necrotic mosaic virus the phylogeny of rna-dependent rna polymerases of positive-strand rna viruses evolution and taxonomy of positive-strand rna viruses: implications of comparative analysis of amino acid sequences cleavage of structural proteins during the assembly of the head of bacteriophage t4 functions of the 126-and 183-kda proteins of tobacco mosaic virus rubella virus rna replication is cis-preferential and synthesis of negative-and positive-strand rnas is regulated by the processing of nonstructural protein the nucleotide sequence and gene organization of red clover necrotic mosaic virus rna-2 roles of the sequence encoding tobacco etch virus capsid protein i genome amplification: requirements for the translation process and a cis-active element capindependent translation mechanism of red clover necrotic mosaic virus rna2 differs from that of rna1 and is linked to rna replication capindependent translational enhancement by the 3¢ untranslated region of red clover necrotic mosaic virus rna1 the 3¢-untranslated region of rna1 as a primary determinant of temperature sensitivity of red clover necrotic mosaic virus canadian strain the p92 polymerase coding region contains an internal rna element required at an early step in tombusvirus genome replication cis-acting functions of alfalfa mosaic virus proteins involved in replication and encapsidation of viral rna coupling between genome translation and replication in an rna virus a conformational switch at the 3¢ end of a plant virus rna regulates viral replication replication of red clover necrotic mosaic virus rna in cowpea protoplast: rna1 replicates independently of rna2 uncoupled expression of p33 and p92 permits amplification of tomato bushy stunt virus rnas independent replication of red clover necrotic mosaic virus rna-1 in electroporated host and nonhost nicotiana species protoplasts analysis of the vpg-proteinase, (nia) encoded by tobacco etch potyvirus: effects of mutations on subcellular transport, proteolytic processing, and genome amplification rna-mediated trans-activation of transcription from a viral rna a plant rna virus suppresses rna silencing through viral rna replication the red clover necrotic mosaic virus rna2 trans-activator is also a cis-acting rna2 replication element mutational analysis of cis-acting sequences in the 3¢-and 5¢-untranslated regions of rna2 of red clover necrotic mosaic virus cis-and trans-acting elements in cowpea mosaic virus rna replication composition of alphavirus-like replication complexes: involvement of virus and host encoded proteins accumulation of alfalfa mosaic virus rnas 1 and 2 requires the encoded proteins in cis cis-preferential replication of the turnip yellow mosaic virus rna genome immunodetection, expression strategy and complementation of turnip crinkle virus p28 and p88 replication components coding capacity determinants in vivo accumulation of a defective rna of clover yellow mosaic virus the complete nucleotide sequence and genome organization of red clover necrotic mosaic virus rna-1 the roles of the red clover necrotic mosaic virus capsid and cell-to-cell movement proteins in systemic infection synthesis of the putative red clover necrotic mosaic virus rna polymerase by ribosomal frameshifting in vitro the authors thank s. a. lommel for the kind gift of prc1|g and prc2|g, which are original cdna clones of rna1 and rna2 of rcnmvaustralian strain. we thank c. p. coloma for the construction of several cdna clones. this work was supported in part by a grant-in-aid for scientific research (a) (18208004) from the japan society for the promotion of science, and in part from a grant-in-aid for scientific research on priority area (a) 'spatiotemporal network of rna information flow' from the ministry of education, culture, sports, science and technology, japan. key: cord-253024-b393ea2u authors: fu, kaisong; baric, ralph s. title: evidence for variable rates of recombination in the mhv genome date: 1992-07-31 journal: virology doi: 10.1016/0042-6822(92)90684-h sha: doc_id: 253024 cord_uid: b393ea2u abstract mouse hepatitis virus has been shown to undergo rna recombination at high frequency during mixed infection. temperature-sensitive mutants were isolated using 5-fluorouracil and 5-azacytidine as mutagen. six rna+ mutants that reside within a single complementation group mapping within the s glycoprotein gene of mhv-a59 were isolated which did not cause syncytium at the restrictive temperature. using standard genetic techniques, a recombination map was established that indicated that these mutants mapped into two distinct domains designated f1 and f2. these genetic domains may correspond to mutations mapping within the s1 and s2 glycoproteins, respectively, and suggest that both the s1 and s2 domains are important in eliciting the fusogenic activity of the s glycoprotein gene. in addition, assuming that most distal is alleles map roughly 4.0 kb apart, a recombination frequency of 1 % per 575–676 by was predicted through the s glycoprotein gene. interestingly, this represents a threefold increase in the recombination frequency as compared to rates predicted through the polymerase region. the increase in the recombination rate was probably not due to recombination events resulting in large deletions or insertions (>50 bp), but rather was probably due to a combination of homologous and nonhomologous recombination. a variety of explanations could account for the increased rates of recombination in the s gene. mouse hepatitis virus (mhv), a member of coronaviridae, contains a single-stranded nonsegmented plus-polarity rna of about 32 kb in length (lee et a/., 1991; pachuk et a/., 1989) . the genomic rna is arranged into seven or eight coding regions and is encapsidated within multiple copies of a 50-kda nucleocapsid protein (n) (lai, 1990; siddell, 1983) . the n protein forms a helical nucleocapsid structure that is probably associated with the transcriptional complex compton et a/., 1987; stohlman and lai, 1979; stohlman eta/., 1988; sturman eta/., 1980) . the nucleocapsid structure is surrounded by a lipid envelope and contains two or three virus-specific glycoproteins. the s glycoprotein has a molecular weight of 180 kda and is frequently cleaved into two 90-kda glycoproteins designated sl and s2 (frana et a/., 1985; sturman et a/., 1980 . the n-terminal signal sequence is contained within the sl domain while the c-terminus is contained within the s2 region (boireau et al., 1990; luytjes eta/., 1987; rasschaert et al., 1990; schmidt et al., 1987) . the sl domain can undergo significant amino acid alteration without losing its function while the s2 domain is more highly conserved and contains a hydrophobic heptad repeat element that probably forms a complex ' to whom correspondence should be addressed. coiled coil (luytjes et a/., 1987; schmidt et a/., 1987) . the s glycoprotein is responsible for virus binding to the specific cellular receptor, induction of cell fusion, and elicitation of neutralizing antibody and cell-mediated immunity (collins et al., 1982; fleming et a/., 1986; sturman et a/., 1980 . in addition to s, a 23-kda m glycoprotein is present in the virion that probably functions in assembly and release (sturman et a/., 1980 . in some strains of mhv, a 65-kda hemagglutinin (he) that shares considerable sequence homology with the influenza c virus hemagglutinin is also present (luytjes et al., 1988; shieh et al., 1989; yokomori et al., 1989) . upon entry into susceptible cells, the genomic rna is translated into one or more polyproteins that act as an rna dependent rna polymerase (mahy et a/., 1983; brayton et al., 1982 brayton et al., , 1984 . while the exact mechanism for mhv transcription is still under study, the preponderance of data suggest that the genome is initially transcribed into a full-length minus strand rna, which acts as template for the synthesis of a genomelength rna and six subgenomic mrnas by "leaderprimed" transcription (baker et a/., 1990; baric et al., 1983 baric et al., , 1985 makino et a/., 1986a makino et a/., , 1991 . in turn, the subgenomic mrna then act as template for the synthesis of subgenomic minus strands, which participate in successive rounds of mrna amplification (sawicki and sawicki, 1990; sethna et a/., 1989 sethna et a/., , 1991 . homologous recombination among viruses with nonsegmented rna genomes has been reported among picornaviruses (cooper, 1968 (cooper, , 1977 cooper et al., 1975; king et al., 1982 king et al., , 1985 king et al., , 1987 lake et al., 1975) , brome mosaic virus (bujarski and kaesberg, 1986) alphaviruses (hahn et al., 1988; weiss and schlesinger, 1991) , cowpea chlorotic mottle virus 3a (allison et al., 1990) , and coronaviruses (keck et al., 1988a,b; lai et a/., 1985; makino et al., 1986b) . during mhv replication, the rna recombination frequency is unusually high and approaches 25% or more during mixed infection . recently, biochemical analysis has revealed extensive polymorphisms and deletions in the sequence of the s glycoprotein gene of different mhv variants. the variant viruses were probably derived from nonhomologous recombination because no consensus or conserved sequences flanked the deletions (banner et al., 1990; parker et a/., 1990) . sequence analysis of rna recombinant viruses also suggests the presence of a recombination "hot spot" in the mhv s glycoprotein gene (banner el al., 1990) . however, in the absence of selection pressure, rna recombination sites in this region are random, suggesting that this preferred site reflects the selection for biologically more efficient recombinants . recombination analysis provides a powerful tool for mapping genetic loci and determining the recombination frequency between individual temperature sensitive (ts) mutations cooper, 1968 cooper, , 1977 lake et al., 1975) . in this study, several group f rna+ ts mutants were used to establish a genetic recombination map in the s glycoprotein gene of mhv-a59. temperature-sensitive mutants representing the group f rna+ mutants (la7, la1 2, nc6, nc1 4, nc1 6, nc1 7) and group e rna-mutants (la1 8) of mhva59 were used throughout the course of this study. all virus stocks were propagated at 32" in 1 50-cm2 flasks containing dbt cells as described previously . plaque assays were performed at either the permissive (32") or nonpermissive (39.5") temperatures in dbt cells in dulbecco's modified essential medium (dmem) (sigma) containing 10% nu-serum (collaborative research, inc.), 1% gentamycin/kanamycin (gibco), and 0.8% agarose (brl). all plaque assays were stained 28-36 hr postinfection by the addition of neutral red for 2 hr. cloned 17ci-1 cells were kindly provided by dr. sue baker (loyola university, chicago) and maintained at 37" in dmem containing 7% newborn calf serum (gibco), 1% gentamycin/kanamycin, 2% glucose (sigma), and 5% ttyptose-phosphate broth (sigma) . most of the mutants used in this study have been characterized previously (la1 8, la7, la1 2, nc6) . to increase the accuracy of our recombination mapping data, additional mutants residing within complementation group f were isolated. briefly, cultures of mhv-a59-infected cells were mutagenized with either 20 pug/ml 5-azacytidine or 100 pg/ ml 5-fluorouracil for 12 hr. supernatant fluids containing mutagenized virus were plaqued at permissive temperature and individual plaques were isolated. each plaque was diluted into 0.5 ml ice-cold pbs, screened at 32" and 39.5" by plaque assay, and isolates displaying ts phenotypes were repurified two to three times at 32". virus stocks were assayed at the permissive and restrictive temperatures and only those isolates displaying reversion frequencies of less than 1 om3 were used for future studies. complementation and rna phenotype analysis were performed as described previously (leibowitz et al., 1982; martin et al., 1988; , and three additional group f rna+ mutants were isolated and designated nc14, nc1 6, and nc1 7. nc1 4 and nc1 6 were isolated using 5-fluorouracil, while nc 17 was isolated using 5-azacytidine. to select for revertants from the group f rna+ ts mutants, virus stocks were plaque assayed at 39.5", and individual plaques were isolated and repurified by plague assay at the restrictive temperature. individual stocks of revertant virus were grown in 1 50-cm2 flasks at 37" and the reversion frequency of each was determined by plague assay at 32 and 39.5". at least three independent revertants from each rna+ mutant were isolated. to isolate rna recombinant virus, dbt cells were coinfected with two different ts mutants at an m.o.i. of 10 each at 32". the medium was harvested at 16 hr postinfection and stored at -70". recombinant viruses were isolated at the restrictive temperature, repurified at 39.5", and grown in dbt cells at 37". culture of 17cl-1 cells (1 x 106) in 25-mm six-well dishes (costar) were infected with various group f ts mutants, revertants, or recombinant viruses at a multiplicity of infection of 10 for 1 hr at room temperature. following adsorption, the inoculum was removed, and the cultures were rinsed with pbs and incubated in complete dmem for 4 hr at 32". actinomycin d (2 pgi ml) was added for 1 hr and one-half the cultures shifted to the restrictive temperature at 5 hr postinfection by the addition of prewarmed media. viral progeny were harvested at varying intervals and analyzed by plaque assay. to determine whether these mutants could transcribe viral mrna, cultures of cells were infected with various ts mutants and maintained at the permissive or restrictive temperature for 8 hr. the media were removed and the cultures washed with 2 ml of iso-tkm (10 m/w tris-hci, ph 7.5, 150 mni kci, and 1.5 mm mgci,). the rna was isolated as described by sawicki and sawicki, 1990 , and extensively purified by successive rounds of phenol (u.s. biochemical company), phenol/chloroform/isoamy alcohol, and chloroform extraction. the rna was bound to nitrocellulose filters and probed with radiolabeled rna probes specific for the mhv n gene . the blots were washed and exposed to xar-5 film for 24 hr. recombination analysis and establishing a genetic recombination map of the group f rna+ mutants recombination analyses were performed as previously described and the recombination frequencies calculated as the percentage of ts+ virus present in the progeny from the following formula: rf = vw,,., -ta + b)3~.s x , ooo,o, . . -* bw32 (ab),,., was the titer of the cross at nonpermissive temperature while (ab),, represented the titer of the same cross at permissive temperature. (a + b)39.5 was the sum of the revertants of each parent strain assayed at the nonpermissive temperature. the formula only measured single or odd-number cross-over events resulting in the ts+ phenotype and did not account for recombination events that resulted in the doublets mutant phenotype. all recombination frequencies were standardized to a standard cross [la7 x nc1 6; recombination frequency 3.0 f 1.21 as previously described . the mutants were aligned according to their standardized recombination frequencies and positioned from la7, la1 8, and nc1 6. cultures of dbt cells in loo-mm2 dishes were infected at a m.o.i. of 5 with different ts mutants, revertant viruses, or recombinant viruses, and intercellular rna was harvested at 8-l 2 hr postinfection. the precipitate was resuspended in te buffer (10 mni tris-hci, ph 7.2, 1 mm edta) and used as template for reverse transcription and taq amplification reactions. cdna synthesis was carried out in a 20-~1 reaction mixture containing 1 pg intercellular rna, 5 mm mgci,, 10 m/l/l tris-hci ph 8.4, 50 mm kci, 0.1% triton x-l 00 (sigma), 1 mm dntp each (promega), 0.5 pg random hexamer (perkin elmer cetus), 1 u/pi rnasin (promega), 15 u amv reverse transcriptase (promega) and incubated at 42" for 1 hr. the products were extracted and precipitated in ethanol and resuspended in 50 ~1 h,o prior to successive rounds of polymerase chain amplification (pcr). one-tenth of the cdna was amplified in a 50 ~1 reaction mixture containing 10 mm tris-hci (ph 9.0) 50 mm kci, 3 mm mgci,, 0.1 o/o triton x-l 00, 1.25 u taq dna polymerase (promega), 0.2 mm dntp mixture and the appropriate primer pairs (100 ng of each). pcr amplification was performed for 30 successive cycles at 1.5 min at 94" to denature the dna, 2 min at 55" for primer annealing, and 4 min at 72" for primer extension. the products were loaded onto 1% agarose gels in tae (40 mltltris, 9 mm naci, 1 mm edta, (ph 8.0)) separated electrophoretically, stained with ethidium bromide, and visualized under uv light. the dna products were transferred to nitrocellulose filters, and confirmed by hybridization with 32p-labeled oligomer probes specific for internal sequences in each gene fragment amplified. three sets of overlapping primer pairs were obtained from highly conserved sequences in the mhv-a59 and jhm s glycoprotein genes (banner et a/., 1990; luytjes et al., 1987; parker et a/., 1990) . set a was derived from sequences spanning nucleotides -1 to 15 (cat gct ggt cgt gtt t) and 662-677 (aac gta gta gcg gag g), respectively, and should result in a dna fragment of 0.67 kb in length. set b was derived from nucleotides 642-662 (gcg tac tat tcg gat aaa cc) and 2 103-2 115 (ccc acg acc gaa tac g) and were used to amplify a -1.5-kb fragment encompassing the hypervariable region in the sl domain (banner et a/., 1990; parker et a/., 1990) . set c was derived from nucleotides 2067-2083 (acg gat gag gcg ctt c) and 4055-4070 (gtc llt cca gga gag g) and were used to amplify a -2.0-kb fragment spanning the c terminus of the s glycoprotein gene. internal oligodeoxynucleotides were used as probe to demonstrate the specificity of the pcr reaction. six complementation group f rna+ mutants and one group e rna-mutant were used during the course of this study. three of the rna' mutants (la7, la1 2, 3.0 x lo7 1.0 x 103 3.3 x 1o-4 1.1 x lo* 1.0 x lo2 1.0 x 10-6 5.5 x 107 1.3 x lo* 2.4 x 1o-6 5.5 x lo7 9.5 x lo4 1.7 x 10-3 3.5 x 10' 4.3 x lo3 1.2 x 1o-4 7.8 x 10' 1.4 x lo4 1.8 x 1o-4 6.9 x 10' 8.7 x 10' 1.2 x loo 7.3 x lo7 7.2 x 10' 1.0 x loo 9.1 x 10' 9.5 x lo7 1.0 x loo 2.1 x lo7 2.0 x 10' 9.5 x 10-l 8.5 x lo* 5.5 x 10" 6.4 x lo-' 9.1 x lo7 7.2 x 10" 7.9 x 10-z 2.2 x 10' 2.4 x lo7 1.1 x loo 4.0 x 10' 2.0 x lo6 5.0 x 10-2 8.6 x 10' 8.4 x 10' 9.7 x 10-l 1.2 x lo8 1.2 x lo8 1.0 x lo0 1.2 x lo8 1.2 x 108 1.0 x loo 9.4 x 10' 1.2 x 108 1.2 x loo 8.2 x lo7 9.2 x lo7 1.1 x lo0 1.0 x 108 9.0 x lo7 9.0 x 10-l 9.2 x 10' 8.5 x 10' 9.2 x 10-l 2.8 x 10' 3.2 x 10' 1.1 x loo 5.2 x lo7 4.5 x 10' 8.6 x 10-j 4.7 x 10' 4.6 x 10' 9.7 x 10-l 5.9 x 10' 4.8 x 10' 8.1 x 10-l + + + + + + + + "calculated as 39.5132" nc6) and one rna-mutant (la1 8) have been characterized previously (schaad el al., 1990) . to increase the accuracy of the recombination map and obtain more representative mutants in the s glycoprotein gene, three additional rna+ mutants were isolated by mutagenesis as previously described . two mutants were isolated following mutagenesis with 100 pg/ml 5-fluorouracil (nc14, nc16) and one mutant with 20 pg/ml 5-azacytidine (nc17). all mutants had reversion frequencies of less than 10e3, and did not complement each others defect at the restrictive temperature (table 1 , data not shown). all of the rna+ mutants were capable of transcribing viral mrna when maintained at the restrictive temperature (data not shown, table 1 ). while nc14 produced a small amount syncytium, the remainder of the mutants were not capable of giant cell formation at the restrictive temperature (fig. 1 ). to assist in precisely defining the location of these mutations and examining the mechanism for mhv rna recombination within the s glycoprotein gene, a panel of revertants were obtained from six of the rna+ mutants (table 1) . revertant viruses had similar titers, produced syncytium, and were of the rna+ phenotype when assayed at both permissive and restrictive temperatures. to conclusively document the revertant phenotype and determine if growth characteristics were similar to parental controls, growth curves were compared at the permissive and restrictive temperatures. cultures of cells were infected with nc1 6 or its revertant, nc1 6-rl, at 32" and shifted to 39.5" at 5.0 hr postinfection. viral progeny were harvested at different times after infection and assayed by plaque assay at the permissive temperature. similar growth curves were evident between ts nc1 6 and its revertant nc1 6-rl in cultures maintained at the permissive temperature (fig. 2) . however, following temperature shift, growth of ts nc16 was inhibited significantly under conditions in which the revertant replicated normally. similar results have also been obtained with other mutants and their revertants (data not shown). to assess the ability of these mutants to be used in recombination studies, we performed a series of crosses between different group f ts mutants and examined the differences between the recombination and reversion frequencies. cultures of cells were infected at a m.o.i. of 10 each with two different ts mutants and maintained at 32" for 14 hr postinfection. the progeny were harvested and titered at the permissive and restrictive temperatures. recombination frequencies ranged from as little as 0.1% to >4.00/0 suggesting that the mutants map in different locations in the gene. the ratio of recombinants to revertants ranged from 9 to 3 140 times higher than the sum of the spontaneous reversion frequencies of each individual ts mutant used in the cross and indicated that recombination mapping was feasible throughout this region ( table 2 ). the extent of the difference in the ratio of recombinants to revertants probably reflected the stability of the mutants used in the cross and the distance between the individual ts lesions. establishing a recombination map for the group f rna+ mutants in contrast to poliovirus and apthovirus ts mutants, mhva59 ts mutants were amenable to complementation analysis, providing strong evidence that all of the rna+ ts mutants used in this study contain defects in a single gene or a noncomplementable function (koolen et al., 1983; leibowitz et al., 1982; martin et al., 1988; schaad et al., 1990) . all mutants were originally crossed three to five times with the reference mutants la7, nc16, or a group e rna-mutant la18 and standardized to the reference cross la7 x nc16. to obtain a more detailed map of the group f mutants, crosses were also performed among each of the viruses used in this study and standardized to the reference cross. the results of these experiments are shown in table 3 . the distances between different ts mutants were within statistical limits and permitted the construction of a genetic map (fig. 3) . on the basis of their genetic recombination frequencies, the group f rna+ ts mutants appeared to map into two distinct domains, designated fl (la7, la12, nc6) and f2 (nc14, nc16, nc17, la1 2). ts la1 2 mapped closely to representative mu-tants within each subgroup suggesting that it may be a double mutant. assuming that crossover events occur in both directions and that nc6/la7 and nc 14/nc 16 map at different ends of the 4.0-kb s glycoprotein gene, a 1% recombination frequency occurred over 575-667 nucleotide pairs of rna. since these mutants do not produce syncytium at the restrictive temperature ( fig. 1) and sequence analysis of rna recombinant viruses suggested that the defect in la7 mapped within the first 1 .l kb of the s glycoprotein gene (banner et al., 1990; keck eta/., 1987 keck eta/., , 1988a makino eta/., 1986b makino eta/., , 1987 , these data provided an anchor for mapping the location of the remaining group f rna+ mutants. these data predicted that the fl mutants map in the sl glycoprotein while the f2 mutants map within the s2 glycoprotein sequences (fig. 4) . comparisons between the recombination frequencies in the polymerase and s glycoprotein genes using well characterized ts mutants of mhv-a59, we have predicted that the recombination frequency between the group f rna' mutant la7, and the group a rna-mutants la6/la3 to be approximately l%/ 1300-l 400 nucleotides of dsrna ( fig. 5) . to determine if recombination frequencies vary in different portions of the genome, we compared the maximum recombination frequencies between mutants spanning the polymerase genes of mhv (group a (la3/la6) x group e (la1 8)) across gene b encoding the p30 and he genes (group e (la18) x group f (la7/la12)) or through the s glycoprotein gene (group f (la7 x nc16inc14)). all mutants were crossed three to five times and average recombination frequencies calculated as previously described (fig. 5) . interestingly, the maximum recombination frequency predicted between the two most distant ts mutants in complementation groups a (la3, la6) and e (la18) in the 22-kb polymerase region ranged from about 1%/l 800 (la1 8 x la3) to 1 %i2500 (la1 8 x la6) nucleotides of ds rna. the maximum recombination frequency predicted between the group e and f mutants that spanned the p30 and he genes ranged from about 1%/l 100 (la1 2 x la1 8) to 1 %/1400 (la7 x la18) nucleotides of dsrna. since the maximum predicted recombination frequency across the 4.0-kb s glycoprotein gene ranged from about 1 o/0/575 (la7 x nc 14) to 1 o/o/667 (la7 x nc 16) nucleotides of dsrna, these data suggested that variable rates of recombination occurwithin different portions of the mhv genome ( figure 5 ). are deletions contributing to the increased rates of recombination among the group f rna+ mutants? extensive amounts of polymorphism and deletion have been detected in both the he and the s glycoprotein gene sequences of different mhv strains and in the sequence of other coronaviruses (banner et al., 1990; boireau et a/., 1990 parker et al., 1990; rasschaert et a/., 1990) . it is possible that the increased rate of recombination within the s glycoprotein gene may represent the sum of homologous recombination and recombination (homologous or nonhomologous) resulting in deletions. to test this possibility, cultures of cells were infected with various combinations of ts mutants as shown in fig. 4 and rna recombinant viruses isolated 14 hr postinfection. since the ratio of recombinants/revertants ranged from 9 to 3 140 in these crosses (table 2) these data suggested that the majority of ts+ isolates represented true rna recombinants and not revertants. based on the recombination rate in the s glycoprotein gene, the predicted distance between the ts alleles used in these crosses ranged from -100 bp to >l 000 nucleotides (fig. 4) . to determine if deletions were present within the s gene of rna recombinant viruses, a series of overlapping primers were synthesized that span the entire s glycoprotein gene of mhv-a59. the location of these primers as well as the size of predicted pcr products are shown in fig. 4 . cultures of cells were infected with various rna recombinant viruses and intercellular rna was harvested at 8-12 hr postinfection. following cdna synthesis and 30 rounds of amplification with the taq polymerase and specific oligonucleotide primer pairs, the dna products were separated on 1% agarose gels and visualized by uv light or southern blotting techniques (fig. 6) . in fig. 6a , pcr products spanning the hypervariable region of mhv-a59, jhm, and mhv2 clearly demonstrate the 89 amino acid deletion that is present in the jhm s glycoprotein gene (banner eta/., 1990; parker eta/., 1990) . southern blotting techniques using an internal oligomer probe demonstrate that the -1.5-kb fragment is specific for the mhv s glycoprotein sequences spanning nucleotides 642-21 15 and is not present in uninfected cells (fig. 6b) . no obvious deletions were detected through the hypervariable region (fig. 6c) , n-terminus, or c terminus in the s glycoprotein gene of over 20 different rna recombinant viruses tested (fig. 6d) . since the distance between the group fl and f2 mutants would have required sizable deletions (>loo bp) to result in ts+ virus (fig. 4) these data suggested that large deletions were rare events in these crosses, and did not contribute to an increase in the recombination frequency within the s glycoprotein gene. these studies cannot rule out the possibility of small deletions in one ts parent contributing to an increase in the ts+ progeny. however, no obvious deletions were detected in the s glycoprotein gene of 19 different revertant viruses. in fig. 6e , pcr products spanning the hypervariable region of nc17 and its four revertants are presented. while these data cannot conclusively rule out the possibility of small deletions (~50 bp) that would not be detected under these conditions, the data suggested that most revertants probably contain a single nucleotide reversion. although homologous recombination occurs during the replication strategy of several nonsegmented rna viruses (bujarski and kaesberg, 1986; cooper, 1968; hahn et al., 1988; king el al., 1987; weiss and schlesinger, 1991) , high frequency rna recombination is probably a unique phenomenon associated with mhv and perhaps the replication strategies of other coronavirus keck et al., 1987 keck et al., , 1988a makino et a/., 1986b makino et a/., , 1987 lai et a/., 1985) . using highly characterized ts mutants of mhv-a59, we established a genetic recombination map in the polymerpredrcted nucleotide domarns of the group f rna' mutants. assumrng a recombination frequency of lo/01575 bp of dsrna and that la7 maps roughly 0.5 kb from the 5' end of the s glycoprotern gene, the tentatrve nucleotide domains of the mutants mapping in the fl and f2 subgroups were predrcted. the hatched boxes represent the locatron of the region of polymorphism and putative hot spot of recombination in the mhv genome (banner et a/., 1990; parker et al., 1989) . arrows represent the approxrmate locatron of neutralizing epitopes in the s glycoprotein (routledge et al., 1991; weissmiller et a/., 1990) . to determine if recombinatron events resulting in deletions were contributing to the increased recombinatron rate in the s glycoprotern gene, a series of crosses were obtained between different rna+ mutants as shown at the top of the figure. pcr products spannrng different portions of the mhv s glycoprotein gene were obtained using specific oligomer products and the taq polymerase. the locatron of these primer pairs and their predicted products are shown in the bottom of the figure. to predict the maximum recombination frequency at the 5'end of the genome, we calculated the recombination frequency between the three most distant rna-mutants (group a rna-mutants: la3. la6; group f rna-mutant: la1 8) assuming that these mutants map at the most distant ends of the 22.kb polymerase region (-21 kb apart). to map the maximum recombination rate across the he and p30 genes, recombination rates were determined between mutants mapping approximately 3.5 kb apart at the 3' end of the polymerase gene (la1 8) and the 5' end of the s glycoprotein gene (group f rna+ mutants: la7, la1 2). recombination rates were calculated through the s glycoprotein gene, assuming the group f rna+ mutants la7 and nc1 4/nc16 map roughly 4.0 kb apart. ase genes of mhv. assuming that the recombination frequency was equivalent throughout the entire genome, these data suggested that the recombination frequency approached 25% or more . in this study, we have demonstrated that the ts mutants from a rna+ complementation group of mhv-a59 can also be arranged into an additive, linear, genetic map. several lines of evidence strongly suggest that the group f rna+ mutants map in the s glycoprotein gene of mhv-a59. first, the group f mutants do not produce syncythium when maintained at the restrictive temperature. induction of cell fusion has been mapped to either the s2 domain of the bovine coronavirus s glycoprotein gene (yoo eta/., 199 1) or the mhv sl and s2 glycoprotein domains (gallagher et al., 199 1; routledge et al., 1991; weismiller eta/., 1990) . second, tl fingerprint analysis of rna recombinant viruses derived from crosses between ts la7 or la1 2 and mhv-jhm demonstrated that the mhv-a59 sl domain was always replaced by heterologous mhv-jhm sequences. since no other region in the mhv-a59 genome was uniformly replaced in recombinant viruses, these data strongly suggested that the mutations in these ts viruses reside within the sl coding sequences (keck et al., 1987 (keck et al., , 1988a makino et al., 1986b makino et al., , 1987 . finally, sequence analysis of rna recombinant viruses derived from these mutants place the ts allele in la7 within the 5' most 1.1 kb of the sl glycoprotein gene (banner et a/., 1990) . while these data do not definitively prove that the group f mutants map in the s glycoprotein gene sequence, these data have strongly supported the localization of these mutations within this coding region. on the basis of recombination mapping data, the group f rna+ mutants appear to map into two discrete domains in the s glycoprotein gene sequence (fig. 4) . data from our laboratory and others suggest that the fl mutants probably map within the sl glycoprotein while the f2 mutants map within the 52 glycoprotein sequences (banner et a/., 1990; keck et al., 1987 keck et al., , 1988a makino et al,, 1987 makino et al,, , 1988 . sequence analysis of the group f rna+ mutants and revertant viruses will be required to definitively map the location of these alleles. however, since mutants from both subgroups are incapable of producing syncytium at the restrictive temperature, these data suggest that both domains are important in eliciting cell fusion. currently, it is unclear whether the alterations in the fl and f2 conditional lethal mutants result in a temperature-sensitive fusogenic domain or alter the synthesis, transport, and surface expression of the s glycoprotein gene. the recombination frequency for the entire mhv genome has been estimated to approach 25% or more . these estimates were based on the assumption that the recombination frequency was uniform and approached 10/0/l 300-1400 nucleotides of dsrna throughout the entire 32-kb mhv genome. however, recombination rates measured within the polymerase region and s glycoprotein gene were estimated to occur at frequencies of about 1%/l 800-2500 and 1%/575-667 bp of dsrna, respectively (fig. 5) . recombination rates across the p30/he genes were estimated to occur at about 19/o/l 100-l 400 bp of dsrna. currently, our best estimate for an mhv recombination frequency was measured within the s glycoprotein gene because these mutations must have resided within a 4.0-kb stretch of rna. moreover, the frequency of recombination was internally consistent between several independent crosses within a single complementation group (table 3) . while definitive proof will require the identification of the exact location of the ts alleles used in these studies, these data suggest that the recombination frequency varies in different portions of the genome and is roughly threefold higher in the s glycoprotein gene. several explanations could account for an increase in the recombination frequency within the s glycoprotein gene. first, the recombination rate through the fig. 6. pcr ampliflcatron and size analysrs of the s glycoprotein gene of wildtype, ts, recombmant, and revertant viruses. cultures of cells were infected with different strains of mhv ts mutants, recombinant viruses, or revertant viruses. the intercellular rna was extracted and used as template for reverse transcription and 30 rounds of pcr amplification usrng the taq polymerase and different primer parrs. (a) pcr products spanning the hypervariable region of mhva59, jhm, and mhv-2 (642-2155) (lanes 2-4, respectively; lane 5, uninfected control); (b) southern blot analysis of the pcr products shown in (a) using an internal ollgomer probe; (c) pcr products spanning the hypervariable region in the s glycoprotein gene of mhv-a59 (lane 2) and rna recombinant viruses derived from nc1 7 x nc1 6 [lanes 3-5) nc6 x la7 (lanes 6-8) and nc1 6 x la7 (lanes 1 o-l 2); (d) pcr products spanning the c (lanes 2-1 1) and n (lanes 12-21) terminl of mhv-a59 (lanes 2, 12) and rna recombinant viruses derived from nc1 7 x nc1 6 (lanes 3-5 and 13-l 5). nc6 x la7 (lanes 6-8 and 16-l 8) and nc1 6 x la7 (lanes 9-l 1 and 19921); (e) pcr products spanning the hypervariable region of nc 17 (lane 2) and four different revertant viruses (lanes 3-6). lane 1 contains dna markers in all panels. polymerase region of mhv-a59 may be grossly under-la6) was unclear. this seems unlikely because a uniestimated because the exact location of the ts allele in form recombination frequency of lo/o/575 nucleotides the group e mutant (la1 8) and group a mutants (la3/ of dsrna throughout the genome would: (1) predict the recombination frequency for the mi-iv genome to approach -56% or more; (2) place rna-complementation groups a, b, and c in orf 1 b and rna-complementation groups d and e in the p30 /he nonstructural proteins, and (3) suggest that conditional lethal mutants were not isolated within orfla at the 5'most three-fifths (-14 kb) of the genome. this is highly unlikely since several proteolytic, hydrophobic membrane-anchoring and cysteine-rich domains have been identified in orf 1 a, which should be amenable to mutagenesis (baker et a/., 1989; lee et a/., 1991) . more importantly, tl fingerprint analysis of rna recombinant viruses derived from ts mutants in complementation groups c, d, and e clearly place each genetic function in the polymerase gene (keck et a/., 1987; lai et al., 1985) . since rna-complementation groups c, d, and e have also been demonstrated to function in mrna synthesis and map within orf 1 b, which contains polymerase, helicase, and metal-binding sequence motifs, it is extremely unlikely that these mutants map in the he or p30 subgenomic orfs bredenbeck et a/., 1990; lee et al., 1991; schaad et al., 1990) . in contrast, the recombination rate predicted through the polymerase region was not inordinately high (1%/1800-2500 bp of dsrna), but rather closely approximated the recombination frequencies estimated to occur in apthovirus and poliovirus infections (cooper, 1977; king, 1987) . thus, it seems likely that the frequencies measured between the polymerase and s glycoprotein genes actually represent true differences in the recombination rate. since the enhanced recombination frequencies in mhv were observed within a physically smaller (4.0-kb) region as compared to the 22-kb polymerase region, it is possible that a mechanism similar to high negative interference in dna viruses may account for the increased rates of recombination (chase and doermann, 1958) . this seems unlikely since high negative interference is probably mediated by dna polymerase repair mechanisms, which have not been demonstrated in the mhv rna polymerase (glickman and radman, 1980) . the increase in the recombination rate in the s glycoprotein gene could also not be attributed to a combination of true homologous recombination and recombination resulting in large deletions. nonhomologous recombination probably explains the appearance of defective interfering rnas of sindbis virus, vesicular stomatitis virus, and mhv (holland, 1987; makino et a/., 1988 monroe and schlesinger, 1983) . since deletions have been demonstrated in the mhv s and he glycoprotein genes as well as in the nonstructural genes encoded in mrna 4 and 5 and in the tgev s glycoprotein gene and other subgenomic orfs (ban-ner el al., 1990; la monica et al., 1991; luytjes et al., 1987; parker et al., 1990; rasschaert et al., i 990; schwartz et al., 1990; yokomori and lai, 1991) , these data have suggested that the subgenomic orfs may be very amenable to frequent deletions and insertions. insertions in the mhv s glycoprotein gene have also been reported previously (taguchi et al., 1987) . analysis of 20 rna recombinant viruses derived from different crosses between the group f mutants suggested that large deletions or insertions were rare and occurred at <&the rate of true homologous recombination. while these data could not conclusively rule out the presence of very small deletions, this seems unlikely since the predicted distances between the ts alleles used in these crosses should have resulted in deletions ranging from -100 bp to > 1000 bp in length. it is also unlikely that the ts+ virus isolated from these crosses were revertants containing small deletions (~50 bp) in a singlets parent, since the ratio of recombinants: revertants was at least 9: 1 or greater in each of the crosses and none of the revertants analyzed from each parental ts mutant had evidence of deletions. since revertants of sb ts mutants usually contain single nucleotide reversions at the site of mutation (hahn et al., 1989a,b) , the most likely interpretation from these data is that the increase in the rate of recombination in the s glycoprotein gene probably reflects an increase in the rate of true homologous rna recombination. if the mechanism for homologous recombination and recombinations resulting in deletions are similar as suggested by banner et al., 1990 , these data suggest that the frequency of these two recombination events is very different. alternatively, the regions flanked by the ts mutations used in these crosses may be critical for s glycoprotein gene function or plaque formation and could not be deleted. two possible mechanisms could explain variable rates of recombination in the mhv genome. the first possibility is a preferred recombination site in the mhv s glycoprotein gene. a clustering of rna recombination sites adjacent to the hypervariable region in the s glycoprotein region suggest the presence of a preferred site of recombination (banner eta/., 1990) . however, in the absence of selection, crossover sites spanning the hypervariable region of the mhv s glycoprotein gene were random, suggesting that the preferred site of recombination reflects in vitro selection for certain types of recombinants . although the majority of data support non-site-specific homologous recombination throughout the entire poliovirus genome (kirkegaard and baltimore, 1986; sarnow et al,, 1990) brian, 1989, and sawickr and sawicki, 1990 , have clearly demonstrated the presence of subgenomic minus strands in mhv and tgev infected cells. if the subgenomic minus strands partrcipate in template switching with full-length genomrc rnas, recombrnation rates should increase from the 5' to the 3' end of the genome. panel a demonstrates how template switching could occur during negative-strand synthesis between a full-length and subgenomic-length plus strand rna and result in a full-length negative-stranded rna recombinant molecule. panel b demonstrates how template switching between full-length and subgenomic negative strands during positrve-strand synthesis could result in recombinant genome-length molecules, switching occur after the synthesis of uu in sites chosen to minimize the adverse free energy change involved in switching to a heterotypic template (king, 1988) . in addition, a nonrandom distribution of recombination sites has also been reported among intertypic poliovirus recombinants (tolskaya et al., 1987) . if mhv rna recombination is mediated by freely segregating rna segments that are generated by transcriptional pausing during rna synthesis, preferred sites of recombination may also exist in au-rich regions and/or in regions of secondary structure in the mhv genome (baric et al., 1987) . however, it is difficult to envision how these types of preferred sites of recombination would result in higher intratypic recombination frequencies between mhv-a59 ts mutants since extensive secondary structure is found throughout the mhv genome and the g:c:a:u and aa/uu dimer ratios are roughly equivalent in the polymerase and s glycoprotein regions (banner et al., 199 1; baric et al., 1987; soe et al., 1988; fu et al., unpublished) . a more likely mechanism to explain the increase in the recombination rate in the s glycoprotein gene is based on the basic replication strategy of coronaviruses (fig. 7) . recently, subgenomic minus-strand and replicative intermediate rnas have been demonstrated during tgev and mhv infection (sethna et a/., 1989 (sethna et a/., , 1991 sawicki and sawicki, 1990) . these data indicate that the amount of negative-strand template rna is unequal and increases from the 5' to 3' end of the genome. because of the availability of more negative strand template, recombination rates should increase proportionately from the 5' to 3' end of the genome and be highest in the n gene coding region contained within mrna 7. for example, recombination events in the polymerase region can only involve template switching between full-length negative-or positive-strand rna templates. however, recombination events in the s glycoprotein gene or other subgenomic orfs could not only occur between full-length rna templates but also involve subgenomic mrna and subgenomic negative-strand rna templates as well. several findings support this hypothesis. first, recombination frequencies between the polymerase gene and s glycoprotein genes of mhv may vary threefold. second, in vitro transcribed subgenomic-length di rnas rapidly undergo rna recombination with fulllength (or subgenomic) rnas supporting the notion that recombination events can occur between different-sized template rnas . third, analysis of sepharose 2b-cl column purified full-length ri rna has demonstrated the presence of mrna 7 subgenomic nascent-plus strands bound to the fulllength negative-strand rna (baric et al., 1983) . while the original interpretation of these data supported "leader-primed" transcription, an alternative explanation for these findings is that these subgenomic nascent-plus strands have disassociated from subgenomic ri rna template and recombined with the fulllength negative-strand rna. finally, a large number of rna recombinant viruses selected with markers in the s glycoprotein gene of mhv contain additional crossover sites in the m and n structural genes encoded at the 3' end of the genome (keck et al., 1988b) . these findings are consistent with the idea that subgenomic minus strands function in rna recombination and suggest that recombination events may increase in frequency toward the 3' end of the genome. high-frequency rna recombination is a unique property associated with mhv replication. our data suggest that the frequency of rna recombination is mediated in part by the large size of the mhv genome (32 kb) and its novel mechanism for rna synthesis involving "leader primed" discontinuous transcription and mrna replication via subgenomic minus strands (baric et al., 1983 (baric et al., , 1987 sethna et a/,, 1989 sethna et a/,, , 1991 sawicki and sawicki, 1990) . the possibility that the mhv recombination rate increases proportionately from the 5' to 3' ends of the genome may also contribute to the evolution, genetic diversity, and organization of coronavirus genomes. for instance, the gene order of ibv is different from other coronaviruses and it contains an additional gene between the m and n genes at the 3'end of the genome (cavanagh eta/., 1991) . tgev also contains an additional gene, designated x, which is located at the 3'end of the genome (kapke and brian, 1986) . more remarkably, so,me coronaviruses, but not all, contain a p30 nonstructural protein and an he glycoprotein that is related to the influenza c virus he (luytjes et al., 1988) . coronaviruses and toroviruses may also have diverged by nonhomologous recombination events in their polymerase and envelope proteins (snijder et al., 1991) . these findings are consistent with the fact that the coronavirus genome has a remarkable ability to evolve by homologous and nonhomologous recombination, especially in the genes encoded by subgenomic mrna. regeneration of a functional rna virus genome by recombination between deletion mutants and requirement for cowpea chlorotic mottle virus 3a and coat genes for systemic infection identification of a domain required for autoproteolytic cleavage of murine coronavirus gene a polyprotein an in vitro system for the leader-primed transcription of coronavirus mrnas. f/&y0 a clustering of rna recombination sites adjacent to a hypervariable region of the peplomer gene of murine coronavirus random nature of coronavirus rna recombination the absence of selection pressure characterization of replicative intermediate rna of mouse hepatitis 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coronavirus infected cells leader sequences of murine coronavlrus mrnas can be freely reassorted. evidence for the role of free leader rna in transcription high frequency rna recombination of munne coronaviruses rna recombination of coronaviruses; localization of neutralizing epitopes and neuropathogenlc determinants on the carboxy terminus of peplomers prlmary structure and translation of a defective interfering rna of murlne coronavtrus high frequency leader sequence switching during coronavirus defective rna replication. i viroi a system for study of coronavlrus mrna synthesis: a regulated, expressed subgenomlc defective interfering rna results from lntergenlc insertion temperature sensitive mutants of mouse hepatitis virus type 3(mhv-3): isolation, biochemical and genetic characterization rnas from two independently isolated defective interfering parttcles of sinbdis virus contain a cellular rna sequence at their 5'ends molecular cloning of the gene encodlng the putative polymerase of mouse hepatltls coronavirus, strain a59 sequence analysis reveals extensive polymorphism and evidence of deletions wlthin the e2 glycoprotetn gene of several strains of munne hepatitis virus mroiogy porcine respiratory coronavirus differs from transmissible gastroenteritis virus by a few genomic deletions analysis of murine coronavirus surface glycoproteins functions by using monoclonal antibodies. 1. viral poliovirus genetics coronavirus transcription: subgenomic mouse hepatitis virus replicative intermediates function in rna synthesis genetics of murine coronavirus transcription: identification of a cistron required for mhv negative strand synthesis nucleotide sequence of the gene encoding the surface projection glycoprotein of coronavirus mhv-jhm murine coronavirus nonstructural protein ns2 is not essential forvirus replication in transformed cells coronavirus subgenomic minus-strand rnas and the potential for mrna replicons minus-strand copies of replicating coronavirus mrnas contain antileaders identification of a new transcriptional initiation site and the corresponding functional gene 2b in the murine coronavirus rna genome coronavirus jhm: coding assignments of subgenomic mrnas comparison of the genome organization of toro-and coronaviruses: evidence for two nonhomologous rna recombination events during berne virus evolution sequence and translation of the murine coronavirus 5'.end genomic rna reveals the n-terminal structure of the putative polymerase specific interaction between the coronavirus leader rna and nucleocapsid protein phosphoproteins of murine hepatitis virus isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid. 1. viral the novel glycoproteins of coronaviruses proteolytic cleavage of the e2 glycoprotein of murine coronaviruses: activation of cell fusing activity of virions by trypsin and separation of two different 90 k cleavage fragments characterization of jhmv variants isolated from rat brain and cultured neural cells after wild type jhmv infection studies on the recombination between rna genomes of poliovirus: the primary structure and nonrandom distribution of crossover regions in the genomes of intertypic poliovirus recombinants monoclonal antibodies to the peplomer glycoprotein of coronavirus mouse hepatitis virus identify two subunits and detect a conformational change in the subunit released under mild alkaline conditions recombination between sindbis virus rnas biosynthesis, structure, and biological activities of envelope protein gp65 of murine coronavirus mouse hepatitis virus s rna sequence revealed that nonstructural proteins ns4 and ns5a are not essential for murine coronavirus replication. 1. viral the s2 subunit of the spike glycoprotein of bovine coronavirus mediates membrane fusion in insect cells we thank sheila peel, lorraine alexander and mary schaad for helpful comments and criticisms. this work was supported by american heart association grant (aha 87-l 135, aha 90-4635) and a grant from the national institutes of health (a123946). this work was done during the tenure of an established investigator of the american heart association (aha 89-0913) (r.s.b.). key: cord-270487-m770a1rl authors: wurch, t.; guidasci, t.; geldreich, a.; lebeurier, g.; mesnard, j.-m. title: the cauliflower mosaic virus reverse transcriptase is not produced by the mechanism of ribosomal frameshifting in saccharomyces cerevisiae date: 1991-02-28 journal: virology doi: 10.1016/0042-6822(91)90103-i sha: doc_id: 270487 cord_uid: m770a1rl abstract the capsid protein and the reverse transcriptase of cauliflower mosaic virus (camv) are encoded by two genes (orf iv and orf v) that lie in different translation reading frames. a comparison can be drawn between the synthesis of both camv proteins and the fusion protein in a yeast retrotransposon, ty, resulting from a +1 frameshifting event which fuses two out-of-phase orfs encoding the structural protein and the reverse transcriptase of ty. for this reason, we constructed a yeast expression vector containing camv orf vii fused to camv orf iii by a fragment of 452 bp including the overlapping region of orf iv and orf v, orf vii and orf iii being used as reporter genes. we characterized two proteins (22 and 50 kda) synthesized from this plasmid in the yeast expression system. we demonstrated that the 50-kda polypeptide is not synthesized from a +1 frameshifting event but is probably a dimeric form of the 22-kda protein. from this result we conclude that the camv reverse transcriptase is not produced by a mechanism of ribosomal frameshifting. cauliflower mosaic virus (camv) has a 8000 bp long crrcular double-stranded dna as genetrc material (for recent reviews, see (i, 2)). the genome contains seven open reading frames (orfs). they are closely spaced (orfs i, ii, and iii) or they overlap (orfs iii, iv, and v). small intergenomic regions separate orfs vii and i (60-odd bp) and orfs v and vi (loo-odd bp) while a larger one (about 600 bp) exists between orfs vi and vii. the translation strategy of these orfs has not yet been fully elucidated except for orf vi which is translated from a 19 s mrna (3) . for orfs vii, i, ii, and iii it has been suggested that the proteins are synthesized from a 35 s polycistronic rna, the translation being initiated at the atg of orf vii (4). translation of downstream cistrons (orfs i, ii, and ill) would be the result of a termination-reinitiation mechanism (4, 5) . little is known, however, about the expression of orfs iv and v which code for the capsid protein (6, 7) and for the reverse transcriptase, respectively (8, 9) . these orfs overlap (by 79 bp for strain cabb-s (10)) and orf v is in the +l reading frame with respect to orf iv. such an organization has been described for the yeast retrotransposon ty. this transposon possesses two genes tya and tyb which code for a structural protein and for an enzymatic polypeptide with reverse transcriptase activity, respectively (17, 12) . tya and tyb overlap by 38 bp and tyb is in the +l reading frame with respect to tya. tyb is expressed as a tya' to whom requests for reprints should be addressed. tyb fusion protein (13) . the production of this fusion polypeptide is achreved via a frameshifting event that leads to the translational avoidance of the tya termination codon and a shift into the tyb reading phase. this expression strategy is analogous to the production of the gag-pol fusion protein in the retroviruses, gag coding for structural proteins and pol coding for the reverse transcriptase. however, in the case of the retroviruses the gaggpol protein is synthesized via -1 frameshift (74, 75) or readthrough (16) mechanisms. a camv orf iv-orf v fusion protein has not been detected either in infected plants (7, 17) or in in vitro translation systems (18) . the absence of a fusion polypeptide could be due to the immediate processing of the protein by the proteolytic activity located at the nterminal part of the orf v product (79) . likewise some factors necessary for a +l frameshifting event could be absent in the in vitro translation systems. for this reason, we decided to study the expression strategy of camv orf iv-orf v in the yeast system. this system is a powerful tool since it was used with success to demonstrate a +l frameshift mechanism for the yeast retrotransposon ty (73). to exclude the possibility that the fusion protein was processed by the camv protease we removed a portion of orf v indispensable for the proteolytic activity (19). for this we selected a fragment of 452 bp (from nucleotide 3461 to nucleotide 3913 of strain cabb-s of camv (70)). this fragment contains the region necessary for a frameshifting event: orf iv ends at nucleotide 3670 and orf v begins at nucleotide 3591. positive controls for our assay system were provided by 837 0042 fusing (i) camv orfvii in frame with the end of orf iv, and (ii) the beginning of orf v in frame with the camv orf iii (fig. 1) . we used this construction because we had antisera raised against the n-terminal extremities of orf iii product (anti-p3 (20) ) and orf vii product (anti-p7 (21)) and against the c-terminal part of orf iv product (anti-p4 (7)). in the case of a +l frameshifting event between orf iv and orf v two proteins should appear in a western blot: one protein of 18.5 kda corresponding to the orf vii-iv product (p7-p4) and another one of 41 kda corresponding to the orfs vii-iv-v-iii product-containing fusion protein (see fig. 1 ). the 18.5kda polypeptide would be detected by anti-p7 and anti-p4 sera whereas the 41 -kda protein would be detected by anti-p7, antilp4, and anti-p3 sera. a plasmid construction useful for this approach is pfs450 which was constructed from pmw701 (21) . pmw701 is a yeast expression vector containing orf vii (see fig. 1 nase). we inserted into the bsu361 site of orf vii located at nucleotide 277 (orf vii starts at nucleotide 13 and ends at nucleotide 303) the camv frameshift sequence fused to orf iii (see fig. 1 ) and then introduced pfs450 into saccharomyces cerevisiae strain cl 3-abys86 (pra l-1, prb l-1 ) prc l-1, cps l-3, ura a3, leu 2-3, 112 his). this strain is particularly suitable because it is deficient in several vacuolar proteinases (23) . yeast cultures containing either pfs450 or pmw701 were grown to an a,,, of 1.80 and the proteins were extracted as already described (24) . the polypeptides were separated on a 0.1% sds, 15% polyacrylamide gel, electroblotted onto a nitroceilulose sheet, and incubated with either anti-p7 or anti-p4 sera. yeast cells containing pmw701 synthesized a protein of 14 kda immunodetected by anti-p7 serum (fig. 2 , slot 1) but not by anti-p4 serum (fig. 2, slot 3) . this protein corresponds to the native p7 and has already been described (21) . cells containing pfs450 synthesized a protein of 22 kda immunodetected by anti-p7 and anti-p4 sera (fig. 2, slots 2 and 4) . this is slightly greater than the theoretical molecular weight of p7-p4 (18.5 kda). it is not surprising since p7 and p4 are known to have apparent molecular weights on sds-page greater than their theoretical molecular weights (18, 21) . as our hybrid protein was detected by sera raised against the n-terminal extremity of p7 and the c-ter-minal part of p4 we can conclude that orf vii is fused in frame with the end of orf iv. to verify this, the integrity of the orf v-orf iii junction was confirmed by dna sequencing (data not shown). to look for a +l frameshifting event we used the anti-p7 serum which detected the protein p7-p4 more efficiently than did the anti-p4 serum (see fig. 2 ). total proteins of yeast containing pfs450 were extracted, separated on a 0.1% sds, 8-l 5% gradient polyacrylamide gel, electroblotted onto nitrocellulose and incubated with anti-p7 serum. two products were immunodetected (fig. 3, slot 1) . one of these is 22 kda and is the simple translation product of orf vii-orf iv (p7-p4; see above). the other, less abundant product is about 50 kda. this larger species could correspond to either a dimeric form of p7-p4 or a fusion product which can only be produced by a +1 frameshift event. for this reason we constructed a new plasmid pfs451 which corresponds to pfs450 deleted in camv orf iii. the construction of pfs451 is shown in fig. 4 . in the case of a +l frameshifting event, yeasts containing pfs451 should still be able to synthesize the 22-kda protein (p7-p4) but not the 50-kda protein (see fig. 4 ). in these new conditions, a 40-kda protein should be synthesized. when we compared yeast extracts containing either pfs450 or pfs451, we were unable to detect any differences with anti-p7 serum (fig. 5) product of 50 kda corresponds probably to aggregates of p7-p4 and is not synthesized by a +l frameshift event. yeast proteins were also tested with the anti-p3 serum and no protein was immunodetected (data not shown). the background of the aspecific immunoreactions was reduced by immunoabsorbing all the tested antisera against a mixture of bacteria and yeast cells. except for the 22-and 50-kda proteins no other proteins were detected (result not shown). from these results, we conclude that the 50-kda polypeptide is probably a dimeric form of p7-p4. during the course of these experiments we observed that yeast containing pfs451 expressed more p7-p4 than yeast containing pfs450 (compare slots 1 and 2 of fig. 5) . in order to address the question of whether the overlapping sequence between camv orf iv and orf v was able to direct a +l frameshifting event, we introduced this sequence between camv orf vii and camv orf iii. this construct was then expressed in a yeast system known to be able to carry out the frameshifting mechanism. however, no fusion protein synthesized from orf vii and orf iii could be detected. three reasons could explain this result. (i) the cloned fragment contained only 452 bp (see results), possibly lacking a potential stem-loop structure positioned downstream of this fragment which might be important for expression. however, in coronaviruses a stretch of 86 nucleotides (25) and for rous sarcoma virus one of 147 nucleotides (14) are sufficient for efficient -1 frameshifting. moreover, for hiv -1 frameshifting (15) and ty +l frameshifting (73) no stem-loop structures downstream from the frameshifting site are necessary. (ii) the absence of a fusion protein could be due to the presence of a transcriptional stop signal located just after the stop codon of orf iv. however, by northern blotting we detected full-length rnas capable of expressing all the proteins from the orf vii to the orf iii (result not shown). (iii) the camv frameshifting event may not be possible in yeast because some necessary plant or viral factors are lacking. while this work was in progress, schultze et al. (26) demonstrated that a camv mutant in which orf iv and orf v are separated by stop codons in all three reading frames is viable and stable. they proposed that the orf v is translated separately from the orf iv. our results confirm this hypothesis. such a mechanism is not unusual and has already been proposed. it has been demonstrated that synthesis of hepadnavirus reverse transcriptase does not require formation of a capsid-polymerase fusion protein (27, 28) . how the camv reverse transcriptase is synthesized is still unknown. it will be of interest to introduce ourconstruction into plant protoplasts to understand how the reverse transcriptase is synthesized. we thank mr. l. kremer for excellent technical assistance. the collaboration of dr. d. h. wolf in providing us with s. cerevisiae strain cl 3-abys86 was much appreciated. plant dna lnfectlous agents chromosomes: eukaryotic, prokaryotic, and viral proc. nat/. acad. sci. usa proc. nat/. acad. sc;. usa 81 proc. nat/. acad. sci. usa key: cord-262574-gu0930s3 authors: slagle, betty l.; butel, janet s. title: identification and characterization of a mouse mammary tumor virus protein uniquely expressed on the surface of balb/cv mammary tumor cells date: 1985-05-31 journal: virology doi: 10.1016/0042-6822(85)90102-3 sha: doc_id: 262574 cord_uid: gu0930s3 abstract a unique subline of balb/c mice, designated balb/cv, exhibits an intermediate mammary tumor incidence (47%) and harbors a distinct milk-transmitted mouse mammary tumor virus (mmtv). the balb/cv subline was used to study the molecular basis of potential virus-host interactions involving cell surface-expressed mmtv proteins. cell surface iodination identified virus-specific proteins expressed on balb/cv primary mammary tumor cells grown in culture. in contrast to (c3h)mmtv-producing cell lines which expressed mmtv gp52, balb/cv tumor cells lacked gp52 and expressed instead a 68k, env-related protein. the 68k env protein was also detected on the surface of metabolically labeled balb/cv tumor cells by an external immunoprecipitation technique. the expression of 68k env was restricted to mammary tissues of balb/cv mice that also expressed other mmtv proteins. biochemical analysis established that 68k env was not modified by n-linked glycosylation. 125i-labeled 68k env was rapidly released into the media of tumor cell cultures and was recovered both in the form of a soluble protein and in a 100,000 g pellet. the biologic function of this cell surface-expressed viral protein remains unknown. balb/c mice are commonly used as a model system for the study of mammary tumorigenesis because they exhibit a low incidence of spontaneous mammary tumors, they lack the exogenous milk-transmitted mouse mammary tumor virus (mmtv), and they are susceptible to tumor induction by a variety of exogenous factors (michalides et az., 1979; pauley et az., 1979; butel et az., 1981; bentvelzen, 1982) . the endogenously transmitted mmtv sequences of balb/c mice are organized into three proviruses, designated units i, ii, and iii (cohen et ah, 1979; cohen and varmus, 1980) , or mtv-6, -8, and -9, respectively (traina et ak, 1981) . the expression of the balb/c endogenous ' author to whom reprint requests should be addressed. proviruses is generally limited to 3' long terminal repeat (ltr) sequences (dudley et al, 1978; wheeler et al, 1983; van ooyen et al, 1983; breznik et al, 1984) . a unique subline of balb/c mice, designated balb/cv, has recently been described (drohan et al, 1981; slagle et cd., 1984) . whereas balb/cv mice have a spontaneous mammary tumor incidence of 47%, the parental balb/ccrlmed mice from which the balb/cv subline was derived maintain a tumor incidence of ~1%. the milk-transmitted (balb/cv)-mmtv shares group-specific antigenic determinants with (c3h)mmtv on each of the virus structural proteins (slagle et cd, 1984) , but it reportedly can be distinguished from all known strains of mmtv by both immunological and molecular criteria (drohan et al, 1981) . the origin of the balb/cv isolate remains unknown. although it could have originated by infection of a balb/c mouse with a unique exogenous variant, the possibility also exists that it may represent an activation of one of the balb/c endogenous proviruses. expression of endogenous mmtv has been documented in c3h mice (deome et al, 1959; van nie and verstraeten, 1975; vacquier et al, 1981; puma et al, 1982) . mmtv-related antigens have been detected at the surface of mammary tumor cells in several mouse strains (for a review, see bentvelzen and hilgers, 1980) . the expression of viral-specific antigens at the surface of virus-infected or -transformed cells may be important for several reasons. surface-associated structural proteins are frequently involved in the maturation pathways of viruses which bud from the cell. additionally, surface-expressed viral antigens are more likely to be detected by host immune surveillance systems than are viral proteins localized inside the cell. thus, immunization strategies would be most logically directed against those exposed antigens. finally, the possibility exists that virus-specific cell surface antigens might be shed from the cell and serve as tumor-blocking factors, with subsequent effects on the host immune regulation of growing tumor cells. we have used the balb/cv subline of mice to investigate the molecular basis of potential virus-host interactions involving surface-associated viral proteins in the mammary system. we first used cell surface iodination to identify balb/cv proteins expressed at the surface of tumor cells in primary cultures. in contrast to c3h-producing cell lines, balb/cv tumor cells lacked detectable levels of cell surface gp52 and expressed instead a 68k, envrelated protein. we then examined the basis for the aberrant cell surface localization of this protein. 68k"" does not appear to be modified by glycosylation and was highly unstable at the cell surface. labeled 68ken" shed into the media was present both as a soluble protein and in a form that could be pelleted by highspeed centrifugation. although the biologic role of balb/cv surface 68k"" remains obscure, several intriguing possibilities are discussed. materials and methods viruses and ceus. concentrated (c3h)-mmtv (lot no. p-1033) was obtained from the biological carcinogenesis branch, division of cancer cause and prevention, national cancer institute. mm5mt/cl cells (owens and hackett, 1972; fine et al, 1974) and h-l cells (scolnick et al, 1976 ) produce (c3h)mmtv, while mtv-l cells from a balb/cv animal (butel et az., 1977) are virus free. the cells were cultivated in dulbecco's minimum essential medium (d-mem) containing 10% heat-inactivated fetal bovine serum (fbs; grand island biological co., grand island, n. y.), 0.1 pg/ml gentamicin sulfate, 10 pg/ml insulin (sigma chemical co., st. louis, mo.), 2 pg/ml dexamethasone (sigma), and 0.3% sodium bicarbonate in a humidified atmosphere of 10% co2 at 37'. antisera. antisera against detergentdisrupted (c3h)mmtv [anti(c3h)mm-tvd], affinity-purified (c3h)mmtv gp52/ gp36 (anti-gp52/gp36), and gel-purified (c3h)mmtv p28 (anti-p28) were prepared in rabbits. the specificities of these antisera have been detailed previously (slagle et ad, 1984) . adsorption experiments have demonstrated that the anti-gp52/gp36 serum reacts specifically with mmtv glycoproteins and envelope-related precursors and does not react with normal cell proteins of balb/c mammary tissue (slagle et aq, 1985) . mice. all mice were from a conventional closed mouse colony housed in the department of cell biology, baylor college of medicine. the balb/cv substrain was derived from a balb/ccrlmed mouse, as described (drohan et al, 1981; slagle et al, 1984) . balb/ccrlmed mice were used for the transplantation of cv-2 han outgrowths as previously described (slagle et al., 1984) . establishment of primary tumor cell cultures. primary cell cultures of balb/cv tumor cells were established as reported (slagle et ul, 1984) and grown in the media described above. only primary tumors arising spontaneously from transplants of the cv-2 han outgrowth line (slagle et al., 1984) were analyzed in these experiments, with the exception of a serially transplanted balb/cv tumor included as a control in fig. 5 . lactoperoxidase-catalyzed cell surface iodination. intact cell monolayers were iodinated according to the procedure of soule et az. (1982) . previous studies from our laboratory have established the surface specificity of this iodination procedure (soule et al, 1982; santos and butel, 1982; lanford and butel, 1982) . cells grown in loo-mm plates were rinsed three times with tris-buffered saline (tbs, 2 mmtris, ph 7.4, 140 mlm nacl, 5 mlm kcl, 0.4 mm nazhp04, 6 mm dextrose, 0.5 mm mgcl,, and 0.7 mm caclz), and a fourth time with dulbecco's phosphate-buffered saline (d-pbs; dulbecco and vogt, 1954) . one milliliter of d-pbs containing 1 mci lz51-na (>350 mci/ml; amersham, arlington heights, ill.) and 28 ~1 of a 1 mg/ml solution of freshly prepared lactoperoxidase (calbiochem-behring corp., la jolla, calif.) were added per plate. each plate then received 28 ~1 of a 10e4 dilution of 30% hzoz (fisher, dallas, tex.) at 0, 2, 4, and 6 min, with gentle rotation of plates during the 2-min intervals. at the end of the 8-min labeling period, the d-pbs/l? was removed and the cell monolayers either were rinsed in cold tbs and extracted or were rinsed with warm tbs, serumfree media added, and the cells incubated at 37" for a chase period before extraction. analysis of iodinated protein(s) shed into culture jluid. media collected from iodinated cell monolayers after a 15-min chase period were clarified by centrifugation at 15,000 rpm for 30 min. the supernatant was recovered and subjected to a second centrifugation for 1 hr at 100,ooog through a 30% sucrose cushion. the supernatant of the high-speed centrifugation was immunoprecipitated using rabbit antisera. the pellet was dissolved in extraction buffer (eb) and then immunoprecipitated. eb consisted of 50 mm tris-hcl, ph 8.0, 100 mm naci, 1% np-40, and 1% trasylol (mobay chemical co., new york, n. y.). labeled cells were extracted in eb and immunoprecipitated as previously de-scribed (lanford and butel, 1979; slagle et ac, 1984) . immune complexes were dissociated using gel disruption buffer (0.5 m tris-hci, ph 6.8, 2% sds, 2% 2-mercaptoethanol, 10% glycerol, and 0.002% bromophenol blue) and then analyzed by sds-polyacrylamide gel electrophoresis (sds-page). sds-page. discontinuous sds-page was performed as described by lanford and butel (1979) . the stacking gel was 5% acrylamide using a 30:0.8 acrylamide-tobisacrylamide ratio. the separating gel was lo%, using a 1oo:l acrylamide-tobisacrylamide ratio. electrophoretic transfer of proteins from gels to nitrocellulose. proteins were electrophoretically transferred from sds gels to nitrocellulose filters and detected by antibody and 1251-protein a as previously described (slagle et al., 1984) . iodination of (c3h)mmtv by enxymobead method detergent-solubilized (c3h)-mmtv was iodinated using enzymobeads (bio-rad laboratories, richmond, calif.), an immobilized preparation of lactoperoxidase and glucose oxidase, utilizing the procedure described by soule et al. (1982) . (c3h)mmtv (100 pg), 25 @i enzymobeads, 1 mci '251-na (>500 mci/ml, amersham), and 50 ~1 1% p-d-glucose were added to a small test tube, and the reaction mixture was incubated for 10 min at room temperature. the reaction was then quenched by running the mixture over a bovine serum albumin-pretreated pdlo column (pharmacia, piscataway, n. j. . at the end of a 3-hr labeling period at 37", cells were rinsed and extracted (eb, 4", 4 hr). clarified extracts were analyzed for trichloroacetic acid (tca) counts (see below). external immunoprecipitation of cell surface mmtv proteins. confluent cell monolayers were first starved for 2 hr in methionine-deprived media (d-mem containing 0.1x methionine, 2% dialyzed fbs, 0.1 pg/ml gentamicin sulfate, 10 pg/ml insulin, 2 pg/ml dexamethasone, and 0.3% sodium bicarbonate), and then labeled for 1 hr in 1.5 ml per loo-mm plate of the same media supplemented with 130 &i/ ml of @s]methionine (amersham/ searle corp.). radiolabeled cell monolayers were then subjected to external antibody immunoprecipitation as described by santos and butel (1982) . briefly, cell monolayers were rinsed with cold tbs, placed on ice, and incubated with 1 ml media containing 50 ~1 heat-inactivated rabbit antiserum (30 min, 4") as described in the legend to fig. 7 . unattached antibody was removed by extensive rinsing with cold tbs, and cells were disrupted in eb. immune complexes (representing surface-exposed antigens complexed with antibodies) were removed from clarified extracts by the addition of heat-inactivated, formalin-fixed staphylococcus aureus cowan strain i (saci; kessler, 1975) as described previously (santos and butel, 1982) . final immunoprecipitates were analyzed by sds gels and autoradiography. for chase experiments, cells were incubated in media containing excess unlabeled methionine for variable time periods prior to the antibody adsorption step. tunicamycin inhibition. tunicamycin (tm; calbiochem-behring corp.) was resuspended to 100 pg/ml in distilled water, ph 8.0. primary cell cultures were then incubated for 19 hr in media containing 0, 0.5, 1.0, or 1.5 pg/ml tm, as previously described (jarvis and butel, 1985) . metabolic labeling (described above) was performed during the final 3 hr of the 19-hr incubation and was done in the presence of the appropriate tm concentration. tca precipitation. forty microliters of labeled whole cell extracts (3h or ?s, described above) were spotted onto triplicate glass fiber filters (whatman, no. 934-ah; fisher scientific co., pittsburgh, pa.), and the filters dried (80", 20 min). proteins were then precipitated by incubating the filters sequentially in cold (4") 10% tca, 5% tca, 5% tca, and 95% ethanol for 5 min each. filters were then dried (so', 30 min), placed in liquiscent (national diagnostics, somerville, n. j.), and the radioactivity was determined using a beckman ls-250 liquid scintillation spectrometer. external labeling of cell surface carbchydrate by tritiated sodium bwohydride method. primary cultures of balb/cv tumor cells were labeled in situ with tritiated sodium borohydride [nab3h4; >20 ci/mmol; amersham] using a modification of the procedure previously described (gahmberg and hakomori, 1973; gahmberg, 1978) . briefly, monolayers were rinsed three times with tbs and a fourth time with d-pbs, ph 7.0. two milliliters of tbs containing 50 units of heat-inactivated neuraminidase (calbiochem-behring corp.) and 40 units of heat-inactivated galactose oxidase (millipore corp., freehold, n. j.) were added per 75-cm' flask, and the culture was incubated for 30 min at 37". monolayers were then washed three times in d-pbs, and 2.0 ml of tbs containing 1 mci nab3h4 was added to each flask and incubated for 30 min at room temperature. monolayers were then rinsed three times with cold tbs, extracted in eb, immunoprecipitated, and analyzed by sds-page. gels were then impregnated with autofluor (national diagnostics), dried, and exposed to x-ray film at -70". electwn microscopy. random fragments of a balb/cv tumor were removed and washed three times in phosphate-buffered saline (40 mm sodium phosphate, ph 7.2, 150 mm nacl), fixed in 3% glutaraldehyde in 0.1 m pipes buffer (sigma; ph 7.4), and postfixed in 2% osmium tetroxide in 0.1 m pipes. tissues were then stained en bloc with 2% aqueous uranyl acetate, and embedded in epon (ems):araldite (polysciences). samples were sectioned, stained with lead citrate, and examined in an rca emu3 transmission electron microscope at 100 kv. three different established mouse mammary tumor cell lines were examined for cell surface expression of mmtv antigens. cells grown to near confluence were rinsed three times with tbs and intact monolayers were iodinated using the lactoperoxidase-catalyzed reaction described under materials and methods. after labeling, cell monolayers were rinsed with cold tbs, extracted, immunoprecipitated using rabbit anti-(c3h)mmtvd, and the immunoprecipitates were analyzed on 14% sds gels. mm5mt/cl and h-l cells, both lines which produce (c3h)mmtv, were found to express mmtv gp52 at the cell surface ( fig. 1, lanes 1 and 3) . (the faint band visible at 68k in lanes 1 and 3 was not obtained in repeated experiments.) mtv-l cells, which do not produce virus particles, were shown to lack detectable amounts of gp52 on the cell surface ( proteins were detected. these results confirm previous observations that mmtv gp52 is expressed on the surface of mmtvpositive cells (yang et a& 1977; schochetman et al, 1978; massey and schochetman, 1979) . primary cultures of a balb/cv tumor were analyzed for cell surface expression of viral proteins using the same iodination procedure. in contrast to the mmtv-producing cell lines, balb/cv cells lacked detectable gp52 at the cell surface. instead, a 68k protein was identified (fig. 2 , lane 2). monospecific antisera prepared against mmtv gp52/gp36 (fig. 2, lane 3) or p28 (fig. 2, lane 4) identified the 68k protein as being env related (fig. 2, lane 3) . several organs from adult balb/cv mice were analyzed for the presence of 68k"" (table 1) . 68k"" was present in lactating mammary gland (lmg), preneoplastic cv-2 and cv-4 mammary tissue, balb/cv tumor tissue, and mtv-l cells. all other organs, including the mammary gland from a virgin balb/cv mouse (virgin mammary gland, vmg), were negative for the expression of 68kav, as well as for correctly processed mmtv proteins, gp52 and p28 (table 1) . thus, the expression of 68k" appears to be restricted to mammary tissues that also express ,other mmtv-specified proteins. the cell surface localization of the 68k" protein, in the absence of mature gp52, was unexpected. therefore, the protein was extensively characterized to understand its aberrant cell surface expression. it is known that the mmtv env precursor, as well as gp52 and gp36, are modified by glycosylation (anderson et al, 1979; dickson and atterwill, 1980) . we used three different approaches to investigate whether the surface 68k"" was undergoing biochemical modifications similar to those reported for the mmtv env precursor in other systems. the first approach used in characterizing the surface 68k"" protein involved the use of endoh, a glycosidic enzyme known to cleave at the site of attachment of asparagine-linked glucosamine to the core oligosaccharide tarentino et d, 1974) . since it has previously been shown that the env precursor is modified by n-linked, high-mannose glycosylation, it was predicted that a similarly modified 68k"" protein would be sensitive to endoh digestion. primary tumor cells were iodinated, extracted, immunoprecipitated, and the immunoprecipitates subjected to endoh digestion as described under materials and methods. the mobility of balb/cv surface 68k"" in sds gels was not affected by incubation with endoh (fig. 3, lane 4) . as a control for enzyme activity, '%i-labeled (c3h)-mmtv was immunoprecipitated and digested under identical conditions. gp52, which is modified by n-linked, high-mannose glycosylation (dickson and atterwill, 1980; jarvis and butel, 1985) , showed a decrease in molecular weight upon treatment with endoh (fig. 3, lane 7 ; see arrow), consistent with the loss of carbohydrate. the migration of gp36, an nlinked, complex-type glycoprotein (dickson and atterwill, 1980) , was not affected "tissues and cells were extracted as described previously (slagle et a& 19&q , and an aliquot was separated by sds-page. *separated proteins were then transferred to nitrocellulose (699 ma, overnight, 4") and probed using anti-(c3h)mmtv, anti-gp52/gp36, and ia?protein a. a positive result indicates that the protein band was visible on the autoradiogram. a negative result indicates that no protein band was visible on the autoradiogram following prolonged exposure of film (sensitivity of detection, 5 ng; unpublished obsewation). ' one of seven balb/cv lmg extracts contained 68kw" only and lacked detectable levels of ~23 and gp52. d nd = not done. 'the mtv-l cell line was established from a virus-positive balb/cv mammary tumor (butel et d, 1977) . by endoh treatment (fig. 3, lane 7) . the glycosidic specificity of the enzyme was demonstrated by the fact that nonglycosylated p28 and p14 were not affected by endoh digestion (fig. 3, lane 7) . these results indicate that surface 68k"" is not modified by n-linked, high-mannose-type glycosylation. it has been demonstrated that, although most of the mmtv env-precursor proteins are processed as high-mannose glycoproteins that are subsequently cleaved into gp52 and gp36, a small population of the precursor polyprotein is converted to a complex oligosaccharide by the addition of fucose and galactose (dickson and atterwill, 1980; sarkar and racevskis, 1983) . complex oligosaccharides, which are endoh resistant, are sensitive to the inhibitor of glycosylation, tunicamycin (tm), that inhibits the en bloc transfer of preassembled oligosaccharides from a lipid carrier to the newly synthesized protein (leavitt et al, 1977) . it was possible that the balb/cv 68k"" surface protein may have been modified in that way. therefore, mmtv proteins expressed at the cell sur-face in the presence of tm were identified. primary cultures of balb/cv tumor cells were grown for 19 hr in the presence of tm. during the final 3 hr of incubation, cells were starved in glucose-free media (30 min) and metabolically labeled with either [3hlglucosamine or [%]met, as described under materials and methods. at the end of the labeling period, half of the duplicate plates were extracted and processed for tca-precipitable counts. cells in the remaining duplicate plates were iodinated, extracted, immunoprecipitated, and the immunoprecipitates analyzed on sds gels. cells grown in the presence of 1.5 yg/ ml tm showed a 50% decrease in [3hlglucosamine incorporation (as compared to control, untreated cells), while [*s]met incorporation into tca-precipitable counts was unaffected at this concentration of tm (fig. 4b) . the cell surface expression of 68k"" was monitored in the tm-inhibited cells (fig. 4a) , and no decrease in the molecular weight of 68k" was noted (fig. 4a, lane 4b) . the amount of surface 68k"" present did not appear to decrease in the presence of tm, although this procedure did not allow 'precise quantitation of 68k"" synthesis. these data are consistent with the endoh results and suggest that surface 68k" is not modified by the addition of n-linked, complex-type oligosaccharides. dickson and atterwill (1980) have demonstrated that the subpopulation of the mmtv env precursor that is expressed at the cell surface contains galactose. that protein can, therefore, be detected by a cell surface labeling procedure that involves treating cells with galactose oxidase, followed by a reduction in the presence of [3h]sodium borohydride. this method was employed in a final effort to determine if surface 68k"" was glycosylated. primary cultures of balb/cv tumor cells were labeled as described under materials and methods, extracted, immunoprecipitated, and the immunoprecipitates analyzed by sds-page, fluorography, and autoradiography. as a control, a serially transplanted tumor known to express both gp52 and 68k" on the cell cultures were treated as above and, during the final 3 hr of the 19-hr tm incubation, were metabolically labeled with both mlucosamine and ["6sjmet. cells were then extracted, and clarified extracts were analyzed for tca-precipitable counts. 'h and % cpm obtained from cells grown in the presence of tm were compared to those obtained from control (non-tm-treated) cells. cell surface (slagle et al, 1981; fig. 5, lane 2) was subjected to this labeling procedure in parallel. we were able to identify galactose-containing gp52 (fig. 5, lane 5) , but not 68k" on the surface of these control cells. we were unable to identify either 68k" or gp52 on the surface of balb/cv primary tumor cells using this procedure (data not shown). the specificity of the oxidation-reduction reaction was demonstrated by the fact that galactose oxidase was required for the labeling of gp52 (fig. 5, lane 4) . these data provide additional evidence that the balb/cv surface 68k"" is not modified by glycosylation. the gp52 expressed on the plasma membranes of virus-producing cells is quite stable, substantiating its proposed function of providing a cell surface budding site for immature intracellular core particles during the virus maturation process (for a review, see schochetman et al., 1980) . since we considered the possibility that balb/cv surface 68k" might provide a similar function, the stability of 68k" in the plasma membrane was determined. primary balb/cv tumor cells were grown as monolayer cultures and iodinated. at the end of the labeling period, some cultures were extracted immediately while companion cultures were rinsed, fresh serum-free media added, and the cells reincubated for variable chase periods before extraction and immunoprecipitation. the gp52 present on the cell surface of control mm5mt/cl cells was found to be stable during a 30-min chase period (fig. 6) . longer chase periods established that gp52 was stable on these cells for at least 2 hr (data not shown). in contrast, the -risen), -1 fig. 5. nabah, labeling of cell surface carbohydrates. primary cell cultures of a control serially transplanted balb/cv tumor previously shown to express both surface 63k" and gp52 (slagle et al, 1981) were iodinated or labeled by nap&. cells were then extracted, immunoprecipitated, and the immunoprecipitates analyzed by 14% sds-page and autoradiography or fluorography. sera used for immunoprecipitation included normal rabbit serum (lanes 1 and 3) 68ke"" protein present on balb/cv tumor cells was rapidly lost from the cell surface and was completely absent after only a 15min chase (fig. 6) . newly synthesized 68k" was rapidly reinserted into the plasma membrane and could be iodinated on cells that had been previously iodinated and then chased for 30 min (fig. 6 , see asterisk). a different experimental approach was used to address the possibility that the instability of 68k"" might be induced by the iodination process per se, rather than being an intrinsic property of the protein. primary cultures of balb/cv tumor cells were starved for 2 hr in methionine-free media and were then metabolically labeled for 1 hr with r5s]met. intact cell monolayers were rinsed with cold tbs, placed on ice, and reacted with specific antisera to detect 35s-labeled mmtv proteins expressed on the cell surface. excess antibody was rinsed away, the cells were extracted, and sac1 was added to remove immune complexes from the clarified ex-tracts. final immunoprecipitates were analyzed by sds-page and autoradiography. three high-molecular-weight mmtvspecific proteins were detected by the external antibody technique: 79km (fig. 7, lane 3) , 77kgw (fig. 7, lane 3) , and 68k" (fig. 7, lanes 3 and 4) . the gag precursors detected by this procedure, which were not accessible for cell surface iodination (fig. 2) , were probably precipitated as part of budding virus at the cell surface. the stability of these three proteins within the plasma membrane was demonstrated by chasing the pulse-labeled cells in unlabeled media prior to the antibody reaction. whereas the 77km was stable during the chase periods examined (fig. 7 , lanes 5-7), both the 79km and the 68k"" were turned over rapidly and were almost completely absent following a 45min chase (fig. 7, lanes 5-7) . the longer half-life of 68k" at the cell surface in this experiment, as compared to iodinated 68k" (fig. 6) , is possibly explained by the additional time needed for 35s-labeled intracellular 68k"" to move to the cell surface. these data, based on metabolic labeling (lane 4), or anti-(c3h)mmtv (lanes 3, 5, 6, and 7) for 30 min at 4'. nonbound antibody was removed by rinsing, and the cells were extracted and processed as described under materials and methods. '"c-labeled molecular-weight markers are shown in lane 1. note that both 79kw and 68k" are rapidly turned over at the cell surface, in contrast, '77k@'q remains stable during the chase periods examined. coupled with external immunoprecipitation, indicate that the 68k" synthesized by balb/cv tumor cells is rapidly turned over at the cell surface, and confirm results obtained by the iodination procedure (fig. 6) . the fate of the '=i-labeled 68k" released from the cells was investigated by centrifugations of media collected at the end of a 30-min chase period. media containing lz51-labeled 68k"" were clarified (15,000 rpm, 30 min), followed by centrifugation at 100,ooog (1 hr) onto a 30% sucrose cushion. l%i-labeled 68k" was immunoprecipitated from the supernatant of the high-speed centrifugation (fig. 8, lanes 2 and 3) , an observation compatible with this protein existing in soluble form. 'si-labeled 68k"" also was present in the 100,ooog pellet. the presence of other viral proteins in this pellet (data not shown) provides indirect evidence that some of the surface 68k""" may be incorporated into virus particles. however, numerous attempts to localize shed '%i-labeled 68k"" into material banding at a density of 1.16-1.18 g/cc on a sucrose gradient have been unsuccessful. since the exclusive localization of the mmtv env precursor at the cell surface is usually associated with a block in virus 1 2 3, 4 fig. 8. recovery of '2si-labeled 68p" released into the media. balb/cv tumor cells were iodinated, and the media from a 30-min chase were collected and clarified (15,000 rpm, 30 min). 'zi-labeled 68k"" was then immunoprecipitated from the supernatant (lanes l-3) and the pellet (lane 4) of a high-speed centrifugation (100,000 g, 1 hr, onto a 30% sucrose cushion). sera used for immunoprecipitation maturation (nusse et az., 1979; racevskis and sarkar, 1982; slagle et al, 1985) , we next determined if balb/cv tumor cells were producing mature b-type mmtv particles. random segments of a balb/ cv primary tumor were fixed, sectioned, and examined by electron microscopy. the remainder of the tumor was established as a primary cell culture, iodinated, and shown to express surface 68k"" (data not shown). electron micrographs revealed numerous intracytoplasmic a-type particles (fig. 9a) , as well as virus particles budding into intercellular spaces (fig. 9b , see arrows). type-b morphology, typical of mmtv, was noted with the extracellular virus particles (fig. 9c, see arrows) . discussion this report describes a thorough analysis of the expression of mmtv-specific proteins on the surface of balb/cv mammary tumor cells. in contrast to other mmtv-producing systems in which gp52 is the main viral cell surface protein detected (for a review, see schochetman et al, 1980) , the balb/cv tumor cells lack detectable levels of mmtv gp52 on the cell surface. instead, we identified a 68k env-related protein. the finding of a highmolecular-weight form of the mmtv env protein on the cell surface in the absence of properly processed gp52 is not unique to the balb/cv system, having been reported for gr lymphoma cells , dba/b leukemia cells (racevskis and sarkar, 1982) , and balb/c d-2 preneoplastic mammary cells (slagle et cd., 1985) . in those reports, the aberrant expression of an unprocessed env precursor at the cell surface was associated with a lack of virus production. therefore, the balb/cv system differs from those in that type b virus particles are readily detected by electron microscopy in balb/ cv tumors (see fig. 8 ). the normal maturation pathway for the mmtv env gene has been well defined. the 24 s env-specific mrna is translated on membrane-bound ribosomes (dickson and atterwill, 1980) , resulting in a 66k-68k polyprotein (robertson and varmus, 1979; dudley and varmus, 1981; arthur et al, 1982) from which a leader sequence is cotranslationally removed (dickson et cd, 1982; arthur et al, 1982) . the 60k apoprotein (dickson and atterwill, 1980; arthur et az., 1982) is cotranslationally modified by glycosylation, resulting in the mature env precursor, designated pr70av (sarkar and racevskis, 1983) or pr73"" (dickson and atterwill, 1980; robertson, 1984) . recent studies have demonstrated the existence of at least two populations of pr70"". the majority of pr70""" is cleaved into gp52 and gp36 en route to the cell surface; once at the cell surface, only gp52 is accessible to iodination (yang et al, 1977; schochetman et al, 1978; massey and schochetman, 1979) . a second population of pr70"" is not cleaved into gp52 and gp36, but instead is modified further by complex-type glycosylation (anderson et al, 1979; dickson and atterwill, 1980; racevskis and sarkar, 1982; sarkar and racevskis, 1983) . this population, now designated pr75""" (sarkar and racevskis, 1983) or pr73"" (dickson and atterwill, 1980) , can be detected at the cell surface (dickson and atterwill, 1980) as well as in the media (sarkar and racevskis, 1983) of mmtv-producing cells. although the precise nature of the surface 68k" processing defect noted in this study is unknown, the size of the protein is compatible with at least four possibilities, based on the above information. first, the 68ke"" protein may represent an mmtv env precursor from which the leader sequence has not been removed. the size of the predicted mmtv leader sequence varies (11,000,7000, or 5700 da), depending on which of the three potential methionine starts is utilized in viva (redmond and dickson, 1983; majors and varmus, 1983) . thus, the balb/cv 68k" is approximately the size expected of a 60k apoprotein 'plus an uncleaved 7000-da leader. the molecular process that might allow a protein to retain its leader sequence is unclear. one possible explanation involves the intracellular location of env mrna translation. in the avian sarcoma virus system, 10% of the pp60wcspecific mrna is translated on membrane-bound ribosomes (presumably resulting in plasma membrane-localized pp60wc), while the remaining 90% is translated on free ribosomes (resulting in cytoplasmic localization of the protein; purchio et al., 1980) . any mmtv env mrna similarly translated on free ribosomes might be expected to retain its leader sequence. however, the mechanism by which the 68k"" would then get transported to the cell surface is unknown. a second explanation for the processing defect of 68k"" is based on the observation that the env gene of the endogenous mtv-8 provirus of gr mice has been shown to be defective. a mutation giving rise to a stop codon results in a truncated 68k"" precursor which is not processed into gp52 and gp36 (groner et al, 1984; g. knedlitschek and n. kennedy, personal communication) . such a truncated env protein would lack the hydrophobic "membrane anchor" region of gp36 (redmond and dickson, 1983; majors and varmus, 1983) . it remains to be determined whether the env gene of mtv-8 in balb/c mice contains the same termination codon as observed in mtv-8 of gr mice. such a mutation conceivably could result in the phenomena of aberrant processing and instability of the protein in the plasma membrane reported for 68k" in this paper. a similarly truncated env precursor in the balb/cv system would have to retain an 11k leader sequence to achieve the observed 68k size. the third possibility for the origin of the 68k" processing defect is that 68k"" is the normal, glycosylated env precursor, which does not get cleaved into gp52 and gp36 and is inappropriately expressed at the cell surface. however, the data presented in this paper are not consistent with this possibility. 68kav was shown to be resistant to both endoh (fig. 3) and tm (fig. 4) , suggesting that 68k" is not modified by n-linked glycosylation. we are unable to rule out the possibility that 68k" may be modified by the less well understood o-type glycosylation, which has been reported for a glycoprotein of coronaviruses (holmes et al, 1981; niemann and klenk, 1981) , as well as for sv40 tumor (t) antigen (jarvis and butel, 1985) . o-linked glycosylation, which is tm and endoh resistant, has not been reported for a glycoprotein of mmtv. finally, it is possible that 68ken" represents a fusion protein consisting of some env sequences and those from another gene, either viral or cellular in origin. since we used env-specific antisera to characterize 68k" rather than individual antisera monospecific for gp52 and gp36, we have not demonstrated unequivocally that 68k" is indeed the bona jide mmtv env precursor. such a phenomenon, resulting in the generation of a fusion protein, has not been described for the mmtv system. the inability to detect gp52 on the surface of balb/cv tumor cells is unexpected in view of the fact that intracellular gp52 and gp36 can be identified (slagle et az., 1984) and virus particles can be seen budding from the cell surface (see fig. 9 ). several possible explanations for this observation can be considered. mature gp52 may indeed be in the plasma membrane, but oriented such that it is inaccessible not only to surface iodination (figs. 2-4, 6) but also to labeling by the nab3h., technique. alternatively, gp52 may be present in its normal conformation, but may be interacting with 68k" such that it is hidden by the. larger protein and unavailable for labeling. such an interaction would involve a gp36 portion of 68k", since gp52 and gp36 have been shown to associate in forming the spikes of the viral envelope (dion et al, 1979; westenbrink and koornstra, 1979; racevskis and sarkar, 1980) . it is also possible that properly oriented surface gp52 is present, but at levels below detection using the available techniques. finally, we must consider the possibility that balb/cv tumor cells lack cell surface gp52 and that 68k" is providing the function of serving as the cell surface budding site for maturing virus particles (discussed below). the cell surface expression of 68k", in the absence of detectable surface gp52, appears to be a defect in the provirus, rather than in the ability of the cells to correctly process the env precursor. the latter phenomenon has been described for the env gene of akr virus-infected rat cells (van der hoorn et al, 1983 ) and the gag and env genes of mulv-infected rat cells (fitting et al, 1981) . in balb/cv tumor cells, however, the intracellular glycosylated forms of pr70"", gp52, and gp36 are present (slagle et az., 1984; unpublished observations) , suggesting that the cells contain the enzymes necessary to correctly process a normal mmtv env gene. since balb/cv tumors contain several mmtv proviruses (drohan et al, 1981) , it is difficult to determine which provirus is serving as the template for 68k"" expression. we must consider the possibility that the mmtv expression observed in balb/cv tumors is coming from more than one proviral template. for example, the 68k" might be expressed from a defective provirus, while the virus particles are produced from the proviral template of the milk-transmitted (balb/ cv)mmtv. alternatively, the milk-transmitted proviral template might also be defective. in this scenario, the properly processed env-gene products found in balb/cv tumor cells could be explained by occasional readthrough of a termination codon in the env gene of mtv-8. a final consideration in determining the template for 68k"" expression is that the primary tumors in this study arose from a dimethylbenzanthracene (dmba)-induced preneoplastic han outgrowth line (cv-2; slagle et al, 1984) . the aberrant processing of the balb/cv env gene is not unique to this particular outgrowth line, nor is it due to a mutagenic effect of dmba treatment, because the same surface 68k"" can be detected on normal mammary tissue from lactating balb/ cv mice and in hormone-induced preneoplastic balb/cv tissue (table 1) . the biologic role, if any, of surface 68ke"" is unknown, although several interesting possibilities can be envisioned. first, 68k"" might be involved in virus maturation. the incorporation of viral precursor proteins into rapid-harvest virus has been reported for other oncornaviruses (jamjoon et al, 1975; oskarsson et al, 1975; shapiro and august, 1976) . however, the marked instability of 68k"" in the plasma membrane, as compared to the stability of cell surface gp52 of mm5mt/ cl cells (see figs. 6, '7), suggests that 68k" is not involved in virus maturation. the recovery of some shed 68k" in a 100,000 g pellet provides circumstantial evidence that 68k" might be virus associated. although we have been unable to definitively demonstrate the presence of 68k" in the balb/c virus particle, we cannot rule out the possibility that some 68k" is occasionally incorporated into virus. a second putative function for surface 68k"" centers on the fact that much of the lz51-labeled 68k" shed from cultured balb/cv tumor cells can be recovered as a soluble protein (see fig. 8 ). this shed 68k" is stable and is not converted to a lower molecular-weight form during the several hours of chase period examined (data not shown). the shedding of proteins from the surface of tumor cells has been proposed as a mechanism by which growing tumor cells escape elimination by the host immune system (alexander, 1974; nordquist et ak, 1977; grossman and berke, 1980; van blitterswijk et al, 1975) . it is conceivable that shed 68k" might provide just such a biologic function in balb/cv mice. mmtv antigens have been identified in the serum of tumor-bearing mice (hilgers et al, 1973; verstraeten et ak, 1975; ritzi et al., 1976; zangerle et al., 1977; schochetman et ak, 1979) . however, since antibody to mmtv is not protective against tumors (muller et ac, 1971; ihle et al., 1976; miller et al, 1977; arthur et al., 1978) and since mmtv antigens can be detected concurrently with mmtv antibodies in the sera of tumor-bearing mice (arthur et al, 1978) , the possibility that shed viral proteins serve as blocking factors in modulating the host immune response to growing mammary tumors remains intriguing. the authors gratefully acknowledge the help and advice of daniel medina. we also thank ed calomeni for the electron micrographs in this report. this study was supported in part by research grants ca 25215 and ca 33369 from the national cancer institute, by national service research award ca 09197 from the national institutes of health, and by an american association of university women predoctoral fellowship (awarded to b.l.s.). escape from immune destruction by the host through shedding of surface antigens: is this a 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of mouse leukemia cells 3yl rat cells are defective in processing of the envelope precursor protein of akr virus studies of genetic transmission of mtv by c3hf mice structural analysis of a 1.7-kilobase mouse mammary tumor virus-specific rna quantitative estimation of mouse mammary tumor virus (mtv) antigens by radioimmunoassay the purification and characterization of a major glycoprotein of the murine mammary tumor virus transcription of mouse mammary tumor virus: identification of a candidate mrna for the long terminal repeat gene product identification of the mammary tumor virus envelope glycoprotein (gp52) on mouse mammary epithelial cell surface radioimmunoassay for glycoprotein gp47 of murine mammary tumor virus in organs and serum of mice and key: cord-274673-tjzlssal authors: de groot, raoul j.; van leen, robert w.; dalderup, mieke j.m.; vennema, harry; horzinek, marian c.; spaan, willy j.m. title: stably expressed fipv peplomer protein induces cell fusion and elicits neutralizing antibodies in mice date: 1989-08-31 journal: virology doi: 10.1016/0042-6822(89)90619-3 sha: doc_id: 274673 cord_uid: tjzlssal abstract we have established bovine papilloma virus (bpv)-transformed mouse c127 cell lines that synthesize the peplomer protein of the feline infectious peritonitis virus (fipv) strain 79-1146. for this purpose, a new cassette expression vector phsl, which carries the drosophila hsp70 promotor and the polyadenylation signal of the moloney murine leukemia virus long terminal repeat, was constructed. cocultivation of the bpv-transformed cell lines with fipv-permissive feline fcwf-d cells resulted in polykaryocyte formation. since it depended on the presence of fcwf-d cells, binding of e2 to the cell receptor may be required for membrane fusion. e2 was synthesized as a core-glycosylated protein of 180k which was only slowly transported from the endoplasmic reticulum to the medial golgi: of the e2-molecules labeled during a 1-hr pulse about half was still completely sensitive to endoglycosidase h after a 2-hr chase, while the remaining e2 had been chased into multiple, partially endoglycosidase h-resistant forms. immunofluorescence studies also indicated that most e2 was retained intracellularly. mice immunized with whole lysates of the transformed cells produced fipv-neutralizing antibodies as shown by plaque reduction. coronaviruses, a group of positive-stranded, enveloped rna viruses, cause considerable economical losses by infecting livestock and other domestic animals. the molecular biology of these viruses is studied not only because of their unusual replication strategy (for review see siddel et a/., 1983; spaan et a/., 1988) but also for the development of effective vaccines by recombinant dna technology. coronaviruses are characterized by large petalshaped peplomers protruding from the viral membrane. these surface projections, which consist of the peplomer protein e2, play an important role during the infection process, since they mediate receptor binding and membrane fusion (sturman and holmes, 1983) . e2 is a large (180k to 200k) glycoprotein (sturman and holmes, 1983) . sequence analyses of the e2 genes of several coronaviruses revealed that its protein moiety is 1 150 to 1450 amino acid residues in length and contains an n-terminal signal sequence, a c-terminal transmembrane anchor, and 21 to 35 potential nglycosylation sites (binns et al., 1985; de groot et a/., 1987b,c; jacobs eta/,, 1987; luytjes eta/., 1987; niesters eta/., 1986; rasschaer-t and laude, 1987; schmidt eta/., 1987) . besides glycosylation, synthesis of e2 entails acylation and for some coronaviruses, like mouse ' present address: gist brocades n.v., postbus 1,260o ma delft. *to whom requests for reprints should be addressed. hepatitis virus (mhv) and avian infectious bronchitis virus (ibv), cleavage into two subunits (frana et a/., 1985; stern and sefton, 1982; sturman eta/., 1985) . in the infected cell some e2 is not incorporated into virions but transported to the plasma membrane where it can induce polykaryocyte formation holmes, 1983, 1985) . cavanagh (1983) provided evidence that each peplomer is a dimer or trimer of e2. the presence of two regions with heptad periodicity in the c-terminal half of the protein suggests that the monomers are held together by a complex interchain coiled coil (de groot et a/., 1987c) . several observations suggest that e2 is the principal antigen eliciting protective immunity. the n-terminal half of e2 was required for a protective immune response against ibv in chickens (cavanagh et a/., 1986) while mice could be protected against a lethal challenge with mhv by vaccination with purified e2 (hasony and macnaughton, 1981) or e2-derived synthetic peptides (talbot et a/., 1988; m. koolen, personal communication) . passive immunization with e2specific monoclonal antibodies also provided protection against mhv (buchmeier et a/., 1984; wege et al., 1984) . however, the situation is much more complex in the case of feline infectious peritonitis virus (fipv), which causes a fatal disease in cats (fip), involving antibody-mediated early death (pedersen and boyle, 1980; weiss and scott, 198 1) . mammalian cell lines expressing the fipv peplomer gene would provide a convenient source of protein to dissect the role of e2 in fip. moreover, such cell lines could be used to study virus assembly and membrane fusion. here we report synthesis of fipv e2 in bovine papilloma virus (bpv)-transformed cl 27-cells. it is shown that the expression product induces fusion of fipv-permissive feline cells and is immunogenic in mice. in addition, we describe two new bpv "cassette" expression vectors allowing (i) easy cloning of genes between promotor and termination sequences, (ii) easy replacement of promotor and termination sequences, and (iii) both transient and stable expression. plasmid pdbpv-mmtneo(342-12) (law et al,, 1983 ) was obtained from the american type culture collection. plasmids pa4 ltr and pbn247 were kindly provided by dr. a. j. m. berns (dutch cancer institute, amsterdam). the cl 27 cells used for transfection experiments were obtained from dr. p. howley (nih, bethesda). cells were propagated in dmem with 10% fetal calf serum. transfection was carried out using the calcium phosphate precipitation technique (graham and van der eb, 1973) . to enhance the transfection efficiency, the cells were treated with 25% dmso (stow and wilkie, 1976) or 20% glycerol (frost and williams, 1978) 4 hr after transfection. cell lines were established by isolating foci as described by law et al. (1983) . fe/is catus whole fetus (fcwf)-d cells were obtained from dr. n. c. pedersen (school of veterinary medicine, university of california, davis). for northern blot analysis rna was isolated as described by spaan et al. (1981) . glyoxal-denatured rna was fractionated on 0.8% agarose gels in 10 mm sodium phosphate, ph 8.0 (mcmaster and carmichael, 1977) transferred to nylon membranes (genescreen plus; nen, boston), and hybridized according to the manufacturer's recommendations. standard recombinant dna techniques were performed as described . cytoplasmic dot hybridization was performed according to white and bancroft (1982) . the expression vector phsl was derived from the vector pdbpv-mmtneo(342-12). in order to facilitate the construction of this new vector (see fig. l) , the large bamhl fragment (7945 bp) representing the entire bpv-1 genome was deleted from pdbpv-mmtneo. the remaining fragment, designated pmtn, was recircular-ized. as terminator sequences we chose the long terminal repeat (ltr) of moloney murine leukemia virus (van beveren et a/., 1980) . the 0.6-kb pvulllhindlll fragment containing the ltr was isolated from plasmid pa4 ltr. by linker addition the pvull site was converted into a bg/ll site. similarly, the hindill site was changed into a bamhl site. the resulting fragment was ligated to the large fragment of bglliibamhi-digested pmtn, yielding plasmid pmtl. the 0.7-kb ecoril bamhl fragment, carrying the drosophila heat-shock (hsp70) promotor (torok and karch, 1980) was excised from pbn247 and, after converting the barnhi site into a bg/ll site by linker addition, ligated to the large ecoriibglli fragment from pmtl. the resulting vector, phsl, was used in our experiments. a full-length cdna copy of the e2 gene of fipv strain 79-1 146 (see below) was inserted into the 8g/ll site of phsl, while the bpv-1 genome was inserted into the bamhl site. the construct containing the bpv genome in the same transcriptional orientation as the fipv e2 gene was designated phsfilb(+); the construct containing the bpv genome in the opposite orientation was named phsfllb(-). cell lines made with these plasmids were indicated by rm(+) and rm(-), respectively. tailoring of the fipv peplomer gene plasmid pb1 contains a full-length cdna copy of the e2 gene of fipv 79-l 146 (de groot et al., 1987b) . before cloning this gene into the expression vectors redundant noncoding sequences located at the 5' and 3' ends were removed. for trimming of the 5'end (extending 350 nucleotides from the e2 initiation codon), 16 ng of the synthetic oligonucleotide ytgtgccatgat-tgtgct 3'(corresponding to position -6 to +l 1 of the e2 gene) was annealed in 10 m/l/l tris-hci, ph 7.5, 50 mlvl nacl (total volume 30 ~1) to 1 pg of single-stranded dna from a recombinant m 13 mp8 phage containing the (-)sense strand of the 5' terminal 1.4-kb pstl fragment of pb1. double-stranded dna was synthesized by incubation with 0.05 mm dntps and 3 units klenow dna polymerase in a final volume of 40 ~1, for 15 min at room temperature. the dna was precipitated with isopropanol, washed with 70% ethanol, and resuspended in 10 /*i 10 m/l/l tris-hci, ph 8, 1 mm edta. subsequently, 15 ~1 of sl buffer (30 mm potassium acetate, ph 4.6,0.25 n/l naci, 1 mmznso,, 5% glycerol) containing 7 units sl nuclease (pharmacia) was added; digestion was for 20 min at room temperature (eghterdarzadeh and henikoff, 1986) . after phenol extraction and ethanol precipitation, the dna was digested with pstl. thus a 1058-bp fragment was generated corresponding to the 5' end of the e2 gene. this dna was cloned into hindill (blunt-ended with klenow)/pstl-digested puc8 in the presence of barnhi linkers. the ligation mixture was used for transformation of jm 109 according to hanahan (1983) . recombinant clones were selected by colony hybridization. the fipv-specific inserts were sequenced. plasmid pl a contained the 5' end sequence of the fipv peplomer gene, starting 5 nucleotides upstream of the aug co-don (1 nucleotide was lost probably by nibbling of the sl nuclease), preceded by a barnhi linker. plasmid pl a was digested with pstl and the 3000-bp pstl fragment of b 1 was inserted in the correct orientation. the accl site located 98 nucleotides downstream of the stop codon was used to remove the redundant sequences at the 3' end. as a result, the complete fipv e2 gene was contained in a 4.3-kb barnhi fragment. to monitor e2-induced cell fusion, 1 o6 fcwf-d cells and 1 o5 bpv-transformed cl 27 cells were seeded into a 35-mm-diameter petri dish and allowed to adhere and grow for 16 hr at 37". for induction of the heat-shock promotor the cells were incubated for 2 hr at 42" and then reincubated at 37". cell fusion was assessed by light microscopy. protein analysis fipv 79-l 146-infected cells were labeled with [35s]methionine (amersham) from 6 to 6.5 hr after infection as described previously (de groot et a/., 1987a). after 2 hr incubation at 42', rm(+)19 cells were labeled at 37" by growing in cysteine-free dmem, containing 100 $ci/ml [35s]cysteine (amersham). cells were lysed in pbs, 0.5yotriton x-l 00. e2 was immunoprecipitated using ascitic fluid from an fipv-infected cat (de groot et a/., 1987a). digestion with endoglycosidase h was performed as described by rose and bergmann (1983) . proteins were denatured in laemmli sample buffer, containing 5% 2-mercaptoethanol, and subsequently analyzed on 5% sds-polyacrylamide gels. lmmunofluorescence indirect surface immunofluorescence was carried out with rm(+)l9 cells, fixed in 3% paraformaldehyde as described by rose and bergmann (1983) . for intracellular immunofluorescence, the cells were fixed for 20 min at -20" in 95% methanol, 5% acetic acid. sera (1: 100) obtained from a cat before and after experimental infection with fipv 79-l 146 were used as the primary antibodies, followed by fitc-conjugated rabbit anti-cat immunoglobulin g (1:80, nordic). immunization rm(+)19 and am2 cells were grown to confluence, subjected to heat shock, followed by a 5-hr incubation at 37". the cells were washed three times with pbs and harvested with a cell scraper. they were then suspended in pbs (10' cells/ml) and disrupted by freezethawing followed by 1 0-min sonification (bath sonicator, type rk102h, bandelin, west germany). the cell suspensions were stored at -70". the concentration of the cell lysates was adjusted to an equivalent of io' cells/mouse/dose. the first dose, consisting of lysate mixed with an equal volume of complete freund's adjuvant, was applied subcutaneously. the mice were boosted intraperitoneally on days 10 and 21 with lysates suspended in pbs (marchioli et al., 1987) . twofold serial dilutions of heat-inactivated sera (150 ~1) were mixed with about 120 pfu of fipv 79-l 146 in an equal volume of dmem, 10% fetal calf serum. incubation was for 1 hr at 37". these samples were used to infect monolayers of fcwf-d cells. after 1 hr absorption at 37", the cells were washed twice with pbs and finally maintained for 20 hr under an agar overlay. plaques were counted after staining with 0.02% neutral red in pbs. antibody titers, expressed as the reciprocal serum dilution causing 50% plaque reduction, were estimated according to the method of reed and munch (1938) . at the time we started this research, no convenient bpv expression vectors were available. therefore, we constructed the expression vector pmtl. this vector carries the mouse metallothioneine (mmt) i promotor (hamer and walling, 1982) while the moloney murine leukemia ltr (van beveren et al., 1980) provides a polyadenylation signal. pmtl has been designed with the aim to allow for easy exchange of promotor and termination sequences. to construct vector phsl the mmt promotor was replaced by the drosophila hsp70 heat-shock promotor (torok and karch, 1980) (fig. 1) . any protein coding sequence can be cloned into the unique bg/ll site downstream of the promotor. vectors for stable expression were constructed by inserting the bpv-1 genome sarver et al., 1981) into the unique barnhi site downstream of the ltr. the versatility of these vectors was demonstrated by stable expression of the vesicular stomatitis virus (vsv) membrane glycoprotein and the nucleocapsid protein of mhv (not shown). a bpv-transformed cell line am2, which expresses the mhv nucleocapsid protein under the control of the mmt promotor (bredenbeek et a/., unpublished), served as a negative control in the experiments described below. a full-length cdna copy of the fipv e2 gene was obtained from plasmid pb1 (de groot et al., 1987b) . to avoid possible interference with transcription and translation, noncoding sequences at the 5' and 3' end of the gene were removed. to anticipate possible adverse effects related to the transcriptional orientation of the bpv sequences in the vector, we used both phsfilb(+) and phsfilb(-) (fig. 1) 2. (a) screening for e2 transcripts. cytoplasmic rna was extracted from 2 x 1 o5 bpv-transformed cells and spotted on nitrocellulose as described by white and bancroft (1982) (rows b-f). about 1 pg of rna isolated from fipv-infected cells and 50 ng of an 1800bp hindill fragment derived from the fipv e2 gene were spotted as positive controls (a2 and a4, respectively). a duplicate filter was incubated for 30 min at 37" in 2x ssc, containing 10 @/ml rnase a. filters were hybridized to a nick-translated fipv e2-specific probe. (b) glyoxal-denatured rna extracted from noninduced (lane 2) and heat-shock-induced (lane 3) rm(-)i 7 cells was separated on 0.8% agarose gels, transferred to a nylon membrane, and hybridized to a nick-translated 4.5-kbbamhl fragment containing the complete fipv e2 gene. to estimate molecular weights a glyoxal-denatured ecoril hindill digest of phage x dna was induced (lane 1). rm(-)] were isolated, established as continuous cell lines, and tested for accumulation of ez-specific rna by cytoplasmic dot hybridization (white and bancroft, 1982) after a heat shock (fig. 2a) . about 20% of the cell lines gave a positive reaction. there was no difference in the percentage of positive cell lines generated by phsfilb(+) or (--). to characterize the e2 transcripts, cytoplasmic rna was extracted from some of these cell lines and analyzed by northern blotting. the nick-translated 4.3-kb bamhl restriction fragment containing the complete e2 gene (fig. 1 ) was used as a probe. a prominent rna species of 4.9 kb in length was detected. this size is consistent with a full-length transcript initiated at the heat-shock promotor and terminated at the polyadenylation site in the ltr. the 4.9-kb rna species was also observed in noninduced cells, indicating that the heatshock promotor is leaky (fig. 2b) . transcription was enhanced about two-to fivefold after induction. in one cell line, rm(+)15, the 4.9-kb rnawas not present; instead an e2-specific rna of about 2 kb was found (not shown). to test for e2 synthesis and its transport to the cell surface, we used an assay based on e2-induced fusion activity. the eight "best hybridizing" cell lines were cocultivated with fcwf-d cells (fipv permissive feline cells) and found to induce cell fusion (fig. 3) . syncytia were only formed after addition of fcwf-d cells. fusion did not occur in fcwf-d cells cocultivated with am2 cells (which express the mhv nucleocapsid protein) or rm(+)15 cells (which produce an aberrant e2 transcript; not shown). for most cell lines heat shock was required to induce cell fusion; after 16 hr of cocultivation at 37" only few syncytia were observed. syncytia appeared and increased in number 4 to 5 hr after heat shock. in contrast, one cell line rm(+)19 caused widespread cell fusion even without induction. differences in fusion potential were taken to reflect differences in the level of e2 expression. using this criterium the rm(+)19 cell line was considered the best producer and therefore selected for further study. the low-producing cell line rm(-)17 was included for comparison. to analyze e2 synthesized in these cell lines, [35s]cysteine-labeled cell lysates were subjected to immunoprecipitation with an fipv-specific antiserum. a protein of 180k was detected in rm(+)19 cells. this product was also present in rm(-)i7 cells, although in very low amounts, but it was not detected in lysates of am2 cells (fig. 4a) . the 180k protein comigrated with pulse-labeled e2 produced in fipv-infected fcwf-d cells (fig. 4b) . these results indicated that the 180k product was indeed the e2 protein. the bands >200k were observed in both the lysates of e2-producing cell lines and the am2 cell line indicating that these products are nonspecifically precipitated by the fipv polyclonal antiserum. e2 could not be detected on the plasma membrane of rm(+)l9 cells by immunofluorescence microscopy using the serum of an fipv-infected cat (not shown), but in permeabilized rm(+)l9 cells perinuclear and granular cytoplasmic staining was observed (fig. 5) ; staining was not observed in am2 cells (not shown). these results suggested that most e2 was retained intracellularly. to study the transport of e2 in the transformed cells, we analyzed the sensitivity of its oligosaccharide (3). cells were labeled with [%]cysteine from 0 to 5 hr after heat shock. lysates were subjected to immunoprecipitation using an e2-specific serum. samples were analyzed on 5% sds-polyacrylamide gels. the 180k product is indicated by an arrow; m: molecular weight marker. (b) pulse-chase labeling of e2 in rm(+)l9 cells. cells were labeled from 1 to 2 hr after heat shock with [%]cysteine. subsequently, cells were either lysed immediately ("pulse") or after a 2-hr chase with dmem containing 4 mniunlabeled cysteine ("chase"). e2 was immunoprecipitated. half of each sample was digested with endo h (+); the other half was not digested (-). samples were analyzed on 59/o polyacrylamide gels. e2 immunoprecipitatedfrom pulse-labeled fipwnfected cells was used as marker. relative amounts of e2 were estimated by densitometry of the autoradiogram using a scanning densitometer (type dd2, kipp and sons, the netherlands). chains to endoglycosidase h (endo h). endo h-sensitive high-mannose oligosaccharides are added to the protein backbone during translocation across the membrane of the endoplasmic reticulum (er). these precursor sugar chains are processed to endo h-resistant "complex" oligosaccharides in the medial golgi (dunphy and rothman, 1985; hubbard and ivatt, 1981) . figure 4b shows that e2 produced during a lhr pulse-labeling of rm(+)l9 cells did not contain complex oligosaccharide side chains. digestion with endo h yielded a 160k product comigrating with pulse-labeled, endo h-treated e2 from fipv-infected cells. about 50% of e2 in rm(+)19 cells was still fully endo h-sensitive after a 2-hr chase. the remaining e2 molecules appeared to be chased into products ranging in length from 160k to 180k after endo h-treatment. prolonged chasing did not result in the appearance of a discrete endo h-resistant product. rather, the material accumulated as multiple diffuse bands ranging in length from 180k to 200k (not shown). to determine whether the bpv-transformed cell lines synthesize enough e2 for use as immunogen, mice were immunized on day 1 and boosted on days 10 and 21 with lysates of rm(+)19 and am2 ceils. sera collected at days 1 (preserum), 21, 28, and 40 were tested for the presence of e2-specific antibody in a plaque reduction assay. table 1 shows that mice immunized with rm(+)l9 cell lysates developed rising titers of fipv neutralizing antibodies. no plaque reducfig. 5 . detection of the e2 protein by indirect immunofluorescence microscopy. rm(+)19 cells grown on coverslips were fixed with 95% methanol, 5% acetic acid and incubated with serum obtained from a cat before (a) and after(b) experimental infection with fipv 79-1 146. tion was observed with sera from mice immunized with am2 cell lysates. expression vectors containing either the complete genome or subgenomic fragments of bpv-1 have been used to establish continuous cell lines producing large quantities of exogenous proteins (hsiung et al., 1984; pavlakis and hamer, 1983; reddy et a/., 1987; sambrook er al., 1985; sarver et a/., 1985 sarver et a/., , 1987 zinn et al., 1982) . in these vectors the bpv sequences carry the functions for plasmid replication and stable mainte-nance in the transformed cells (law eta/., 1981; sarver eta/., 1981) . we have set out to establish a number of bpv-transformed mammalian cell lines which express coronavirus structural proteins. such cell lines may provide a continuous source of protein for use as vaccine, but may also aid in the study of protein function and virion assembly. to facilitate plasmid construction we have developed the cassette vectors pmtl and phsl containing the mouse mmt-1 and drosophila hsp70 promotor, respectively. both vectors possess the moloney murine leukemia virus ltr to provide a polyadenylation signal. the unique bg/ll site downstream of the promoa mice were immunized with disrupted rm(+)19 or am2 cells (five mice per group). doses of either preparation were given on days 0 (subcutaneously), 10, and 21 (intraperitoneally). heat-inactivated sera were pooled and tested in a plaque reduction assay. 'the neutralizing titer is indicated as the reciprocal of the serum dilution causing 50% plaque reduction when incubated with 120 pfu of fipv 79-l 146. tor elements can be used to insert any gene of interest. for stable expression, the bpv genome can be introduced into the unique barnhi site. however, the vectors are equally suited for transient expression; e.g., by inserting the sv40 origin into the barnhi site they can be used for expression in cos cells (subramani and southern, 1983 ). here we have used phsl-derived plasmids to establish cell lines synthesizing the peplomer protein of fipv strain 79-l 146. southern blot analysis of total cellular dna extracted from our best producing cell line rm(+)l9 showed the presence of 100-l 50 integrated copies of phsfilb(-t) dna per equivalent of diploid mouse genome (not shown). the cell lines induced syncytium formation when cocultivated with fipv-permissive feline cells. this effect was concluded to be e2 specific, since it was not induced by the bpv-transformed cell line am2 synthesizing the mhv nucleocapsid protein nor by the rm(+)15 cell line producing truncated e2 transcripts. in addition, syncytia formation was inhibited in the presence of fipv antiserum; preimmune serum from the same cat did not inhibited cell fusion (data not shown). fusion did not occur between murine cells but depended upon the presence of feline cells, suggesting that for membrane fusion to occur binding of e2 to the fipv cell receptor is required. a similar observation was made for cell lines expressing the envelope glycoprotein of human immune deficiency virus: polykaryocyte formation was restricted to cells bearing the cd4 receptor (sodroski et al., 1986) . in rm(+)19 ceils e2 was synthesized as a core-glycosylated protein of 180k. removal of the oligosaccharide side chains by endo h reduced the size of the expression product to 160k (about the size of the fipv e2 apoprotein) (de groot et a/., 1987b). as shown by the fusion assay some of the e2 was transported to the plasma membrane. however, immunofluorescence studies suggested that most e2 was retained intracel-lularly. this was confirmed in pulse-chase experiments: of the e2 molecules labeled during a 1 -hr pulse about half was still completely sensitive to endo h treatment after a 2-hr chase. the remaining material was chased into immature endo h-resistant products 160k to 180k in length, probably representing partially processed medial golgi forms (dunphy and rothman, 1985) . these results indicate that in rm(+)19 cells e2 is only slowly transported from er to golgi. for comparison, the vsv glycoprotein expressed in bpv-transformed cl 27 cells is processed to the mature endo hresistant form in less than 30 min (florkiewicz et a/., 1983) . the slow maturation of e2 in rm(+)19 cells could be host cell determined: the influenza virus hemagglutinin (ha) acquired terminal glycosylation with a half-time of more than 2 hr in bpv-transformed cl27 cells (sambrook eta/., 1985) , while in simian cv-1 cells terminal glycosylation was completed within 40 min (copeland et a/., 1986) . however, the slow rate of transport was also observed in various cell lines infected with a vaccinia virus recombinant expressing e2 of fipv (vennema et al., submitted). since coronaviruses bud through pre-golgi membranes (tooze et a/., 1988) a delayed intracellular transport of free e2 is likely to be a prerequisite for efficient virus assembly. to explore the potential of our bpv-transformed cell lines as immunogen, we immunized mice with lysates of rm(+)19 cells. we did not determine the amount of e2 in these lysates, since quantitative assays are not yet available. however, there was enough e2 to elicit fipv neutralizing antibodies. although the titers were modest, we trust that higher titers can readily be obtained by using larger amounts of cell lysate and optimized immunization protocols. we would like to use rm(+)19 cells for immunization experiments in cats to further 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sequence of the 5' long terminal repeat and adjacent cellular sequences hybridoma antibodies to the murine coronavirus jhm: characterization of epitopes on the peplomer protein (e2) antibody-mediated enhancement of disease in feline infectious peritonitis: comparisons with dengue hemorrhagic fever cytoplasmic dot hybridization regulated expression of an extrachromosomal human b-interferon gene in mouse ceils key: cord-255773-b4re5bky authors: zhang, qingzhan; shi, kaichuang; yoo, dongwan title: suppression of type i interferon production by porcine epidemic diarrhea virus and degradation of creb-binding protein by nsp1 date: 2016-01-14 journal: virology doi: 10.1016/j.virol.2015.12.010 sha: doc_id: 255773 cord_uid: b4re5bky type i interferons (ifn-α/β) are the major components of the innate immune response of hosts, and in turn many viruses have evolved to modulate the host response during infection. we found that the ifn-β production was significantly suppressed during pedv infection in cells. to identify viral ifn antagonists and to study their suppressive function, viral coding sequences for the entire structural and nonstructural proteins were cloned and expressed. of 16 pedv nonstructural proteins (nsps), nsp1, nsp3, nsp7, nsp14, nsp15 and nsp16 were found to inhibit the ifn-β and irf3 promoter activities. the sole accessory protein orf3, structure protein envelope (e), membrane (m), and nucleocapsid (n) protein were also shown to inhibit such activities. pedv nsp1 did not interfere the irf3 phosphorylation and nuclear translocation but interrupted the enhanceosome assembly of irf3 and creb-binding protein (cbp) by degrading cbp. a further study showed that the cbp degradation by nsp1 was proteasome-dependent. our data demonstrate that pedv modulates the host innate immune responses by degrading cbp and suppressing isgs expression. porcine epidemic diarrhea (ped) is a highly contagious acute enteric disease characterized by vomiting, watery diarrhea, and severe dehydration of up to 80-100% mortality in suckling piglets (song and park, 2012; sun et al., 2012a; debouck and pensaert, 1980; junwei et al., 2006) . ped was first reported in england in feeder and fattening pigs during 1970s (wood, 1977) , and reemerged in asia since 2010 with greater virulence and economic losses li et al., 2012; puranaveja et al., 2009; yang et al., 2013) . in the us, pedv appeared for the first time in 2013 and severely affected most pig-producing states marthaler et al., 2013; mole, 2013; stevenson et al., 2013) . the causative agent is porcine epidemic diarrhea virus (pedv), which belongs to the alphacoronavirus genus in the family coronaviridae (http://ictvonline.org/virustaxonomy.asp). pedv is an enveloped virus with a single-stranded positive-sense rna genome of approximately 28 kb in length with the 5 0 -cap and the 3 0polyadenylated tail. the pedv genome is arranged with orf1a, orf1b, s, orf3, e, m, n, in order with both termini flanking with the 5 0 -and 3 0 -untranslated regions (utrs) (duarte et al., 1993) . orf1a codes for the large polyprotein pp1a, while orf1b is always expressed as a fusion protein pp1a/b with pp1a through a ribosomal frameshifting. pp1a and pp1a/b are further processed to 16 nonstructural proteins, nsp1 through nsp16. orf3 codes for an accessory protein which is likely an additional nonstructural protein, whereas s, e, m and n genes code for four structural proteins (song and park, 2012) . during viral infection, the sensing of foreign nucleic acids in the cytosol leads to the activation of an innate immune response to produce type i interferons (ifn-α/β) and establishes an antiviral state. the type i ifns and ifn-mediated response provide a first line of defense against viral infection. the host innate immune system deploys the pattern-recognition receptors (prrs) to sense and respond to the pathogen-associated molecular patterns (pamps) of virus (kawai and akira, 2011) . this recognition triggers the activation of retinoic acid-inducible gene i (rig-i) or melanoma differentiation gene 5 (mda5), which further binds to the mitochondrial adapter protein mavs/ips-1 and recruits tnf receptor-associated factor 3/6 (traf3 and traf6). traf3 activates iκb kinase (ikk)-related kinases such as tank-binding kinase 1 (tbk1) and ikkε for phosphorylation of interferon regulatory factors 3 and 7 (irf3/irf7) and type i ifn production (fitzgerald et al., 2003; sharma et al., 2003) . traf6 leads to tank1 activation, followed by nf-kb activation and cytokine production (rajsbaum and garcia-sastre, 2013) . upon tbk1 activation, phosphorylated irf3 undergoes homodimerization and unveils the nuclear localization signal leading to the nuclear translocation, where it forms a complex with the transcription co-activator creb (camp responsive element binding)-binding protein (cbp)/p300 (dragan et al., 2007; lin et al., 1998; panne et al., 2007) . the irf3-cbp/p300 complex further binds to the positive regulatory domain (prd) i-iv regions of the ifn-β promoter to assemble the enhanceosome together with nf-κb and other factors to turn on the transcription of type i ifn genes (honda and taniguchi, 2006) . the irf3-cbp/ p300 interaction is crucial for ifn transcription. following production and secretion, ifn molecules bind to the cell surface receptors and trigger the activation of janus kinase-signal transducers and activators of transcription (jak-stat) signaling cascade. phosphorylated stat1 and stat2 associate to form a heterodimer, which in turn recruits the ifn-regulatory factor 9 (irf9) to form the ifn-stimulated gene factor 3 (isgf3). isgf3 translocates to the nucleus and induces genes regulated by ifnstimulated response elements (isre), resulting in expression of hundreds of antiviral genes and establishment of an antiviral state (stark and darnell, 2012) . in turn, many viruses have evolved to counteract the host innate immune defense and such viral functions are often redundant. for nidoviruses, eleven and six viral proteins have been described as ifn antagonists for severe acute respiratory syndrome coronavirus (sars-cov) and porcine reproductive and respiratory syndrome virus (prrsv), respectively kindler and thiel, 2014; shi et al., 2014; sun et al., 2012b; totura and baric, 2012) . for betacoronaviruses, nsp1 has been reported as a multifunctional viral antagonist for innate immune response (huang et al., 2011b; narayanan et al., 2008; wang et al., 2010) . for pedv, the viral modulation of innate immune signaling is poorly understood. pedv infects vero cells, but these cells are type i ifndeficient due to a chromosomal deletion (desmyter et al., 1968 ). in the present study, we identified marc-145 cells as a suitable line of cells for pedv infection and for study of innate immune modulation. we showed that pedv suppressed the type i interferon production and isgs expression in these cells, and identified nsp1, nsp3, nsp7, nsp14, nsp15, nsp16, e, m, n and orf3 as the viral ifn antagonists. we showed that pedv nsp1 caused the cbp degradation by the proteasome-dependent pathway. the cbp degradation is a novel mechanism of coronavirus nsp1 for ifn suppression and our study provides a new insight into the immune modulation and evasion strategy of pedv. pedv replicates in the cytoplasm of villous epithelial cells of the small and large intestines (debouck and pensaert, 1980; sueyoshi et al., 1995) . the viral antigen is also detectable in the macrophages that infiltrated the lamina propria (lee et al., 2000) . histological studies showed that pedv replicates in the porcine respiratory tract in vivo and transformed alveolar macrophages (3d4) in vitro (park and shin, 2014) . vero cells are widely used for pedv for diagnosis, virus isolation, and research, but these cells are type i ifn-deficient due to the chromosomal deletion (desmyter et al., 1968) . to study a possible regulation of innate immune signaling by pedv, various cell lines were examined for susceptibility. cells were infected with pedv at an moi of 0.1 in various trypsin concentrations and cpe was examined daily for up to 5 days. in vero and marc-145 cells, apparent cpe of multinucleation was observed by 24 h post-infection ( fig. 1a and b, left panel). trypsin activates the cleavage of s protein and induces membrane fusion to trigger infection (park et al., 2011; wicht et al., 2014) . pedv infection was characterized by syncytia formation (hofmann and wyler, 1988) and infection foci were visualized by anti-pedv m and n antibodies, indicating the susceptibility of both cell types for pedv infection ( fig. 1a and b, middle and right panel). the viral proteins were detected using specific antibodies by western blot and the specific bands were corresponding to the m and n proteins, further confirming the productive infection of these cells by pedv (fig. 1c ). the optimal trypsin concentration for pedv propagation was 5 μg/ml and 2 μg/ml for vero and marc-145 cells, respectively. marc-145 cells have been used to study type i ifn signaling of porcine arterivirus (kim et al., 2010; overend et al., 2007; patel et al., 2010) , and thus infection of these cells one μg of each of the cloned genes was transfected to hela cells in 12-well plates, and protein expression was determined by immunofluorescence (b) and western blot (c) for each gene using anti-flag antibody. (d and e) regulation of poly(i:c)-induced ifn-β promoter activity by individual pedv proteins. hela cells were seeded in 12-well plates and co-transfected with pifn-β-luc along with individual pedv genes and prl-tk at a ratio of 1:1:0.1. since the expression levels of nsp3 and nsp16 were low, three-times more plasmids were transfected for these genes to ensure the comparable level of protein expression. prrsv nsp1α (p-nsp1α) is a known type i ifn suppressor, and the ifn-suppression of its mutant p-nsp1α(m) was lost. both constructs were included as controls. at 24 h post-transfection, cells were stimulated with poly (i:c) (0.5 μg/ml) for 12 h and the luciferase activities were measured. the reporter experiments were repeated three times, each time in triplicate. asterisks indicate the statistical significance. statistical analysis was performed by student's t test using gst as a control. n p o0.05, nn po 0.01 and nnn p o 0.001. (f) vsv-gfp bioassay. the cell culture supernatants for ifn-β promoter luciferase assays were collected and diluted serially by 2-folds up to 1:64. fresh marc-145 cells were grown in 96-well plates and incubated with each dilution of supernatants for 24 h, and then infected with vsv-gfp at an moi of 0.1 for 16 h. vsv replication was measured by monitoring the fluorescence by gfp expression using fluorescent microscopy. data were presented as log 2 sample dilution folds. (g) inhibition of irf3 promoter activation by pedv proteins. the ifn antagonists were further examined for irf3 activities by luciferase reporter assays. statistical analysis was performed by student's t test using gst as a control. by pedv allowed us to study ifn modulation and signaling cascade. to determine whether pedv infection antagonized the type i ifn response, ifn-β mrna was determined in virus-infected cells. marc-145 cells were infected with pedv and stimulated with poly (i:c) followed by qrt-pcr for ifn-β mrna using total rna. as shown in fig. 2a , pedv infection did not induce the level of ifn-β mrna expression whereas poly(i:c) alone induced the ifn-β gene expression effectively, indicating the suppression of ifn-β response by pedv. to further evaluate the ifn-β response in pedv-infected cells, a dual luciferase assay was performed. the results showed the suppression of ifn-β promoter activity in pedv-infected cells upon poly(i:c) stimulation (fig. 2b) , demonstrating the modulation of ifn production by pedv infection. irf3 was additionally examined for its role for pedv-mediated ifn-β suppression. the irf3 promoter activity was found to be inhibited (fig. 2c ). the suppression of ifn-β production was confirmed by bioassay using vsv-gfp. vsv is sensitive to type i ifn treatment and thus commonly used for ifn bioassays. culture supernatants were collected from pedv-infected cells and were uv-irradiated, followed by incubation with marc-145 cells and infection with vsv-gfp. vsv grew normally (fig. 2d) , whereas it did not grow with supernatants collected from poly(i:c)-treated cells for up to 1:8 dilution. vsv-gfp also grew normally with supernatants from both pedv-infected cells with or without poly(i:c) stimulation, confirming the suppression of type i ifn production by pedv. the viral ifn antagonism is often redundant, and at least 11 viral proteins have been identified as ifn antagonists for sars-cov (kindler and thiel, 2014; shi et al., 2014; totura and baric, 2012) . to identify such proteins for pedv, we cloned pedv genes representing nsps 1 through 16, and structural genes for s, e, m, and n including the orf3 accessory protein gene (fig. 3a ). among these, nsp11 is a small oligopeptide generated from pp1a when ribosomal frameshifting does not occur and so was not included in this study. each gene was inserted into the pxj41 expression vector with the flag tag at either n-or c-terminus, and examined for ifn suppression. the protein expression of cloned genes was examined by immunofluorescence (fig. 3b ) and western blot (fig. 3c ) using anti-flag antibody. all genes were expressed as anticipated. hela cells were then co-transfected with an individual gene along with pifn-β-luc and prl-tk, and reporter assays were conducted. prrsv nsp1α (p-nsp1α) is known as an ifn-β suppressor, and its cystine mutant p-nsp1α(m) (c28s) is an ifn suppression revertant (han et al., 2013; song et al., 2010) , and so they were included as positive and negative controls, respectively. poly(i:c) upregulated the ifn-β transcription in cells expressing pxj41, gst, and p-nsp1α (m), while p-nsp1α suppressed the ifn-β promoter activity as expected ( fig. 3d and e). of the nsps, nsp1, nsp3, nsp7, nsp14, nsp15 and nsp16 were shown to down-regulate the ifn-β activity (fig. 3d ). for structural proteins, e, m, and n were found to suppress the ifn induction (fig. 3e) , and orf3 was additionally identified as an ifn suppressive protein (fig. 3e ). the findings from the reporter assays were validated by vsv-gfp bioassays. the dilution corresponding to 50% of cells exhibiting gfp expression was determined as the end-point inhibition. for pxj41, gst, and nsp9, gfp expression was evident and their end-point inhibitions were determined as 1:64 (fig. 3f ). in contrast, the viral proteins identified as the luciferase suppressors showed an apparent inhibition of vsv-gfp replication and their end-points were determined to be 1:4 to 1:16 (fig. 3f ). these titers represent 4-to 16fold lower than those of controls. taken together, these data demonstrate that pedv has the ability for ifn suppression, and nsp1, nsp3, nsp7, nsp14, nsp15, nsp16, orf3, e, m and n are the viral ifn antagonists. to determine the target for ifn inhibition, the irf3 pathway was examined for individual viral proteins using the irf3 luciferase reporter constructs. upon stimulation, the irf3-dependent luciferase expression was reduced by nsp1, nsp3, nsp7, nsp14, nsp15, nsp16, orf3, e, m, and n, comparing to those of pxj41 and gst (fig. 3g ). this suggests that the irf3 signaling pathway was interfered by these viral proteins for the suppression of the ifn-β production. pedv nsp1 antagonism is a nuclear event sars-cov is a betacoronavirus and its nsp1 triggers inhibition of type i ifn induction and downstream signaling, host mrna decay and cleavage, and inhibition of protein translation (huang et al., 2011b; lokugamage et al., 2012; narayanan et al., 2008; tanaka et al., 2012) . transmissible gastroenteritis virus (tgev) is an alphacoronavirus and its nsp1 inhibits host protein expression (huang et al., 2011a) . nsp1 of alphacoronavirus and betacoronavirus lacks the overall sequence similarity (narayanan et al., 2015) , and thus alphacoronavirus nsp1 may have a distinct basis for its biological function. since nsp1 appeared the most potent suppressor in our study on pedv, nsp1 was chosen to study the molecular basis for the ifn suppression. the subcellular localization was first examined by confocal microscopy in transiently expressing cells. the nsp1 distribution was evident in the both nucleus and cytoplasm ( fig. 4a) , which is consistent with tgev nsp1 (narayanan et al., 2015) . co-expression of nsp1 with either the endoplasmic reticulum or mitochondrial marker showed the site for cytoplasmic nsp1 in the endoplasmic reticulum (fig. 4b ). quantitative rt-pcr was conducted to evaluate ifn-β suppression in nsp1-gene transfected cells. the expression of nsp1 significantly suppressed the ifn-β mrna transcription (fig. 5a) , further validating the nsp1 antagonism against ifn-β production. subsequently, the ifnmediated antiviral gene expression was examined for isg15 and isg56 by qrt-pcr. pedv nsp1 reduced the poly(i:c)-stimulated mrna levels of both isg15 (fig. 5b ) and isg56 (fig. 5c ), indicating the suppression of ifn signaling by nsp1. the suppression of ifn-β, irf3, and nf-κb activations raises a possibility that nsp1 may target a component of the rig-i like receptor (rlr) signaling pathway. to examine this premise, nsp1 was co-expressed with one of the main components in the rlr signaling pathway, and ifn luciferase activities were determined at 24 h post-transfection. the over-expression of ips-1, or irf3 led to the robust activation of the ifn-β promoter as anticipated, whereas the activation was significantly inhibited by nsp1 ( fig. 6a fig. 4 . subcellular localization of pedv nsp1. hela cells were seeded on slides in 6-well plates and transfected with pedv nsp1 gene (a), or co-transfected with nsp1 and pdsred2-er or pdsred2-mito (b). at 24 h post-transfection, cells were fixed and permeabilized with triton x-100. cells were then incubated with rat anti-flag mab for 1 h, followed by alexa fluor 488-conjugated goat anti-mouse (green) secondary antibody to visualize nsp1. the er and mitochondrial proteins were fused with the er and mitochondria targeting sequence (clontech) and so directly visualized (red). nuclei (blue) were stained with dapi. images were collected using a zeiss lsm-510 meta confocal laser-scanning microscope and processed with the lsm image browser (zeiss). irf3 is a resident protein in the cytoplasm. when stimulated, it is phosphorylated and homodimerized, leading to the translocation to the nucleus (dragan et al., 2007) . to determine whether pedv nsp1 targeted the irf3-dependent pathway, the irf3 phosphorylation was first examined. nsp1-gene transfected cells were stimulated with poly(i:c), and the irf3 phosphorylation was examined by western blot. as anticipated, the poly(i:c) stimulation led to irf3 phosphorylation in pxj41-transfected cells, and similarly, in nsp1-expressing cells, the irf3 phosphorylation was evident and comparable to that of control (fig. 7a , top panel, lane 4), suggesting that pedv nsp1 exerts its suppression downstream of the irf3 phosphorylation. thus, the nuclear translocation of irf3 was next examined. prrsv nsp1α is known not to block the irf3 nuclear localization, and so was used as a control in this study. endogenous irf3 was normally diffused and distributed in the cytoplasm, but translocated to the nucleus when stimulated by poly(i:c) (fig. 7b, second panel) . similarly to prrsv nsp1αexpressing cells (fig. 7b , fourth panel), irf3 also localized normally in the nucleus after stimulation in pedv nsp1-expressing cells (fig. 7b, bottom panel) , suggesting that the ifn suppression by pedv nsp1 may be a nuclear event. the irf3 nuclear translocation in nsp1-expressing cells was further confirmed by cell fractionation and western blot analyses. while irf3 was phosphorylated and localized in the nucleus after stimulation, pedv nsp1 did not inhibit the irf3 phosphorylation and nuclear translocation (fig. 7c ), further indicating that the nsp1-mediated ifn suppression was a nuclear event. interruption of irf3 and cbp association by nsp1 after nuclear translocation, an irf3 dimer associates with the creb-binding protein (cbp). this complex then binds to the prd i-iii 6 . disruption of irf3-mediated ifn signaling by nsp1. hela cells were seeded in 12-well plates and co-transfected with pmavs/ips-1 (a) or pirf3 (b) along with the nsp1 gene, prl-tk, and ifn-β-luc reporter for 24 h. cells were harvested to measure the firefly and renilla luciferase activities. relative luciferase activity was defined as a ratio of the firefly luciferase to renilla luciferase activities. data are presented as mean value 7 standard deviation from three independent experiments. statistical analysis was performed by student's t test. n po 0.05, nn p o0.01, and nnn po 0.001. immunofluorescence staining for irf3 nuclear translocation by nsp1. hela cells were transfected with the nsp1-expressing plasmid (2 μg/well) in 6-well plates for 24 h and stimulated by poly(i:c) for 8 h. cells were fixed and incubated with rabbit anti-irf3 pab and rat anti-flag mab for 1 h. prrsv nsp1α does not inhibit the irf3 nuclear localization and was used as a control. alexa fluor 594-conjugated goat anti-rabbit and 488-conjugated goat anti-mouse secondary antibodies were used to visualize irf3 (red) and viral nsp1 (green), respectively. nuclei (blue) were stained with dapi. yellow arrows indicate irf3 localization in the nucleus in the absence of nsp1 expression. white arrows indicate irf3 localization in the nucleus in nsp1-expressing cells. (c) phosphorylation and nuclear localization of irf3 by nsp1. hela cells were transfected with the pedv nsp1 gene for 24 h, then stimulated with poly(i:c) for 8 h. cells were lysed for nuclear-cytoplasmic fractionations and subcellular distribution of irf3 and pirf3. hsp90 was used as a cytosolic protein marker and parp was used as a nuclear protein marker. regions of the ifn-β promoter to assemble the basal transcription machinery complex together with nf-κb and other transcription factors to turn on the transcription of type i ifn genes (honda and taniguchi, 2006) . thus, the irf3-cbp/p300 interaction for the assembly of enhanceosome is crucial for ifn expression. since pedv nsp1 did not block the irf3 phosphorylation and nuclear translocation in our study, it was hypothesized that nsp1 might disrupt the formation of enhanceosome in the nucleus. to address this, the irf3/ cbp association was first examined in nsp1-expressing cells. cells were transfected with the nsp1 gene and stimulated with poly(i:c) followed by co-immunoprecipitation using anti-irf3 antibody and immunoblot with anti-cbp antibody. in unstimulated cells, cbp was undetectable due to the absence of irf3/cbp association (fig. 8a , left lane), but irf3/cbp association became evident upon stimulation (fig. 8a, middle lane) . in nsp1-expressing cells however, the association of irf3 and cbp disappeared even upon stimulation (fig. 8a , right lane) and the detectable level of irf3 remained unchanged ( fig. 8a, second panel) . absence of the association of cbp/irf3 may occur when nsp1 binds to either irf3 or cbp, or when irf3 is unstable in the presence of nsp1. since pedv nsp1 was found to be a nuclear protein (figs. 4a, b and 7b, c), nsp1 in the nucleus might interact with either irf3 or cbp. however, neither the interaction between irf3 and nsp1, nor between cbp and nsp1 was observed by co-immunoprecipitation in our study. irf3 was also stable in the presence of nsp1 ( fig. 7a and c) , indicating that the absence of irf3/ cbp association was not due to the instability of irf3. interestingly, the level of cbp was found to decrease in nsp1-expressing cells (fig. 8a) , leading us to investigate the degradation of cbp by nsp1. some viruses including htlv, adenovirus, and an orthormyxovirus thogoto interact with cbp to modulate type i ifn induction, suppress protein expression, or promote virus infection (ferrari et al., 2014; jain et al., 2015; jennings et al., 2005; wurm et al., 2012; zhang et al., 2008) . degradation of cbp has been described for the porcine arterivirus prrsv as a strategy for ifn antagonism . since the level of cbp was found to decrease in pedv nsp1-expressing cells (fig. 8a) , cbp expression was validated in pedv-infected cells by co-staining using anti-cbp antibody and anti-pedv m pab. in uninfected cells, cbp was predominately localized in the nucleus in marc-145 and vero cells (fig. 8b, yellow arrows) . in contrast, cbp was depleted in virus-infected cells (fig. 8b , white arrows), demonstrating that the cbp was degraded by pedv. we further sought to study whether the cbp degradation by pedv was mediated by nsp1 protein. cbp was exclusively nuclear in control cells, whereas it was depleted in nsp1-expressing cells (fig. 9a) . prrsv nsp1α is known to degrade cbp in the nucleus , and in prrsv nsp1αexpressing cells, cbp was significantly depleted (fig. 9a ). the cbp degradation was quantified by examining the ratio of nsp1expressing cells showing cbp degradation out of the chosen number of nsp1-expressing cells (fig. 9b ). approximately 92% of prrsv nsp1α-expressing cells showed more than 80% reduction of cbp, which is in consistent with the previous report (han et al., 2013) . for pedv nsp1-expressing cells, approximately 90% cells showed more than 80% reduction of cbp, while no cbp reduction was observed in control cells. this finding was confirmed by western blot. in pedv nsp1-expressing cells, cbp degradation was evident compared to that of control cells (fig. 9c , top panel, lane 3). to eliminate a possibility that the reduction of cbp might be due to the short half-life of cbp, cyclohexamide (chx) treatment was conducted (fig. 9d) . at 24 h post-transfection, cells were treated with chx to shut down the new protein synthesis for indicated times followed by western blot. in nsp1-expressing cells, cbp reduction was evident at the beginning of chx treatment, and further decreased by 2 h post-treatment. the cbp degradation was complete by 4 h post-treatment, whereas nsp1 and β-actin remained stable (fig. 9d) . together, our data show that pedv nsp1 was the viral protein contributing to the cbp degradation. unlike prrsv nsp1α, pedv nsp1 does not contain a proteinase activity, and no direct interaction between cbp and nsp1 was identified in our study. it is thus unlikely that cbp would be a direct substrate of pedv nsp1. therefore, it was of interest to examine whether the cbp degradation was a proteasomedependent process. the treatment with mg132 blocked the cbp degradation by nsp1. as little as 5 μm of mg132 was sufficient to inhibit the cbp degradation, and 10 μm was able to restore the cbp level back to the control level (fig. 9e) . to eliminate the cbp degradation by nsp1 was cell-type specific, we further tested the cbp degradation by nsp1 in pig intestinal epithelial cell line (ipec-j2 cells), which reported to be susceptible to pedv (zhao et al., 2014) . cbp degradation in nsp1-expressing cells was evident comparing to control cells (fig. 9f, top panel) . additionally, the cbp degradation by nsp1 was also blocked by mg132 treatment in ipec-j2 cells (fig. 9f, bottom panel) . this study indicates that the cbp degradation by pedv nsp1 was proteasome-dependent in the nucleus. the innate immune system is the first line of host defense in response to viral infection. it initiates the production of type i ifns and proinflammatory cytokines through the recognition of pamps by prrs and establishes antiviral states which are highly effective on resisting and controlling infections. in turn, many viruses have developed strategies to counteract the host innate immune response to establish productive infection. previous studies have shown that pedv infection fail to induce the ifn-β promoter activation and that plp2 (papain-like proteinase 2) of pedv antagonizes the ifn response by deubiquitinating rig-i and sting (xing et al., 2013b) . the pedv n protein suppresses the irf3 and nf-κb activities and antagonizes the ifn-β production by disrupting the interaction between irf3 and tbk1 (ding et al., 2014) . on the contrary, a recent study shows that pedv infection induces nf-κb activation in intestinal epithelial cells with the n protein as the activator (cao et al., 2015b) . in the present study, we have identified marc-145 as pedv permissive cells, and used these cells as a model to study the innate immune modulation for pedv. we have shown the suppression of type i ifn production by pedv, which is consistent with the recent finding in iecs (cao et al., 2015a) . we also have identified multiple viral proteins responsible for this suppression. we have further determined pedv nsp1 as the viral component promoting cbp degradation in the nucleus via the proteasome-dependent pathway. many viruses in the order nidovirales are able to modulate the host innate response, which plays an important role for their pathogenesis. in the family arteriviridae, equine arteritis virus suppresses type i ifn production in equine endothelial cells (go et al., 2014) , and prrsv also suppresses ifn production (albina et al., 1998) . prrsv is susceptible to type i ifns in cells and the suppression of type i ifn varies for different isolates (albina et al., 1998; lee et al., 2004; overend et al., 2007) . mouse hepatitis virus (mhv), which is a betacoronavirus, induces a high level of ifn-α secretion by plasmacytoid dendritic cells (pdcs) during infection (cervantes-barragan et al., 2007) . however, other cell types infected by mhv such as macrophages, microglia, and oligodendrocytes produce extremely low-levels of type i ifns (li et al., 2010; roth-cross et al., 2008; zhou and perlman, 2007) . the mhv ns2 protein is dispensable for virus replication in cells but is required for induction of hepatitis in mouse (schwarz et al., 1990) . the 2 0 ,5 0 -phosphodiesterase (pde) activity of ns2 mediates the cleavage of 2 0 ,5 0 -oligoadenylate and prevents the activation of rnase l, while enhancing viral growth and pathogenesis, thus ns2 is a viral ifn antagonist (zhao et al., 2012) . sars-cov, which is another member virus in the genus betacoronavirus, impairs the ifn response in virus-infected cells, and an ifn therapy has been suggested to be efficacious for sars patients (cinatl et al., 2004; spiegel et al., 2005) . mers-cov is also a betacoronavirus, and both mers-cov and sars-cov do not induce a pronounced ifnresponse in polarized airway epithelial cells (calu-3), alveolar adenocarcinoma cells (a549) and human monocyte-derived macrophages (lau et al., 2013; zhou et al., 2014; zielecki et al., 2013) . even though the acute infection of tgev induces a highlevel of ifn-α in newborn pigs (la bonnardiere and laude, 1981) , protein 7 counteracts the host antiviral response and influences viral pathogenesis (cruz et al., , 2013 . the 7a protein of an alphacoronavirus feline infectious peritonitis virus is a type i ifn antagonist (dedeurwaerder et al., 2014) . type i ifns of chickens inhibits viral replication and respiratory illness of the gammacoronavirus infectious bronchitis coronavirus (ibv) (pei et al., 2001) . ibv delays the ifn response and the 3a and 3b accessory proteins have been identified as the ifn antagonists (kint et al., 2015) . thus, modulation of type i ifn response seems to be a common evasion strategy of viruses in the order nidovirales. we have shown in the present study the direct evidence that pedv indeed downregulates type i ifns production during infection. pedv suppresses the ifn-β and irf3 activities. since irf3 is a key element in the production of type i ifns, our finding leads to a hypothesis that pedv modulation of type i ifns production targets the irf3 signaling pathway. interestingly, pedv normally activates the nf-κb activity in vero e6 cells (xing et al., 2013b) . a recent study confirms that pedv infection in intestinal epithelial cells induces nf-κb activation (cao et al., 2015b) . in that study, nuclear localization of p65 increases by pedv after 12 h through 48 h. however, activation of nf-κb during viral infection is generally an early event. for prrsv, nf-κb is activated 30 min after infection (fu et al., 2012) . thus, how pedv modulates nf-κb activation during early time of infection needs to be further investigated. we have identified at least ten viral ifn antagonists and all ten proteins inhibit the irf3 activity. whether these ifn antagonists modulate the nf-κb activity needs to be further investigated. the pedv n protein suppresses sendai virus-induced nf-κb activity in a dose-dependent manner (ding et al., 2014) . in other study, n protein activates nf-κb in intestinal epithelial cells (cao et al., 2015b) . a possible explanation is that the nf-κb activation may be time-dependent and cell type-dependent. together, the irf3 signaling is likely the target by pedv for type i ifns modulation. at least eleven viral proteins have been identified as ifn antagonists for sars-cov (kindler and thiel, 2014; shi et al., 2014; totura and baric, 2012) , whereas ten proteins have been identified for pedv in our study. thus, coronaviruses seem to arm with multiple antagonists. a possible explanation for such a functional redundancy is that coronavirus genomes are the largest rna known to biology and undergo continuous genetic evolution. when a functional mutation occurs in a major antagonist, other antagonists may complement the function to ensure efficient replication and adaptation in hosts. for sars-cov, nsp1 is a multifunctional protein with the suppressive activity for ifn and blocks the phosphorylation of stat1 and degrades host cell mrna (totura and baric, 2012) . sars-cov nsp14 and nsp15 works as exoribonuclease and endoribonuclease, respectively, thus specific digestion of dsrnas and the consequent removal of rna-pamps may lead to an inadequate activation of ifn response (kindler and thiel, 2014) . sars-cov nsp16 contains 2 0 -omethlytransferase activity and modifies the cap of viral rnas in order to evade the detection by the host immune system (totura and baric, 2012) . the sars-cov m protein impedes the formation of traf3 á tank á tbk1/ikkϵ complex for suppression of type i ifn production (siu et al., 2009 (siu et al., , 2014 . the plp2 domain of sars-cov nsp3 negatively modulates type i ifn pathway and functions as a viral deubiquitinase. in our study, the full length pedv nsp3 indeed inhibit the ifn activity. all ten antagonists identified for pedv correspond to the respected antagonists of sars-cov. the corresponding proteins of pedv may share the similar motifs and functions with those of sars-cov. additionally, sars-cov encodes several accessory proteins. they are nonessential for viral replication but function as innate immune antagonists. for pedv, orf3 is the sole accessory protein, and a previous report shows that orf3 functions as an ion channel protein and is relevant to infectivity and pathogenicity (wang et al., 2012) . orf3 is nonessential for viral replication in vitro as shown by targeted rna recombination . in our study, orf3 is a potent ifn antagonist. the viral antagonists may target different pathways of the host innate immune signaling and their synergistic effects may shut down the host innate immune response more efficiently during the course of infection. cbp is a histone acetyltransferase and plays a key role in transcription regulation. the cbp/p300 coactivators interact with hundreds of transcription factors including stats, c-myc, pias1, p53, nf-κb, and irf family (bedford and brindle, 2012; goodman and smolik, 2000; long et al., 2004) . for ifn expression, the assembly of an enhanceosome consisting of nf-κb, irfs, atf2/c-jun, and the architectural protein hmg i(y) is required in response to virus infection. the ifn enhanceosome recruits cbp/p300 for synergistic activation of transcription (merika et al., 1998) . some viruses modulate the cbp activity for viral evasion. two distinct regions in the simian virus 40 t antigen can independently alter the levels and loading of cbp/p300 transcripts onto polysomes for cell immortalization and transformation (robles et al., 2013) . african swine fever virus nuclear protein a238l inhibits the expression of tnf-α by displacing the cbp/p300 coactivators (granja et al., 2006) , and herpes simplex virus 1 (hsv-1) icp0 protein recruits activated irf3 and cbp/p300 to the nuclear foci, which may result in reduced transcription of ifn-β and inhibition of the host response (melroe et al., 2007) . hsv-1 vp16 protein inhibits nf-κb activation and interferes the recruitment of irf3 to cbp to block the ifn-β production (xing et al., 2013a) . the ml protein of thogoto virus interferes with irf3 function without blocking its nuclear translocation but interrupts the association of irf3 with cbp (jennings et al., 2005) , which is similar to the function of pedv nsp1. the ml protein was later found to interact with the rna polymerase ii transcription factor iib (tfiib), however, this interaction hardly interferes the host general gene expression but strongly suppresses both the irf3-and nf-κbregulated promoter activities (vogt et al., 2008) . thus, it is hypothesized that the virus-mediated cbp degradation may play a specific and key role for ifn modulation with a little impact on general cellular gene transcriptions. the degradation of cbp is a novel strategy for ifn modulation and has been extensively studied in the family of arteriviridae, especially for prrsv . for prrsv nsp1α, cbp degradation is associated with the zinc-finger motif and is likely the key mechanism for ifn suppression (han et al., 2013) . for pedv, nsp1 is the most potent ifn suppressor among all viral antagonists without affecting the irf3 phosphorylation and nuclear localization. in line with this, pedv infection depletes the endogenous cbp. furthermore, pedv nsp1 disrupts the association of cbp-irf3 and degrades cbp in a proteasome-dependent manner. sars-cov nsp1 inhibits type i ifn production, induces host mrna degradation, and suppresses host protein translation (narayanan et al., 2008) . however, the domains of sars-cov nsp1 responsible for suppression of host gene expression and type i ifn production are absent in pedv nsp1 (huang et al., 2011b; narayanan et al., 2008) . even though nsp1s of alphacoronavirus and betacoronavirus share similar functions, they lack an overall sequence similarity and neither conserved motifs nor domains exist in viruses of alphacoronaviruses. thus, it is plausible that nsp1 of alphacoronaviruses may have a distinct function regulating host innate immune responses and gene expression. tgev nsp1 suppresses protein translation in cells and cell-free extracts. however, the suppression of protein translation by pedv nsp1 may not be a general event since the β-actin shows the similar level of expression after infection and transfection. the lack of association of cbp-nsp1 and irf3-nsp1 suggests that the cbp degradation by nsp1 is an indirect event that needs to be further determined. similar to tgev nsp1, the subcellular localization of pedv nsp1 is nuclear-cytoplasmic. the sequence of pedv nsp1 does not harbor any known nuclear localization signal, and thus nsp1 may piggy-bag a nuclear protein to enter the nucleus. the proteasome-dependent cbp degradation seems a unique viral tactic utilized to inhibit ifn-β production. it is of interest to study whether this is a common evasion strategy for coronaviruses. cbp localizes in the pml nuclear bodies, which are discrete nuclear foci that are disrupted in acute promyelocytic leukemia (boisvert et al., 2001; doucas et al., 1999; lamorte et al., 1998) . the pml nuclear bodies dynamically colocalize with numerous proteins including cbp, pml, p53, rb, sp100, daxx, eif4e, and sumo (jensen et al., 2001) . upon inhibition of proteasome activity, pml, sp100, ebna-5, sumo-1, and the 20s proteasome subunit move to the nucleolus, suggesting that proteasomal degradation occurs at the nuclear loci (boddy et al., 1996) . hausp, the ubiquitin-specific hydrolase in the pml nuclear bodies, removes ubiquitin moieties from proteins prior to proteasomal degradation (everett et al., 1998) . thus, pml nuclear bodies may represent the sites where ubiquitinated proteins are processed by enzymes such as hausp prior to degradation in the nucleolus (st-germain et al., 2008) . valproic acid, a histone deacetylase inhibitor, could induce cbp degradation through the ubiquitin-proteasome pathway, while increasing the colocalization of cbp with ubiquitin nuclear speckles and with pml nuclear bodies (st-germain et al., 2008) , suggesting that pml nuclear bodies may be the sites for the ubiquitin-dependent degradation of cbp. it is of interest to examine whether pedv nsp1 promotes ubiquitination of cbp for degradation in the nucleus and whether this degradation associates with pml nuclear bodies. pedv infects vero cells and marc-145 cells. porcine amino peptidase n (papn) has been identified as the major cell entry receptor for pedv (li et al., 2007; nam and lee, 2010) . transient expression of papn confers pedv non-permissive canine kidney cells (mdck) to be permissive for pedv infection. papn also increases the pedv infectivity in porcine small intestine epithelial cells (iecs) (cong et al., 2015) . the respiratory tract may support pedv infection in pigs and the virus infects and replicates in transformed alveolar macrophages (3d4) in vitro (park and shin, 2014) . primate apn or receptor-independent pathways in vero and marc-145 cells may complement the function of papn for pedv infection (taguchi and matsuyama, 2002) . in summary, we have shown the suppression of type i ifn production by pedv and have identified specific viral ifn antagonists. among these antagonists, nsp1 is the most potent protein and functions to degrade cbp in the nucleus. our data provides a novel insight into the understanding of the immune evasion strategy of pedv. hela cells (nih aids research and reference reagent program, germantown, md) and marc-145 cells (kim et al., 1993) reagent was purchased from invitrogen (carlsbad, ca). qiaamp viral rna mini kit and rneasy mini kit were purchased from qiagen (venlo, limburg). power sybr green pcr master mix was purchased from life technologies (carlsbad, ca). alexa fluor 594conjugated (goat anti-rabbit, red) and 488-conjugated (goat antimouse, green) secondary antibodies and pierce™ ecl western blotting substrate were purchased from thermo scientific (waltham, ma). the firefly luciferase genes were used as reporters with its expression under the control of various promoters as indicated below. the plasmid pifn-β-luc contains the entire ifn-β enhancer-promoter. the plasmid p4 â irf3-luc contains four copies of irf3 binding region prd i-iii of the ifn-β promoter. pifn-β-luc and p4 â irf3-luc were obtained from dr. stephan ludwig at heinrich-heine-universität, düsseldorf, germany (ehrhardt et al., 2004) . the renilla luciferase plasmid prl-tk (promega) contains the herpes simplex virus thymidine kinase (hsv-tk) promoter and was included in all experiments to serve as an internal control. active stimulator pmavs/ips-1 was obtained from dr. j. shisler (university of illinois, urbana, il). pirf3 was kindly provided by dr. b. gotoh (university of fukui, fukui, japan). pdsred2-er and pdsred2-mito were purchased from clontech. plasmids with the flag tag for expression of nsp1 through nsp16, and the s, s1, s2, orf3, e, m and n genes were cloned from the viral genomic rna by standard reverse transcription and pcr techniques using indicated primers (table 1) . twenty-three viral genes were amplified and cloned into the eukaryotic expression vector pxj41 using indicated restriction enzymes. the nsp1 to nsp16, orf3, and n genes were expressed as fusion proteins with the n-terminal flag tag, and the s, s1, s2, e, and m genes were expressed as fusion proteins with the c-terminal flag tag to avoid the functional disruption of the signal sequence. the constructs were confirmed by sequencing, immunofluorescence, and western blot. prrsv nsp1α and its cystine mutant p-nsp1α(m) (c28s) are described elsewhere (han et al., 2013; song et al., 2010) . hela cells were seeded in 12-well plates and grown to 80% confluency prior to transfection. individual viral protein genes, luciferase reporters, and prl-tk as an internal control were transfected at a ratio of 10:10:1 in a total of 1.05 μg/well using lipofectamine 2000 according to the manufacturer's instruction (invitrogen). at 24 h post-transfection, cells were stimulated by transfection with 0.5 μg/well of poly(i:c) for 12 h. cells were then lysed and luciferase assays were performed using the dual luciferease assay system according to the manufacturer's instructions (promega). values were normalized using the renilla luciferase activity as the internal control and presented in fold-changes. three independent assays were performed with each assay in triplicate. total rna was extracted from hela or marc-145 cells using rneasy mini kit according to the manufacturer's instructions (qiagen). the rna was treated with dnase i to remove contaminating genomic dna. reverse transcription (rt) reaction was performed with 1 mg of total rna using random primers and m-mlv reverse transcriptase (invitrogen). sybr green real-time pcr was conducted in the abi 7500 real-time pcr system according to the manufacturer's instructions (life technologies). the real-time pcr primers for ifn-β, isg15, isg56 and β-actin were listed in table 2 . for each sample, the β-actin gene was amplified and used as an internal control. specific amplification was confirmed by sequencing pcr products. the threshold cycle for target genes and the difference between their c t values (δc t ) were determined. the relative transcript levels of target gene are equal to 2 à δδct threshold method (livak and schmittgen, 2001) and are shown as fold changes relative to the respective untreated control samples. hela cells were seeded in 6-well plates and transfected with 2 μg of plasmid. at 24 h post-transfection, cells were stimulated by transfection with 1 μg of poly(i:c) for 12 h. supernatants were harvested for bioassay. for pedv, marc-145 cells were infected with pedv at an moi of 1 for 12 h prior to poly(i:c) stimulation. supernatants from virus-infected cells were uv-irradiated for 30 min to remove infectivity prior to bioassay. the supernatants were then serially diluted by 2-fold. marc-145 cells were freshly grown in 96-well plates and incubated with 100 μl of each dilution for 24 h. cells were then infected in 100 μl of vsv-gfp at 10 4 pfu/ ml for 16 h and gfp expression was examined by inverted fluorescence microscopy (nikon eclipse ts100, 10 â 10). each dilution was examined twice in triplicate each. indirect immunofluorescence assay (ifa) and confocal microscopy cells were seeded on coverslips and transfected with plasmids or infected with pedv. for transfection of hela cells, total 2 mg of individual plasmids were transfected for 24 h using lipofectamine table 1 primers used for the cloning of pedv nonstructural and structural genes (pedv strain usa/colorado/2013). restriction enzyme recognition sequences are underlined. the flag tag is italicized and underlined. primers used for relative quantitative real-time rt-pcr. ifn-β-f gatttatctagcactggctgg ifn-β-r cttcaggtaatgcagaatcc isg15-f caccgtgttcatgaatctgc isg15-r ctttatttccggcccttgat isg56-f cctccttgggttcgtctaca isg56-r ggctgatatctgggtgccta β-actin-f atcgtgcgtgacattaag β-actin-r attgccaatggtgatgac 2000 according to the manufacturer's instructions (invitrogen). cells were then either treated with poly(i:c) for 12 h or ifn-β for 40 min. cells were fixed with 4% paraformaldehyde in pbs overnight at 4°c and permeabilized using 0.1% triton x-100 for 15 min at room temperature (rt). after blocking with 1% bsa in pbs at rt for 30 min, cells were incubated with a primary antibody in pbs for 1-3 h. cells were then washed three times with pbs and incubated with alexa fluor 488-labeled anti-mouse secondary antibody, or alexa fluor 594-labeled anti-rabbit secondary antibody (thermo scientific) for 1 h at rt in the dark. cells were incubated with dapi for 5 min at rt for nuclear staining. after washing with pbs, cover slips were mounted on microscope slides using fluoromount-g mounting medium (southern biotech, birmingham, al), and visualized by fluorescence microscopy (nikon eclipse ts100). confocal microscopy was conducted as described elsewhere (kannan et al., 2009) . hela cells were seeded in 6-well plates to 80% confluency and transfected with 2 μg/well of nsp1 plasmid for 24 h. cells were stimulated with 1 μg of poly(i:c) for 12 h and fractionated using the nuclear/cytosol fractionation kit (biovision, milpitas, ca) with minor modifications. briefly, cells were washed with cold pbs and collected using cell scrapers in 1 ml of cold pbs. cell pellets were resuspended in 200 μl ceb-a buffer and incubated on ice for 10 min. after addition of ceb-b, tubes were vortexed and incubated on ice for 1 min. the cell lysates were then centrifuged at 4°c 5 min at 16,000g and supernatants were collected as the cytosolic fraction. the cell pellets were suspended in neb buffer and vortexed for 30 s and repeated 5 times every 10 min. the nuclear pellets were finally centrifuged for 10 min at 4°c 16,000g and kept the supernatants as the nuclear fraction. cells were harvested in ripa buffer [20 mm tris (ph 7.5), 150 mm nacl, 1 mm edta, 1 mm phenylmethanesulphonyl fluoride (pmsf), 0.1% sds, 0.5% sodium deoxycholate, 1% np-40] containing the proteinase inhibitors cocktail (promega). cells were frozen-thawed, collected in the pre-cold tubes, and centrifuged to remove insoluble components. total protein concentration was determined using pierce bca protein assay kit (thermo scientific). equal amounts of proteins were resolved by sds-page and blotted to pvdf membranes (millipore). after blocking with 5% nonfat dry milk in tbst (0.05% tween-20) for 1 h, membranes were incubated with a primary antibody in tbst containing 5% nonfat dry milk overnight at 4°c, followed by washing and incubation with horseradish peroxidase (hrp)-conjugated secondary antibody for 1 h at rt. the membrane was visualized using pierce ecl western blotting substrate (thermo scientific) and images were taken by fluorchem™ r system according to the manufacturer's instructions (proteinsimple). co-immunoprecipitation (co-ip) was performed as described previously with modifications (kim et al., 2010) . gene-transfected cells were lysed in lysis buffer [50 mm tris (ph 8.0), 150 mm nacl, 5 mm na 3 vo 4 , 1 mm pmsf, 100 mg/ml leupetin, 1% np-40, 10% glycerol] supplemented proteinase inhibitors cocktail (promega). cell lysates were clarified by centrifugation at 4°c for 10 min at 16,000g. supernatants were transferred to fresh tubes and incubated with either flag-or irf3-antibody at 4°c overnight, followed by incubation with protein g agarose beads (fast flow, millipore) at 4°c for 4 h. pellets were collected by centrifugation and washed for five times. the final pellets were eluted with laemmli sample buffer (bio-rad) and were subjected to western blot. student's t-test was used for all statistical analyses. asterisks indicate the statistical significance. *po0.05, **p o0.01 and ***p o0.001. interferon-alpha response 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diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase genetic variation analysis of reemerging porcine epidemic diarrhea virus prevailing in central china from human t-cell leukemia virus type 1 tax modulates interferon-alpha signal transduction through competitive usage of the coactivator cbp/p300 antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns2 protein is required for virus replication and liver pathology transmissible gastroenteritis virus and porcine epidemic diarrhoea virus infection induces dramatic changes in the tight junctions and microfilaments of polarized ipec-j2 cells mouse hepatitis virus does not induce beta interferon synthesis and does not inhibit its induction by double-stranded rna active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus key: cord-266018-8bhnlsgy authors: trifilo, matthew j.; lane, thomas e. title: the cc chemokine ligand 3 regulates cd11c(+)cd11b(+)cd8α(−) dendritic cell maturation and activation following viral infection of the central nervous system: implications for a role in t cell activation date: 2004-09-15 journal: virology doi: 10.1016/j.virol.2004.06.027 sha: doc_id: 266018 cord_uid: 8bhnlsgy the role of cc chemokine ligand 3 (ccl3) in activation of dendritic cells (dcs) following mouse hepatitis virus (mhv) infection of the central nervous system (cns) was examined. the results indicate that ccl3 participates in an effective host response to mhv infection by contributing to cd11c(+)cd11b(+)cd8α(−) dc maturation, activation, and migration to cervical lymph nodes (cln). diminished cd8α(−) dc activation correlated with reduced ifn-γ expression by virus-specific t cells accompanied by increased il-10 production suggesting that ccl3 contributes to an effective host response to viral infection by enhancing the t cell activation potential of dc. the cc chemokine ligand 3 (ccl3-macrophage inflammatory protein-1a) is capable of activating monocytes and lymphocytes and serves an important role in the initial recruitment of these cells to tissues following microbial infection (cook et al., 1995; domachowske et al., 2000) . in support of the importance for ccl3 in imparting functional signals to t cells are data from our laboratory demonstrating that instillation of mouse hepatitis virus (mhv) into the brains of ccl3 à/à mice results in an inability to clear virus from the central nervous system (cns) (trifilo et al., 2003) . mhv-infected ccl3 à/à mice exhibited a significant reduction in the numbers of infiltrating virus-specific cd8 + t cells present within the brain indicating that trafficking was impaired. moreover, the ability to produce ifn-g as well as the cytolytic activity of virus-specific cd8 + t cells was dramatically reduced in the absence of ccl3 signaling. taken together, these data indicate that ccl3 signaling significantly enhances the differentiation of primed cd8 + t cells into effector cells that allows their release from secondary lymphoid organs into circulation and effective migration to the cns. the present study was undertaken to characterize potential mechanisms by which ccl3 signaling imparts effector function to antigen-specific t cells following mhv infection of the cns. to further understand the relationship between ccl3 signaling and t cell activation, ccl3 +/+ and ccl3 à/à mice were infected with mhv and the presence and activation state of dc-like cells within the brain and draining cervical lymph nodes (cln) determined. our results delineate a ccl3-dependent pathway of t cell activation that involves the maturation and activation of a subpopulation of (dendritic cells) dcs (cd11c + cd11b + cd8a à ) within the cns as well as influencing the accumulation of these cells within the cln following mhv infection of the cns. characterization of cd11c + cells within the cns following mhv infection of ccl3 +/+ and ccl3 à/à mice to characterize the populations of cells present within the cns of mhv-infected mice, cells were harvested at days 0, 2, and 5 post-infection (p.i.) and immunophenotyped by flow cytometry. we chose to focus our attention on markers that are associated with professional antigen presenting cells such as dc as recent studies have indicated dc-like cells can be detected within the brains under inflammatory conditions (fisher and reichmann, 2001; fischer et al., 2000) . furthermore, we have previously determined that ccl3 mrna expression is detected within the cns early (b3 days) and therefore may participate in the appearance of cd11c + cells within the brain following mhv infection (trifilo et al., 2003) . therefore, we sought to characterize the populations of cd11c + cells within the brain following mhv infection of ccl3 +/+ and ccl3 à/à mice. results in fig. 1a indicate the frequency of cd11c + cells present within the cns of narve ccl3 +/+ and ccl3 à/à mice is b1%. however, within 2 days following intracranial infection with mhv, there is a marked increase in the frequency of cd11c + cells within the brains of both strains of mice (fig. 1a) . analysis of cd11b expression revealed that approximately 75% of cd11c + cells in both ccl3 +/+ and ccl3 à/à mice were also cd11b + (fig. 1a) . cd11c + cd11b + cells isolated from the cns of either ccl3 +/+ or ccl3 à/à mice expressed little to no cd8a or dec205 suggesting a phenotype similar to myeloid derived dc (cd11c + , cd11b + , cd8a à , dec205 à ) ( fig. 1b ) (anjuere et al., 1999; henri et al., 2001) . increased cd8a and dec205 expression on cd11c + cd11b à cells indicated that the majority of the remaining cd11c + cells present within the brain were similar phenotypically to lymphoid derived dc (cd11c + , cd11b à , cd8a + ) (fig. 1b) (anjuere et al., 1999; henri et al., 2001) . although the fig. 1 . analysis of dendritic cells (dcs) within the brain following mhv infection. (a) cells were isolated from the brains of uninfected (narve) or infected (day 2 p.i.) ccl3 +/+ and ccl3 à/à mice and stained for cd11c + and cd11b + expression. gated populations represent cd11c + cd11b + (upper-right quadrant) or cd11c + cd11b à (lower-right quadrant) and numbers indicate frequencies of gated cells within the isolated population. (b) cd8a and dec205 expression on cd11c + cells. cd11c + cd11b + cells from either mhv-infected ccl3 +/+ or ccl3 à/à mice at day 2 p.i. did not express detectable levels of either cd8a or dec205 while expression of both cd8a and dec205 was readily detectable on cd11c + cd11b à cells present within the brains of both populations of mice at day 2 p.i. (c) total numbers of cd8a + and cd8a à dcs within the brains of mhv or sham-infected ccl3 +/+ and ccl3 à/à mice at days 2 and 5 p.i. data presented represent an average cell number derived from two separate experiments with a minimum of 10 mice analyzed per experimental group. (d) cd8a à cells isolated from brains of ccl3 +/+ or ccl3 à/à mice at day 2 p.i. were gated upon and cd80, cd86, and cd40 expression was determined by flow cytometry. the mean fluorescence intensity (mfi) for cells obtained from either ccl3 +/+ or ccl3 à/à mice is indicated. flow data shown in panels a, b, and d are representative of two separate experiments with a total of 10 mice for each experimental condition. remaining cd11c + cd11b à cd8a à population was not further characterized, it is likely that these cells may be plasmacytoid in origin (cd11c + cd11b à cd8a à b220 + ) or consist of a yet to be defined population of dc. comparison of the total numbers of cd8a + cells within the brains of mhv-infected ccl3 +/+ and ccl3 à/à mice revealed no dramatic differences between the two populations of mice at either 2 or 5 days p.i. (fig. 1c) . in contrast, numbers of cd8a à cells were increased by approximately 30% within the brains of mhv-infected ccl3 +/+ mice as compared to ccl3 à/à mice at day 2 p.i. however, by day 5 p.i., there were increased numbers of cd8a à cells present in the brains of ccl3 à/à mice when compared to ccl3 +/+ mice. in attempt to better evaluate the activation state of cd11c + cells within the brains of mhv-infected mice, we next determined the expression levels of co-stimulatory molecules cd80 (b-7.1), cd86 (b-7.2), and cd40 on cd8a à cells within the cns of ccl3 +/+ and ccl3 à/à mice. we chose to focus on this subpopulation of dcs in more detail as this clearly was the predominant dc population within the brains of infected mice suggesting a potentially more important role in defense. analysis of cd8a à cells isolated from the brains of ccl3 +/+ mice at day 2 p.i. revealed these cells expressed detectable levels cd80, cd86, and cd40 as determined by measuring the mean fluorescence intensity (mfi) (fig. 1d ). although mhv infection of ccl3 à/à mice also resulted in enhanced expression of cd86 on the surface of cd8a à cells, the mfi for both cd80 and cd40 was dramatically reduced as compared to cd8a à cells within the brain of ccl3 +/+ mice at day 2 p.i. (fig. 1d ). together, these results indicate that although ccl3 signaling is not required for the appearance of dc-like cells within the brain, expression of the co-stimulatory molecules cd40 and cd80 is muted in the absence of ccl3 signaling. characterization of cd11c + cells within the cln of mhv-infected ccl3 +/+ and ccl3 à/à mice following mhv infection of the cns, virus-specific t cells are present within the cln suggesting that the bulk of virus-specific t cells are generated in the periphery (marten et al., 2003) . therefore, the accumulation of cd11c + cells within the cln of infected ccl3 +/+ and ccl3 à/à mice was determined. before infection, cd11c + cells expressing a myeloid dc phenotype (cd11c + cd11b + ) and lymphoid dc phenotype (cd11c + cd11b à ) were present within the cln of both ccl3 +/+ and ccl3 à/à mice at an approximate 1:2 ratio, respectively ( fig. 2a) . however, within 2 days of mhv infection of the cns, the frequency of cd11c +-cd11b + dcs, but not cd11c + cd11b à dcs, dramatically increased within the cln of ccl3 +/+ mice ( fig. 2a) . further analysis revealed that the cd11c + cd11b + cell population was cd8a à dec205 à while the cd11c +-cd11b à population was cd8a + dec205 + (fig. 2b) . although the total number of cd8a à cells increased within the cln of ccl3 à/à mice, there was an approximate 4-fold reduction in total numbers of cd8a à cells as compared to ccl3 +/+ mice at days 2 and 5 p.i. (fig. 2c ). similar to the brain, no difference in numbers of cd8a + cells within the cln was detected at either days 2 or 5 p.i. (fig. 2c ). examination of co-stimulatory molecule expression on cd8a à dcs present within the cln of ccl3 +/+ mice correlated with the increased expression of co-stimulatory molecules cd40, cd80, cd86 as well as increased mhc i and ii expression when compared to sham-infected mice (figs. 2d and e) . these data suggest that this population of activated cd8a à cells within the cln is able to present antigen and induce t cell differentiation within the cln following mhv infection of the cns. although cd8a à dcs isolated from the cln of ccl3 à/à mice expressed similar levels of cd80 and cd86 as compared to ccl3 +/+ (determined by mfi), expression of cd40 as well as mhc i and ii were reduced as compared to cd8a à cells isolated from mhv-infected ccl3 +/+ mice (figs. 2d and e). one mechanism by which dcs influence the t cell response to infection is through the secretion of cytokines that can subsequently polarize the immune response towards either a th1 or th2 phenotype depending on the antigenic challenge. to determine if cd8a à dcs present within the cln of infected ccl3 +/+ and ccl3 à/à mice were capable of secreting either chemokines or cytokines following mhv infection of the cns, these cells were isolated and production determined by elisa. ccl3 was readily detectable from cd8a à dcs obtained from ccl3 +/+ mice while ccl3 was not detected in supernatants collected from ccl3 à/à cd8a à dcs (fig. 3) . the ccl3 +/+ cd8a à dc population secreted il-12p70 with low-level production of il-10 ( fig. 3) . in contrast, the cd11c + cd11b + cd8a à cells isolated from ccl3 à/à mice secreted approximately 3-fold less il-12p70 while il-10 secretion was increased by 5-fold as compared to cells from ccl3 +/+ mice (fig. 3) . altered cytokine production in ccl3 à/à t cells we next evaluated the ability of t cells obtained from the cln of either mhv-infected ccl3 +/+ or ccl3 à/à mice to synthesize cytokines following exposure to defined viral antigens. t cells were isolated from the cln of ccl3 +/+ and ccl3 à/à mice at days 7 and 12 p.i. following intracranial infection with mhv and stimulated with peptides corresponding to either the immunodominant cd4 epitope present within the matrix (m) glycoprotein at residues 133-147 (m133-147) or the immunodominant cd8 epitope in the surface (s) glycoprotein spanning residues 510-518 (s510-518) and cytokine production by t cells determined by intracellular cytokine staining (castro and perlman, 1995; xue et al., 1995) . the results presented in table 1 indicate similar frequencies of cd4 + and cd8 + t cells from ccl3 +/+ mice produced ifn-g at days 7 and 12 p.i. both cd4 + and cd8 + ccl3 +/+ t cells also secreted il-2 following specific peptide exposure at day 7, although expression was limited to the acute stage of mhv infection as the frequency of il-2producing cells was reduced at day 12 p.i. (table 1 ). in contrast, the frequency of cd4 + and cd8 + t cells isolated from ccl3 à/à mice secreting ifn-g following peptide stimulation was dramatically reduced. expression of il-2 by ccl3 à/à t cells was comparable with ccl3 +/+ mice at day 7 p.i. however, by day 12 p.i., the frequency of ccl3 à/à t cells expressing il-2 remained elevated as compared to ccl3 +/+ t cells (table 1 ). in addition, only limited frequencies of ccl3 +/+ cd4 + and cd8 + t cells produced il-10 following mhv infection whereas ccl3 à/à cd4 + and cd8 + t cells displayed an overall increase in the frequency of il-10 ( table 1) . the major findings of this study are (i) mhv infection of the cns results in the appearance of two distinct populations of cd11c + cells each expressing markers characteristic of lymphoid (cd11c + cd11b à cd8a + dec205 + ) and myeloid dendritic cells (cd11c + cd11b + cd8a à dec205 à ), (ii) the accumulation of cd8a à dcs within the draining cln is reduced in the absence of ccl3 signaling, (iii) expression of co-stimulatory molecules such as cd40 by cd8a à dcs within either the brain and cln of mhv-infected ccl3 à/à mice is diminished suggesting that ccl3 signaling enhances expression of these molecules, and (iv) absence of ccl3 signaling results in the re-direction of the t cell response to viral antigens as determined by cytokine production. these data support and extend recent studies from our laboratory demonstrating an important role for ccl3 in generating effector anti-viral t cells capable of migrating to the brain in response to viral infection (trifilo et al., 2003) . i.) ccl3 +/+ and ccl3 à/à mice and stained for cd11c + and cd11b + expression. gated populations represent cd11c + cd11b + (upper-right quadrant) or cd11c + cd11b à (lower-right quadrant) and numbers indicate frequencies of gated cells within the isolated population. (b) cd8a and dec205 expression on cd11c + cells. cd11c + cd11b + cells from either ccl3 +/+ or ccl3 à/à at day 2 p.i. did not express detectable levels of either cd8a or dec205 while expression of both cd8a and dec205 was readily detectable on cd11c + cd11b à cells present within the brains of both ccl3 +/+ and ccl3 à/à mice at day 2 p.i. (c) total numbers of cd8a + and cd8a à dcs within the clns of mhv or sham-infected ccl3 +/+ and ccl3 à/à mice at days 2 and 5 p.i. data presented represent an average cell number derived from two separate experiments with a minimum of 10 mice analyzed per experimental group. (d) cd8a à cells obtained from mhv-infected (day 2 p.i.) or sham mice were gated and the level of cd80, cd86, and cd40 expression was determined by flow cytometry. the mfi for cells obtained from either ccl3 +/+ or ccl3 à/à mice is indicated. (e) cd8a à cells obtained from mhv-infected or sham mice were evaluated for expression of mhc i and ii. the mfi for staining of either mhc i or ii is indicated in the histogram. flow data shown in panels a, b, d, and e are representative of two separate experiments with a total of 10 mice for each experimental condition. the activation of dc and their mobilization to secondary lymphoid organs is thought to be a key step in the initiation of an adaptive immune response (banchereau and steinman, 1998). recent studies have indicated that following infection of the cns with toxoplasma gondii, cd11c + cells are present within the brain and these cells were able to stimulate the proliferation of narve t cells (fisher and reichmann, 2001) . similarly, our results also indicate an increase in cd11c + cells within the cns following viral infection, suggesting that these cells are likely critical for successful t cell priming following migration to draining lymph nodes. whether these cells are present within the cns by differentiation of local antigen presenting cells, or through migration of immature dc has not been determined and is currently under investigation. regardless, our data imply that ccl3 expression and signaling contributes to the migration of cd8a à cd11c + cells to secondary lymphoid tissue where they participate in priming of t cells. in support of this, we have shown that ccl3 is important in arming these cells with the capability to optimally stimulate antigen-specific t cells with the ability to fully differentiate into effector cells (trifilo et al., 2003) . the data presented in this study support and extend these observations and indicate that these results may be the result of a combination of diminished expression of both mhc class i and ii, reduced expression of cd40, as well as a shift in cytokine production by cd8a à cells. indeed, cd40l:cd40 mediated interactions between t cells and apc can enhance il-12 production by dc and blockade of this interaction has been shown to result in reduced autoimmunity by down-regulating th1 differentiation (cella et al., 1996; macatonia et al., 1995) . accumulating evidence indicates that in addition to driving virus-specific t cell proliferation, the activation state of dcs can also directly influence the effector function of t cells through the secretion of proinflammatory cytokines (fischer et al., 2000; maldonado-lopez et al., 1999 pulendran et al., 1999) . for example, following several viral and bacterial infections, cd8a + dcs have been shown to be able to secrete large amounts of the proinflammatory cytokine il-12 both in vitro and in vivo and this results in a preferential expression of th1-associated cytokines, such as ifn-g by responding t cells (aliberti et al., 2000; maldonado-lopez et al., 1999 pulendran et al., 1999; reis e sousa et al., 1997) . until recently, the prevailing thought was that cd8a + dcs were primarily responsible for production of il-12 and contributing to a th1 response. however, recent studies have indicated that cd8a à dcs also have the potential for secreting il-12 and influencing the t cell response (doxsee et al., 2003) . our studies clearly indicate that cd8a à dcs isolated from the draining cln of mhv-infected ccl3 +/+ mice secrete il-12 suggesting that these cells help influence a protective th1-mediated immune response characterized by the majority of antigen-specific t cells expressing ifn-g rather than il-10 (table 1 ). in stark contrast is the data indicating that cd8a à dcs present in the cln of infected ccl3 à/à mice predominantly secrete il-10 and this correlates with limited ifn-g expression and enhanced expression of the th2-associated cytokine il-10 (table 1) . therefore, the data indicate that cytokine production, rather than the type of cd11c + cell, may control the predominant t cell immune response within the cln. taken together, these data point to an important role in ccl3 expression in linking innate and adaptive immune responses following viral infection of the cns by contributing to the activation fig. 3 . cytokine and chemokine secretion by cd11c + cd11b + cd8a à cells isolated from mononuclear cells pooled from the cln of mhv-infected ccl3 +/+ and ccl3 à/à mice at day 7 p.i. supernatants were analyzed for the production of il-12p70, il-10, and ccl3 by elisa 24 h following culture. a minimum of three to six mice per group were used for isolation of cells and data presented indicate the average f sd. *p v 0.001. table 1 frequency of cytokine-producing t cells present within draining cervical lymph nodes following mhv infection a day p.i. ifn-g il-2 il-10 cd4 + t cells ccl3 +/+ 7 2 0f 4 35f 5 5f 2 12 data represent two separate experiments with at least three mice per group, n = 6. data are presented as average f sd. a cytokine expression determined by pooling cells from draining cln of mhv-infected mice at defined times p.i. and pulsing with defined cd4 + epitope (m133-147) and cd8 + epitope (s510-518). b p v 0.01. decreased frequency of ifn-g secreting t cells and increased frequency of il-2 producing t cells following mhv infection of ccl3 à/à mice as compared to ccl3 +/+ mice. c p v 0.002. increased frequency of il-10 producing t cells in ccl3 à/à mice as compared to ccl3 +/+ mice following mhv infection. of dcs through regulating the migration of cells from the cns to lymphoid tissues as well as the expression of both co-stimulatory molecules and cytokine production. however, it is important to note that there is the possibility of other chemokines and chemokine receptors participating in dc responses following viral infection. indeed, the cc chemokine receptor 2 (ccr2) is expressed on professional apc including macrophages and dcs and is thought to contribute to defense following microbial challenge by enhancing recruitment as well as production of antimicrobial products such as tnf-a and no by these cells (luster, 2002; mccoll, 2002; serbina et al., 2003) . in addition, the absence of ccr2 signaling results in diminished trafficking and accumulation of dendritic cells within secondary lymphoid tissues following antigenic challenge (peters et al., 2000 (peters et al., , 2001 sato et al., 2000) . studies are currently in progress to evaluate the contributions of additional chemokine signaling pathways that may also participate in dc activation and migration following coronavirus infection. in conclusion, the studies presented support and extend previous work from our laboratory indicating an important role for chemokines in the migration of t cells into the cns following mhv infection glass et al., 2001; liu et al., 2001) . here, we have demonstrated a novel role for the chemokine ccl3 in enhancing the accumulation and activation of cd8a à dcs within secondary lymphoid tissue and this correlates with altered t cell activation and differentiation following viral infection. these results indicate that cd8a à dcs likely function in an apc-like role within the cln following mhv infection and that these cells rely upon chemokine instruction to activate t cells. at a more fundamental level, the results presented demonstrate that chemokines serve as critical upstream signals in the innate immune response that later is important with regards to the initiation of a protective adaptive immune responses to viral infection. mhv j2.2v-1 was kindly provided by j. fleming (u. wisconsin). ccl3 à/à and ccl3 +/+ mice (c57bl/6, h-2 b ) were purchased from jackson laboratories (bar harbor, me). mice were anesthetized by inhalation of methoxyflurane (pitman-moor inc., washington crossing, nj) and injected intracranially (i.c.) with 1000 pfu mhvj2.2v-1 suspended in 30 al sterile pbs. control (sham) mice were injected with 30 al sterile pbs alone. mononuclear cells were obtained from the brains and cervical lymph nodes [two draining cervical lymph nodes (cln) per mouse] of either ccl3 +/+ or ccl3 à/à mice at defined times post-infection (p.i.) using a previously described protocol (trifilo et al., 2003) . cell surface expression of phenotypic markers was examined using the following reagents for flow cytometric analysis: apcconjugated rat anti-mouse cd8; percp-conjugated rat anti mouse cd4 (pharmingen, san diego, ca). pe conjugated d b /s510-518 mhc class i tetramer (beckman coulter, san diego, ca) was utilized for identification of cd8 + t cells specific for viral spike protein antigen (trifilo et al., 2003) . to determine the presence of dendritic-like cells within the cns and lymph nodes, cells were stained using fitcconjugated rat anti-mouse cd11c (serotec, oxford, england) in combination with percp-conjugated rat anti-mouse cd8a (pharmingen), rat anti-mouse apc-conjugated cd11b (pharmingen), and rat anti-mouse dec205 (pharmingen). the maturation and activation state of dc were determined using fitc-conjugated rat anti-mouse cd40, cd80, cd86, mhc i, and mhc ii (pharmingen). isotypematched antibodies were used as controls for all staining conditions described. isolation of cd11c + cells from the cln ccl3 +/+ and ccl3 à/à mice were infected intracranially with 1000 pfu of mhv and brains and cln were removed at defined times post-infection for analysis. brain samples were minced and homogenized into a single cell suspension followed by fractionation on a 70/30% percoll gradient at 1300 â g for 30 min. for isolation of mononuclear cells from the cln, lymph nodes were homogenized using frosted glass slides and the resulting single cell suspension was treated with sterile h 2 o to lyse red blood cells. due to the low frequency and numbers of cd11c + cells within the brain and cln, samples from three to six mice were pooled for each experiment. to enrich for cd11c + cd11b + cd8a à dcs, both brain and cln samples were separately magnetically sorted by negative selection against cd8a using macs microbeads coated with anti-cd8a (miltenyi biotec, auburn, ca) according to the manufacturer's instructions. cd11c + cells were then magnetically selected from the cd8a à fraction using macs microbeads coated with anti-cd11c (miltenyi). the resulting population was n90% pure for cd11c + cells that were subsequently determined to be cd11b + and cd8a à by flow cytometry (data not shown). macs enriched cells were then resuspended in dmem supplemented with 10% fbs. cytokine and chemokine expression from cd11c + cd8a à cells freshly prepared cd11c + cd8a à cells were isolated from the cln from ccl3 +/+ and ccl3 à/à mice at day 7 p.i. and seeded into 96-well plate at a cell density of 2 â 10 5 cells/200 al in dmem containing 10% fetal bovine serum (fbs, biowhittaker, walkersville, md). following isola-tion, cell viability was n95%. after 24 h, supernatants from the samples were collected, and the level of il-12, il-10, and ccl3 were determined using quantikine m mouse immunoassays kits (r&d systems, minneapolis, mn) according to manufacturer's specifications. assays had a minimum sensitivity of 5 pg/ml (il-10 and il-12p70) and 10 pg/ml (ccl3). mononuclear cells were obtained from the clns of mhv-infected mice at defined times post-infection and cytokine production by t cells determined by intracellular cytokine staining to defined viral antigens using a previously described protocol (trifilo et al., 2003) . statistically significant differences between experimental groups was determined by the mann-whitney rank sum test, and p values of v0.05 were considered significant. ccr5 provides a signal for microbial induced production of il-12 by cd8a+ dendritic cells definition of dendritic cell subpopulations present in the spleen, peyer's patches, lymph nodes, and skin of the mouse dendritic cells and the control of immunity cd8+ t-cell epitopes within the surface glycoprotein of a neurotropic coronavirus and correlation with pathogenicity ligation of cd40 on dendritic cells triggers production of high levels of interleukin-12 and enhances t cell stimulatory capacity: t-t help via apc activation lack of ccr2 results in increased mortality and impaired leukocyte activation and trafficking following infection of the central system with a neurotropic coronavirus requirement of mip-1 alpha for an inflammatory response to viral infection the chemokine macrophage-inflammatory protein-1 alpha and its receptor ccr1 control pulmonary inflammation and antiviral host defense in paramyxovirus infection the immune response modifier and toll-like receptor 7 agonist s-27609 selectively induces il-12 and tnf-alpha production in cd11c+cd11b+cd8-dendritic cells brain dendritic cells and macrophages/ microglia in central nervous system inflammation phenotype and functions of brain dendritic cells emerging during chronic infection of mice with toxoplasma gondii reduced macrophage infiltration and demyelination in mice lacking the chemokine receptor ccr5 following infection with a neurotropic coronavirus the dendritic cell populations of mouse lymph nodes the t cell chemoattractant ifninducible protein 10 is essential in host defense against viral-induced neurologic disease the role of chemokines in linking innate and adaptive immunity dendritic cells produce il-12 and direct the development of th1 cells from narve cd4+ t cells cd8alpha(+) and cd8alpha(à) subclasses of dendritic cells direct the development of distinct t helper cells in vivo cytokines regulate the capacity of cd8alpha(+) and cd8alpha(à) dendritic cells to prime th1/th2 cells in vivo kinetics of virus-specific cd8+-t-cell expansion and trafficking following central nervous system infection chemokines and dendritic cells: a crucial alliance a mechanism for the impaired ifn-gamma production in c-c chemokine receptor 2 (ccr2) knockout mice: role of ccr2 in linking the innate and adaptive immune responses chemokine receptor 2 serves an early and essential role in resistance to mycobacterium tuberculosis distinct dendritic cell subsets differentially regulate the class of immune response in vivo in vivo microbial stimulation induces rapid cd40 ligand-independent production of interleukin 12 by dendritic cells and their redistribution to t cell areas cc chemokine receptor (ccr)2 is required for langerhans cell migration and localization of t helper cell type 1 (th1)-inducing dendritic cells. absence of ccr2 shifts the leishmania major-resistant phenotype to a susceptible state dominated by th2 cytokines, b cell outgrowth, and sustained neutrophilic inflammation tnf/inos-producing dendritic cells mediate innate immune defense against bacterial infection the cc chemokine ligand 3 (ccl3) regulates cd8+ t cell effector function and migration following viral infection identification of a cd4+ t cell epitope within the m protein of a neurotropic coronavirus this work was supported by national institutes of health grant 41249 and national multiple sclerosis society grant 3278-a-3 to t.e.l. key: cord-274424-juj71nc5 authors: pulford, david j.; britton, paul title: intracellular processing of the porcine coronavirus transmissible gastroenteritis virus spike protein expressed by recombinant vaccinia virus date: 1991-06-30 journal: virology doi: 10.1016/0042-6822(91)90617-k sha: doc_id: 274424 cord_uid: juj71nc5 abstract the spike (s) protein from a virulent british field isolate of porcine transmissible gastroenteritis virus (tgev) fs772/70 was constructed from cdna and inserted into the vaccinia virus (vv) thymidine kinase gene locus under the control of the vv early/late gene p7.5k promoter. recombinant s protein was synthesized as an endo-β-n-acetylglucosamini-dase h (endo h)-sensitive glycoprotein with high mannose simple oligosaccharides (gp190) that underwent post-translational modification to an endo h-resistant glycoprotein with complex oligosaccharides (gp210). immunofluorescence analysis demonstrated that the majority of recombinant s protein was retained at the golgi but some s protein was expressed on the plasma membrane. monoclonal antibodies (mabs) raised against native s protein reacted with this recombinant s protein; also, mice infected with the recombinant vaccinia virus (rvv) expressing the s protein induced tgev neutralizing antibodies. a truncated s protein (sδ) was also expressed in rvv-infected cells by introducing a deletion into the s protein cdna that removed 292 amino acids from the c-terminus. the sδ protein (gpl70) was shown to be antigenically similar to tgev s protein by immunofluorescence and immunoprecipitation tests but was retained in the endoplasmic reticulum and not expressed on the cell surface. transmissible gastroenteritis virus (tgev) belongs to the family coronaviridae, a large group of enveloped viruses with a positive-stranded rna genome. the virus causes gastroenteritis in neonatal pigs, resulting in a high mortality and morbidity. tgevvirions are composed of three structural proteins; a basic phosphorylated nucleoprotein (n) m, 47,000 was shown to associate with the viral genomic rna to form the nucleocapsid and interact with a glycosylated membrane protein (m) observed as a series of polypeptides n/r, 28-31,000, and the peplomer or spike (s), a surface h/l, 200,000 glycoprotein (garwes and pocock, 1975) . the tgev s protein has been shown to elicit a neutralizing antibody response (laude et a/., 1986; jimenez et al., 1986; gatwes et a/., 1987) capable of conferring some protection to suckling pigs (garwes eta/., 1978/79) . by analogy to the s protein of mouse hepatitis virus (mhv), the tgev s protein may possess the cell receptor binding components (collins et a/., 1982) and virulence determinants of the virus (fleming et al., 1986) . the s protein of tgev and coronaviruses antigenically related to tgev such as feline infectious peritonitis ' present address: department of pathology and microbiology, school of medical sciences, university of bristol, university walk, bristol, 858 ltd, uk. *to whom all correspondence and reprint requests should be addressed. virus (fipv), canine coronavirus, and porcine respiratory coronavirus, are not cleaved (for a review see spaan et al., 1988) . however, the s proteins from mhv, infectious bronchitis virus, bovine coronavirus, human coronavirus (oc43 and 229e), and porcine hemagglutinating encephalomyelitis virus which are not antigenically related to tgev are proteolytically cleaved into two subunits (spaan et a/., 1988) . some coronavirus s proteins have been demonstrated to induce cell fusion (collins et a/., 1982; sturman et al., 1985; de groot eta/., 1989) , generating multinucleated syncytia. the complete amino acid sequences for the s proteins of the virulent fs772/70 strain ) and the avirulent purdue strain (jacobs et al., 1987; rasschaert and laude, 1987) have been published and compared with other coronaviruses. the fs772/70 strain s protein has a 1449-amino acid precursor polypeptide with 33 potential n-linked glycosylation sites and 97% sequence homology at the nucleotide and the amino acid level with the purdue strain. from the deduced amino acid sequence the coronavirus s protein was shown to contain characteristic features: a 16-amino acid cleavable secretory signal; a heptad repeat sequence that forms a-helical structures which may interact with other subunits to form a coiled-coil oligomeric structure ; a hydrophobic sequence near the c-terminus that is probably responsible for anchoring the s protein to the virion envelope. this membrane anchor region is imme-diately followed by a cysteine-rich domain, a feature common to all other coronaviruses, that may stabilize protein-lipid interactions. in this paper we report the construction of a fs772/ 70 cdna s gene and its expression by a recombinant vaccina virus (rvv) to study the antigenicity and cellular localization of the s protein. the role of the membrane anchor domain was investigated by the introduction of a c-terminal gene deletion downstream of the heptad repeat sequence. tgev, strain fs772/70, was cultivated in a porcine continuous cell line (llc-pkl) maintained with medium containing 10 pg ml-' trypsin (hofmann and wyler, 1988) . cv-1 and human 143 thymidine kinase negative (htk-) cells were grown in eagles mem medium (flow labs) containing 10% heat-inactivated fetal calf serum. transfections were performed on subconfluent monolayers of cv-1 cells previously infected with wild-type vaccinia virus (vv) (wr strain) using plasmid dna calcium phosphate precipitates (mackett et a/., 1985) . recombinant vaccinia viruses were cultivated in htk-cells in the presence of 25 pg ml-' 5-bromodeoxyuridine (budr; sigma) as described by mackett et al. (1985) . of the tgev s and sa genes from cdna cloning procedures were as described by maniatis et a/. (1982) . enzymes were used according to manufacturers' instructions (new england biolabs, bethesda research labs). plasmids ptg47, ptg25, and ptg30 were used to reconstruct a dna copy of the tgev s gene; the procedure is outlined in fig. 1 . essentially barnhi linkers (pcggatccg, no. 1021 biolabs) were added to the hoal site known to be 11 bp upstream of the s gene initiation codon ) and the 1.29kb hindill fragment was cloned into puc9 and excised as a 0.85-kb xbal-barnhi fragment. the barnhi-sty1 fragment derived from ptg47, the styl-kpnl fragment derived from ptg25, the kpnl-xbal fragment derived from ptg30, the 0.85-kb xbal-barnhi fragment, and barnhi dephosphorylated pbr322 were mixed, ligated in a five-way reaction mixture, and transformed into dhl escherichia co/i cells. as a result of the different cohesive ends an insert of 4.6 kb would correspond only to the complete s gene. an aprtcs transformant was found to contain a plasmid, ppbp1, with a 4.6-kbp insert in pbr322, corresponding to the tgev s barnhi gene cassette. the reconstructed gene was verified by sequencing the junction regions. the tgev cdna s gene was subcloned from ppbp1 and inserted into the barnhi cloning site of the vv plasmid insertion vector pgs20 (mackett et al., 1984) downstream of the w early/late p 7,5k promoter. the recombinant plasmid, pgsp-1, containing a correctly orientated s gene, was identified by restriction endonuclease digestion with sall. the s protein with a c-terminal deletion was generated by cloning a 3.7-kbp sali fragment from pgsp-1 into the sali site of pucl2. restriction endonuclease digestion of recombinant plasmids produced either a 0.25-or 3.45-kbp barnhi fragment and the latter was recloned back into barnhi-digested pgs20. the recombinant plasmid insertion vector pgspa-38 was found to contain a correctly orientated sa gene by sryl restriction endonuclease digestion of plasmid dna. a synthetic oligonucleotide primer 5'-gtgtgcggctac-tataacta-3', that binds to the complementary dna strand 43 bp downstream of the barnhi cloning site in pgs20, verified the position of the sa protein stop codon in pgspa-38 by sequencing ( fig. 2a) . the rvvs, vts-1 and vtsa-1, were generated with pgsp-1 and pgspa-38 using methods described by mackett et al. (1984) . thymidine kinase negative viruses were screened by dna dot blot and dna from vts-1 and vtsa-1 infected cells was analyzed by southern blot using a [32p]-labeled s gene cdna probe as described in . groups of three female balb/c mice were immunized by intraperitoneal (ip) inoculation with l-2 x 1 o5 pfu/ mouse of partially purified recombinant or wildtype vv as described by . after 4 weeks, one animal from each group was sacrificed to obtain convalescent serum, the remaining animals were hyperimmunized with homologous virus by ip inoculation and bled after a further 4 weeks. mice initially inoculated with either phosphate-buffered saline (pbs) or wr strain vv were inoculated after 4 weeks with a dose of vts-1 to establish if age or a previous w infection affected the stimulation of tgev neutralizing antibody. tgev neutralizing antibody was assessed by plaque reduction assay as described by garwes et a/. (1987) . viral proteins were routinely radiolabeled by incubating tgev-infected llc-pkl cells or rvv-infected htk-cells, at 8 hr p.i. or 17 hr p.i., respectively, in methionine-free medium for 1 hr and then in the presence of 50-l 00 &i ml-' l-[35s]methionine (amersham international; see figure legends for further details). after pulse labeling, cells were washed in pbs (10 mm potassium phosphate, 150 m/l/l naci, ph 7.2) and chased in medium supplemented with 2 mm l-methionine. cells were washed in pbs before treating with test lysis buffer (20 mm tris-hci, ph 7.6, 2 mm edta, 100 mm naci, 1% triton x-l 00, 0.1% aprotinin). nuclei and ceil debris were pelleted from infected cell lysates by centrifugation at 100,000 g using a beckman tla-100 ultracentrifuge. tissue culture medium was clarified by low speed centrifugation. immunoprecipitations were performed by mixing 1 vol of cell lysate with l/l 00th vol of porcine tgev hyperimmune antiserum at 4' for 1 hr and the resulting immune complexes were incubated overnight with formalinfixed staphy/ococcus aureus cells (sac; brl) previously washed three times in test buffer. immune complexes were washed three times with test buffer and resuspended in laemmli sample buffer, and proteins separated on 6% sds-polyacrylamide gels (laemmli et a/., 1970) . 14c-methylated proteins (amersham, code cfa.626) were routinely used as molecular weight markers. proteins were detected by fluorography after immersing gels in 0.8 m sodium salicylate for 30 min. pelleted immune complexes were washed as described above, resuspended in 40 ~i50 mll/l tris-hci, ph 6.8, containing 0.25% sds, and boiled for 2 min. the solubilized proteins were incubated in the presence or absence of 1 mu endo h (boehringer-mannheim) in 90 mmsodium citrate, ph 5.5, at 37" for 18 hr. the proteins were analyzed on 6% polyacrylamide gels and detected by fluorography. glass coverslips with subconfluent monolayer cultures of htk-or llc-pkl cells were infected with tgev and wild-type or rvvs. at 20 hr p.i. cells were either fixed with cold 80% acetone and air dried or washed in cold pbs and maintained at 4" for surface staining. cells were incubated for 1 hr with porcine tgev hyperimmune antiserum diluted 1 :loo in pbs and then extensively washed with pbs. cells were then incubated for 30 min with fluorescein isothiocyanate-conjugated rabbit anti-pig immunoglobulin g (nor-die immunology) diluted 1:40 in pbs. coverslips were washed, air dried, and mounted on glass slides with 80% glycerol. fluorescent cells were observed and photographed with a leitz wetzlar uv microscope. a 4.6-kbp barnhi s gene cassette was constructed from tgev fs772/70 cdna and used to generate ppbp1 (fig. 1) . the 4350-bp s gene was capable of encoding a precursor polypeptide of 1449 amino acids with a m, 159,811. after cleavage of the n-terminal signal peptide, shown to be absent in virion-associated purdue strain s protein (rasschaert and laude, 1987) the protein would consist of 1433 amino acid residues with a ai, 157,891. the s gene barnhi gene cassette was subcloned into the vv insetion vector pgs20 to give pgsp-1 as described under materials and methods. the sa gene, contained in pgspa-38, was 3474 bp long, capable of encoding a 1157-amino acid polypeptide, corresponding to a deletion of 292 residues from the complete s protein and also included eight amino acid residues derived from the pucl2 polylinker and pgs20 sequences. the sa gene terminated in a new tga stop codon contained within the vv tk sequences (fig. 2) . the predicted size of the precursor sa polypeptide was m, 126,748, which when modified by the removal of the 16-amino acid n-terminal signal peptide was reduced to m, 124,916. the c-terminal deletion also resulted in removal of eight potential nlinked glycosylation sites predicted for the s protein. the s gene initiation codon was 54 bp away from the vv early promoter rna start site for both pgsp-1 and pgspa-38 insertion vectors. the tgev s genes were inserted into the vv genome by homologous recombination into the tk locus, and southern blot analysis was used to confirm that the resulting rvvs vts-1 and vtsa-1 contained the 4.6-and 3.45-kbp tgev barnhi gene fragment, respectively. two plaque-purified rvv clones, vts-1 and vts-2, were used to immunize balb/c mice, and the level of tgev neutralizing antibodies was measured by a plaque reduction assay (table 1 ). both recombinant virus clones induced tgev neutralizing antibodies that were boosted with a second inoculation. mice previously inoculated with the wild-type vv and inoculated with vts-1 produced a 22-fold lower level of tgev neutralizing antibody compared to the convalescent serum of vts-1. a primary infection with wild-type vv could have stimulated the mouse immune system fig. 1. construction of the tgev s gene from the fs772/70 cdna. the complete tgev s gene was generated by a four-way ligation into pbr322. the thin lines represent tgev cdna and the thick lines vector dna sequence. the top line represents the three cdna clones used to generate specific cdna fragments. if more than one enzyme was used they are listed in a descending order next to the appropriate arrow. the 0.4-bp hpal-pstl fragment from ptg47 had barnhi linkers added and was then digested with barnhi and sty1 to generate a 0.2.kb fragment with a barnhi site upstream of the s gene initiation codon. the 1.29.kb hindill-hindill fragment from ptg30 was initially cloned into puc9 and removed as a xbal-barnhi fragment, with the barnhi site derived from the puc9 polylinker sequence, to provide a barnhi site within the tgev orf-3a gene 3'to the end of the s gene. the relevant fragments were then ligated together with pbr322 such that only the correct alignment of the cohesive ends would give a fragment of 4.6 kb in pbr322 corresponding to the tgev s gene bamhl cassette. into producing a rapid clearance of a subsequent rw infection and prevented the expression of large amounts of s protein. mice given a single inoculation with vts-1 4 weeks after the other mouse group had a fourfold reduction in the level of tgev neutralizing antibody. this suggested that age may have a significant effect on the induction of tgev neutralizing antibodies, but further animal studies are needed to make any firm conclusions. expression of the recombinant spike antigens tgev gene products synthesized by vts-1 and vtsa-1 were analyzed by pulse labeling rvv-infected htk-cells with l-[35s]methionine in the presence or absence of 10 pg ml-' tunicamycin for 6 hr. tgev s protein was immunoprecipitated from cell lysates or tissue culture fluids using porcine tgev hyperimmune antiserum. this resulted in s polypeptides of aj 190,000 (gpl90) and aj 210,000 (gp210) being immunoprecipitated from vts-1 -infected cell lysates in the absence of tunicamycin (fig. 3 , lane 2) while in the presence of tunicamycin, a aa, 160,000 (~160) precursor s polypeptide was synthesized (fig. 3, lane 3) . cells infected with vtsa-1 expressed a m, 170,000 glycosylated polypeptide (gpl70) only (fig. 3, lane 4) that was expressed as a aa, 130,000 (~130) precursor protein (fig. 3, lane 5) in the presence of tunicamycin. it was noted that the levels of s protein expressed were appreciably less in the presence of tunicamycin (fig. 3) . comparison of the nonspecifically precipitated proteins in the presence or absence of tunicamycin (fig. 3) indicated that the reduction in s protein synthesis was not due to inhibition of viral replication in the presence of tunicamycin. these observations suggested therefore that either glycosylation was required for efficient synthesis of the s protein or the majority of the anti-s antibodies were directed against the glycosylated form of the protein resulting in less efficient immunoprecipitation of the nonglycosylated form. although gp210 was immunoprecipitated from the vts-1 -infected cell culture medium in the absence of tunicamycin (fig. 4 , lane 3) no detectable extracellular sa protein (gpl70) was recovered from medium of vtsa-l-infected cells (fig. 4, lane 6) suggesting that the membrane anchor is required for export out of cells. the lack of detectable ~160 or ~130 in the culture medium of vts-l-and vtsa-l-infected cells, respectively (fig. 4 lanes 4 and 7) , could result from the levels being too low to be detected by the antibodies due to reduction in synthesis or the lack of glycosylation of the s protein in the presence of tunicamycin. the rate of s protein intracellular transport was compared by measuring the acquisition of endo h resisnote. tgev neutralizing antibody was titrated by a 50% plaque reduction assay and expressed as the reciprocal of the antiserum dilution. tance in tgev-, vts-i-, and vtsa-1 -infected cells. after a i-hr pulse, the tgev s protein consisted of a major gp190 component and a minor gp210 component (fig. 5a) . after a 1-hr chase, gpl90 and gp210 were present in approximately equal proportions but after a 2-hr chase, gp210 was the dominant s protein component and the majority was endo h resistant. tgev s protein steadily became endo h resistant with time such that after a 4-hr chase virtually all s protein had been modified in the golgi. s protein was also detected in the tissue culture medium of tgev-infected cells after a 1 -hr chase and accumulated steadily with time. digestion of gp210 from extracellular or intracellular sources with endo h reduced the size of the s protein to m, 205,000 . this observation has also been made for the fipv s protein from fipv-infected feline cells (vennema eta/., 1990) and suggested that coronavirus s proteins still have high mannose or hybrid oligosaccharide structures following golgi processing. the recombinant s protein was completely endo h sensitive after a 1-hr pulse and after chasing became partially endo h resistant with approximately half of the protein observed as unresolved components of high molecular weight (fig. 5b ). extracellular s protein was not detected in the culture medium after chasing for 4 hr because s protein transport from the rer through the golgi stack and out of the cell was significantly disrupted in the absence of coronaviral morphogenesis. infection of llc-pkl cells with vts-1 produced a discrete mr 2 10,000 endo h-resistant form of s protein (data not shown). this was in contrast to the partial endo h-resistant forms observed in vts-1 -infected htk-cells, implying that the tgev s protein may be post-translationally modified to a different extent depending on the origin of the cell line. the sa protein remained completely endo h sensitive, even after a 4-hr chase, demonstrating that the truncated s protein was glycosylated with high mannose oligosaccharides only and was not subject to golgi-specific modifications (fig. 5b ). the cellular location of the s and sa protein in rvvinfected cells was analyzed by indirect immunofluorescence microscopy (fig. 6) . acetone-fixed tgev-infected llc-pkl cells, probed with anti-s-specific serum, had a granular appearance with occasional bright accumulations in localized parts of the cell (data not shown). in vts-l-infected htk(fig. 6a ) and llc-pkl (fig. 6c) an intracellular compartment in all infected cells (represented with arrows), while vtsa-1 -infected cells had a uniform distribution of sa protein throughout the cytoplasm ( fig. 6b and 6d ). recombinant s protein was also observed on the cell surface of unfixed infected htk-cells (fig. 6e ), unlike sa protein (fig. 6f) , demonstrating that the full-length protein has all the intrinsic properties for cell surface transport and does not require the cooperative effect of other tgev proteins. the possibility that soluble s protein released from human cells may bind back to cell surface receptors and give the appearance of cell surface fluorescence can be disregarded as tgev s protein only binds porcine cell surface receptors. studies described in this paper with tgev recombinant s protein have shown that it is an n-linked glyco-protein with a m, 190-210,000. mice inoculated with two separate clones of vts demonstrated that the recombinant s protein was immunogenic and elicited tgev neutralizing antibodies. this was in contrast to previous studies with tgev n and m proteins expressed by rvvs that induced an immune response but no neutralizing antibodies . the tgev s protein neutralizing epitopes and major antigenic sites were retained by the sa protein but this protein was not transported to the plasma membrane or through the golgi stack and must therefore be accumulated in the endoplasmic reticulum. hu et al. (1984) constructed an incomplete tgev s protein gene that initiated 8 bp downstream of a hpal site but ended 80 bp upstream of a xbal site. this gene construct expressed an endo h sensitive m, 180,000 polypeptide that was not transported to the cell surface in rvv-infected cells (hu et al., 1985) . sequence analysis of the tgev purdue-l 15 (jacobs et al., 1987; rasschaet-t et al., 1987) and fs772/70 strains demonstrated that the xbal site was 3776 bp from the s protein initiation codon and within an orf of 4350 bp, implying that the s gene product expressed by hu et a/. (1985) was a truncated protein. coronavirus s proteins are known to be highly glycosylated with n-linked oligosaccharides and undergo modification with complex sugars at the medial compartment of the golgi stack during virus maturation (niemann et al., 1982) . simple high mannose and hybrid structures can be removed from glycoproteins by digestion with endo h but glycoproteins modified with complex sugar structures are resistant to cleavage with endo h (hubbard and ivatt, 1981; dunphy and rothman, 1985) . the rvv vts-1 produced two s protein species in human cells with different oligosaccharide composition, assigned gpl90 and gp2 10, while the sa protein was expressed in human cells as a single glycoprotein species designated gp170. the gp210 was partially resistant to endo h, suggesting that gpl90 was modified to gp210 by the addition of complex oligosaccharides at the golgi. the sa protein remained endo h sensitive even after extensive chasing, implying that it was not transported to the golgi stack. pulse-chase analysis of tgev-or vts-1 -infected cells demonstrated that s protein was initially synthesized as gpl90 but gradually accumulated as gp210 due to post-translational modifications. endo h digestion of recombinant s protein expressed in htk-produced a range (n/l, 160-210,000) of partially resistant polypeptides. both of these observations were made for the fipv (79-l 146) s protein expressed in a bovine papilloma virus transformed mouse cell line (de groot et a/., 1989) . however, the tgev s gene expressed by a rvv in porcine llc-pkl cells produced an endo h-resistant gp210 product with no partially resistant endo h intermediates. a similar observation was also made for the fipv s gene expressed by a rw in feline cells (vennema et al., 1990) , suggesting that expression of coronavirus s proteins in a cell line compatible with their origin may have profound effects upon the extent of post-translational processing of these proteins. the acquisition of endo h resistance was significantly retarded for recombinant s protein compared to s protein expressed during a tgev infection. retardation of coronavirus s protein intracellular transport has also been observed for the fipv and mhv s proteins expressed by rvvs (vennema et al., 1990) . in this study, retardation between the rer and the golgi in the absence of virus budding was interpreted as a transient accumulation of the s protein at or near the site of virus budding and suggested that the s protein may have a role in defining the site of virus budding, a func-tion previously ascribed to the m proteins of coronaviruses (booze et al., 1985) . lmmunofluorescence studies with vts-infected cells demonstrated that recombinant s protein accumulated in a polar perinuclear compartment of the cell, a similar observation was seen for the cellular localization of rvv expressed tgev m protein (pulford and britton, 1990 ) and at the cell surface. the presence of tgev gp210 in the culture medium of vts-infected cells suggested that gp210 may be released from the cell. proteins can be transported from the rer to the golgi by interacting with carrier proteins or following membrane incorporation, by the flow of membrane from the er to the cell surface. this membrane flow may be responsible for the cell surface presence of coronavirus s proteins. the sa protein was not processed in the golgi apparatus, nor found on the cell surface nor found to be exported into the extracellular medium, indicating that the c-terminal membrane anchor domain of tgev s protein may be required for all of these functions. puddington eta/. (1986) used a mutant vsv g protein to establish that the cytoplasmic tail of the g protein was essential for its transport through the golgi stack. in addition, bray eta/. (1989) used rws to demonstrate that a dengue virus envelope protein truncated at the c-terminus, unlike the wild-type protein, was not secreted into the extracellular medium. however, sveda et a/. (1982) found that c-terminal sequences of the influenza virus hemagglutinin (ha) were essential for cell surface expression and that mutants with a disrupted anchor domain were secreted, suggesting that influenza virus ha, unlike tgev, vsv, and dengue virus envelope proteins, underwent a different protein maturation pathway. evidence suggests that unless a membrane glycoprotein folds into a native or near-native conformation it will not be exported from the rer (lodish, 1988) . however, the general conformation and antigenicity of the sa protein appeared to be retained as it reacted with both polyclonal and monoclonal tgev s antisera in contrast to denatured s protein or s protein expressed in e. co/i cells (correa et a/., 1990; delmas et a/., 1990; pulford, unpublished observations) . the results presented in this paper suggest that the sa protein was retained in the rer possibly due to the removal of essential c-terminal transport signals. we would like to thank miss k. mawditt for synthesizing the oligonucleotide used in the sequencing, mrs. a. waite for carrying out the animal inoculations and dr d. pocock for helpful comments on the manuscript. this research was supported by the biotechnology action programme of the commission of the european communities contract n" [bap-0235.uk(hl)]. mice immunised with recombinant vaccinia virus expressing dengue 4 virus structural proteins with or without nonstructural protein nsl are protected against fatal dengue virus encephalitis sequence of the s gene from a virulent british field isolate of transmissible gastroenteritis virus monoclonal antibodies to murine hepatitis virus-4 (strain jhm) define the viral glycoprotein responsible for attachment and cell-cell fusion localization of antigenic sites of the e2 glycoprotein of transmissible gastroenteritis coronavirus cdna cloning and sequence analysis of the gene encoding the peplomer protein of feline infectious peritonitis virus stably expressed fipv peplomer protein 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proteins: implications for virus assembly key: cord-269862-krcu3hfa authors: wang, shui-mei; wang, chin-tien title: apobec3g cytidine deaminase association with coronavirus nucleocapsid protein date: 2009-05-25 journal: virology doi: 10.1016/j.virol.2009.03.010 sha: doc_id: 269862 cord_uid: krcu3hfa we previously reported that replacing hiv-1 nucleocapsid (nc) domain with sars-cov nucleocapsid (n) residues 2–213, 215–421, or 234–421 results in efficient virus-like particle (vlp) production at a level comparable to that of wild-type hiv-1. in this study we demonstrate that these chimeras are capable of packaging large amounts of human apobec3g (ha3g), and that an hiv-1 gag chimera containing the carboxyl-terminal half of human coronavirus 229e (hcov-229e) n as a substitute for nc is capable of directing vlp assembly and efficiently packaging ha3g. when co-expressed with sars-cov n and m (membrane) proteins, ha3g was efficiently incorporated into sars-cov vlps. data from gst pull-down assays suggest that the n sequence involved in n–ha3g interactions is located between residues 86 and 302. like hiv-1 nc, the sars-cov or hcov-229e n-associated with ha3g depends on the presence of rna, with the first linker region essential for ha3g packaging into both hiv-1 and sars-cov vlps. the results raise the possibility that ha3g is capable of associating with different species of viral structural proteins through a potentially common, rna-mediated mechanism. apolipoprotein b mrna-editing enzyme-catalytic polypeptide-like 3g (apobec3g) is a member of the apobec3 family of cytidine deaminases (cullen, 2006) . human apobec3g (ha3g) can be packaged into hiv-1 virions to mediate c-to-u editing on nascent proviral minus strands during reverse transcription, resulting in the inhibition of viral replication mangeat et al., 2003; yu et al., 2004; zhang et al., 2003) . accordingly, ha3g as an anti-viral factor confers innate immunity to hiv-1. however, hiv-1 encodes vif, which counteracts ha3g by inducing ha3g degradation via the ubiquitin-proteasome pathway, thereby blocking its incorporation into budding virions (mehle et al., 2004; yu et al., 2003) . the ability of vif to negate ha3g function is both dependent on physical association and partly species-specific-in other words, vif cannot counteract a form of a3g from a different species. this inefficient vif-ha3g interaction means that hiv-1 vif is capable of counteracting human and chimpanzee a3g, but not mouse a3g (ma3g) or african green monkey a3g (mariani et al., 2003) . other researchers have shown that ha3g or other apobec family members can confer innate immunity to a wide range of retroviruses as well as hbv (which is similar to retroviruses in that it also goes through a reverse transcription step during genomic replication) (cullen, 2006) . the incorporation of a3g into hiv-1 virions is mediated by the gag nucleocapsid (nc) domain (alce and popik, 2004; cen et al., 2004; khan et al., 2005; luo et al., 2004; schafer et al., 2004; zennou et al., 2004) . in addition to playing a key role in viral rna packaging (berkowitz et al., 1993 (berkowitz et al., , 1995 poon et al., 1996; zhang and barklis, 1997) , nc contains an i domain that is responsible for gag-gag interactions (bennett et al., 1993; bowzard et al., 1998) . heterologous polypeptides capable of self-association have been shown to confer the ability to efficiently produce chimeric vlps when substituted for hiv-1 nc (accola et al., 2000; burniston et al., 1999; johnson et al., 2002; zhang et al., 1998) . however, replacing nc with a leucine-zipper motif that does not encapsidate rna abolishes ha3g packaging without significantly affecting hiv-1 virion production (zennou et al., 2004) , suggesting rna involvement in ha3g incorporation. this is consistent with the proposal that rna is required for either ha3g viral incorporation or gag-ha3g interaction (khan et al., 2005; schafer et al., 2004; svarovskaia et al., 2004; zennou et al., 2004) . we previously demonstrated that hiv-1 gag mutants containing severe acute respiratory syndrome coronavirus nucleocapsid (sars-cov n) coding sequences as nc substitutes can effectively assemble vlps . while completely unrelated, the sars-cov n protein is similar to hiv-1 nc in that it contains putative protein-protein interaction domains (he et al., 2004; narayanan et al., 2003; surjit et al., 2004; yu et al., 2005) and plays a role in viral rna packaging (hsieh et al., 2005; huang et al., 2004a) . given that sars-cov n possesses a rna-binding property, it is likely that assembly-competent chimeras containing a replacement of hiv-1 nc by an sars-cov n sequence may support the incorporation of ha3g into vlps. here we demonstrate that the carboxyl-terminal half of the sars-cov or human coronavirus 229e (hcov-229e) n protein not only enables efficient vlp production, but also confers the ability to efficiently package human apobec3g (ha3g) when substituted for hiv-1 nc. direct evidence comes in the form of ha3g being efficiently packaged into sars-cov vlps. the interaction between coronavirus n and ha3g is also rnadependent. this implies that in addition to retroviruses, sars-cov and other rna viruses may be capable of packaging ha3g via nucleocapsid domains. we previously reported that the hiv-1 gag chimeras containing the sars-cov n carboxyl-terminal 215-421 [nc(n2)] or 234-421 [nc (n4)] codons in the deleted nc region (fig. 1) are capable of assembling vlps as efficiently as wt gag . this raises the question of whether the inserted sars-cov n sequence confers the chimeric ability to package ha3g. we therefore coexpressed assembly-competent nc(n2) and nc(n4) with ha3g in 293t cells and used western immunoblotting to detect vlpassociated ha3g. our controls were the hiv-1 nc-deleted mutants δnc, δpc, and delnc, all with different numbers of deleted nc codons ( fig. 1) . our results show that nc(n2) and nc(n4) were capable of packaging ha3g at the same efficiency level as the wild-type ( fig. 2a , lanes 6 and 7 vs. lane 2). low but detectable vlp-associated ha3g was observed in the nc-deletion mutants (lanes 4 and 5). since hiv-1 vif inhibits ha3g virion incorporation by triggering ha3g degradation (conticello et al., 2003; kao et al., 2003; liu et al., 2004; mariani et al., 2003; marin et al., 2003; mehle et al., 2004; sheehy et al., 2003; stopak et al., 2003; yu et al., 2003) , the ha3g expression level or the presence of vif may affect the level of virus-associated ha3g. to test whether vif or ha3g expression level affects ha3g packaging into chimeric vlps, we introduced mutation-blocking vif expression into wt and nc(n2) and co-transfected each vif-deficient (δvif) result with different amounts of the ha3g-coding plasmid. in the absence of vif, we observed a relatively higher steady-state ha3g expression level accompanied by an increased level of vlp-associated ha3g (fig. 2b, right top panel) . members of the δvif group occasionally expressed higher vlp levels than their vif-intact counterparts (fig. 2b, . however, we also repeatedly observed δvif group members producing higher levels of vlp-associated ha3g. in the presence or absence of vif, nc(n2) displayed a level of vlpassociated ha3g comparable to that displayed by wt. these results suggest that the sars-cov n carboxyl-terminal region can be substituted for hiv-1 nc without affecting vlp assembly or ha3g packaging functions. to determine the required sars-cov n sequences for conferring ha3g packaging ability, we co-expressed ha3g with wt or with chimeras containing a variety of n coding regions as hiv-1 nc substitutes. as illustrated in fig. 1 , full-length sars-cov n sequences were inserted into the δnc (δnc [con] ) and delnc (nc[con]) constructs. nc(n1), nc(n3), nc(n5), nc(n6), and nc(n7) respectively contain n codons 2-213, 215-359, 302-421, 2-168, and 2-86 in the deleted nc region. we again found that both nc(n2) and nc(n4) were capable of efficiently packaging ha3g at vlp-associated ha3g levels comparable to or higher than those of wt (fig. 2c, lanes 5 and 7) . even though δnc(con) and nc(con) were found to be defective in vlp production, they incorporated ha3g at an efficiency level comparable to that of wt (lanes 2 and 3). further, despite producing substantial amounts of vlps, the nc(n1) was incapable of packaging ha3g as efficiently as wt (i.e., it produced a vlp-associated ha3g level approximately 60% that of wt) (lane 4). the barely detectable level of vlp-associated ha3g found in nc(n6) may be due to lower levels of vlps in the blot and/or lower levels of ha3g expression (fig. 2c) . however, in a separate blot containing higher vlp and ha3g expression levels, vlp-associated ha3g in nc(n6) was approximately 50% that measured in wt (data not shown). both ha3g viral incorporation and gag-ha3g interaction require the presence of rna (khan et al., 2005; schafer et al., 2004; svarovskaia et al., 2004; zennou et al., 2004) . we tested whether the association between sars-cov n and ha3g has the same requirement by fusing the sars-cov n amino-terminal (n1, n6, and n7) or carboxyl-terminal sequence (n2, and n5) to the carboxyl terminus of gst and co-expressing each gst fusion construct with ha3g. cell lysates containing ha3g and gst fusions were treated with rnase prior to performing a gst pull-down assay. results indicate that gst-con, gst-n1, gst-n2, and gst-n6 were capable of pulling down ha3g, whereas the amount of ha3g associated with gst fusion was markedly reduced following rnase treatment (fig. 3) . neither gst-n5 nor gst-n7 was capable of pulling down ha3g, regardless of whether or not they were treated with rnase (fig. 3, lanes 7, 9, 16 and 18 ). these results suggest that (a) the ha3g-binding domain of sars-cov n is largely contained within the sequence between residues 86 and 302, and (b) efficient n-ha3g interactions occur in a rna-dependent manner. , nucleocapsid (nc), p6, and amino acid residues in sp1-nc-sp2 junction. δnc has ten hiv-1 nc residues remaining in the deleted region; δpc has nc almost deleted, with sp1 partially removed. delnc has the two methionine residues (bracketed) in the sp1-nc junction removed. pcr-amplified fragments containing various portions of sars-cov n coding sequences were inserted into the deleted nc regions. numbers denote codon positions at inserted sars-cov n protein sequence boundaries. arrows indicate sp1-nc and nc-sp2 junction sites. remaining hiv-1 nc residues in deleted regions are underlined. altered or foreign amino acid residues inserted in juncture area are italicized. all constructs were expressed in hivgptd25 (a hiv-1 pr-defective expression vector). to test whether ha3g can be efficiently packaged into sars-cov, we transfected 293t cells containing the ha3g-coding plasmid with sars-cov m and n expression vectors. sars-cov vlps were generated by co-expressing m and n proteins (huang et al., 2004b) . results indicate that the amount of sars-cov vlp-associated ha3g was comparable to that of hiv-1 (fig. 4a , lane 4 vs. lane 5). medium ha3g was not detected when co-expressed with m or n alone. extracellular n was also undetectable without m co-expression (data not shown). results from co-immunoprecipitation experiments indicate that n was capable of associating with ha3g but m was not (data not shown). the data suggest that ha3g packaging into sars-cov vlps largely depends on ha3g-n association. to further confirm an association between released ha3g and sars-cov vlps formed by m and n, supernatant pellets derived from incorporation of human apobec3g into vlps. 293t cells were co-transfected with a human apobec3g (ha3g) expression vector and indicated plasmid. at 48-72 h posttransfection, cells and supernatant were collected and subjected to western immunoblotting. ha3g plasmid dna (2 or 8 μg) was used for co-transfection as indicated. δvif is a vifdeficient mutation introduced into wt and nc(n2) (panel b, lanes 6-9). wild-type gag and chimeric proteins were probed with a monoclonal antibody directed against hiv-1 ca; an anti-ha monoclonal antibody was used to detect ha3g. vif antiserum was used to detect vif. relative levels of vlp-associated ha3g are indicated along the bottom (panel c). ha3g, wt gag, and chimeric proteins were quantified by scanning ha3g and p24ca-associated band densities from immunoblots. ratios of ha3g to p24 gag were determined and normalized to that of wt. cells co-transfected with ha3g, m, and n expression vectors were subjected to sucrose density gradient fractionation experiments. the results shown in fig. 4b indicate that both m and n had fraction 6 peaks at a density of 1.18 g/ml, which is consistent with a previous report on sars-cov virus-like particle density (huang et al., 2004b) . a ha3g peak was also found at fraction 6, suggesting an association between ha3g and sars-cov vlps generated via m and n coexpression. members of the apobec3 protein family share a highly conserved zinc-coordinating motif: his-x-glu-x 23-28 -pro-cys-x 2-4 -cys (chen et al., 2007b) . similar to apobec1, apobec3g contains two zinccoordinating motifs, with the carboxyl-terminal zinc-coordinating motif possessing catalytic cytidine deaminase activity. to identify ha3g regions responsible for ha3g incorporation into sars-cov vlps, we co-transfected 293t cells with m and n expression vectors and a plasmid encoding wt or a ha3g mutant containing various carboxyl-or amino-terminus deletions (fig. 5a) . the results indicate that ha3g mutants lacking residues 1-104, 157-384, or 246-384 were capable of being incorporated into vlps, and that ha3g mutants lacking residues 1-156 or 309-384 were undetectable in the medium samples (fig. 5b, lanes 4 and 7) . in the cases of δ246-384 and δ309-384, poor particle incorporation may be due in part to low expression levels. in particular, δ309-384 was barely detectable in repeat experiments. low steady-state expression levels of the carboxylterminal truncated ha3g mutant have been reported previously (cen et al., 2004) . in comparison, the δ157-384 mutant expressed greater steady-state stability and a higher level of vlp-associated ha3g (lanes 5 and 12). to test whether the mutations had similar effects on ha3g packaging into chimeric vlps, wt or each ha3g mutant was individually co-expressed with nc(nc2) (which contains the carboxyl-terminal half of the n coding sequence and is known to efficiently produce vlps and package ha3g) (figs. 1 and 2). our results indicate that the ha3g mutations exerted similar effects on ha3g packaging into nc(n2) vlps with no detectable vlp-associated δ1-156 or δ309-384 (fig. 5c) . combined, these results suggest that the essential region for ha3g packaging into sars-cov vlps is located between residues 104 and 156, corresponding to the first linker region. to test whether the deletion mutations exerted similar effects on ha3g incorporation into hiv-1 vlps, we co-expressed wt or each ha3g deletion mutant . equal amounts of cell lysates were treated with (lanes 10-18) or without (lanes 1-9) 0.2 mg/ml dnase-free rnase a for 30 min at 25°c, followed by mixing with glutathione-agarose beads for 2 h at 4°c. complexes bound to the beads were pelleted, washed, and subjected to western immunoblotting with anti-ha and anti-gst antibodies. individually with hiv-1 gag. in agreement with previous reports (cen et al., 2004; luo et al., 2004) , the δ1-156 mutation (with a deletion involving the first linker region) blocked ha3g incorporation (fig. 5d, lane 4) . overall, the deletion mutations had similar effects on ha3g packaging into hiv-1, nc(n2), and sars-cov vlps. specifically, the essential domain for incorporating ha3g into hiv-1 and sars-cov vlps was located in the same (first) linker region. to test whether the sars-cov n ability to confer ha3g packaging is dependent on n-ha3g interaction and therefore distinct from its selfinteraction capacity, we co-expressed ha3g with an assemblycompetent gag containing a leucine-zipper domain as a nc substitute. inserting a wild-type leucine-zipper (wtzip) domain into the deleted nc region restored vlp production to wt level (fig. 6a, lane 4) . a mutant leucine-zipper motif (kzip) replacement was incapable of rescuing the δnc assembly defect (fig. 6a, lane 5) , but in spite of producing a wt-comparable level of vlps, δnc(wtzip) did not package ha3g as efficiently as wt (lane 4 vs. lane 2). we also examined whether human coronavirus 229e (hcov-229e) n protein (having approximately 30% homology with sars-cov n at the amino acid level) (tswen-kei tang, 2005) is capable of conferring the ability to package ha3g when substituted for hiv-1 nc. to perform this test, we constructed nc(229en2) (an nc[n2] counterpart) by inserting the hcov-229e carboxyl-terminal half nucleocapsid coding sequence into the deleted hiv-1 nc region. the resulting construct was transiently co-expressed with ha3g in 293t cells. the results indicate that nc(229en2) can also package ha3g to a degree comparable to that of wt when released vlps are at a similar level (data not shown). to mitigate the impact of overexpressed ha3g on this conclusion, we repeated the experiment by co-expressing ha3g with wt or the chimeric construct in 293 cells. as the results in fig. 6b indicate, the carboxyl-terminal half of hcov-229e n (which also contains a putative self-association domain) (tswen-kei tang, 2005) was capable of replacing the hiv-1 nc function with respect to vlp assembly and ha3g packaging-that is, substantial amounts of vlps and ha3g were detected in nc(229en2) transfectant supernatant (lane 4). to determine whether the interaction between ha3g and hcov-229e n also requires rna, we performed a gst pull-down assay in the presence or absence of rnase. as expected, the gst fusions containing the full-length (gst-229en), amino-terminal half (gst-229en1), or carboxyl-terminal half (gst-229en2) of the hcov-229e n sequence were capable of efficient ha3g pull-down, which is also dependent on the presence of rna (fig. 7) . these data suggest that hcov-229e n and sars-cov n may share a propensity for ha3gassociation. open boxes indicate the zinccoordinating motif, hxe-x 23-28 -cx 2-4 c. the asterisk denotes the cytidine deaminase catalytic site. domains that previously referred to the "linker" peptides are depicted by arrowheads. numbers denote amino acid positions in ha3g. indicated positions of deleted ha3g amino acid residue boundaries were used to designate mutant ha3g constructs. note that each ha3g deletion mutant was tagged with a single copy of ha at the carboxyl terminus and that the wt ha3g was triple-tagged with ha. this may have caused a higher signal for the detected wt ha3g, thus leading to underestimates of the vlp-associated ha3g deletion mutants. (b-d) incorporation of wt and mutant ha3g into vlps. 293t cells were cotransfected with m and n, nc(n2) or an hiv-1 gag expression vector (hivgptd25) plus each of the ha3g expression vectors. to avoid effects from vif on ha3g expression level, nc (n2) and hivgptd25 were expressed in vif-deficient (δvif) contexts. at 48-72 h post-transfection, supernatant and cells were harvested, prepared, and subjected to western immunoblotting. m and n were probed with m antiserum and an anti-n monoclonal antibody. ha3g was probed with an anti-ha antibody and wt gag and chimeric proteins were detected with an anti-p24ca antibody. levels of p24ca-and n-associated proteins and virus-associated ha3g in each sample were quantified by scanning immunoblot band densities. ratios of total ha3g versus p24ca-associated or n-associated protein levels were calculated for each sample and normalized to that of wt ha3g in parallel experiments. dashes (-) denote ratios below 0.01. similar results were observed in repeat and independent experiments. the immunoblots presented here were intentionally overexposed to allow for visualization of the individual ha3g mutants. any conclusion that sars-cov n enables efficient ha3g packaging into vlps when substituted for hiv-1 nc must be tempered when over-expression systems are considered. still, the evidence presented in this paper suggests an association between sars-cov n and ha3gthat is, the leucine-zipper domain is incapable of conferring the ability to package ha3g when substituted for hiv-1 nc even though it enables efficient vlp production. moreover, assembly-competent chimeras with various sars-cov n protein sequences inserted into the deleted hiv-1 nc did not have equal ha3g packaging capabilities: nc(n2) and nc(n4) exhibited a higher level of vlp-associated ha3g than nc(n1). results from a gst pull-down assay suggest that the sars-cov n domain associated with ha3g is largely contained within the central n region between residues 86 and 302 (fig. 3) . although gst-n1 and gst-n6 can pull down ha3g, we found that nc(n1) and nc(n6) are incapable of packaging ha3g as efficiently as wt or nc (n2). therefore, insufficient ha3g incorporation into nc(n1) and nc (n6) may be due in part to steric hindrance in a chimeric protein context. evidence that a carboxyl-terminal self-association domain of sars-cov n or hcov-229e n (as opposed to the amino-terminal rna-binding domain) confers the ability to efficiently package ha3g suggests that for this specific purpose, hiv-1 nc can be replaced by a protein sequence that does not encapsidate hiv-1 genomic rna. given the number of studies demonstrating that viral genomic rna is dispensable for efficient a3g packaging (alce and popik, 2004; cen et al., 2004; khan et al., 2005; luo et al., 2004; navarro et al., 2005; schafer et al., 2004; svarovskaia et al., 2004; zennou et al., 2004) , any genomic rna packaging defect that does exist should not exert a significant impact on the ha3g package. the finding that efficient sars-cov n association with ha3g is rna-dependent (fig. 3) agrees with the proposal that rna (viral or nonspecific) is required for mediating ha3g-gag association and ha3g incorporation into virions (svarovskaia et al., 2004; zennou et al., 2004) . we assumed that the chimeras considered competent in ha3g packaging are capable of packaging considerable amounts of rna, since the sars-cov n (hsieh et al., 2005; luo et al., 2006) and hiv-1 ma domains (burniston et al., 1999; ott et al., 2005) both possess nucleic acid binding properties that may confer rna packaging ability. using a rna quantification assay as described by chang et al. (2008) , we found that nc(n2) vlps contain rna at approximately 80% of the level measured in hiv-1 gag vlps (data not shown). recent studies have suggested that ha3g preferentially binds with cellular 7sl rna, which in turn facilitates an association between rna-bound ha3g and nc for viral incorporation (bogerd and cullen, 2008; wang et al., 2007) . accordingly, 7sl rna apparently plays a key role in determining ha3g packaging into hiv-1 virions. additional experiments are required to determine whether sars-cov or nc(n2) are as capable as hiv-1 in terms of packaging 7sl rna. to date, one in vitro study has demonstrated that a3g binds to rna prior to hiv-1 nc association (bogerd and cullen, 2008) . accordingly, it may be that the interactions required for a3g/rna/nc ternary complex formation involve specific contacts between nc and both a3g and a3g-bound rna. sars-cov n association with ha3g may in large part depend on n binding to ha3g-bound rna, since the sars-cov n contains clusters of basic residues in the amino-and carboxyl-terminal regions that may confer rna-binding ability (hsieh et al., 2005) . extensive genetic analyses indicate that sars-cov n rna-binding domains are largely located between amino acid residues 45-181 (huang et al., 2004a) , 248-280 (chen et al., 2007a; luo et al., 2006) , and 363-382 equal amounts of cell lysates were treated with (lanes 6-10) or without (lanes 1-5) 0.2 mg/ml dnasefree rnase a, followed by gst pull-down assay as described in the legend of fig. 3 . (luo et al., 2006) , all of which contain large quantities of basic residues. it is conceivable that basic residue clusters within sars-cov n make a significant contribution to ha3g-n association by enhancing n binding to ha3g-associated rna. compatible with this hypothesis, we found that the gst fusions gst-con, gst-n1, gst-n2 and gst-n6, all of which contain most of the residues involved in rna-binding, are competent in their associations with ha3g (fig. 3) . the exception to this rule is gst-n5, which is unable to pull down ha3g despite bearing a putative rna-binding domain (residues 363-382). our finding that the essential region for ha3g packaging into hiv-1 or sars-cov vlps resides between codons 104 and 156 is compatible with a genetic analysis demonstrating that residues 124 to 127 are responsible for ha3g viral incorporation (huthoff and malim, 2007) . a very recent study suggests that ha3g contains a necessary and sufficient cytoplasmic retention signal located between residues 113 and 128 (bennett et al., 2008) . consistent with this report, our immunofluorescence experiment results indicate that cells expressing the ha3g deletion mutant δ1-156 show fluorescence staining in both the nucleus and cytoplasm; in contrast, fluorescence in cells expressing wt or ha3g mutants δ1-104, δ157-384, or δ246-384 is predominantly present in cell cytoplasm (data not shown). however, the mislocalization of δ1-156 does not account for its defective incorporation into hiv-1 or sars-cov vlps, since gst pull-down assay results suggest that cytoplasmic ha3g(δ1-156) is still incapable of associating with sars-cov n (data not shown). hcov-229e n and sars-cov n share two conserved sequences: one from residues 50 to 170 and the other from 250 to 360 (tswen-kei tang, 2005) . combined, the ability of nc(229en2) to package ha3g and the rna-dependent association of hcov-229e n with ha3g ( fig. 7) imply that in addition to sars-cov n, coronaviral n products may associate with ha3g via binding with rna. however, human tcell leukemia virus type 1 (htlv-1) has been found to prevent ha3g packaging through gag nc domain action (derse et al., 2007) , suggesting that some rna viruses are incapable of packaging a3g. our results indicate that the presence of the ha3g first linker region is required for ha3g to be packaged into either hiv-1 gag or sars-cov vlps. it is possible that an rna/ha3g complex, either alone or in combination with other cellular factors, is an acceptable structure for an association between hiv-1 nc and sars-cov n. here we demonstrated for the first time that sars-cov is capable of packaging ha3g via the sars-cov n protein. it remains to be determined whether chimeric nc(n2) or sars-cov can package endogenous a3g as efficiently as hiv-1. the biological significance of a sars-cov n association with ha3g also requires further exploration. according to one report, ha3g is capable of editing virus-associated hiv-1 rna (bishop et al., 2004) ; the ha3g-mediated inhibition of viral replication may be independent of its cytidine deaminase activity (rösler et al., 2005; turelli et al., 2004) . since humans are not natural hosts for sars-cov , human susceptibility implies that cells infected by sars-cov may not express a3g, or sars-cov may have evolved to counteract a3g anti-viral activity. further studies are required to address this issue. the parental hiv-1 proviral plasmid dna used in this study was hxb 2 (ratner et al., 1985) . the cdna clone of the sars-cov n (sars coronavirus strain twc, genbank accession number ay321118) was provided by the centers for disease control of the department of health, taiwan. hiv-1 nc-deletion mutants δnc, delnc, and δpc and chimeras δnc(con), nc(con), nc(n1), nc(n2), nc(n3), nc(n4), nc (n5), nc(n6), nc(n7), δnc(wtzip) and δnc(kzip) were all as previously described . primers used for making nc(229en2) were 5′-cgcaatcgattcatgaaggcagttgct-3′ (for-ward) and 5′-cttcggatcccgtttacttcatcaat-3′ (reverse) using a coronavirus 229e (hcov-229e) nucleocapsid expression vector ptre-hn (schelle et al., 2005) as template (kindly provided by volker thiel). to construct a vif-deficient (δvif) mutation, vif-encoding plasmid dna was digested with ndei, filled in, and relegated, resulting in a shift in the vif open reading frame. all of the engineered constructs were cloned into the pr-defective hiv-1 proviral expression vector hivgptd25 (wang et al., 2000) . human apobec3g (ha3g) expression vector pcdna3.1-apobec3g-ha (sheehy et al., 2002; stopak et al., 2003) was obtained through the nih aids research and reference reagent program. ha3g deletion mutants were gifts from s. cen (cen et al., 2004) . mammalian expression vectors encoding sars-cov m and n were provided by g.j. nabel (huang et al., 2004b) . to construct gst fusions, amplicons containing sars-cov n or hcov-229e n coding sequences were digested with bamhi and clai and fused to the c-terminus of gst, which is directed by a mammalian elongation factor 1a promoter (cortes et al., 1996) . primers for cloning gst fusions were gst-con, 5′-taaaggatcctctgataatggaccc-3′ (forward), 5′-tcatatcgatttatgcctgagttgaatc-3′ (reverse); gst-n1, 5′-taaaggatcctctgataatggaccc-3′ (forward), 5′-gctcat-cgattagctagccattcgagc-3′ (reverse); gst-n2, 5′-tggctggat-ccggtggtgaaactgcc-3′ (forward), 5′-tcatatcgatttatgcct-gagttgaatc-3′ (reverse); gst-n5, 5′-attacggatcctggccg-caaattgca-3′ (forward), 5′-tcatatcgatttatgcctgagttgaatc-3′ (reverse); gst-n6, 5′-taaaggatcctctgataatggaccc-3′ (forward), 5′-gctcatcgattatgttgttccttgagg-3′(reverse); gst-n7, 5′-taaaggatcctctgataatggaccc-3′ (forward), 5′-gctcatcgat-tagccaatttggtcat-3′ (reverse); gst-229en, 5′-cgggatccgcta-cagtcaaatgg-3′ (forward), 5′-ccatcgattagtttacttcatcaat-3′ (reverse); gst-229en1, 5′-cgggatccgctacagtcaaatgg-3′ (forward), 5′-ccatcgattattcctgaggcttgtc-3′ (reverse); and gst-229en2, 5′-cgggatccatgaaggcagttgct-3′ (forward), 5′-ccatc-gattagtttacttcatcaat-3′ (reverse). mutations were confirmed by restriction enzyme digestion or dna sequencing. 293t and 293 cells were maintained in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal calf serum (gibco). confluent 293t or 293 cells were trypsinized and split 1:10 onto 10cm dishes 24 h prior to transfection. for each construct, cells were transfected with 20 μg of plasmid dna using the calcium phosphate precipitation method; 50 μm chloroquine was added to enhance transfection efficiency. unless otherwise indicated, 10 μg of each plasmid was used for co-transfection. culture supernatant and cells were harvested for protein analysis 2-3 d post-transfection. at 48-72 h post-transfection, supernatant from transfected 293t or 293 cells was collected, filtered, and centrifuged through 2 ml of 20% sucrose in tse (10 mm tris-hcl [ph 7.5], 100 mm nacl, 1 mm edta plus 0.1 mm phenylmethylsulfonyl fluoride [pmsf]) at 4°c for 40 min at 274,000 ×g. pellets were suspended in ipb (20 mm tris-hcl [ph 7.5], 150 mm nacl, 1 mm edta, 0.1% sds, 0.5% sodium deoxycholate, 1% triton x-100, 0.02% sodium azide) plus 0.1 mm pmsf. next, cells were rinsed with ice-cold phosphate-buffered saline (pbs), collected in ipb plus 0.1 mm pmsf, and microcentrifuged at 4°c for 15 min at 13,700 ×g to remove cell debris. supernatant and cell samples were mixed with equal volumes of 2x sample buffer (12.5 mm tris-hcl [ph 6.8], 2% sds, 20% glycerol, 0.25% bromophenol blue) and 5% β-mercaptoethanol and boiled for 5 min. samples were resolved by electrophoresis on sds-polyacrylamide gels and electroblotted onto nitrocellulose membranes. membrane-bound gag or chimeric proteins were immunodetected using a mouse monoclonal antibody directed against hiv-1 p24ca (wang et al., 1998) or the sars-cov nucleocapsid . sars-cov m was detected with a rabbit anti-m polyclonal antibody (rockland). hatagged ha3g was probed with a mouse anti-ha (sigma) monoclonal antibody at a dilution of 1:5000. for hiv-1 vif detection, a rabbit anti-vif polyclonal antibody (goncalves et al., 1994) was used at a 1:2000 dilution. the secondary antibody was a sheep anti-mouse or donkey anti-rabbit horseradish peroxidase-(hrp) conjugated antibody (invitrogen), both at 1:5000 dilutions. to detect gst and gst fusions, anti-gst hrp conjugate (amersham) was used at a 1:5000 dilution. horseradish peroxidase activity was detected according to the manufacturer's protocol. gst pull-down assay 293t cells either mock transfected or transfected with gst fusion expression vectors were collected, lysed in ripa buffer (140 mm nacl, 8 mm na 2 hpo 4 , 2 mm nah 2 po 4 , 1% np-40, 0.5% sodium deoxycholate, 0.05% sds) containing complete protease inhibitor cocktail (roche), and microcentrifuged at 4°c for 15 min at 13,700 ×g (14,000 rpm) to remove cell debris. aliquots of post-nuclear supernatant (pns) were mixed with equal amounts of 2× sample buffer and 5% β-mercaptoethanol and held for western blot analysis. ripa buffer was added to the remaining pns samples to final volumes of 500 μl. each sample was mixed with glutathione-agarose beads (30 μl) (sigma) and rocked for 2 h at 4°c. bead-bound complexes were pelleted, washed tree times with ripa buffer, twice with pbs, eluted at 1× sample buffer with 5% β-mercaptoethanol, boiled for 5 min, and subjected to sds-10% page as described above. supernatant cultures of transfected 293t cells were collected, filtered, and centrifuged through 2 ml 20% sucrose cushions as described above. viral pellets were suspended in pbs buffer and laid on top of a pre-made 20-60% sucrose gradient consisting of 1 ml layers of 20, 30, 40, 50 and 60% sucrose in tse that had been allowed to sit for 2 h. gradients were centrifuged in an sw50.1 rotor at 40,000 rpm (274,000 ×g) for 16 h at 4°c; 500 μl fractions were collected from top to bottom. sucrose density was measured for each fraction. proteins in each fraction were precipitated with 10% trichloroacetic acid (tca) and subjected to western immunoblotting. confluent 293 cells were split 1:80 onto coverslips 24 h before transfection. two days post-transfection, cells were fixed at 4°c for 20 min with ice-cold pbs containing 3.7% formaldehyde, washed once with pbs and once with dmem plus 10% heat-inactivated calf serum (dmem/calf serum), and permeabilized at room temperature for 10 min in pbs plus 0.2% triton x-100. samples were incubated with the primary antibody for 1 h and secondary antibody for 30 min. following each incubation, samples were subjected to three washes (5 to 10 min each) with dmem/calf serum. the primary antibody was an anti-ha (sigma) at a 1:200 dilution. a rabbit antimouse rhodamine-conjugated antibody at a 1:100 dilution served as the secondary antibody (cappel, icn pharmaceuticals, aurora, ohio, usa). after the final dmem/calf serum wash, the coverslips were washed three times with pbs and mounted in 50% glycerol in pbs for viewing. images were taken using an epifluorescence microscope (olympus ax-80) or laser scanning confocal microscope (olympus fv300). efficient particle production by minimal gag constructs which retain the carboxy-terminal domain of human immunodeficiency virus type 1 capsid-p2 and a late assembly domain apobec3g is incorporated into virus-like particles by a direct interaction with hiv-1 gag nucleocapsid protein functional chimeras of the rous sarcoma virus and human immunodeficiency virus gag proteins nuclear exclusion of the hiv-1 host defense factor apobec3g requires a novel cytoplasmic retention signal and is not dependent on rna binding specific binding of human immunodeficiency virus type 1 gag polyprotein and nucleocapsid protein to viral rnas detected by rna mobility shift assays retroviral nucleocapsid domains mediate the specific recognition of genomic viral rnas by chimeric gag polyproteins during rna packaging in vivo apobec-mediated editing of viral rna single-stranded rna facilitates nucleocapsid: apobec3g complex formation importance of basic residues in the nucleocapsid sequence for retrovirus gag assembly and complementation rescue human immunodeficiency virus type 1 gag polyprotein multimerization requires the nucleocapsid domain and rna and is promoted by the capsid-dimer interface and the basic region of matrix protein the interaction between hiv-1 gag and apobec3g hiv-1 matrix protein repositioning in nucleocapsid region fails to confer virus-like particle assembly structure of the sars coronavirus nucleocapsid protein rna-binding dimerization domain suggests a mechanism for helical packaging of viral rna extensive mutagenesis experiments corroborate a structural model for the dna deaminase domain of apobec3g the vif protein of hiv triggers degradation of the human antiretroviral dna deaminase apobec3g in vitro v(d)j recombination: signal joint formation role and mechanism of action of the apobec3 family of antiretroviral resistance factors resistance of human t cell leukemia virus type 1 to apobec3g restriction is mediated by elements in nucleocapsid subcellular localization of the vif protein of human immunodeficiency virus type 1 dna deamination mediates innate immunity to retroviral infection analysis of multimerization of the sars coronavirus nucleocapsid protein assembly of severe acute respiratory syndrome coronavirus rna packaging signal into virus-like particles is nucleocapsid dependent structure of the n-terminal rna-binding domain of the sars cov nucleocapsid protein generation of synthetic severe acute respiratory syndrome coronavirus pseudoparticles: implications for assembly and vaccine production identification of amino acid residues in apobec3g required for regulation by human immunodeficiency virus type 1 vif and virion encapsidation nucleic acid-independent retrovirus assembly can be driven by dimerization the human immunodeficiency virus type 1 vif protein reduces intracellular expression and inhibits packaging of apobec3g (cem15), a cellular inhibitor of virus infectivity viral rna is required for the association of apobec3g with human immunodeficiency virus type 1 nucleoprotein complexes bats are natural reservoirs of sars-like coronaviruses influence of primate lentiviral vif and proteasome inhibitors on human immunodeficiency virus type 1 virion packaging of apobec3g amino-terminal region of the human immunodeficiency virus type 1 nucleocapsid is required for human apobec3g packaging carboxyl terminus of severe acute respiratory syndrome coronavirus nucleocapsid protein: self-association analysis and nucleic acid binding characterization broad antiretroviral defence by human apobec3g through lethal editing of nascent reverse transcripts species-specific exclusion of apobec3g from hiv-1 virions by vif hiv-1 vif protein binds the editing enzyme apobec3g and induces its degradation vif overcomes the innate antiviral activity of apobec3g by promoting its degradation in the ubiquitin-proteasome pathway characterization of n protein self-association in coronavirus ribonucleoprotein complexes complementary function of the two catalytic domains of apobec3g redundant roles for nucleocapsid and matrix rna-binding sequences in human immunodeficiency virus type 1 assembly charged amino acid residues of human immunodeficiency virus type 1 nucleocapsid p7 protein involved in rna packaging and infectivity apobec-mediated interference with hepadnavirus production complete nucleotide sequence of the aids virus, htlv-iii specific packaging of apobec3g into hiv-1 virions is mediated by the nucleocapsid domain of the gag polyprotein precursor selective replication of coronavirus genomes that express nucleocapsid protein isolation of a human gene that inhibits hiv-1 infection and is suppressed by the viral vif protein the antiretroviral enzyme apobec3g is degraded by the proteasome in response to hiv-1 vif hiv-1 vif blocks the antiviral activity of apobec3g by impairing both its translation and intracellular stability the nucleocapsid protein of the sars coronavirus is capable of self-association through a c-terminal 209 amino acid interaction domain human apolipoprotein b mrna-editing enzymecatalytic polypeptide-like 3g (apobec3g) is incorporated into hiv-1 virions through interactions with viral and nonviral rnas biochemical and immunological studies of nucleocapsid proteins of severe acute respiratory syndrome and 229e human coronaviruses inhibition of hepatitis b virus replication by apobec3g analysis of minimal human immunodeficiency virus type 1 gag coding sequences capable of virus-like particle assembly and release assembly and processing of human immunodeficiency virus gag mutants containing a partial replacement of the matrix domain by the viral protease domain 7sl rna mediates virion packaging of the antiviral cytidine deaminase apobec3g severe acute respiratory syndrome coronavirus nucleocapsid protein confers ability to efficiently produce virus-like particles when substituted for the human immunodeficiency virus nucleocapsid domain induction of apobec3g ubiquitination and degradation by an hiv-1 vif-cul5-scf complex single-strand specificity of apobec3g accounts for minusstrand deamination of the hiv genome recombinant severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein forms a dimer through its c-terminal domain apobec3g incorporation into human immunodeficiency virus type 1 particles effects of nucleocapsid mutations on human immunodeficiency virus assembly and rna encapsidation analysis of the assembly function of the human immunodeficiency virus type 1 gag protein nucleocapsid domain the cytidine deaminase cem15 induces hypermutation in newly synthesized hiv-1 dna we thank y.-p. li for the reagents and technical assistance, cen shan for providing the ha3g deletion mutant plasmids, volker thiel for the hcov-229e n cdna, and g. nabel for the sars-cov m and n expression vectors. the following reagents were obtained through the nih aids research and reference reagent program: pcdna3.1-apobec3g-ha from warner c. greene and hiv-1 vif antiserum from d. gabuzda. this work was supported by grants vgh94-314 from the taipei veterans general hospital and nsc94-2320-b-010-035 from the national science council, taiwan, republic of china. key: cord-274480-aywdmj6o authors: song, wenfei; wang, ying; wang, nianshuang; wang, dongli; guo, jianying; fu, lili; shi, xuanling title: identification of residues on human receptor dpp4 critical for mers-cov binding and entry date: 2014-10-21 journal: virology doi: 10.1016/j.virol.2014.10.006 sha: doc_id: 274480 cord_uid: aywdmj6o middle east respiratory syndrome coronavirus (mers-cov) infects host cells through binding the receptor binding domain (rbd) on its spike glycoprotein to human receptor dipeptidyl peptidase 4 (hdpp4). here, we report identification of critical residues on hdpp4 for rbd binding and virus entry through analysis of a panel of hdpp4 mutants. based on the rbd–hdpp4 crystal structure we reported, the mutated residues were located at the interface between rbd and hdpp4, which potentially changed the polarity, hydrophobic or hydrophilic properties of hdpp4, thereby interfering or disrupting their interaction with rbd. using surface plasmon resonance (spr) binding analysis and pseudovirus infection assay, we showed that several residues in hdpp4–rbd binding interface were important on hdpp4–rbd binding and viral entry. these results provide atomic insights into the features of interactions between hdpp4 and mers-cov rbd, and also provide potential explanation for cellular and species tropism of mers-cov infection. middle east respiratory syndrome (mers), a novel coronavirus which causes severe respiratory illness, was first reported in a patient from saudi arabia in 2012 (de groot et al., 2013) . to date, individual cases as well as small clusters and large outbreaks have been reported in several countries and the mortality rate is estimated at 30% among laboratory-confirmed cases (organization, 2014) . phylogenetic analysis demonstrates that the mers coronavirus (mers-cov) is genetically closest to clade 2c betacoronavirus found in camels and insectivorous bats (ithete et al., 2013) although the true viral reservoir remains uncertain. the clinical symptoms caused by mers-cov are similar to those caused by severe acute respiratory syndrome coronavirus (sars-cov) although the two viruses use two distinct receptors; mers-cov uses dipeptidyl peptidase 4 (dpp4) while sars-cov uses angiotensin-converting enzyme 2 (ace2). other coronaviruses use other receptors and perhaps this provides partial explanation for their cellular and species tropism. mers-cov can replicate in a range of cell lines derived from human, non-human primate, porcine, and bat (de wit et al., 2013) . traditional small laboratory animals, such as mice (coleman et al., 2014) , hamsters (de wit et al., 2013) , and ferrets (raj et al., 2014) , were shown to resist mers-cov infection. the finite host range of mers-cov has seriously restricted the development of appropriate animal models to study the pathogenesis of this virus and to assess the efficacy of potential therapeutic strategies. raj et al. (2014) demonstrated that human receptor dpp4 (hdpp4) domain (residues 246 to 505) could confer the susceptibility of ferret dpp4 to mers-cov infection. zhao et al. (2014) are the first to describe a method of developing a small-animal model for mers-cov in which an adenovirus expressing hdpp4 was utilized to transiently transduce mouse airway cells and make mice susceptible to mers-cov infection. recently van doremalen et al. (2014) showed that dpp4 played an important role in the observed species tropism of mers-cov infection and identified residues in dpp4 responsible for this restriction. these results indicate that the insusceptibility to infection is primarily determined by the inability of mers-cov binding to dpp4 of a non-permissive cell line. previous findings have shown that hdpp4 extracellular domain consists of a variable n-terminal eight-blade β-propeller domain and a conserved c-terminal α/β-hydrolase domain (engel et al., 2003; rasmussen et al., 2003) . however, our understanding of critical residues of hdpp4 on mers-cov interaction and entry is quite limited. we and others have previously characterized rbd-hdpp4 crystal structure lu et al., 2013; wang et al., 2013) . the rbd-hdpp4 crystal structure showed that the viral rbd recognized blades iv and v of the dpp4 β-propeller domain. the atomic interaction details of the binding interface revealed that the rbd receptor recognition was predominantly mediated by several amino-acid residue interactions, including rbd residue d539 with dpp4 residue k267, rbd y499 with dpp4 r336, rbd residues d510 and e513 with dpp4 residues r317 and q344, rbd l506, w553 and v555 with dpp4 l294 and i295. previously, we have generated a panel of mers-cov mutant rbd proteins at the residues d539, y499, d510, e513, l506, w553 and v555 to characterize their impacts on binding activity to hdpp4 and the entry efficiency into target cells. however, the impacts of the corresponding residues on hdpp4 have not been well characterized. here, through structure-guided mutagenesis, we identified several key residues in hdpp4 that were critical for rbd binding measured by both real-time surface plasmon resonance (spr) and pseudovirus entry. these residues included k267 and r336 on binding patch 1, and l294, i295, r317 and q344 on binding patch 2. the mutations of three positively charged residues k267, r336 and r317 perhaps interfere with the interaction of the negatively charged residues on the surface of rbd; the mutations of l284, i295 and q344 may lead to the change of hydrophobic or hydrophilic properties of hdpp4 at the interface with rbd. our previous findings have shown that the binding interface between hdpp4 and mers-cov rbd is mainly composed of two binding patches, patch 1 and patch 2 (fig. 1a) . the patch 1 interface is characterized by interactions between c-terminal end of the long linker connecting the rbd β6/β7 strands and the hdpp4 blade 4. the contact in patch 1 is critically determined by the polar interactions among a group of hydrophilic amino-acid residues, including rbd e536, d537, d539 and y499 and hdpp4 k267 and r336. in this patch, dpp4 residue k267 interacts with rbd d539 by salt bridge (fig. 1b) , while dpp4 residue r336 forms hydrogen bond with rbd residue y499 (fig. 1c) . patch 2 has a hydrophobic core surrounded by a hydrophilic periphery. in the hydrophobic core, rbd and hdpp4 contacts are critically dependent on a few 'hot spot' residues including rbd l506, w553 and v555, and dpp4 l294 and i295. however, the surrounding hydrophilic surface consists of rbd residues d510, e513 and y540, and dpp4 residues h298, r317 and q344. among these hydrophilic residues, the salt bridge and hydrogen bond between d510 and r317, e513 and q344 contribute to the maintenance of rbd-receptor contact (fig. 1d) . to study the impacts of the substitutions of the critical residues on hdpp4 described above on the interaction between mers-cov rbd and hddp4, we determined the binding efficiency between these two proteins by employing spr technique. first, we constructed a series of hdpp4 mutants guided by the rbd-hdpp4 complex crystal structure information . the wide-type and mutant hdpp4 were introduced into baculovirus expression system. all wide-type and mutant forms of hdpp4 were expressed efficiently (data not shown). second, the binding efficiency was measured by spr. as shown in fig. 2 and table 1 , mutations at several hdpp4 residues, in individual or combination, resulted in a significant attenuation in binding to mers-cov rbd. in patch 1, residue k267 mutation (k267a and k267e) presumably damaged the salt-bridge interaction, completely abrogated the binding between hdpp4 and rbd, while r336a reduced rbd and hdpp4 binding about 100 fold. in patch 2, double mutations at l294 and i295 (l294a þ i295a and l294d þi295d) completely eliminated the binding between rbd and hdpp4, presumably by disrupting hydrophobic interactions with rbd l506, w553 and v555. in contrast, the single-residue substitution of r317a and q344a in the hydrophilic surface of patch 2 had negligible effect on binding efficiency. to further study the importance of the critical residues on hdpp4 on viral entry, we measured the entry efficiency of pseudovirus into cos7 cells expressing the wide-type and mutant forms of hdpp4. the expression levels of the wide-type and mutant hdpp4 were analyzed by fluorescence-activated cell sorting (facs) using goat anti-hdpp4 polyclonal antibody. all of the wide-type and mutant hdpp4 proteins could be expressed on the surface of cos7 cells with the similar expression efficiency (fig. 3a) . forty-eight hours later, these cells were exposed to pseudovirus infection and their entry efficiency was measured by luciferase activity 48 h later. as showed in fig. 3b , the residue mutations located at patch 1 (k267a, k267e and r336a) and hydrophobic region of patch 2 (l294a þi295a and l294aþ i295d) fig. 1 . the amino-acid residue interaction details at the binding interface. (a) two patches of the binding interface. patch 1 interface is characterized by interactions between the c-terminal end of the long linker connecting the rbd β6/β7 strands (light magenta) and the hdpp4 blade 4 (cyan). in patch 2, a gently concaved outer surface in rbd (light magenta) accommodates a linker containing a short α helix between hdpp4 blades 4 and 5 (cyan). (b) and (c) hydrophilic residues of rbd and hdpp4 interact through polar contacts in patch 1. rbd d539 has salt-bridge interaction with hdpp4 residue k267 (b). dpp4 residue r336 forms hydrogen bond with rbd residue y499 (c). the polar contacts (salt-bridge and hydrogen bond) are drawn as black dashed sticks. (d) hot spot residues in the hydrophobic core and hydrophilic periphery of patch 2. resulted in significantly reduction in viral entry. this is consistent with the binding results described previously. in the hydrophilic region of patch 2, residue substitution r317 led to partial loss of viral infection (41.4%), while the mutation q344 modestly increased viral infection (22.8%). in summary, we have identified several key residues in hdpp4 critical for viral binding and entry into target cells. these residues include positively charged residues of patch 1 (k267 and r336) and hydrophobic zone of patch 2 (l294 and i295). in contrast, the mutations at hydrophilic zone of patch 2 (r317 and q344) had little influence on binding and virus entry efficiency. these results showed that the positively charged residues at the outer surface of blade 4 and the hydrophobic regions of blade 5 may play an important role in mediating viral binding and entry into the target cells, while the impact of mutations at hydrophilic region of patch 2 was barely detectable. this is consistent with our earlier findings where residue mutations at the corresponding negatively charged and hydrophobic core positions on rbd of mers-cov could significantly reduce both binding and viral entry efficiency. sequence analysis of dpp4 from multiple animal species (fig. 4 ) showed that mers-cov susceptible animals, such as macaque, camel and bat, shared the same sequence with hdpp4 at blades iv and v. in contrast, those mers-cov resistant animals, such as mouse, rat and ferret, have residues at l294, i295 and r366 that are all different from hdpp4. raj et al. (2014) reported that when these sites of hdpp4 were changed to the residues of ferret, the binding and viral infection efficiency could also be decreased. van doremalen et al. (2014) found 5 residues involved in the hdpp4-rbd interaction which were important to determine the susceptibility to mers-cov infection, in which i295 and r336 were included. these results are consistent with our findings and suggest these residues play an important role in rbd binding and viral entry, and determining the tropism to mers-cov infection. mers-cov rbd (residues 367-606) and the extracellular domain of hdpp4 (residues 39-766) were expressed using a bacto-bacs baculovirus expression system (invitrogen). in brief, the dna encoding rbd and hdpp4 were respectively cloned into the pfastbac™ dual vector (invitrogen) incorporating an n-terminal gp67 signal peptide to facilitate secretion and a c-terminal hexa histidine-tag for purification. the constructed dna was then transformed into the bacterial dh10bac competent cells and the recombined bacmid dna was extracted and transfected into sf9 cells using cellfectin ii reagent (invitrogen). after 5-7 days of incubation at 300 k, the low-titer viruses were harvested and then amplified. the amplified high-titer viruses were then used to infect sf9 cells and the cell culture supernatant containing target protein was harvested 60 h after infection, concentrated, loaded to nickel (ni)-charged resin (ge healthcare), and eluted with 0.5 m imidazole and further purified using the superdex™ 200 highperformance column (ge healthcare) pre-equilibrated with tris buffer (50 mm tris, ph 8.8, 40 mm nacl). fractions containing the purified protein were collected and applied directly to a preequilibrated resource™ q column (ge healthcare) and then eluted with a 0.05-1 m nacl gradient in 40 mm tris buffer (ph 8.8). fractions containing protein were finally purified using super-dex™ 200 column pre-equilibrated with hbs (10 mm hepes, ph 7.2, 150 mm nacl) and centrifuged to 1 mg/ml. mutants of the extracellular domain of hdpp4 were constructed using a standard pcr-based cloning strategy. and the mutant proteins were expressed and purified in the same way. the spr analyses were carried out using a biacore t200 instrument (ge healthcare) equipped with a research-grade cm5 sensor chip. to measure the affinity binding between rbd and wide-type or mutant hdpp4, the rbd was immobilized on the sensor chip by standard amine coupling procedure. the flow cell 1 was left blank to serve as a reference. purified rbd at a concentration of 5 μg/ml in sodium acetate buffer (10 mm, ph 5.0) was immobilized to a density of 300-400 response units on the flow cell 2. for the collection of binding data, hdpp4 or its mutants in a buffer of 10 mm hepes, ph 7.2, 150 mm nacl, and 0.005% (v/v) tween-20 were injected over the two flow cells at a series of concentration at a 30 μl/min flow rate and 298 k. the rbd-hdpp4 complex was allowed to associate for 60 s and dissociated for 60 s. the surfaces were regenerated with an injection of 5 mm naoh between each cycle if needed. the data was analyzed with the biacore t200 evaluation software by fitting to a 1:1 langmuir binding model. mers-cov pseudovirus was generated by co-transfection of human immunodeficiency virus (hiv) backbone expressing firefly luciferase (pnl43r-e-luciferase) and mers-cov spike glycoprotein expression vector (pcdna3.1 þ , invitrogen) into the 293 t cells. viral supernatants were harvested 48 h later, normalized by p24 elisa kit (beijing quantobio biotechnology co., ltd, china) before infecting the target cos7 cells transiently expressing wide-type or mutant hdpp4. the wide-type and mutant hdpp4 expressing cos7 cells were incubated with goat anti-hdpp4 polyclonal antibody (r&d) followed by incubation with fluorescein phycoerythrin (pe)labeled rabbit anti-goat igg antibody (santa cruz). the expression levels of wide-type and mutant hdpp4 were measured by flow cytometer (bd aria ii) and the mean fluorescence intensity (mfi) was analyzed. the cos7 cells infected by mers-cov pseudovirus were lysed at 48 h post infection and viral entry efficiency was quantified by comparing the luciferase activity between pseudoviruses-infected cos7 cells expressing wide-type and those infected cos7 cells expressing mutant hdpp4. the entry efficiency (%) of pseudovirus was calculated on the basis of luciferase activity. and the percentages of pseudovirus entry efficiency shown for mutant hdpp4 were luciferase activity values versus that of the wide-type hdpp4, as the entry efficiency for wide-type hdpp4 was defined as 100%. data shown were corrected for the expression of different hdpp4 constructs by the parameter of mfi. error bars represent standard errors of the means of three independent experiments. student's t-test; n po 0.05; nn po 0.01. crystal structure of the receptor-binding domain from newly emerged middle east respiratory syndrome coronavirus wild-wide-type and innate immune-deficient mice are not susceptible to the middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters the crystal structure of dipeptidyl peptidase iv (cd26) reveals its functional regulation and enzymatic mechanism close relative of human middle east respiratory syndrome coronavirus in bat molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 middle east respiratory syndrome coronavirus (mers-cov) -update. world health organization adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase 4-mediated entry of the middle east respiratory syndrome coronavirus crystal structure of human dipeptidyl peptidase iv/cd26 in complex with a substrate analog host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 rapid generation of a mouse model for middle east respiratory syndrome we thank drs linqi zhang and xinquan wang for their kind support and helpful suggestions. this work was supported by national natural science fund 81101236, 81471929, ministry of science and technology of china (2014cb542500), the national science and technology major projects (2012zx10001-004). key: cord-273487-nfgjz6f9 authors: xu, zaikun; hobman, tom c. title: the helicase activity of ddx56 is required for its role in assembly of infectious west nile virus particles date: 2012-11-10 journal: virology doi: 10.1016/j.virol.2012.08.011 sha: doc_id: 273487 cord_uid: nfgjz6f9 although flaviviruses encode their own helicases, evidence suggests that cellular helicases are also required for replication and/or assembly of these viruses. by and large, the mechanisms of action for viral and cellular helicases are not known. moreover, in some cases, enzymatic activity is not even required for their roles in virus biology. recently, we showed that expression of the host nucleolar helicase ddx56 is important for infectivity of west nile virus (wnv) particles. in the present study, we demonstrate that the helicase activity of this enzyme is essential for its role in assembly of infectious wnv virions. over-expression of the capsid-binding region of ddx56 also reduces infectivity of wnv suggesting that interaction of ddx56 and capsid protein is an important step in the virion assembly pathway. to our knowledge, this is the first study showing that enzymatic activity of a cellular helicase is critical for infectivity of flaviviruses. west nile virus (wnv) is an important human pathogen that can cause severe neurological disease (reviewed in brinton, 2002) . it is wide spread throughout the globe and is maintained in an enzootic cycle with birds serving as the main reservoir. similar to most other flaviviruses, mosquito-borne transmission is the main mode of human infection. currently, there are no wnv-specific vaccines or therapies that are approved for use in humans. as with all viruses, wnv is an obligate intracellular parasite that is completely dependent upon the host cell for viral entry, replication, assembly and egress. a recent study revealed that more than 300 human genes are required for replication of wnv and the closely related flavivirus, dengue virus (krishnan et al., 2008) . in addition to replication, we anticipate that many other human genes are needed for assembly and secretion of nascent wnv virions. rather than conducting genome-wide screens to identify host factors that are involved in wnv biology, we focused on cellular proteins that interact with the capsid protein. in addition to performing a critical structural role in virus assembly, our data indicate that interactions between this viral protein and the milieu of host proteins may contribute to pathogenesis van marle et al., 2007) . as well as potentially functioning in a negative capacity at the virus-host interface, interaction of wnv capsid with host proteins may be required for replication and/or virus assembly. with respect to the latter, we recently identified the nucleolar helicase ddx56 as a wnv capsid-binding protein (xu et al., 2011) . expression of ddx56 is not required for virus replication and thus could not have been identified in an rnai screen designed to identify host factors that function in this process. however, expression of the nucleolar helicase ddx56 is important for infectivity of wnv virions (xu et al., 2011) . findings from other laboratories indicate that both cellular and viral helicases play critical roles in the biology of flaviviruses (ariumi et al., 2007; krishnan et al., 2008; mamiya and worman, 1999; owsianka and patel, 1999; you et al., 1999) . accordingly, considerable efforts have been directed toward determining the feasibility of targeting cellular and viral helicases as a means to block viral infections (geiss et al., 2009; kwong et al., 2005; maga et al., 2011; stankiewicz-drogon et al., 2010) . to aid in development of anti-viral therapies that target rna helicases which are integral to flavivirus biology, it is critical to understand how these enzymes function in this capacity. indeed, evidence suggests that some rna helicases are required for replication of viral rna whereas others are needed for assembly of infectious virus particles. in addition to serving as co-factors during virus replication and assembly, the mechanism by which a given helicase functions in these processes may vary considerably. moreover, in some cases the enzymatic activity of a helicase may not even be required for its role in the infection pathway. for example, the helicase activity of the ns3 protein is dispensable for its function assembly of infectious yellow fever virions (patkar and kuhn, 2008) . in the present study, we investigated the mechanism by which the nucleolar helicase ddx56 functions in morphogenesis of wnv virions. our data show that inactivating mutations in the dead box motif of ddx56 impair its role in production of infectious wnv virions. similarly, over-expression of the capsid-binding region of ddx56 has a negative impact on wnv infectivity. together, these findings are consistent with a scenario in which the interaction between catalytically active ddx56 and the wnv capsid protein is an important step during packaging genomic rna into nascent virions. as a first step toward determining whether the enzymatic activity of ddx56 is required for infectivity of wnv particles, we constructed ddx56 mutants that lack helicase function. the dead box motif is highly conserved in a subgroup of rna helicases (schmid and linder, 1992) and mutagenesis studies have shown that substitution of asparagine for aspartate or glutamine for glutamate results in complete loss of helicase activity (pause and sonenberg, 1992) . in addition to creating d166n and e167q mutations in ddx56, silent mutations were introduced downstream of the dead box motif in an shrna target site (fig. 1) . the latter mutations allow the expression of the dead box mutants in stable cell lines in which endogenous levels of ddx56 have been reduced by rna interference ( fig. 2a) . hek293t cells stably expressing ddx56-specific or nonsilencing shrnas were transduced with lentiviruses encoding acgfp and myc-tagged wild type ddx56 or dead box mutants. expression of the myc-tagged ddx56 proteins was monitored by immunoblot analyses at 48 h post-transduction. immunoblot analyses show that compared to expression in non-silencing (ns) cells, the level of wild type myc-tagged ddx56 (wt-rnai-s) in ddx56 knockdown cells was just on the threshold of detection with the anti-myc antibody (fig. 2b, lanes 3 and 9) . in contrast, the myc-tagged rnai-resistant forms of wild type ddx56 as well as the d166n and e167q mutants were robustly expressed in the ddx56 knockdown cells (fig. 2b, lanes 10-12) . these data demonstrate that mutant versions of ddx56 are stable in a cellular background in which endogenous ddx56 is depleted by rnai. dead box mutants are correctly targeted to the nucleolus next, it was important to demonstrate that the dead box mutants were correctly targeted to the nucleolus similar to wild type ddx56 (zirwes et al., 2000) . mock-and wnv-infected a549 cells were transfected with plasmids encoding wild type or dead box mutants of ddx56. as evidenced by colocalization with nucleolin, both rnai-sensitive and -resistant forms of myctagged ddx56 are targeted to the nucleoli (fig. 3) . similarly, the dead box mutants d166n and e167q localized to nuclei of a549 cells. these data confirm that mutations in the dead box motif or shrna targeting sites within the ddx56 open reading frame do not alter targeting of the helicase. to determine whether enzymatic activity of ddx56 was important for viral gene expression and/or infectivity of wnv, ddx56 knockdown cells expressing rnai-sensitive or resistant forms of myc-tagged ddx56 proteins were infected with wnv. at 48 h post-infection, cell lysates and supernatants were subjected to immunoblot analyses and plaque assays, respectively. the immunoblot in fig. 4a shows that based on acgfp expression, similar levels of transduction efficiency were achieved for all of the ddx56 constructs. as expected, only the rnai-resistant forms of ddx56 were detected by immunoblotting with antimyc. in agreement with a recent report from our laboratory (xu et al., 2011) , titers of wnv from ddx56 depleted cells were reduced more than 100 fold compared to those from nonsilencing controls (fig. 4b) . however, infection of cells expressing rnai-resistant ddx56 resulted in normal viral titers, which were well over 1 â 10 8 pfu/ml. in contrast, infection of cells expressing rnai-sensitive ddx56 or dead box mutants produced viral titers that were similar to ddx56 knockdown cells that had been transduced with a lentivirus encoding acgfp only. capsid protein was efficiently co-immunoprecipitated with d166n and e167q mutants (fig. 4c ) and thus the failure of these dead box mutants to complement the production of fig. 1 . construction of rnai-resistant forms of ddx56 with mutations in the dead box motif. aspartate (d) and glutamate (e) residues in the dead box motif of ddx56 were changed to asparagine (n) and glutamine (q) to produce d166n and e167q mutants, respectively. in addition, silent mutations were introduced into sirna-binding sites to make the resulting constructs resistant to rnai. all constructs contain a myc tag at the c-terminus. infectious wnv virions was not due to an inability to bind capsid. despite the large decrease ( 4 100-fold) in viral titers, expression of dead box mutants did not interfere with replication of wnv. this conclusion is based on the observation that levels of wnv capsid protein were normal in these cells (fig. 4a) . the stable hek293t cell lines were transduced with lentiviruses encoding normal (rnai-sensitive) or rnai-resistant versions of myc-tagged wt and d166n and e167q mutants. the lentiviruses also encode acgfp, which is used to monitor transduction of cells. forty-eight hours post-transduction, levels of endogenous and myc-tagged ddx56 proteins were determined by immunoblotting. fig. 3 . mutations in the dead box active site of ddx56 do not affect targeting to the nucleolus. a549 cells were transfected with plasmids encoding myc-tagged wild type (wt), d166n, and e167q mutants as well as a vector. twenty-four hours post-transfection, cells were infected with wnv (moi ¼5) for 24 h and then fixed and processed for indirect immunofluorescence using mouse anti-myc, rabbit anti-nucleolin, and guinea pig anti-wnv capsid antibodies. primary antibodies were detected using donkey anti-mouse alexa546, donkey anti-rabbit alexa647 and goat anti-guinea pig alexa488 secondary antibodies. arrowheads indicate colocalization between capsid, ddx56 proteins and the nucleolar resident protein nucleolin. nuclei were stained with dapi. rnai-sensitive (rnai-s) and rnai-resistant (rnai-r) forms of wt and ddx56 mutants are indicated. images were captured using a leica tcs sp5 confocal scanning microscope. size bar ¼10 mm. our recent studies suggest that ddx56 may function in packaging viral genomic rna into nascent virions (xu et al., 2011) . to establish whether the helicase activity of ddx56 is important for this process, we assayed the relative amounts of genomic rna in wnv particles secreted from cells expressing d166n and e167q mutants. loss of ddx56 helicase activity did not affect expression of wnv capsid protein (fig. 4a ) nor its secretion from infected cells in the form of virus particles (fig. 5a ). to determine if packaging of viral rna was affected, total rna was extracted from wnv particles and the relative amounts of genomic rna (normalized to capsid protein) were determined by rt-pcr. data in fig. 5b show that virus particles isolated from infected cells expressing helicase dead mutants d166n and e167q contained 3-4 times less genomic rna than those isolated from non-silencing cells or cells expressing rnai-resistant wild type ddx56. these data indicate that the enzymatic activity of ddx56 is important for packaging genomic rna into wnv virions. over-expression of the capsid-binding region of ddx56 reduces infectivity of wnv the data shown above together with results from our previous study (xu et al., 2011) are consistent with a scenario in which interaction between wnv and catalytically active ddx56 plays an important role in assembly of infectious virions. if this is indeed the case, blocking interaction between capsid and ddx56 should also reduce the yield of infectious wnv. our approach was to over-express the capsid-binding region of ddx56 in order to prevent interaction of endogenous ddx56 with capsid. to identify the region of ddx56 that binds to capsid, myc-tagged constructs encoding the n-and c-terminal regions of ddx56 were coexpressed with wnv capsid in transfected cells followed by coimmunoprecipitation and immunoblotting. data in fig. 6 show that in transfected or infected cells, the wnv capsid does not bind to the dead box helicase-containing region of ddx56 (ddx56-nt-myc), but rather, the c-terminal part of the protein, which was produced in cells expressing ddx56-ct-myc. this region of ddx56 also contains the targeting information that is required for localization to the nucleolus (fig. 7) . next, lentiviruses encoding myc-tagged ddx56 n-and cterminal regions were used to transduce hek293t cells, which were then infected with wnv. at 48 h post-infection, cell lysates and culture supernatants were subjected to immunoblot and plaque assays, respectively. data in fig. 8a show that expression of full length, n-or c-terminal regions of ddx56 do not affect viral gene expression based on the observation that similar levels of capsid protein were detected in all of the infected cell lysates. however, expression of ddx56-ct-myc reduced viral titers approximately 50-fold compared to expression of full length ddx56-myc or nt-ddx56-myc (fig. 8b ). while these results suggest that interaction between wnv capsid and ddx56 is important for a post-replication step in the wnv infection cycle, assembly and secretion of wnv particles was not affected (fig. 8c) . but the fact that significantly less viral rna was detected in the virus particles is consistent with our hypothesis that capsid-ddx56 interactions are important for incorporation packaging viral genome. conventional antiviral therapies involve targeting virusencoded enzymes that are required for replication, processing of polyproteins or viral egress from host cells. while there have been in the right panel, cell lysates were subjected to immunoprecipitation followed by immunoblotting with antibodies to myc and capsid. the protein marked by n is igg light chain. (c) hek293t cells were co-transfected with plasmids encoding gst-capsid together with wt, nt and ct ddx56 constructs or vector alone. in the left panel, relative levels of myc-tagged ddx56 proteins and capsid were determined in whole cell lysates (wcl) by immunoblotting with antibodies to the myc epitope and gst. in the right panel, cell lysates were subjected to gst-pulldown followed by immunoblotting with antibodies to myc and gst. (d) hek293t cells were co-transfected with plasmids encoding ddx56 constructs and then infected with wnv. in the left panel, relative levels of myc-tagged ddx56 proteins and capsid were determine in whole cell lysates (wcl) by immunoblotting with antibodies to the myc epitope and wnv capsid protein. in the right panel, cell lysates were subjected to immunoprecipitation by anti-capsid antibody followed by immunoblotting with antibodies to myc and capsid. notable successes using this approach, it suffers from a number of limitations. first, viruses almost invariably develop resistance to these types of drugs. this is particularly true of rna viruses whose polymerases do not possess proof reading activity and are therefore by nature, error prone and mutable. second, the choice of targets is very limited due to the small number of enzymes encoded by most viruses. in contrast, targeting host factors as means to control viral infection is a burgeoning area that has shown promise, particularly for flaviviruses. replication and assembly of infectious flavivirus particles requires hundreds of host genes (krishnan et al., 2008) . accordingly, it may be possible to target a variety of host proteins, particularly enzymes, as a means to control viral infection. although this approach may also have drawbacks, viral resistance to small molecules that block host enzymes should be minimal. rna helicases are one class of host enzymes that have been proposed as anti-viral targets (geiss et al., 2009; kwong et al., 2005; maga et al., 2011; stankiewicz-drogon et al., 2010) . among the helicase superfamily, dead box proteins form the largest group of these enzymes. these helicases are thought to play multiple roles in rna metabolism including mrna splicing and transport, transcription, translation and remodeling of ribonucleoprotein complexes. in most cases, the physiological substrates of dead box helicases have not been defined but it is clear that a number of pathogenic rna viruses require these enzymes for replication and/or assembly. for example, at least two cellular helicases, ddx1 and ddx3 function as cofactors that promote replication of hiv (fang et al., 2004; yedavalli et al., 2004) . ddx1 and ddx3 both appear to facilitate export of hiv rna from the nucleus to the cytoplasm through interaction with rev. ddx1 also stimulates replication of coronavirus rna (xu et al., 2010) . however, because coronaviruses replicate in the cytoplasm, it would appear that the role ddx1 plays in replication of these large rna viruses is distinct from its function as a cofactor for hiv. yeast two hybrid and rna interference screens have also identified dead box helicases that play significant roles in the biology of flavivirus infections (ariumi et al., 2007; krishnan et al., 2008; mamiya and worman, 1999) . many of these enzymes likely function in replication of viral rna however recent evidence suggests that helicases are needed for efficient virus assembly too. for example, we recently determined that the nucleolar helicase ddx56 is needed for a critical post-replication step in wnv morphogenesis (xu et al., 2011) . ddx56 appears to function in assembly of pre-ribosomal particles, but it is not associated with mature ribosomes (zirwes et al., 2000) . by analogy, it is tempting to speculate that ddx56 functions similarly in assembly of other large ribonucleoprotein particles such as viral nucleocapsids. the fact that ddx56 is not detected in mature wnv virions (xu et al., 2011) is consistent with this scenario. a related study by another laboratory revealed that ddx24 performs a similar function during assembly of hiv virions (ma et al., 2008) . ddx56 is not required for viability of cells in vitro (xu et al., 2011) and therefore, targeting the activity of this enzyme could prove beneficial in controlling wnv and potentially other flavivirus infections. rather than using rnai to down-regulate expression of ddx56 in cells, it would be advantageous to use small molecule inhibitors that target the enzymatic activity of this fig. 7 . nucleolar targeting information of ddx56 is contained in the c-terminal 329 amino acid residues. a549 cells were transfected with plasmids encoding myc-tagged wild type (wt) ddx56, ddx56-nt or ddx56-ct. twenty-four hours post-transfection, cells were infected with wnv (moi ¼ 5) for 24 h and then fixed and processed for indirect immunofluorescence using mouse anti-myc, rabbit anti-nucleolin, and guinea pig anti-wnv capsid antibodies. primary antibodies were detected using donkey anti-mouse alexa546, donkey anti-rabbit alexa647 and goat anti-guinea pig alexa488 secondary antibodies. arrowheads indicate colocalization between capsid, ddx56 constructs and the nucleolar resident protein nucleolin. nuclei are stained with dapi. images were captured using a leica tcs sp5 confocal scanning microscope. size bar ¼10 mm. protein. if this is to be a feasible approach, it was important to demonstrate that the helicase activity of ddx56 is in fact required for its role in wnv infectivity. while this may seem intuitive, the enzymatic function of a ''helicase'' is not always required for its role in virus biology. indeed, among highly related viral helicases, the mode of action during virus assembly can vary greatly. for example, assembly of infectious kunjin virus, a close relative of wnv, requires expression of the viral ns3 protein in cis (liu et al., 2002) . in contrast, during assembly of yellow fever virus particles, the helicase activity of ns3 is not required for this process (patkar and kuhn, 2008) . moreover, yellow fever virus ns3 can perform its virus assembly function when expressed in trans unlike kunjin virus ns3 which must be expressed in cis. the helicase activity of ddx56 is not essential for replication or assembly of wnv virions per se but our data indicate that it is critical for infectivity of virus particles. moreover, because overexpression of the capsid-binding region of ddx56 has a similar effect as reducing expression of ddx56 or knocking out its helicase activity, we hypothesize that interaction between capsid and this host enzyme facilitate loading and/or organization of viral rna during virus assembly. through an unknown mechanism, wnv infection causes relocalization of ddx56 from the nucleolus to cytoplasmic structures that are enriched in capsid protein (xu et al., 2011) . it is likely that these capsid-positive cytoplasmic elements are virus assembly sites on the endoplasmic reticulum. next, it will be of interest to develop small molecule inhibitors that can be used to target the helicase activity of ddx56. these inhibitors would not be expected to block replication of wnv, but by reducing the infectivity of nascent virions, viral spread would be minimized thereby allowing the immune system to contain the infection. laboratory reagents were purchased from the following sources: complete tm edta-free protease inhibitor cocktail from roche diagnostics (laval, quebec); protein g-sepharose from ge healthcare bio-sciences ab (piscataway, nj); per-fectin transfection reagent from genlantis (san diego, ca); transit s -293 transfection regent from mirusbio (madison, wi); pierce bca protein assay kit from thermo scientific (rockford, il); human full-length verified ddx56 cdna (clone id 3456547) and human gipz lentiviral shrnamir individual clone (clone id v3lhs_353980) targeting ddx56 mrna from open biosystems (huntsville, al); hek 293t, a549 and bhk21 cells from the american type culture collection (manassas, va); perfecta tm sybr s green supermix low rox from quanta biosciences (gaithersville, md); superscript iii reverse transcriptase cdna synthesis kit, random primers, prolong s gold anti-fade reagent with 4 0 -6-diamidino-2-phenylindole (dapi) and all other reagents for mammalian cell culture were obtained from invitrogen (carlsbad, ca). antibodies were obtained from the following sources: ddx56specific mouse monoclonal from progen biotechnik (heidelberg, germany); rabbit and guinea pig polyclonal antibodies to wnv capsid protein were generated in this laboratory xu et al., 2012) ; myc-specific mouse monoclonal antibodies were purified from 9e10 hybridoma cells from atcc (manassas, va); b-actin-specific mouse monoclonal antibody, gst-specific rabbit polyclonal antibody and nucleolin-specific rabbit antibodies from abcam (cambridge, ma); green fluorescent protein (gfp)-specific rabbit polyclonal antibody from dr. l. berthiaume (university of alberta, edmonton, canada); goat anti-rabbit and goat anti-mouse igg conjugated to horseradish peroxidase from jackson immuno-research laboratories (west grove, pa); donkey anti-mouse igg conjugated to alexa fluor 546, donkey anti-rabbit igg conjugated to alexa fluor 647 and goat anti-guinea pig igg conjugated to alexa fluor 488 from invitrogen (carlsbad, ca). all plasmids were constructed using pcr and standard subcloning techniques. prior to use in experiments, the newly constructed plasmids were verified by diagnostic restriction endonuclease digestion and dna sequencing. constructs encoding myc-tagged rnai resistant forms of ddx56 were generated using the overlap extension pcr method. for the first pcr, the plasmid pcdna3.1(-)-ddx56-myc (xu, anderson, and hobman, 2011) served as the template. primer pairs used for the first pcr were noti-f and rnai-resistant-r and mlui-r and rnai-resistant-f (table 1 ). the rnai-resistant mutations were designed based on the target sequence recognized by the ddx56specific shrnamir-353980 targeting sequence as shown in fig. 1 . the product of the overlap pcr which was amplified using primers noti-f and mlui-r was subcloned into the not i and mlu i sites of plvx-tight-puro to produce plvx-ddx56-wt-rnai-r. to generate rnai-resistant dead box mutants of ddx56, the wild type rnai resistant ddx56 cdna produced as described above was subjected to pcr using the primer pair noti-f/mlui-r together with primers that introduce mutations into the dead box motif (d166n-f and d166n-r; e167q-f and e167q-r). the resulting cdnas (d166n and e167q) were then subcloned into the not i and mlu i sites of plvx-tight-puro to produce plvx-ddx56-d166n-rnai-r and plvx-ddx56-e167q-rnai-r, respectively. the cdnas encoding rnai-resistant wild type, d166n and e167q ddx56 were excised from the plvx plasmids and subcloned into the spe i and xho i sites of the lentiviral vector ptrip-cmv-mcs-ires-acgfp. the resulting plasmids ptrip-acgfp-ddx56-wt, d166n, and e167q-rnai-r, respectively, direct independent expression of acgfp and ddx56 wild type and mutants. the plasmid ptrip-acgfp-ddx56-wt-rnai-s (sensitive) was created by ligation of a cdna encoding wild type myc-tagged ddx56 into the spe i and xho i sites of ptrip-ires-acgfp. dna fragments encoding myc-tagged amino acid residues 1-218 and 219-547 of ddx56 were generated by pcr with primers pairs ddx56-f/ddx56-nt-r and ddx56-ct-f/ddx56-r, respectively, using the plasmid pcdna3.1(-)-ddx56-myc (xu, anderson, and hobman, 2011) as template. the resulting cdnas were ligated into the ecor i and bamh i sites of the mammalian expression vector pcdna3.1(-)-myc. myc-tagged ddx56-nt and ddx56-ct cdnas produced by pcr using primers spei-ecorv-f and xhoi-r were also ligated into the spe i and xho i sites of the lentiviral vector ptrip-cmv-mcs-ires-acgfp. construction of the mammalian expression plasmid encoding the 105 amino acid isoform of wnv capsid, pcmv5-capsid, has been described previously . hek293t and a549 cells were cultured in dmem supplemented with 100 u/ml penicillin/streptomycin, 10% heat-inactivated fetal bovine serum and 10 mm hepes, ph 7.4, at 37 1c and 5% co 2 . dna transfections were performed using perfectin or transit s -293 transfection reagent as described by the manufacturers. a549 cells grown on coverslips were transfected with plasmids encoding myc-tagged full length and truncation ddx56 constructs. at 24 h post-transfection, cells were infected with wnv (moi¼5) for 24 h and then fixed and processed for indirect immunofluorescence microscopy. coverslips were mounted onto microscope slides using prolong gold antifade reagent with dapi, after which the samples were examined using a leica tcs sp5 confocal microscope. in all cases, samples were viewed using 63 â objective lens and images were acquired using identical tct tct tgc tgg tct tgc cat tcc-3 qrt-pcr exposure times for each channel. images were processed using image j and las af lite software. hek293t cells (1.2 â 10 6 ) were seeded into 100-mm-diameter dishes and the next day, were transfected with expression plasmids (8 mg) using perfectin. after 48 h, cells were washed with phosphate-buffered saline (pbs) and then lysed with np-40 lysis buffer (150 mm nacl, 2 mm edta, 1% non-idet p-40, 50 mm tris-hcl [ph 7.2], 1 mm fresh dithiothreitol) containing protease inhibitors on ice for 30 min. lysates were clarified by centrifugation for 10 min at 14,000 rpm in a microcentrifuge at 4 1c. small aliquots of the clarified lysates were kept for loading controls. the remaining lysates were precleared with protein a-or g-sepharose beads for 1 h at 4 1c before sequential incubation with mouse anti-myc antibodies for 3 h and then protein a-or g-sepharose beads for 2 h at 4 1c. for the gst pull-down experiments, cleared lysates were incubated with glutathione-sepharose 4b beads for 2 h at 4 1c. immunoprecipitates and gst pull-downs were washed three times with lysis buffer before the bound proteins were eluted by boiling in protein sample buffer. proteins were separated by sds-page and transferred to polyvinylidene difluoride membranes for immunoblotting. stable hek293t cell lines expressing a ddx56-specific (clone id v3lhs_353980 open biosystems) or a non-silencing shrnamir (catalog number rhs4346; open biosystems) were created by lentiviral transduction followed by puromycin selection (1 mg/ml) as described (xu et al., 2011) . after selection, the resulting polyclonal cell lines were maintained in media containing 0.25 mg/ml puromycin. hek293t cells (2.5 â 10 6 ) in 100 mm dishes were cotransfected with ptrip-acgfp plasmids encoding myc-tagged ddx56 cdnas (wt, d166n, e167q, nt or ct), pgag-pol (5.6 mg) and phcmv-vsvg (1.6 mg) using transit s -293 transfection reagent. forty-eight hours later, polybrene (4 mg/ml) and hepes (20 mm) were added to the lentivirus-containing culture supernatants which were then passed through 0.45 mm filter before aliquotting. stocks were stored at à 80 1c or used immediately to transduce hek293t or a549 cells. typically, lentiviral stocks were diluted 1:10 in dmem containing 3% fbs, polybrene (4 mg/ml) and hepes (20 mm). cells were then spinoculated by centrifugation at 1200 rpm in an eppendorf a-4-62 rotor for 1 h at 37 1c after which the plates were transferred to a 37 1c incubator. after 6 h, the media were replaced with dmem containing 10% fbs. unless otherwise indicated, transduced cells were analyzed 48 h posttransduction. wnv strain ny99 was kindly provided by mike drebot (public health agency of canada, winnipeg, mb). all virus manipulations were performed under level 3 containment conditions. unless otherwise indicated, cultured cells were infected using an moi of 5. plaque assays were conducted using bhk21 cells as described (xu et al., 2011) . to recover wnv particles from infected cells, medium from infected cells was precleared of cell debris by centrifugation for 10 min at 2500 â g, after which the resulting supernatants were passed through 0.45-mm filters. virus was inactivated by exposure to ultraviolet light in a biosafety cabinet for 1 h prior to removal of the material out of the level 3 facility. wnv virions were then recovered from the clarified medium by centrifugation at 100,000 â g for 1 h. total rna from crude wnv virion preparations was isolated with trizol s reagent (invitrogen) according to the manufacturer's instructions. reverse transcription and quantitative pcr analyses of wnv genomic rna was performed as described (xu et al., 2011) . the relative amount of wnv genomic rna in each sample was normalized to the amount of capsid protein which was determined by immunoblotting. ddx3 dead-box rna helicase is required for hepatitis c virus rna replication the molecular biology of west nile virus: a new invader of the western hemisphere a dead box protein facilitates hiv-1 replication as a cellular co-factor of rev focus on flaviviruses: current and future drug targets interactions between the west nile virus capsid protein and the host cell-encoded phosphatase inhibitor rna interference screen for human genes associated with west nile virus infection viral and cellular rna helicases as antiviral targets complementation analysis of the flavivirus kunjin ns3 and ns5 proteins defines the minimal regions essential for formation of a replication complex and shows a requirement of ns3 in cis for virus assembly the requirement of the dead-box protein ddx24 for the packaging of human immunodeficiency virus type 1 rna toward the discovery of novel anti-hiv drugs. second-generation inhibitors of the cellular atpase ddx3 with improved anti-hiv activity: synthesis, structure-activity relationship analysis, cytotoxicity studies, and target validation hepatitis c virus core protein binds to a dead box rna helicase hepatitis c virus core protein interacts with a human dead box protein ddx3 yellow fever virus ns3 plays an essential role in virus assembly independent of its known enzymatic functions mutational analysis of a dead box rna helicase: the mammalian translation initiation factor eif-4a d-e-a-d protein family of putative rna helicases synthesis of new acridone derivatives, inhibitors of ns3 helicase, which efficiently and specifically inhibit subgenomic hcv replication west nile virus-induced neuroinflammation: glial infection and capsid proteinmediated neurovirulence the cellular rna helicase ddx1 interacts with coronavirus nonstructural protein 14 and enhances viral replication the capsid-binding nucleolar helicase ddx56 is important for infectivity of west nile virus west nile virus infection causes endocytosis of a specific subset of tight junction membrane proteins requirement of ddx3 dead box rna helicase for hiv-1 rev-rre export function hepatitis c virus core protein enhances nf-kappab signal pathway triggering by lymphotoxin-beta receptor ligand and tumor necrosis factor alpha a novel helicasetype protein in the nucleolus: protein noh61 we thank eileen reklow and valeria mancinelli for technical support. z.x. is the recipient of a graduate studentship award from alberta innovates health solutions (aihs). t.c.h. holds a scientist award from aihs and is a canada research chair. this work was supported by operating funds from the canadian institutes of health research. key: cord-270534-ebkwv4zo authors: bodmer, bianca s.; fiedler, anna h.; hanauer, jan r.h.; prüfer, steffen; mühlebach, michael d. title: live-attenuated bivalent measles virus-derived vaccines targeting middle east respiratory syndrome coronavirus induce robust and multifunctional t cell responses against both viruses in an appropriate mouse model date: 2018-06-11 journal: virology doi: 10.1016/j.virol.2018.05.028 sha: doc_id: 270534 cord_uid: ebkwv4zo cases of middle east respiratory syndrome coronavirus (mers-cov) continue to occur, making it one of the who´s targets for accelerated vaccine development. one vaccine candidate is based on live-attenuated measles virus (mv) vaccine encoding the mers-cov spike glycoprotein (mers-s). mv(vac2)-mers-s(h) induces robust humoral and cellular immunity against mers-s mediating protection. here, the induction and nature of immunity after vaccination with mv(vac2)-mers-s(h) or novel mv(vac2)-mers-n were further characterized. we focused on the necessity for vector replication and the nature of induced t cells, since functional cd8(+) t cells contribute importantly to clearance of mers-cov. while no immunity against mers-cov or mv was detected in mv-susceptible mice after immunization with uv-inactivated virus, replication-competent mv(vac2)-mers-s(h) triggered robust neutralizing antibody titers also in adult mice. furthermore, a significant fraction of mers cov-specific cd8(+) t cells and mv-specific cd4(+) t cells simultaneously expressing ifn-γ and tnf-α were induced, revealing that mv(vac2)-mers-s(h) induces multifunctional cellular immunity. the middle east respiratory syndrome coronavirus (mers-cov) is a member of the coronaviridae family and emerged in 2012 in the kingdom of saudi arabia (zaki et al., 2012) . coronaviruses typically cause mild infections of the upper respiratory tract, but already in 2002, the severe acute respiratory syndrome cov (sars-cov) with a mortality rate of about 10% among infected patients was introduced in the human population. sars-cov spread world-wide and caused more than 8000 diagnosed infections, but was contained within a year after its emergence (http://www.who.int/csr/sars/country/table2004_04_21/ en/). in contrast, infections with mers-cov are ongoing for more than 5 years, with 2103 laboratory-confirmed cases distributed over 27 countries with at least 733 deaths that were reported to the who by november 2017 (http://www.who.int/emergencies/mers-cov/en/). this apparent case-fatality rate of 35% is of grave concern, because epidemic spread as has been observed for sars-cov could result in a disastrous death toll. mers-cov has been introduced zoonotically by transmission from dromedary camels to human patients (alagaili et al., 2014; haagmans et al., 2014; reusken et al., 2013a) and serological studies indicate wide-spread and early distribution among this animal host (alagaili et al., 2014; reusken et al., 2013b) . therefore, a continuous risk of transmission especially to persons in close contact to camels is evident. fortunately, the human to human transmission rate has remained low. aside of individuals with regular contact to camels, only health care workers or relatives of mers-cov patients have a considerable risk of infection (alraddadi et al., 2016; drosten et al., 2014) , but still at a modest level. nonetheless, the high case fatality rate, the recurrent outbreaks of mers-cov infections, and especially the risk of virus adaptation potentially resulting in epidemic or even pandemic spread make the development of an effective vaccine against mers-cov an international priority. the efficacy of several vaccine candidates has been demonstrated in different animal models up to even dromedary camels (reviewed in (okba et al., 2017) ). one of these candidates, mv vac2 -mers-s(h) (malczyk et al., 2015) , is based on the measles virus (mv) vaccine platform technology (mühlebach, 2017) , and encodes the mers-cov spike protein (s) as an additional antigen in the backbone of recombinant mv vac2 (del valle et al., 2007) resembling vaccine strain moraten that is authorized and in use in the us since 1968. this candidate induces both robust humoral and functional cellular immuneresponses against mers-cov. moreover, mers-cov viral load and inflammation of the lung were significantly reduced in challenged mice that had been vaccinated with mv vac2 -mers-s(h), before (malczyk et al., 2015) . while these experiments provided proof of concept for efficacy of this vaccine candidate, further mechanistic insights into the nature of the induced t cell responses remain to be elucidated. these are of special interest, since it has been shown that t cells are essential for clearance of the infection (coleman et al., 2017; zhao et al., 2014) : depletion of cd8 + t cells increased overall inflammation, bronchiolar inflammation, lymphocyte infiltration, and pleuritis at day 7 post-infection in mice (coleman et al., 2017) , while mers cov-susceptible mice depleted of all t cells were unable to clear the virus . as an alternative to the spike glycoprotein, conserved (internal) structural proteins such as the nucleocapsid protein n are of special interest as putative target of anti-viral t cell responses to be triggered by future mers vaccines . therefore, we have also generated and characterized mers-cov n protein-encoding vaccine candidates based on the mv vac2 vaccine platform, in this study. to further characterize the induction of mers cov-specific immune responses, we first analyzed the necessity for viral replication for the induction of mers cov-and mv-specific immune responses using the highly immunogenic mv vac2 -mers-s(h) vaccine candidate. in addition, we characterized the functionality of cd8 + and cd4 + t cell responses in juvenile (6-12 week old) and adult (7 months of age) mice using flow cytometry and functional assays. vero (african green monkey kidney) (atcc# ccl-81) and 293 t (atcc crl-3216) cell lines were purchased from atcc (manassas, va, usa) and cultured in dulbecco's modified eagle's medium (dmem, biowest, nuaillé, france) supplemented with 10% fetal bovine serum (fbs; biochrom, berlin, germany) and 2 mm l-glutamine (l-gln; biochrom). jawsii dendritic cells (atcc crl-11904) were purchased from atcc and cultured in mem-α with ribonucleosides and deoxyribonucleosides (gibco brl, eggenstein, germany) supplemented with 20% fbs, 2 mm l-gln, 1 mm sodium pyruvate (biochrom), and 5 ng/ml murine gm-csf (peprotech, hamburg, germany). dc2.4 murine dendritic cells (shen et al., 1997) were cultured in rpmi containing 10% fbs, 2 mm l-gln, 1% non-essential aminoacids (biochrom), 10 mm hepes (ph 7,4), and 50 μm 2-mercaptoethanol (sigma-aldrich, steinheim, germany). cells were cultured at 37°c in a humidified atmosphere containing 6% co 2 for a maximum of 6 months of culture after thawing of the original stock. the codon-optimized gene encoding mers-cov-n (genebank accession no. jx869059) flanked with aatii/mlui binding sites in plasmid pma-rq-mers-n was obtained by gene synthesis (invitrogen life technology, regensburg, germany). the antigen and the immediate early cytomegalovirus (cmv) promoter (martin et al., 2006) were inserted into plasmids p(+)br-mv vac2 -atu(p) (del valle et al., 2007) or p(+)mv vac2 -gfp(h) via mlui/aatii and sfii/sacii, respectively, to generate p(+)polii-mv vac2 -mers-n(p) or p(+)polii-mv vac2 -mers-n (h). for construction of lentiviral transfervectors encoding mers-cov-n, the orf of mers-n was amplified by pcr with primers encompassing flanking restriction sites nhei/xhoi and template pma-rq-mers-n. details on primers and pcr are available upon request. pcr products were cloned into pcr2.1-topo (invitrogen life technology) and fully sequenced. intact antigen orf was cloned into pcscw2gluc-ires-gfp (hewett et al., 2007) using nhei/xhoi restriction sites to yield pcscw2-mers-n-ires-gfp. lentiviral vectors were produced and used for the generation of antigen-expressing dendritic cell lines as described, before (malczyk et al., 2015) . in short, hiv-1-derived vectors were generated using a standard 3 plasmid system and the transfer vector plasmid pcscw2-mers-n-ires-gfp by pei transfection. subsequent purification after harvest of transfected 293 t cells yielded virus stocks used to transduce dc cell lines, which were single cell-sorted by facs and selected for antigen expression. mers-n encoding vaccine candidates mv vac2 -mers-n(p) and mv vac2 -mers-n(h) were rescued as described (malczyk et al., 2015; martin et al., 2006) . single syncytia were picked and overlaid onto 50% confluent vero cells cultured in 6-well plates and harvested as "passage 0" (p0) by scraping and freeze-thaw cycle of cells at the time of maximal infection. subsequent passages were generated as described for the following viruses. mers-n encoding vaccine viruses in p3 were used for characterization, viruses in p4 for vaccination. mers-s encoding vaccine virus mv vac2 -mers-s(h), and control virus mv vac2 -atu(p) (malczyk et al., 2015) were also used in p4 for vaccination. both as well as mv vac2 -gfp(p) and mers-cov (isolate emc/2012) (zaki et al., 2012) used for neutralization assays were propagated and titrated on vero cells by the method of spearman and kaerber (hubert, 1984; kärber, 1931) . mv vac2 -mers-s(h) was inactivated by uv-irradiation using a cl-1000 uv crosslinker (uvp, cambridge, uk). 100 μl of virus suspension in 48-well-plates on ice were exposed to uv light of 254 nm at 3 cm distance from the uv source of 1,85 × 10 5 μj/cm 2 for 30 min. inactivation of virus was controlled by incubation of vero cells with a control aliquot inactivated, in parallel. all virus stocks were stored in aliquots at −80°c. cells were lysed and immunoblotted as previously described (funke et al., 2008) . a rabbit anti-mers-cov serum (1:1000) was used as primary antibody for mers-cov-n and a rabbit anti-mv-n polyclonal antibody (1:25,000) (abcam) for mv-n detection. a donkey hrp-coupled anti-rabbit igg (h&l) polyclonal antibody (1:10,000) (rockland, gilbertsville, pa) served as secondary antibody for both. peroxidase activity was visualized with an enhanced chemiluminescence detection kit (thermo scientific, bremen, germany) on amersham hyperfilm ecl (ge healthcare, freiburg, germany). all animal experiments were carried out in compliance with the regulations of german animal protection laws and as authorized by the rp darmstadt. six-to 12-week-old or 7 months old ifnar -/--cd46ge mice (mrkic et al., 1998) deficient for type i ifn receptor and transgenically expressing human cd46 were inoculated intraperitoneally (i.p.) with 1 × 10 5 tcid 50 of recombinant viruses or uv-inactivated vaccine preparations on days 0 and either on day 21 or 28. mice were bled on days 0, 28, and 49 post initial infection (p.i.). serum samples were stored at − 20°c. mice were euthanized on days 32, 42, or 49 p.i., and splenocytes were harvested for assessment of cellular immune responses. quantification of vnts was done as described, before (malczyk et al., 2015) . in brief, mouse sera were serially diluted in 2-fold dilution steps in dmem in duplicates. a total of 50 pfu of mv vac2 -gfp(p) or 200 tcid 50 of mers-cov (strain emc/2012) were mixed with diluted sera and incubated at 37°c for 1 h. virus suspensions were added to 1 × 10 4 vero cells seeded 4 h prior to assay in 96-well plates and incubated for 4 days at 37°c. vnts were calculated as the reciprocal of the highest mean dilution that abolished infection. murine gamma interferon (ifn-γ) enzyme-linked immunosorbent spot (elispot) assays (ebioscience, frankfurt, germany) were performed according to the manufacturer's instructions using multiscreen immunoprecipitation (ip) elispot polyvinylidene difluoride (pvdf) 96well plates (merck millipore, darmstadt, germany). 5 × 10 5 isolated medium inoculated mice served as mock control. vnts were calculated as the highest dilution abolishing infectivity. dots represent single animals (n = 6); horizontal lines represent mean per group. the y-axis starts at the detection limit; all mice at the detection limit had no detectable vnt. (g) secretion of ifn-γ after antigen-specific re-stimulation of splenocytes harvested 32 days post prime immunization and after co-culture with jawsii (left) or dc2.4 (middle) dendritic cells transgenic for mers-n (black) or untransduced controls (nc, white). (right) to analyze cellular responses directed against mv, splenocytes were stimulated with 10 μg/ml mv bulk antigen (mv bulk) or left unstimulated (sham). the reactivity of splenocytes was confirmed by cona treatment (10 μg/ml). tcid 50 , tissue culture infectious dose 50; one-way-anova with tukey multiple comparison. *: p < 0,05; **: p < 0,01; ***: p < 0001; ****: p < 0,0001. splenocytes were co-cultured with different stimuli in 200 μl rpmi + 10% fbs, 2 mm l-gln, and 1% penicillin-streptomycin for 36 h. for re-stimulation of mers n-specific t cells, splenocytes were co-cultivated with 5 × 10 4 jawsii, dc2.4 dendritic cells, or clones of either cell line encoding mers-n. on the other hand, splenocytes were stimulated with 10 μg/ml mv bulk antigen (serion immunologics, würzburg, germany), 10 μg/ml mers s-derived peptide s1165 (biosynthesis inc., lewisville, tx, usa, (channappanavar et al., 2014) ), or 10 μg/ml siinfekl control peptide (sin) of ovalbumin (aa 257-264) (invivogen, san diego, ca, usa), as appropriate. for general t cell stimulation, 10 μg/ml concanavalin a (cona, sigma-aldrich, st. louis, mo, usa) was used. as negative control, splenocytes were left untreated. after 36 h, cells were removed from the plates, and plates were incubated with biotin-conjugated anti-ifn-γ antibodies and avidin-hrp according to the manufacturer's instructions. 3-amino-9ethyl-carbazole (aec; sigma-aldrich) substrate solution for development of spots was prepared according to the manufacturer's instructions using aec dissolved in n,n-dimethylformamide (merck millipore) and used for peroxidase-dependent staining, afterwards. spots were counted using an eli.scan elispot scanner (ae.l.vis, hamburg, germany) and elispot analysis software eli.analyse v5.0 (ae.l.vis). for flow cytometry-based determination of cytokine expression by intracellular cytokine staining (ics), splenocytes of vaccinated mice were isolated, and 2 × 10 6 splenocytes per mouse were cultivated in 200 μl rpmi1640 + 10% fbs, 2 mm l-gln, 1 × non-essential amino acids (biochrom), 10 mm hepes, 1% penicillin-streptomycin, 50 μm βmercaptoethanol, 10 μg/ml brefeldin a (sigma-aldrich), and one of the stimuli also used for elispot analysis. for general t cell stimulation, 0.25 μg/ml tetradecanoylphorbol acetate (tpa, sigma aldrich) and 0.5 μg/ml ionomycin (iono, sigma-aldrich) were used as positive control, and only medium was used as negative control. splenocytes were stimulated for 5 h at 37°c. subsequently, cells were stained with fixable viability dye efluor450 (ebioscience), cd4-pe (1:2000) (bd, franklin lakes, nj, usa), cd8-fitc (1:500) (bd), and, after permeabilization with fixation/permeabilization solution (bd) and perm/ wash buffer (bd), stained with ifn-γ-apc (1:500) (bd) and tnf-α-pe-cy7 (1:500) (bd). cells were fixed with ice-cold 1% paraformaldehyde (pfa) in pbs and analyzed via flow cytometry using an lsrii sorp flow cytometer (bd) and fcs express software (de novo software, glendale, ca, usa). since the nucleocapsid protein (n) of cov is quite conserved, it is regarded as an appropriate target to induce anti-viral t cells. therefore, mers-cov n was chosen as an alternative antigen to be expressed by the recombinant mv vaccine platform. full-length mers-n was cloned into two different additional transcription units (atus) either behind p (post p) or h (post h) cassettes of measles vaccine strain mv vac2 genome, and virus clones were successfully rescued and amplified in vero cells with titers of up to 2 × 10 8 tcid 50 /ml. the essential verification of antigen expression by western blot analysis of vero cells infected with the mv vac2 -mers-n vaccines revealed expression of the n antigen with only little impact of the genomic position of the transgene cassette (fig. 1a) , while growth kinetics showed no impairment of virus replication compared to the respective mv vac2 -gfp(p) control virus ( fig. 1 b, c) . to test the efficacy of the mv vac2 -mers-n candidate in vivo, genetically modified ifnar -/--cd46ge mice were chosen, since they are the prime small animal model for analysis of mv-derived vaccines (mrkic et al., 1998) . thus, 6 mice per group were inoculated via the intraperitoneal (i.p.) route on days 0 and 28 with each time 1 × 10 5 tcid 50 of mv vac2 -mers-n(p), mv vac2 -mers-n(h), or empty control virus mv vac2 -atu(p). medium-inoculated mice served as negative controls. 21 days or four days after boost immunization, sera or splenocytes of immunized mice were sampled, respectively (fig. 1d) . as expected, all mice immunized with recombinant mv (including the control virus) developed high mv virus neutralizing titers (vnt) (512-2048 vnt, fig. 1f ). little evidence for induction of neutralizing antibodies against mers-cov was found in all mice, as expected for the intra-particular antigen (fig. 1e) . no vnts against mv or mers-cov were detected in control mice inoculated with medium alone. to analyze splenocytes of animals vaccinated with mv vac2 -mers-n (h) or control animals inoculated with medium or mv vac2 -atu(p) by elispot assay for antigen-specific ifn-γ secretion, the antigen-specific t cells were re-stimulated in vitro by syngeneic murine dc cell lines (jawsii and dc2.4), which had been genetically modified by lentiviral vector transduction to stably express mers-n protein and thereby to present the respective t cell epitopes on mhc. single cell clones were derived by flow cytometric sorting of single gfp-positive cells. antigen expression by transduced dcs was verified by western blot analysis (data not shown). elispot assays using splenocytes of vaccinated animals in co-culture with jawsii-mers-n or dc2.4-mers-n revealed about 200 ifn-γ secreting cells per 1 × 10 6 splenocytes after immunization with mv vac2 -mers-n(h) (fig. 1g) , which was significant over controls. additionally, cellular immune responses targeting mv antigens were detected upon stimulation with mv bulk antigens in vaccinated mice that had received any recombinant virus, as expected. however, mv bulk antigens stimulated about 930-1500 ifn-γ secreting cells per 1 × 10 6 splenocytes of mv vaccinated animals, as described, before (malczyk et al., 2015) . finally, splenocytes of all mice revealed a similar basic reactivity to unspecific t cell stimulation, as confirmed by similar numbers of ifn-γ secreting cells upon cona treatment (fig. 1g) . thus, the generated mv-based vaccine platform expressing mers-n induces significant mers n-specific cellular immune responses, as desired. in any case, humoral and cellular responses induced by vaccine candidate mv vac2 -mers-s had been considerably higher in previous analyses under similar conditions (malczyk et al., 2015) . therefore, further characterization of anti-mers-cov immunity induced by mv vac2 -derived vaccines proceeded with this mers-s encoding vaccine candidate, which yielded approximately 5-fold higher numbers of reactive t cells after vaccination. since the mers vaccine candidate mv vac2 -mers-s(h) induced robust protective humoral and cellular immune responses in ifnar -/--cd46ge mice (malczyk et al., 2015) , we were interested in the necessity of viral replication of this life-attenuated vaccine for the induction of mers cov-specific immunity. for these analyses ifnar -/--cd46ge mice were chosen as the animal model, again, since these mice are the standard animal model for analysis of mv-derived vaccines (mühlebach, 2017) , their genetic composition is compatible with an established mers-cov challenge model , as shown, before (malczyk et al., 2015) , and their size allows housing under regularly available conditions opposed to dromedary camels, the only know natural host of mers-cov, to date. as all morbilliviruses, the mv-based vaccine virions are highly cellassociated, and transfer of antigenic protein within the vaccine preparation cannot be excluded. therefore, we vaccinated these mv-susceptible mice with either 1 × 10 5 tcid 50 of live or of the same formulation and quantity uv-inactivated mv vac2 -mers-s(h) in a primeboost regimen ( fig. 2a) . mv vac2 -atu(p), which does not encode any additional antigen, was included as vector control. blood was drawn from naïve mice on day 0 before vaccination, and on days 28 and 49 post-immunization. serum samples were tested for their ability to neutralize mv vac2 -gfp(p) (fig. 2b , d, f) or mers-cov (fig. 2c, e, g) . sera of naïve mice had no neutralizing antibodies against either virus (fig. 2b, c) . after the first immunization, both live virus preparations induced neutralizing antibodies against mv, with mv vac2 -atu(p) triggering significantly higher titers (1280-1920 vnt) than mv vac2 -mers-s(h) (480-640 vnt). after the second immunization, anti-mv vnts increased to titers of 640-2560 in both cohorts. in contrast, only one out of four animals in the uv-inactivated vaccine group had a borderline neutralizing antibody titer of 20 after the first immunization, and another animal had a titer of 30 after the boost. while mv vac2 -atu(p) and the uv-inactivated mv vac2 -mers-s(h) vaccine did not induce neutralizing antibodies against mers-cov above background levels over the course of the experiment, the group vaccinated with live mv vac2 -mers-s(h) developed titers around 50 after the first immunization and 40-320 (mean of 300) after the boost. taken together, these data reveal that replication of the vaccine is necessary to induce functional antibody responses against mv and the additional antigen mers-s. to assess the capacity of the different mv vac2 -mers-s(h) vaccine preparations to induce mers-cov s-specific cellular immune responses, splenocytes of mice, which had already been tested for humoral responses ( fig. 2a) , were isolated and analyzed 49 days after immunization for antigen(ag)-dependent ifn-γ secretion using elispot assay. the isolated splenocytes were re-stimulated with mers-s immunodominant peptide s1165 (channappanavar et al., 2014) or mv bulk antigen (mv bulk) to analyze mv-specific cellular immune responses. ovalbumin-derived siinfekl-peptide (sin) served as peptide negative control, or cells were left untreated (mock). stimulation with concanavalin a (cona) was used to confirm general t cell reactivity in splenocyte preparations (fig. 2h) . while splenocytes of all mice responded to cona with 1000 to 1500 spots per 1 × 10 6 splenocytes, only those from animals vaccinated with live mv vac2 -mers-s(h) could be stimulated with mers s-specific peptide s1165 reaching mean values of 1040 spots per 1 × 10 6 splenocytes. in contrast, splenocytes of the uv-inactivated group or control virus mv vac2 -atu(p) could not be restimulated to secrete ifn-γ. furthermore, only replication-competent vaccine viruses induced mv-specific cellular immune responses in vaccinated mice. re-stimulation with mv bulk ag induced a mean of 800 and 970 spots per 1 × 10 6 splenocytes for mv vac2 -mers-s(h) or mv vac2 -atu(p) vaccinated mice, respectively. consequently, replication of the vaccine candidate is essential to induce both arms of the immune system with responses against mv as well as the additional mers-s antigen. usually, 6-12 weeks old juvenile mice are used for our mers-cov neutralizing antibodies in sera of (b, c) naїve mice, or in sera of mice after (d, e) prime-or (f, g) boost-immunization. one-way anova with tukey multiple comparison. * : p < 0,05; * *: p < 0,01; ***: p < 0001; ****: p < 0,0001. (h) secretion of ifn-γ after antigen-specific re-stimulation of splenocytes. ifn-γ elispot analysis using splenocytes of mice vaccinated on days 0 and 28 with indicated vaccines isolated 21 days after boost immunization and after incubation with indicated stimuli (mers-s peptide s1165, mv bulk antigen (mv bulk), immunodominant ovalbumin-derived siinf-ekl-peptide (sin) as a peptide negative control) or untreated (mock). the reactivity of splenocytes was confirmed by concanavalin a (cona) treatment (10 μg/ml). the number of cells per 1 × 10 6 splenocytes represent the amount of cells expressing ifn-γ upon re-stimulation. dots represent individual animals, horizontal bars mean. one-way anova with tukey multiple comparison. ****: p < 0,0001. b.s. bodmer et al. virology 521 (2018) 99-107 immunization studies. to evaluate if there is an age-dependent change in vaccination efficacy, approximately 7 months-old mice were vaccinated with mv vac2 -mers-s(h) in a prime-boost vaccination scheme with 3 weeks between prime and boost vaccination (fig. 3a ). mice were sacrificed at day 42 post-immunization, and splenocytes were re-stimulated with mv-antigens or mers-s peptide s1165. we found that reactive ifn-γ-secreting t cells were also specifically induced in mice of this age (fig. 3b) . a mean of 520 spots per 1 × 10 6 splenocytes was detected upon re-stimulation with mv bulk antigen, whereas 900 spots per 1 × 10 6 splenocytes were induced by re-stimulation with the mers s-derived peptide s1165, illustrating that mv-and mers-cov-specific cellular immune responses are effectively induced in adult mice. to gain more detailed insights in the quality of the observed t cell responses, we further characterized the responsive t cell populations by flow cytometry, determining the expression of cd8 + and cd4 + surface markers as well as ifn-γ and tnf-α upon re-stimulation with s1165 or mv bulk antigen. as a positive stimulus for t cell activation tetradecanoylphorbol-acetate and ionomycin (tpa/iono) were used. exocytosis of cytokines was blocked by addition of brefeldin a (10 μg/ ml) during stimulation. cells were permeabilized, labelled, and fixed for flow cytometry. the gating strategy excluded duplicates (not shown), selected for living cells (fig. 4a, upper panel) , and separated cd8 + and cd4 + t cells (fig. 4a, lower panel) . selected cd8 + t cells were then analyzed for their expression of ifn-γ (fig. 4b left panel) , tnf-α (fig. 4b middle panel) , or both ( fig. 4b right panel) as exemplarily shown for splenocytes re-stimulated with mers-s peptide s1165. likewise, cd4 + t cells expressing ifn-γ (fig. 4c, left panel) , tnf-α (fig. 4c, middle panel) , or both (fig. 4c , right panel) are depicted after re-stimulation with mv bulk antigen. vaccination with mv vac2 -mers-s(h) induced a significant amount of mers s-specific cd8 + t cells expressing either ifn-γ (fig. 4d, left panel) or tnf-α (fig. 4d, middle panel) , with means of 0.6% and 0.4% of total positive cells, respectively. among those, a significant fraction of cells revealed to be multifunctional, with a mean of 0.3% of all cd8 + cells or 75% of the tnf-α − responsive cells being positive for both cytokines (fig. 4d, right panel) . moreover, vaccination induced a significant fraction of vector-specific cd4 + t cells expressing ifn-γ (fig. 4e, left panel) , or tnf-α (fig. 4e , middle panel) upon re-stimulation with mv bulk antigen. among those, multifunctional cd4 + t cells expressing both cytokines were induced with a mean of about 0.1% (fig. 4e, right panel) . to conclude, vaccination with mv vac2 -mers-s(h) induces not only ifn-γ or tnf-α expressing t cells directed against mers-cov and mv, but also a significant fraction of multifunctional cytotoxic t cells specific for mers-s and cd4 + t cells specific for mv antigens, illustrating that a broad and robust mers-covspecific immune response is induced by vaccination with mv vac2 -mers-s(h). in this study, we aimed to understand the induction of immunity and the functionality of induced t cell responses after vaccination with mv vac2 -mers-s(h), a vaccine candidate that induces protective immunity against mers-cov in an appropriate animal model. in parallel, we generated and tested also alternative mv-based vaccine candidates expressing mers-cov n protein as conserved t cell antigen. mv vac2 -mers-n vaccine candidates indeed induced significant antigen-specific cellular immune responses in vaccinated transgenic mice, revealing that also mers n-expression by recombinant mv may have a useful role to combat mers-cov. since the immune responses induced by mers-s expressing candidates had been nevertheless considerably higher, we proceeded with s-expressing vaccine virus to characterize the induction of anti-mers-cov immunity by mv-based vectors. using mv vac2 -mers-s(h), robust anti-mers cov immune responses were induced also in older mice, while replication of the vaccine vector was necessary to induce either arm of adaptive immunity against vector or pathogen. furthermore, vaccination with mv vac2 -mers-s(h) triggered significant numbers of multifunctional mers s-specific cd8 + t cells and mv-specific cd4 + t cells, simultaneously producing ifn-γ and tnf-α upon stimulation with respective antigens. since not only numbers, but also the quality of the induced mers cov-specific t cell responses might be relevant for protection against mers-cov, it is quite encouraging to see that approx. 50% of ifn-γ reactive cd8 + t cells also expressed tnf-α, whereas in reverse 75% of tnf-α-reactive cd8 + t cells co-expressed ifn-γ upon stimulation with the immune-dominant mers-s peptide. this induction of multifunctional t cells is quite in accordance with previous studies, since the potential of mv during natural infection or the recombinant mv to induce multifunctional, antigen-specific t cells has already been demonstrated. infection of macaques with wild-type mv induces polyfunctional t cells specific for mv proteins with increasing numbers of cells secreting il-2, tnf-α, as well as ifn-γ over time (nelson et al., 2017) , and polyfunctional t cells directed against mv-h can be expanded from pbmc of human donors (ndhlovu et al., 2010) . likewise, hiv-vaccine candidates mv1-f4, which encode gag, rt, and nef of an hiv-1 clade b or a clade c strain as foreign antigen, induce antigenspecific multifunctional t cells simultaneously expressing ifn-γ, tnf-α, and il-2 in mice and also macaques (stebbings et al., 2013 (stebbings et al., , 2012 . while the combination of ifn-γ and tnf-α indicates functional t cells fig. 3 ) were re-stimulated and subjected to intracellular staining (ics) for ifn-γ and tnf-α and stained for extracellular t-cell markers cd4 and cd8 for flow cytometry analysis. (a -c) gating strategy for analysis of cd8 + or cd4 + t-cells expressing ifn-γ or tnf-α within splenocytes stimulated with (b) s1165 peptide or (c) mv-bulk ag. duplicates (not shown) and dead cells (a) were excluded from analysis. (b, c) cd8 + and cd4 + cells were separately subjected to analysis for ifn-γ-(left panels), tnf-α-(middle panels) or double-positive cells (right panels). quantification of flow cytometry data of (d) cd8 + -and (e) cd4 +positive cells after incubation with indicated stimuli (mers s-specific peptide s1165, mv bulk ag (mv bulk), immunodominant ovalbumin-derived siinf-ekl-peptide (sin) as a peptide negative control, or untreated cells (mock); reactivity of splenocytes was confirmed by tetradecanoylphorbol-acetate and ionomycin (tpa/iono) treatment (10 μg/ml). dots represent individual animals, horizontal bars mean. repeated-measures one-way anova with tukey multiple comparison. *: p < 0,05. with higher potency, in general, expression of il-2 is a sign of the induction of cd8 + memory t cells (williams et al., 2006) . in our study, the strong correlation of ifn-γ and tnf-α expression thus indicates a high functionality of induced t cell responses. extension of the antibody panel for il-2 detection could yield further insight into the durability of these t cell responses induced by the mv vaccine platform in future studies. such multifunctional cd8 + t cells specific for mers-cov may become especially important, since mouse studies have shown that cd8 + t cells are crucial for clearance of mers-cov infection (coleman et al., 2017; zhao et al., 2014) . noteworthy, for other viral infections such as human immunodeficiency virus (hiv), modified vaccinia virus ankara (mva), or cytomegalovirus (cmv) the amount of just ifn-γ producing t cells does not correlate with ctl killing effectivity, but the multifunctionality of antigen-specific t cells inversely correlated with viral load (betts et al., 2006; lichterfeld et al., 2004; precopio et al., 2007; sandberg et al., 2001) , further underlining the importance of multifunctionality. besides these cellular immune responses, also considerable humoral immunity was induced in vaccinated animals, here. the mean vnt was somewhat lower than expected (malczyk et al., 2015) , but still quite high. an alternative vaccine candidate derived from modified vaccinia virus ankara, mva-mers-s, revealed protection in dromedary camels, the natural host for mers-cov (haagmans et al., 2016) . passive immunotherapy with dromedary immune sera significantly reduced mers-cov titers in lung tissue of challenged mice, starting with a vnt of 160 (zhao et al., 2015) . neutralizing antibody titers in reconvalescent plasma of human patients diagnosed with mers were determined by microneutralization tests in two previous studies, and were on average at 175 (arabi et al., 2016) or 58.3 (zhao et al., 2017) . furthermore, a prnt 50 titer of at least just 50 was required to lower virus titers by more than 0.5 log in mice challenged after transfer of human reconvalescent plasma (zhao et al., 2017) . these titers were exceeded in this study. in addition, mv vac2 -mers-s(h) induced higher anti-mers-s titers in c57/bl6 mice than mva-mers-s in balb/c mice, when comparing studies that used similar virus titers for vaccination (malczyk et al., 2015; volz et al., 2015) , thus indicating an at least comparable efficacy. thus, also vnt determined here indicate efficacy and were anyway not statistically different from those published before for mv vac2 -mers-s(h) (malczyk et al., 2015) . nevertheless, the exact correlates of protection for mers-cov remain to be determined in future studies, since it will be essential to evaluate the efficacy of the different vaccine candidates against this most important benchmark. in contrast, uv-inactivated mv vac2 -mers-s(h) did not induce any antibodies able to neutralize mers-cov or mv. while neutralizing antibodies can in principle also be induced by inactivated vaccines or proteins, e.g. full-length or truncated mers-s protein in combination with adjuvant (wang et al., 2015) . obviously, the amount of mers-s antigen within the mv vac2 -mers-s(h) vaccine formulation or the adjuvant effect of the inactivate were not sufficient during application. therefore, replication of the mv-derived mers vaccine candidate is necessary for the induction of immune responses both against vector and antigen of interest in vaccinated animals. indeed, the induction of cellular immunity is usually more efficient by de novo expression of antigen after immunization. consequently, the application of a replication competent vaccine platform is justified here to robustly induce potent responses of both arms of the adaptive immune system. these powerful immune responses were not only induced in juvenile mice 6-12 weeks of age, but also in adult mice older than half a year of age. this is quite of interest, since adult vaccinees are also the target group for vaccination in response to the mers-cov outbreak, as defined in the target product profile by the who (http://www.who.int/ blueprint/what/research-development/mers_cov_tpp_ 15052017.pdf). remarkably, mv vaccine strain virus encoding chikungunya virus (chikv) antigens was already tested in a phase i clinical trial in adult human vaccinees (18-45 years old) (ramsauer et al., 2015) . these adult test subjects all developed significant humoral immunity against chikv, despite their adult age and most interestingly also independent from measles pre-immunity. taken together, these study shows that mv vac2 -mers-s(h) induces surprisingly high numbers of multifunctional t cells specific for mers-s also in adult test subjects, as a result from replication of the recombinant vector. therefore, high quality cellular immune responses are induced in addition to the robust antibody responses by this vaccine candidate, further qualifying mv vac2 -mers-s(h) for evaluation as vaccine candidate against mers-cov. in parallel, mers-n encoding mv can be a further option to generate protection against mers in future studies and constructs. evaluation of serologic and antigenic relationships between middle eastern respiratory syndrome coronavirus 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efficacy of recombinant modified vaccinia virus ankara delivering middle east respiratory syndrome coronavirus spike glycoprotein evaluation of candidate vaccine approaches for mers-cov interleukin-2 signals during priming are required for secondary expansion of cd8+ memory t cells isolation of a novel coronavirus from a man with pneumonia in saudi arabia recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses rapid generation of a mouse model for middle east respiratory syndrome passive immunotherapy with dromedary immune serum in an experimental animal model for middle east respiratory syndrome coronavirus infection this work was supported by the german center for infection research (dzif; ttu 01.802). the authors would like to thank vivian scheuplein, jürgen schnotz, and daniela müller for excellent technical assistance. the authors are indebted to ron fouchier for providing mers-cov strain emc/2012, kenneth rock for dc2.4 cells, roberto cattaneo for providing the pb(+)mvvac2 construct, and urs schneider for providing the polii rescue system originally used to generate and to rescue recombinant mv vectors. the authors would further like to thank bakhos tannous for providing pcscw2gluc-ires-gfp. moreover, the authors would like to thank veronika von messling for critically reading the manuscript. key: cord-263302-z5uhrta5 authors: zhang, xuming; liu, runzhong title: identification of a noncanonical signal for transcription of a novel subgenomic mrna of mouse hepatitis virus: implication for the mechanism of coronavirus rna transcription date: 2000-12-05 journal: virology doi: 10.1006/viro.2000.0637 sha: doc_id: 263302 cord_uid: z5uhrta5 abstract subgenomic rna transcription of coronaviruses involves the interaction between the leader (or antileader) and the intergenic (ig) sequences. however, it is not clear how these two sequences interact with each other. in this report, a previously unrecognized minor species of subgenomic mrna, termed mrna5–1, was identified in cells infected with mouse hepatitis virus (mhv) strains jhm2c, jhm(2), jhm(3), a59, and mhv-1. sequence analysis revealed that the leader-body fusion site of the mrna is located at approximately 150 nucleotides (nt) downstream of the consensus ig sequence for mrna 5 and did not have sequence homology with any known ig consensus sequences. to determine whether this sequence functions independently as a promoter, we cloned a 140-nt sequence (from ≈70 nt upstream to ≈70 nt downstream of the fusion site) from viral genomic rna and placed it in front of a reporter gene in the defective-interfering (di) rna-chloramphenicol acetyltransferase (cat) reporter vector. transfection of the reporter rna into mhv-infected cells resulted in synthesis of a cat-specific subgenomic mrna detected by reverse transcription-polymerase chain reaction (rt-pcr). the strength of this promoter was similar to that of the ig7 (for mrna 7) as measured by the cat activity. deletion analysis showed that the sequence as few as 13 nt was sufficient to initiate mrna transcription, while mutations within the 13-nt abolished mrna transcription. in vitro translation study confirmed that the envelope (e) protein was translated from mrna5–1, which encodes the open reading frame (orf) 5b at its 5′-end, indicating that mrna5–1 is a functional message. furthermore, when the orf5b was replaced with the cat gene and placed in the di in the context of viral mini-genome, cat was expressed not only from the first orf of mrna5–1 but also from the second and third orf of mrna5 and genomic di rna, respectively, suggesting that more than one mechanism is involved in regulation of orf5b expression. our findings thus support the notion that base-pairing between the leader (or antileader) and the ig is not the sole mechanism in subgenomic rna transcription. mouse hepatitis virus (mhv), a prototype of murine coronavirus, contains a single-strand, positive-sense rna genome of ϸ32 kb in length (lee et al., 1991; pachuk et al., 1989) . upon virus infection into susceptible cells, the viral genomic rna serves both as an mrna for translation of the putative rna-dependent rna polymerase polyprotein, which is required for subsequent rna transcription and replication, and as a template for the synthesis of the genome-length, negative-strand rna that in turn is used for the synthesis of the viral genome. six to seven subgenomic mrnas (mrnas 2 to 7) are found in mhv-infected cells (lai et al., 1981; leibowitz et al., 1981) . they are co-nested at the 3ј-ends (lai et al., 1981; leibowitz et al., 1981) . each mrna contains a leader sequence of approximately 70 nucleotides (nt) at the 5ј-end, which is identical to the leader sequence of the genomic rna (lai et al., 1983 (lai et al., , 1984 spaan et al., 1983) . depending on mhv strains, there are two to four consensus ucuaa repeats with the last repeat being ucuaaac, at the 3ј-end of the leader (makino and lai, 1989a,b) . an identical or similar consensus sequence is present between each gene, termed intergenic (ig) sequence (budzilowicz et al., 1985; shieh et al., 1989) , which serves as a transcription initiation signal (promoter) for subgenomic mrna synthesis (based on the leader-primed transcription model) or a termination signal for subgenomic negative-strand rna synthesis (based on the discontinuous transcription on the negative-strand rna) (lai and cavanagh, 1997, and references therein) . the ig is the cis-acting sequence absolutely required for subgenomic rna transcription (makino et al., 1991) ; it serves as a joining point between the leader (or antileader) and the remaining body part of each subgenomic rna. for simplicity and consistency, we use the terminology according to the leader-primed transcription model (lai and cavanagh, 1997 ) throughout this report. direct sequencing of viral mrnas have revealed varying numbers of ucuaa repeats at the 3ј-end of the leader among certain subgenomic mrna species of jhm and jhm2c (makino et al., 1988 (makino et al., , 1989a , the latter of which is a naturally occurring small plaque mutant of jhm (makino et al., 1984) . further sequencing on cdna clones derived from jhm2c mrnas by reserve transcription-polymerase chain reaction (rt-pcr) has shown that the leader-body joining sites in subgenomic mrna2-1 are more heterogeneous . in addition to the varying numbers of repeats, the joining sites of some mrnas are located either upstream or downstream of the authentic consensus ig sequence, and they appear to be randomly distributed. the degree of such heterogeneity varies among jhm strains and mrna species. such phenomenon has also been observed in a recombinant mhv a59 expressing a green fluorescence protein (fischer et al., 1997) . two mrna species smaller than mrna7 were also identified in mhv-infected cells; they initiate from ig-like sequences uccaaac and ucuaaau, respectively, within the nucleocapsid gene (schaad and baric, 1993) . thus, heterogeneity of leaderbody joining sites in subgenomic mrnas may be a common phenomenon for mhv. the genomic and subgenomic mrnas of coronaviruses are capped and contain multiple open reading frames (orfs). in general, only the 5ј-most orf of each mrna is translated into a protein via the cap-dependent ribosomal scanning mechanism, while the downstream orfs are not translatable (lai and cavanagh, 1997 , and references therein). an exception was found with the orf5b of mhv, which encodes the e (envelope) protein. the e is a structural protein (yu et al., 1994) and is essential for virion assembly (vennema et al., 1996) . it was found to be translated from the second orf of the bicistronic mrna5 in vitro siddell, 1994, 1995) , suggesting that translation of the e protein is cap-independent, possibly via an internal ribosomal entry site (ires). subsequently, the ires has been roughly mapped through a series of deletion mutants to be located approximately between 100-nt upstream and 180-nt downstream of initiation codon for orf5b (jendrach et al., 1999) . the counterparts of the e gene in other coronaviruses include orf5b of bovine coronavirus (bcov), orf4 of transmissible gastroenteritis virus (tgev) and human coronavirus (hcov) 229e, and orf3c of avian infectious bronchitis virus (ibv) (godet et al., 1992; smith et al., 1990; siddell, 1995) . interestingly, while orf5b of bcov and orf4 of tgev and hcov are translated from the first orf of mrna5-1 and mrna4, respectively, the orf3c of ibv and orf5b of mhv are translated from the third and second orf of mrna3 and mrna5, respectively. more intriguingly, within tgev strains, the orf3b of muller strain is translated from the second orf of mrna3 via an ires, whereas that of the purdue strain is translated from the first orf of mrna3-1 via cap-dependent mechanism (o'connor and brian, 2000). it is not known, however, why strains of the same tgev or of various coro-naviruses evolved such distinct mechanisms in regulation of their gene expression. during the course of studying mhv transcriptional regulation, we unexpectedly identified a minor species of subgenomic mrna in jhm2c-infected cells. subsequently, the same mrna species was identified in other mhv strains. we found that the leader-body joining site for this mrna is located approximately 150-nt downstream rather than the authentic consensus ig sequence for mrna5. we thus named mrna5-1 for this novel mrna species, and accordingly, ig5-1 for this transcription initiation site. when it was placed in front of the chloramphenicol acetyl-transferase (cat) gene in the defective-interfering (di) rna-cat reporter plasmid, the ig5-1, which is devoid of any known ires sequence, can direct the synthesis of a subgenomic cat-containing mrna and expression of the cat activity, thus confirming that the ig5-1 serves as a promoter for transcription of a subgenomic mrna. deletion analyses identified that a 13-nt sequence is sufficient for driving the reporter gene transcription. furthermore, in vitro translation study confirmed that the e protein is translated from mrna5-1, suggesting that mrna5-1 is likely a functional message in virus-infected cells. previous studies have shown that subgenomic mrna2-1 of a small plaque mutant jhm2c is more heterogeneous in the leader-body joining site than that of its parental jhm strain, and that, even within the same jhm2c strain, mrna2-1 is more heterogeneous than mrna7 . to understand whether such heterogeneity is virus-strain specific or mrna-species specific, we have undertaken systematic analyses on the structure of all subgenomic mrna species of jhm2c. dbt cells were infected with jhm2c at a multiplicity of infection (m.o.i.) of 5 in the presence of actinomycin d. intracellular rnas were extracted from infected cells at 8 h postinfection (p.i.). cdnas were amplified by reverse transcription-polymerase chain action (rt-pcr) with a sense primer specific to the leader sequence and an antisense primer specific to a sequence downstream of the ig for each subgenomic mrna. pcr products were directly cloned into the ptopo2.1 ta vector. inserts were then released from the vector by digestion with a restriction endonuclease ecori and were analyzed by agarose gel electrophoresis. we found that heterogeneity of subgenomic mrnas occurred in all mrna species (fig. 1 , and further data not shown). while it was consistent with the previous finding that the heterogeneity at the leader-body fusion site appeared to be randomly distributed , transcripts amplified from mrna5-specific primers exhibited another distinct subset. as shown in fig. 1b , three types of cdna inserts for mrna5 were identified. fourteen of the 20 clones analyzed had a size corresponding to mrnas initiating at the authentic consensus ig5 site. one had a size larger than the authentic mrna5. surprisingly, a quarter (5/20) of clones had a size smaller than the authentic mrna5, suggesting that these transcripts possibly initiated at a downstream sequence. these results indicate that mrna 5 of jhm2c is also heterogeneous, but that a second initiation site might be present at a downstream location. a noncanonical sequence downstream of the consensus intergenic sequence for mrna5 likely serves as a leader-body fusion site for a smaller species of subgenomic mrna. to confirm that the cdna clones shown in fig. 1 indeed represent mrna5, and if so, what are the structural feature of these clones at the leader-body joining sites, all 20 clones were sequenced with an automated dna sequencer. as shown in fig. 2a , all 20 clones contained sequences corresponding to mrna5 of jhm2c, indicating the specificity of the rt-pcr products. consistent with the data from agarose gel analysis ( fig. 1) , the leaders of the 14 clones fused to the body at the authentic consensus ig sequence (ucuaaac) ( fig. 2a) . in one clone, the leader fused to a sequence upstream of the consensus ig sequence. this clone was not pursued for further experiments due to its singularity. all the other five smaller clones had a leader fused to a nonconsensus sequence approximately 150-nt down-stream of the authentic consensus ig sequence, with only three nucleotides complementary to the template (figs. 2a and 2b). these sequence data indicate that the noncanonical downstream sequence possibly serves as a site for leader-fusion for a smaller subgenomic mrna species, which is termed mrna5-1. to further determine whether mrna5-1 is transcribed also in other mhv strains, dbt cells were infected with jhm(2), jhm(3), a59, and mhv-1 at m.o.i. of 5. intracellular viral rnas were isolated. the primer pair 5ј-l9 and 3ј-ig6-50 was used for amplifying the 5ј-end of mrna5-1 in rt-pcr. two rounds of pcr for a total of 65 cycles were performed. the pcr fragments of ϸ600 nt in length were gel-purified and used for direct sequencing. as shown in figs. 2c-2f, the dna fragments of the four mhv strains contain the same leader-body junction site that is identical to that of the ig5-1 of jhm2c (see fig. 2b ). these results thus confirm the existence of mrna5-1 in all five mhv strains studied that uses the downstream ig5-1 sequence as the leader-fusion site. it is worth noting that minor species of mrnas other than mrna5-1 might exist, which could not be identified by direct sequencing of pcr fragments. the noncanonical transcriptional signal is a functional promoter for the synthesis of a subgenomic mrna in a di rna-cat reporter system. to test whether this noncanonical sequence can serve as a transcription initiation site (promoter) for a reporter gene, a sequence of 140-nt encompassing approximately 70 nt each of the upstream and downstream of the leader-fusion site was placed in front of a cat reporter gene in a di rna vector . the di rna-cat reporter system has been extensively used for studying mhv replication, transcription, and gene expression liao et al., 1995; zhang et al., , 1997 zhang et al., , 1998 . if the noncanonical signal serves as a promoter, a cat-containing subgenomic mrna would be transcribed in helper mhv-infected cells and the cat activity would be expressed. as shown in fig. 3a , the cat activity expressed from decat5-1-transfected cells was as high as that from decat7, which contains the promoter sequence for transcription of mrna 7 . this result indicates that the 140-nt noncanonical sequence drives the expression of the cat gene in the di rna reporter plasmid. because it is reported that mhv orf5b is translated from mrna5 via an ires sequence siddell, 1994, 1995) , it is important to determine whether the cat activity resulted from translation of the bicistronic di rna or of a separate subgenomic mrna, even though this 140-nt sequence does not contain the ires sequence recently identified by jendrach et al. (1999) . to directly identify the cat-containing subgenomic mrnas, rnas were isolated from mhv-infected and decat5-1 rnatransfected cells. rnas isolated from decat7 rnatransfected cells and from mock-transfected cells were used as positive and negative controls, respectively. subgenomic mrnas were then amplified by rt-pcr with a cat-specific antisense primer and a leader-specific sense primer. as shown in fig. 3b , a specific subgenomic mrna containing the cat gene was identified in decat5-1 rna-and decat7 rna-transfected cells but not in the mock-transfected cells, indicating that a cat-containing subgenomic mrna was specifically transcribed from the 140-nt sequence of decat5-1. this result demonstrates that the noncanonical sequence can serve as an initiation signal (promoter) for subgenomic mrna transcription in a di rna-reporter system. mutational analysis of the transcriptional initiation signal. to identify the minimal sequence required for subgenomic mrna transcription, three deletions within the 140-nt sequence were made by pcr, and the deletion fragments were cloned into the di rna-cat reporter vector in place of the wild-type, full-length (140-nt) se, and mhv-1 (f), respectively. dna sequencing was carried out in the automatic dna sequencer (abi prism model 377) using the cloned plasmid dnas with a t7 promoter primer (b) or directly using gel-purified pcr fragments with primer 3јig6-50 (c-f). the consensus repeat sequence is underlined in green. the leader and mrna body sequences are indicated with arrows in red and blue, respectively. the three nucleotides (aat) at the fusion site are boxed in blue. quence (fig. 4a ). the ability of the deleted sequences in initiating subgenomic mrna transcription was then determined by both the expression of cat activity and the synthesis of cat-containing subgenomic mrnas. as shown in fig. 4a , when an upstream 63-nt sequence was deleted, the cat activity was similar to that of the fulllength [compare decat5-1(140) with decat5-1⌬63]. however, when an additional 13-nt sequence was deleted, the cat activity was significantly reduced to approximately 12-fold above the background (more than 100-fold reduction) [compare decat5-1⌬76 with decat5-1⌬63 in fig. 4a ). when a construct containing only a 13-nt sequence franking the leader-fusion site was transfected [construct decat5-1(13)], 95% of the cat activity was expressed as compared to the full-length [decat5-1(140)], indicating that the 13-nt sequence is sufficient for driving cat expression. consistent with the results of cat activity, subgenomic mrnas corresponding to the respective constructs were identified by rt-pcr in mhv-infected and di-transfected cells (fig. 4b) . the noncanonical transcription initiation signal, therefore, contains not more than 13 nt and encompasses the leader-joining site. to further determine whether this 13-nt core sequence is specifically required for mrna transcription, three mutations (-gguua-to -ggcgc-) were introduced by pcr-based site-directed mutagenesis. when the mutant di rna [decat5-1(13)m] was transfected, cat activity expressed from this rna was drastically decreased (71fold reduction) (fig. 4a) . no cat-containing subgenomic mrna was detectable by rt-pcr (fig. 4b) . these results indicate that the three nucleotides in this core sequence are required for its transcription activity. subgenomic mrna5-1 is a functional message for translation of the e protein in an in vitro translation system. to determine whether mrna 5-1 is a functional message, the open reading frame prediction program of software macvector (version 3.5) was used to analyze the sequence and to predict the possible orfs in mrna5-1. our analysis indicated that orf5b is the most probable orf at the 5ј-end of mrna5-1 (fig. 5a ). to confirm that orf5b can be expressed from mrna5-1, a fig. 3. the noncanonical sequence is a functional promoter for subgenomic mrna transcription in the defective-interfering (di) rna chloramphenicol-acetyltransferase (cat)-reporter system. (a) the names of the di rna cat reporters are shown on the left and their structures in the middle. cat activities expressed from these constructs, shown on the right, represent fold-increase against the background, which is set as onefold (1ϫ). the cat activities are representative of three independent experiments. ig5-1 and ig7 indicate the transcription initiation sites for mrna5-1 and mrna7, respectively. orf, di open reading frame. (b) reverse-transcription pcr was performed to detect cat-containing subgenomic mrnas using cat-and leader-specific primers. pcr products were analyzed by electrophoresis on a 1% agarose gel. lanes mock-tx, decat5-1(140), and decat7 are the rt-pcr products from helper jhm-infected and mock-, decat5-1(140) rna-, and decat7 rna-transfected cells, respectively. m, molecular mass marker in base pair (bp) is shown on the left. cat activities expressed from these constructs, shown on the right, represent fold-increase against the background, which is set as onefold (1ϫ). the cat activities are representative of three independent experiments. ig5-1 indicate the transcription initiation sites for mrna5-1. only the 13-nt core sequence of the ig5-1 is shown. orf, di open reading frame. thick lines denote sequence of viral origin, while the thin lines indicate their di origin. (b) reverse-transcription pcr was performed to detect cat-containing subgenomic mrnas using catand leader-specific primers as described under materials and methods. intracellular rnas were isolated from mhv-infected and various di cat rna-transfected cells as indicated at the top. pcr products were analyzed by electrophoresis on a 1% agarose gel. m, molecular mass marker in base pair (bp) is shown on the left. cdna containing the authentic 5ј-end of mrna5-1 (including the leader and orf5b) was synthesized and cloned into a plasmid vector (fig. 5a ). rnas were then synthesized by in vitro transcription with t7 rna polymerase. expression of orf5b from mrna5-1 was determined in an in vitro translation system. as shown in fig. 5b , a protein with a molecular mass of approximately 9-12 kda, which corresponds to the predicted size of orf5b, the e protein (yu et al., 1994) , was synthesized in the rabbit reticulocyte lysate (lane ivt). this protein was precipitated by an e-specific antibody (lane ivt ϩ ␣e). the reaction was specific because the same protein could not be precipitated by an unrelated antibody (the m2 monoclonal antibody against the flag-epitope) (lane ivt ϩ ␣flag), or by protein g agarose beads alone (lane ivt ϩ g). these results demonstrate that mrna5-1 is a functional message and that it translates into the e protein. translational regulation of orf5b in the di rna expression system. it was previously reported that the orf5b of mhv was expressed from the second orf of mrna5 via an ires siddell, 1994, 1995) . our current data showed that orf5b could also be expressed from the first orf of mrna5-1 in an in vitro translation reaction (fig. 5 ). an obvious question then is how orf5b is expressed in vivo (in virus-infected cells). to address this question, we made two sets of di cat reporter constructs, both of which contain the authentic orf5a and the cat gene in place of orf5b. in the first set, the ig5 consensus sequence and the 5ј-utr are present, so that orf5a would be expressed from mrna5, while in the second set the orf5a would not be expressed due to the absence of mrna5 transcription (see their structures in fig. 6 ). we then transfected these di rnas into jhm-infected cells. detection of the cat activity would indicate the expression of the orf5b from these reporter di rnas. as expected, high cat activity was detected in decat5-rna-transfected cells (1384fold increase over the background) (fig. 6a) . interestingly, the cat activity expressed from decat5m rnatransfected cells remained at a high level (921-fold above background level), though it was slightly lower than that of decat5. because there was no subgenomic di mrna5-1 transcribed from decat5m (fig. 6b) , it is conceivable that the cat activity must have been expressed cat activities expressed from these constructs, shown on the right, represent fold-increase against the background, which is set as onefold (1ϫ). the cat activities are representative of three independent experiments. ig5 and ig5-1 indicate the transcription initiation sites for mrna5 and mrna5-1, respectively. ig5-1m indicates two nucleotide mutations in the ig5-1 sequence. orf, di open reading frame. thick lines denote the sequences derived from the viral genome; dashed lines indicate deletions. (b) reverse-transcription pcr was performed to detect cat-containing subgenomic mrnas using cat-and leaderspecific primers. rnas were isolated from cells infected with a helper mhv and transfected with various di cat rnas as indicated at the top. lane mock-tx, mock-transfection. pcr products were analyzed by electrophoresis on a 1% agarose gel. the solid arrow denotes pcr products representing mrnas transcribed from ig5 site, and the unfilled arrow indicates mrnas transcribed from ig5-1 site. m, molecular mass marker in base pair (bp) is shown on the left. from either di mrna5 or genomic di rna, in the latter of which the cat gene is the third orf. in both cases, the expression of the cat activity must be mediated via internal entry of ribosome. when the ig5 consensus sequence was removed and the tis5-1 was mutated (construct decat5-1lm), the cat activity was drastically reduced (164-fold above the background). because neither di mrna5 nor di mrna5-1 was transcribed from decat5-1lm (fig. 6b) , the low level of cat activity was likely expressed from the genomic di rna via ribosomal internal entry. however, when the wild-type ig5-1 sequence was restored (decat5-1l), cat activity was significantly increased (fig. 6a) . because the wild-type ig5-1 allowed the transcription of di mrna5-1 (fig. 6b) , this result indicates that a large part of the cat activity was expressed from di mrna5-1 (compare the structure and cat activity of decat5-1l with those of decat5-1lm in fig. 6 ). taken together, these results indicate that mhv orf5b can be expressed from mrna5, mrna5-1, and even the genomic rna in vivo using the di rna expression system. in this study, we have identified a noncanonical sequence that allows the transcription of a novel subgenomic mrna species (mrna5-1) of jhm2c, jhm(2), jhm(3), a59, and mhv-1. using the mhv di rna-cat reporter system, we were able to demonstrate that the noncanonical sequence serves as a signal for transcription of a subgenomic mrna (fig. 3) . although the precise sequence for this transcription signal was not determined, a 13-nt sequence including the leader-joining site was shown to be sufficient for the expression of the di cat reporter gene (fig. 4) . furthermore, the three nucleotides at the leader-joining site appear to be required for its transcription activity, since mutations of these nucleotides abolished subgenomic mrna transcription (fig. 4) . the computer program macvector predicts that this novel mrna species encodes the orf5b of mhv at its most 5ј-end. in vitro translation studies with the rabbit reticulocyte lysate confirmed that the e protein is translated from this mrna (fig. 5) , indicating that this mrna is a functional transcript. our results thus establish for the first time that a noncanonical sequence, which has no sequence resemblance to the consensus ig sequence, can serve as a signal for transcription of a functional mrna in mhv. this finding will have important implications not only in the mechanisms of coronavirus rna transcription but also in the regulation of coronavirus gene expression both at the transcription and translation levels. the major feature in coronavirus rna transcription is the discontinuous process. regardless of whether the discontinuous transcription occurs during (ϩ)-or (ϫ)strand synthesis, subgenomic rna synthesis always in-volves the interaction between the leader (antileader) and the intergenic sequence. it has been suggested that this interaction is probably mediated by direct rna-rna interaction between the complementary sequences (see review by lai and cavanagh, 1997 , and references therein). using mhv jhm2c, we previously showed that mrna2-1 of jhm2c is very heterogeneous at its leaderbody joining site . in addition to those mrnas whose leader-body fusion occurs at the consensus ig sequence, some mrnas have a leader fused at either upstream or downstream of the consensus ig sequence. because these upstream or downstream regions do not have complementary sequences between the leader and the consensus ig site, it is less likely that direct rna-rna interaction through complementary sequences is the major determinant for this discontinuous process. while the previous observation reveals a random fashion of the heterogeneous leaderbody fusion sites, our present data show a distinct, conserved site, even though both studies have identified noncanonical sequences for leader-body fusion. thus, our current finding reinforces the notion that direct rna-rna base-pairing between the leader (antileader) and the ig region is not the sole mechanism in regulating coronavirus rna transcription. if base-pairing guides subgenomic rna transcription as shown in arterivirus (van marle et al., 1999) , then why the remaining consensus sequences that are base-paired with the leader and are distributed throughout the genomes of arteriviruses and coronaviruses do not transcribe subgenomic mrnas. this fact combined with previous fischer et al., 1997) and current findings further suggests that other mechanisms, i.e., protein-rna and protein-protein interactions and/or rna secondary structures zhang and lai, 1995; lai, 1998) , are likely involved in this discontinuous transcription process. with respect to the translation of orf5b, our data clearly show that, when the orf was placed at the 5ј-most end of an mrna (pbs-mrna5-1), it could be translated efficiently both in the rabbit reticulocyte lysate in vitro translation system (construct pbs-mrna5-1 in fig. 5 ) and in vivo in the di rna cat-reporter system (construct decat-5-1l in fig. 6) . these data suggest that orf5b is possibly expressed from mrna5-1 via the 5ј cap-dependent mechanism. however, we cannot rule out the possibility of the cap-independent pathway because part of the ires (jendrach et al., 1999) is also present in these mrnas. when the cat gene was placed in the second orf of mrna5 (construct decat5m in fig. 6 ) or in the third orf of genomic di rna (construct decat5-1lm in fig. 6) , the cat gene could also be expressed in vivo with the di cat-reporter system. these results thus clearly indicate that orf5b can be expressed via internal ribosomal entry, consistent with the finding by siddell (1994, 1995) . jen-drach et al. (1999) reported that the ires encompassing a region from approximately 100-nt upstream to 180-nt downstream of the orf5b aug start codon mediated the translation of orf5b. our results showed that the presence of the upstream sequence alone is able to mediate its expression, albeit with low efficiency (fig. 6) . it is important to point out that, although our experimental design was not intended to address the mechanism of translation, the results presented here (fig. 6 ) strongly suggest that orf5b can be expressed from mrna5, mrna5-1, and genomic rna. this finding raises an intriguing question: why does mhv use multiple pathways to regulate the expression of a single gene? to date, there is no clear answer to this question, but it is known that the expression of the e protein is diverse. for example, the e gene is expressed from the third orf of mrna3 in ibv, but from the first orf of mrna5-1 of bcov and of mrna4 of tgev and hcov 229e (siddell, 1995) . apparently, various coronaviruses have the ability to use different mechanisms for expressing their e protein. recently, o'connor and brian (2000) reported that in tgev, orf3b of the purdue strain is expressed from the first orf of mrna3-1, whereas that of the muller strain is expressed from the second orf of mrna3. our results offer another interesting possibility that orf5b may also be expressed from mhv genomic rna and subgenomic mrnas 2-4, since they contain the ires sequence. when the cat gene was placed as the third orf in the di, a low level of cat activity was expressed (fig. 6a) . one possible explanation is that a low amount of cat-containing subgenomic di rna was transcribed from the mutated ig5-1 site. however, our sensitive rt-pcr could not detect any such mrna (fig. 6b) . thus, a more reasonable explanation is that the cat activity is expressed from the genomic rna via internal entry of ribosome. this is further supported by the data obtained with the decat5m construct. however, by comparing decat5-1l with decat5-1lm (fig. 6) , one could conclude that translation of the cat gene from the genomic di rna via internal entry of ribosome must be very inefficient. by analogy to the di system, our results suggest that the e gene can also be expressed from the genomic rna and subgenomic mrnas 2-4, since they all contain the ires, but that the efficiency of such translation is low. further investigation on the mechanisms of translation of orf5b is needed. it is worth noting that the detection of mrna5-1 in jhm2c-infected cells is unlikely the result of rt-pcr artifact. we employed the same procedure for amplifying various mrna species (mrnas 2-7) and did not identify such conserved mrna species (zhang, 2000) . by contrast, we were able to amplify mrna5-1 repeatedly and in an amount proportional to mrna5 (data not shown). however, mrna5-1 is a minor species relative to mrna5 (figs. 1 and 2) . interestingly, when makino et al. (1984) analyzed the intracellular viral mrnas of jhm2c and its parental jhm strains, they found that a minor mrna species (named rna c) migrated slightly faster than mrna5 on 1% agarose gel. they noted that it was difficult to separate the major (mrna5) from the minor (rna c) rna species, but possible mixtures were noticeable. although rna c was identified in both jhm and jhm2c, it was predominantly present in jhm2c (makino et al., 1984) . sequence comparison also revealed that the ig5-1 is conserved between jhm and jhm2c (data not shown). thus, it is tempting to speculate that mrna5-1 identified in this study might represent rna c detected by makino et al. (1984) . however, rna c-like mrna species was not found in a59 and mhv-1. thus, mrna5-1 may represent a novel mrna species. cells, virus, and antibody. the murine astrocytoma cell line dbt (hirano et al., 1974) was used for virus growth, virus infection, and rna transfection. the naturally occurring small plaque mutant jhm2c, the parental jhm(2), and jhm(3) strains (makino et al., 1984 (makino et al., , 1988 , a59, and mhv-1 were used in this study. the goat antiserum specific to mhv e protein was kindly provided by dr. julian leibowitz, university of texas in college station, and its specificity was confirmed previously (yu et al., 1994) . the m2 monoclonal antibody specific to an eightamino acid flag-epitope was purchased from babco, inc. reverse transcription and polymerase chain reaction and cloning of viral subgenomic mrnas. for detection of viral subgenomic mrnas, dbt cells were infected with jhm2c at a multiplicity of infection of 5. virus grew in the presence of actinomycin d (10 g/ml). intracellular rnas were isolated from cells at 7 h postinfection by the nonidet p-40 method as described previously and used for cdna synthesis by rt with an antisense primer 3јig5-300 (5ј-gcg tag gcc gtg aag cta-3ј). this primer is complementary to a sequence approximately 300 nucleotides downstream of the ig consensus sequence between genes 6 and 5 of viral rnas. an additional sense primer (5јl9) specific to the leader was used for the subsequent pcr amplification. the conditions for the rt-pcr were essentially the same as described previously . briefly, the rt reaction was carried out at 42°c for 90 min, and the pcr was performed in a thermocycler (dna engine ptc-200, m.j. research) for 30 cycles. the condition for each cycle was denaturation at 95°c for 30 s, annealing at 62°c for 1 min, and extension at 72°c for 1 min. for detection of subgenomic mrnas containing the cat reporter gene, rt-pcr was carried out using a cat-specific antisense primer 3ј-cat542 (5ј-tta cgc ccc gcc ctg cca ctc atc gc-3ј, complementary to the 3ј-end of the cat orf) and the leader-specific sense primer 5ј-l9. pcr products were analyzed by agarose gel electrophoresis either directly or after cloning and restriction enzyme digestion as indicated. pcr products were directly cloned into the ptopo2.1 ta cloning vector (in vitrogen). for detecting mrna5-1 in other mhv strains [jhm(2), jhm(3), a59 and mhv-1], the antisense primer 3ј-ig6-50 (5ј-gct gtc cat tgg tag acg-3ј, complementary to a sequence at nt 30-47 of the orf6) was used for rt reaction and the primer pair 5ј-l9 and 3ј-ig6-50 for pcr. analysis and sequencing of cdna clones. all cdna clones for jhm2c were analyzed by restriction enzyme digestion and agarose gel electrophoresis. rt-pcr products representing cdna fragments derived from jhm(2), jhm(3), a59, and mhv-1 were purified from agarose gel with the gel extraction kit (gelexii, qiagen) and used directly for dna sequencing. sequences were determined with the automatic dna sequencer (model prizm 377, abi) in the core facility of the department of microbiology and immunology, uams. for sequencing of the cloned dna, either the t7 promoter primer or the m13 reverse primer was used; for direct sequencing of pcr fragments, primer 3ј-ig6-50 was used. plasmid constructions. for generating a di cat reporter plasmid containing the ig5-1 sequence (upstream of orf5b), a three-step jumping pcr was performed. in the first pcr, pta-ig5-370 dna was used as a template. pta-ig5-370 contains the 5ј-end 370 nt of mrna5, which was cloned after rt-pcr amplification (see figs. 1 and 2). the sense primer was 5ј-speig5-1 (5ј-tta cta gtt gtg agt gac gcc t-3ј), which contains a spei site at the 5ј-end (italic) and a sequence at nt 81-95 downstream of the consensus ig5 site. the antisense primer was 3ј-ig5-1 cat (5ј-att ttt ttc tcc ata ccc tgg ttg cta ca-3ј), which contains a sequence from nt 206 to 221 downstream of the consensus ig5 and the first 14 nt of the cat orf (italic). in the second pcr, the cat orf was amplified from the pdecat2-1 dna template with the primer pair 5ј-cat (5ј-atg gag aaa aaa at-3ј) and 3јcat542. products from these two pcr reactions were purified with the gel elution kit (qiagen) following agarose gel electrophoresis and were used as templates for a third pcr with the primer pair 5јspeig5-1 and 3ј-cat542. the final pcr products were digested with spei and bspei, and they were directionally cloned into the spei and bspei sites of pdecat2-1, generating pdecat5-1(140). this construct contains 140 nt of the ig5-1 sequence in front of the cat orf. for making two deletion constructs, pdecat5-1(140) was used as a template for pcr amplification with the primer pair 5ј-speig5-1⌬63 and 3јcat542, and 5ј-speig5-1⌬76 and 3јcat542, respectively. 5ј-speig5-1⌬63 (5ј-tta cta gtg ctt cca att taa-3ј) contains a spei site (italic) and a sequence at nt 144-156 downstream of the ig5 consensus sequence. 5ј-speig5-1⌬76 (5ј-tta cta gtt att gcc aac ccc ga-3ј) contain a spei site at the 5ј-end (italic), and a sequence at nt 157-172 downstream of ig5 consensus sequence. the pcr products were digested with spei and bspei, and directionally cloned into the spei and bspei sites of pdecat2-1, resulting in pdecat5-1⌬63 and pdecat5-1⌬76, respectively (fig. 4) . for creating di cat reporter constructs containing the 13-nt core sequence and its mutant, dnas were synthesized from the pdecat2-1 dna templates by pcr with the primer pairs 5ј-speig5-1(13)cat (5ј-tta cta gtg ctt cca att taa atg gag aaa aaa at-3ј) and 3ј-cat542, and 5ј-speig5-1(13)cgc-cat (5ј-tta cta gtg ctt ccc gct taa atg gag aaa aaa at-3ј) and 3ј-cat542, respectively. both 5ј-primers contain a spei site at the 5ј-end (italic) and the first 14 nucleotides of the cat orf at the 3ј-end. while 5ј-speig5-1(13)cat contains the wild-type, core 13-nt sequence downstream of the ig5 consensus sequence, 5ј-speig5-1(13)cgc-cat has 3-nt mutations within this core sequence (doubleunderlined). pcr fragments were digested with spei and bspei and directionally cloned into pdecat2-1, resulting in pdecat5-1(13) and pdecat5-1(13)m, respectively (fig. 4) . to construct pdecat5 (fig. 6) , rt-pcr was performed to generate cdna fragments encoding the 5a gene from jhm2c rna with the sense primer 5ј-ig5-70 (5ј-gtc tac ctt ggt agt tca a-3ј, corresponding to nt 70 to 53 upstream of the ig5 consensus sequence) and the antisense primer 3ј-ig5abcat [5ј-att ttt ttc tcc att aaa tta aac att tc-3ј, complementary to the first 14 nt of cat orf (italic) and a sequence at the overlapping junction between jhm2c orf 5a and 5b]. in the second pcr, the cat orf was synthesized with primers 5ј-cat and 3ј-cat542. pcr products were purified with the gel elution kit (qiagen) following agarose gel electrophoresis and used as templates for a third pcr with the primer pair 5ј-ig5-70 and 3ј-cat542. the pcr products were digested with spei (a natural spei site present immediately upstream of the ig5 consensus sequence) and bspei, and the digested fragments were directionally cloned into the spei and bspei sites of pdecat2-1, generating pdecat5. to create mutant pdecat5m (fig. 6) , an upstream dna fragment was amplified from pdecat5 dna templates with the sense primer 5ј-ig5-70 and an antisense primer 3ј-ig5bmcg [5ј-aac ggg aag caa aaa tct-3ј, complementary to a sequence at the ig5-1 site with 2-nt mutations (doubleunderlined) ]. an overlapping downstream dna fragment was amplified from the same templates with the sense primer 5ј-ig5bmcg [5ј-uuu ugc uuc ccg uuu aau uau ugc ca-3ј, corresponding to a sequence at ig5-1 site with 2-nt mutations (double-underlined)] and the antisense primer 3ј-cat542. these two pcr fragments were purified and used as templates for the third pcr, in which the primer pair 5ј-ig5-70 and 3ј-cat542 was used. products were cloned into the pdecat2-1 in the same manner as for pdecat5. for constructing pdecat5-1l and pdecat5-1lm, pcr was performed with the pair of primers (5ј-speig5-1 and 3ј-cat542) on the template dnas of pdecat5 and pdecat5m, respectively. the pcr products were digested with spei and bspei and directionally cloned into the pdecat2-1. mutations of these clones were confirmed by dna sequencing. for in vitro transcription and translation study, a plasmid containing the 5ј-end authentic mrna5-1 sequence was constructed in three steps. first, a cdna was synthesized with rt-pcr with the sense primer 5јspeig5-1 and the antisense primer 3јig6-50. pcr products were digested with drai to remove the 5ј-end sequence of ϸ115 nt. second, pcr was performed to generate a cdna containing the 5ј-end of mrna5-1 using pta-mrna5-1 dna as a template, which was cloned in the ptopo2.1 ta vector (see figs. 1 and 2) . the primer pair was t7 promoter primer and 3јig5-300. third, dna fragments from the first and second pcr were mixed and used as templates for a third pcr, in which the primer pair (the sense t7 promoter primer and the antisense primer 3јig6-50) was used. pcr fragments were digested with snabi and blunt-ended with t4 dna polymerase and used for cloning. pdecat2-1 dna was digested with snabi and xbai to remove all but the 5ј-end 24-nt of the di rna, blunt-ended, and used as a vector for cloning of the above pcr fragments. the resultant plasmid pbs-mrna5-1 contains the 5ј-end of mrna5-1 with the complete orf5b (fig. 5) . in vitro transcription and rna transfection. for generating di rna for transfection, plasmid dnas were linearized with xbai. rnas were transcribed with t7 rna polymerase using the megascript in vitro transcription kit according the manufacturer's instruction (ambion). for generating mrnas for in vitro translation study, plasmid dnas were linearized with xbai. capped mrnas were transcribed with the megascript kit in the presence of cap-analog (brl-gibco). dna templates were digested with rq dnase i (promega). mrnas were purified with g25 column (ambion). the in vitro transcribed di rnas were transfected into mhv-infected dbt cells with the transfection reagent dotap according to the manufacturer's instruction (boehringer mannheim) as described previously . extraction of cell lysate and cat assay. infected and transfected dbt cells from a 60-mm petri dish were harvested at 8 h p.i. in most experiments, resuspended in 150 l of 0.25 m tris-hcl (ph 8.0), and lysed by freezing and thawing three times. cellular lysate was incubated at 60°c for 10 min. following a brief centrifugation, 50 l of each sample was assayed for cat activity using an assay kit according to the manufacturer's instructions (promega). the cat reaction was carried out for 12 h as described previously . in vitro translation. the in vitro translation reaction was carried out in the nuclease-treated rabbit reticulocyte lysate system in the presence of 35 s-methionine using the in vitro transcribed rnas according to the manufacturer's recommendations (promega). immunoprecipitation. immunoprecipitation was carried out in 200 l of radioimmunoprecipitation assay (ripa) buffer (50 mm tris, ph 7.4, 150 mm nacl, 0.5% np-40, 0.1% sds, 1 mm pmsf) containing 10 l of the in vitro translation products and 1 l of the goat-anti-e polyclonal antiserum by constant rocking on a rocking platform at 4°c overnight. the antibody-antigen complexes were then precipitated with protein g-agarose beads (boehringer mannheim) at 4°c for 2 h. agarose beads were washed 3-5 times with ripa buffer. protein complexes were denatured by boiling for 3 min in lammeli's sample loading buffer (100 mm tris, ph 6.8, 200 mm dtt, 4% sds, 0.2% bromphenal blue, 20% glycerol) and analyzed by sds-polyacrylamide gel electrophoresis. the gels were exposed to x-ray film and autoradiographed. three intergenic regions of coronavirus mouse hepatitis virus strain a59 genome rna contain a common nucleotide sequence that is homologous to the 3ј-end of the viral mrna leader sequence analysis of a recombinant mouse hepatitis virus expressing a foreign gene reveals a novel aspect of coronavirus transcription tgev coronavirus orf4 encodes a membrane protein that is incorporated into virions replication and plaque formation of mouse hepatitis virus (mhv-2) in mouse cell line dbt culture characterization of an internal ribosome entry site within mrna5 of murine hepatitis virus cellular factors in the transcription and replication of viral rna genomes: a parallel to dna-dependent rna transcription characterization of leader rna sequences on the virion and mrnas of mouse hepatitis virus-a cytoplasmic rna virus the molecular biology of coronaviruses mouse hepatitis virus a59: messenger rna structure and genetic localization of the sequence divergence from the hepatotropic strain mhv 3 the presence of leader sequences in the mrna of mouse hepatitis virus the complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and rna polymerase the virusspecific intracellular rna species of two murine coronaviruses: mhv-a59 and mhv-jhm requirement of the 5ј-end genomic sequence as an upstream cis-acting element for coronavirus subgenomic mrna transcription coronavirus defective-interfering rna as an expression vector: the generation of a pseudorecombinant mouse hepatitis virus expressing hemagglutinin-esterase evolution of the 5ј-end of genomic rna of murine coronaviruses during passages in vitro high-frequency leader sequence switching during coronavirus defective interfering rna replication a system for study of coronavirus mrna synthesis: a regulated expressed subgenomic defective interfering rna results from intergenic site insertion discontinuous transcription generates heterogeneity at the leader fusion sites of coronavirus mrnas analysis of genomic and intercellular viral rnas of small plaque mutants of mouse hepatitis virus downstream ribosomal entry for translation of coronavirus tgev gene 3b molecular cloning of the gene encoding the putative polymerase of mouse hepatitis coronavirus strain a59 evidence for new transcriptional units encoded at the 3ј end of the mouse hepatitis virus genome identification of a new transcriptional initiation site and the corresponding functional gene 2b in the murine coronavirus rna genome the small membrane protein identification of a new membrane-associated polypeptide specified by the coronavirus infectious bronchitis virus coronavirus mrna synthesis involves fusion of non-contiguous sequences internal ribosome entry in the coding region of murine hepatitis virus mrna5 translation of the mhv sm protein is mediated by the internal entry of ribosomes on mrna5 arterivirus discontinuous mrna transcription is guided by base pairing between sense and antisense transcription-regulating sequences nucleocapsidindependent assembly of coronavirus-like particles by co-expression of viral envelope protein genes mouse hepatitis virus gene 5b protein is a new virion envelope protein heterogeneity of subgenomic mrnas of a mutant mouse hepatitis virus strain jhm2c unusual heterogeneity of leader-mrna fusion in a murine coronavirus: implications for the mechanism of rna transcription and recombination interaction between the cytoplasmic proteins and the intergenic (promoter) sequence of mouse hepatitis virus rna: correlation with amount of subgenomic mrna transcribed coronavirus leader rna regulates and initiates subgenomic mrna transcription both in trans and in cis expression of gamma interferon by a coronavirus defectiveinterfering rna vector and its effect on viral replication, spread and pathogenicity expression of hemagglutinin/esterase by a mouse hepatitis virus coronavirus defective-interfering rna alters viral pathogenesis this work was supported by the united states public health services grants ai 41515 and ai 47188 from the national institutes of health (to x.m.z.). we thank dr. julian leibowitz for kindly providing the anti-e protein antiserum. key: cord-272260-88l9bq4i authors: han, l.y.; cai, c.z.; ji, z.l.; chen, y.z. title: prediction of functional class of novel viral proteins by a statistical learning method irrespective of sequence similarity date: 2005-01-05 journal: virology doi: 10.1016/j.virol.2004.10.020 sha: doc_id: 272260 cord_uid: 88l9bq4i the function of a substantial percentage of the putative protein-coding open reading frames (orfs) in viral genomes is unknown. as their sequence is not similar to that of proteins of known function, the function of these orfs cannot be assigned on the basis of sequence similarity. methods complement or in combination with sequence similarity-based approaches are being explored. the web-based software svmprot (http://jing.cz3.nus.edu.sg/cgi-bin/svmprot.cgi) to some extent assigns protein functional family irrespective of sequence similarity and has been found to be useful for studying distantly related proteins [cai, c.z., han, l.y., ji, z.l., chen, x., chen, y.z., 2003. svm-prot: web-based support vector machine software for functional classification of a protein from its primary sequence. nucleic acids res. 31(13): 3692–3697]. here 25 novel viral proteins are selected to test the capability of svmprot for functional family assignment of viral proteins whose function cannot be confidently predicted on by sequence similarity methods at present. these proteins are without a sequence homolog in the swissprot database, with its precise function provided in the literature, and not included in the training sets of svmprot. the predicted functional classes of 72% of these proteins match the literature-described function, which is compared to the overall accuracy of 87% for svmprot functional class assignment of 34 582 proteins. this suggests that svmprot to some extent is capable of functional class assignment irrespective of sequence similarity and it is potentially useful for facilitating functional study of novel viral proteins. the complete genomes of 1536 viruses have been sequenced (viral genomes at ncbi http://www.ncbi.nlm. nih.gov/genomes/static/vis.html). knowledge of these genomes has facilitated mechanistic study of viral infections and provided important clues for searching molecular targets of antiviral therapeutics (herniou et al., 2003; marra et al., 2003; miller et al., 2003) . the function of over 15% of the putative protein-coding open reading frames (orfs) in these viral genomes is unknown (herniou et al., 2003; marra et al., 2003; miller et al., 2003) . determination of the function of these unknown orfs is important for a more comprehensive understanding of the molecular mechanism of specific virus and for searching novel targets for antiviral drug development. the sequence of many of these unknown orfs has no significant similarity to proteins of known functions, and their functions are difficult to probe on the basis of sequence similarity. for instance, 50%, 100%, 20%, and 67% of the unknown orfs in the recently determined genomes of ferde-lance virus (makeyev and bamford, 2004) , grapevine fleck virus (sabanadzovic et al., 2001) , indian citrus ringspot virus (rustici et al., 2002) , and sars coronavirus (he et al., 2004) are without a homolog in swissprot database (boeckmann et al., 2003) based on blast search against all swissprot entries as of september 2004. this suggests that a significant percentage of new viral proteins are likely to have no known sequence homolog. it is thus desirable to explore alternative methods or combination of methods for providing useful hint about the function of unknown viral orfs. various alternative methods for probing protein function have been developed. these include evolutionary analysis (benner et al., 2000; eisen, 1998) , hidden markov models (fujiwara and asogawa, 2002) , structural consideration (di gennaro et al., 2001; teichmann et al., 2001) , protein/gene fusion (enright et al., 1999; marcotte et al., 1999) , proteinprotein interactions (bock and gough, 2001) , motifs (hodges and tsai, 2002) , family classification by sequence clustering (enright et al., 2002) , and functional family prediction by statistical learning methods (cai et al., 2003 han et al., 2004; jensen et al., 2002; karchin et al., 2002) . in the absence of clear sequence or structural similarities, the criteria for comparison of distantly related proteins become increasingly difficult to formulate (enright and ouzounis, 2000) . moreover, not all homologous proteins have analogous functions (benner et al., 2000) . the presence of shared domain within a group of proteins does not necessarily imply that these proteins perform the same function (henikoff et al., 1997) . therefore, careful evaluation is needed to determine which method or combination of methods is useful for facilitating functional study of novel proteins with no homology to proteins of known function. the web-based software svmprot (http://jing.cz3.nus. edu.sg/cgi-bin/svmprot.cgi) to some extent has shown some potential for assigning the functional class of distantly related proteins and homologous proteins of different functions as well as homologous proteins (cai et al., 2003 . it classifies proteins into functional classes defined from activities or physicochemical properties rather than sequence similarity (bock and gough, 2001; cai et al., 2003 cai et al., , 2004 han et al., 2004; karchin et al., 2002) . in developing svmprot, proteins in a training set, represented by their sequence-derived physicochemical properties, are projected onto a hyperspace where proteins in a class are separated from those outside the class by a hyperplane. by projecting a new sequence onto the same hyperspace, svmprot determines whether the corresponding protein is a member of that class based on its location with respect to the hyperplane. the accuracy of svmprot depends on the diversity of the protein samples, the quality of the representation of protein properties, and the efficiency of the statistical learning algorithm. to some extent, no sequence similarity is required per se. thus svmprot may be potentially explored for facilitating functional assignment of proteins whose function cannot be assigned on the basis of sequence similarity. this work evaluates the usefulness of svmprot for predicting the functional class of viral orfs of unknown function. it is assessed by using novel viral proteins that are without a single homolog in the swissprot database (boeckmann et al., 2003) , with their precise function described in the literature, and are not included in the training sets of svmprot. these proteins are collected from an unbiased search of medline (wheeler et al., 2003) and swissprot database (boeckmann et al., 2003) . the svmprot predicted functional classes of these proteins are compared with the function described in the literature and databases to evaluate to what extent svmprot are useful for functional class assignment of novel viral proteins. the prediction accuracy for assignment of these novel proteins is compared with the overall accuracy of the svmprot assignment of a large number of proteins to examine the level of sequence similarity independence of svmprot classification. table 1 gives svmprot ascribed functional classes for each of the 25 novel viral proteins together with literaturedescribed function. more than one class may be characterized by svmprot and the probability of correct prediction for each class is also given in table 1 . there are 18 proteins with the top hit of the svmprot assigned functional class matching the literature-described function, representing 72% of the novel viral proteins studied in this work. these proteins are mota protein of bacteriophage t4 (gerber and hinton, 1996) , outer capsid protein vp4 of bovine rotavirus (serotype 10/strain b223) (hardy et al., 1992) , adometase of bacteriophage t3 (hughes et al., 1987) , r.cviji of chlorella virus il3a (skowron et al., 1995) , exonuclease of bacteriophage lambda (sanger et al., 1982) , r.cviaii of paramecium bursaria chlorella virus 1 (zhang et al., 1992) , orf13 of haemophilus phage hp1 (esposito et al., 1996) , protein kinase of enterobacteria phage t7 (dunn and studier, 1983) , dna-directed rna polymerase of african swine fever virus (strain ba71v) (yanez et al., 1995) , agt (miller et al., 2003) , bgt (miller et al., 2003; tomaschewski et al., 1985) , dnk (broida and abelson, 1985) , endonuclease ii (sjoberg et al., 1986) , endonuclease v (valerie et al., 1984) , gp61.9 (valerie et al., 1986) , irf protein (chu et al., 1986) , and i-tevii (tomaschewski and ruger, 1987) of enterobacteria phage t4. mota protein of bacteriophage t4 has been found to be a transcription activator that binds to dna (gerber and hinton, 1996) and the far-c-terminal region of the sigma70 subunit of escherichia coli rna polymerase (pande et al., 2002) . the top hit of svmprot predicted functional class for this protein is the dna-binding, which matches with literature-described functions. bovine rotavirus is a double-stranded rna virus that is naked. thus, the outer capsid protein vp4 of bovine rotavirus (serotype 10/strain b223) is located at the viral surface acting as part of the viral coat (hardy et al., 1992) . this protein is predicted by svmprot as a coat protein that is consistent with literature-described function. the other 14 proteins are enzymes, and these are all correctly assigned by svmprot to the respective enzyme ec class. because these proteins have no homolog of known function in the swissprot entries of swissprot database based on psi-blast search, our study suggests that svmprot has certain level of capability for providing useful hint about the functional class of novel proteins with no or low homology to known proteins, and this capability is not based on sequence similarity or clustering. the overall accuracy of 72% for the assignment of the novel viral proteins is smaller, but not too far away, than that of 87% for svmprot functional class assignment of 34 582 proteins. this indicates certain level of the sequence-similarityindependent nature of svm protein classification. several factors may affect the accuracy of svmprot for functional characterization of novel plant proteins. one is the diversity of protein samples used for training svmprot. it is likely that not all possible types of proteins, particularly those of distantly related members, are adequately represented in some protein classes. this can be improved along with the availability of more protein data. not all distantly related proteins of the same function have similar structural and chemical features. there are cases in which different functional groups, unconserved with respect to position in the primary sequence, mediate the same mechanistic role, due to the flexibility at the active site (todd et al., 2002) . this plasticity is unlikely to be sufficiently described by the physicochemical descriptors currently used in svmprot. therefore, svmprot in the present form is not expected to be capable of classification of these types of distantly related enzymes. some of the svmprot functional classes are at the level of families and superfamilies that may include a broad spectrum of proteins. it has been shown that svm works not as well as hmm for distinguishing proteins in a superfamily, but may be more accurate with subfamily discrimination (karchin et al., 2002) . thus, the use of some large families and superfamilies as the basis for classification may affect the prediction accuracy of svmprot to some extent. svmprot prediction may be further improved by using protein subfamilies as the basis of classification, more comprehensive set of protein samples, and more refined protein descriptors. svmprot optimization procedure and feature vector selection algorithm may also be improved by adding additional constraints, and by incorporating independent component analysis and kernel pca in the preprocessing steps. svmprot shows certain level of capability for predicting functional class of a number of novel viral proteins. this suggests that svmprot is potentially useful to a certain extent for providing useful hint about the function of distantly related proteins in viruses as well as in other organisms. further improvements in protein functional family coverage, sample collections, and svm algorithm may enable the development of svmprot into a practical tool for facilitating functional study of unknown orfs in virus genomes and other genomes. the key words, bnovel protein virusq or bnovel viral proteinq, are used to search the medline (wheeler et al., 2003) and the swissprot database (boeckmann et al., 2003) for finding viral proteins that are both described as novel and with their precise function provided. as the search of the medline is confined to the abstracts, those proteins whose function is not explicitly hinted in an abstract are not selected. thus, the selected proteins likely account for a portion of the known novel viral proteins with available functional information. psi _ blast (altschul et al., 1997) sequence analysis is subsequently conducted on each of these novel viral proteins against all swissprot entries in the swissprot protein database (boeckmann et al., 2003) so that those with at least one sequence homolog of known function (including that of the same protein in different species) are removed. the commonly used criterion for homologs, the similarity score e-value b the inclusion threshold value of 0.005 (altschul et al., 1997) , is used in this work. finally, those proteins that are in the training sets of svmprot are removed. a total of 25 novel viral proteins are identified in this process, which together with their protein accession number and literature-described functional indications and related references are given in table 1 . svmprot is based on a statistical learning method support vector machines (svm) (burges, 1998) . in addition to the prediction of protein functional class (cai et al., 2003 han et al., 2004; karchin et al., 2002) , svm has also been used for a variety of protein classification problems including fold recognition (ding and dubchak, 2001) , analysis of solvent accessibility (yuan et al., 2002) , prediction of secondary structures (hua and sun, 2001) , and protein-protein interactions (bock and gough, 2001) . as a method that uses sequence-derived physicochemical properties of proteins as the basis for classification, svm may be particularly useful for functional classification of distantly related proteins and homologous proteins of different functions (cai et al., 2003 . there are 75 protein functional classes currently covered by svmprot. these include 46 enzyme families, 13 channel/transporter families, 4 rna-binding protein families, dna-binding proteins, g-protein-coupled receptors, nuclear receptors, tyrosine receptor kinases, cell adhesion proteins, coat proteins, envelope proteins, outer membrane human herpesvirus 6 chemokine like (luttichau et al., 2003) no function predicted nm (continued on next page) proteins, structural proteins, and growth factors. two broadly defined families of antigens and transmembrane proteins are also included. the majority of known types of viral proteins are included in these classes. representative proteins of a particular functional class (positive samples) and those do not belong to this class (negative samples) are needed to train a svmprot classifier for this class. the positive samples of a class are constructed by using all of the known distinct protein members in that class. because of the enormous number of proteins, the size of negative samples needs to be restricted to a manageable level by using a minimum set of representative proteins. one way for choosing representative proteins is to select one or a few proteins from each protein domain family. the negative samples of a class are selected from seed proteins of the 7316 curated protein families (domain-based) in the pfam database excluding those families that have at least one member belong to the functional class. pfam families are constructed on the basis of sequence similarity. the purpose of using pfam proteins is to ensure that the negative samples are evenly distributed in the protein space. sequence similarity is not required for selecting positive samples. in this sense, svmprot is to some extent independent of sequence similarity. the svmprot training system for each family is optimized and tested by using separate testing sets of both positive and negative samples. while possible, all the remaining distinct proteins in each functional family (not in the training set of that family) are used as positive samples and all the remaining representative seed proteins in pfam curated families are used to construct negative samples in a testing set. the performance of svmprot classification is further evaluated by using independent sets of both positive and negative samples. there is no duplicate protein in each training, testing, or independent evaluation set. data set construction can be demonstrated by an illustrative example of viral coat proteins. the key word bvirus coat proteinq is used to search the swissprot, which finds 3012 entries. these entries are checked to remove noncoat proteins, redundant entries, and putative proteins, which gives 848 positive samples. these positive samples cover 140 pfam families; thus, 14 758 seed proteins of the remaining 7176 pfam families are used as the negative samples. these positive and negative samples are further divided into 346 and 1474 training, 305 and 8370 testing, and 197 and 4914 independent evaluation sets using the procedure described above. not all of the svmprot classes are at the same hierarchical level. these classes are mixtures of subfamilies, families, and superfamilies. some classes, such as antigen, need to be more clearly defined into specific subclasses. while it is desirable to define all of the classes at the same level, this is not yet possible because of insufficient data for the subhierarchies of some families and superfamilies. effort is being made to collect sufficient data so that svmprot classification systems can be constructed on the basis of a more evenly distributed family structures. transferase (wilfred et al., 2002) no function predicted nm splt13 (np _ 258405) spltmnpv virus a noval envelope protein (yin et al., 2003) no function predicted nm trl10 (aal27474) human cytomegalovirus (hcmv) structural envelop glycoprotein (spaderna et al., 2002) transmembrane ( nonetheless, prediction on the basis of the current structures provides useful hint about the function of a protein. svmprot is trained for protein classification in the following manner. first, every protein sequence is represented by specific feature vector assembled from encoded representations of tabulated residue properties including amino acid composition, hydrophobicity, normalized van der waals volume, polarity, polarizability, charge, surface tension, secondary structure, and solvent accessibility for each residue in the sequence (cai et al., 2003) . the feature vectors of the positive and negative samples are used to train a svmprot classifier. the trained svmprot classifier can then be used to classify a protein into either the positive group (protein is predicted to be a member of the class) or the negative group (protein is predicted to not belong to the class). the theory of svm has been described in the literature (burges, 1998) . thus, only a brief description is given here. svm is based on the structural risk minimization (srm) principle from statistical learning theory (burges, 1998) . in linearly separable cases, svm constructs a hyperplane that separates two different groups of feature vectors with a maximum margin. a feature vector is represented by x i , with physicochemical descriptors of a protein as its components. the hyperplane is constructed by finding another vector w and a parameter b that minimizes twt 2 and satisfies the following conditions: where y i is the group index, w is a vector normal to the hyperplane, |b| / twt is the perpendicular distance from the hyperplane to the origin and twt 2 is the euclidean norm of w. after the determination of w and b, a given vector x can be classified by: in nonlinearly separable cases, svm maps the input variable into a high dimensional feature space using a kernel function k(x i , x j ). an example of a kernel function is the gaussian kernel that has been extensively used in different protein classification studies (bock and gough, 2001; burges, 1998; cai et al., 2002; ding and dubchak, 2001; hua and sun, 2001; karchin et al., 2002; yuan et al., 2002) : linear support vector machine is applied to this feature space and then the decision function is given by: where the coefficients a i 0 and b are determined by maximizing the following langrangian expression: under conditions: a i z 0 and x l ià1 a i y i ¼ 0 a positive or negative value from eq. (3) or eq. (5) indicates that the vector x belongs to the positive or negative group, respectively. to further reduce the complexity of parameter selection, hard margin svm with threshold instead of soft margin svm with threshold is used in svmprot. scoring of svm classification of proteins has been estimated by a reliability index and its usefulness has been demonstrated by statistical analysis (cai et al., 2003; hua and sun, 2001) . a slightly modified reliability score, r value, is used in svmprot: where d is the distance between the position of the vector of a classified protein and the optimal separating hyperplane in the hyperspace, d n 0 indicates the sample belongs to the positive group and d b 0 the negative group. there is a statistical correlation between r value and expected classification accuracy (probability of correct classification) (cai et al., 2003; hua and sun, 2001) . thus, another quantity, p value, is introduced to indicate the expected classification accuracy. p value is derived from the statistical relationship between the r value and actual classification accuracy based on the analysis of 9932 positive and 45,999 negative samples of proteins (cai et al., 2003) . identification and characterization of a filament-associated protein encoded by amsacta moorei entomopoxvirus gapped blast and psi-blast: a new generation of protein database search programs functional inferences from reconstructed evolutionary biology involving rectified databases-an evolutionarily grounded approach to functional genomics predicting protein-protein interactions from primary structure the swiss-prot protein knowledgebase and its supplement trembl in 2003 sequence organization and control of transcription in the bacteriophage t4 trna region a tutorial on support vector machine for pattern recognition support vector machines for predicting hiv protease cleavage sites in protein svm-prot: web-based support vector machine software for functional classification of a protein from its primary sequence enzyme family classification by support vector machines characterization of the intron in the phage t4 thymidylate synthase gene and evidence for its self-excision from the primary transcript enhanced functional annotation of protein sequences via the use of structural descriptors multi-class protein fold recognition using support vector machines and neural networks complete nucleotide sequence of bacteriophage t7 dna and the locations of t7 genetic elements phylogenomics: improving functional predictions for uncharacterized genes by evolutionary analysis generage: a robust algorithm for sequence clustering and domain detection protein interaction maps for complete genomes based on gene fusion events an efficient algorithm for large-scale detection of protein families the complete nucleotide sequence of bacteriophage hp1 dna identification of a novel protein encoded by the bamhi a region of the epstein-barr virus protein function prediction using hidden markov models and neural networks an n-terminal mutation in the bacteriophage t4 mota gene yields a protein that binds dna but is defective for activation of transcription prediction of rna-binding proteins from primary sequence by a support vector machine approach amino acid sequence analysis of bovine rotavirus b223 reveals a unique outer capsid protein vp4 and confirms a third bovine vp4 type analysis of multimerization of the sars coronavirus nucleocapsid protein gene families: the taxonomy of protein paralogs and chimeras the genome sequence and evolution of baculoviruses 3d-motifs: an informatics approach to protein function prediction a novel method of protein secondary structure prediction with high segment overlap measure: support vector machine approach nucleotide sequence and analysis of the coliphage t3 s-adenosylmethionine hydrolase gene and its surrounding ribonuclease iii processing sites prediction of human protein function from post-translational modifications and localization features classifying g-protein coupled receptors with support vector machines a highly selective ccr2 chemokine agonist encoded by human herpesvirus 6 evolutionary potential of an rna virus detecting protein function and protein-protein interactions from genome sequences the bacteriophage t4 transcription activator mota interacts with the far-c-terminal region of the sigma70 subunit of escherichia coli rna polymerase nucleotide sequence, genome organisation and phylogenetic analysis of indian citrus ringspot virus complete nucleotide sequence and genome organization of grapevine fleck virus nucleotide sequence of bacteriophage lambda dna the bacteriophage t4 gene for the small subunit of ribonucleotide reductase contains an intron cloning and applications of the two/three-base restriction endonuclease r.cviji from il-3a virus-infected chlorella identification of glycoprotein gptrl10 as a structural component of human cytomegalovirus determination of protein function, evolution and interactions by structural genomics plasticity of enzyme active sites nucleotide sequence and primary structures of gene products coded for by the t4 genome between map positions 48.266 kb and 39.166 kb t4-induced alpha-and beta-glucosyltransferase: cloning of the genes and a comparison of their products based on sequencing data identification, physical map location and sequence of the denv gene from bacteriophage t4 nucleotide sequence and analysis of the 58.3 to 65.5-kb early region of bacteriophage t4 characterization of spodoptera exigua multicapsid nucleopolyhedrovirus orf17/18, a homologue of xestia c-nigrum granulovirus orf129 database resources of the national center for biotechnology analysis of the complete nucleotide sequence of african swine fever virus identification of a novel protein associated with envelope of occlusion-derived virus in spodoptera litura multicapsid nucleopolyhedrovirus prediction of protein solvent accessibility using support vector machines characterization of chlorella virus pbcv-1 cviaii restriction and modification system key: cord-274172-3dctmmfe authors: lucas, alexandra; coulter, marion; anderson, robert; dales, samuel; flintoff, wayne title: in vivo and in vitro models of demyelinating diseases ii. persistence and host-regulated thermosensitivity in cells of neural derivation infected with mouse hepatitis and measles viruses date: 1978-07-15 journal: virology doi: 10.1016/0042-6822(78)90289-1 sha: doc_id: 274172 cord_uid: 3dctmmfe abstract following inoculation of continuous cell lines of neural and other derivations, persistent infections are established with facility by mouse hepatitis and measles viruses. this occurs equally with the prototype mhv3 and its neurotropic variant jhm as well as with the edmonston vaccine and sspe hallé measles variants. in almost every instance that the infection becomes persistent at 32.5°, virus replication is found to be thermosensitive at 39.5°; however, progeny virus derived from such infections at 32.5° is itself thermostable when replicating in the indicator, fully permissive cell lines. the new data, therefore, reveal the existence of a host-conferred interrelationship between persistence and virus restriction at elevated temperature. they indicate that the two agents with neurotropic potential, when they become established as pathogens in the nervous system, could be under close host cell regulation involving as yet unknown mechanisms. sles associated with a progressive and fatal human disease, subacute sclerosing panen-there is abundant evidence implicating cephalitis (sspe) (hortabarbosa et al., viruses in the etiology of neurological diseases in both animals and man. studies in 1971) , and the jhm strain of murine hepvitro and in vivo using spontaneous and atitis virus pathogenic for rodents bailey et al., 1949; weiner, selected virus mutants have shown that 1973). the sspe agent known to prevail when the infection becomes defective, persistent, or latent, the agent sometimes persistently in the brain of afflicted individspreads along neuronal tracts, causes exten-uals has been evoked by growing brain explants in continuity with permissive cells sive cytopathology including demyelina(horta-barbosa et al., 1969; burnstein et tion, or may establish slow and chronic al., 1974) . the emergent virus has close infections. the mechanisms underlying serological identity with measles virus such virus-cell interactions in the nervous system have been elucidated in part for the (baublis and payne, 1963) (herndon et al., 1975) , suggesting this as a useful model for certain human demyelinating diseases. in a recent publication we described an in vitro correlate to this in uiuo model (lucas et al., 1977) , involving a rat schwannoma rn2-2 cell line, which functions as a discriminating host for the jhm virus. jhm readily established a persistent infection in rn2-2 cells during which there occurred cyclical variations in the titer of virus released into the medium and selflimiting syncytiogenesis. in contrast, infection of the rn2-2 cells with a prototype murine hepatitis virus, mhvr, was aborted immediately. in this report we describe further experiments on the two virus models in which we have compared replication of jhm versus mhv3 and the halle versus edmonston strains of measles virus in continuous cell lines of neural and other origin. cells and cell culture. the rat astrocytoma c6 cells were obtained from dr. a. marks (banting and best institute, toronto), the rat hepatoma htc cells from dr. k. yamamoto (university of california, san francisco), and the rat myoblast l6 cell line from dr. d. b. sanwal (university of western ontario, london). dr. s. e. pfeiffer (university of connecticut, farmington) kindly supplied the murine glioblastoma cell lines, g26-20 and g26-24. the murine neuroblastoma cl300 cells were obtained in 1971 from dr. j. p. broome (new york university medical school) and the african green monkey kidney vero cells from dr. l. hatch (st. joseph's hospital, london). the sources of the rat schwannoma rn2-2 cell line and the murine fibroblast l-2 line were previously described (lucas et al., 1977) . prior to use, the cells were cloned by limit dilution in 96-well microtest ii trays (falcon plastics), and a representative clone of each cell line was used in this particular study. the cells were routinely propagated as monolayers in eagle's minimal essential medium (mem) supplemented with 10% heat-inactivated fetal bovine serum (fbs) (microbiological associates), sodium bicarbonate (2 g/liter), penicillin (100 u/ml), and streptomycin (100 mg/ml) at 37" in a humidified atmosphere containing 5% coz. viruses. the source and routine propagation of the mhvs and jhm strains of mouse hepatitis virus have been previously described (lucas et al., 1977) . the hall4 strain of sspe and the edmonston strain of measles virus were obtained from the american type culture collection at passage levels of hela/2-vera/s and hk/24-ham 141, respectively. both viruses were routinely maintained by passage on vero cell monolayers. diluted virus was allowed to adsorb at a multiplicity of infection (m.0.i.) of 0.01 at 32.5 or 37" for 1 hr before medium was added. plaque assay. mhva and jhm virus production was determined by a plaque assay on l-2 cell monolayers as previously described (lucas et al., 1977) . measles virus production was ascertained using a similar plaque assay on vero cell monolayers. for plaque assays after overlaying with mem containing 5% fbs and 0.5% methyl cellulose, 4000 cps, the infected monolayers were incubated at 32.5" for 4 days in the case of halle virus and 5 days when assaying the edmonston strain, then were fixed and stained. the titer is expressed as plaqueforming units per milliliter (pfu/ml). infectious center assay. to determine the fraction of cells able to release pfu during the state of persistence, the monolayers were washed, and the cells were released from them by trypsinization and then plated at serial dilutions on either l-2 monolayers in the case of jhm and mhvs infections or on vero monolayers when hall& and edmonston infections were involved. after 3 to 5 hr attachment period at 32.5", the plates were overlaid with the methyl cellulose-mem mixture and incubated until plaques developed as above. infection of cell lines. various cell lines grown into monolayers were inoculated at a m.o.i. of 0.01 at 32.5 or 39.5' with one of mhv3, jhm, hall& or edmonston viruses. following adsorption for 1 hr, unadsorbed virus was removed, and the cultures were overlaid with nutrient medium. the infected monolayers were examined for development of virus-induced cytopathology and monitored daily for production of free plaque-forming units per milliliter necessitating the complete replacement of the medium each day. determination of cerebroside sulfate. confluent monolayers of the various cell lines were incubated for 48 hr at 37" in mem supplemented with 2.5% fbs and containing 40 pci/ml of [35s]sulfate (43 ci/mg; new england nuclear). cells were harvested by scraping and washed three times with phosphate-buffered saline. aliquots were kept for protein determination by the method of lowry et al. (1951) using bovine serum albumin as standard, but the bulk of material was extracted with chloroformmethanol according to the method of bligh and dyer (1959) . the lower phase lipids were subjected to mild alkaline hydrolysis (kates, 1972) and chromatographed in chloroform:methanol:water (65:25:4, v/v/v), on silica gel h thin-layer plates using cerebroside sulfate as a standard. after preparative isolation of the cerebroside sulfate band and elution with chloroform:methanol:water (95:95:10, v/v/v), the counts per minute of [35s]sulfate incorporated were determined by liquid scintillation counting in a beckman ls-350 instrument. the origin and nature of continuous cell lines used in the present study are shown in table 1 . l-2 cells were found to be satisfactory as a fully permissive host in which mhvs and jhm can be grown to high titer and assayed. in the case of measles, vero cells originating from monkey kidney served as the host for virus production and quantification. cells of neural origin are identified according to the type of tumor from which they originated and by salient biochemical properties which they have retained. thus, lines c6 (benda et al, 1968) , rn2-2 (pfeiffer and wechsler, 1972; lucas et al., 1977) , g26-20, and g26-24 (sundarraj et al., 1975) continue to synthesize in vitro the nervousspecific sloo protein and contain high concentrations of the 2':3'-cyclic nucleotide 3'phosphohydrolase. furthermore, g26-20, g26-24 (dawson et al., 1977) , and rn2-2 cells (lucas et al., 1977) contain cerebroside sulfate, a lipid characteristically present in myelin. absence of this sulfated glycolipid from c6 astrocytoma and mouse neuroblastoma cl300 cells is not surprising since neither originated from myelin-producing cells; however, the presence of detectable levels of this sulfolipid in vero cells from monkey kidney and the htc line of rat hepatoma shows that occurrence of cerebroside sulfate is not per se an adequate diagnostic marker for cells of glial origin. cell lines the previously established tropism of the jhm agent for rn2-2 cells (lucas et al., 1977) led us to investigate in detail whether a similar interaction occurs with other neural lines. following inoculation of monolayers with either jhm or mhv3 viruses, persistent infections could be readily established. in the case of the mouse hepatitis viruses, infection in rn2-2 cells was rapidly aborted with mhvz but became cyclical in the case of jhm, as shown in fig. 1a and previously described (lucas et al., 1977) . thus the rn2-2 cell line is able to discriminate between the two coronaviruses. associated with the jhm virus release, one could observe development of syncytia which were self-limiting in area, disappearing and reappearing continually, without ever completely destroying the monolayer. infectious center assays demonstrated that during persistence there was also a cyclical increase in the fraction of virus-producing cells, ranging between 0.01 and 1%. of the c6 rat astrocytoma failed to elicit any evidence for virus-induced cytopathology (cpe) or virus replication. within 1 day after inoculation, the titer commenced to decline and become nil within 3 to 5 days. by contrast, infection of murine oligodendroglioma lines, g26-20 and g26-24, and neuroblastoma cl300 cell lines with mhv3 and jhm readily initiated persistent infections, characterized also in each case by a cyclical rise and fall in titer (figs. lb, c, and d). unlike the cpe present with jhm in rn2-2 cells, the infection of g26-20 or g26-24 cells did not elicit any syncytiogenesis, despite the fact that high titers comparable to those found in lytic infections of l-2 cells were recorded. throughout the 50-day duration of the experiments, the fraction of cells scored as infectious centers also fluctuated between 0.1 and 100% in the case of jhm and between 0.06 and 100% for mhvs infecting either g26-20 or g26-24 cells. inoculation of the cl300 neuroblastoma with either coronavirus produced a more extensive and destructive cpe than observed when jhm infects rn2-2 cells. most of the cells in cl300 monolayers were eventually involved. the remaining cells not recruited into the massive syncytia could repopulate the cultures but thereafter the cpe was again manifested. over long term, cultures of cl300 continue to generate pfu for 3 or more months indicating that the infection is continually held in balance between a lytic and persistent state. duration of infection could also be prolonged deliberately by reseeding almost completely destroyed cultures with uninfected cl300 cells. the number of infectious centers could not be determined because the cells released by trypsinization that remained as intact units and could be plated on indicator l-2 monolayers were not virus producers. perhaps use of trypsin in this case eliminated all c1300-infected cells. lines not originating in the nervous system it is, apparent that both the jhm and mhv, strains readily establish persistent infections in several neural cell lines, al-though the ability to discriminate in favor of jhm, with complete suppression of mhv3, was an unique property of the rn2-2 rat schwannoma.. it remained possible that this type of host regulation might be a species-related characteristic, although this seemed unlikely in view of the inability of either virus to replicate in the c6 rat astrocytoma. to further investigate species-related control of infectiousness, monolayers of rat htc hepatoma and l6 myoblast cells were inoculated with jhm and mhva. both corona agents elicited the characteristic cpe within 2 days postinfection. the syncytiogenesis was limited, never resulting in the complete destruction of the monolayers, despite the continuous elaboration of pfu in cyclical waves (figs. 1e and f). throughout the period of the experiments, the fraction of cells scored as infectious centers fluctuated between 0.7 and 22.4% in the case of htc infected with jhm, and between 3 and 45% for the mhvz infection. the infectious centers with l6 cells remained relatively constant between 6.3 and 11.5% when jhm was the infecting agent and between 14 and 30% if inoculated with mhv3. from this it becomes apparent that the capacity for discriminating between jhm and mhv3 is an unique property of rn2-2 schwannoma cells and is not species-related. these data also show that establishment of persistence by jhm and mhvs is not exclusive to neural cells but may also occur readily in cells originating from a hepatoma and in myoblasts. the relative ease in establishing persistence with the coronaviruses led us to investigate in the same cell lines comparable associations with measles virus. for this purpose, the hall6 and edmonston strains were used. when fully permissive, vero cell monolayers were inoculated with either strain at 32.5; at an m.o.i. of 0.01, large polykaryocytes first became apparent within 24 hr using the hall6 virus and within 36 hr with the edmonston virus. titers as high as lo8 pfu/ml were determined on cell-free me-dium removed 4 days postinfection with the ha116 strain. at this time the entire monolayers had become virtually one continuous syncytium. infection with the edmonston strain produced lower titers, only lo7 pfu/ml, of cell-free virus and at a slower rate since the peak was not reached until 6 to 7 days postinfection. to investigate the replication of measles in the other non-neural and neural continuous cell lines, monolayer cultures were inoculated at 32.5" and examined for cytopathology, virus production, and development of infectious centers. infection of g26-20 and g26-24 cells proved to be aborted since there was no cpe or infectious virus formation. instead, commencing 1 day after infection, there was a progressive eclipse of the inoculum to zero titer within 5 days. restriction on virus production was maintained for longer than several weeks. by comparison, inoculation of rn2-2, c6, htc, and l6 rat cell lines with either ha& or edmonston virus readily established persistent infections. as illustrated in figs. 2a-d, in all cases tested infectious particles were produced and released into the medium in a cyclical manner over a period of 1 to 2 months. initially, the titers were relatively low, but with time higher virus yields were recorded. in none of the above associations was there any evidence of cpe, despite the fact that in some instances virtually all cells became infectious centers. usually the fraction of cells shown to be infectious centers fluctuated between 5 and 80% with c6 cells, 0.05 and 60% with rn2-2 cells, 0.1 and 0.5% in the case of htc cells and 1 and 10% with the l6 cells as the host for either virus. from the foregoing it is evident that inoculation of these particular rat cell lines at low m.o.i. with hal16 or edmonston measles viruses leads directly to the establishment of persistent but inapparent infections. outgrowth of or selection for a variant progeny virus that is temperature-sensitive for growth (haspel et al., 1973; preble and youngner, 1973; shenk et al., 1974; gould and linton, 1975; kimura et cd., 1975; youngner and quagliana, 1975; youngner et al., 1976; armen et al., 1977; truant and hallum, 1977) . to determine whether a similar appearance of thermolabile progeny occurred in the cell-virus systems being examined here, temperature-shift experiments were conducted after initiating persistent infections. in some instances, cultures actively producing virus at 32.5" were moved to incubators set at 39.5", then the medium was changed daily and assayed at 32.5' for pfu per milliliter. with the halle and edmonston persistent infections of rn2-2 cells, the elevated temperature caused an abrupt cessation of virus release. as indicated in figs. 3a and b, the titer declined within 1 day, and no virus at all was present within 2 or 3 days following shift-up of the temperature, while yield pfu in the characterisic cycling fashion. overall, very similar effects were observed with other persistently infected cell lines. one exception was l6 myoblasts carrying the edmonston measles virus which continued to elaborate pfu at 39.5", although the yield of virus was 1 log lower than at 32.5". the production of pfu from the majority of the coronavirus persistent infections was inhibited or profoundly reduced at the elevated temperature. upon shift to 39.5", the titer declined rapidly and production ceased completely within 4 or 5 days (data not shown). coincidentally the cytopathology evident with several of the infections at 32.5" was absent at 39.5". only partial reduction of replication of jhm in htc, l6, and g26-24 cells was demonstrated at 39.5", whereby the titer was 2 log units lower than at 32.5". with neuroblastoma cl300 there was no appreciable difference in jhm titer at the two temperatures. it should be remembered that coronavirus infection of cl300 cells borders between the persistent and lytic type. restriction of virus replication at 39.5' could also be instituted from the time of inoculation if infection was also made at 39.5' as shown in figs murine hepatitis viruses. in most cases, for some of which the data are not shown, the results were like those with jhm infection of rn2-2 cells at 39.5" in which cpe was absent and virus production declined sharply until none was being made within 4 to 5 days. as in the exceptions described above for shift-up experiments, jhm infection of the g26-24, htc, and l6 cells which commenced at 39.5' was incompletely suppressed and that of cl300 cells not at all inhibited. parallel infections commenced and maintained at 39.5" with the hall6 or edmonston measles viruses in rn2-2, c6, htc, and l6 cells gave results identical to those obtained after shift of carrier cultures from 32.5 to 39.5". in all cases except that of the edmonston virus infection of l6 cells, no pfu were formed. it was possible that thermosensitivity of the replication process described above was due to the selection for and appearance of thermolabile variants of the measles and coronaviruses employed. this idea seemed unlikely, however, because thermosensitivity of infections carried out at 39.5" was immediate. otherwise all four agents used must themselves have been thermolabile. an answer to these alternatives was sought by plaque assays of progeny derived from lytic or persistent infections. titers were compared on indicator cultures incubated at 32.5 or 39.5". a summary of the data shown in table 2 revealed that, with two partial exceptions, none of the progeny was thermolabile, whether originating from long-term or newly established cultures, nor was there any evident alteration in the plaque morphology which might have implied a change of the virus during passage in carrier cells. one exception concerned the hall& virus which was minimally temperature-sensitive whether it originated from permissive or partially restrictive host cells. following long-term mhv, infection of cl300 cells, it appears that there may have been some selection for somewhat temperature-sensitive progeny viruses as indicated by reduction of 1 log in titer at 39.5 versus 32.5'. it thus appears that in the majority of the carrier cultures, the host was conferring thermosensitivity upon the replication process without altering the genotype of the progeny. furthermore, as summarized in table 3 , there was an almost absolute ' the virus being produced by cultures at 32.5" was assayed for temperature sensitivity. aliquots of the medium were removed and assayed for the ability to form plaques on monolayers of either l cells (for jhm and mhv,,) or vero cells (for ha116 and edmonston) at both 32.5 and 39.5' as described in materials and methods. r(-) indicates replication negative. correlation between the ability of specific cell types to undergo chronic infection and thermosensitivity of the replication process. the host-regulated temperature sensitivity observed in cells persistently infected with the hepatitis and measles viruses raised questions concerning the reversibility of the thermosensitive restriction. to study this, we utilized persistent infections of rn2-2 cells by the jhm or the ha116 viruses. long-term cultures actively producing virus at 32.5" were shifted to 39.5" and incubated until no pfu were being formed, usually 3 to 5 days. then such cultures were returned to 32.5", and the medium was changed and assayed daily for pfu per milliliter. with the jhm infection, if cultures were kept at 39.5' for up to 6 days, virus production recovered in most cases in 2 to 3 days following shift-down to 32.5". in some instances when cultures were kept at 39.5' for 10 days, production of pfu resumed after shift-down. this recovery, however, required a longer duration at 32.5" before progeny reappeared. analogous data were derived from the measles infection. in most instances, virus production was resumed several days after shift-down of cultures that had been kept for as long as 10 days at the elevated temperature. to discount the possibility that resumption of virus replication after temperature reversal from 39.5 to 32.5" was due to uneclipsed virus adsorbed at the cell surface, cultures were inoculated with jhm or hall6 viruses at 32.5 or 39.5". after 1 hr to allow for adsorption, extracellular virus remaining was neutralized by adding specific antisera to cultures maintained at their respective temperatures. some cultures were kept at 39.5" for several days with daily changes of medium, then were moved to an incubator set at 32.5". treatment with antiserum did not alter the recovery following shift-down indicating that intracellular genome expression must have been involved in resumption of virus production. these results further imply that both the jhm and ha116 viruses were maintained in a latent form at the restrictive temperature. the relative ease with which persistent infections with the coronaviruses and measles strains were established and the associated thermosensitivity of the replication suggested that infections with many virus lowing 1 hr of adsorption, unadsorbed virus types may indiscriminately become persist-was washed away, and cultures were covent. to investigate this possibility mono-ered with nutrient medium. after 24 hr of layers of the various cell types were infected incubation at 32.5 or 39.5", the concentraat 32.5 and 39.5" with ihd-w vaccinia virus tion of vsv in the medium was determined at an m.o.i. of 5 pfu or vesicular stomatitis by plaque titration on l-2 cell monolayers. virus (vsv) at an m.o.i. of 0.01 pfu. fol-with vaccinia virus, combined cell-associ results from these studies focus attention on three salient findings: (1) the strains of measles and mouse hepatitis viruses used can readily become established in a persistent form of infection in cell lines of neural and non-neural origin; (2) almost invariably when the infection is of the persistent type, virus replication becomes thermosensitive due to unknown factors under host control; the virus progeny from persistent infections are themselves not thermolabile; and (3) among the many cell types tested a rat rn2-2 schwannoma has the unique ability to discriminate between the prototype mhv, and the neurotropic variant, jhm, supporting the persistence of only the latter. the usual outcome of infecting a spectrum of cells originating from neural and non-neural tissue with any one among jhm, mhv3, hall& or edmonston viruses is the development of persistence as documented in table 3 . the systems under investigation here differ from some of those examined previously by others in the directness, speed, simplicity, and reproducibility in establishment of chronic infections after inoculation at low m.o.i. the latent period may be less than 1 day in duration, and no additional manipulation is required, such as repeated virus passage at high titer or the addition of neutralizing antibody. although each type of cell-virus association examined here exhibits some individual peculiarity, one feature characteristic of all is the cyclical rise and fall of low virus titers extending over a period of at least several weeks and perhaps continuing indefinitely. the murine hepatitis viruses can infect chronically both murine and rat cell lines, but a small fraction of the cells in culture become infectious centers. the cytopathology produced may be self-limiting as in the case of rat cells rn2-2, htc, and l6, or verge on the lytic when cl300 neuroblastoma is the host, or exhibit no evident cpe as with murine oligodendroglioma lines g26-20 and g26-24. the ha116 and edmonston strains are incapable of infecting the murine cells tested indicating that in this case there may exist a species-related restriction. a large fraction of measles-infected cells in a culture can become infectious centers without any apparent relationship to the extent of cpe, if any, associated with the infectious process. although the molecular events underlying the maintenance of persistence in the systems under study here remain to be elucidated, in other virus carrier states chronic infections have been explained by effects involving the formation of and competition from defective interfering (di) particles for host synthetic machinery (holland et al., 1976; kawai and matsumoto, 1977) , or by inhibition of virus production due to interferon, sometimes in a cyclical fashion (wiktor and clark, 1972) , or due to selection of genotypically changed variants of the virus (thacore and youngner, 1969; kawai et al., 1975; youngner et al., 1976; truant and hallum, 1977) . involvement of di particles in the present studies seems to be unlikely because the inoculation was made at low m.o.i. and medium was changed daily which should have minimized the production or accumulation of di particles, unless such particles can somehow be maintained in a cellassociated state. nevertheless, some direct proof for discounting any role for di particles has not been provided. evidence with the jhm persistent infec-tion of rn2-2 cells for the involvement of during suppression remain unknown. the an interferon-like mechanism comes from data already in hand strongly indicate that the partial resistance to superinfection with at elevated temperatures both the corona vsv (lucas et al., 1977) . it was not possi-and measles agents can be maintained in ble, however, to transfer interference some latent form; however, preliminary reagainst vsv to uninfected rn2-2 cells by sults based on infectious center assays with material in medium taken from persistently the measles-infected cells suggest that un-jhm-infected rn2-2 cultures. clearly ad-der the nonpermissive conditions, the virus ditional work is required to ascertain information is segregated unequally among whether interferon or some other factors all the daughter cells. function in the chronic infectious process the ability to distinguish specifically beunder study by us. tween the jhm and mhv, viruses de-as indicated earlier, a common feature of scribed previously (lucas et al., 1977) and other virus-carrier systems is the outgrowth in this report appears to be an unique propof or selection for a variant progeny virus erty of the rat rn2-2 cell line. the capathat is temperature-sensitive for growth. bility for discrimination between the pro-but, for a majority of chronic infections totype and neurotropic variant is not spewith mouse hepatitis and measles viruses cies-related because neither virus can repexamined currently, the situation is quite licate in the rat c6 astrocytomas, yet both different. despite a drastic suppression of agents readily establish persistence in the virus production at the elevated tempera-rat l6 myoblasts and htc hepatoma cell ture, the progeny with one exception, are lines. some preliminary experiments indinot thermolabile when tested in permissive cate, however, that the restriction on mhv3 cells. the thermal restriction instead ap-multiplication can be circumvented if rn2pears to be host-controlled. in fact, there is 2 cells are inoculated at an m.o.i. of 10 or a close correlation between the capability greater. under such circumstances, the for establishment of persistence and ther-cpe becomes extensive and rapid, resulting mosensitivity of the virus replication proc-in a total destruction of the monolayer culess (table 3 ). the basis for temperature ture. progeny from this infection when inrestriction at 39.5" remains unknown. it jected into mice cause necrotizing meninshould not be forgotten that control leading gitis essentially identical to that characterto persistence and thermosensitivity of the izing infection with mhv3 propagated in replication process with other agents can the permissive l cells (lucas et al., 1977) . be virus-as well as host-specified as indi-thus, the restriction on mhvj appears to cated in the case of vw which has a full involve some quantitative equilibrium conproductive cycle at 32.5 and 39.5' in the trolled by both the virus and its host. rn2-2, c6, c1300, or htc cells, but is taken together, the results described in restricted to a chronic infection in g26-20 this report suggest that the host cell has a or g26-24 cells. similarly, replication of profound influence in regulating the replivaccinia is fully productive at 39.5" in the cation process of agents with neurotropic rn2-2 and htc cells and is only partially potential. the relative ease with which perdepressed in c6 and cl300 cells. the host sistence and thermosensitivity develop in function(s) controlling the temperature re-the cell lines examined provides new sysstriction of virus production do not appear tems for inquiries into the molecular events to be involved in the growth and viability and mechanisms by means of which certain of host cells as shown by growth rates or viruses operate as slowly acting pathogens plating efficiencies which are similar at 32.5 in the nervous system. and 39.5" for several of the lines tested (unpublished observations). the present experimental data indicate that thermosensitivity is a reversible proc temperature sensitive mutants of measles virus produced from persistently infected hela cells a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin: ii measles antigen and syncytium formation in brain cultures from subacute sclerosing panencephalitis (sspe) differentiated rat glial cell strain in tissue culture a rapid method of total lipid extraction and purification. canad persistent infection of bsc-1 cells by defective measles virus derived from subacute sclerosing panencephalitis a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin: i. isolation and biological properties of the virus synthesis of myelin glycosphingolipids (galactosyl-ceramide and galactosyl (3-o-sulfate) ceramide (sulfatide)) by cloned cell lines derived from mouse neurotumors the production of a temperature sensitive persistent measles virus infection activation of a latent measles virus infection in hamster cells mouse hepatitis virus induced recurrent demyelination long-term persistent vesicular stomatitis virus and rabies virus infection of cells in vitro subacute sclerosing panencephalitis: isolation of measles virus from a brain biopsy subacute sclerosing panencephalitis: isolation of suppressed measles virus from lymph node biopsies techniques of lipidology" (t characteriaztion of rabies viruses recovered from persistently infected bhk cells interfering and noninterfering defective particles generated by a rabies small plaque variant virus temperature sensitive virus derived from bhk cells persistently infected with hvj (sendai virus) mechanism of demyelination in jhm virus encephalomyelitis. electron microscope studies protein measurement with the folin phenol reagent in viuo and in vitro models of demyehuating diseases: tropism of the jhm strain of murine hepatitis virus for cells of glial origin biochemically differentiated neoplastic clone of schwann cells selection of temperature sensitive mutants during persistent infection: role in maintenance of persistent newcastle disease virus infections in l cells latent infection of sensory ganglia with herpes simplex virus: efficacy of immunization temperature sensitive virus from aedes albopiditus cells chronically infected with sindbis vi-ns biochemically differentiated mouse gliel lines carrying a nervous system specific cell surface antigen (ns-1) celis persistently infected with newcastle disease virus. i. properties of mutants isolated from persistently infected l cells a persistent infection of baby hamster kidney -21 cells with mumps virus and the role of temperature sensitive variants pathogenesis of demyelination induced by a mouse hepatitis virus chronic rabies virus infection of cell cultures temperature sensitive mutants isolated from hamster and canine cell lines persistently infected with newcastle disease virus role of temperature sensitive mutants in persistent infections initiated with vesicular stomatitis virus the occurrence of vacuolated neurons in the brains of hamsters affected with subacute sclerosing encephalitis following measles or langat virus infection. &it research council scholar. key: cord-271526-14nfqusv authors: molenkamp, richard; spaan, willy j.m. title: identification of a specific interaction between the coronavirus mouse hepatitis virus a59 nucleocapsid protein and packaging signal date: 1997-12-08 journal: virology doi: 10.1006/viro.1997.8867 sha: doc_id: 271526 cord_uid: 14nfqusv abstract the coronavirus mouse hepatitis virus (mhv) is an enveloped positive stranded rna virus. in infected cells mhv produces a 3′ coterminal nested set of subgenomic messenger rnas. only the genomic rna, however, is encapsidated by the nucleocapsid protein and incorporated in infectious mhv virions. it is believed that an rna packaging signal (ps), present only in the genomic rna, is responsible for this selectivity. earlier studies mapped this signal to a 69-nt stem–loop structure positioned in the 3′ end of orf1b. the selective encapsidation mechanism probably initiates by specific interaction of the packaging signal with the nucleocapsid protein. in this study we demonstrate thein vitrointeraction of the mhv-a59 nucleocapsid protein with the packaging signal of mhv using gel retardation and uv cross-linking assays. this interaction was observed not only with the nucleocapsid protein from infected cells but also with that from purified virions and from cells expressing a recombinant nucleocapsid protein. the specificity of the interaction was demonstrated by competition experiments with nonlabeled ps containing rnas, trna, and total cytoplasmic rna. the results indicated that no virus specific modification of the n-protein or the presence of other viral proteins are required for thisin vitrointeraction. the assays described in this report provide us with a powerful tool for studying encapsidation (initiation) in more detail. gion a domain (from here on called ps) of 69 nt could be identified that is probably required for the encapsidation the murine coronavirus mouse hepatitis virus (mhv) of defective genomes (fosmire et al., 1992) . this signal is an enveloped virus containing a positive stranded rna is present in genomic rna, but not in sgrnas and it is genome of about 31 kb (holmes, 1991; likely that it has also a similar function in the encapsida-1988). the virion envelope is composed of a lipid bilayer tion of genomic rna. recently, it was demonstrated that derived from an internal compartment of the host cell the encapsidation of a heterologous rna by mhv was and three or four virus-encoded structural membrane fully dependent on the presence of this ps (woo et al., proteins (luytjes, 1995) : the spike protein (s), the mem-1997). furthermore, it was shown by bos et al. (1997) that brane protein (m), the small membrane protein (e), and transferring the ps to a sgrna resulted in the specific the optional hemagglutinin-esterase protein (he). the viencapsidation of this sgrna, though with reduced effiral envelope surrounds a nucleocapsid with helical symciency. metry composed of the genomic rna and multiple copies the nucleocapsid protein of mhv is a basic phosphoof the nucleocapsid protein (n). evidence for the presprotein of 454 amino acids and has an apparent molecuence of a fifth structural envelope protein translated from lar weight of approximately 55 kda (armstrong et al. , an internal open reading frame (orf) within the n gene 1983; parker and masters, 1990 ; laude and masters, has been published recently (fischer et al., 1997 (fischer et al., ). 1995 . it is phosphorylated exclusively on serine residues in infected cells mhv produces a 3 coterminal nested (stohlman and lai, 1979) . the n protein contains 41 of set of subgenomic mrnas (sgrnas) which possess an these potential phosphorylation sites, but the exact numidentical 5 leader sequence derived from the 5 end of ber and location of phosphoserines have not been identithe genome (lai et al., 1984; spaan et al., 1983 spaan et al., , 1988 . fied yet. the basic amino acids are not clustered in only genomic length rna is packaged into virus partistrings, but local densities of positive charge can be cles; however, trace amounts of sg rnas are sometimes found, particularly in two regions in the middle of the n detected in purified virus (makino et al., 1988) . earlier protein (laude and masters, 1995) . in contrast, the cstudies (fosmire et al., 1992; most et al., 1991) have terminus is quite acidic. the mhv n protein does not mapped a region in the 3 end of orf1b that is essential contain known rna binding motifs, like the arginine-rich for encapsidation of defective genomes. within this remotif (arm) or zinc fingers (draper, 1995; holmes and behnke, 1981; burd and dreyfuss, 1994) . leader rna has been reported although there is some analysis. all enzyme incubations and biochemical reactions were performed according to the instructions of the discrepancy about the specificity of this interaction (stohlman et al., 1988; bredenbeek, 1990) . a leader-rna manufacturers. binding domain in the n protein was mapped and com-construction of plasmids prises the two basic regions mentioned above (nelson and stohlman, 1993; masters, 1992) . furthermore, spe-(i) pps290. a 204-nt fragment containing ps was obcific interaction of the coronavirus infectious bronchitis tained by polymerase chain reaction (pcr) using pmidivirus (ibv) nucleocapsid protein with the 3 terminus of c as a template (most et al., 1991) and oligonucleotide the genome has recently been reported (zhou et al., primers c060 and c061 (table 1) . to obtain pps290 this 1997). fragment was cloned in pcrii using the ta cloning kit the 69-nt ps is able to form a stable secondary struc-(invitrogen) according to the instructions of the manufacture and the integrity of this structure is essential for turer. the encapsidation of defective genomes (fosmire et al., (ii) pemcv-n. a ncoi restriction site was created at 1992). it has been postulated (fosmire et al., 1992) that the position of the aug start codon of the n gene by the ps functions as an encapsidation initiation site, prob-pcr mutagenesis using oligonucleotide primers c261 ably by interacting specifically with the n protein. initiaand c224 (table 1 ). the pcr fragment was digested with tion of encapsidation by packaging signal/(nucleo)capsid ncoi and apai and fused to the remaining 3 sequences protein interactions has been observed for several other of the n gene. a consequence of this procedure was rna viruses, including alphaviruses, retroviruses, and that the second amino acid of the n protein was changed escherichia coli bacteriophages. (owen and kuhn, 1996; from a ser to an ala. the reconstituted n gene was berkowitz et al., 1995; zhang and barklis, 1995; schlethen exchanged with the ncoi-bamhi fragment of pl1a singer et dupraz and spahr, 1992; witherell et (snijder et al., 1996) and the sequence of the ncoi-apai al., 1991; aldovini and young, 1990; weis et al., 1989) . fragment was confirmed by sequence analysis. the final however, a specific interaction of the mhv n protein expression vector, pemcv-n, contained a t7 promoter with the ps has not been demonstrated yet. and emcv-ntr, followed by the entire n gene and a t7 in this report we have used gel retardation and uv terminator sequence. cross-linking assays to study the in vitro interaction of mhv-a59 nucleocapsid protein and a small rna con-preparation of riboprobe taining the ps domain. we observed specific interaction in order to serve as template dna, pps290 was linearbetween in vitro transcripts containing the ps and n proized with bamhi, extracted with phenol/chloroform, and tein isolated from infected cells, but also with n protein precipitated with ethanol. alternatively, templates for the extracted from purified viruses. furthermore, we were production of ps180 and psdhp rna were produced by able to identify a similar interaction with recombinant pcr using a 5 oligonucleotide containing the t7 pronucleocapsid protein expressed in the vaccinia t7 exmoter sequence in addition to mhv specific sequences pression system. these experiments underline the possi(table 1) . radiolabeled rna was synthesized by t7 tranbility of studying the encapsidation of mhv-a59 rna at scription for 1.5 h at 37њ. reactions contained 1 mg of a molecular basis and allow us to map important dolinearized plasmid dna, 1 mm (each) atp, ctp, and mains in both the nucleocapsid protein as well as the gtp, 50 mm utp, 10 mci of [a-32 p]utp (3000 ci/mmol), rna packaging signal. and 25 units of t7 polymerase in a final volume of 25 ml transcription buffer (gibco brl). the reaction products materials and methods were extracted with phenol/chloroform, purified on a sephadex g50 collumn and precipitated by adding 1/10 cells and viruses volume of 5 m nh 4 ac (ph 5.2) and 2 vol of ethanol. the mouse l cells were grown in dulbecco's modified eaamount of incorporated label was determined by tca gle's medium (dmem; gibco) supplemented with 8% fetal precipitation. calf serum. mhv-a59 stocks were grown as described (spaan et al., 1981) phenylmethylsulfonyl fluoride, 0.5 mm dithiothreitol 10 ml binding buffer (5 mm hepes (ph 7.9), 50 mm kac, 2.4 mm mgac 2 , 0.1 mm edta, 0.01 mm dtt, 1 mm atp, (dtt)) (dignam et al., 1983) . subsequently, the cells were disrupted by freezing (080њ) and thawing (37њ) once or 0.4 mm gtp). where necessary specific or nonspecific competitor rna was added as indicated in the figure by 15 strokes of a dounce homogenizer. cellular debris and nuclei were removed by centrifugation for 30 min at legends. in supershift experiments, 1 ml of the n-specific monoclonal antibody 5b188.2 (talbot and buchmeier, 13,000 rpm at 4њ. the supernatant was aliquoted and stored at 080њ. the protein concentration was deter-1985) or b-galactosidase monoclonal antibody (boehringer mannheim) and 18 units of rnaguard (pharmacia) mined using the bicinchoninic acid protein assay kit (sigma). were added. the mixtures were incubated at room temperature for 20 min, after which 5 ml of 50% glycerol (ii) protein lysates from purified viruses. virus was purified as described before (luytjes et al., 1997) . briefly, was added to each reaction. the mixtures were then separated by electrophoresis on a 5% polyacrylamide/ viruses harvested from 8 1 10 7 cells were precipitated with polyethylene glycol and loaded on top of a 20 to 50% 10% glycerol gel (mono:bis å 37.5:1) in 0.51 tbe (45 mm tris-cl (ph 7.5)), 45 mm boric acid, 1 mm edta) for 6 linear sucrose gradient. the gradient was centrifuged for 16 h at 16,000 rpm at 4њ in a sw40ti rotor. subsequently h at 5 ma (fixed). subsequently, the gel was dried and exposed to x-ray film with an intensifying screen at 080њ. the gradient was fractioned into 16 fractions. all fractions were assayed for viral proteins by western blot using uv cross-linking the rabbit polyclonal mhv-a59 antiserum k134. the two fractions corresponding to the virus peak and two bottom rna binding reactions were performed as described fractions (control) were combined, and their volumes above. subsequently the samples were irradiated at a were adjusted to 5 ml with tesv (20 mm tris (ph 7.4), 2-cm distance with 1.8 j/cm 2 of 254-nm uv light in a 1 mm edta, 100 mm nacl). virus was pelleted by centrif-stratalinker 1800 (stratagene). the complexes were then ugation in a sw55ti rotor for 3 h at 35,000 rpm at 4њ. incubated with a mixture of 500 ng rnase a (pharmacia) the resulting virus pellet was lysed in 100 ml buffer c and 2.5 units rnase t1 (gibco brl) for 20 min at 37њ. the supplemented with 0.5% np-40. complexes were analyzed by sodium dodecyl sulfate-(iii) recombinant n protein lysates. vaccinia virus inpolyacrylamide gel electrophoresis on 15% gels. fections and dna transfections were performed as de-alternatively, cross-linked and rnase treated samples scribed previously . briefly, rk13 cells were immunoprecipitated prior to electrophoresis as dewere grown to subconfluency in 5-cm-diameter petri scribed earlier with monoclonal antidishes (4 1 10 6 cells per dish) and infected with the t7 body 5b188.2 (talbot and buchmeier, 1985) or rabbit rna polymerase expressing vaccinia virus recombinant polyclonal mhv-a59 antiserum k134. (vtf7.3) at a m.o.i. of 5. at 1 h postinfection the cells were transfected with 20 ml lipofectin (gibco brl) containing 5 results mg of pemcv-n. after an incubation of 16 h protein ly-ps290 rna binds specifically to proteins from mhvsates were made as described above. infected and mock-infected cells gel mobility shift assays in the studies described here we have analyzed the interaction between the mhv-a59 n protein and rnas rna binding reactions contained 10 ng of radiolabeled riboprobe and 4 mg of protein lysate in a final volume of containing the 69-nt ps signal. in order to identify this 15). these observations indicate that there is a specific interaction between ps290 rna and proteins from mhvinfected cells as well as with proteins from mock-infected cells. the mhv-a59 nucleocapsid protein interacts specifically with ps290 rna to investigate whether the n protein is part of the protein-ps290 rna complex, a supershift assay using a n-specific antibody was performed. if the n protein is indeed part of the complex, binding to n of a n-specific antibody should result in the formation of a large complex composed of ps290 rna, n protein, and n-specific antibody. this complex is expected to migrate slower in the gel as compared to the protein-ps290 rna complex and meier, 1985) was used to analyze the protein-ps290 rna complex and the monoclonal b-galactosidase-specific antibody was used as a control. when n-specific antiinteraction we have first used gel mobility shift assays body 5b188.2 was added to the complex formed between using protein lysates from both mhv-infected and mock-ps290 rna and proteins from the i-lysate, a second cominfected cells and an in vitro transcript containing the ps plex, migrating slightly slower than the protein-ps290 (fig. 1) . from pilot experiments using 10 ng of labeled rna complex was readily observed (fig. 3, lane 5) . in rna we determined that approximately 4 mg of protein contrast, addition of 5b188.2 to the complex formed belysate was required to observe a distinct retarded band tween ps290 rna and proteins from the mi-lysate did (data not shown). we therefore used these amounts as not result in a supershift (fig. 3, lane 13) . this demonstandard in all binding experiments. strates the presence of the mhv-a59 n protein in the an interaction between ps290 rna and proteins from complex formed between ps290 rna and proteins from infected cells (i-lysate), as well as from mock-infected the i-lysate. cells (mi-lysate) was readily observed (fig. 2, lanes 2 the b-galactosidase-specific antibody was not able to and 9). however, the complex formed between ps290 shift the complex formed between ps290 rna and pro-rna and proteins from the mi-lysate migrated slightly teins from the i-lysate or from the mi-lysate (fig. 3 , lanes slower in the gel and appeared to be more diffuse, indi-11 and 19). furthermore, incubation of ps290 rna and cating that there are some differences in binding activity antibody 5b188.2 or b-galactosidase-specific antibody between i-and mi-lysates. without protein lysate, did not result in the formation of in order to determine the specificity of the interaction, competition experiments were performed. competition of the interaction between ps290 rna and proteins from the i-lysate with a 10-fold molar excess of nonlabeled ps290 rna resulted already in a decrease of intensity of the retarded band (fig. 2, lane 5) . at a level of 30fold molar excess of nonlabeled ps290 rna the retarded band was no longer visible and all of the labeled rna migrated at the position of the unbound rna probe (fig. 2, lane 7) . competition with a 100-fold molar excess of yeast trna did not affect the intensity or the mobility of the retarded band, indicating that the observed interaction between ps290 rna and proteins from the i-lysate is specific for the ps290 rna (fig. 2, lane 8) . competition of the interaction between ps290 rna and proteins from the mi-lysate with non-labeled ps290 rna or yeast trna beled ps290 rna competed entirely for protein binding formed using a 1-to 30-fold molar excess of unlabeled ps290 rna or (fig. 2, lane 14) , whereas a 100-fold molar excess of a 1-to 100-fold molar excess of yeast trna. the positions of the unbound rna (probe) and complexed rna (complex) are indicated. yeast trna had no effect on the interaction (fig. 2, lane tor rna. cross-linked and rnase treated samples were immunoprecipitated with the monoclonal n-specific antibody 5b188.2 or the rabbit polyclonal mhv-a59 antiserum k134 as indicated in the figure legends. an interaction between the n protein and ps180 rna was readily observed (fig. 4, lane 1) , but no interaction was observed between the n-protein and psdhp rna (fig. 4, lane 2) . complexes formed between proteins from the mi-lysate and ps180 rna or psdhp rna could not be immunoprecipitated with the n-specific antibody (fig. 4, lanes 3 and 4) . when the ps180 and psdhp rnas were used in a competitive gelretardation experiment with radiolabeled ps290 and the i-lysate, complete competition was observed with the ps180 rna, whereas no immunoprecipitation and that this interaction is dependent on the presence of the 69-nt ps hairpin. to determine the specificity of the nucleocapsid pro-rna-protein complexes (fig. 3, lanes 8-10 and 12) , tein-ps180 rna interaction in uv cross-linking assays, clearly indicating that the observed supershift was not competition experiments were performed. addition of a the result of an interaction between the rna probe and 100-fold molar excess of nonlabeled ps180 rna resulted the n-specific antibody. in the complete inhibition of n protein binding (fig. 5 , addition of a 100-fold molar excess of nonlabeled lane 2), whereas, a 100-fold molar excess of trna had ps290 competitor rna resulted in the complete inhibition no effect on the interaction between the n protein and of protein binding to the rna probe (fig. 3, lane 6) . in ps180 rna (fig. 5, lane 3) . this clearly demonstrates the contrast, a 100-fold molar excess of trna did not affect specificity of the interaction between the n protein and the formation of rna-protein and rna-protein-anti-ps180 rna. body complexes (fig. 3, lane 7) , which demonstrates the specificity of the interaction between the n protein and ps290 rna. nucleocapsid protein from purified viruses and recombinant expressed nucleocapsid protein interact specifically with ps180 rna uv cross-linking demonstrates specific nucleocapsid protein ps180-rna interaction during rna encapsidation or particle formation, the n protein might undergo structural changes or other modifi-another assay for studying the interaction between the cations. it would therefore be interesting to see if the n n protein and ps containing rnas is uv cross-linking. protein incorporated in virus particles is still able to inter-to exclude the possibility that the non-mhv-specific sequences present in ps290 rna are involved in the interaction between the nucleocapsid-protein and ps290 rna, an rna probe (ps180) containing only mhv-specific sequences was constructed. furthermore, in order to investigate whether the 69-nt hairpin of ps180 (and ps290) rna is responsible for specific nucleocapsid protein binding, psdhp rna was constructed. this rna is identical to ps180 rna except that the largest part of the 69-nt ps hairpin has been removed. the u contents of ps180 and psdhp are similar (approx. 25%). pilot experiments were performed to estimate the optimal uv dose. from these experiments it was determined that an uv dose of 1.8 j/cm 2 gave the most distinct bands with the least background (data not shown). from uninfected cells was added as nonspecific competi-observed using the vtf7-lysate (fig. 6c, lane 2) . these experiments clearly demonstrate that no virus specific modifications of the n protein or any other viral proteins are required for the specific interaction with ps180. initiation of encapsidation by a specific interaction between the (nucleo-) capsid protein and a rna packaging signal is a common mechanism used by positive stranded rna viruses. such an interaction has already weis et al., 1989) , retroviruses (zhang were immunoprecipitated with n-specific monoclonal antibody and barklis, 1995; berkowitz et al., 1995; dupraz and 5b188.2 . competition was performed with unlabeled ps180 rna or yeast trna as indicated above the lanes. the position of the n-protein spahr, 1992; aldovini and young, 1990) but also in e. coli specific band is indicated. bacteriophages like r17 and ms2 (witherell et al., 1991) . in this report we describe the specific in vitro interaction of the mouse hepatitis virus a59 n protein with tranact specifically with ps180. in order to address this quesscripts containing the rna packaging signal of mhv. tion, virus particles were purified on a sucrose gradient. this interaction was studied by gel retardation and uv lysates were prepared from the virus peak fractions (pcross-linking assays using an rna probe containing the lysate) and from the gradient bottom fractions (b-lysate; 69-nt ps and the n protein from mhv-a59 infected cell negative control). uv cross-linking was performed using lysates. a similar interaction was identified between the both lysates, radiolabeled ps180 rna, and competitor n protein from purified virus and recombinant n-protein rna (5 mg of total cytoplasmic rna extracted from uninexpressed by the vtf7 expression system. fected cells; fig. 6b ). a specific interaction was observed in addition to the n-ps rna complex we also observed between the n-protein from the p-lysate and ps180 rna that cellular proteins bind to ps containing rnas. the (fig. 6b, lanes 2 and 4) . in contrast, no complex could nature of these cellular proteins and the significance of be immunoprecipitated with n-specific antibodies when this interaction, however, remains unclear. the b-lysate was used (fig. 6b, lanes 1 and 3) . this indicates that any structural change of the n protein which might occur during virus assembly does not affect the ability of the n protein to interact with ps180 rna. in mhv-infected cells, the n protein might also undergo virus-specific or virus-dependent posttranslational modifications. these modifications might play a role in the specific interaction with the ps. furthermore, it is not yet known if the interaction between the n protein and the ps involves other viral proteins as well. in order to investigate this, recombinant n protein was expressed by the vtf7.3 expression system. a n protein expression vector (pemcv-n) was transfected in vtf7.3-infected rk13 cells and protein lysates were made 16 h posttransfection (vtf7pn lysate). as a control protein lysates from mock-transfected but vtf7.3-infected rk13 cells were made (vtf7-lysate). uv cross-linking experiments, followed by immunoprecipitation with n-specific antifig. 6c ). using the vtf7pn lysate, a clear band was fraction lysate and p denotes the peak-fraction lysate. immunoprecipitaobserved migrating at the expected position of the nution with 5b188.2 and k134 were performed as indicated above the lanes. the position of the n-protein-specific band is indicated. cleocapsid protein (fig. 6c, lane 1) . this band was not the nature of the nucleocapsid protein-ps rna cells (woo et al., 1997) . although it is not known whether this encapsidation was efficient, it might be possible that interaction the heterologous flanking sequences have increased the the n-protein of mhv does not posess any well known flexibility of the rna molecule, allowing it to adapt the rna binding domains, such as the arm (lazinski et al., proper structure for encapsidation. 1989; talbot and buchmeier, 1985) or zinc fingers (draper, 1995) . it interacts, although, specifically with significance of the nucleocapsid protein-ps rna leader-rna (stohlman et al., 1988; baric et al., 1988) and interaction an rna binding domain has been identified (nelson and stohlman, 1993) . this domain comprises the two major since mhv virions contain only mhv genomic rna, it hydrophobic basic regions in the middle of the protein. is evident that rna encapsidation is a highly specific since this central part of the n protein contains a high process. the location of the encapsidation signal in degree of basic amino acids, it is possible that the ps orf1b ensures the specific encapsidation of only genorecognition domain might also be positioned somewhere mic rna. it is unclear if the mhv ps is by itself sufficient within this part of the n protein. for efficient encapsidation. it is known that rous sarcoma in infected cells the n protein of mhv is very rapidly virus has multiple encapsidation sites, which are rephosphorylated on serine residues (stohlman and lai, quired to interact for efficient packaging of the genome 1979) and part of it concomitantly becomes associated (sorge et al., 1983; pugatsch and stacey, 1983) . recently, with a cell membrane fraction (stohlman et al., 1983; it was shown that a subgenomic rna of mhv containing anderson and wong, 1993) . it is still unknown if phosthe ps can be encapsidated specifically but the efficiency phorylation is carried out by a host cell or viral encoded was much lower as compared to the encapsidation effiprotein kinase and its (biological) role remains unclear. ciency of a defective interfering rna (bos et al., 1997) . it has been suggested that it might govern the tightness preassembled nucleocapsids have never been obof the interaction between n and rna (laude and masserved in coronavirus infected cells, but an electronters, 1995). it will be interesting to compare the phosphordense structure, which may correspond to the nucleoylation of the n protein from infected cells and the recomcapsid, can be found at the cytoplasmic face of the binant n protein and to investigate the possible role of budding site (dubois-dalcq et al., 1982; holmes, 1991) . phosphorylation in rna binding. phosphorylation and nucleocapsid incorporation in budding virions is exlikewise dephosphorylation could also induce a major pected to be mediated by m-n protein interactions. asconformational change of the n protein. it has been sugsociation of the m protein to the nucleocapsid in np-40gested that dephosphorylation of the nucleocapsid after disrupted virions has been reported (sturman et al., virus entry is involved in uncoating of the viral rna (ma-1980) ; however, the same study demonstrated that the hondas and dales, 1991) . m protein was able to bind rna in the absence of n. it in general the recognition of an rna signal by a protein is still unclear if the interaction between m and n is a involves secondary structure elements in addition to the prerequisite for rna encapsidation in vivo. this interacprimary sequence of the rna signal (draper, 1995) . tion could position the n protein in a favorable way to there is accumulating evidence for an induced-fit mechinteract with the genomic rna. this must then be folanism in rna-protein interactions affecting both the lowed by interaction with additional n proteins, forming rna and the protein (beck and nassal, 1997 ; allain et the helical nucleocapsid. involvement of other viral proal., 1996) . sufficient flexibility in the rna molecule could teins in ps rna binding, however, was not observed in be a major determinant in protein binding. from that our in vitro studies. recombinant nucleocapsid protein, perspective, the recognition of the ps by the n protein expressed in the vtf7 expression system, interacts also could be highly dependent on the secondary structure specifically with the ps rna (fig. 6c) , although the effiof the ps domain. when a short rna probe consisting ciency of this interaction was not determined. a striking of only the 69-nt ps was used in our studies, no gel feature of coronaviruses is that the nucleocapsid has a retardation or uv cross-linking to the n protein was obhelical symmetry (macnaughton et al., 1978; holmes and served (unpublished observations). computer prediction behnke, 1981) , in contrast to the nucleocapsids of all of the secondary structure of this small 69-nt rna moleother positive stranded rna viruses, which are icosaecule showed that the secondary structure was entirely dral or spherical (murphy et al., 1995) . however, electron different from that of the ps in the orf1b context. this microscopy studies on the transmissible gastroenteritis suggests that the flanking orf1b sequences are necescoronavirus and mhv (risco et al., 1996) have revealed sary to force the ps in its specific structure or that the recently a spherical core structure inside the virion (intersmall rna probe lacks the flexibility to form the specific nal core) and this structure reacted with m-and n-spestructure. recently, it has been shown that the 69-nt ps, cific antibodies. a structural model for coronaviruses was flanked by non-mhv sequences could confer specific proposed, in which a spherical core, composed of a combination of n and m proteins, was present in addition to encapsidation to a heterologous rna in mhv infected interaction between nucleocapsid protein and packaging signal of a murine coronavirus in the absence of helper virus. virology 218, a helical nucleocapsid. it will be of interest to study the 52-60. possible relationship between the morphology of the nu-bos, e. c. w., dobbe, j., luytjes, w., and spaan, w. j. m. (1997) . a subcleocapsid or internal core and the mechanism of encapgenomic mrna transcript of the coronavirus mouse hepatitis virus sidation initiation. strain a59 defective interfering (di) rna is packaged when it contains it has been shown that, as opposed to alphavirus asthe di packaging signal. j. virol. 71, 5684-5687. bredenbeek, p. j. (1990) . ''nucleic acid domains and proteins involved sembly, nucleocapsid formation and rna encapsidation in the replication of coronaviruses.'' ph.d. thesis, university of are not strictly required for virion formation (vennema et utrecht, utrecht, the netherlands. al., 1996; strauss and strauss, 1994) . burd, c. g., and dreyfuss, g. (1994) . conserved structures and diversity coexpression of m and e protein was sufficient for partiof functions of rna-binding proteins. science 265, 615-621. cle formation. however, incorporation of nucleocapsids dignam, j. d., lebowitz, r. m., and roeder, r. g. (1983) . acurate transcription initiation by rna polymerase ii in a soluble extract from during the budding process could greatly increase the isolated mammalian nuclei. nucleic acids res. 11, 1475 res. 11, -1489 efficiency of virion formation. draper, d. e. (1995) . protein-rna recognition. annu. rev. biochem. 64, the study of the encapsidation of coronaviruses is 593-620. greatly hampered by the absence of an infectious clone. dubois-dalcq, m. e., doller, e. w., haspel, m. v., and holmes, k. v. the in vitro binding assay, described in this report, can (1982) . cell tropism and expression of mouse hepatitis viruses (mhv) greatly enhance our understanding of the encapsidation in mouse spinal cord cultures. virology 119, 317-331. dupraz, p., and spahr, p. f. (1992) . specificity of rous sarcoma virus mechanism. we have shown that recombinant nucleonucleocapsid protein in genomic rna packaging. j. virol. 66, capsid protein expressed in the vtf7 expression system 4670. is able to interact with the ps in a similar fashion as the fischer, f., peng, d., hingley, s. t., weiss, s. r., and masters, p. s. nucleocapsid protein produced in infected cells. muta(1997) . the internal open reading frame within the nucleocapsid gene tional analysis of both the nucleocapsid protein and the of mouse hepatitis virus encodes a structural protein that is not essential for viral replication. j. virol. 71, 996-1003. ps should provide us with information that allows us to fosmire, j. a., hwang, k., and makino, s. (1992) . identification and understand the encapsidation mechanism in more detail. characterization of a coronavirus packaging signal. j. virol. 66, 3522-3530. holmes, k. v. (1991) . in ''fundamental virology'' (b. n. fields and d. m. sequence specific sequences involved in human immunodeficiency virus type 1 packagrecognition of rna hairpins by bacteriophage antiterminators reing result in production of noninfectious virus 646-characterisation of two temperature-sensitive mutants of coronavi-650 ribo-232. nucleoprotein-like structures from coronavirus particles sequence of 39 endosomal association of a acids res cascoronavirus nucleocapsid protein nucleocapsid protein and viral rnas: implications for viral transcrip-primary structure and translation of a defective interfering rna of tion localization of an rna-binding domain in the minantsin the interaction between the rna encapsidation signal and nucleocapsid protein of the coronavirus mouse hepatitis virus. arch. reverse transcriptase of avian hepatitis b viruses retrovimain at the 3 end of the polymerase gene is essential for encapsidaral nucleocapsid domains mediate the specific recognition of genotion of coronavirus defective interfering rnas virus taxonomy, classification and nomenclature of viruses binding domain of mouse hepatitis virus nucleocapsid protein. j. synthesis and subcellular localization of the murine coronavirus nu-gen and sindbis virus nucleocapsid protein that is involved in specificity of deans phosphoproteins of murine n genes of five strains of the coronavirus mouse hepatitis virus hepatitis viruses relikely to be required for avian retroviral packaging the navirus envelope glycoproteins and interaction with the viral nucleotransmissible gastroenteritis coronavirus contains a spherical core capsid antigenic variation among molecular cloning: murine coronaviruses: evidence for polymorphism on the peplomer a laboratory manual nucleocapsidelements and trans-acting proteins involved in the assembly of rna independent assembly of coronavirus-like particles by co-expression viruses the arterivirus nsp4 protease is the protofor specificity in the encapsidation of sindbis virus rnas cis-acting rna packaging action between rna phage coat proteins and rna. prog. nucleic locus in the 115-nucleotide direct repeat of rous sarcoma virus nucleocapsid protein effects on the 424-434. specificity of retrovirus rna encapsidation the infectious bronchitis virus nucleocapsid protein binds navirus mrna synthesis involves fusion of non-contiguous se-rna sequences in the 3 terminus of the genome key: cord-270814-krw8zmr5 authors: rao, pasupuleti v.; kumari, suman; gallagher, thomas m. title: identification of a contiguous 6-residue determinant in the mhv receptor that controls the level of virion binding to cells date: 1997-03-17 journal: virology doi: 10.1006/viro.1997.8446 sha: doc_id: 270814 cord_uid: krw8zmr5 abstract murine carcinoembryonic antigens serve as receptors for the binding and entry of the enveloped coronavirus mouse hepatitis virus (mhv) into cells. numerous receptor isoforms are now known, and each has extensive differences in its amino terminal immunoglobulin-like domain (ntd) to which mhv binds via its protruding spike proteins. some of these receptor alterations may affect the ability to bind viral spikes. to identify individual residues controlling virus binding differences, we have used plasmid and vaccinia virus vectors to express two forms of mhv receptor differing only in their ntd. the two receptors, designated biliary glycoproteins (bgp) 1aand 1b ntd, varied by 29 residues in the 107 amino acid ntd. when expressed from cdnas in receptor-negative hela cells, these two bgp molecules were displayed on cell surfaces to equivalent levels, as both were equally modified by a membrane-impermeant biotinylation reagent. infectious center assays revealed that the 1aisoform was 10 to 100 times more effective than 1b ntdin its ability to confer sensitivity to mhv (strain a59) infection. bgp1awas also more effective than bgp1b ntdin comparative virus adsorption assays, binding 6 times more mhv (strain a59) and 2.5 times more mhv (strain jhmx). bgp1awas similarly more effective in promoting the capacity of viral spikes to mediate intercellular membrane fusion as judged by quantitation of syncytia following cocultivation of spike and receptor-bearing cells. to identify residues influencing these differences, we inserted varying numbers of 1bresidues into the bgp1abackground via restriction fragment exchange and site-directed mutagenesis. analysis of the resulting chimeric receptors showed that residues 38 to 43 of the ntd were key determinants of the binding and fusion differences between the two receptors. these residues map to an exposed loop (c-c′ loop) in a structural model of the closely related human carcinoembryonic antigen. nized mhv strains (barthold et al., 1986; barthold, 1987) . these receptors are type i single-pass transmembrane coronaviruses are attractive objects for studies of aniglycoproteins containing an ectodomain structure made mal virus entry into cells because there exists a remarkup of four immunoglobulin-like domains (dveksler et al. , able level of variation in the ligands that engage virions 1991). the variability between 1 a and 1 b lies primarily in with cells. several strains of mhv are now recognized the most membrane-distal domain (yokomori et al., (siddell, 1995) and several receptors are also known 1992b, dveksler et al., 1993a) . this amino terminal do(dveksler et al., 1991; yokomori and lai, 1992b; nedellec main (ntd) is essential for virus binding in vitro (dveksler et al., 1994; chen et al., 1995) . this abundant natural et al., 1993b) and is similarly required to render cells variation provides the opportunity to compare different susceptible to infection by mhv (dveksler et al., 1995) . receptors for their ability to interact with different virus initial studies on the coronavirus-binding functions of strains. such comparative analyses will allow one to bgp1 a and 1 b indicated that they differ substantially in identify the determinants responsible for differences in virion adsorptive capacity -when the two receptors virus:cell interactions. were immobilized on nitrocellulose filters, only the 1 a in this study, we have focussed attention on two allelic isoform supported detectable virus-binding (dveksler et isoforms of the mhv receptor, biliary glycoprotein (bgp) al., 1993a) . this finding suggested that one or more of isoforms 1 a (dveksler et al., 1991; the 29 amino acid differences between the 1 a and 1 b 1992a) and 1 b (yokomori and lai, 1992b; ntds (yokomori and lai, 1992b ) are responsible for 1993a; mccuaig et al., 1993) , and we have examined controlling virion adsorptive capacity, either because their capacity to interact with purified enveloped corothey provide direct receptor:virion contact points or benavirions. the 1 a isoform is expressed in mhv suscepticause they control the overall conformation of the recepble mice, while mouse strains homozygous for the 1 b tors. if the ntd differences were indeed critical, then isoform are generally resistant to many currently recogidentification of the relevant residues could be achieved by constructing 1 a /1 b hybrid cdnas and then measuring virus binding to the protein products. we have used duce chimeras that have been useful in identifying resi-changed. 1 b 84-122 and 1 b 70-122 were made by exchange of acci -psti and bamhi-psti fragments, respec-dues controlling the virus-adsorptive capacity of the mhv receptor. tively (see fig. 7 ). additional exchanges were performed after creation of a ndei site at codon 53 using the megaprimer mutagenesis procedure (aiyar and leis, 1993) . ndei site creation altered codon 53 (n53h) in bgp1 a and cells and viruses codons 53 and 54 in bgp1 b ntd (n53h, k54m). 1 b 54-122 and 1 b 10-54 were made by exchange of ndei -ndei baby hamster kidney (bhk), rabbit kidney (rk13), and and ncoi -ndei fragments, respectively (see fig. 7 ). rehuman carcinoma (hela) cells were grown in dulbecco combinant 1 b 38-43 was made by oligonucleotide-dimodified eagle medium (dmem) supplemented with 10% rected pcr mutagenesis using a primer spanning coheat-inactivated fetal bovine serum (dfbs). murine 17 dons 38-43 and the ndei site (scharf, 1990) . nucleotide cl 1 cells (sturman and takemoto, 1972) were grown in sequencing methods (sanger et al., 1977) were employed dmem containing 5% tryptose phosphate broth (tpb; to confirm that bgp1 a and 1 b clones matched those re-difco laboratories, detroit, mi) and 5% dfbs. murine ported for these genes (mccuaig et al., 1993) and to astrocytoma (dbt) cells (hirano et al., 1978) were grown verify the isolation of all of the mutant receptors dein minimal essential medium (mem) containing 10% tpb scribed herein. and 5% dfbs; these cells were used to measure mhv infectivities by plaque assay. murine coronavirus strains generation of vaccinia virus recombinants. a59 and jhmx (makino et al., 1984) (felgner et al., (icn radiochemicals, irvine, ca) . clarified supernatants 1987) . at 3 days postinfection, cultures were frozen at were overlaid above 30% (w/w) sucrose cushions in pbs 080њ. after three freeze -thaw cycles, debris was recontaining 0.01% bsa and subjected to ultracentrifugamoved by centrifugation and virus in the clarified matetion (beckman-spinco sw28 rotor for 3 hr at 28,000 rpm). rial was plated on rk13 cells. plaques arising from spe-virion-containing pellets were resuspended in ice-cold cific amplification of recombinant virus were revealed pbs containing 0.01% bsa and stored in silanized glass by overlay of cells with mycophenolic acid-containing vials at 080њ. medium, as described by falkner and moss (1988) . virus from well isolated plaques was grown in rk13 cells synthesis and cloning of mhv receptor cdna to generate stocks of recombinant virus (vbgp1 a , constructs vbgp1d, vbgp1 b ntd , vbgp1 a q10g,v123g , and additional 1 a /1 b methods for construction of vaccinia virus insertion/ chimeras). expression plasmids (ptm3; moss et al., 1990) encoding mhv receptor isoform bgp1 a or bgp1 a lacking codons immunoblot analysis of bgp proteins 10-122 (bgp1d) have been described previously (gal-cell monolayers were infected with the respective lagher, 1996). these plasmid vectors were further modi-vbgp recombinants (m.o.i. å 10) and coinfected with fied by reinsertion of bgp ntd sequences into bgp1d. vtf7.3 (m.o.i. å 1). at 20 hr postinfection, lysis buffer (10 to this end, the bgp1 a and 1 b ntd sequences were mm tris -hcl (ph 7.5), 100 mm nacl, 1 mm edta, 0.5% amplified from outbred cd1 mouse liver rna by rt-pcr np-40, 0.1 mm pmsf, and 0.3 u/ml aprotinin) was added, (kawasaki, 1990) using primers based on the reported and 10-ml aliquots (equivalent to 2 1 10 4 cells) were bgp1 a sequence (mccuaig et al., 1993) and products subjected to western immunoblotting. receptor was encoding for the 1 a and 1 b ntd were identified by identified using antireceptor antibody 874 (a gift of dr. restriction mapping and by sequencing (sambrook et m. j. buchmeier, scripps research institute, la jolla, ca) al., 1989) . 1 b ntd sequences were cloned in-frame into which was directed against conserved carboxy-terminal the unique xmai site of bgp1d to generate the chimeric residues 443-458. bgp1 b ntd . two undesired codon alterations that resulted at the junctions of the insertion (q10g and v123g) quantitation of relative bgp cell surface levels were introduced into a control bgp1 a construct by performing a parallel insertion of the bgp1 a ntd into ptm3-at 20 hr postvaccinia infection, hela cells expressing the bgp molecules were washed three times with ice-bgp1d. the control recombinant was designated as bgp1 a q10g,v123g . cold pbs and incubated for 1 hr at 4њ with pbs containing 1 mg/ml nhs-lc biotin (sulfosuccinimidyl-6-(biotina-to construct additional bgp chimeras for use in identifying virus binding determinant(s), restriction fragments mido) hexanoate; pierce co., rockford, il). unreacted reagent was then quenched by the addition of 20 mm between ptm3-bgp1 a and ptm3-bgp1 b ntd were ex-tris-hcl (ph 7.5) for 15 min at 4њ. monolayers were virus binding assays washed thoroughly with pbs and lysed (2 1 10 6 cells/ hela cells overexpressing bgp receptors from vacml in lysis buffer). clarified cytoplasmic extracts (250 ml) cinia vectors were washed twice with ice-cold sfm at were then mixed with antipeptide antibody 874 (12.5 ml) 20 hpi, then radiolabeled mhv particles were diluted in and left overnight at 4њ. twenty microliters of gammabind sfm and added at varying multiplicities. after incuba-g-sepharose (pharmacia biotech) was then added for 1 tions at 4њ, unadsorbed virions were removed and cells hr at 4њ. sepharose beads were collected by centrifugawere rinsed five times with ice-cold pbs containing 0.5% tion, washed twice in ripa buffer (10 mm tris-hcl (ph bsa and 0.05% tween 20. ripa buffer was added, and 7.2) 150 mm nacl, 1% sodium deoxycholate, 0.1% sds, radioactivities associated with the cell lysates and superand 1% triton x-100), then once in 10 mm tris -hcl (ph natant media were quantified by scintillation counting. 7.2) containing 0.1% v/v nonidet p-40. immunoprecipitated receptors were then subjected to western blotting, intercellular fusion assay and the biotinylated fraction of each sample was identithe cell fusion-dependent reporter gene (b-galactosified by incubation of blots for 1 hr with 0.1% v/v streptavidase) activation assay of nussbaum et al. (1994) was din-horseradish peroxidase (hrp) conjugate (pierce adapted for studies of mhv fusion as described pre-co.) in pbs/3% bsa. immobilized hrp was detected by viously (gallagher, 1996) , with minor modifications. in incubation in pbs containing 0.5 mg/ml 4-chloro-1-naphbrief, stably transfected hela-bgp1 a cells were infected thol, 17% v/v methanol, and 0.015% hydrogen peroxide. with mhv-a59 (m.o.i. å 10) and with vaccinia virus strain blots were photographed (polaroid 665 film) and signal wr (m.o.i. å 10). after 1 hr at 37њ, these cells were further intensities were quantified with an lkb ultrascan xl dentransfected by lipofection with the reporter gene consitometer. struct pg1nt7b-gal (kindly provided by dr. richard a. morgan, national center for human genome research, bethesda, md). five hours later (before the onset of mhv vaccinia-infected bhk cells were challenged with a59-induced syncytium formation yet after pg1nt7b-gal mhv-a59 (m.o.i. å 10) at 10 hr postvaccinia infection. dna amplification via vaccinia-encoded polymerases) fourteen hours later, cells were washed with pbs, fixed the cells were trypsinized, suspended in dmem 10% in acetone, and incubated for 1 hr at 22њ with a 1:250 dfbs, and 0.6-ml (3 1 10 5 cell) aliquots were overlaid dilution of antispike mab 4b11.6 (collins et al., 1982) on to confluent 5-cm 2 monolayers of hela cells that were in pbs/2% bsa. bound mab was detected with fitccoinfected 20 hr earlier with vtf7.3 and vbgp recombiconjugated goat ig directed against mouse ig (cappell, nants. after 3 hr at 37њ, the mixed monolayers were fixed durham, nc) and cells were photographed using a leitz and stained with x-gal for in situ localization of b-gal fluorescence microscope. activity (macgregor et al., 1989) . alternatively, the monolayers were lysed by addition of 0.5% np-40 in pbs and infectious center assays the quantity of b-galactosidase in each lysate was calculated using a colorimetric enzyme assay involving hydro-cdna constructs encoding bgp1 a and bgp1 b ntd were lysis of chlorophenyl red b-galactopyranoside (nussinserted into plasmid expression vector puhd-10-3 baum et al., 1994). od 590 values were normalized by (gossen and bujard, 1992) and the plasmids (0.5 mg) comparing the hydrolysis rates for each sample with that were transfected by lipofection into aliquots of 3 1 10 5 obtained for a standard preparation of e. coli b-galactosi-hela-tta cells (gossen and bujard, 1992) . to determine dase (calbiochem, la jolla, ca) and were expressed as transfection efficiencies, parallel cultures were cotransnanograms/well. fected with each puhd-bgp construct in conjunction with 0.05 mg of the b-galactosidase-encoding pcmv-b (clon-results tech labs, inc.). in situ x-gal staining (macgregor et al., 1989) at 36 hr posttransfection revealed 11.8 and 12.5% synthesis of chimeric mhv receptors from vaccinia b-gal-positive cells for the 1 a and 1 b ntd transfectants, revirus vectors spectively. to assess susceptibilities to mhv infection, cells were washed at 36 hr posttransfection with cold a method for receptor overexpression on cell surfaces was necessary to reproducibly measure the binding of serum-free dmem (sfm) and inoculated with mhv-a59 at 4њ for 1 hr. unbound virus was removed by washing mhv particles to a series of different receptors. hela cell lines in which the mhv receptor gene was stably and with pbs containing 0.5% bsa and 0.05% tween 20 and cells were then incubated in dmem 10% dfbs for 2 hr constitutively expressed (hela-bgp1 a cells) have been developed (gallagher, 1996) ; however, we found that at 37њ. cells were then trypsinized, washed twice with dmem 10% dfbs, and serial dilutions were plated on clones varied dramatically in receptor levels (data not shown). therefore we anticipated difficulty in identifying dbt indicator monolayers. plaques were visualized after a 2-day incubation period. a series of stable hela cell transfectants in which differthe carboxy-terminal 16 amino acids are identical for to increase the likelihood of equivalent receptor producall four receptors; and an antipeptide antibody (874) tion, different receptor cdnas were expressed from vacraised against these cytoplasmic residues was available cinia virus vectors. such vectors have historically produced to us for use in monitoring receptor levels. initial tests high levels of surface glycoprotein (broder et al., 1994; nususing this anti-c-terminal antibody were performed by sbaum et al., 1995) and their preparation is known to be immunoblotting bhk and hela cell lysates collected 20 relatively straightforward (falkner and moss, 1988) . hr after coinfection with vtf7.3 and vtm3-bgp. the re-a set of cdnas capable of encoding four distinct sults (fig. 2 ) revealed identical patterns of immunoreacforms of mhv receptor were each recombined into tive protein in lysates expected to contain bgp1 b ntd , the vaccinia virus genome using insertion vector ptm3 1 a q10g,v123g , and 1 a (lanes 1-3, respectively). a sharp band (elroy-stein and moss et al., 1990) and at c. 55 kda was detected, which likely represent underrecombinant viruses (designated vtm3-bgp) were seglycosylated form(s) of the 424 amino acid proteins. the lected as described under materials and methods. the series of bands ranging from 80 to 120 kda indicated use of the ptm3 vector places the cdnas under the that equivalent levels of the various glycoforms of receptranscriptional control of a bacteriophage t7 promoter; tor were present in each infected culture. lysates exthus expression of mhv receptors required coinfection pected to contain bgp1d had a similar, slightly more of hela cells with vtm3-bgp and vtf7.3, which encodes bacteriophage t7 rna polymerase (fuerst et al., 1986) . the four translation products predicted from this expression scheme are depicted in fig. 1 . the mature bgp1 a , after removal of its signal peptide, has an ectodomain composed of four immunoglobulin-like domains, designated d1 to d4 (dveksler et al., 1991) . a deletion mutant of bgp1 a (bgp1d) was prepared by excision of nucleotides encoding the bulk of the virus-binding d1 domain as well as 15 amino acids of the d2 domain. corresponding sequences from the allelic bgp1 b (mccuaig et al., 1993) were then placed back into the truncated cdna to generate a hybrid capable of encoding the chito test the contribution that these two changes might an antipeptide antibody directed against conserved carboxy-terminal have on interaction with virions, cdna for a bgp1 a con(4) taining the mutations (designated bgp1 a q10g,v123g ) was bgp1d, (5) vtf7.3 alone, (6) molecular weight markers, kilodaltons. prepared by ligating the ntd sequences of bgp1 a into identical profiles of immunoreactive protein were observed in similarly infected hela cell lysates. the deletion mutant. sion vector puhd-10-3 and transfected into hela-tta cells (gossen and bujard, 1992) tantly, the results of fig. 4 showed that bgp1 only 1 to 2 log 10 higher. moreover, these differences in receptor effectiveness could not be eliminated by increasing input multiplicities during virus challenge. fiintense immunoreactive protein profile, with the apparent nally, we observed a straightforward correlation between molecular weights of the bands lower by approximately infectious centers and virus yields; 24 hr after challenge 20 kda (lane 4). of the bgp transfectants with mhv a59 at 10 pfu per to determine whether the four different mhv receptors cell, yields were 69000, 3000, and 200 pfu/ml for the 1 a , were displayed on the cell surface at similar levels, we 1 b ntd , and control transfectants, respectively. thus, when first made the assumption that each receptor contains expressed in the hela-tta cells, bgp1 a was utilized as similar numbers of exposed amines available for conjuan mhv-a59 receptor much more effectively than gation with the membrane-impermeant biotinylation re-bgp1 b ntd . agent sulfo-nhs-lc biotin. with this in mind, we derivatized the proteins on intact cells at 20 hr postvaccinia infection with the reagent and then we lysed cells and virus binding capacities of the chimeric mhv immunoprecipitated the receptor proteins. the relative receptors proportion of biotinylated receptor among the immuno-with the hela cell monolayers each expressing a difprecipitates was then assessed by western blotting usferent mhv receptor on the cell surface, straightforward ing streptavidin-horseradish peroxidase as a detection virus adsorption assays became feasible. thus we colreagent. by this method, the relative abundance of biotinlected particles from supernatants of mhv-infected 17cl1 ylated receptors were similar in all cultures; densitometcells that had been incubated for 4 hr in the presence ric scans of the blots revealed less than 10% variation of trans[ 35 s] label, and we concentrated the radiolabeled in signal intensities ( percentage of added radioactivity remaining bound to the monolayers was determined. the results (fig. 5) re-spike antigen (fig. 3, panels 1, 2, and 3) while the truncated receptor failed to support mhv-a59 infection vealed a gradual increase in the levels of nonspecific adsorption of virions to cells displaying bgp1d, from 0.8% (panel 4), in concert with the results of dveksler et al. (1993b) . after 15 min, to 1.6% by 1 hr, and 3.5% by 8 hr. mhv-a59 bound far more avidly to cells with bgp1 a or 1 b ntd ; 9.5 additional examination of the various receptors for their ability to confer susceptibility to infection involved and 3%, respectively, after 1 hr. subtraction of nonspecific adsorption values revealed that the bgp1 b ntd was 6-fold infectious center assays. to this end, cdnas encoding bgp1 a or bgp1 b ntd were introduced into plasmid expres-less effective than bgp1 a in virion adsorptive capacity. this difference in efficiency of binding was maintained identify the residues involved in virus binding by systematically exchanging 1 a and 1 b residues. this was accomthroughout the 8-hr, 4њ incubation period. moreover, this plished through a series of restriction fragment ex-pattern whereby bgp1 a bound more virus than 1 b ntd rechanges to produce recombinant receptors containing mained constant over a 10-fold range of virus input multivarying numbers of 1 b residues within a bgp1 a backplicities (data not shown). ground (fig. 7) . these receptors were expressed in in all subsequent assays, we chose to interact [ 35 s] hela cells from vaccinia vectors and then tested for virions with receptor-bearing cells for 1 hr at 4њ, as bindtheir ability to bind [ 35 s] labeled mhv particles. chimeric ing differences between the two receptor isoforms were receptors containing 1 b residues from positions 54 to 122 readily discernable under these conditions. our next were as effective as 1 a in binding, while the reciprocal assay involved testing the related strain jhmx for bindchimeras containing 1 b residues 10 to 54 were ineffective ing. for jhmx, a similar pattern of virus adsorption (table 2) . emerged (fig. 6) . the amount of virion adsorption, how-to further narrow down residues controlling receptor ever, was lower for jhmx and the difference between binding efficiency, we focused on codons 38 to 43, as bgp1 a and bgp1 b ntd in jhmx adsorptive capacity was only this region is the most variable among the presently se-2.5-fold, comparatively lower than the 6-fold difference quenced bgps (see fig. 8 ; boxed region). additionally observed when strain a59 was used. this stretch of six residues is predicted to form a protruding loop connecting two b strands that form the frame-mapping the mhv receptor determinants that control work of the immunoglobulin-like cea domain (bates et virus adsorption al., 1992) . thus the bgp1 a residues 38 to 43 were the two receptors contain a limited number of amino changed to 1 b residues by oligonucleotide-directed mutaacid differences and show measurable differences in genesis. the resulting chimera (1 b 38-43) exhibited a weak binding efficiency that was equivalent to that of virus binding capacity. this provided an opportunity to (table 2) . thus a key determinant of binding differences between the isoforms was present within enhance intercellular fusion with s-expressing cells. to these six residues. do this, we employed a cell fusion-dependent reporter gene activation assay (nussbaum et al., 1994) . in brief, in performing these experiments comparing the efficiency of bgp1 a and bgp1 b ntd receptors, we readily identified a correlation between virus binding capacity and syncytium formation. upon infection by mhv-a59, stable hela-bgp1 a transfectants fused rapidly into polykaryons, while the corresponding hela-bgp1 b ntd cells were much less susceptible. this was readily evident by simple microscopic examination of infected cell monolayers anywhere from 10 to 30 hr postinfection. however, previously published information suggested that measurements of additional receptor mutants for virus binding and fusion promotion capacities might not reveal a direct relationship between these two properties. first, evidence for the uncoupling of virus binding and fusion activation functions has been demonstrated in studies of mutant retrovirus receptors (james et al., 1996; lifson et al., 1988; truneh et al., 1991) . second, two reports focusing on the the results obtained from a subset of these tests is shown in fig. 9 . when cells were fixed and stained with transfectants with vaccinia virus (strain wr) and mhv strain a59 and then loading a fraction of the cells with x-gal (macgregor et al., 1989) , intense blue clusters were abundantly evident in monolayers expressing bgp1 a (fig. a transcriptionally silent b-galactosidase gene under the control of the phage t7 promoter. these cells, which 9a, panel a) and the bgp1 a q10g,v123g mutant. fewer blue syncytia were seen in the bgp1 b ntd -expressing mono-bear s proteins on their surface, were then overlaid at subconfluent densities onto monolayers of hela cells layers (fig. 9a, panel c) , while blue cells were rare in the cultures lacking the bgp ntd altogether (fig. 9a , coinfected with vtf7.3 and the respective vtm1-bgp recombinants. transfer of the t7 polymerase to the s-ex-panel b). quantitation of the b-galactosidase activity in detergent lysates prepared from each monolayer at vari-pressing cells causes b-galactosidase expression, which can be measured and taken as an indicator of the ous times after cell mixing revealed the potency of each receptor in promoting fusion (fig. 9b) . from 2 to 4 hr after amount of fusion between receptor and spike-bearing cells. cocultivation, specific b-galactosidase expression in cultures harboring bgp1 a was 5-6 times the level found in bgp1 b ntd -containing cultures, in concert with the sixfold previous qualitative assays in which virions were al-bound to bgp1 b ntd . when identical assays were performed using [ 35 s] mhv strain jhmx, 2.8% bound specifi-lowed to interact with denatured and immobilized mhv receptors showed that allelic receptor isoforms termed cally to bgp1 a , while 0.8% bound bgp1 b ntd (fig. 6 ). these differences between the two receptors in virus binding bgp1 a and 1 b do indeed differ in virion adsorptive capacity (boyle et al., 1987; dveksler et al., 1993a) , thereby capacity impact the outcome of mhv infection. we found that transfection of either bgp1 a or bgp1 b ntd genes into providing the opportunity for determining which nonhomologous region(s) control the differences. our initial hela cells conferred sensitivity to mhv a59 infection, but bgp1 b ntd was inefficiently utilized as revealed by goal was to establish assays in which we could accurately measure the relative virus binding capacities of quantitation of infectious centers (fig. 4) . this latter finding was generally consistent with a variety of previous these two receptors. we anticipated that the success of these assays might require abundant receptor levels observations. chen and baric (1996) reported that challenge of stable bhk-bgp1 a transfectants with mhv a59 because previous attempts to measure virion binding to murine cells endogenously expressing bgps were not led to titers of 10 8 pfu/ml after 1 day, while parallel challenge of bhk-mmcgm2 (bgp1 b ) transfectants sensitive enough to reveal any specific adsorption (yokomori et al., 1993; wilson and dales, 1988) . our binding yielded only 10 4 pfu/ml. in similar tests, compton (1994) found analagous (but less dramatic) differences between assays therefore involved interaction of radiolabeled virions to native receptors that were overexpressed on the the two stably transfected bhk cell lines in their support of mhv a59 infection. finally, yokomori and lai (1992b) surface of hela cells from vaccinia vectors. by this method, specific binding measurements were obtained, found that cos7 cells transiently transfected with bgp1 a or 1 b supported very low levels of mhv a59; the 1 a even though the proportions of cell-associated [ 35 s] virions were relatively low (fig. 6) . adsorption might be lim-transfectants were marginally superior as virus hosts. we have exchanged portions of bgp1 a cdna with ited in part by slow diffusion to cell monolayers, as prolonged incubation periods gradually increased binding those from bgp1 b and have assessed the resulting hybrid gene products for virus binding capacity. our most infor(fig. 5) . ligand densities also likely play a role-recombinant bgp1 a that is immobilized onto sepharose at ex-mative recombinant receptor was a bgp1 a in which only residues 38 -43 were specific to 1 b . this chimera was traordinarily high densities will bind the majority of [ 35 s] virions in our recently developed in vitro receptor binding indistinguishable from the complete bgp1 b ntd in its support of virus binding (table 2 ; see data highlighted by assays (gallagher, 1997) . we established conditions in which parallel hela cell bold type), thus strongly suggesting that these six residues are the critical determinants controlling virus ad-monolayers displayed equivalent amounts of either bgp1 a or a chimeric bgp1 b ntd on the cell surface. these sorption levels. despite this compelling data, it must be fig. 9 . quantitation of intercellular fusion between spike-and receptor-bearing hela cells. stable hela-bgp1 a transfectants were infected with mhv strain a59 (m.o.i. å 2) and concomitantly transfected by lipofection with pg1nt7-bgal, which produces the b-gal product only in the presence of t7 rna polymerase. 5 hr later, the spike-bearing cells were trypsinized and overlaid onto hela cell monolayers that had been infected 20 hr earlier with vtf7.3 and the indicated vtm3-bgp recombinants. the resulting intercellular fusion levels were quantitated by measuring b-gal enzyme activities produced by the mixing of t7 rna polymerase (in vtf7.3/vtm3-bgp cytosol) with pg1nt7-bgal (in mhv-a59 cytosol). (a) cocultivated cells were fixed after a 3-hr incubation and incubated with x-gal substrate to reveal the b-gal product in situ (a) bgp1 a , (b) bgp1d, (c) bgp1 b ntd , (d) bgp1 a q10g,v123g . (b) at the indicated times after overlay, cell monolayers lysed with pbs containing 0.5% np-40. b-gal activity in the lysates was measured by a colorimetric assay and the od 590 nm values were normalized by comparison with od590 nm produced by a preparation of purified bgalactosidase. remembered that a tremendous number of possible re-the binding capacities of the receptors. in fact we have come to similar conclusions using mhv jhm in place of ceptor chimeras can be generated, each with a unique combination of 1 a and 1 b residues. it therefore remains a59-while receptor independent fusion was observed with jhm, enhancement of this endogenous activity was possible that investigations of additional 1 a /1 b combinations will reveal a role for residues outside the 38-43 most pronounced in the presence of receptors with 1 a residues 38 -43. at present we have no evidence of sepa-stretch in binding, perhaps because some patterns will impact overall receptor conformation. rate domains on the bgp molecule that are individually responsible for binding and induction of membrane fu-exchange of bgp1 a residues 38-43 with those from 1 b also eliminates a potential n-linked glycan addition site sion. however, such separate domains may eventually be identified, as studies of both the hiv and poliovirus at residue 37. however it has been established from thorough mutagenesis studies in the holmes laboratory receptors have revealed specific regions that induce changes in virion conformation required for membrane that a carbohydrate at this position does not contribute to virus binding (dveksler et al., 1995) . identification of penetration (james et al., 1996; morrison et al., 1994) . finally, the results from these quantitative binding these critical amino acid residues led us to attempt blockade of binding with synthetic peptides. to date we assays may shed light on the determinants of murine coronavirus tropism in vivo. jhmx can infect murine brain have been unable to interfere with the virus:receptor interaction using synthetic bgp1 a peptide 33-45, implying tissue more readily than a59 (lavi et al., 1986; robb and bond, 1979) , and the overall abundance of bgp1 a and 1 b a requirement for the immunoglobulin-like framework region in presenting a defined conformation of the resi-in brain is very low lai, 1992a, 1992b) . given that jhmx is actually less avid than a59 in the dues. according to a predicted three-dimensional model for bgp binding properties, it appears likely that the different in vivo tropisms of these strains are not dictated simply the corresponding human cea ntd (bates et al., 1992) , residues 38 to 43 would represent a ''loop'' that is held by their capacity to bind these bgps but rather by utilization of alternative receptors or by additional events oc-in place by two of the beta strands (strands c and c) that comprise a portion of the framework region (fig. 8) . curring subsequent to binding. this c-c loop is further predicted to protrude from the internal framework and thus it is reasonable to infer its choe and sodroski, 1992; harrison, 1994) . pogossen and herman bujard for the hela-tta cells. this work was liovirus binding is influenced by mutations in the c-cљ, supported by nih grant r29-ns-31616 and by a grant from the cold spring harbor using recombinant vaccinia virus vectors a pregnancy-specific glycoprotein is expressed in the brain and serves as a receptor for mouse hepatitis fusion mutants are attenuated and display altered hepatotropism utility of mouse cell line dbt for propagation and assay of mouse hepatitis virus persistence: coevolution of increased host cell resistance and virus virulence contribution of charged amino acids in the cdr2 region of cd4 to hiv-1 gp120 binding pcr protocols'' (m. a. monoclonal antibodies to murine hepatitis virus-4 (strain jhm) define the viral glycoprotein responsible for attachment and cell-cell fusion the organ tropism of mouse hepatitis virus a59 in mice is dependent on dose in their use of murine carcinoembryonic antigen-related glycoprotein receptors synthetic cd4 peptide derivatives that inhibit hiv infection and cytopathicity. science 241, 712-716. the mouse hepatitis virus (mhv) receptor: expression in human and hamster cell lines confers susceptibility to mhv expression in vivo analysis of genomic and intracellular viral rnas of small plaque mutants of related glycoprotein family are functional receptors for the coronavirus mouse hepatitis virus-a59 virus strain a59 and blocking antireceptor monoclonal antibody bind to the n-terminal domain of cellular receptor quantitative equilibrium and kinetic binding studies of mutants in conjunction with a high resolution cd4 atomic structure homolog-scanning mutagenesis reveals poliovirus receptor residues important for virus binding and replication escherichia coli gpt gene provides dominant selection for vaccinia virus open reading frame expression lipofection: a highly efficient, lipid-mediated dna-transfection procedure. mouse hepatitis virus infection proc. natl. acad. sci. usa bgp2, a new member of the carcinoembryonic antigen-related gene family, encodes an transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage t7 rna polymerase fusogenic mechanisms of enveloped-virus glycoproteins analyzed by a novel recomindependent infection by a neurotropic murine coronavirus murine coronavirus membrane fusion is blocked by modification of thiols buried within the spike protein a role for naturally occurring variation of the measles virus hemagglutinin and cd46 pathogenic murine coronaviruses. murine coronavirus spike protein in stabilizing association with the cellular receptor characterization of biological behavior in vitro and virus-specific intracellular rna of strongly neurotropic jhmv and weakly neuro-gombold molecular cloning: protein cleavage signal tight control of gene expression in a laboratory manual in vivo and in vitro models of demyelinating disease: efficiency of virus spread and formation of 5467 cloning with pcr. in ''pcr protocols'' (m. a. innis, infectious centers among glial cells is genetically determined by the murine host mouse hepatitis virus utilizes two carcinoembryonic antigens as alternative receptors tis virus in the resistant mouse strain sjl is functional: implications for the requirement of a second factor for viral infection 5942-spike protein-dependent cellular factor other than the viral receptor is required for mouse hepatitis virus entry cљ-d, and d-e loops (aoki et al., 1994; morrison et al., schweppe foundation of chicago. 1994 ). additional comparisons of receptor structure and function will require definitive resolution of the bgp1 a references receptor through crystallographic methods. this is fast becoming a realistic possibility as a system for producaiyar, a., and leis, j. (1993) . modification of the megaprimer method tion and purification of reasonably large quantities of of pcr mutagenesis: improved amplification of the final product. 14, 366-368. that the relative fusion levels corresponded directly with key: cord-275234-t6e7vr9y authors: leone, gustavo; duncan, roy; mah, david c.w.; price, angela; cashdollar, l.william; lee, patrick w.k. title: the n-terminal heptad repeat region of reovirus cell attachment protein σ1 is responsible for σ1 oligomer stability and possesses intrinsic oligomerization function date: 1991-05-31 journal: virology doi: 10.1016/0042-6822(91)90677-4 sha: doc_id: 275234 cord_uid: t6e7vr9y abstract the oligomerization domain of the reovirus cell attachment protein (σ1) was probed using the type 3 reovirus of synthesized in vitro. trypsin cleaved the α1 protein (49k molecular weight) approximately in the middle and yielded a 26k n-terminal fragment and a 23k c-terminal fragment. under conditions which allowed for the identification of intact σ1 in the oligomeric form (∼200k) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the n-terminal 26k fragment was found to exist as stable trimers (80k) and, to a less extent, as dimers (54k), whereas the c-terminal fragment remained in the monomeric form. a polypeptide (161 amino acids) containing the n-terminal heptad repeat region synthesized in vitro was capable of forming stable dimers and trimers. using various criteria, we demonstrated that the stability of the intact σ1 oligomer is conferred mainly by the n-terminal heptad repeat region. our results are summarized in a model in which individual heptad repeats are held together in a three-stranded α-helical coiled-coil structure via both hydrophobic and electrostatic interactions. the reovirus cell attachment protein (protein ~1) is strategically located at the twelve vertices of the outer capsid of the viral icosahedron (lee et al., 1981; furlong eta/., 1988) and plays a pivotal role in viral infectivity and tissue tropism (sharpe and fields, 1985) . in electron microscopy, this protein can sometimes be seen as lollipop-shaped structures with proximal fibrous tails and distal globular heads projecting from the surfaces of viral particles (furlong et a/., 1988) . protein al purified from reovirions or from a vaccinia expression system also has a similar morphology (furlong et a/., 1988; banerjea et a/., 1988; fraser et a/., 1990) . the observations that the c-terminal half of al contains the receptor-binding domain (nagata et al., 1987; yeung et a/., 1989) and the n-terminal onequarter possesses intrinsic virion-anchoring property (mah et a/., 1990) suggest that the fibrous tail and the globular head represent the n-and c-terminal portions, respectively, of this protein. these findings concur with sequence analysis of the reovirus sl gene (encoding (rl), which predicts the existence of distinct structural domains in the al protein (bassel-duby et a/., 1985; duncan et al., 1990; nibert et al., 1990) . the ' present address: mcintyre medical sciences building, mcgill cancer centre, 7/f, 3655 drummond street, montreal, quebec, canada h3g ly6. ' to whom requests for reprints should be addressed. n-terminal one-third of al is highly a-helical and contains a heptapeptide repeat of hydrophobic residues, suggestive of a coiled-coil structure. this is followed by a middle region composed largely of p-sheets. the cterminal one-third of 01 does not possess any distinct patterns and is therefore predicted to assume a complex globular structure. the oligomeric nature of al has also been examined (bassel-duby et al., 1987; banerjea et a/., 1988) . when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) under dissociating conditions (boiled in sds-containing sample buffer), the 455 amino acid long al migrates as a monomeric 44k molecular weight protein. however, if the boiling step is omitted, al migrates as an oligomer (-200k molecular weight) . this observation, coupled with the identification of a total of four bands upon chemical cross-linking of purified al (with the largest species migrating at a position corresponding to approximately 200k), has led to the suggestion that al is a tetramer (bassel-dubyetal., 1987) . the oligomerization state of al is apparently closely linked to its function since of the two al species (monomeric and oligomeric) synthesized in an in vitro transcription and translation system, only the oligomeric form is capable of binding to cell receptors (g. leone, r. duncan, and p. w. k. lee, unpublished data) . this observation prompted us to examine the nature of al oligomerization, an understanding of which should lead to better definitions of structure-function relationships of this protein. in this report, we used trypsin treatment as an initial step to identify regions that are important for maintaining and stabilizing the al oligomeric structure. of the two fragments generated by such treatment (yeung et a/., 1989) , only the n-terminal fragment, but not the c-terminal fragment, was found to exist as stable trimers (and dimers to a less extent) upon sds-page analysis. subsequent in vitro transcription and translation experiments revealed that the n-terminal one-third of al, which harbors the heptapeptide repeat region, possesses intrinsic dimerization and trimerization function. we further demonstrated that ionic interactions, in addition to hydrophobic interactions, within the heptapeptide repeat region are also responsible for stabilizing the ul oligomer. these observations have led to a structural model of the oligomerization domain of protein al. the plasmid (pg4t3) used in the present study was derived from our prokaryotic sl -gene expression vector psp4 (masri et al., 1986) in which the sl gene contained additional sequences at the 3'-end derived from pbr322 (pstl-ecori) during the original subcloning procedure. this plasmid was cleaved with sstll, which cuts the sl gene at position 1397, and the synthetic linker 5'-ggcactggggcat-ftcatcggtac-3' 3'-cgccgtgaccccgtaaagtagc-5' was added which contained the authentic 3'-terminal sl gene sequence from the sstll site to the 3'-end of the gene where a unique kpnl site was added. a barnhi-kpnl fragment was isolated (from the barnhi site at position 15, which cuts immediately after the atg initiation codon of al to the newly introduced kpnl site at the 3'-end of the gene) and ligated to an ecori-bamhl translation initiation linker (pharmacia): the resultant ecori-kpnl fragment was cloned into the ecori-kpnl site of pgem-4 (promega biotec) to produce pg4t3. the plasmid pg4t3 was linearized with either hindill or ecorv restriction endonucleases and used in in vitro transcription reactions to generate full-length or truncated sl mrnas. the mrnas were then translated in vitro in rabbit reticulocyte lysates according to the manufacturer's specifications (promega). a typical translation mixture contained 50-l 00 ng of rna and 20 &i of [35s]methionine in a total volume of 25 ~1. after incubation at 37" for 45 min, reactions were stopped by the addition of 200 ~1 phosphate-buffered saline (pbs). protein sample buffer (5x) was then added (final lx concentration was 50 mn/r tris, ph 6.8, 1% sds, 2% p-mercaptoethanol, 10% glycerol, and 0.01% bromphenol blue) and the mixtures were further incubated at 37" for 30 min (nondissociating condition) or boiled for 5 min (dissociating condition) prior to sds-page. upon completion of the in vitro translation reaction, tlck-treated trypsin (sigma) dissolved in pbs (0.05 mg/ml) was added to the translation mixtures to a final concentration of 0.005 mg/ml (unless otherwise stated). after incubation at 37" for 30 min, trypsin inhibitors (soybean and egg white trypsin inhibitors, sigma) were added and the mixtures were further incubated in protein sample buffer prior to sds-page (see above). the monoclonal anti-al antibody g5 has been previously described (burstin et al., 1982) and shown to interact with the c-terminal tryptic fragment of 01 (yeung et a/., 1989) . the n-terminal specific polyclonal anti-u1 antiserum was prepared in rabbits using the sds-page-purified trpe-al fusion protein (containing trpe and residues l-l 58 of al) expressed in escherichia co/i using the path3 vector (cashdollar et a/., 1989). aliquots of lysates or trypsin-treated lysates were mixed with an equal volume of appropriate dilutions of the antibodies and incubated at room temperature for 1 hr. fixed staphylococcus aureus (10% suspension) that had been preadsorbed with bsa (5 mg/ ml) was then added to the mixture and incubated for an additional 30 min. lmmunoprecipitates were washed three times with wash buffer (50 mmtris, ph 7.4, 150 mm naci, 0.1% sds, 1% triton x-l 00) resuspended in 140 ~1 high-ph buffer (50 mm tris, 0.1 m h,po,, 2 mm dtt, 0.1% sds, 6 m urea, ph to 1 1.6 with naoh), and incubated at 37" for 45 min. suspensions were then pelleted and supernatants neutralized with 7 ~1 neutralizing solution (0.1 m h,po,, 1 n/r tris, ph 7.4). protein sample buffer was then added to the samples and either incubated at 37" for 30 min (nondissociating condition) or boiled for 5 min (dissociating condition) prior to sds-page. discontinuous sds-page was performed using the protocol of laemmli (1970) . both 10 and 12.5% acryl[35s]methionine-labeled reovirus-infected cell lysates (s-45) prepared as previously described (lee er a/., 1981) and [%]methionine-labeled in vitro translation products of the reovirus sl mrna in rabbit reticulocyte lysates (sp6), were precipitated with an anti-01 monoclonal antibody (g5). after being released from the immunoadsorbent, precipitated proteins were mixed with protein sample buffer and were either boiled for 5 min (6) or incubated at 37" for 30 min (37) prior to sds-page. r (lane 1) represents purified [?s]methionine-labeled reovirus. amide gels were used. gels containing 35s-labeled proteins were fixed and then treated with dmso-ppo, dried under vacuum, and exposed to kodak xar-5 film at -70". gels to be used for band excision were dried under vacuum without prior treatment, and then exposed to film. developed x-ray film was superimposed on dried gels and bands to be excised were marked with a pouncer. marked protein bands were excised from gels and rehydrated in laemmli running buffer. proteins from excised bands were electroeluted into electroelution cups in a volume of 200 jj. protein sample buffer was added to electroeluted proteins and boiled for 5 min prior to sds-page. since some of the following studies on al oligomerization involved the use of genetically truncated protein ct~ , it was necessary to express ~1 in an in vitro system and to ascertain that oligomeric al was indeed generated in such a system. to this end, t3 reovirus sl mrna was prepared in vitro using sp6 rna polymerase and translated in a rabbit reticulocyte lysate. translation products were then immunoprecipitated with an anti-al antibody (g5) and analyzed by sds-page under conditions that would not cause the disruption of ~1 oligomers (see materials and methods). the results, illustrated in fig. 1 , show that stable ~1 oligomers were indeed produced in vitro (lanes 4 and 5) and their migration rate was identical to that of authentic ~1 from t3 reovirus-infected cells (lanes 2 and 3). like the authentic protein, al oligomers synthesized in vitro were capable of binding to cell receptors (r. duncan, g. leone, and p. w. k. lee, unpublished observations) , and were cleaved by trypsin to yield a well-defined pattern (see below). some al monomers (44k) were also produced in this system, but they were not precipitable by the anti-al antibody, did not manifest cell-binding function, and were highly susceptible to degradation by trypsin or chymotrypsin even when these proteases were used at very low concentrations (data not shown). subsequent analysis exclusively of the oligomeric form of ~1 was therefore possible. previously it was found that trypsin cleaves the ~1 oligomer near the middle to generate two fragments of approximately equal size (banerjea et al., 1988; yeung et a/., 1989) . the cleavage site has now been determined to be between arg245 and lle246 (duncan and lee, unpublished data) . thus the monomeric forms of the n-terminal fragment (245 amino acids) and the cterminal fragment (210 amino acids) have molecular weights of approximately 26k and 23k, respectively, corresponding to their migration rates in sds-page under dissociating conditions (yeung et a/., 1989) . such a cleavage pattern was also obtained with al synthesized in vitro (fig. 2, lane 3 ). an additional minor band of approximately 21 k molecular weight was also observed sometimes. using n-and c-terminal-specific r37b xc sera, we have identified the 26k and 21 k tryptic fragments to be of n-terminus origin, and the 23k fragment to be of c-terminus origin (fig. 4) . to see whether any of the tryptic fragments could be identified in the oligomeric state, trypsin-treated al in sds-containing sample buffer was incubated at 37" (rather than boiled) prior to sds-page. it was found that the 26k n-terminal fragment was replaced quantitatively by a band migrating at approximately 80k molecular weight (fig. 2, lane 2) . the migration rate of the 23k c-terminal fragment remained unchanged. the 2 1 k n-terminal fragment was absent; instead two faint bands of approximately 48k and 54k molecular weight appeared. protein bands of 80k, 54k, and 48k molecular weight were excised from a gel, eluted, boiled in sample buffer, and subjected to sds-page (fig. 3) . both the 80k and 54k proteins were found to be converted to the 26k n-terminal fragment, whereas the 48k protein was converted to the 2 1 k n-terminal fragment. the most reasonable interpretation of these results would be that the 80k and 54k bands represent the trimer and dimer forms, respectively, of the 26k n-terminal fragment, and the 48k band represents the dimer of the 21 k n-terminal fragment. the region in the 26k n-terminal fragment that is absent in the 21 k nterminal fragment may therefore be involved in stabiliz-ing the third subunit of the 80k n-terminal trimer. the identity of this region is presently unknown. it is important to note that under conditions where the n-terminal tryptic fragment exists as stable trimers (and dimers), the c-terminal fragment was consistently found to be in the monomeric state. this is in contradiction to the findings of banerjea et a/. (1988) who reported that both the n-and c-terminal tryptic fragments exist as stable oligomers (tetramers) in sds at 37". to reconcile such differences, we decided to examine the effects of varying the trypsin concentration on al cleavage pattern. at the lowest concentration (0.005 mg/ml) of trypsin that cleaves al oligomers completely, an identical pattern to that shown in fig. 2 was observed (fig. 4a, lane 2) . increasing the concentration of trypsin to 0.05, 0.1, and 0.5 mg/ml, and subsequently boiling the samples in sample buffer, resulted in the gradual disappearance of the 26k n-terminal fragment with the concomitant appearance, in almost stoichiometric amounts, of a 25k molecular weight band (fig. 4a, lanes 3-5) . thus the 25k band is a cleavage product of the 26k band. the 23k c-terminal fragment remained unchanged. when identical samples were instead incubated at 37" in sample buffer prior to sds-page, both the 26k and 25k bands were replaced by bands with molecular weights of approximately 80k and 75k, respectively (fig. 4a, lanes 6-9) . again, the mobility of the 23k cterminal fragment remained unchanged. radioimmunoprecipitation of the samples with the n-and c-terminus-specific antibodies confirmed that the 25k and 75k proteins, like the 26k and 80k proteins, were of n-terminus origin (fig. 48) and that the 23k protein was of c-terminal origin (fig. 4c) . it is possible to explain the discrepancies between our present findings and those reported by banerjea et al. (1988) on the basis of the methods used for protein al detection. a polyclonal anti-t3 reovirus serum was used by the aforementioned investigators to identify ~1 tryptic fragments on western blots as opposed to radioimmunoprecipitation with anti-al n-and c-terminal-specific antibodies used in our studies. in our hands, polyclonal anti-native al serum is incapable of recognizing the presumably denatured c-terminal tryptic fragment on a blot, although the n-terminal fragment is easily detectable. these observations, together with the fact that the trypsin concentration (1 mg/ml) used by banerjea et al. was within the concentration range where we found both the 26k and the 25k n-terminal cleavage products, have led us to conclude that the two bands previously identified by these investigators as n-terminal and c-terminal oligomers correspond to our 80k and 75k bands, and are therefore in fact both n-terminal oligomers. to determine the extent of involvement of the n-terminal portion in stabilizing the al oligomer, native al and trypsin-treated al were subjected to various treatments prior to sds-page and the relative stability of native al oligomers and n-terminal trimers (80k protein) was compared. whereas dissociation of oligomers by heat was used as a general measure of oligomer stability, the effects of ph variation and presence or absence of urea and @-mercaptoethanol were also examined. under all conditions tested, native al oligomers and n-terminal trimers behaved identically (fig. 5 ). both al oligomers and n-terminal trimers were stable at 50" in sample buffer but dissociated at 60" (fig. 5a ). both were stable under alkaline (ph 1 1.6) to mild acidic (ph 6.0) conditions, but dissociated at ph 5.5 (fig. 5b) . urea (6 iv) apparently had no effect on either oligomerit species (fig. 5c) . it was previously reported that al oligomers were rendered less stable when the concentration of p-mercaptoethanol in the sample buffer was reduced (bassel-duby et a/., 1987). consistent with these findings, we observed that both native al oligomers and n-terminal trimers were less stable in the total absence of p-mercaptoethanol (fig. 5c ). in summary, the stability of the n-terminal trimer was found to be very similar to that of the native al oligomer. these data suggest that interactions between al subunits responsible for stabilizing the oligomeric structure occur mainly, although by no means solely, within the n-terminal half of the al protein. the absence of cysteine residues within the n-terminal tryptic fragment of al suggests that the stabilizing effect of p-mercaptoethanol could not be directly due to its reducing properties. indeed, enhanced oligomer stability was not observed when another reducing agent, dithiothreitol, was used in place of ,& mercaptoethanol (fig. 6a) . a less well-characterized property of p-mercaptoethanol is that of chelation (mcmichael and ou, 1977) . if /3-mercaptoethanol indeed stabilizes oligomers by chelating divalent cations, addition of divalent cations should destabilize oligomers. indeed, mgci, at concentrations above 1 mm was found to destabilize n-terminal trimers (fig. 6b ) as well as native al oligomers (data not shown). other divalent cations (zinc and calcium) had a similar effect (data not shown). the effects of divalent cations and p-mercaptoethanol on oligomer stability are clearly antagonistic. thus the destabilizing effect of 5 mm and 20 ml\/l mgci, could be reversed by the inclusion of 1 and 5% ,l3-mercaptoethanol, respectively, in the sample buffer (fig. 6b) . similar results were obtained using edta as the chelator in place of ,&mercaptoethanol, except that at edta concentrations above 5 mm, oligomer stability was inconsistent (data not shown). the disruption of ~1 oligomers by divalent cations suggests that ionic interactions are involved in stabilizing the n-terminus trimer (and hence the 01 oligomer). indeed, the distribution of charged residues in the nterminal coiled-coil region was found to highly favor the (c) samples were incubated in regular protein sample buffer which contained 2% fl-mercaptoethanol (-u+p, control) , or in protern sample buffer containing 6 m urea in addition to 2% p-mercaptoethanol (+u), or in protein sample buffer lacking both urea and p-mercaptoethanol (-p), at 37" for 30 min prior to electrophoresis. native and trypsin lanes in each experiment originate from the same gel. formation of salt bridges between al subunits (see discussion). however, the presence of the extended heptad repeat in the same region suggests that hydrophobic interactions must play a major role in oligomer stabilization. in this regard, the aforementioned ionic interactions are presumed to serve an augmentative function. in agreement with the above hypothesis, temperature stability experiments indicated that in the presence of divalent cations (abolishing ionic interac-tions), oligomers were stable up to a temperature of 20", but in the absence of divalent cations (maximizing ionic interactions), oligomers were stable up to a temperature of 50" (data not shown). properties of the heptad repeat region of al it is believed that sequences with heptapeptide repeats of apolar residues are involved in stabilizing coiled-coil structures through hydrophobic interactions between the a-helices. in the case of al, the heptad repeat region spans the n-terminal one-third of the protein (from residue 28 to 164). it was then of interest to see whether this region possesses intrinsic dimerization and, in view of the trimeric nature of the n-terminal tryptic fragment of ~1, trimerization functions. to this end, the plasmid pg4t3 was treated with the restriction endonuclease ecorv, which cuts the sl gene at nucleotide 497 (encoding the n-terminal 161 amino acids). run-off mrna transcripts were then prepared and translated in vitro, and the ability of the translational products to oligomerize was then determined by sds-page. the results are shown in fig. 7 . when the samples were boiled in sample buffer prior to sds-page, closely migrating bands of approximately 18k molecular weight were found (fig. 7, lane 2) , as was predicted from the amino acid sequence. the lack of absolute homogeneity in size of the translational products was due to the absence of a translation stop codon in the mrna transcripts, necessitating translation termination to occur by the falling off of ribosomes close to, but not precisely at, the 3'ends of the mrnas. when identical samples were instead incubated at 37" in sample buffer (fig. 7, lane 3) , there was a noticeable decrease in the intensities of the bands migrating at approximately 18k. concomitantly, two additional bands with estimated molecular weights of 36k and 54k appeared. these two bands corresponded to the dimeric (36k) and trimeric (54k) forms of the ecorv translation products. clearly, the dimer was the predominant oligomeric form identified. whether trimer formation was inefficient or whether trimers were less stable in our sds-page system is not known. none-theless, for the first time, direct evidence is presented that demonstrates the intrinsic ability of a heptad repeat (18 repeats in this case) to form dimers and trimers, an ability that may very well depend on the number of repeats present in the polypeptide. to determine the extent to which the heptad repeat region is involved in stabilizing the n-terminal tryptic trimers, the ecorv translational products were subjected to the various treatments previously applied to the n-terminal tryptic fragment and the native ~1 oligomer. the results are shown in fig. 8 . both the dimeric and trimeric forms of the heptad repeat were unstable at 50", a temperature at which the n-terminal tryptic trimer was found to be stable. however, like the n-terminal trimer, heptad dimers and trimers were both stable under alkaline (ph 11.6) to mild acidic (ph 6.0) conditions (but dissociated at ph 5.5) and in 6 m urea, and were destabilized by the absence of p-mercaptoethanol or by the presence of divalent cations in the sample buffer. again, as observed for the n-terminal tryptic trimer, the destabilizing effect of divalent cations could be neutralized by p-mercaptoethanol. fig. 8. oligomer stability of the n-terminal 161 amino acid long polypeptide. the polypeptide synthesized was subjected to various treatments as described in the legends to fig. 5 and 6 . protein sample buffer was then added and the samples were incubated at 37" for 30 min prior to sds-page. reo represents the reovirus marker. molecular weights are indicated at right. servation that receptor-binding function is manifested by the oligomeric form, but not by the monomeric form of al, prompted us to examine the region(s) on ~1 that is responsible for the stability and the formation of the ~rl oligomer. our present analysis of the two tryptic fragments of al has revealed that the stability of the ~1 oligomer is maintained mainly via the n-terminal fibrous portion, rather than the c-terminal globular portion of al. under nondissociating conditions (preincubation in sds-containing sample buffer at 37" for 30 min), the n-terminal half of al, like intact ~1, migrates in sds-page as an oligomer (trimer), whereas the cterminal half migrates as a monomer. we have evidence, however, that the latter ttyptic fragment is also a trimer (albeit a less stable one) which dissociates under the assay conditions used in the present study. the additional observation that both the n-terminal tryptic fragment and the full-length 01 oligomer respond to temperature and ph changes, and to p-mercaptoethanol in a similar manner, further suggests that the n-terminal half of al plays a major role in stabilizing the ul oligomer. that the n-terminal portion of al possesses oligomerization potential was first suggested by bassel-duby eta/. (1985) from sequence analysis of the type 3 reovirus sl gene. first of all, the n-terminal one-third of al was found to be highly a-helical. the additional presence, in the same region, of an extended heptad repeat (a-b-c-d-e-f-g)n, where a and d are characteristically apolar residues, further indicates the propensity of this region to adopt a coiled-coil rope-like structure. such theoretical considerations are clearly compatible with the fibrous morphology of al as revealed by electron microscopy (furlong et al., 1988; banerjea et al., 1988; fraser et a/., 1990) and with the present demonstration that the heptad repeat region alone, when synthesized in an in vitro system, is capable of forming dimeric and trimeric structures whose stability is remarkably similar to that of the trimeric nterminal tryptic fragment or of the native al oligomer. in view of the recent speculations on the structural and functional aspects of the "leucine zipper," we consider our direct demonstration that the heptad repeat possesses intrinsic dimerization and trimerization function to be significant (see also note added in proof). there is little doubt that, in the case of the trimer, apolar residues at positions a and d contribute to hydrophobic interactions that are responsible for holding the three strands together in a coiled-coil configuration (figs. 9a and 9b). in addition, ionic interactions were also found to play a role since the dimers and trimers were destabilized by divalent cations but were restabilized by the addition of a chelator. indeed, an examination of the distribution of charged residues in positions e and g reveals that when the three cu-helices are placed in parallel and in register to each other, residues of opposite charge on adjacent cu-helices invariably lie in juxtaposition (fig. 9c ) (see also . at locations where a corresponding residue of opposite charge is not present on an adjacent a-helix, a spatially close residue on the same helix is present to neutralize the charge. analysis of analogous regions on the al proteins of the other two serotypes reveals a similar, albeit less perfect, distribution pattern of charge residues (data not shown). it is important to point out that whereas the present study clearly indicates that the 01 oligomer is stabilized mainly via the n-terminal half of the protein, the implications of the intrinsic oligomerizing function of the heptad repeat region on the oligomerization process of the intact al oligomer need to be viewed with caution. recent evidence suggests that intracellular protein folding and oligomerization are mediated by chaperones and that these events are atp-dependent. in the case of ~1, we have recently observed that whereas the oligomerization of the n-terminal one-third of ~1 occurs spontaneously, that of full-length al is an atpdependent event (unpublished data), which further suggests that, as has been reported for a number of oligomeric proteins, the formation of the al oligomer is chaperone-mediated. this would in turn imply that the intrinsic oligomerizing function of the heptad repeat region is necessary, but not sufficient, for intact al oligomerization, and that a domain(s) downstream of this region must also be involved. we are currently probing the interactions between the three governing factors, namely, the heptad repeat region, a downstream domain(s), and chaperones, in the al oligomerization process, the revelation of which may have general and fundamental implications. our demonstration that the n-terminal tt-yptic fragment is a trimer and the n-terminal heptad repeat region possesses intrinsic trimerization function suggests that native protein al is most likely also a trimer, although the possibility of al being a multiple of a trimer (e.g., dimer of a trimer) cannot be ruled out based on the present data alone. either model contradicts a recent suggestion, based on sequence analysis and computer-processed electron microscopy, that ~1 is a tetramer (fraser et al., 1990) . although the precise reasons for this discrepancy remain to be revealed, we have recently obtained data from biophysical studies and in vitro ~1 assembly experiments that are compatible with a trimeric, but not a tetrameric (or a hexamerit), model of al (unpublished data). a trimeric ~1 would theoretically migrate at a position corresponding to 150k in sds-page, rather than at -2ook. how-0155 fig. 9. analysis of sequences involved in 01 trimerization. (a) a-helical wheel depiction of the heptad repeat region (amino acids 28 to 158) of ~1. note the predominant presence of hydrophobic residues at positions a and d (see also nibert et al., 1990) . charged residues at positions e and g are also indicated. (b) cross-section of three parallel a-helices depicting plausible hydrophobic interactions between amino acids at positions a and d of adjacent helices. (c) schematic diagram showing interactions between negatively charged (white) and positively charged (black) residues at positions e (helix at left) and g (helix at right) on two adjacent helices (see also ever, our present studies suggest that under the conditions used for its detection (incubation at 37" in sds), the n-terminal fibrous tail of the al trimer would still be intact, whereas the c-terminal globular head would be totally unfolded. the resulting ~1 structure would accordingly resemble a "hydra," rather than a "lollipop," and would therefore manifest a somewhat retarded migration rate. results from more refined experiments correlating incubation temperatures (in sds) with migration rates of intact al and al tr-yptic fragments concur with such a rationale (unpublished observation). it is noteworthy that, in terms of oligomerization status, ~1 is not unlike other viral cell-attachment proteins such as the influenza hemagglutinin the vsv g protein (doms et a/., 1987; kreis and lodish, 1986 ) the adenovirus fiber (van oostrum et a/., 1987; devaux et al., 1990 ) the envelope glycoprotein (gpl20) of the human immunodeficiency virus (weiss et al., 1990) and the coronavirus spike protein (delmas and laude, 1990) all of which appear to be trimeric, and the trimeric state may reflect a unifying concept, hitherto unidentified and perhaps pertinent to structural and/or functional requirements, in the virus attachment process. this work was supported by the medical research council of canada. l.w.c. was supported by a grant from the national science foundation. g.l., d.c.w.m., and a.p. are recipients of alberta heri-tage foundation for medical research (ahfmr) studentships. r.d. is an ahfmr fellow and p.w.k.l. is an ahfmr scholar. note added in proof. shortly after submission of our manuscript, banerjea and joklik (1990) reported the intrinsic oligomerizing property of the n-terminal heptad repeat region of 01. high-level synthesis of biologically active reovirus protein 01 in a mammalian expression vector system reovirus protein ~1 translated in vitro, as well as truncated derivations of it that lack up to two-thirds of its c-terminal portion, exists as two major tetrameric molecular species that differ in electrophoretic mobility sequence of reovirus hemagglutinin predicts a coiled-coil structure evidence that the ~1 protein of reovirus serotype 3 is a multimer evidence for functional domains on the reovirus type 3 hemagglutinin identification of the ~1 s protein in reovirus serotype 2-infected cells with antibody prepared against a bacterial fusion protein assembly of coronavirus spike protein into trimers and its role in epitope expression structure of adenovirus fibre. i. analysis of crystals of fibre from adenovirus serotypes 2 and 5 by electron microscopy and x-ray crystallography role for adenosine triphosphate in regulating the assembly and transport of vesicular stomatitis virus g protein trimers identification of conserved domains in the cell attachment proteins of the three serotypes of reovirus molecular structure of the cell-attachment protein of reovirus: correlation of computer-processed electron micrographs with sequence-based predictions sigma 1 protein of mammalian reoviruses extends from the surfaces of viral particles oligomerization is essential for transport of vesicular stomatitis viral glycoproteins to the cell surface cleavage of structural proteins during the assembly of the head of bacteriophage t4 protein 01 is the reovirus cell attachment protein the n-terminal quarter of reovirus cell attachment protein al possesses intrinsic virion-anchoring function functional expression in fscherichia co/i of cloned reovirus sl gene encoding the viral cell attachment protein al metal ion dependence of a heat-modifiable protein from the outer membrane of fscherichia co/i upon sodium dodecyl sulfate-gel electrophoresis analysis of functional domains on reovirus cell attachment protein ~1 using cloned sl gene deletion mutants structure of the reovirus cell attachment protein: a model for the domain organization of ~1 pathogenesis of viral infections. basic concepts derived from the reovirus model the structure of the adenovirus capsld. ill. hexon packing determined from electron micrographs of capsid fragments. 1 oligomeric organization of gpl20 on infectious human immunodeficiency virus type 1 particles structural identification of the antibody-binding sites of hong kong influenza hemagglutinin and their involvement in antigenic variation structure of the hemagglutinin membrane glycoprotein of influenza virus at 3a resolution the cell attachment proteins of type 1 and type 3 reovirus are differentially susceptible to trypsin and chymotrypsin key: cord-277838-931sco95 authors: erles, kerstin; toomey, crista; brooks, harriet w; brownlie, joe title: detection of a group 2 coronavirus in dogs with canine infectious respiratory disease date: 2003-06-05 journal: virology doi: 10.1016/s0042-6822(03)00160-0 sha: doc_id: 277838 cord_uid: 931sco95 an investigation into the causes of canine infectious respiratory disease was carried out in a large rehoming kennel. tissue samples taken from the respiratory tract of diseased dogs were tested for the presence of coronaviruses using rt–pcr with conserved primers for the polymerase gene. sequence analysis of four positive samples showed the presence of a coronavirus with high similarity to both bovine and human coronavirus (strain oc43) in their polymerase and spike genes, whereas there was a low similarity to comparable genes in the enteric canine coronavirus. this canine respiratory coronavirus (crcv) was detected by rt–pcr in 32/119 tracheal and 20/119 lung samples, with the highest prevalence being detected in dogs with mild clinical symptoms. serological analysis showed that the presence of antibodies against crcv on the day of entry into the kennel decreased the risk of developing respiratory disease. canine infectious respiratory disease (cird) is a highly contagious disease, especially in dogs housed in groups in rehoming centers and boarding or training kennels. many dogs suffer from a mild cough and recover after a short time; however, in some cases a severe bronchopneumonia can develop (appel and binn, 1987) . in addition to causing distress to the dogs, cird also delays rehoming at rescue centers and causes disruption of schedules in training kennels as well as incurring considerable treatment costs. cird is considered to be a multifactorial disease because it usually occurs when dogs from different origins are brought together, a situation that exposes the dogs to a variety of different microorganisms as well as an unfamiliar environment. the infectious agents that have been considered the major causative pathogens involved are canine parainfluenzavirus (cpiv) (binn et al., 1967) , canine ade-novirus type 2 (cav-2) (ditchfield et al., 1962) , and the bacterium bordetella bronchiseptica (bemis et al., 1977a; keil et al., 1998) . also, canine herpesvirus, human reovirus, and mycoplasma species have been isolated from dogs with symptoms of cird (karpas et al., 1968; lou and wenner, 1963; randolph et al., 1993) . experimental infection of dogs with single infectious agents has been shown to cause only mild respiratory symptoms but failed to reproduce the severe disease that can be seen in natural outbreaks, supporting the theory that the pathogenesis of cird is multifactorial (appel and percy, 1970; karpas et al., 1968; bemis et al., 1977b) . vaccines are available against some of the infectious agents that have been found to be associated with this disease, namely, bordetella bronchiseptica as well as cpiv and cav-2. despite the use of these vaccines, cird is still prevalent in kennels worldwide, suggesting that additional viruses or bacteria may be involved in the disease. members of the family coronaviridae are enveloped viruses, 80 -160 nm in diameter, containing a linear positive-stranded rna genome. the structural proteins of coronaviruses are the spike glycoprotein, the membrane glycoprotein, and the nucleocapsid protein. the hemagglutinin/ esterase glycoprotein is found only on the surface of group 2 coronaviruses (e.g., bovine coronavirus and murine hepatitis virus) (spaan et al., 1988) . the polymerase gene of coronaviruses is known to be highly conserved. it has therefore previously been used for phylogenetic analysis of this virus family (stephensen et al., 1999) . a possible role of coronaviridae in the pathogenesis of cird was investigated in this study because members of this family are known to cause respiratory disease in humans as well as cattle, swine, and poultry (mäkelä et al., 1998; pensaert et al., 1986; ignjatovic and sapats, 2000) . in cattle, bovine respiratory coronavirus is associated with shipping fever, a multifactorial respiratory disease similar to cird (storz et al., 2000) . canine coronaviruses are reported to cause acute diarrhea mainly in young dogs (tennant et al., 1993) . however, one study reports the detection of canine coronavirus in dogs with respiratory disease and describes the isolation of the virus from one lung sample and three intestinal samples (binn et al., 1979) . this investigation sought to detect coronaviruses associated with cird in a large kenneled dog population with a history of endemic respiratory disease, using virus culture and pcr techniques as well as serology on paired serum samples. using the primers conscoro5 and conscoro6, we analyzed the cdna obtained from 40 tracheal samples by rt-pcr. of these, 7 were found to be positive by pcr and subsequent hybridization (17.5%). the pcr products were cloned and sequenced and the sequence data were compared to available viral sequences using the fasta similarity search program (pearson, 1990) . comparison of the coronavirus cdna polymerase sequence obtained from four of the canine tracheal samples to other coronavirus sequences revealed that they were most similar to sequence data from bovine coronavirus (genbank accession no. af 220295) and human coronavirus strain oc 43 (genbank accession no. af 124989). the identity in the analyzed 251-bp sequence was 98.8% for the bovine and 98.4% for the human coronavirus polymerase gene, whereas it was only 68.53% for canine coronavirus (strain 1-71). an alignment of the novel sequence with the corresponding sequences of 11 coronaviruses and phylogenetic analysis using the maximum parsimony method resulted in the consensus tree shown in fig. 1 . the cdna sequence obtained from a tracheal sample (t101) was found on a common branch with bovine coronavirus, human coronavirus-oc43, and hemagglutinating encephalomyelitis virus. the virus was provisionally called canine respiratory coronavirus (crcv). for further analysis of the rna sequence of crcv, an alignment of the rna for the spike gene of the bovine coronavirus ly 138 strain and the human coronavirus oc43 strain was performed. consensus regions were chosen for the selection of four primer pairs amplifying the complete spike gene in four overlapping fragments; the primer sequences are shown in table 1 . the cdna obtained from tracheal sample t101 was used to perform rt-pcr and subsequent sequencing of the obtained spike fragments. the analysis of the sequencing data showed that the spike gene of crcv is 4092 nucleotides long, corresponding to 1093 amino acids. it was determined that the cdna sequence obtained from tracheal sample t101 had a 97.312% nucleotide identity with the spike gene of bovine coronavirus strain ly138 (genbank accession no. af058942) and 96.921% identity with that of human coronavirus strain oc43 (genbank accession no. z32768) in an overlap of 4092 nucleotides. when comparing the amino acid sequence obtained by translation of the cdna sequence from t101 to the amino acid sequence of the bcv, hcv-oc43, and canine enteric coronavirus spike proteins the identities were 96, 95.2, and 21.2%. bovine coronavirus and other group 2 coronaviruses contain an additional structural protein, the hemagglutinin/ esterase (he). because of the high similarity of crcv with bcv, we analyzed the presence of a he gene in crcv. an alignment of the he gene sequences of bcv and hcv oc43 was used to design the primers he1 and he2 (table 2 ). four tracheal samples that had previously been identified as positive for coronavirus rna by rt-pcr with primers for the spike gene were tested by rt-pcr with the primer set for the he gene. all four samples showed a pcr band of the expected size (fig. 2) . using a nested set of primers for the spike gene (sp1-2 and sp3-4), tracheal and lung samples from 119 dogs were analyzed for crcv. of these, 42 were from dogs with no respiratory signs (grade 1), 18 dogs had shown mild respiratory signs (grade 2), 46 had shown moderate (grade 3), and 13 severe respiratory signs (grades 4 and 5). grades 4 and 5 were merged due to the low case numbers in these groups. in total 32 tracheal samples (26.9%) and 20 lung samples (16.8%) were found positive by nested rt-pcr. for eight dogs a positive pcr result was obtained for both trachea and lung. table 3 shows the pcr results for coronavirus in dogs with different grades of respiratory disease. sequence analysis of the nested pcr products obtained from tracheal tissues of six different dogs showed identical dna sequences for all six cdna samples. because of the homology of the spike gene region of crcv to the spike region of bovine coronavirus, an elisa antigen for bcv was used for serological analysis. sera from 5 dogs with no history of infectious respiratory disease that had not been housed in the investigated kennel were tested. the od values ranged from ϫ0.013 to 0.39, with an average od value of 0.154. furthermore, sera from 30 dogs admitted to a veterinary clinic for various reasons were tested for antibodies to coronavirus. of these, 20 samples showed an od of ͻ0.4 (ϫ0.46 to 0.396) and 10 samples showed an od of ͼ1.0 (1.012 to 1.949). samples with an od of 0.6 or above were subsequently considered positive. the bcv antigen elisa was performed using paired sera of 111 dogs from the study kennel. of these, 81 dogs had shown symptoms of respiratory disease during a period of 21 days and 30 had remained healthy. of the group of dogs which developed respiratory disease, 17 were positive for antibodies to crcv on the day of entry into the kennel and 64 were negative. of the 64 dogs in this group which had no detectable antibodies to crcv on day 1, 63 tested positive on day 21. all 46 dogs of these 63 for which a sample on day 7 was available tested negative on day 7. therefore 63 dogs showed a seroconversion during the study period, whereas only 1 dog remained negative. of the 30 dogs that had remained healthy, 17 had antibodies to crcv on the day of entry. all of the 13 dogs that were negative on day 1 tested negative on day 7 but showed a seroconversion until day 21. thus, of 34 dogs that were positive for antibodies to crcv on arrival in the kennel, 17 developed respiratory disease (50%), whereas of 77 dogs that were negative on arrival, 64 developed respiratory signs during the study period (83.1%) (fig. 3) . therefore dogs that had no antibodies to crcv on entry into the kennel had an increased probability of developing respiratory disease (p ͻ 0.001). only 1 of the 77 dogs that were negative on arrival remained negative during the study period of 21 days, whereas 76 dogs showed a seroconversion. an elisa assay using a canine coronavirus antigen was performed to investigate whether crcv showed a serological cross-reaction to canine enteric coronavirus. sera from 27 dogs, previously tested for antibodies to crcv using the bcv antigen, were selected. it was found that 8 dogs had antibodies to cecv on the day of entry into the kennel; of these 4 also had antibodies to crcv. nineteen dogs were found to be negative for cecv on day 1; 17 of these were also negative for crcv. of the 19 negative dogs, 5 showed a seroconversion to cecv during the 21-day period of the investigation and 17 showed a seroconversion to crcv. analysis of the prevalence of respiratory disease in this group showed that 6 of the 8 dogs (75%) that were positive for antibodies to cecv on day 1 developed respiratory disease. of the group of 19 dogs that had no detectable antibodies to cecv on day 1, 15 showed signs of respiratory disease (78.9%) (p ϭ 0.594). tracheal tissue samples from five dogs that had been identified as positive for coronavirus rna by rt-pcr were inoculated on cell cultures of canine adult lung fibro-blasts and mdck cells. for three samples virus isolation was also attempted on a72 cells. the cultures showed no signs of a cytopathic effect during three passages. after the third passage, rna was extracted from the cultures and found to be negative for coronavirus by rt-pcr. this study reports the detection of a canine coronavirus, provisionally called crcv, in kenneled dogs with respiratory disease. coronaviruses have been reported to cause respiratory disease of man, cattle, swine, and poultry, but their presence in the respiratory tract of dogs and a possible association with cird has not been determined. only one study has mentioned the isolation of canine coronavirus from four dogs with symptoms of respiratory disease (binn et al., 1979) . therefore it was investigated in this study whether coronaviruses could be detected in dogs from a kennel with a high prevalence of cird. the disease was endemic in this kennel and could not be controlled by the use of vaccines recommended against "kennel cough," strongly suggesting that additional agents were contributing to the disease syndrome observed. samples taken from the respiratory tract of these dogs were examined using rt-pcr primers directed to the conserved polymerase gene of coronaviruses (stephensen et al., 1999) . analysis of the cdna sequences obtained from the canine samples revealed that crcv had the highest similarity with the polymerase gene of bovine coronavirus (98.8%) and human coronavirus oc43 (98.4%) but only a very low similarity to the polymerase gene of the enteric canine coronavirus (strain 1-71, 68.53% similarity). phylogenetic analysis of the polymerase sequences of 11 coronaviruses showed crcv to be located on a common branch with 3 group 2 viruses: bovine coronavirus (bcv), human coronavirus strain oc43 (hcv-oc43), and hemagglutinating encephalomyelitis virus. however, canine enteric coronavirus, a group 1 coronavirus, was shown to be only distantly related. canine respiratory coronavirus therefore is a novel coronavirus of dogs that is most closely related to bcv and hcv-oc43, both of which are known to cause respiratory disease. sequence analysis of the spike gene confirmed the high similarity of crcv with bcv and hcv-oc43, both of which are members of the group 2 of the coronaviridae family. group 2 coronaviruses contain an additional structural protein, he. we were able to demonstrate the presence of a he gene in the cdna obtained from canine tracheal samples by pcr. crcv therefore has a he gene and belongs to group 2 of the coronavirus family. attempts to isolate crcv from tissue of the respiratory tract, using either canine lung fibroblasts or a kidney epithelial cell line, have been unsuccessful so far. however, the isolation of crcv may require the use of fetal canine cells or tracheal organ culture. also it is possible that the virus was inactivated during storage or due to freezing and thawing. by pcr, crcv was detected in tracheal and lung tissue and therefore appears to infect the upper and lower respiratory tracts of dogs. the possible presence of viral rna in other tissues needs to be analyzed in further studies and the target cells of crcv have to be identified using in situ hybridization or immunohistochemistry. the presence of antibodies to crcv was analyzed using an elisa based on a bcv antigen because of the high sequence similarity of the two viruses in the spike gene and because crcv antigen could not be obtained by virus culture. the elisa results confirmed the presence of a virus similar to bcv in the study population. the prevalence of antibodies was 30% at the time of entry into the kennel and 99% after 21 days. almost all dogs negative on the day of entry into the kennel showed a seroconversion to crcv within 3 weeks, indicating that the virus is highly contagious. serology using an antigen for cecv showed a much lower prevalence of antibodies to cecv on day 21. therefore the bcv elisa results did not reflect an infection with canine enteric coronavirus and the cross-reactivity between the two antigens seems to be low. serum antibodies to crcv were present in about 30% of dogs of various origins, including dogs entering a rehoming kennel as well as pet dogs. the presence of crcv is therefore not limited to the investigated kennel and the virus seems to be established in the dog population. within the kenneled population, crcv rna was detected in 27.3% of dogs with all grades of respiratory disease as well as in 26.2% of dogs that were apparently healthy at the time of euthanasia. however, crcv rna was most frequently found in the trachea of dogs with mild cough (55%). studies using the human coronavirus strain 229e have shown that coronaviruses can cause disruption of the respiratory epithelium and ciliary dyskinesia (chilvers et al., 2001) . if one supposes that an infection with crcv may have a similar effect then the virus could play an important role in the early stages of the pathogenesis of cird. by damage of the respiratory epithelium and disruption of the ciliary clearance crcv could facilitate the entry of other viral or bacterial pathogens causing the more severe respiratory symptoms. the less frequent detection of crcv in tissue samples from dogs with more severe disease may be explained by the destruction of the respiratory epithelium by other microorganisms in the advanced stages of cird. at the same time it is likely that the immune response that is induced by crcv in almost all dogs would help to clear the infection, causing the prevalence of crcv rna to be lower in later stages of the disease. furthermore, serological analysis revealed that dogs with antibodies to crcv on the day of entry into the kennel developed respiratory disease less frequently than dogs rt-pcr results from tracheal and lung samples of 119 dogs with different respiratory signs (none to severe) using a nested pcr directed against the coronavirus spike gene. the table shows the number of positive samples out of total sample number and the percentage of positive samples in parentheses. fig. 3 . comparison of the prevalence of respiratory disease in two groups of dogs: dogs in group 1 were positive for serum antibodies to respiratory coronavirus on the day of entry into the kennel; dogs in group 2 were negative. the graph shows the percentage of dogs developing respiratory disease in group 1 compared to group 2 (p ͻ 0.001). n is the total number of dogs in each group. without antibodies (p ͻ 0.001). therefore the presence of antibodies to crcv had a protective effect against respiratory disease in this population, indicating a possible role of the virus in the pathogenesis of cird. however 50% of the dogs that were seropositive for crcv on day 1 still developed respiratory disease. it was not determined if dogs which developed disease had lower antibody titers than those that stayed healthy as the elisa was performed using a single serum dilution. alternatively, the disease in seropositive dogs may well have been caused by other respiratory pathogens present in this population. studies determining the prevalence of other pathogens are currently under way. as cird is a complex disease involving a variety of microorganisms, further epidemiological studies are required to determine the exact role of crcv in this syndrome. it is likely that infections with crcv alone may cause only subclinical or mild respiratory symptoms but in conjunction with other pathogenic agents severe respiratory disease may occur. alternatively, the presence of crcv may exacerbate disease caused by other agents. this study describes the detection of a canine respiratory coronavirus in dogs with cird, which is genetically and antigenically distinct from the previously described enteric coronavirus. the pathogenesis of "kennel cough" has not been thoroughly investigated since the 1970s, when b. bronchiseptica, canine adenovirus type 2, and canine parainfluenza were determined to be the main causes of the disease. however, the vaccination of all dogs against cpiv, cav-2, and distemper virus did not help to control the disease in this kennel despite evidence that the majority of dogs responded to the vaccine within 21 days (data not shown). a b. bronchiseptica vaccine had been used in the past prior to this study but was discontinued because it failed to protect against respiratory disease. most rehoming and training kennels frequently have to deal with outbreaks of cird despite regular vaccination of all dogs. the aetiology of cird therefore needs to be reevaluated and the role of novel microorganisms or microorganisms previously not associated with the disease should be established. dogs from a well-established rehoming kennel with a history of endemic respiratory disease were monitored for this study. on entry into the kennel, all dogs were vaccinated with kavak da 2 pip69 (fort dodge), a live attenuated vaccine for distemper virus, canine adenovirus type 2, canine parainfluenzavirus, and canine parvovirus. also, a killed leptospirosis vaccine was used (fort dodge). the health status of each dog was assessed twice a day by a veterinary clinician and the respiratory signs were graded as follows: (1) no respiratory signs, (2) mild cough, (3) cough and nasal discharge, (4) cough, nasal discharge, and inappetence, (5) evidence of bronchopneumonia. the overall health status of the dogs was graded as follows: (1) good health, (2) poor health, (3) very poor health. the age, breed, and sex of the dogs were recorded. one hundred and nineteen dogs from the kennel population were euthanased for welfare reasons, ranging from behavioral problems to signs of severe respiratory disease for these dogs, a full postmortem examination was performed. the tissue samples were stored at ϫ70°c until further use. serum samples were collected from 111 dogs on the day of entry into the rehoming kennel. for 81 dogs a follow-up serum was available on day 7 and for 111 dogs a serum was available on day 21 after entry. of the 111 dogs, 30 remained healthy during the 21 days between the first and the last serum samples, whereas 81 dogs developed respiratory disease. sera from 35 dogs housed elsewhere were obtained from the diagnostic service of the royal veterinary college. these sera had been submitted for biochemical analysis for various reasons. five of these sera were from 18-month-old beagles with no history of respiratory disease. sera were routinely stored at ϫ20°c. rna was extracted from tracheal and lung tissue of 119 dogs using trireagent (sigma). approximately 25-50 mg of homogenized tissue was used and rna was extracted as recommended by the manufacturer. synthesis of cdna was performed using random hexameres (roche) and impromii reverse transcriptase (promega). for the detection of coronaviruses a modification of the primers 2bp and 4bm directed against the polymerase gene as described by stephensen et al. (1999) were used (con-scoro5, 5ј-act-car-atg-aat-ttg-aaa-tat-gc; con-scoro6, 5ј-tca-cac-tta-gga-tar-tcc-ca). pcr was performed using taq polymerase (promega) in the provided reaction buffer containing a final concentration of 2.5 mm mgcl 2 and 0.5 m primers. for pcr with the primers conscoro5 and conscoro6 the following temperature profile was used: after denaturation at 95°c for 5 min, 10 cycles were carried out at 95°c for 1 min, annealing at 37°c for 1 min, and extension at 72°c for 1 min. this was followed by 10 cycles using an annealing temperature of 45°c, 10 cycles at an annealing temperature of 50°c, and 10 cycles at an annealing temperature of 53°c, followed by a final extension at 72°c for 10 min. a 20-l fraction of the pcr product was analyzed on a 1.5% agarose gel and blotted onto a nylon membrane after electrophoresis. the nylon membrane was hybridized with an oligonucleotide probe specific for the pcr product at 37°c overnight (probe conscoro, aag-ttt-tat-ggy-ggy-tgg-ga). the probe was 3јa-tailed with digoxi-genin-dutp and was detected using anti-digoxigenin conjugate and cspd chemoluminescent substrate (roche). primers for the he gene were chosen using an alignment of the he genes of bcv strain ly-138 (genbank accession no. m84486) and hcv strain oc43 (genbank accession no. m76373). the sequence and location of the primers are shown in table 2 . the following temperature profile was used for the pcr: denaturation at 95°c for 5 min, followed by 35 cycles of 95°c for 1 min, 50°c for 40 s, and 72°c for 1 min, followed by a final extension at 72°c for 10 min. the expected size of the pcr product was 497 bp. primer sequences specific for the spike gene were derived from an alignment of the spike region of bovine coronavirus strain ly-138 (genbank accession no. af 058942) and human coronavirus strain oc43 (genbank accession no. l14643). for sequencing of the complete spike gene the primers sp1-sp8, spf, and spr were designed. table 1 shows the primer sequences. a pcr was performed using pfu polymerase (promega) and the following temperature profile: denaturation at 95°c for 5 min, followed by 35 cycles of 95°c for 1 min, 50°c for 40 s, and 72°c for 3 min. the final extension was performed at 72°c for 10 min. the pcr products were separated on an agarose gel and purified using the qiaquick gel purification kit (qiagen). pcr products were cloned into the pt7blue2 blunt vector (novagen) and sequenced using the thermo sequenase fluorescent labeled primer cycle sequencing kit with 7-deaza-dgtp (amersham pharmacia) using cy5-labeled primers. for the detection of coronavirus rna in tissue samples, the cdna was tested by pcr with the primers sp1 and sp2, followed by a nested pcr using the primers sp3 and sp4 and 2 l of the product of the first amplification. the temperature profile used was denaturation at 95°c for 5 min, followed by 35 cycles of 95°c for 1 min, 55°c for 40 s, and 72°c for 1 min. the final extension was performed at 72°c for 10 min. the nested pcr produced a 442-bp fragment. nucleic acid similarity searches were performed using fasta (pearson, 1990) , with a gap open penalty of 16 and a gap extension penalty of 4. protein similarities were determined using fasta or gcg (genetics computer group, wisconsin, usa) with a gap open penalty of 8 and a gap extension penalty of 2. sequence alignments were performed using clustalx (thompson et al., 1997) . the phylogenetic relationship to known coronaviruses was analyzed using the phylip 3.6 package (felsenstein, 1989) . the alignments were followed by a bootstrap analysis using the seqboot program. the data sets obtained were used for a maximum parsimony analysis using the dnapars program and a consensus tree was calculated using con-sense. the resulting trees were drawn using the treeview program (page, 1996) . the crcv polymerase partial sequence has been assigned genbank accession no. ay150273; the crcv spike gene sequence has been assigned genbank accession no. ay150272. elisa antigen for bovine coronavirus or canine coronavirus (churchill applied biosciences, huntingdon, uk) was resuspended in pbs at the concentration recommended by the manufacturer and incubated on 96-well plates (falcon) overnight at 37°c. the plates were washed with pbs and blocked with pbs containing 5% skimmed milk powder for 30 min. the sera were diluted 1:100 in blocking buffer and incubated on the plates for 1 h. after washing with pbs/0.05% tween 20 (sigma), a peroxidase-labeled rabbit anti-dog igg conjugate (sigma) was added (1:5000 in pbs/ 0.05% tween 20) for 1 h. the plates were incubated with color substrate (opd, sigma) for 10 min and the reaction was stopped by adding 2 m h 2 so 4 . the adsorption was determined in an elisa photometer at 492 nm. virus isolation was attempted on canine adult lung fibroblasts (passages 3 to 7) and mdck and a72 cells. the lung fibroblasts were maintained in mem with 20% fetal calf serum (fcs); mdck and a72 cells were maintained in mem with 5% fcs. tracheal tissue samples (approx 25 mg) were homogenized using a scalpel and mixed vigorously in 1 ml of mem containing penicillin (100 u/ml), streptomycin (0.1 mg/ml), amphotericin b (2.5 g/ml), and trypsin (1 g/ml). the samples were centrifuged at 13,000 rpm for 10 min and the supernatant was used to inoculate cell cultures. after 30 min at 37°c the supernatant was removed and maintenance medium was added to the cultures. the cultures were passaged three times in the absence of a cytopathic effect. then, rna was extracted from the cells and rt-pcr for coronavirus was performed using the nested pcr for the spike gene. the data were analysed using the 2 test or fisher's exact test and p values below 0.05 were considered statistically significant. and guidance. we thank the veterinary nurses and clinicians at the dogs home battersea for technical assistance. we also thank dr. v. chalker and dr. m. collins for critical reading of the manuscript and s. oliver for providing a control sample for bovine coronavirus. canine infectious tracheobronchitis short review: kennel cough sv-5-like parainfluenza virus in dogs naturally occurring respiratory disease in a kennel caused by bordetella bronchiseptica pathogenesis of canine bordetellosis studies of respiratory disease in random-source laboratory dogs: viral infections in unconditioned dogs viruses recovered from laboratory dogs with respiratory disease the effects of coronavirus on human nasal ciliated respiratory epithelium association of a canine adenovirus (toronto a 26/61) with an outbreak of laryngotracheitis phylip-phylogeny inference package (version 3.2c) avian infectious bronchitis virus canine tracheobronchitis: isolation and characterization of the agent with experimental reproduction of the disease role of bordetella bronchiseptica in infectious tracheobronchitis in dogs natural and experimental infection of dogs with reovirus, type1: pathogenicity of the strain for other animals viruses and bacteria in the etiology of the common cold treeview: an application to display phylogenetic trees on personal computers rapid and sensitive sequence comparison with fastp and fasta isolation of a porcine respiratory, non-enteric coronavirus related to transmissible gastroenteritis prevalence of mycoplasmal and ureaplasmal recovery from tracheobronchial lavages and prevalence of mycoplasmal recovery from pharyngeal swab specimens in dogs with or without pulmonary disease coronaviruses: structure and genome expression phylogenetic analysis of a highly conserved region of the polymerase gene from 11 coronaviruses and development of a consensus polymerase chain reaction assay isolation of respiratory bovine coronavirus, other cytocidal viruses, and pasteurella spp. from cattle involved in two natural outbreaks of shipping fever studies on the epizootiology of canine coronavirus the clustalx windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools the authors are most grateful to the dogs home battersea for funding to professor j. brownlie and for support key: cord-269866-3tpyj04y authors: liu, d. x.; inglis, s. c. title: identification of two new polypeptides encoded by mrna5 of the coronavirus infectious bronchitis virus date: 1992-01-31 journal: virology doi: 10.1016/0042-6822(92)90094-6 sha: doc_id: 269866 cord_uid: 3tpyj04y abstract the second smallest subgenomic messenger rna, mrna5, of the coronavirus infectious bronchitis virus includes in its “5′ unique region” two separate open reading frames (5a and 5b), whose coding function has not so far been established, and thus it may represent a dicistronic messenger rna. we report here that two polypeptides with the sizes expected for the 5a and 5b products can be synthesised by in vitro translation of a single artificial mrna containing both the 5a and 5b orfs. to establish whether these polypeptides represent genuine virus gene products, both the 5a and 5b coding sequences were expressed as bacterial fusion proteins, and these were used to raise monospecific antisera. antisera raised against both the 5a and 5b-specific sequences recognized specifically proteins of the expected size in infectious bronchitis virus-infected chicken kidney and vero cells, indicating that 5a and 5b do represent genuine virus genes, and suggesting that mrna5 is indeed functionally dicistronic. the second smallest subgenomic messenger rna, mrna5, of the coronavirus infectious bronchitis virus includes in its " 5' unique region" two separate open reading frames (5a and 5b), whose coding function has not so far been established, and thus it may represent a dicistronic messenger rna. we report here that two polypeptides with the sizes expected for the 5a and 5b products can be synthesised by in vitro translation of a single artificial mrna containing both the 5a and 5b orfs. to establish whether these polypeptides represent genuine virus gene products, both the 5a and 5b coding sequences were expressed as bacterial fusion proteins, and these were used to raise monospecific antisera. antisera raised against both the 5a and 5b-specific sequences recognized specifically proteins of the expected size in infectious bronchitis virus-infected chicken kidney and vero cells, indicating that 5a and 5b do represent genuine virus genes, and suggesting that mrna5 is indeed functionally dicistronic. 0 1992 academic press, inc. infectious bronchitis virus (ibv), a pathogen of chickens, is the prototype virus of the coronaviridae, a family of enveloped viruses with a large positivestranded rna genome. deduction of the complete nucleotide sequence of the ibv genome, recently accomplished through cdna cloning (4), has indicated that the virion rna is 27 kilobases (kb) in length, and contains at least 10 separate open reading frames (orfs) with the capacity to encode proteins of between 6.7 and 440k. although the genomic rna is capped and polyadenylated and is infectious (21, 23) , it appears that it does not act as messenger rna for most of the known virus proteins; available evidence indicates that all but the two large orfs nearest to the 5'end of the genome are translated from subgenomic mrna species. five such subgenomic mrnas have been identified in virus-infected cells, and these form a 3' co-terminal 'nested' structure (28, 29) . recently these mrnas have been redesignated mrna 2 to 6 in decreasing order of size (9)) with the genomic mrna representing mrna1. for mrnas 2, 4, and 6, the 5'-"unique" region (i.e., the region which is not present in the next smallest mrna species) contains a single orf, suggesting that these mrnas are functionally monocistronic. this is supported by in vitro translation studies which have shown that mrnas 2,4, and 6 encode the major virion structural proteins spike (s), membrane ' to whom reprint requests should be addressed (m), and nucleocapsid (n), respectively (30). however, the other three mrnas 1, 3, and 5, all contain more than one orf in their 5'-"unique" region, suggesting that they may be functionally polycistronic. previous studies in this laboratory have indicated that the two 5' proximal orfs present on the genomic rna are expressed in infected cells (6) and can be translated from a single mrna through an efficient ribosomal frame-shift mechanism (5, 7) . similarly all three of the orfs present on mrna 3 encode proteins in infected cells and can be translated from a single mrna (20, 26) although by a different mechanism involving independent initiation. as yet however no information is available regarding the coding capacity of mrna5. nucleotide sequence analysis (3) has shown that the 5'-"unique" region of mrna5 contains two orfs, designated 5a and 5b, which have the potential to encode two polypeptides of molecular weight 7.4 and 9.5k, respectively. we report here the identification of two products encoded by this mrna in ibv-infected chicken kidney (ck) and monkey kidney (vera) cells. in addition we show, by in vitro translation of synthetic mrna, that both polypeptides may be synthesized from a single mrna, suggesting that this mrna, like mrna 3, is functionally polycistronic. as a first step toward identification of 5a and 5b gene products, we tested the coding potential of these orfs by in vitro translation of synthetic mrna; successful expression of the orfs would support the idea that they represented genuine gene products, and would further provide markers for identification of 5a in vitro transcription and translation was carried out as described previously (5, 15 ). and 5b proteins in virus-infected cells. for this purpose, two plasmids (plbs6 and plbs7) were constructed. plasmid plbs6 contains the 5b orf adjacent to the sp6 phage rna polymerase promoter, and plasmid plbs7 is identical to plbs6 except that it contains both the 5a and 5b orfs (with 5a adjacent to the sp6 promoter). details of these plasmid constructions are given in the legend to fig. la . as shown in fig. 1 a, plasmids plbs6 and plbs7 may be transcribed in vitro using the sp6 phage rna polymerase into mrna containing either both orfs (plbs7) or 5b alone (plbs6). transcripts were therefore prepared from hindlll-digested plbs6 and plbs7, incorporating the dinucleotide 7mgpppg to provide a 5' cap structure ( io), purified as detailed in fig. 1 b, and then translated in a cell-free system derived from wheat germ ( 15). the results of this experiment are shown in fig. 1 b. messenger rna from plbs6 directed the synthesis of a single major translation product, with an apparent molecularweight of about 1 ok, which was consistent with the predicted mw of 9.5k for the product of 5b. two major products were produced by plbs7-derived transcripts. the larger one comigrated exactly with the 1 ok protein synthesized in response to plbs6-derived rna, identifying it as the product of 5b. the more abundant product however was not present among those directed by plbs6 transcripts and migrated with the expected molecular weight for 5a (7.5k), strongly suggesting that it does indeed represent the product of the 5a orf. these data are therefore consistent with the idea that both the 5a and 5b orfs could encode polypeptides in the virus-infected cells. furthermore the observation that both 5a and 5b-encoded proteins could be synthesized in vitro in response to a single mrna (i.e., the plbs7-derived transcripts) raises the possibility that the natural mrna5 may be functionally dicistronic. to investigate these possibilities further we therefore set out to examine virus-infected cells for the presence of 5a-and sb-encoded polypeptides. in order to identify the products of the 5a and 5b orfs in virus-infected cells, we first sought to express the appropriate sequences in bacteria and to raise monospecific antisera against the resulting protein products. the bacterial expression system chosen (based on the pex series of plasmids) was that developed by stanley and luzio (27) . in this system, foreign sequences are inserted in the correct reading frame, at the c terminus of a ,&galactosidase gene, which is itself fused to the promoter, operator and n terminal region of the cro gene of bacteriophage x. synthesis of the p-galactosidase fusion protein in bacteria carrying these plasmids is therefore repressed in cells carrying a functional x repressor, but can be induced in escherichia co/i pop21 36 cells (which carry a temperaturesensitive repressor) simply by increasing the culture temperature to 42". we have used this system previously to identify three proteins encoded by the ibv mrna3 (20, 26) . two plasmids were constructed for bacterial expression of the 5a and 5b orfs. one of these, pexl/eia, contains the 5a orf fused in frame with the p-galactosidase gene of the vector, while the other, pex2/5b, contains the 5b orf fused in frame with the ,&galactosidase gene of the vector. details for the construction of these plasmids are given in the legend to fig. 2 . following heat induction, bacteria harboring the pexl/ 5a and pex2/5b plasmids produced fusion proteins that were larger than the wild-type fl-galactosidase by the expected amount (data not shown), indicating successful expression of the viral coding sequences. these fusion proteins were purified by electroelution, and then inoculated into rabbits to produce monospecific antisera. the specificity and reactivity of the monospecific antisera generated by this route were then tested by immunoprecipitation studies using radiolabeled 5a-and 5b-encoded proteins synthesized by in vitro translation of synthetic mrna transcribed from plbb4 using the t7 phage rna polymerase. the results of this experiment (fig. 2) indicated that both sera are indeed capable of recognising specifically the appropriate target sequence, although the relative efficiency of precipitation of the two proteins suggested that the avidity of the anti-5b serum may be considerably higher. the two specific antisera were next used to immunoprecipitate 5a and 5b orf-related proteins from [35s] methionine labeled ibv-infected ck and vero cell extracts. for this purpose, newly confluent monolayers of ck cells and vero cells were infected with the beaudette strain of ibv, labeled with [35s]methionine, and harvested as previously described (20) . immunoprecipitation was carried out as previously described (20) . the results of these experiments (figs. 3a and 3b) 12.5 -+ +5b +5a fig. 2. lmmunoprecipitation of in vitro synthesized 5a and 5b polypeptides with anti-5a and anti5b antibodies. [35s]methionine-labeled in vitro translation products of mrna derived from hindill digested plbb4 were analysed by electrophoresis on a 20% polyacryamide gel and detected by fluorography either before (lane plbb4/hindlll) or after immunoprecipitation with anti-5a antiserum, anti-5b antiserum, or control rabbit antiserum (raised against a bgalactosidase/influenza np fusion protein). lmw, molecular weight markers. in vitro transcription and translation was carried out as before (5, 15) . anti-5a and anti-5b antibodies were prepared by immunisation of rabbits with purified @galactosidase fusion proteins expressed from the plasmids pex1/5a and pex2/5b, respectively (described in the text), and were affinity purified from crude sera by affinity chromatography as described (26) . construction of the plasmids was as follows: for insertion of a cdna fragment containing the 5a orf into a pex-based plasmid, it was necessary to introduce a suitable restriction enzyme site immediately upstream of the 5a coding sequence. to construct a plasmid suitable for this manipulation, an ecorl fragment containing the 5a and 5b orfs was excised from plbs7. end-repaired with dna poll, redigested with hindlll, and cloned into plbt4 (20) in vitro translation products from plbb4-derived rna (fig. 4c) were also included as markers for the 5a and 5b products. (b) detection of polypeptides encoded by the 5a and 5b orfs in ibv-infected and mock-infected chicken krdney (ck) cells by immunoprecipitation. cell lysates were prepared and the polypeptides were analyzed as indicated above. indicated that antisera raised against the sa-specific fusion protein could specifically recognize in each labeled lysate, although faintly, a polypeptide with the expected mw for 5a (i.e., 7.4k) and with the same electrophoretic mobility as the in vitro translation product of the 5a orf. likewise the anti-5b antisera was able to precipitate specifically a protein of the size expected for the 5b product (9.5k), as judged both by gel mobility and by comparison with in vitro translated 5b. thus it appears that both the 5a and 5b orfs are indeed expressed in ibv-infected cells. to provide additional evidence for the presence of products from the 5a and 5b orfs, and to investigate the subcellular location of the proteins in virus-infected cells, indirect immunofluorescence was carried out on ibv-infected vero cells using anti-5a and anti-5b antibodies purified by affinity chromatography as previously described (26) . as shown in fig. 4 , both antisera gave weak but clear positive staining in infected cells at 18 hr postinfection (p.i.) (figs. 4a and 4c) but not in mock-infected cells (figs. 4b and 4d ). using the anti-5a antibodies (fig. 4a) diffuse fluorescence was observed throughout the cell, with some apparent concentration around the nucleus. with the anti-5b anti-bodies (fig. 4c) , the staining was also distributed over the whole cell, but in this case the pattern of fluorescence appeared more granular and was more obviously perinuclear in location. thus the results reported here clearly indicate that ibv-infected ck and vero cells contain polypeptides that are recognized specifically by antisera raised against bacterial fusion proteins containing either 5a or 5b amino acid sequences. these polypeptides are of the sizes expected for the products of the 5a and 5b orfs, and each comigrates during sds-gel electrophoresis with its in vitro-translated counterpart. thus both the 5a and 5b orfs are expressed in infected cells. the function of these polypeptides however remains unclear. the deduced amino acid sequence of 5a is unusual in that 26% (17 out of 65) of its residues are leucines (3), and its obvious hydrophobic nature suggests that it may be membrane-associated, although it does not appear to contain a conventional "membrane-spanning" domain. such an association would be consistent with the general pattern of staining observed in our immunofluorescence studies using anti-5a antibodies, and preliminary cell fractionation experiments suggest that it is indeed associated with fig. 4 . indirect immunofluorescence of ibv-infected (a and c) and mock-infected (b and d) vero cells at 18 hr p.i. using anti-5a (a and b) and anti-5b (c and d) antisera. cells were permeabilized with 1% triton xl 00, and staining was carried out as described previously (26) . the membranes of infected cells (data not shown), but it has not so far proved possible to detect the protein in purified virions. the deduced amino acid sequence of the 5b protein however betrays no obvious clue as to its function, although our immunofluorescence studies suggest that it concentrates in the perinuclear region of infected cells. available evidence suggests that the 5a and 5b polypeptides are expressed from a single subgenomic mrna, mrna5, which consists of a 65 nucleotide leader sequence corresponding to the 5' end of the genomic rna, fused to a "body" which initiates 25459 nucleotides from the genomic 5'end, and runs through to its extreme 3'terminus. this is based on the observation that the next smallest subgenomic mrna, mrna6, which encodes theviral nucleocapsid protein, does not contain the 5a and 5b orfs, and the next largest, mrna4, encodes the viral membrane protein, but not the products of 5a and 5b orfs as indicated by in vitro translation of viral mrna (30). how could these two products be translated from a single messenger rna? the most obvious possibility is by a "leaky scanning" mechanism, in which ribosomes bind at the capped 5' end of the mrna and scan in a 3' direction until an initiation codon is en-countered; occasionally however they may fail to recognize the initiation codon for the upstream 5a orf and continue onward to the 5b orf (17). the sequence context around the initiation codon of the upstream 5a orf (cggacgug) conforms well to that preferred for functional eukaryotic initiation codons ( 16) with an a at the -3 position and g at +4, suggesting that translational initiation of this orf should occur efficiently. our in vitro cell free translation results using synthetic mrnas bearing the 5a and 5b orfs are consistent with this idea; these mrnas directed efficient synthesis of a product corresponding to the 5a polypeptide (fig. 3b) . expression of the 5b orf from such synthetic dicistronic mrnas in vitro was by contrast relatively inefficient, which would be expected if its synthesis relies on leaky scanning ( 17). furthermore synthesis of both proteins from the dicistronic mrna was sensitive to inhibition by the cap analogue 7mgtp in vitro (data not shown) suggesting that translation of each required ribosome entry at the 5' end. however 5b synthesis in vitro appeared to be inefficient even when the 5b orf was placed proximal to the 5'end of a synthetic mrna (fig. 1 b) , in spite of the fact that sequences around the 5b initiation codon (gcugg-c/j&a) would seem to present a favorable context for translation with a g at position -3 and a at +4. it is perhaps surprising therefore that expression of 5b was relatively easy to detect in virus-infected cells in comparison to that of 5a. this could represent a genuine difference in the relative expression ratio of the two proteins in viva from that observed by in vitro translation, as has been reported previously for the products of other di-cistronic mrnas ( 11, 12, 32) . if this were the case, it would undermine somewhat the argument for leaky scanning as a mechanism to account for 5b translation in vivo. however, the relative ease of 5b detection may simply reflect differing avidities of our mono-specific antisera; indeed, the anti-5b serum clearly recognized its target protein more efficiently than did the anti-5a serum when tested against in vitro translated material (fig. 2) . at present then the mechanism by which mrna5 can function dicistronically in infected cells remains unclear, although our in vitro translation data would favor a leaky scanning hypothesis. most eukaryotic mrnas studied to date encode only a single protein, but in recent years, more and more animal viruses have been found to encode di-or polycistronic mrnas. these include adenovirus (the el b mrna encodes two tumor antigens starting at different aug triplets of two overlapping orfs (2))) the papovaviruses (the late 19s rna of simian virus 40 expresses both the vp2 and vp3 proteins (24)), the paramyxoviruses (for example, the measles virus and sendai virus p/c mrnas (1, 13)), and the influenza viruses (the influenza b virus rna segment 6 encodes both the nb and na glycoproteins in different overlapping reading frames (31)). among the coronaviruses, there are also some examples of functionally polycistronic mrnas. one of the other ibv subgenomic mrnas, mrna3, can function as a tricistronic messenger (20) , encoding a 6.7k proteins (3a), a 7k protein (3b), and a membrane-associated virion protein (3~). murine hepatitis virus also appears to encode a dicistronic mrna. messenger rna5 contains two orfs in its 5'-unique region, and a product from the second of these has been detected in virus infected cells (8, 79, 25) . it seems therefore that the translation of small, often briefly overlapping orfs from di-or polycistronic mrnas may not be an uncommon feature of coronaviruses. coronaviruses and their diseases gem viral key: cord-272051-arz8r204 authors: federico, maurizio title: hiv-protease inhibitors block the replication of both vesicular stomatitis and influenza viruses at an early post-entry replication step date: 2011-08-15 journal: virology doi: 10.1016/j.virol.2011.05.002 sha: doc_id: 272051 cord_uid: arz8r204 the inhibitors of hiv-1 protease (pis) have been designed to block the activity of the viral aspartyl-protease. however, it is now accepted that this family of inhibitors can also affect the activity of cell proteases. since the replication of many virus species requires the activity of host cell proteases, investigating the effects of pis on the life cycle of viruses other than hiv would be of interest. here, the potent inhibition induced by saquinavir and nelfinavir on the replication of both vesicular stomatitis and influenza viruses is described. these are unrelated enveloped rna viruses infecting target cells upon endocytosis and intracellular fusion. the pi-induced inhibition was apparently a consequence of a block at the level of the fusion between viral envelope and endosomal membranes. these findings would open the way towards the therapeutic use of pis against enveloped rna viruses other than hiv. hiv protease inhibitors (pis) are a family of small molecules specifically designed for blocking the activity of the hiv aspartylprotease (for a review, see flexner, 1998) . the pi treatment leads to the block of hiv maturation with consequent release of noninfectious viral particles (kaplan et al., 1993) . pis exert their inhibitory effect by disabling the enzyme before it can cleave the gag-pol polyprotein into its essential products. pis, together with anti-hiv compounds targeting alternative steps of the virus life cycle, are part of the most advanced anti-hiv therapies allowing millions of infected people to co-exist with the virus experiencing a good quality of life. however, it is now clear that pis can also directly or indirectly inhibit the activity of many cell proteases. in particular, pis can inhibit the activity of both 20s (andré et al., 1998) and 26s (pajonk et al., 2002) proteasome subunits, as well as that of caspases (badley, 2005) , with alterations in the susceptibility to apoptosis stimuli. both expression and release of matrixmetalloproteinases (mmps) can be also targeted by pis (bourlier et al., 2005; de barros et al., 2007) with consequences in the extracellular matrix modeling and, more in general, in the cell-cell communication. in addition, pis have been found inhibiting cell signaling pathway involving both nf-κb (dewan et al., 2009 ) and akt (kumar et al., 2009; srirangam et al., 2006) . from a clinical point of view, the pi treatment may induce beneficial effects other than those against hiv, as in the case of kaposi sarcoma regression (sgadari et al., 2002) , but can also have detrimental consequences, as for the dyslipidemia occurring in pi-treated hiv patients (calza et al., 2004) . to enter target cells, enveloped rna viruses fuse their envelope either at the cell surface in a ph-independent way, or upon internalization in intracellular vesicles through a ph-dependent mechanism (steven and spear, 2006) . in this latter case, low ph is required to induce the fusion between viral envelope and endosomal membranes, a phenomenon leading to the release of virion contents into cytoplasm. although the viral fusion process is guided by the presence of viral envelope proteins, the contribution of cell proteins is critical. using rna interference as a tool for gene expression inhibition, it was demonstrated that several cell proteins are involved in the mechanism of entry of many viruses including hiv-1 nguyen et al., 2006) , influenza (hao et al., 2008) , west nile (krishnan et al., 2008) , borna disease (clemente et al., 2010) , human hepatitis c (ng et al., 2007) , and vesicular stomatitis (vsv) (pelkmans et al., 2005) viruses. ideally, these cell products might represent potential antiviral therapeutic targets. here, the pi-induced inhibition of replication of both vsv and influenza virus is described for the first time. this was most likely a consequence of a pi-dependent block of the viral envelope fusion at the endosomes. considering that pis are well tolerated drugs in vivo, and that many relevant human pathogens belong to the family of rna viruses infecting cells through an endocytic pathway, this finding would open the way towards a broader therapeutic use of pis. hiv virions emerging from cells treated with pis remain immature viral particles as a consequence of the block of gag polyprotein cleavage. hence, pis do not affect the amounts of hiv release from infected cells, but dramatically decrease their infectivity. surprisingly enough, however, the hiv-1 release from cells infected with hiv-1 pseudotyped with the envelope protein from vsv (vsv-g) has been found inhibited by pis in single cycle replication assays. in fact, when cem gfp cells, i.e., a human t cd4 + lymphoblastoid cell line expressing gfp under the control of hiv-1 ltrs (gervaix et al., 1997) , were infected with 100 ng hiv-1 cap24 equivalent/10 5 cells of (vsv-g) δenv hiv-1, decreased percentages of hiv-1 expressing cells were observed in ritonavir-treated cells as compared to control conditions (fig. 1a) . the outcome did not change by challenging the cells with doses up to 300 ng hiv-1 cap24 equivalent/10 5 cells (not shown). conversely, and as expected, the pi-treatment had no apparent effects on single cycle replication of non-pseudotyped hiv-1, as tested in pi-treated cultures of cem gfp cells infected with wt hiv-1 (fig. 1b) . in these experiments, the presence of t-20 (i.e., a potent inhibitor of the hiv-1 env-mediated fusion) (kilby et al., 1998) added 16 h after challenge ensured that the wt hiv-1 replication was limited to a single cycle. as anticipated, ritonavir blocked the spread of wt hiv-1 in multiple-cycle replication assay (fig. 1b) . to exclude that the observed effect was a consequence of the inhibition of some yet unidentified function of the hiv-1 protease at an early step of the virus life cycle, the assay was reproduced using a vsv-g pseudotyped, pi-resistant hiv-1 strain (here referred to as pm4). this viral mutant expresses a pi-resistant viral protease as a consequence of the four amino acid substitutions in the protease gene, i.e., m 46 i , l 63 p , v 82 t , and i 84 v (condra et al., 1995) . first, the real resistance to pis of pm4 hiv-1 was checked by evaluating the percentages of cem gfp infected cells 6 days after the infection in the presence or not of ritonavir (fig. 1c) . then, cem gfp cells treated with t-20 were infected with (vsv-g) pm4 hiv-1 in the presence or not of ritonavir. this experimental setting was representative of a singlecycle replication assay since t-20 disabled both hiv-1 env-mediated viral entry and possible cell re-infections. similarly to what observed with (vsv-g) δenv hiv-1 strain, a significant inhibitory effect of ritonavir on the hiv-1 expression was detected in (vsv-g) pm4 challenged cells (fig. 1d ). this strongly suggests that the viral protease activity was not involved in the pi-induced inhibition of (vsv-g) hiv-1 expression. overall, these results can be a consequence of a still unrecognized inhibitory effect of pis on some step of the viral entry driven by vsv-g. next, the possibility that the pi inhibitory effect was operative also against vsv was investigated. to this end, hela cells were pre-treated overnight with different concentrations of six pis, i.e., ritonavir, indinavir, saquinavir, nelfinavir, amprenavir and atazanavir. then, the cells were challenged with 0.2 m.o.i. of vsv. after 1 h of adsorption in a small volume, the viral inoculum was removed, the cells extensively washed, and incubated for additional 8 h in the presence of pis. afterwards, the supernatants were harvested and titrated for the amounts of infectious vsv. as depicted in figs. 2a-b, a strong reduction of vsv replication (more than 2 logs) was observed when the cells were treated with 2.5 μm saquinavir and nelfinavir. the inhibitory effect appeared more potent (up to about 4 logs) by increasing the concentration of these pis to 10 μm, and remained significant (i.e., more than 1 log) by increasing the m.o.i. up until 1 (not shown). no differences were observed when the pis pretreatment was carried out for 2, 4, 6, or 8 h (not shown). as calculated through a wide dose-response curve, the 50% inhibitory concentration (ic 50 ) values of saquinavir and nelfinavir were 1.4 and 1.1 μm, respectively. on the other hand, significant inhibition of viral replication were detected in ritonavir and indinavir treated cells starting to the concentrations of 20-30 μm (fig. 2b) . the ic 50 of ritonavir and saquinavir were 16 and 14 μm, respectively. the idea that pis negatively affect the vsv replication was further strengthened by performing a set of more stringent challenge experiments. in detail, hela cells were pre-treated with optimal concentrations of the most effective pis, and then infected with 2-fold decreasing amounts of vsv starting to m.o.i. 2. the cytopathic effect was monitored 24 h post challenge. the outcome of this assay was scored in terms of the viral dilutions no more able to induce cytopathic effect. notably, in these conditions an about 30-fold reduction of vsv infectivity was observed in cells treated with saquinavir or nelfinavir (fig. 2c ). since the average replication time of vsv is 6-8 h (whelan et al., 2004) , in this experimental setting vsv was expected to complete at least three replication cycles in control cells. then, it was controlled whether the antiviral effect was a consequence of a general cytotoxicity of pis. to this aim, cells were treated with different pi concentrations for 8 h, then labeled with carboxyfluorescein diacetate succinimidyl ester (cfse), and thereafter refed with complete medium in the presence of pis. cfse was expected to be equally distributed upon cell division, then resulting in a halving of the overall cell fluorescence after cell duplication. cytotoxic effects were measured in terms of inhibition of the cell duplication as detectable by the impairment of the halving of the cfse-associated cell fluorescence. fig. 2d reports the mean fluorescence intensities (mfis) measured in cell cultures treated with different concentrations of the pis active against vsv replication. block of cell duplication with cell mortality over 20% has been detected using 100 μm ritonavir, saquinavir, and nelfinavir, while indinavir showed a much lower cytotoxic effect. the 50% cytotoxic concentration (cc 50 ) values of pis ( fig. 2d , insert) were calculated as the doses inhibiting by 50% the fluorescence halving, expectedly a consequence of the block of the duplication in 50% of cells. the therapeutic indexes (i.e., cc 50 divided by ic 50 ) appeared higher than 22 for both saquinavir and nelfinavir. this is suggestive of specific antiviral effects for these pis. taken together, these data reveal a previously unrecognized effect of pis against vsv. in addition, these results support the idea that the pi-induced reduction of (vsv-g) hiv-1 expression is a consequence of the inhibition on some step of the viral entry involving the vsv-g envelope protein. to add relevance to our findings, the possibility that the inhibitory effect of pis applied also to alternative virus species infecting by phdependent fusion was investigated. to this aim, the well characterized influenza virus/mdck cell system was considered. mdck cells were treated overnight with different pis, and then challenged with m.o.i. 0.1 and 0.5 of the pr8 influenza virus. after challenge, the cells were extensively washed, and reseeded in medium without serum in the presence of both pis and tosylamido-2-phenil ethyl chloromethyl ketone (tpck)-treated trypsin. twenty-four h later (i.e., the expected time for the completion of the replication cycle of influenza virus) (sidorenko and reichl, 2004; smith and ribeiro, 2010) , the supernatants were harvested and titrated for the contents of infectious particles. a strong inhibition of the replication of influenza virus (up to 4 logs at the lower m.o.i.) has been detected with 10 μm saquinavir and 5 μm nelfinavir (fig. 3a) . ritonavir inhibited the viral replication less efficiently (fig. 3a) , while indinavir, amprenavir and atazanavir appeared ineffective (not shown). in the interpretation of these data, it should be emphasized that the inhibitory effect of pis cannot be a consequence of a pi-induced impairment of the activation of the hemagglutinin envelope protein (ha) of the influenza virus, which is a step required for productive infection (steinhauer, 1999) . in fact, influenza virus preparations used for challenges were exclusively recovered from the allantoic fluid of embryonated chicken eggs, where ha is cleaved by egg proteases. consistently, tpck-trypsin did not influence the infectivity of the virus preparations in single cycle replication assay (not shown). the cell cytotoxicity of pis active against influenza virus was then evaluated in mdck cells similarly to what above described for hela cells (fig. 3b ). block of cell duplication in the presence of cell mortality over 20% has been detected with 100 μm ritonavir and saquinavir, and with 50 μm nelfinavir. the therapeutic indexes appeared higher than 15 for both saquinavir and nelfinavir. the assay gave similar results when the cells were analyzed 24 h after cfse labeling (not shown). taken together, these data suggest that pis could target the replication of diverse enveloped viruses infecting by a ph-dependent endocytic pathway. next, it was investigated whether the pis most effective against vsv and influenza virus replication counteract also the replication of viruses infecting by a ph-independent mechanism. as a proof of principle, the pis were assayed against the replication of newcastle disease virus (ndv), i.e., a paramixovirus infecting upon fusion at the cell surface. mdck cells were pre-treated with 2.5 to 10 μm saquinavir or nelfinavir, and then infected with 2-fold decreasing amounts of ndv starting to m.o.i. 1. the cytopathic effect was monitored 48 h post challenge. no significant reductions in the ndv infectivity have been noticed in pi-treated as compared to control cell cultures (fig. 4) . these results indicate that pis do not affect the replication of viruses entering by a ph-independent way. the inhibitory effect is operative when pis are co-administered with the infecting virus to identify the virus replication step targeted by pis, the effects of pis added before and/or after viral challenge were evaluated. the assays were carried out using both hela/vsv and mdck/influenza virus systems. in a first set of experiments, the pi treatment was discontinued just before the virus challenge. then, the cells were infected with vsv or influenza virus at m.o.i. of 0.2 and 0.5, respectively. after 1 h of adsorption, the cells were extensively washed and refed in the appropriate medium. in these conditions, no inhibition of the replication of both vsv and influenza virus was detectable (fig. 5a) . alternatively, pis were added soon after the virus adsorption or at different times after the challenge within a timeframe of 8 h (for vsv infection) and 24 h (in the case of challenge with influenza virus), i.e. the respective replication times. the supernatants were harvested 8 h (for vsv) and 24 h (for influenza virus) post-infection, and titrated for the amounts of infectious virus particles. it appeared that pis inhibited the virus replication also when added early after challenge (fig. 5b) . the inhibitory effect dropped shortly in the case of vsv infection, and more gradually when cells were infected with influenza virus. saquinavir and nelfinavir have shown similar inhibitory efficiencies (not shown). these results indicate that the presence of pis at early times after virus challenge is mandatory for the inhibitory effect, consistently with the idea that pis target an early event of the replication cycle of both viruses. vsv, and (vsv-g) hiv-1 in an hiv-1 protease independent manner, further supported the idea that pis would influence some common early replication step, e.g., virus attachment, endocytosis, and/or endosomal fusion. to dissect among these different possibilities, the intracellular fate of challenging virus was followed using gfp-labeled hiv-1-based vlps (muratori et al., 2006) pseudotyped with either wt or fusiondefective vsv-g (fig. 6a) . the fluorescence of these vlps relies on the high incorporation levels of the product of fusion between gfp and a hiv-1 nef mutant acquiring a palmitoylation site at its n-terminus as the consequence of the g to c substitution at the amino acid 3. first, the possible effects of pis on the virus attachment on target cells were investigated. to this end, 10 5 hela cells treated or not with pis were challenged with 500 ng of (wt vsv-g) nef g3c -gfp vlps for 1 h at 4°c. afterwards, the cells were extensively washed and facs analyzed. to control that the cell-associated fluorescence was not a consequence of endocytosed vlps, a part of the cell samples was treated with trypsin. it appeared that the treatment with pis did not significantly affect the cell binding of fluorescent vlps (fig. 6b ). this strongly suggests that pis do not interfere with the binding of viral particles on the cell membrane. next, the influence of pis on virus endocytosis was investigated. to this aim, gfp-fluorescent vlps incorporating a vsv-g mutant (here referred to as fd vsv-g) unable to support the ph-dependent fusion were used. the impaired fusion activity was a consequence of the a to k amino acid substitution at the position 133 (fredericksen and whitt, 1995) . the use of this envelope protein mutant was associated with the trypsin treatment just before the facs analysis to ensure that the cell-associated fluorescent signal exclusively referred to the intracellular accumulation of vlps. hela cells treated or not with pis were challenged with 500 ng hiv-1 cap24 equivalents of (fd vsv-g) nef g3c -gfp vlps/10 5 cells, and incubated for 3 h at 37°c in the presence or not of pis. then, the cells were treated with trypsin, and the cell-associated gfp fluorescence was evaluated by facs analysis. as control, vlp-challenged cells were incubated at 4°c before the trypsin treatment. again, no apparent differences in the cellassociated fluorescence were detected between control and pi-treated cells (fig. 6c) , suggesting that pis have no influence on the efficiency of the endocytosis of viral particles. this conclusion was enforced by the results obtained through an alternative experimental approach where control or pi-treated hela cells were infected with 0.2 pfu/cell of vsv. thirty and 60 min later, the cells were extensively washed, treated with trypsin to leave out non-adsorbed viral particles, lysed, and analyzed by western blot for the presence of vsv-g. no significant reduction of the vsv-g specific signals was detectable at both time points in pi-treated cells as compared to control conditions (fig. 6d) . considering that the neosynthesis of vsv-g starts at least 90 min post-infection (rothman and lodish, 1977) , these results, consistently with the data obtained with fluorescent vlps, suggest that pis do not affect the endocytosis of viral particles. the results from these experiments indicate that the inhibitory effect of pis is not the consequence of impaired attachment and endocytosis of viral particles. next, the possibility that the inhibitory effect of pis was a consequence of a defect in the endosome to cytoplasm virus delivery was investigated. to this end, the differences in the cell-associated fluorescence levels in cells internalizing wt or fd vsv-g pseudotyped nef g3c -gfp vlps were exploited. in fact, using the same experimental conditions described for the endocytosis assay, it was reproducibly observed that, early after vlp challenge, the mean fluorescence intensity (mfi) of cells challenged with (wt vsv-g) vlps was near 2-fold higher than that of cells treated with (fd vsv-g) vlps. more significantly, in the latter condition both percentages of fluorescent cells and mfi heavily dropped 16 h after challenge. on the contrary, both facs parameters appeared only slightly reduced in cells challenged with (wt vsv-g) vlps (fig. 7a ). these differences can be explained by the fact that, due to the inability of fd vsv-g to induce fusion and delivery of the vlp contents into cytoplasm, the nef g3c -gfp molecules incorporated in (fd vsv-g) vlps were addressed to rapid degradation into the endosomal/lysosomal compartment. conversely, the efficient fusion between viral envelope and endosome membranes induced by wt vsv-g allowed the release of nef g3c -gfp molecules into cytoplasm, where they are expected to be degraded with a kinetic much slower than that operating in endosomes/lysosomes. next, pi-treated or untreated hela cells were challenged with 500 ng hiv-1 cap24 equivalent of either (wt vsv-g) or (fd vsv-g) nef g3c -gfp vlps/10 5 cells, and 3 and 16 h later, the cells were treated with trypsin. facs analysis of the cell-associated gfp fluorescence revealed that the pi treatment did not affect the fluorescence levels in cells challenged with (fd vsv-g) nef g3c -gfp vlps at both time points. on the contrary, after 3 h it was observed a relevant reduction in the mfi within pi-treated cells challenged with (wt vsv-g) nef g3c -gfp vlps as compared with untreated cells. more strikingly, a strong decrease of both mfi and percentage of fluorescent cells was detectable in pi-treated cells at 16 h post-challenge (fig. 7b) . as expected, among the different pi tested, saquinavir and nelfinavir produced the strongest inhibitory effect (data not shown). next, dose-response endocytosis assays using different concentrations of nelfinavir were carried out by analyzing the cell-associated fluorescence 16 h after the challenge. the results showed that, within the cells challenged with (wt vsv-g) nef g3c -gfp vlps, the pi treatment reduced both mfi and percentages of fluorescent cells in a dose-dependent manner. at the highest pi concentrations, both parameters reached levels similar to those detectable in cells challenged with (fd-vsv-g) nef g3c -gfp vlps (fig. 7c) . hence, it seemed that the pi treatment diverted the fate of endocytosed (wt vsv-g) towards that of (fd vsv-g) vlps. this strongly suggests that pis negatively affect the fusion of viral envelope with the endosomal membranes. together, these results support the idea that the inhibitory effect of pis acts at the level of the delivery of the viral particles from endosomes to cytoplasm of infected cells. a possible explanation for the apparent block of viral envelope fusion could be that pis may increase the ph in endosomes in a way to inhibit the low ph-dependent conformational changes needed for both vsv-g and influenza ha to switch the fusion process. to test whether pis affect intracellular ph, both hela and mdck cells were labeled with lysosensor green dnd-189. this reagent becomes fluorescent only in acidic intracellular compartments, meanwhile exhibiting decreased fluorescence intensity upon ph increase. thus, it represents a useful reagent for detecting possible ph variations in endosomes. cells were treated overnight with the pi doses most effective against vsv and/or influenza virus replication, then labeled with lysosensor green dnd-189 for 30 min in the presence of pis, and finally analyzed by facs. as control, cells treated with bafilomycin a1, i.e., a powerful inhibitor of the vacuolar atpase proton pump, were also tested. as depicted in fig. 8 , no significant variations in the cellassociated fluorescence have been detected in pi-treated cells as compared with control conditions, indicating that pis do not affect the intracellular ph. these results strongly suggest that the pi-induced block of virus delivery in cytoplasm would not be a consequence of the increase of endosomal ph. viruses hijack cell functions to replicate in host cells. hence, the selective targeting of cell products supporting virus replication could represent a successful antiviral strategy. the results from the here described investigations indicate that drugs designed to counteract the activity of the hiv protease show a strong inhibitory effect against activity(ies) of target cells. the antiviral potency of the most effective pis reached about 4 logs in the reduction of virus yields, with therapeutic indexes higher than 15. the pi-induced effect against vsv appeared comparable in magnitude to that observed in cell treated with type-1 interferons (masters and samuel, 1983) . on the other hand, the potency of pis against the influenza virus replication appeared comparable to that of most potent inhibitors. among these, t-705 (furuta et al., 2002) reduced the viral yield of the most susceptible influenza strain tested of at best 2.7 logs at the concentration of 5 μm upon mdck cell challenge with 0.01 m.o.i. (sleeman et al., 2010) . das181, i.e. a sialidase fusion protein, blocked the influenza virus replication at sub-micromolar concentrations, however when mdck cells were challenged with 0.001-0.005 m.o.i. (triana-baltzer et al., 2009) . conversely, micromolar concentrations of pis induced more than 3 logs of reduction of the influenza virus yield from mdck cells challenged with 0.5 m.o.i. in the challenge experiments where pis were added soon after the adsorption of vsv or influenza virus inocula, the antiviral effect appeared slightly reduced as compared with control conditions where pis were maintained throughout. considering also the results obtained in the endocytosis assays, it is conceivable that this subtle difference could be a consequence of the accession to cytoplasm of a small amount of viral particles during the adsorption time and before the pi treatment. the possibility that pis affect some virion structural component in a way to hinder the process of fusion in endosomes appeared unlikely. in fact, the treatment of 10-fold concentrated vsv or influenza virus preparations with up to 500 μm pis for 30 min before challenge did not produce significant decrease of the viral yields (not shown). however, we cannot formally exclude that pis inhibit some yet unidentified protease activity associated with vsv-g or influenza virions. in this regard, it has been proposed that pis could negatively affect the chymotrypsin-like protease activity of the pa subunit of the rna-directed rna polymerase of influenza virus (savarino, 2005) . furthermore, it was reported that nelfinavir reduces the replication of sars coronavirus (yamamoto et al., 2004) likely as a consequence of the inhibition of the 3c-like viral protease (for a review, see 35). the results from here reported experiments indicate that pis act on early events of viral replication, but not on virus attachment and endocytosis. this latter finding was consistent with previously reported results indicating that pis do not affect the endocytosis of hiv-1 particles in dendritic cells (muratori et al., 2009) . rather, it appeared that pis interfere with the delivery of viral particles from endosomes to cytoplasm. this did not seem to depend on a pi-induced increase of the endosomal ph possibly inhibiting the low phdependent conformational changes of viral envelope proteins required for viral fusion. the concept that the activity of cell proteases is part of the mechanisms underlying the replication of many virus species is widely accepted. for example, the cleavage of ha generated by cell proteases is required for the activation of the viral envelope protein preceding the viral fusion of influenza virus (klenk et al., 1975; lazarowitz and choppin, 1975) . similarly, the activation of envelope proteins of ebola (schornberg et al., 2006) , nipah (pager and dutch, 2005) , and corona viruses (bosch et al., 2008) is regulated by cathepsins, i.e., a family of cell aspartyl-proteases. the identification of host factors, in particular aspartyl-proteases, involved in the here described pi-induced inhibition of viral entry deserves further investigations. the results from functional genetic screens have demonstrated that alternative cell proteases act as co-factors in the replication of different viruses clemente et al., 2010; hao et al., 2008; krishnan et al., 2008; ng et al., 2007; nguyen et al., 2006; pelkmans et al., 2005) . concerning vsv, the comparison of these data with those regarding the cell proteases known to be sensitive to pis identifies both proteasome subunits as possible relevant pi targets (clemente et al., 2010 ). this appears consistent with the recently reported evidence that mg132 (i.e., a proteasome inhibitor) inhibits vsv replication (neznanov et al., 2008) . alternative functional genetic screens revealed that also mmp-21 could be part of the mechanism of vsv replication (clemente et al., 2010) . considering that the activity of mmp-21, like other mmps, might be affected by pis, it would be of interest investigating the role of this mmp in the here described antiviral effect of pis. the here reported findings would open the way towards preclinical assays designed to test the potency of pis against in vivo infections sustained by orthomyxo-and rabies viruses. it will be also of interest extending the investigations on additional pathogenic enveloped viruses infecting by endocytosis. vsv-g pseudotyped hiv-1 preparations were obtained from supernatants of 293t cells 48 h after co-transfection with a cmv immediate-early promoted vsv-g-expressing vector and vectors expressing nl4-3 hiv-1, its δenv derivative, or pm4 hiv-1 (molar ratio 1:5) performed by the lipofectamine 2000-based method (invitrogen). supernatants were clarified and concentrated by ultracentrifugation as described (federico et al., 2001) . virus preparations were titrated by measuring hiv-1 cap24 contents by quantitative enzyme-linked immunosorbent assay (elisa; innogenetic). both preparations and assays of vsv (indiana strain) have been performed basically as described (gresser et al., 1968) . briefly, high titer stocks of vsv have been prepared upon infection of hela cells with at low multiplicity of infection (m.o.i.), i.e. b0.01 plaque forming unit (pfu)/cell. supernatants were harvested 24 h later, clarified, stocked, and frozen. vsv titrations of high titer stocks have been carried out by plaque method. the a/puerto rico (pr)/8/34 h 1 n 1 human influenza virus was grown in 11-day-old embryonated chicken eggs. the allantoic fluid was clarified by centrifugation at 5000×g for 15 min at 4°c. the virus was pelleted by centrifugation at 65,000×g for 1 h at 4°c and resuspended in 1 ml of phosphatebuffered saline. portions of the solution were stored as aliquots at −20°c. influenza preparations were titrated by standard plaque assay on mdck cells. virus titers for these stocks ranged from 1 to 8 × 10 7 pfu/ml. preparations of the hertz strain of ndv were recovered after 48 h of incubation in 9-11-day-old embryonated chicken eggs inoculated in the allantoic cavity. after harvesting, the allantoic fluid was clarified by centrifugation at 10,000×g, and titrated as infectious units/ml through the end-dilution method by assessing the cytopathic effect on mdck cells 48 h post infection. the titer of the viral stock used was 2.1 × 10 7 infectious units/ml. cem gfp cells were grown in roswell park memorial institute (rpmi) medium supplemented with 10% heat-inactivated fetal calf serum (fcs). hela, mdck, 293t and 293/gpr inducible hiv-1 packaging cells (sparacio et al., 2001) were grown in dulbecco's modified eagle's medium plus 10% fcs. infections of cem gfp cells with hiv-1 or pseudotyped derivatives were carried out by spinoculation at 400 × g for 30 min at room temperature (r.t.) using 200 ng and 50 ng cap24 equivalent of hiv-1 and (vsv-g) hiv-1/10 5 cells, respectively. then, virus adsorption was prolonged for additional 2 h at 37°c and, finally, cells were washed and refed with the complete medium. hela cells were infected with vsv by adsorbing the viral inoculum for 1 h at 37°c in a small volume (e.g., 0.1 ml of serum free medium for 2 × 10 5 cells in 12 well plates). thereafter, the cells were extensively washed, and refed with appropriate complete medium. mdck cells were used as targets of pr8 influenza virus. the challenges were carried out as for vsv infection, except that after virus adsorption, cells were refed with medium without serum in the presence of 1 μg/ml of tpck-treated trypsin (worthington biochemical corporation). hela and mdck cells served to titrate infectious vsv and influenza virus in supernatants harvested after challenge experiments. the titrations were carried out by the end-dilution method with triplicate conditions by challenging the cells with 3-fold scaled supernatant dilutions (in the presence of 1 μg/ml tpck-trypsin for influenza virus titrations), and by evaluating the cytopathic effect after 36 h (for vsv) and 72 h (for influenza virus). concentrated virus preparations previously titrated by the plaque assay were used as standards. for ndv infections, mdck cells were challenged with 2-fold decreasing viral dilutions starting to m.o.i. 1, and the cythopatic effect was assessed 48 h later. t-20, ritonavir, indinavir, saquinavir, nelfinavir, amprenavir and atazanavir were obtained from the nih aids research and reference reagent program. both control and pi-treated cells were labeled with 1 μm cfse (molecular probes, invitrogen) following the manufacturer's recommendations. a cell sample was immediately processed to determine the fluorescence levels at the zero time. thereafter, the remainder cell cultures were refed with complete medium and, at the time of completion of one cell duplication (on the average, 12 h for both hela and mdck cells at their logarithmic phase), were harvested, and the fluorescence measured by facs analysis. dead cells were identified upon labeling with 5 μg/ml of propidium iodide (sigma-aldrich). preparation of fluorescent vlps, challenge, and detection assays fluorescent vlps were obtained as previously described (muratori et al., 2009) . briefly, 293/gpr hiv-1 packaging cells were cotransfected with vectors expressing the green-fluorescent protein (gfp) fused at its n-terminus with a g 3 c hiv-1 nef mutant, together with a vector expressing wt or fusion-defective (fd) vsv-g (fredericksen and whitt, 1995) . supernatants were harvested 2 days later, concentrated by ultra-centrifugation on 20% sucrose cushion, and titrated for the hiv-1 cap24 contents. for hela cell challenge, 500 ng cap24 equivalent of fluorescent (vsv-g) hiv-1 vlps/10 5 cells were adsorbed for 1 h at 4 or 37°c in a volume of 0.1 ml in 48 well plates. thereafter, 0.1 ml of complete medium was added and, finally, the cells were extensively washed and analyzed by facs. in the endocytosis assays, the facs analysis was carried out after incubation with trypsin for 15 min at 37°c. both cells and purified vlp preparations were lysed in pbs, 1% triton x-100 in the presence of anti-proteolytic agents. for the preparation of cytoplasmic extracts, whole cell lysates were centrifuged at 6000×g for 10 min at 4°c, and the supernatants frozen at −80°c. aliquots of 200 ng hiv-1 cap24 equivalent of vlps and of 30 μg of total cell proteins were separated in 10% sds-page, and then transferred by electroblotting on nitrocellulose membranes (sartorius ag) for 60 min at 100 v with a bio-rad transblot. nitrocellulose membranes were blocked in 3% bovine serum albumin (bsa) fraction v (sigma) in ttbs/edta (10 mm tris-hcl, ph 7.4; 100 mm nacl; 1 mm edta; 0.1% tween-20) for 30 min at room temperature, then incubated for 1 h at r.t. with specific antibodies diluted in 1% bsa/ ttbs-edta. the following abs served for the revelation of both vlpand cell-associated products: arp 444 sheep anti-nef antiserum from mark harris, university of leeds, leeds, uk; rabbit polyclonal anti-vsv-g abs from immunology consultant laboratories; monoclonal anti human β-actin from amersham pharmacia biotech. immune complexes were detected through horseradish peroxidase-conjugated goat anti-sheep, anti-rabbit (both from calbiochem) and antimouse abs (nen), followed by enhanced chemioluminescence reaction (euroclone). the lysosensor probe dnd-189 (molecular probes, invitrogen) served to detect possible changes in the endosomal ph upon pi treatment. this is an acidotropic probe accumulating in acidic organelles which exhibits decreasing fluorescence upon ph increase. cells pre-treated with pis or, as control, with bafilomycin a1 (sigma-aldrich) were labeled with 1 μm of lysosensor dnd-189 in complete medium for 30 min in the presence of the drugs. afterwards, cells were extensively washed, fixed, and fluorescence evaluated by facs analysis. when appropriate, data are presented as mean + standard deviation values (sd). in some instances, statistical analysis was performed according to paired student's t-test, and confirmed using the non-parametric wilcoxon rank sum test. p-values b0.05 were considered significant. an inhibitor of hiv-1 protease modulates proteasome activity, antigen presentation, and t cell responses in vitro and in vivo effects of hiv protease inhibitors on apoptosis cathepsin l functionally cleaves the severe acute respiratory syndrome coronavirus class i fusion protein upstream of rather than adjacent to the fusion peptide protease inhibitor treatments reveal specific involvement of matrix metalloproteinase-9 in human adipocyte differentiation identification of host proteins required for hiv infection through a functional genomic screen dyslipidaemia associated with antiretroviral therapy in hiv-infected patients identification of host factors involved in borna disease virus cell entry through a small interfering rna functional genetic screen in vivo emergence of hiv-1 variants resistant to multiple protease inhibitors inhibition of human preadipocyte proteasomal activity by hiv protease inhibitors or specific inhibitor lactacystin leads to a defect in adipogenesis, which involves matrix metalloproteinase-9 an hiv protease inhibitor, ritonavir targets the nuclear factor-kappab and inhibits the tumor growth and infiltration of ebvpositive lymphoblastoid b cells hiv-1 nef activates stat1 in human monocytes/macrophages through the release of soluble factors hiv-protease inhibitors vesicular stomatitis virus glycoprotein mutations that affect membrane fusion activity and abolish virus infectivity vitro and in vivo activities of anti-influenza virus compound t-705 a new reporter cell line to monitor hiv infection and drug susceptibility in vitro effect of repeated inoculation of interferon preparations on infection of mice with encephalomyocarditis virus drosophila rnai screen identifies host genes important for influenza virus replication partial inhibition of the human immunodeficiency virus type 1 protease results in aberrant virus assembly and the formation of noninfectious particles potent suppression of hiv-1 replication in humans by t-20, a peptide inhibitor of gp41-mediated virus entry activation of influenza a viruses by trypsin treatment rna interference screen for human genes associated with west nile virus infection ritonavir blocks akt signaling, activates apoptosis and inhibits migration and invasion in ovarian cancer cells enhancement of the infectivity of influenza a and b viruses by proteolytic cleavage of the hemagglutinin polypeptide mechanism of interferon action: inhibition of vesicular stomatitis virus replication in human amnion u cells by cloned human leukocyte interferon. i. effect on early and late stages of the viral multiplication cycle generation and characterization of a stable cell population releasing fluorescent hiv-1-based virus like particles in an inducible way human immunodeficiency virus type 1 (hiv-1) protease inhibitors block cell-to-cell hiv-1 endocytosis in dendritic cells different effect of proteasome inhibition on vesicular stomatitis virus and poliovirus replication identification of host genes involved in hepatitis c virus replication by small interfering rna technology unpaking" human immunodeficiency virus (hiv) replication: using small interfering rna screening to identify novel cofactors and elucidate the role of group i paks in hiv infection cathepsin l is involved in proteolytic processing of the hendra virus fusion protein the human immunodeficiency virus (hiv)-1 protease inhibitor saquinavir inhibits proteasome function and causes apoptosis and radiosensitization in non-hiv-associated human cancer cells genome-wide analysis of human kinases in clathrin-and caveolae/raftmediated endocytosis synchronised transmembrane insertion and glycosylation of a nascent membrane protein expanding the frontiers of existing antiviral drugs: possible effects of hiv-1 protease inhibitors against sars and avian influenza role of endosomal cathepsins in entry mediated by the ebola virus glycoprotein hiv protease inhibitors are potent antiangiogenic molecules and promote regression of kaposi sarcoma structured model of influenza virus replication in mdck cells in vitro antiviral activity of favipiravir (t-705) against drug-resistant influenza and 2009 a(h1n1) viruses modeling the viral dynamics of influenza a virus infection generation of a flexible cell line with regulatable, high-level expression of hiv gag/pol particles capable of packaging hiv-derived vectors effects of hiv protease inhibitor ritonavir on akt-regulated cell proliferation in breast cancer role of hemagglutinin cleavage for the pathogenicity of influenza virus biochemistry. viral glycoproteins and an evolutionary conundrum novel pandemic influenza a(h1n1) viruses are potently inhibited by das181, a sialidase fusion protein transcription and replication of nonsegmented negative-strand rna viruses hiv protease inhibitor nelfinavir inhibits replication of sars-associated coronavirus this work was supported by grants from the aids project of the ministry of health, rome, italy. t-20 and pis were obtained from the nih aids research and reference program. i thank p. borghi, department of cellular biology and neuroscience, istituto superiore di sanità, rome, italy for kindly supplying both vsv and ndv preparations. i also thank a.r. castrucci and a.m. ciccaglione, department of infectious, parasitic and immunomediated diseases, istituto superiore di sanità, rome, italy, for kindly providing influenza virus preparations and the vector expressing fd vsv-g, respectively. i'm indebted to g. fornari luswergh for her excellent editorial assistance. key: cord-284646-fhruiw23 authors: jaeger, anna s.; weiler, andrea m.; moriarty, ryan v.; rybarczyk, sierra; o'connor, shelby l.; o'connor, david h.; seelig, davis m.; fritsch, michael k.; friedrich, thomas c.; aliota, matthew t. title: spondweni virus causes fetal harm in ifnar1(-/-) mice and is transmitted by aedes aegypti mosquitoes date: 2020-05-24 journal: virology doi: 10.1016/j.virol.2020.05.005 sha: doc_id: 284646 cord_uid: fhruiw23 spondweni virus (sponv) is the most closely related known flavivirus to zika virus (zikv). its pathogenic potential and vector specificity have not been well defined. sponv has been found predominantly in africa, but was recently detected in a pool of culex quinquefasciatus mosquitoes in haiti. here we show that sponv can cause significant fetal harm, including demise, comparable to zikv, in a mouse model of vertical transmission. following maternal inoculation, we detected infectious sponv in placentas and fetuses, along with significant fetal and placental histopathology, together suggesting vertical transmission. to test vector competence, we exposed aedes aegypti and culex quinquefasciatus mosquitoes to sponv-infected bloodmeals. aedes aegypti could efficiently transmit sponv, whereas culex quinquefasciatus could not. our results suggest that sponv has the same features that made zikv a public health risk. zika virus (zikv) was originally isolated over seventy years ago, and was thought to cause a 14 mild, self-limiting, febrile illness (dick et al., 1952; simpson, 1964) . not until the outbreak 15 in the americas in 2015 and 2016 was zikv identified as a cause of significant adverse 16 pregnancy outcomes (johansson et al., 2016; melo et al., 2016) . before the definition of congenital zika syndrome (czs) in 2016, gestational arbovirus infection was not associated with birth defects. spondweni virus (sponv) is the closest known relative to zikv, but 19 whether sponv is an emerging threat to pregnant women and their babies is unknown. it 20 was previously thought that sponv was geographically confined to africa and caused only 21 mild disease in rare human infections, reminiscent of the consensus around zikv in the 22 decades following its discovery, but recent data suggest that it may be spreading beyond 23 africa (white et al., 2018). sponv may therefore be poised to harm pregnancies in new, 24 immunologically naive populations. to do this, sponv would need to fulfill two major 25 criteria: it would need to be vertically transmitted and cause fetal harm, and be transmitted 26 between humans by the urban mosquito vector aedes aegypti, which is associated with 27 large-scale outbreaks of related arboviruses. 28 the first identification of sponv was thought to have occurred in 1955 in south africa 29 (theiler and downs, 1973; wolfe et al., 1982) . however, it was later recognized that sponv 30 was in fact isolated three years earlier in nigeria, but was misidentified at the time as a 31 strain of zikv because of serological cross-reactivity (haddow et al., 1964; simpson, 1964; 32 draper, 1965) . serological cross-reactivity with zikv and other flaviviruses likely still 33 confounds accurate diagnostics today. as a result, only six well-documented clinical cases of 34 draper, 1965) . it is likely that many infections have gone unrecognized-serosurveys have 36 detected evidence of sponv infection in 10 countries throughout sub-saharan africa 37 (kokernot et al., 1965a; kokernot et al., 1965b; brottes et al., 1966; ardoin et al., 1976; wolfe 38 sponv caused fetal harm, similar to what is observed from zikv infection in this model. 69 vector competence experiments showed that ae. aegypti could transmit sponv when 70 exposed to bloodmeal titers that approximate physiological titers, while cx. quinquefasciatus nonpregnant, mixed sex 6-to 11-week-old mice lacking type i interferon signaling (ifnar1 -/-) ar94 (this is the only strain used in these studies, so it will be referred to hereafter as 86 sponv); or 10 2 pfu of the highly pathogenic african-lineage zikv strain dak ar 41524 87 (zikv-dak) (jaeger et al., 2019) . since contemporary sponv isolates from haiti do not exist, 88 we used the only available low-passage isolate, sponv strain sa ar94. this strain is 98.8% 89 nucleotide identical with the sponv genome recovered from mosquitoes in haiti 90 (genbank:mg182017). serum was collected at 2, 4, and 6 days post-inoculation (dpi) to 91 confirm infection and determine the replication kinetics of sponv in nonpregnant ifnar1 -/-92 mice. we also collected and tested serum at 7, 14, and 21 days from mice surviving sponv 93 inoculation, because sustained vrna loads were observed with the ifnar1-blocking mab 94 model (salazar et al., 2019) . sponv viral titer in the serum peaked at 4 dpi (fig. 1a) , and in 95 surviving animals there was no detectable viremia at 7, 14, or 21 dpi. higher serum titers 96 were observed in animals inoculated with the lowest dose of sponv (10 2 pfu). we 97 postulate that this could be the result of higher inoculating doses causing a rapid initial rise 98 in viremia, which in turn induces a more robust immune response, leading to more rapid 99 clearance of virus from the serum, but confirmation will require further studies. zikv-dak 100 viremia also peaked at 4 dpi and reached significantly higher titers at 4 dpi than either 101 pfu of sponv or 10 2 pfu zikv-dak. based on our preliminary experiments with sponv in 120 nonpregnant animals, and the results from our past studies (jaeger et al., 2019), we chose 121 this dose to minimize the potential confounding impacts of maternal illness on fetal 122 outcomes. we collected serum samples from dams at 2 and 4 dpi to confirm maternal 123 infection. all dams were productively infected, with detectable viremia for all groups by 4 124 dpi (fig. 2a) . zikv-dak replicated to significantly higher titers at 4 dpi as compared to 125 sponv (student's t-test p-value = 0.0008, t = 5.641, df = 7). dams were monitored daily pregnancies and with uninfected counterparts. in general, fetuses appeared either grossly 135 at the time of necropsy, we observed high rates of resorption from both zikv-dak-and 137 sponv-infected pregnancies. resorption rates from zikv-dak-and sponv-infected 138 pregnancies were not significantly different (zikv-dak: 76.92% vs. sponv: 68.29%, fisher's 139 exact test, p = 0.457). resorption rates for both sponv and zikv-dak were significantly 140 higher than pbs-inoculated controls (p <0.0001). despite significantly higher maternal 141 viremia observed at 4 dpi with zikv-dak-infected dams, the fact that resorption rates did 142 not significantly differ between the two groups indicates that both zikv-dak and sponv 143 have a propensity to harm the developing fetus that is independent of the amount of 144 replication in maternal blood. surprisingly, and in contrast to the results described by to further characterize the range of pathogenic outcomes of congenital sponv infection 150 and to assess differences between models, we repeated experiments by treating dams with inoculation with zikv-dak or sponv (sheehan et al., 2006) . this model has been used 153 previously for assessing both zikv and sponv pathogenesis during pregnancy, but does confirm infection, and all dams were productively infected with sponv or zikv-dak 159 following treatment with either dose of mab (fig. 2d) . maternal viremia did not significantly 160 differ between treatment groups (sponv/1mg vs. sponv/2mg: p=0.996; zikv/1mg vs. zikv/2mg: p=0.35; one-way anova with tukey's correction for multiple comparisons). zikvdak titers, however, were significantly higher than sponv titers (sponv/1mg vs. zikv/1mg: 163 p=0.04; sponv/2mg vs. zikv/2mg: p=0.006). next, adhering to our previously established 164 experimental timeline, dams were necropsied on e14.5 to assess and compare fetal 165 outcomes. at the time of necropsy, we observed no significant resorption from either zikv 166 or sponv infected pregnancies, after either dose of mab (fig. 2e) , consistent with the 167 results described by salazar et al. observed after e6.5 virus challenge and e13.5 or e18.5 dam sacrifice (salazar et al., 2019) . resorption rates from zikv-dak-and sponv-infected 169 pregnancies were not significantly different (fisher's exact test, p>0.06 for all comparisons). it is possible that the differences in outcomes in these two models may be due to the closely related to both zikv and sponv and it is not known to cause adverse pregnancy 179 outcomes in humans. to examine whether maternal denv-2 infection is sufficient to induce 180 fetal resorption, we s.c. inoculated pregnant dams on e7.5 with 7.5 x 10 4 pfu of denv-2. 181 prior to studies in pregnant animals we confirmed that this route and dose would result in 182 productive infection in nonpregnant animals (fig. 3a) . all dams were productively infected 183 with denv-2 with detectable vrna loads at 2 and 4 dpi (fig. 3a) . importantly, fetuses 184 continued to develop as examined on e14.5, and rates of resorption were not significantly 185 exact test, p = 0.665) (fig. 3b) . these observations confirm that fetal harm was specifically 187 associated with zikv-dak and sponv infection, but because denv-2 infected mice do not 188 show clinical signs, we cannot exclude the possibility that the more severe fetal outcomes 189 to begin to understand the potential for sponv to be vertically transmitted, a subset of 194 placentas and fetuses were collected for plaque assay at time of necropsy from all virus 195 treatment groups. from the ifnar1 +/tissues, infectious virus was detected in 100% of zikvdak placentas and fetuses screened (fig. 2c) . virus was detected in all but one sponv 197 placenta and 35% of fetuses (fig. 2c) . viral titers were significantly higher in sponv 198 placentas than their corresponding fetuses (one-way anova with tukey's multiple 199 comparisons; p < 0.0001), as were zikv-dak placenta viral titers as compared to zikv-dak 200 fetuses (p = 0.006). in addition, zikv placenta and fetal viral titers were significantly higher 201 than sponv titers (p < 0.0001). 202 placental tissues from dams treated with anti-ifnar1 mab (fig. 2f) . antibody dose did not 204 affect the viral titer present in fetuses or placentas after either sponv-or zikv-dak-205 inoculation (p > 0.9 for all comparisons; one-way anova with tukey's multiple 206 comparisons). in general, fetal and placenta tissue titers were significantly higher in zikv-207 dak challenge groups as compared to sponv challenge groups, with a more significant 208 difference in placenta tissue titers than fetal tissue titers (fig. 2f) . of note, infectious sponv 209 was detected in fetuses from both mab treatment groups, which is in contrast to the placental tissues from denv-2 infected pregnancies were also screened for infectious virus 215 via plaque assay. infectious virus was not detected in any of the screened fetal and placental 216 tissues, further suggesting the specificity of fetal harm to zikv and sponv (fig. 3c) . 217 to better understand the impact of in utero sponv exposure, tissues from the developing 218 ifnar1 +/placenta and fetus were evaluated microscopically. in pbs-and denv-inoculated fetal blood spaces (fig. 4) . in contrast, zikv-dak-and sponv-inoculated dams displayed 221 varying degrees of placental pathology with severe effects predominantly observed in the 222 the labyrinth zone, including vascular injury involving maternal and/or fetal vascular spaces, 223 infarction (obstructed blood flow), necrosis, apoptosis, and hemorrhage (fig. 4) . overall, the 224 severity of the vascular injury in the labyrinth zone was similar between zikv-dak and 225 in the fetuses, there was no significant microscopic pathology from pbs-and denv-227 inoculated dams. in contrast, fetuses from zikv-dak-and sponv-inoculated dams 228 demonstrated varying degrees of pathology. in fetuses from the sponv-inoculated dams, 229 fetal injury was evident as mild pulmonary inflammation and mild to moderate segmental 230 necrosis of the brain and spinal cord (fig. 5) . these data provide indirect evidence that 231 vertical transmission did occur. pathologic findings were more widespread and severe in 232 fetuses from zikv-dak-inoculated dams and included severe necrosis and inflammation of 233 the lung, liver, kidney, brain, and spinal cord. 234 because sponv rna was detected in a pool of cx. quinquefasciatus in haiti, we compared 237 the relative abilities of ae. aegypti and cx. quinquefasciatus from florida to transmit sponv 238 in the laboratory. sponv titers in naturally infected hosts-to which feeding mosquitoes 239 might be exposed in nature-are undefined. therefore, we conducted our experiments with 240 blood meal titers ranging from ~10 6 -10 8 pfu/ml. we considered these doses to be 241 physiologically relevant based on studies with denv (50% mosquito infectious doses = 242 10 5.68 -10 7.21 viral cdna copies/ml) (duong et al., 2015) and zikv (50% mosquito infectious 243 doses = 10 6.1 -10 7.5 pfu/ml) (ciota et al., 2017) . to assess vector competence, mosquitoes 244 were exposed to viremic bloodmeals via water-jacketed membrane feeder maintained at table 1 ). ae. aegypti that had been exposed to we speculate that the difference in outcomes between these two models could be due to displayed the most severe histologic phenotype that corresponded with higher placenta and 329 fetus titers in both pregnancy models (fig. 2) . sponv histopathology was more following inoculation with sponv, zikv, or pbs, mice were sacrificed at e14.5. tissues were 506 carefully dissected using sterile instruments that were changed between each mouse to 507 minimize possible cross contamination. for all mice, each organ/neonate was evaluated 508 grossly in situ, removed with sterile instruments, placed in a sterile culture dish, and further 509 processed to assess viral burden and tissue distribution or banked for future assays. briefly, 510 uterus was first removed, and then dissected to remove each individual conceptus (i.e, fetus 511 and placenta when possible). fetuses and placentas were either collected in pbs 512 supplemented with 20% fbs and penicillin/streptomycin (for plaque assays) or fixed in 4% 513 pfa or 10% neutral buffered formalin for imaging. we characterized an embryo as in the 514 resorption process if it met the following criteria: significant growth retardation compared to 515 litter mates and controls accompanied by clearly evident developmental delay, i.e., 516 morphology was ill defined; or visualization of a macroscopic plaque in the uterus (flores et 517 al., 2014) . 518 tissues were fixed in 4% paraformaldehyde for 24 hours and transferred into cold, sterile 520 dpbs until alcohol processed and embedded in paraffin. paraffin sections (5 μm) were 521 stained with hematoxylin and eosin (h&e). pathologists were blinded to gross pathological findings when tissue sections were evaluated microscopically. the degree of pathology at the maternal-fetal interface was rated on a scale of 0-4: 0 -no lesions (normal); 1 -mild 524 changes (1-2 focal lesions or 10-15% of zone involved); 2 -mild to moderate changes (3-4 525 focal lesions or 10-15% of zone involved); 3 -moderate to severe changes (4-6 focal lesions 526 or 15-25% of zone involved); 4 -severe (>6 focal lesions or >25% of zone involved). the 527 final score was dependent upon the greater of two parameters (# of lesions or % zone 528 involved). this was an identical scoring system to what we reported previously (jaeger et al., 529 2019). the final scores were determined as a consensus score of two independent 530 pathologists. for each zone in the placenta (myometrium, decidua, junctional zone, labyrinth, 531 and chorionic plate/membranes) a 'general' overall score was determined, a score for the 532 amount of 'inflammation', and a score for direct 'vascular injury'. the 'general' score was 533 based on an interpretation of the overall histopathologic findings in each placenta, which 534 included features of necrosis, infarction, apoptosis, hemorrhage, thrombosis, mineralization, 535 vascular injury, and inflammation. the 'inflammation' score quantified the amount of 536 inflammation in that layer. the 'vascular injury' score assessed vascular wall injury (fibrinoid 537 necrosis, endothelial swelling), dilatation of the vessels or spaces, necrosis, loss of vascular 538 lumen diameter, and intraluminal thrombi. the myometrial layer representing the uterine 539 therefore meaningful comparisons between strains could not be assessed. the decidual layer 541 (maternal in origin), the junctional zone composed of fetal giant cells and 542 spongiotrophoblast, and the labyrinth layer (the critical layer for gas and nutrient exchange 543 between the fetal and maternal vascular systems) were scored. since the percentage of 544 injured/pathologic labyrinth zone is a predictor of poor fetal outcome, we also 545 independently scored the labyrinth zone based only on the percentage of fetal and maternal 546 vascular injury/loss using the following scoring system: 0-5%-0 (background); 5-15%-1 547 (mild); 15-30%-2 (moderate); 30-50%-3 (moderate to severe); and >50%-4 (severe). 548 photomicrographs were obtained using a bright light microscope olympus bx43 and 549 olympus bx46 (olympus inc., center valley, pa) with attached olympus dp72 digital camera 550 (olympus inc.) and spot flex 152 64 mp camera (spot imaging), and captured using 551 commercially available image-analysis software (cellsens dimensionr, olympus inc. and spot 552 software 5.2). 553 all mosquitoes used in this study were maintained at the university of minnesota, twin 555 cities as described (christensen and sutherland, 1984) vector competence studies 564 mosquitoes were exposed to sponv-or zikv-infected bloodmeals via water-jacketed 565 membrane feeder maintained at 36.5 °c (rutledge et al., 1964) . bloodmeals consisted of 566 defibrinated sheep blood (hemostat laboratories, inc.) and fresh virus supernatant, yielding 567 infectious bloodmeal titers ranging from ~10 6 -10 8 pfu/ml. bloodmeal titer was determined 568 after feeding. infection, dissemination, and transmission rates were determined for individual 569 mosquitoes and sample sizes were chosen using long established procedures (aliota et al., fisher's exact test was used to determine differences in rates of normal vs. abnormal 582 concepti. 583 virus stock sequence data have been deposited in the sequence read archive (sra) with 585 accession codes pending. the authors declare that all other data supporting the findings of 586 this study are available within the article. 587 compared to pbs controls (fisher's exact test). ***p < 0.0005; **p < 0.005. 827 -------culex 589 pipiens and aedes triseriatus mosquito susceptibility to zika virus wolbachia reduces transmission of zika virus by aedes aegypti the wmel strain of wolbachia reduces transmission of chikungunya 595 virus in aedes aegypti louis encephalitis virus in mice epidemiologic study of arboviruses in 599 the arba-minch district of ethiopia isolations of arboviruses in the lagos area of nigeria, and a survey of antibodies to them in man and animals human to mosquito transmission of 606 dengue viruses antiviral immunity backfires: pathogenic effects of 608 type i interferon signaling in fetal development exsheathment and midgut penetration 610 in aedes aegypti effects of zika virus strain and aedes mosquito species on 613 zika virus. i. isolations and 615 serological specificity infection via 622 mosquito bite alters zika virus tissue tropism and replication kinetics in rhesus macaques asymptomatic 626 humans transmit dengue virus to mosquitoes evidence of 630 vertical transmission and co-circulation of chikungunya and dengue viruses in field 631 populations of aedes aegypti (l.) from guerrero early detection and 634 staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine 635 pregnancy a diversified portfolio virol 3, vi-viii genomic epidemiology reveals 650 multiple introductions of zika virus into the united states dengue, urbanization and globalization: the unholy trinity of the 652 21(st) century genetic characterization of spondweni and zika viruses and 655 susceptibility of geographically distinct strains of aedes aegypti culex quinquefasciatus (diptera: culicidae) to spondweni virus distinguishing between zika and spondweni 659 viruses a gorilla adenovirus-based vaccine against zika virus induces durable immunity and confers protection in pregnancy zika virus infection in pregnant rhesus macaques causes placental 673 dysfunction and immunopathology protective efficacy of nucleic acid vaccines against transmission of zika virus during 682 pregnancy in mice gestational stage and ifn-λ signaling zika and the risk of microcephaly survey 689 for antibodies against arthropod-borne viruses in the sera of indigenous residents of studies on 692 arthropod-borne viruses of tongaland. viii. spondweni virus, an agent previously unknown, 693 isolated from taeniorhynchus (mansonioides) uniformis survey for 695 antibodies against arthropod-borne viruses in the sera of indigenous residents of the caprivi 696 strip and bechuanaland protectorate quantitative genetics of aedes aegypti vector competence for 698 dengue viruses: towards a new paradigm discovery and full genome characterization of two highly 702 divergent simian immunodeficiency viruses infecting black-and-white colobus monkeys 703 (colobus guereza a mouse model of zika virus pathogenesis shared and distinct 708 functions of type i and type iii interferons zika virus: a report on three cases of human infection during an 710 epidemic of jaundice in nigeria pathogenesis and sexual 712 transmission of spondweni and zika viruses further isolations of the arboviruses 714 from mosquitoes collected in tongaland, south africa, 1960-1968 isolation 717 of spondweni virus from four species of culicine mosquitoes and a report of two laboratory 718 infections with the virus the interaction of glutamic and aspartic acids with 720 excitatory amino acid receptors in the mammalian central nervous system congenital zika virus infection: beyond neonatal microcephaly increased yellow fever virus infection and dissemination rates in aedes 735 aegypti mosquitoes orally exposed to freshly grown virus virus infection during pregnancy in mice causes placental damage and fetal demise zika virus: following the path of 742 dengue and chikungunya host and viral features of human 747 dengue cases shape the population of infected and infectious aedes aegypti mosquitoes zika virus-related neurotropic flaviviruses infect human placental explants and cause 751 fetal demise in mice no evidence of zika, dengue, or chikungunya virus 754 infection in field-caught mosquitoes from the recife metropolitan region zika virus 757 and birth defects--reviewing the evidence for causality vaccine mediated protection against zika virus-induced congenital disease studies on the feeding response of 765 mosquitoes to nutritive solutions in a new membrane feeder dengue and zika virus cross-reactive human monoclonal antibodies protect against spondweni virus infection and pathogenesis 770 in mice neutralizing human antibodies 774 prevent zika virus replication and fetal disease in mice blocking monoclonal antibodies specific for mouse ifn-alpha/beta receptor subunit 1 778 (ifnar-1) from mice immunized by in vivo hydrodynamic transfection zika virus infection in man the arthropod-borne viruses of vertebrates. an 783 account of therockefeller foundation virus program zika virus and the nonmicrocephalic fetus: why we should still worry emerg 790 spondweni virus infection in a 792 foreign resident of upper volta the incidence of arthropod-794 borne viruses in a population of culicine mosquitoes in tongaland type i interferons instigate fetal demise after zika virus infection. sci 799 immunol 3, 800 analyzed data and drafted the manuscript o. developed and performed the deep sequencing pipeline declaration of interests ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work the authors acknowledge the university of minnesota, twin cities bsl3 program for facilities 802 and neal heuss for support. we thank natalie benett for her contribution in mosquito the authors declare no competing financial interests. 816 collected from mice during three independent replicates at 2, 4, and/or 6 days post 820 inoculation and titered via plaque assay. assay limit of detection was 100 pfu. viremia 821 peaked at 4dpi for all virus groups, with zikv-dak replicating to significantly higher titers at 822 4dpi than sponv (one-way anova). ****p < 0.0001; ***p < 0.0005; **p < 0.006 (b) survival 823 curves of six-to eleven-week old ifnar1 -/mice s.c. inoculated with 10 3 pfu of sponv, 10 2 824 pfu of sponv, 10 2 pfu zikv-dak, or a pbs control. sponv 10 3 : n=14; sponv 10 2 : n=13, 825 key: cord-289712-w1y0lc5c authors: flintoff, wayne f. title: replication of murine coronaviruses in somatic cell hybrids between murine fibroblasts and rat schwannoma cells date: 1984-04-30 journal: virology doi: 10.1016/0042-6822(84)90312-x sha: doc_id: 289712 cord_uid: w1y0lc5c abstract the replication of the murine coronaviruses mhv3 and jhm has been studied in somatic cell hybrids formed between murine fibroblast l2 cells which support lytic infections with both these agents, and rat rn2 schwannoma cells which support the replication of jhm in a temperature-sensitive, persistent manner but are restrictive to the replication of mhv3. the results described in this report indicate that the totally permissive state is dominant over the persistent or restricted state since the hybrid cells permit the replication of both these viral agents in a lytic manner. the replication of the murine coronaviruses mhv, and jhm has been studied in somatic cell hybrids formed between murine fibroblast l2 cells which support lytic infections with both these agents, and rat rn2 schwannoma cells which support the replication of jhm in a temperature-sensitive, persistent manner but are restrictive to the replication of mhvs. the results described in this report indicate that the totally permissive state is dominant over the persistent or restricted state since the hybrid cells permit the replication of both these viral agents in a lytic manner. murine hepatitis viruses are members of the group of rna-containing enveloped viruses termed coronaviruses (tyrrell et al, 1978) . the agents cause a wide variety of diseases in rodents including hepatitis, enteritis, and encephalomyelitis, the disease type varying with the strain of virus, and age and genetic background of the host (wege et al, 1982) . in some virus-host combinations, persistent infections occur resulting in chronic hepatitis or chronic demyelination of the central nervous systems (wege et al, 1982) . this later observation, centered mainly on studies with the jhm strain of the virus (cheever et al, 1949; weiner, 1973; sorensen et al., 1980) or a temperature-sensitive mutant of it (knobler et al, 1982) , has received considerable interest as a potential model relevant for the study of slowly degenerative neuropathies (weiner, 1973; weiner et al, 1973; sorensen et al, 1980) . these agents can also readily establish persistent infections in vitro in both neural and nonneural cell lines (lucas et ak, 1977 (lucas et ak, , 1978 stohlman and weiner, 1978) , without requirements of viral modifications or en-' presented in preliminary form at the coronavirus workshop in zeist, the netherlands, june 1983. vironmental manipulations such as the presence of viral antibody or interferon. our previous results have indicated that when the infection was of the persistent type, virus replication was almost invariably thermosensitive and that this was due to unknown factors under host control, since the progeny virions themselves were not temperature sensitive (lucas et ak, 1977 (lucas et ak, , 1978 . in addition, one cell line, the rn2 schwannoma, had the unique ability to discriminate between the mhv, strain and the more neurotropic strain jhm (lucas et al, 1977) . jhm was replicated persistently in this line, whereas, mhv, replication was aborted. at present, it is unclear as to the mechanism of persistence or restriction in this cell line. these observations coupled with others (stohlman and weiner, 1978; holmes and behnke, 1982) strongly imply that the host cell has a profound influence in regulating the replication of these agents. as a further approach to analyzing the host functions involved in viral persistence and restriction, somatic cell hybrids have been formed between mouse l2 cells, a cell line totally permissive for both mhv3 and jhm infection, and the rat rn2 cells. the results described in this report indicate that the l-cell functions appear to be dominant over the rn2 controlled ones since both viral agents replicated lytically in the somatic cell hybrids. cells and virus. the sources and routine propagation of the l2 and rn2-2 cell lines, and the mhv3 and jhm strains of mouse hepatitis virus were as previously described (lucas et al, 1977) except that alpha medium (stanners et al, 1971 ) was used in place of eagle's minimal essential medium. virus production was monitored by a plaque assay on lz-cell monolayers as previously described (lucas et a& 1977) . yields are expressed as pfu/ml (plaque-forming units/mililiter). to determine the fraction of cells able to release virus, an infectious center assay was performed (lucas et al, 1978) . selection of genetica& wmrked ia? cells. l2 cells were treated for 3 hr in the presence of 0.2 pg/ml. n-methyl-n'-nitro-nnitrosoguanidine at 34", washed, and resuspended in fresh medium. survival was usually about 50%. the cells were allowed to grow 6 days to allow for expression of putative mutations before selections were carried out. the basic procedure for mutant selections is described elsewhere (flintoff et al., 1976) . cells, at 5 x 105/100-mm tissue culture dish, were exposed to 0.2 pg/ml 6-thioguanine (tg) (sigma chemical co.) for 8 days at 34" with replacement of drug and medium every 2 days. colonies surviving at a frequency of 4 x lop6 were picked, cloned by limit dilution, and tested for resistance. one clone, l2 tgr7, was resistant to at least 50 pg/ml tg, a concentration lo3 higher than was cytotoxic for the wild-type cells, and contained ~0.1% of the wild-type hypoxanthine phosphoribosyl transferase activity as determined by the assay described by chasin and urlaub (1976) . the l2 tgr7 cells were exposed to 3 mm ouabain (oua) to select for oua-resistant cells (baker et al, 1974) . colonies surviving at a frequency of loa were isolated, cloned, and shown to be resistant to at least 3 mm oua. wild-type cells were unable to grow at concentrations above 0.5 mm. one doublely marked clone, l2 tgrouar7-1, was used in the hybridization experiments. neither resistance to tg, nor oua, nor the presence of both of these markers affected the ability of the cells to support the replication of either the mhv, or jhm strains of mouse hepatitis virus. cell-cell hybridization. somatic cell hybrids were formed between the l2 tgrouar7-1 and rn2-2 cells by exposure to polyethylene glycol (peg) 6000 (british drug house) for 1 min using the procedure described by pontecorvo (1975) . cells were plated in complete medium for 1 day to allow recovery from the fusion process prior to the addition of hat + oua selection medium (7 x 1o-5 m hypoxanthine, 2 x lop8 m methotrexate, 4 x lop5 m thymidine, 2 x lop3 m oua). using these selective conditions, neither parental cell line survives. the mouse l2 cells are unable to survive in the hat medium and the rat rn2-2 are killed by the oua. table 1 summarizes the hybridization frequencies obtained in two separate experiments. after 8 to 10 days incubation at 34" surviving colonies were picked, expanded, maintained in normal medium, and used for study. subclones of some of these isolates were obtained by limit dilution. there were no differences in the responses of these subclones and the original isolates to virus infection. kaqotgpic analyses. exponentially growing cells were incubated with 0.25 pg/ml colcemid (grand island biological co.) for 1.5 hr at 34". the cells were washed with hypotonic and fixing solutions and chromosome spreads prepared (worton and duff, 1979) . preparations were either treated with 0.05% trypsin for varying periods of time and stained with giemsa or the centromeres were stained using the procedure described by marshall (1975) . for this latter procedure, slides were baked for 48 hr at 55", treated for 1 min with 0.05% trypsin in 0.15 mnacl, treated with formamide-scc (95 ml neutralized formamide and 5 ml 20x ssc (1x ssc is 0.15 m nacl, 1.5 mm na citrate) at 65" for 20 min, rinsed with distilled hzo, stained with 10% giemsa in gurr's buffer, ph 6.8, and ,j hybrids were formed and selected by growth in hat medium containing 2 mm oua as described under materials and methods. from 5 to 10 chromosome spreads were examined for each cell line. lactate dehydrogenuse assag. the preparation of cell extracts and the assay for lactate dehydrogenase were essentially as described by weiss and ephrussi (1966) . briefly, exponentially growing cells were harvested, washed two times in phosphatebuffered saline, resuspended in 10 mm tris-cl, ph 7.0, quick frozen and thawed two times, and homogenized. the homogenates were centrifuged for 1 hr at 30,000 g in a sorval ss34 rotor at 4" and the supernatants used as the enzyme source. in vitro hybridizations were carried out as described by markert (1963) in the presence of 1 mnacl and '70 mmna phosphate, ph 8.0. the preparations were stored at -20" overnight. for electrophoresis, 3-~1 samples were applied with a gelman stainless-steel wire applicator to sepraphore iii cellulose acetate strips (gelman instrument co.) that had been soaked for 30 min in electrophoresis buffer (0.3 m sodium borate, ph 8.6) and blotted dry. electrophoresis was for 100 min at 200 v. to detect lactate dehydrogenase, the strips were stained for 30 min at 37" in the dark with 0.025 m tris-cl, ph 7.4, 0.1 m d + i lactate, 0.005 kcn, 0.001 m nad, 50 pg/ml nitroblue tetrazolium, and 20 pg/ ml phenazine methosulfate. the strips were fixed for 10 min in 50% methanol, 10% acetic acid, 40% hzo, dried at room temperature, and cleared in glycerol at 70'. p-glucuronidase assay. the preparation of cell extracts and assay for @glucuronidase was essentially as described by weiss and ephrussi (1966) and by herrup and mullen (1977) . briefly, cells were harvested, washed two times in phosphate-buffered saline, resuspended in hzo, and homogenized in 0.2% triton x-100. the homogenates were centrifuged for 10 min at 1470 g in a sorval ss34 rotor at 4". the supernatants were used as the enzyme sources. 8-glucuronidase assays were carried out in 2 ml reaction volumes consisting of 0.1 m na acetate, ph 4.6, 0.2 mm pnitrophenol-p-d glucuronic acid, 0.2% triton x-100, and various amounts of enzyme extract equivalent to 2 x ld to lo6 cells. incubations were at 37" for 2 hr. under such conditions, enzyme activity was linear with respect to both extract level and time. reactions were stopped by the addition of 0.8 ml of 17.5% trichloroacetic acid, and centrifuged at 600 g for 10 min to remove the protein precipitate. the clear supernatants were added to tubes containing 1.2 ml of 4 n naoh to develop the color, and the optical density read at 415 nm. all assays were done in duplicate and reagent blanks containing no enzyme were subtracted from the readings. for heat inactivation, samples were incubated in glass tubes at 62" in 0.1 m na acetate, ph 4.6. at various intervals, duplicate samples were removed, and chilled on ice. substrate solution was then added to the tubes and the standard assay per-formed. results are expressed as the percentage of the unheated controls. of hybrid cells. somatic cell hybrids were formed between the genetically marked mouse l2 cells and the rat rn2 cells by fusing with polyethylene gly-co1 and selection in hat + oua medium. although the frequency of hybrid formation was greater than the survival of either the mouse or rat parental cell lines under these selective conditions (table l) , it was conceivable that parental cells might have survived the selection scheme. thus, it was important to distinguish between authentic cell hybrids and parental survivors. hybrid cells formed between these mouse and rat cells could be readily distinguished from the parental cells on the basis of their chromosome content and on the production of species-specific gene products. as shown in table 2 , the mouse-rat hybrid cells had average chromosome numbers that were much greater than those of either the mouse or rat parental cells used to form them. a majority of the hybrid cells contained almost the entire complement of both mouse and rat chromosomes (table 2) , as determined by their characteristic trypsin giemsa or centromeric banding patterns determined as described under materials and methods. although there was variation in the chromosome composition among the hybrids, there was no apparent consistent pattern in the nature and type of chromosomes that were missing. figure 1 illustrates the chromosomes present in the parental and two hybrid cell lines. since rat and mouse cells produce different isozymes of lactate dehydrogenase (weiss and ephrussi, 1966) , it was of interest to determine whether these mouserat cell hybrids produced both isozymes. as shown in fig. 2 , the rat rn2 cells produced a lactate dehydrogenase enzyme that migrated more cathodally than similar enzyme from mouse l2 cells. when a mixture of rat and mouse lactate dehydrogenases was dissociated and reassembled in vitro, four major bands and a faint fifth band of enzymatic activity were obtained (fig. 2, lane d) . such a pattern was absent in a mixture of parental extracts (fig. 2 , lane c). when extracts of hybrid cells were assayed for lactate dehydrogenase activity (fig. 2 , lanes e, f) an isozyme pattern similar to that of the in vitro assembled isocommon property for these isozymes in mouse-rat hybrids (weiss and ephrussi, 1966) . these results are consistent with the presence and association of both mouse and rat forms of lactate dehydrogenase in the hybrid cells. another function that is readily distinguished between rat and mouse cells is @glucuronidase (weiss and ephrussi, 1966) . rat /3-glucuronidase is more sensitive to heat inactivation than the mouse enzyme. to examine whether both mouse and rat forms of the enzyme were being produced in the hybrid cells, cell extracts were prepared from the cell lines, heated at 62" for various periods of time, and assayed for p-glucuronidase activity. as shown in fig. 3 , the enzymatic activity produced in the hybrid cells was inactivated by heat at a rate intermediate between the mouse and rat enzymes. this is consistent with the presence in the hybrids of both mouse and rat fi-glucuronidases since the heat inactivation was similar to that of a l-to-l mixture of cell extracts from the two parental lines. taken together the above results indicate that these cells are authentic mouse-rat hybrids. morphologically, these hybrid cells did not differ dramatically from the two parental lines. some hybrids contained cells displaying the flattened irregularly shaped morphology of the rn2-2 cell line, whereas, others contained cells roughly triangular in shape with well-defined margins characteristic of the l2 cell line. still others contained cells with more variable morphologies, some of which had well-defined but ragged edges. replication of jhm and mhv, in mmserat cell hybrids. previous results indicated that mouse l2 cells supported the replication of both the jhm and mhva virus strains in a lytic fashion involving extensive cell destruction through syncytial formation. when rn2 cells were used as host, jhm replicated persistently with restricted cytopathology in a temperaturesensitive manner. mhv3 replication was totally restricted in the rn2 cells (lucas et al, 1977 (lucas et al, , 1978 . the availability of somatic cell hybrids between these two cell lines permitted an examination of which host cell type dominantly affected the virus replication process. confluent monolayer cultures of several independently selected hybrid cells were infected at a multiplicity of infection (m.o.i.) of 0.05 with either jhm or mhva, maintained at either 32 or 39", and virus production determined after 24 hr. as shown in table 3 , the hybrid cells could replicate both jhm and mhv3 at both temperatures. the virus yields for the most part were similar to those obtained with the l2 tgrouar7-1 cell line as host and considerably higher than those obtained with the rn2 cell. accompanying these high levels of virus production was an extensive cytopathic effect (cpe) resulting from syncytial formation. by 24 hr at 39", essentially all the cells in the monolayer were involved and total destruction and cell lifting had occurred. a similar cpe was observed at 32", however, total destruction was delayed until 30 to 36 hr postinfection. these effects were apparent with the l2 tgrouar7-1, and hybrid lines 1, 2, 3, 4; 5, 7, and 8. hybrid 6 showed some differences. this hybrid, which produced lower yields of virus than the other hybrid lines at 24 hr (table 3) , showed very little, if any, cpe, at both temperatures with jhm virus. if a cpe was present it was restricted to less than 10% of the cells in the population. similar results were also obtained with mhv3 infections, although the cpe was somewhat more extensive, perhaps involving 20 to 30% of the cells in the population. the cpe observed with either virus in this line appeared to be restricted since longer incubation periods up to 7 days did not result in a more extensive cpe even though 30-80% of the cells scored as infectious centers. the extent of the cpe was also not affected by increasing the m.o.i. to 5 even though the number of cells scoring as infectious centers was 80%. initially hybrid 6 cells could produce either jhm or mhv3 virus at 39". however, after about 1 week in culture these infected cells lost the ability to shed virus at 39' even though they continued to produce virus in a cyclical manner at 32". at this time, if cells shedding virus at 32" were shifted to 39" there was a cessation of virus production. the properties of cyclical release of virus and restricted virus replication at the elevated temperature is reminiscent of the jhm infection of rn2 cells in which from 0.1 to 10% of the cells are infected (lucas et al, 19'77, 19'78) . in the case with hybrid 6 cells, however, both jhm and mhv3 were replicated and the number of infected cells was from 50 to 80%. ten subclones of the hybrid 6 cells behaved similarly when challenged with virus. to examine more closely the early phases of virus production, monolayers of either l2 tgrouar'7-1, rn2-2, hybrid 2, or hybrid 6 cells were infected with jhm virus at a m.o.i. of 0.05 at 32 and 39", and virus released into the medium determined at various times. virus production was similar for jhm infections of l2 tgrouar7-1 and hybrid 2 cells (fig. 4) . the cpe was evident at 6-8 and 3-6 hr postinfection for infections initiated at 32 and 39", respectively. virus production from hybrid 6 cells showed slightly different kinetics in that virus replication appeared to lag behind that of both the l2 tgrouar7-1 and hybrid 2 cells. this was clearly evident for the infection at 32" and less so for that at 39". although virus yields reached levels comparable to those from the l2 tgrouar7-1 and hybrid 2 infections, the cpe was never very extensive involving at the most approximately lo-20% of the monolayer. virus replication in the rn2-2 cells progressed at a much slower rate than in the other cell lines and a few small syncytia were apparent at 70 hr postinfection. similar results were obtained for infections with mhv3 at the same m.o.i. (data not shown), except that the cpe was somewhat more extensive in the hybrid 6 line involving 50-80% of the cells, and that mhv3 showed no evidence for replication in the rn2 cells. the availability of cell lines which respond differently when challenged with the coronaviruses jhm and mhv3 has provided the opportunity to examine which host cell functions dominate in the determination of the outcome of the virus infection. this was accomplished by the formation of somatic cell hybrids between the mouse l2 and rat rn2 cells. cells are authentic cell-cell hybrids since they contained both mouse and rat chromosomes and expressed both mouse and rat forms of lactate dehydrogenase and p-glucuronidase. the results indicate that the permissive or lytic state of coronavirus infection characteristic of the infected mouse l2 cells was dominant over the persistent or restrictive host state of the infected rat rn2 cells. this conclusion is based on the demonstration that the hybrids support the replication in a lytic manner of both jhm and mhvb at 32 and 39". the features of these infections are similar to those of l2 cells with a comparable time course, virus yields, and cytopathic effects. such features are not characteristic of the infections of rn2-2 cells (lucas et al, 1977 (lucas et al, , 1978 . it is conceivable, however, that one of the rat chromosomes codes for a dominantly acting factor which is responsible for virus resistance or persistence. since chromosome loss does occur in cell-cell hybrids (francke and francke, 1981) , perhaps this chromosome is frequently lost which would then lead to virus susceptibility in the hybrids. this seems unlikely, however, since although there was variability among the rat chromosomes present in the hybrid cells, there was no apparent preference for the loss of specific rat chromosomes. one hybrid, hybrid 6, and its subclones differed in the response to virus infection. these cells could initially replicate both virus strains at 32 and 39" without extensive syncytial formation. after about 1 week in culture such cells lost the ability to produce virus at 39" but continued to produce virus at 32". the reasons for the difference between this hybrid and the others is at present unclear. it may be related in some way to the chromosome content of this hybrid cell since its chromosome number is lower than that of the others (table 2) . once established these hybrids appear to be phenotypically stable. several of the lines have been kept in continuous culture for up to 8 months and periodically screened for susceptibility to lytic virus infection. to date, all lines after prolonged culture behaved similarly in their responses to infection as they did shortly after isolation. to my knowledge, this is the first study with cell hybrids formed between a cell line that is totally permissive for virus production and one that supports either a persistent infection or is restrictive to infection. the result that the totally permissive state (i.e., the l cell) is dominant over the restrictive or persistent state (i.e., the rn2 cell) is not unlike other host-virus systems where it has been demonstrated that permissive host functions are dominant over nonpermissive ones (miller et az., 1974; garver, et al, 1980; lemons et a& 1980) . mankovitz, ivation infected with mouse hepatitis and measles viruses lactate dehydrogenase isozymes: dissociation and recombination of subunits a method for analysis of chromosomes in hybrid ceils employing sequential g-banding and mouse specific c-banding human chromosome 19 carries a poliovirus receptor gene fusion resistance and decreased infectability as major host cell determinants of coronavirus persistence production of mammalian somatic cell hybrids by means of polythylene glycol treatment viwo and in vitro models of demyeiinating diseases. iii. jhm virus infection of rats. arch. new-01 two types of ribosomes in mouse-hamster hybrid cells stability of neurotropic mouse hepatitis virus (jhm strain) during chronic infection of neuroblastoma cells the biology and pathogenesis of coronaviruses pathogenesis of demyelination induced by a mouse hepatitis virus viral infections and demyeiinating diseases studies of interspecific (rat x mouse) somatic hybrids. ii. lactate dehydrogenase and j3-glucuronidase karyotyping r., thompson, l. h., whitmore, g. f., siminovitch, l., and till, j. e. (1974) . ouabain-resistant mutants of mouse and hamster cells in culture. cell 1,9-21. chasin, l. a., and urlaub, g. (1976) at present it is unclear as to the nature of the host cell functions that are involved in coronavirus persistence. recent evidence suggests that in one system the ability of the host cell to resist infection and withstand cytopathic effects may be important parameters (mizzen et a& 1983) . it is thus unclear as to the nature and role that the l2 functions play in overcoming the rn2-2 controlled persistent, and thermosensitive replication of jhm, and the restriction of mhvb. knowledge of these l-cell functions might prove useful in an understanding of both coronavirus persistence and restriction. it is of interest to note that although mhv3 replication is restricted in rn2 cells, if it is permitted to initiate replication as it does in hybrid 6 then after about 1 week in culture a persistent infection not unlike that of the jhm infection of rn2-2 cells can be obtained. since there are available several different cell lines that can become persistently infected with the jhm and mhv3 virus strains (lucas et al., 1978) , it will be of interest to determine whether these cell lines behave in a manner similar to the rn2 cells when somatic cell hybrids are formed with the mouse l2 cells. such studies are currently in progress. key: cord-272871-gu9ptt9y authors: white, k.andrew; brancroft, j. b.; mackie, george a. title: defective rnas of clover yellow mosaic virus encode nonstructural/coat protein fusion products date: 1991-08-31 journal: virology doi: 10.1016/0042-6822(91)90977-j sha: doc_id: 272871 cord_uid: gu9ptt9y abstract a small group of 1.2-kb rnas present on polyribosoes from clover yellow mosaic virus (cymv)-infected tissue contains sequences from the genomic rna (grna) of cymv and is encapsidated by cymv coat protein. some features of these rnas suggest that they are similar to defective interfering (di) rnas, and would be the first to be reported for the potexvirus group. the prototype 1.2-kb rna is 1172 nucleotides in length excluding a probable poly(a) tail and is composed of two noncontiguous regions corresponding to 757 nucleotides of the 5′ and 415 nucleotides of the 3′ terminal of cymv's grna. the sequence of the prototype 1.2-kb rna reveals that the two terminal grna regions present in this rna encode a single open reading frame (orf) joining the n-terminus of the 191-kda nonstructural product and the c-terminus of the coat protein to form a 35-kda 191-kda/coat protein fusion product. the coding properties of this prototype rna have been confirmed by translation in vitro of native and synthetic transcripts of the 1.2-kb rnas, both of which direct the synthesis of the anticipated 35-kda product which reacts with anti-cymv antiserum. three additional 1.2-kb rna species, each of which contains a unique junction site, have been characterized. in all cases, a fusion orf encoding a 191-kda/coat protein fusion product is encoded on the rna. the presence of a fusion orf in all members of the 1.2-kb rna species analyzed suggests that maintenance of this orf may be important for the survival of this class of rna within the plant. this coding strategy represents a novel property of plant virus defective rnas. small incomplete rnas which are derived from viral genomic rna (grna) are associated with several plant viruses (hillman et al., 1987; burgyan et al., 1989; li et a/., 1989) . these rnas of-ten interfere with viral replication and are incapable of independent replication. for these reasons they are referred to as defective interfering rnas (di rna& di rnas are distinguished from satellite rnas by their high degree of homology to the grna of the virus with which they are associated. in contrast, satellite rnas show little nucleotide sequence homology to the grna of the parent virus (francki, 1985) . although di rnas are commonly found in association with animal viruses, they appear to be much less prevalent in viral infections of plants. the first definitive di rna identified in plants was found associated with the cherry strain of tomato bushy stunt virus (tbsv; hillman et a/., 1987) . di rnas are also associated with cymbidium ringspot virus (cyrsv), another tombusvirus (burgyan et al., 1989; rubino et a/., 1990) . sequence analysis of these di rnas has revealed several stretches of sequence derived from different regions of the grna of the parent virus. di rna g associated with an isolate of the carmovirus turnip crinkle virus (tcv-b) is comprised primarily of two regions corresponding to the 5'-and 3'-terminal regions of the grna of the parent virus, and contains additional nonviral sequences at its 5' end (li et al., 1989) . the tcv-b di rna intensifies symptoms when present in infections, whereas di rnas of the tombus group attenuate them. coinfection of protoplasts with tbsv and di rnas results in reduced synthesis of grna relative to infections lacking the di rnas (jones et al., 1990) . this reduction of grna production may be responsible, at least in part, for symptom attenuation observed in whole plants (jones et a/., 1990) . the potexviruses represent a group of flexuous, filamentous plant viruses which contain a single strand of messenger-sense rna. clover yellow mosaic virus (cymv) possesses a 7015 nucleotide grna which is, to date, the largest rna sequenced from this group (sit et al., 1990) . a 191-kda protein which contains putative consensus sequences for ntpase-helicases as well as for rna polymerases (skryabin et al., 1988; argos, 1988) is encoded by the most 5' open reading frame (orf) in the viral rna. during infections, cymv produces at least two coterminal subgenomic rnas (sgrnas) with approximate lengths of 2.1 and 1 .o kb (bendena et al., 1987) encoding, respectively, a 25-kda protein of unknown function and coat protein (white and mackie, 1990) . a 1.2-kb rna containing cymv sequences was previously identified in polyribosomes extracted from infected plants, but was not extensively characterized (bendena et a/., 1987) . we report here that some of the properties of this rna species are consistent with those of di rnas. virions of cymv were purified from broad bean plants by using the method of bancroft eta/. (1979) and genomic and subgenomic rnas extracted as described by erickson and bancroft (1978) . polyribosomes were prepared from infected or uninfected plants following the method of palukaitis (1984) and the rnas were extracted from the polyribosomes as described by bendena et al. (1987) . rnas were separated on agarose gels using the conditions of mcmaster and carmichael (1977) . the transfer of rna to nylon and hybridization of the blot with probe was as described by mackie (1986) . primer extension experiments were carried out on 1.2-kb rna purified by sucrose gradient centrifugation from total polyribosomal rna or total rna extracted from virions. approximately 1 pmol of a 5' 32p-labeled oligonucleotide complementary to nucleotides 77 to 93) was mixed with either approximately 1 pg of purified 1.2-kb rna from polyribosomes or 0.1 pg of purified 1.2-kb rna from virions or 5 pg of total polyribosomal rna. annealing and reaction conditions have been described by white and mackie (1990) . sequence markers were produced using phosphorylated cy-2 primer annealed to 5 fig of cymv grna. conditions for this reaction are those of sit et al. (1990) . cloning and sequencing of the 1.2-kb rna cymv rna preparations used for cloning the 1.2-kb rnas were either polyribosomal rna from cymv-infected tissue or rna extracted from cymv virions. first-strand synthesis of cdna was primed with oligonucleotide cy-11 complementary to the 3' terminus of cymv rna (5'gagagtcgaclrl-ttatacacccaaaagtctacggg). approximately 100 pmol of this oligonucleotide was mixed with 3 pg of polyribosomal rna from infected tissue or 1 rg of rna purified from cymv virions in 13 ~1 of h,o. the mixture was heated at 90" for 2 min and then annealed sequentially at 45" for 10 min and at ambient tempera-ture for 10 min. extension of the primer was carried out at 50" for 40 min in a 25-~1 volume containing 50 m/l/l tris-hci, ph 8.3, 75 mm kci, 3 mm mgci,, 10 ml\/l dtt, 0.8 mm each of the four deoxyribonucleoside triphosphates, and 200 u of m-mlv reverse transcriptase (brl, inc.). the cdna produced was then used as template for pcr amplification in the following mixture: approximately 100 pmol of a second primer (cy-12; 5'gtgtgtcgactaatacgactcactatagaaaaca-aaacgaaaacaaac), amplification buffer to a final concentration of 1x (promega biotec., inc.), 2.5 u of taq dna polymerase (promega biotec., inc.), and h,o to a final volume of 100 ~1. a total of 30 cycles of amplification was carried out. the products of the reaction were analyzed by agarose gel electrophoresis and a product with a relative mobility of 1.2 kb was eluted from the gel, digested with sali, ligated into the vector psp64 (melton eta/., 1984) and transformed into escherichia co/i mvl 190 (bio-rad, inc.). dideoxy sequencing reactions were performed with t7 dna polymerase (pharmacia) according to the manufacturer's instructions. internal sequences of the cdna which could not be deduced using vector-specific primers were determined using oligonucleotide primers complementary to cymv sequences. in vitro translation ln vitro transcription was carried out with approximately 2 pg of linearized plasmid dna using t7 rna polymerase. subsequently, an aliquot of the unpurified transcription reaction was added directly to a rabbit reticulocyte lysate (promega biotec., inc.) containing [35s]methionine. the reaction was terminated by the addition of an equal volume of twice-concentrated sds sample buffer followed by boiling. in vitro translation was also carried out on 0.25 pg of cymv rna extracted from virions and on 0.25 pg of brome mosaic virus (bmv) rna. the products of translation were separated on an 1 1% polyacrylamide gel containing 0.1% sds (laemmli and favre, 1975) . lmmunoprecipitation of translation products with antisera directed against cymv particles was carried out as previously described (bendena et al., 1985) . (white and mackie, 1990) . both of these sgrnas are also encapsidated (white and mackie, 1990). we have examined more closely the 1.2-kb rna species which is present in viral infections produced by some stocks of cymv. rnas from polyribosomes isolated from infected or control tissue or rna extracted from virions were denatured, separated by electrophoresis, and transferred to nylon membranes. blots were incubated with a probe specific for the 3' portion of the cymv grna. all four cymv rnas, including the 1.2-kb rnas, can be detected in polyribosomal preparations from infected tissue (fig. 1, lane b) . furthermore, the 1.2-kb rnas can be detected in virions prepared from infected leaves indicating that they can be encapsidated (fig. 1 , lane e). virions isolated from cymv infections produced from viral stocks lacking the 1.2-kb rnas did not contain this rna species (fig. 1 , lane f). no additional rna species beyond those listed above have been detected in total rna preparations from infected leaves, regardless of the stock used for inoculation (data not shown). virions isolated from infected tissue which contained the 1.2-kb rnas were subjected to a series of three successive local lesion passages at low multiplic-ity in gomphrena globosa. four virus isolates of the third local-lesion passage were used separately as inocula for a series of 11 serial passages with sap (tissue ground 1:2 (w/v) in diluent) in the systemic host broad bean. the polyribosomal rna from the first systemic passage no longer contained 1.2-kb rnas (fig. 1 , lane c) nor did these rnas arise during subsequent passages (fig. 1, lane d) . therefore, under our conditions these rnas are not readily generated de nova using purified cymv as the initial inoculum and consequently are not obligatory sgrnas. to determine if the presence of the 1.2-kb rnas interfered with virus production, we examined virus yields from infected tissue in which these rnas were present or absent. when virions from equal amounts of tissue from each source were analyzed on sucrose gradients, both preparations were found to contain about the same quantity of nucleoprotein particles. however, the proportion of full-length virus particles in the preparation containing the 1.2-kb rnas was half that of preparations lacking it and was accompanied by an increase in smaller particles (data not shown). this suggests that the presence of the 1.2-kb rnas in viral infections results in a lower virus yield which may be due in part to the efficient encapsidation of the 1.2-kb rnas (fig. 1 , lane e). we could not detect any difference between the symptoms induced in broad bean plants by cymv infections produced from viral stocks lacking or containing the 1.2-kb rnas. the latter, therefore, do not appear to attenuate symptoms drastically in the host tested. in order to determine which regions of the cymv grna are present in the 1.2-kb rnas we analyzed cymv rnas present in polyribosomal preparations from cymv-infected broad bean leaves with a set of consecutive cdna probes spanning the cymv genome (fig. 2) . denatured polyribosomal rnas were separated electrophoretically and transferred to nylon membranes. figure 2 shows northern blots of polyribosomal rna extracted from cymv-infected tissue containing the 1.2-kb rnas. the 7.0-kb grna is identified by all probes (fig. 2 , lanes a, c, e, g, i, and k; probes 1 to 6), but the 1 .o-kb sgrna encoding coat protein hybridizes only to probes corresponding to the 3' region of the grna (fig. 2 , lanes i and k; probes 5 and 6). the 1.2-kb rnas hybridize efficiently only with probes representing the 5' and 3' extremities of the cymv genome (fig. 2 , lanes a, c, and k; probes 1, 2, and 6). probes corresponding to the central region of cymv grna do not hybridize with the 1.2-kb rnas (fig. 2 , lanes e, g, and i) although they do anneal to grna. the results of this analysis are summarized in the lower localization of cymv grna sequences present in the 1.2kb rnas. total polyribosomal rna (5 ag) isolated from healthy plants or from plants infected with a cymv stock containing 1.2-kb rnas was denatured, resolved by electrophoresis, and blotted to nylon. the blots were hybridized with a set of consecutive nicktranslated cdna probes (designated 1 through 6) corresponding to over 90% of cymv's grna. lanes a, c, e, g, i, and k contained identical samples of polyribosomal rna from infected plants hybridized with probes 1, 2, 3, 4, 5, and 6, respectively. lanes b, d, f. h, j, and i contained polyribosomal rna from uninfected plants hybridized with probes 1, 2, 3,4, 5, and 6, respectively. at the bottom are shown the general positions of the probes relative to grna. panel of fig. 2 . furthermore, oligonucleotides complementary to the 5' and 3' ends of cymv grna each hybridized efficiently to the 1.2-kb rnas under stringent conditions suggesting that these rnas do contain the extreme termini of the grna (data not shown). the 1.2-kb rnas, therefore, clearly constitute reasonably discrete entities containing the extremities of the cymv grna, but not internal regions. primer extension analysis of the 5'termini of the 1.2kb rnas was carried out to determine if their structure was similar to that of the grna. the major extension products generated from purified 1.2-kb rnas migrated to the same position as the major products generated when grna was extended with the same primer (fig. 3, compare lane 3 with lanes 4 and 5) .this demonstrates that the 1.2-kb rnas and the grna contain the same number of residues between the primer and their termini. primer extension also indicates that these rnas, similar to the cymv grna and sgrnas, behave as if they were capped. we believe, therefore, that the extreme 5'terminus of the 1.2-kb rnas is identical with that of the grna. cloning and sequencing of the 1.2-kb rnas we have cloned the 1.2-kb rnas of cymv by pcr amplification of cdna produced from cymv rna preparations which contain it. either total polyribosomal rna or total rna purified from virions was used as template for first-strand synthesis. cymv rnas were primed for reverse transcription with oligonucleotide cy-1 1 which is complementary to the first 23 residues upstream of the poly(a) tail of the cymv grna and which also contains a poly(deoxythymidylate) tract and a sali site 5' to the cymv specific region. a second primer, cy-12, containing a sequence identical with the 5'end of the cymv grna was used in conjunction with the first primer for the subsequent pcr reaction. the second primer also included a t7 rna polymerase promoter and a sali site 5'to the cymv sequence. pcr products were analyzed by electrophoresis in agarose gels. only preparations of cymv rna which contained the 1.2-kb rnas (as determined by northern blotting) yielded a product with electrophoretic mobility corresponding to approximately 1.2 kb. following digestion with sell, the 1.2-kb cdna pcr product was ligated into the sali site of the vector psp64. a, c, g, and t contained the products of a dideoxynucleotide sequencing reaction carried out on purified cymv grna using phosphorylated oligonucleotide cy-2 as primer (d, no dideoxynucleotide was added to the sequencing reaction). cap indicates sequencing products corresponding to the 7 mg cap present at the 5'terminus of cymv grna (sit era/., 1990). the partial sequence shown is the complement of the sequencing ladder. the sequence of the prototype 1.2-kb rna was determined by dideoxy nucleotide sequencing of paw1 obtained from polyribosomal rna (fig. 4) . the sequence of this rna is 1 172 nucleotides in length excluding a probable poly(a) tail. it contains 757 nucleotides from the 5'terminus of the cymv grna and 415 nucleotides from the 3' terminus. the predicted size correlates well with the size of authentic 1.2-kb rnas determined by electrophoresis. the sequence also confirms the analysis of the 1.2-kb rnas by northern blotting (fig. 2) which suggested that only the 5' and 3' extremities of the grna are present. twelve cdna clones, six derived from polyribosomes and six from cymv virions obtained from the same viral stock, were sequenced across the junction region (fig. 4, arrow) and all yielded junctions identical with that in fig. 4 . interestingly, this junction maintains the reading frame of the large 191 -kda nonstructural protein encoded at the.5' end-of cymv grna (orf 1) into the coat protein reading frame situated at the 3' end of the grna. the sfltklfarnpki cac"c"ccac""cc"cccacca"ca"""c"~c"~c"cwcgc"cg"~ccc~ga" 600 qtvlatmylptealygltst ccaaacag"g""agc"acga"gg"~"accca~gg~g~a~"a"a~gg~c"~~a"~"~" 660 ypnvyslsyhkpskfrrkal auauccaaacguguacucccucucauaccbuaracccuccgc"~" 720 fsyapgghkgaenwasqnyr c""c"c""a"gcaccagggggacaaaggagcugagc"gaaaac"ggg'cc"cccaaaac"ac~,a t 780 eadrfaafoffegvsssaal rgri\gcagacagg""cgcagca""cgac"uc""cg~ggag"c"ca"cc"ccgc"gccc" 840 sppgglirepspnermanet i\agccccccaggaggcc"i"ccgagaaccaagcccaaac 900 nkn"hlyqt*srgsnlatts "aaci\agai\cg"ccacc"~"ac~-~a~a"cccgag~ag~~"c""g~"ac~~cag 960 tvatkgaystnasnagflit "acgg"aggccaccaaaggagc""a~"~~c~m~gcg"~~~cg~"gg"""c~""a"~ac 1020 g p e l aggcccggag"ai\ccarccaccraccacucac"ca"a"""~"g~"~c~cgcccccgc~ 1060 cggcg"cccac"ggguwagwgc~cvwava"a"vc~g"a"ccc"-c""~"ca 1140 ggac"gcgagacccg"agacw""ggg"g"a"a" sequence predicts a 19 1 -kda/cp fusion protein of approximately 35 kda (fig. 4) which would contain 221 amino acids of the n-terminus of the 191-kda nonstructural protein and 91 amino acids of the c-terminus of coat protein. in an effort to determine whether the 1.2-kb rnas are internally homogeneous, we determined the sequences in their entirety of six other cloned 1.2-kb rnas obtained from a polyribosomal rna source different from that of the prototype. three junction sequences, each differing from that of the prototype, were found among the six cloned 1.2-kb rnas examined (fig. 5, cf. a with b, c and d) . as in the prototype, the juxtaposition of 5' and 3' sequences at the junction created an in-frame fusion between orf 1 and the coat protein orf in each case. the sequence of one of the additional 1.2-kb rnas revealed the presence of a 12-base direct repeat at the junction site (fig. 5,d) . the insertion, however, maintained the open reading frame into the coat protein sequence. the authenticity of the predicted open reading frames in the additional 1.2-kb rnas was verified by in vitro translation of synthetic transcripts from these cloned cdnas (data not shown). the majority of the sequence of the prototype 1.2-kb rna (fig. 4) and of additional 1.2-kb rnas is identical white, bancroft, and mackie with the corresponding region of cymv grna reported by sit et al. (1990) . there are five single-base alterations present in all 1.2-kb rnas examined, two of which map in the 3' end of the coat protein coding region. these changes result in a conversion of the amino acid sequence of the c-terminus of the coat protein from pyhrpe (sit et a/.,. 1990) to litgpe. the other three single-base changes occur in the 3' noncoding' region. these five single-base alterations detected in all 1.2-kb rnas were also present in the grna from our viral stock (data not shown). this indicates that the 3' ends of the 1.2-kb rnas are in fact faithful copies of the grna from which they were derived and that the sequence of the grna of our strain of cymv has diverged from that of sit et al. (1990) . our virus stock containing these differences is infectious and is efficiently replicated and encapsidated. thus, these alterations in the 3' noncoding region and in the extreme c-terminus of coat protein confer no detectable phenotype. the prototype 1.2-kb rna also contains two additional single-base substitutions at positions 193 (u-c) and 574 (c-+u) which would not alter the amino acid sequence of the polypeptide relative to that of orf 1 of cymv. these two single-base substitutions are present only in the prototype 1.2-kb rna and are not found in the grna of our stock. we have confirmed the potential coding properties of both native and in vitro transcripts of the prototype 1.2-kb rna by in vitro translation and immunological techniques. synthetic transcripts of the prototype 1.2kb rna were generated by runoff transcription of cloned cdnas using t7 rna polymerase. cloned cdnas paw1 (prototype) and paw2 used to generate the synthetic 1.2-kb transcripts were obtained from polyribosomal rna or from virions, respectively. the rna purified from virions used to clone paw2 was also the source of the native 1.2-kb rnas which were translated in this experiment. aliquots of the in vitro translation reactions carried out in a rabbit reticulocyte lysate were separated electrophoretically. the major products synthesized from synthetic transcripts of paw1 and paw2 display identical mobilities and an estimated molecular weight of 35 kda (fig. 6 , lanes d and e, respectively) as does an abundant polypeptide translated from preparations of cymv rna containing native 1.2-kb rnas (fig. 6, lane g) . templates lacking the 1.2-kb rnas were unable to direct the synthesis of the 35-kda product (fig. 6, lane f) . for comparison, the translation products synthesized from synthetic transcripts of a cloned cdna encoding coat protein (pbala6174; white and mackie, 1990) were separated in lane c of fig. 6 . the size of the product generated from this rna is consistent with its coding potential (white and mackie, 1990) . since the translation of the rna transcripts of paw1 and paw2 yields a product of the same relative molecular weight as that of native 1.2-kb rnas, this further supports the authenticity of the cloned 1.2-kb rna. furthermore, the size (35 kda) of these in vitro translation products is fully consistent with the predicted coding properties of the prototype 1.2-kb rna. to show that the 35-kda polypeptides produced by in vitro translation of the 1.2-kb rnas contained coat protein sequences, aliquots from the translation reac-tions were immunoprecipitated with an anti-cymv antiserum and separated by electrophoresis (fig. 6 , bottom). coat protein is efficiently immunoprecipitated by this serum (fig. 6, lanes j, m, and n) . the 35-kda product is also immunoprecipitated by this serum but less efficiently (fig. 6, lanes k, i, and n) . the lower affinity of the fusion protein for coat protein antibody may be due to the absence of n-terminal epitopes of coat protein on the 35-kda protein since the fusion protein is predicted to contain only 9 1 of 2 12 amino acids of coat protein. unlabeled coat protein is an effective competitor of precipitation of the 35-kda product indicating that it is coat protein-specific antibodies which are reacting with this polypeptide (fig. 6, lane 0) . non-immune serum is unable to immunoprecipitate the 35-kda protein (fig. 6, lane p) . bands corresponding to approximately 30 kda are visible in fig. 6 , lanes m and n. these represent a previously described coat protein readthrough product whose initiating codon lies 135 nucleotides upstream from the coat protein orf (bendena et a/., 1987; sit et al., 1990; white and mackie, 1990 ). we have used several approaches to characterize a group of 1.2-kb rnas found in some cymv infections of broad bean plants. the structure of these rnas suggest that they represent di-like rnas and establish them as the first such rnas to be found associated with a member of the potexvirus group. a number of features of the 1.2-kb rnas of cymv distinguish them from previously characterized plant virus di rnas. first, the presence of the 1.2-kb rnas in cymv infections of broad bean plants produces no apparent alteration of symptoms whereas the di rnas associated with several other plant viruses can enhance or attenuate symptoms (hillman et al., 1987; burgyan et a/., 1989; li et a/., 1989) . since the mechanism of symptom development is not understood, this is not necessarily problematic. knorr et a/. (1991) found that the appearance of some classes of tbsv di rnas is not immediately accompanied by symptom attenuation, suggesting that different species of di rnas may differ in their ability to interfere with symptoms. the 1.2-kb rnas of cymv may, therefore, represent an example of the inability of a defective rna to attenuate virus-induced plant pathogenesis. a second distinguishing feature of the 1.2-kb rnas is that they are derived entirely from the 5'-and 3'-terminal regions of the cymv grna and encode two partial viral orfs which are fused in-frame to create a single orf (fig. 7) . this fused orf is present in all four 1.2-kb rna species characterized, despite the fact that each rnas. moreover, these rnas are likely translated in vivo since they are present in polyribosomes. the poor reactivity of the fusion protein with coat protein antibody, however, deterred any attempt to identify this protein in extracts from infected tissue. interestingly, dlsse rna associated with mouse hepatitis virus exhibits a structure and coding strategy similar to the 1.2-kb rnas of cymv (makino er al., 1988) . in this case, the di-encoded products have been detected in infected cells. a third feature of the 1.2-kb rnas of cymv is that their coding capacity is much larger than that observed for other characterized plant virus di rnas. about 80% of the cymv prototype 1.2-kb rnaforms a continuous coding sequence (fig. 7) . since all four sequenced members of the 1.2-kb rna family encode a 19 1 -kda/ coat protein fusion protein, maintenance of this fusion orf may be advantageous, if not essential, for these rnas. one possibility is that translation of the 1.2-kb rnas may stabilize them. the influence of translation on the stability of a plant mrna has been described by vancanneyt et al. (1990) who found that reducing the size of the orf on the patatin mrna led to a decrease in its steady-state level. alternatively, the fused product may contain an rna binding domain and could accordingly play a direct role in the replication or survival of the 1.2 kb rnas. the 1.2-kb rnas of cymv are clearly defective as they lack up to 5.8 kb (approx 80%) of the cymv genome. consequently, three entire orfs and most of the "replicase" and coat protein coding regions are deleted (fig. 7) . this would abolish the ability of these rnas to replicate autonomously. indeed, synthetic transcripts of the prototype 1.2-kb rna replicate only when coinoculated with cymv grna (white, bancroft, and mackie, unpublished results). we cannot identify the initial events which led to the contamination of some of our stocks of cymv with defective 1.2-kb rnas. our attempts to regenerate these rnas from purified virus by serial passages from sap were not successful even though a similar approach ultimately succeeded for tbsv (morris et a/., 1988; knorr et a/., 1991). while we cannot rule out the possibility that the conditions under which our passages were performed were suboptimal for generation of this type of rna, it seems more likely that the successful formation of defective cymv rnas may be a rare event. we believe that to a large extent the initial deletion generating a defective rna may be random. subsequent propagation of the cymv defective rna seems to require an exact in-frame fusion of two protein coding sequences within limited regions of the genome, maintenance of encapsidation signals, and conservation of terminal nucleotide sequences which we presume to be necessary for rna replication. a sequence motif in many polymerases the assembly of clover yellow mosaic virus and its protein synthesis in vitro of the coat protein of papaya mosaic virus molecular cloning of clover yellow mosaic virus rna: identification of coat protein coding sequences in viva and in vitro adefective interfering rna molecule in cymbidium ringspot virus infections the self-assembly of papaya mosaic virus plant virus satellites key: cord-272925-xag1yaie authors: carter, gemma c.; bernstone, laura; sangani, dhaval; bee, jessica wynter; harder, thomas; james, william title: hiv entry in macrophages is dependent on intact lipid rafts date: 2009-03-30 journal: virology doi: 10.1016/j.virol.2008.12.031 sha: doc_id: 272925 cord_uid: xag1yaie macrophages are an important natural target cell for hiv-1, but previous studies of virus entry into these cells are limited, and the involvement of membrane cholesterol and lipid rafts is unknown. cholesterol disruption of macrophage membranes using four pharmacological agents acting by different mechanisms: methyl-β cyclodextrin, nystatin, filipin complex and lovastatin, all significantly inhibited productive hiv entry and reverse transcription. the inhibitory effects of these drugs resulted in decreased virus release from infected cells, and could be substantially reversed by the addition of water-soluble cholesterol. the virus bound equally to cholesterol-disrupted cells even though hiv receptor expression levels were significantly reduced. macrophage cd4 and ccr5 were found to partition with the detergent-resistant membranes with a typical raft-associating protein flotillin-1. hiv particles were observed co-localising with a marker of lipid rafts (ctb-fitc) early post infection. these data suggest that macrophage membrane cholesterol is essential for hiv entry, and implicate lipid raft involvement. virus entry into target cells is a complex process involving many cellular and viral proteins with the primary goal of overcoming the barrier of the cellular membrane. some viruses achieve this by acquiring a lipid envelope during exit from the host cell; these enveloped viruses enter cells by fusion of their envelope with the cellular membrane at either the cell surface or within an endosome. viruses without a lipid envelope have acquired mechanisms to penetrate the membrane barrier, entering cells mainly via an endocytic pathway using either clathrin-coated vesicles or caveolae, or by the formation of a pore at the cell surface as has been proposed for poliovirus (hogle, 2002; marsh and helenius, 2006; smith and helenius, 2004) . there is a growing amount of evidence that cholesterol, an important structural component that modulates the fluidity of biological membranes, is essential for the uptake of many viruses. successful virus entry may require cholesterol in the host cell membrane or in the viral envelope. many enveloped viruses, such as vaccinia virus (chung et al., 2005) , herpes simplex virus (bender et al., 2003) and severe acute respiratory syndrome-coronavirus (li et al., 2007) require cholesterol in the target cell membrane. the same is true for many non-enveloped viruses; sv40 (anderson et al., 1996) , foot-and-mouse disease virus and echovirus (marjomaki et al., 2002; pietiainen et al., 2004) require cholesterol to enter cells by the lipid raft-dependent caveolae endocytosis pathway. in the case of influenza virus, the presence of cholesterol in its viral envelope is critical, but it is not essential in the target cell (sun and whittaker, 2003) , whereas the converse has been found for murine leukaemia virus (lu and silver, 2000) and duck hepatitis b virus (funk et al., 2008) . in contrast, various strains of dengue virus and yellow fever virus enter and infect cells independently of cholesterol (umashankar et al., 2008) . cholesterol is concentrated within the fluid mosaic bilayer of the plasma membrane, along with sphingolipids and glycerophospholipids, in specialised dynamic microdomains known as lipid rafts. these lipid assemblies are tightly packaged and ordered, allowing them to float within the rest of the fluid sea of disordered lipid bilayer (simons and ikonen, 1997) . the presence of cholesterol in the lipid rafts confers some resistance to detergents at low temperatures, allowing their separation from detergent-sensitive membranes by ultracentrifugation (wilflingseder and stoiber, 2007) . due to this property, they are often referred to as detergent-resistant membranes (drms). membrane rafts act as mobile platforms within the plasma membrane, and are involved in many important diverse cellular processes. many proteins associate with lipid rafts, and these include gpi-anchored proteins, gtpases and kinases. mounting evidence for the cholesterol dependency of productive virus entry suggests that many viruses may hijack these dynamic lipid raft platforms to utilise them as an entry portal to the cell. cholesterol is already known to be important at multiple stages of the hiv lifecycle. the virus assembles and buds out of lipid raft domains, and in doing so acquires a cholesterol and sphingomyelinrich envelope (aloia et al., 1988 (aloia et al., , 1993 campbell et al., 2004 ) that is resistant to detergents but has a strikingly different composition to host cell membranes chan et al., 2008) . the hiv accessory protein, nef, functions on many levels to manipulate cellular cholesterol, including binding to cholesterol to enhance its trafficking virology 386 (2009) to rafts (zheng et al., 2003) , increasing cellular cholesterol biosynthesis (van 't wout et al., 2005) and inhibiting cholesterol efflux by redistributing a major component of the cholesterol efflux pathway castrillo et al., 2003) . although in contrast, other studies have found that nef specifically enhances the incorporation of sphingomyelin but not cholesterol into budding viral particles (brugger et al., 2007) . the importance of hiv envelope lipid composition is highlighted by the finding that depletion of viral envelope cholesterol causes a dramatic reduction in viral infectivity campbell et al., 2004 campbell et al., , 2002 guyader et al., 2002; liao et al., 2003) . the early events in hiv entry, in which the viral envelope protein, gp120-gp 41, engages with the primary receptor cd4 on target cells, are well characterised. this interaction permits conformational changes within gp120 that allow additional binding to a co-receptor, usually cxcr4 for t-lymphocyte tropic strains, or ccr5 for macrophage tropic isolates. co-receptor binding is the trigger for virus entry, during which the fusion peptide gp41, inserts into the cellular membrane to drive the fusion event. the lipid composition of the host cell is important for hiv entry. cholesterol depletion of target cells using pharmacological agents prevents hiv entry, and render cells resistant to infection (kozak et al., 2002; liao et al., 2001; nguyen and taub, 2002b; popik et al., 2002) . the association of cd4 with lipid raft domains is well accepted (kozak et al., 2002; nguyen et al., 2005; percherancier et al., 2003; popik et al., 2002) , but whether this association is required to support productive virus entry is disputed. one study found that mutant cd4 targeted to non-raft membranes did not permit efficient hiv entry (del real et al., 2002) , whereas alternative cd4 mutants that also prevent cd4 association with lipid rafts were found to support hiv entry (percherancier et al., 2003; popik and alce, 2004) . the membrane localisation of the co-receptor molecules is somewhat controversial. ccr5 has been shown to associate with lipid rafts microdomains in cell lines by its presence in drm fractions and its co-localisation with the raft associated lipid gm1 (manes et al., 1999; popik et al., 2002) . in contrast, studies in primary t cells identified ccr5 in the detergent-soluble membrane (dsm) fractions representing the non-raft membranes (percherancier et al., 2003) . cxcr4 has been reported to be lipid raft-associated in 293 cells (manes et al., 2000) but non-raft-associated in t cell lines (kozak et al., 2002; popik et al., 2002) . hiv-1 gp120 binding to cd4 molecules in lipid rafts has been proposed to cause recruitment of the hiv coreceptor into the lipid rafts or its interface, thus bringing all molecules required for hiv entry together (kozak et al., 2002; nguyen et al., 2005; popik et al., 2002) . a different productive entry pathway for hiv into macrophages has been described, in which virus is taken up by an endocytic route involving macropinocytosis, a constitutive process whereby the cell takes up large quantities of the extracellular fluid (marechal et al., 2001) . numerous intracellular pathogens are taken up into macrophages by macropinocytosis, including brucella (watarai et al., 2002) and afipia felis (schneider et al., 2007) , and in these cases a requirement for lipid rafts has been established. cholesterol is already known to play an important role in hiv replication of macrophages, with specific studies showing that nef affects the normal function of atp-binding cassette transporter a1 to impair cholesterol efflux from infected macrophages (mujawar et al., 2006) . modulation of intracellular lipid metabolism in hiv-infected macrophages may facilitate virus budding from lipid raft associated membranes, but it is tempting to infer that cholesterol might also play an important role in the entry of hiv into macrophages. all of the studies regarding the involvement of cholesterol and lipid rafts in hiv entry have been performed using immortalised cell lines, t cell lines and primary cd4+ lymphocytes. given their critical position in hiv pathogenesis, and their highly specialized cellular architecture, we thought it necessary to investigate the requirement of cholesterol and role of lipid rafts in hiv-1 entry into macrophages. we have assessed the effect of depleting cholesterol in the macrophage plasma membrane, using four different pharmacological agents, on virus binding, entry and hiv receptor levels. here we show that membrane cholesterol is essential for productive hiv entry into macrophages. virus binding to cholesterol-depleted macrophages is not reduced but there are alterations to the surface expression of the receptor molecules. we observed virus co-localising with a marker of lipid rafts early after infection, and isolated macrophage cd4 and ccr5 in the membrane fractions representing raft microdomains. this, together with the sensitivity of virus entry to membrane cholesterol depletion, implies a role for macrophage lipid rafts in hiv-1 entry. to investigate the involvement of lipid rafts in hiv uptake into macrophages, we first sought to visualise virus entry and lipid rafts using fluorescent cholera toxin b subunit (ctb-fitc) the binding of which to gm1 clusters these membrane domains together into larger patches that can be observed microscopically. hiv nl4.3 pseudotyped with jrfl envelope was bound to macrophages on ice, and warmed to 37°c in the presence of ctb-fitc and analysed at various times post entry. a representative experiment taken at 20 min post entry is shown in figs. 1a-d, and shows virus co-localising with ctb-positive areas of the macrophage plasma membrane. the number of particles directly associated with gm1-positive membranes on the cell surface or in endocytic vesicles was quantified by taking 0.8 μm z-slices through the cell and scoring the percentage of the total particles colocalising with ctb-fitc. three consecutive z-slice focal planes are shown in panels a-c. these slices start towards the bottom of the cell (a) and proceed upwards, and show many cell-associated virus particles clearly co-localising with ctb-fitc. the distribution of all virus particles and ctb-fitc is shown in a projection of z-stack slices in panel d. analysis of multiple cells (n = 10) at 20 min post entry shows approximately 34% (±3.72 sem) of total particles are colocalising with ctb-fitc. at later times post entry (40 and 60 min), there is a reduction in association of virus with ctb-fitc perhaps due to productive entry of most virus particles into the cytoplasm or degradation of the virus preventing its detection (fig. 1e ). membrane cholesterol can be disrupted by a number of pharmacological agents; the manipulation of this key component of lipid microdomains is frequently used to implicate these assemblies in virus entry. the effects of methyl-β cyclodextrin (mβcd) are well characterised. this cholesterol-binding agent is not incorporated into the membrane but selectively extracts membrane cholesterol by binding it in a central non-polar cavity (ilangumaran and hoessli, 1998) . we incubated macrophages with various concentrations of mβcd in serum-free media, and measured the depletion of lipid rafts by detecting fluorescent ctb-fitc binding to the raft glycolipid gm1 by flow cytometry. ctb-fitc binding to drug-treated cells on ice was reduced in a dose-dependent manner compared to untreated cells confirming that mβcd effectively disrupts macrophage lipid rafts ( fig. 2a) . the effect of this drug on cell viability was measured using the mts cytotoxicity assay (fig. 2b ) and showed that 1 h treatment with mβcd had no toxic effect on the macrophages. quantification of the amount of free cholesterol and cholesteryl esters in 10 mm mβcdtreated macrophages using amplex red cholesterol quantification assay revealed a significant decrease (p = 0.01 by paired t-test) in all 6 donors tested with an average of 28.7% (±20.4%) reduction compared to untreated control cells. the potential anti-viral properties of mβcd on hiv entry into macrophages was tested by measuring virus reverse transcription in drug-treated macrophages. the hiv envelope acquires cholesterol during budding, and mβcd can deplete this virion cholesterol, resulting in reduced virus infectivity (guyader et al., 2002; liao et al., 2003) . therefore, to avoid this potentially confounding effect, cells were pre-treated with mβcd for 1 h, and washed extensively before the addition of virus, to ensure that any anti-viral effects observed were not due to decreased particle infectivity. mβcd treatment of macrophages was found to significantly reduce the generation of reverse transcription products following challenge with hiv bal, at both concentrations tested, in macrophages derived from six different donors (fig. 2c) . the inhibitory effect of mβcd could be partially reversed by the replenishment of cellular cholesterol with watersoluble cholesterol added for 1 h directly after mβcd treatment. cholesterol replenishment restored virus reverse transcription to 43.5% of the untreated control (fig. 2d ). the inhibitory effects of mβcd were also observed when p24 release from infected cells was measured by elisa (fig. 2e ). reduced p24 release kinetics were observed in the early days post infection for all donors treated with mβcd, but the effects were less pronounced due to the longer length of this experiment and the measurement of multiple rounds of infection. restoration of p24 release kinetics was observed upon cholesterol replenishment. therefore, depletion of macrophage membrane cholesterol with mβcd severely inhibits the early steps of productive hiv infection of macrophages. to further pinpoint the stage at which cholesterol-containing lipid rafts are required in hiv infection, we determined the effect of cholesterol depletion on virus binding to macrophages. equal amounts of hiv bal were added to untreated and mβcd-treated macrophages for 2 h on ice. unbound virions were removed by extensive washing before cells were lysed and the amount of cellassociated virus determined by p24 elisa (fig. 2f) . a slight decrease in virus adsorption to mβcd-treated macrophages compared to untreated control cells was observed. however, this decrease was not significant and would not account for the 8-fold decrease observed in reverse transcription in mβcd-treated cells. therefore, cholesterol and lipid rafts might play a minor role in virus binding but they are more likely to be required for entry or post-entry events. decreased entry of hiv might be attributed to alterations in receptor or co-receptor levels; therefore we sought to determine if mβcd treatment of macrophages alters the levels of cd4, ccr5 and cxcr4. detection of these cell surface molecules using antibody labelling followed by flow cytometry shows that control macrophages express cd4, ccr5 and cxcr4 at detectable levels ( fig. 3a) . treatment of macrophages with mβcd significantly decreases the cell surface expression of all receptors; cd4 by 35%, ccr5 by 100% and cxcr4 by 59%. both ccr5 and cxcr4 expression levels are restored upon cholesterol replenishment, implying that the decrease is due solely to cholesterol depletion. interestingly, replenishment with cholesterol does not restore the cd4 expression levels, but reduces them further to almost undetectable levels. this further reduction in cd4 expression may explain why cholesterol replenishment only partially restores hiv reverse transcription (see above). mβcd treatment of macrophages does not reduce the expression of all membrane proteins because the expression of cd71 (transferrin receptor), a known non-raft associated protein, was not reduced. therefore, mβcd selectively alters the exposure of proteins found in the cholesterolrich lipid raft membranes without effecting non-raft associated proteins. the combined decrease in cd4 and ccr5 receptor levels may account for the decrease in productive hiv entry. the reduction in hiv receptor levels in cholesterol-depleted macrophages and the co-localisation of virus with a marker of lipid rafts may imply that the receptors are localised in these microdomains. traditional methods to isolate lipid rafts rely on cholesterol's property of favouring the formation of membranes that are resistant to detergent at low temperature, and can be separated from soluble membranes by ultracentrifugation. we adapted a procedure for isolating macrophage drms by disrupting cells by a combination of nitrogen cavitation and homogenisation followed by fractionation using an optiprep density gradient. western blot analysis of equal volumes of each fraction shows the expected localisation of the control proteins: flotillin-1 is located in fractions 4 and 5 representing the drms, and transferrin receptor is in fractions 8 and 9 indicative of the dsms (fig. 3b) . the receptor cd4 is present in the detergent-resistant fractions 4 and 5, and has a similar distribution to that of flotillin-1, although it is also present at low levels in fractions 6 and 7. ccr5 is present in detergent-resistant fractions 4-5 only. therefore, the receptors required for macrophage-tropic hiv entry reside in the drm fractions of macrophage membranes, which have properties of lipid rafts and contain raft-associated protein flotillin-1. macrophages for 2 h on ice was measured by determining the amount of cell-associated virus using p24 elisa. data represent mean ± sd of results obtained with multiple independent experiments using several donors (4 donors for ctb, viability and hiv binding assays, 4-6 donors for qpcr and p24 data is representative of 3 donors). ⁎⁎ very significant difference, p b 0.01 and ⁎⁎⁎ extremely significant difference p b 0.001 (paired t-test). to exclude any drug-specific effects of mβcd, we used two other compounds that can bind to cholesterol and disrupt lipid rafts by directly inserting into membranes and sequestering cholesterol into complexes. both nystatin and filipin complex, at concentrations significantly inhibiting ctb-fitc binding (fig. 4a) and not compromising cell viability (fig. 4b) , inhibited hiv infection in a dosedependent manner. nystatin at concentrations of 66 and 50 μg/ml significantly reduced reverse transcription in infected cells by 8.4-and 4.1-fold respectively compared to those treated with the same concentration of dmso vehicle (fig. 4c ). filipin complex significantly inhibited the number of hiv reverse transcripts by 3.2-fold compared to the untreated control (fig. 4d) . similarly, these inhibitory effects can be observed as reduced p24 release kinetics at early time points post infection (fig. 4e) . both nystatin and filipin complex do not reduce virus binding to macrophages (fig. 4f) . this supports the findings obtained by mβcd treatment, implying that cholesterol and lipid rafts are likely to be required for entry and/or post-entry events and not binding. studies with mβcd indicate that the hiv receptors are sensitive to cholesterol depletion so we sought to determine if receptor levels would also be altered when cholesterol organisation is disrupted by another mechanism (fig. 4g) . nystatin significantly decreased cd4 and ccr5 expression by 59% and 91%, respectively. the expression level of cxcr4 was only slightly decreased. however, nystatin did significantly reduce levels of cd71 by 43% indicating that this drug might reduce the cell surface expression of many proteins including those outside lipid rafts. conversely, filipin complex marginally decreased cd4 and ccr5 but had no effect on the levels of cxcr4 or cd71. the anti-viral effects of nystatin might be explained by the lower levels of cd4 and ccr5 but it is likely that filipin complex might inhibit infection by a different mechanism than decreased receptor levels. the cholesterol-binding drugs, mβcd, nystatin and filipin, mechanically disrupt lipid rafts either by actively removing or by sequestering cholesterol. lipid rafts can also be disrupted using statins, such as lovastatin. these drugs inhibit the 3-hydroxy-3methylglutaryl coenzyme a (hmg-coa) reductase enzyme responsible for the production of mevalonate, a precursor in cholesterol biosynthesis. cells were treated with lovastatin for 4 days in the presence of serum-free medium supplemented with lipoproteindeficient serum after which cell viability was unaffected (fig. 5a) . the binding of ctb-fitc to lovastatin-treated cells was reduced by 33% (± 11.1%) compared to the untreated cells (fig. 5b) . lovastatin treatment of macrophages very significantly inhibited hiv reverse transcription in a dose-dependent manner by 16.6-and 5-fold with 12.5 μm and 5 μm lovastatin, respectively (fig. 5c) . a marked decrease the surface expression of hiv receptors after nystatin or filipin complex treatment was determined by staining macrophages with fluorescent antibodies to cd4, ccr5, cxcr4, cd71 or an appropriate isotype control. cell surface binding was measured by flow cytometry, and mean fluorescence intensity values were calculated by subtracting the isotype control fluorescence from the specific antibody fluorescence. data represent mean ± sd of results obtained with multiple independent experiments (3 donors for qpcr, ctb and p24 binding assays, 4 donors for receptor expression, viability and p24 data is representative of at least 2 donors). ⁎ significant difference p b 0.05, ⁎⁎ very significant difference, p b 0.01 (paired t-test). in p24 release kinetics compared to untreated control was also observed (fig. 5d) . the effects of lovastatin on hiv reverse transcription were partially reversed by the addition of mevalonate, the product of hmg-coa reductase, to the culture medium (fig. 5e ). the surface expression levels of all hiv receptors (but not cd71) were decreased after lovastatin treatment but not to significant levels (fig. 5f ). although these effects consistently implicate normal cholesterol synthesis for productive virus entry and receptor expression levels, we were unable to detect significant changes in cholesterol abundance per se in lovastatin-treated cells using the standard assay. here we have demonstrated the critical importance of cholesterol for entry of hiv-1 into macrophages. firstly, we showed that productive virus entry is significantly reduced when the macrophage target cells are treated with mβcd, a cholesterol-depleting drug. by incubating cells with this drug before the addition of virus, only cholesterol from the target cells would be removed allowing virus particle cholesterol and infectivity to remain intact. the addition of exogenous cholesterol to macrophage membranes directly after macrophages were pre-treated with 12.5 μm lovastatin and/or 800 μg/ml mevalonate (whose production is blocked by lovastatin) for 4 days. productive hiv entry was measured by qpcr detection of hiv bal reverse transcription after 30 h of infection. (f) the surface expression of hiv receptors after lovastatin treatment was determined by staining macrophages with fluorescent antibodies to cd4, ccr5, cxcr4, cd71 or an appropriate isotype control. data represent mean ± sd of results obtained with multiple independent experiments (2 donors for ctb and viability assays, 2-5 donors for qpcr, 5-6 donors for receptor expression and p24 data are representative for 2 donors). ⁎⁎⁎ extremely significant difference p b 0.001 (paired t-test). mβcd treatment substantially restored hiv infection, indicating that the decreased infectivity was at least in part due to the depletion of cholesterol from the cell membrane. secondly, we modified the properties of cholesterol-rich macrophage membranes using nystatin and filipin complex to sequester cholesterol into large aggregates. treatment of macrophages with these drugs significantly inhibited productive hiv entry in a concentration-dependent manner. thirdly, depletion of macrophage cholesterol using lovastatin, a compound that inhibits hmg-coa reductase, the rate limiting enzyme in cholesterol biosynthesis, significantly inhibited hiv productive entry after a prolonged 4 day treatment. incubation of macrophages with mevalonate, whose production is prevented by lovastatin, partially restored productive hiv entry into macrophages. therefore, modification of macrophage membrane cholesterol content using 4 different pharmacological inhibitors acting on cholesterol by different mechanisms all significantly inhibited hiv entry. cholesterol is a major component of lipid raft microdomains and manipulation of its localisation or presence within membranes is frequently used to implicate these lipid assemblies in virus entry. here we provide additional evidence for the involvement of macrophage lipid raft microdomains in hiv-1 entry by observing virus colocalisation with a marker (ctb-fitc) of these microdomains at an early time post-infection. furthermore, we have isolated the receptor molecules cd4 and ccr5, the receptors typically utilised by macrophage-tropic viruses, in drm fractions partitioning with a known-raft associated protein flotillin-1. these results confirm observations regarding the importance of target cell plasma membrane cholesterol and lipid raft microdomains in virus entry into t cell lines and primary cd4+ t cells (liao et al., 2001; manes et al., 2000; percherancier et al., 2003; popik et al., 2002) and extend them to macrophages, a critical natural target cell for hiv-1. for viruses to infect target cells, they have to firstly bind to attachment factors or receptor molecules on the cell surface. a decrease in productive virus entry may reflect a reduction in virus binding to cells. we confirmed which stage of infection was being affected by cholesterol depletion by investigating virus binding to the macrophage membranes. no significant decrease in the amount of bound virus after a 2 h incubation on ice was observed between drugtreated and control untreated macrophages. this indicates that cholesterol depletion of macrophages does not perturb the attachment of virus, perhaps to alternative molecules such as heparan sulphate glycosaminoglycans (saphire et al., 2001) , but must alter virus entry events after attachment. the surface expression levels of the hiv receptors were found to be altered to differing extents by the 4 pharmacological agents. mβcd treatment had the most pronounced effect on the receptors, with cd4 and cxcr4 being significantly reduced and ccr5 surface levels disappearing to almost undetectable levels. cholesterol replenishment restored the levels of ccr5 and cxcr4 to similar levels as the untreated macrophages. interestingly, cd4 expression was decreased further by cholesterol replenishment to almost undetectable levels. this complete knockdown in cd4 surface expression may explain why only a partial restoration of hiv reverse transcription was observed upon cholesterol replenishment of mβcd treatment macrophages. the addition of cholesterol may reduce cd4 expression to a level that is not favourable for hiv infection. the mechanism by which cholesterol further decreases cd4 surface expression is unknown but as these cholesterol-treated macrophages have a more granular appearance by flow cytometry, we can speculate that these macrophages actively take up cholesterol via an endocytic mechanism that may simultaneously internalise cd4. it is not uncommon for cholesterol replenishment to give rise to only partial restoration of infection; it has been reported for other viruses with the suggested explanation being that only one form of cholesterol may have been restored (tang et al., 2008) . the reduction in surface expression of hiv receptors following depletion of membrane cholesterol contrasts with findings using different cells. treatment with similar concentrations of mβcd caused no reduction in plasma membrane expression of cd4 on primary tcells (percherancier et al., 2003) , pbls (viard et al., 2002) or pm1 t cell line . the reduction of cd4 surface expression upon mβcd and cholesterol treatment, to the best of our knowledge, seems to be unique to macrophages. this observation may result from the differences in cd4 expression and internalisation on macrophages compared to t lymphocytes. in macrophages, cd4 expression levels are 10 to 20-fold lower than they are in cd4+ t cells (collman et al., 1990; kazazi et al., 1989; lee et al., 1999) , and cd4 internalisation rates are enhanced, likely due to the absence of lck (pelchen-matthews et al., 1998) . reduction in cxcr4 and ccr5 surface expression has been reported after treatment with a cyclodextrin derivative bcd in pm1 and primary t cells (liao et al., 2001) . more importantly, cholesterol has been shown to be essential for the conformational integrity of ccr5 and cxcr4, and in these studies bcd treatment prevented the cell surface binding of antibodies specific for distinct ccr5 epitopes taub, 2002a, 2002b) . molecular simulations, and work with model membranes of defined composition show that the integrity of lipid raft microdomains is very sensitive to minor changes in overall cholesterol concentrations (risselada and marrink, 2008) . most likely, cholesterol depletion in macrophages disrupts lipid rafts, thereby redistributing raft-associated proteins such as ccr5 to other membrane domains. it is possible that, in this new context, their conformation is altered to one that is no longer recognised by antibodies, and unable to sustain hiv entry. nystatin and filipin complex modify cholesterol organisation within cells, but do not affect overall cholesterol concentration, and have a differing effect on hiv receptor levels. nystatin significantly decreases cd4 and ccr5 surface expression but also significantly decreased non-raft associated cd71, implying that effect of nystatin may not be restricted to proteins located in cholesterol rich domains. conversely, filipin complex treatment significantly reduced cd4 expression levels and slightly decreased ccr5 levels but did not appear to have such a dramatic effect on the expression of other membrane proteins. the effects of cholesterol sequesteration on the surface expression of the hiv receptors, after nystatin and filipin treatment, have not been investigated before. disruption of cholesterol biosynthesis with lovastatin also reduced the surface expression of the hiv receptors on macrophages but not by significant levels. this is in contrast to observations with cd4 t lymphocytes, in which lovastatin down-modulated the mrna and cell surface protein expression of ccr5 (but not cd4 or cxcr4), resulting in reduced hiv-1 infection (nabatov et al., 2007) . the sensitivity of virus entry to depletion of cellular cholesterol could be explained by the association of the viral receptors, cd4 and ccr5, with lipid rafts. the tightly structured cholesterol-rich lipid rafts, known as drms, can be isolated in the presence of ice cold 1% triton x-100 and separated by ultracentrifugation. macrophage cd4 and ccr5 were found in the drm fractions along with flotillin-1 whose location in these membranes is well known. in contrast, cd71 was found in the soluble fractions reflecting its association with the non-raft membranes. decreased hiv receptor, but not cd71, expression after mβcd treatment supports the notion that the hiv receptors are located in cholesterol-rich domains in macrophages. we provide additional evidence for the involvement of lipid rafts in virus entry by visualising virus uptake in parallel with a fluorescent marker of lipid rafts. at 20 min post infection, approximately a third of all cellassociated virions were co-localised with lipid raft membranes in punctate structures that appear to have been internalised and may resemble an endosomal compartment. cd4 association with drms has been established in many different cell types and we can show this to be true for macrophage cd4 (kozak et al., 2002; liao et al., 2001; nguyen et al., 2005; percherancier et al., 2003; popik et al., 2002) . ccr5 has been reported to be present in lipid rafts in cell lines artificially over-expressing this receptor (manes et al., 1999; popik et al., 2002) , but not in primary cd4 t lymphocytes, where this receptor was found in non-raft membrane domains (percherancier et al., 2003) . here, we show that in macrophages, ccr5 is also present in or associated with lipid rafts, and both receptors required for macrophage-tropic virus entry are localised in similar membrane domains. this is in agreement with electron micrographs showing cd4 and ccr5 (but not cxcr4) localised on the outer membranes of microvilli and blebs in macrophages, often in closely apposed microdomains (singer et al., 2001) . however, despite existing together in drms it is possible that further aggregation of receptors is still required and may occur upon gp120 binding. the most likely explanation for the inhibition of productive hiv entry into cholesterol-depleted macrophages is that redistribution of (or conformational changes to) the hiv receptors render the macrophages unsusceptible to hiv infection. it is plausible that manipulation of membrane cholesterol may inhibit entry by other mechanisms, of which we suggest five possibilities. 1) depletion of macrophage cholesterol may alter the lipid composition of the cellular plasma membrane so that it is no longer competent for fusion with the hiv lipid envelope. 2) disruption of membrane rafts may prevent receptor migration and co-localisation. both cd4 and ccr5 are mobile within cellular membranes (shown in cho cells) with the chemokine receptor being significantly more mobile than cd4 and requiring membrane cholesterol for this mobility (steffens and hope, 2004) . 3) signal transduction pathways induced upon ccr5 and envelope engagement may be required for virus entry (harmon and ratner, 2008) . cholesterol has been shown to be essential for ccr5 signalling and its depletion may prevent these important signalling events in macrophages (cardaba et al., 2008) . 4) hiv entry into macrophages may proceed by macropinocytosis, and cholesterol-depletion may block this uptake pathway in macrophages, as has been reported in brain microvascular endothelia (liu et al., 2002) . 5) lovastatin may inhibit hiv entry by another mechanism, as it has been shown that blockade of hmg coa reductase impedes prenylation of small rho gtpases that may be involved in post-entry signalling events (del real et al., 2004) , or by diminishing hiv-1 attachment to target cells by suppressing icam1 lfa1 interactions (giguere and tremblay, 2004) . however, addressing these possibilities to determine the exact role of cholesterol will be challenging, especially using infectious macrophage-tropic virus and primary macrophages. our study highlights the importance of cholesterol and lipid rafts in macrophage hiv infection. this provides further support for the use of cholesterol lowering pharmacological agents as anti-retroviral therapy by confirming their anti-viral effect in another pathogenically significant hiv target cell, the macrophage. peripheral blood mononuclear cells (pbmcs) were isolated using ficoll-paque plus (ge healthcare) density gradient centrifugation from the blood of healthy donors. monocytes were isolated by cd14 positive selection using anti-cd14 magnetic beads (miltenyi biotec) according to the manufacturer's instructions. monocytes were seeded at a density of 1.5 × 10 5 cells/cm 2 in 6-well plates, 12-well plates, 24-well plates or t75 flasks. culture media referred to as rpmi fcs m-csf: consisted of rpmi 1640 (paa) with 10% fetal calf serum (fcs; paa), 2 mm l-glutamine (paa), 100 u/ml penicillin and 100 μg/ml streptomycin (paa), supplemented with 100 ng/ml (approximately 1.7 × 10 4 u/ml) recombinant human m-csf (r&d systems). monocytes were differentiated for 7-9 days prior to use. hiv-1 bal was obtained through the aids research and reference reagent program, division of aids, niaid, nih, from s. gartner (gartner et al., 1986) and viral stocks were generated in differentiated, unstimulated pbmcs. infected cell supernatants were harvested 14-21 days after infection and frozen for use in infectivity assays. virus stocks with proviral dna removed were generated for qpcr experiments by treatment with 100 μg/ml dnase i (sigma). to generate single round replication competent virus, 293t cells were regularly passaged in dmem (paa) with 10% fcs, 2 mm l-glutamine, 100 u/ml penicillin and 100 μg/ml streptomycin. cells seeded into t75 flasks were transfected with pnl4.3lucr-e-(2 μg) and pjrfl env (1.5 μg) using fugene 6 according to manufacturers instructions. the nl4.3 jrfl virus was harvested 48 h later, passed through a 0.44 μm filter and frozen. macrophages were washed once with serum-free rpmi, and treated for 1 h at 37°c with varying concentrations of mβcd, filipin complex, or nystatin (all from sigma) diluted in serum-free rpmi. as nystatin is dissolved in dmso (mβcd and filipin in water), to exclude for any effects of this solvent the control cells were incubated in the same dilution of dmso as used for nystatin. after incubation, cells were washed once with serum-free rpmi prior to infection or flow cytometry analysis. following infection, cells were cultured in rpmi fcs m-csf. water soluble cholesterol (400 μg/ml, sigma) was added for a further 1 h where stated. for lovastatin treatment, macrophages were washed once with serum-free rpmi, before the addition of varying concentrations of lovastatin (sigma) with or without mevalonate (sigma), diluted in rpmi with 10% lipoprotein-deficient serum (sigma). cells were left for 4 days at 37°c before infection or flow cytometry analysis. infected cells were cultured in rpmi fcs m-csf following the infection procedure. to determine cell viability following treatment with cholesterol disrupting agents, macrophages were incubated with celltiter 96® aqueous one solution cell proliferation assay reagent (promega) diluted 1/6 in serum-free rpmi or rpmi with lipoprotein-deficient serum containing lovastatin or mevalonate, for 1 h at 37°c. in triplicate, 100 μl samples of the supernatant were then transferred to a 96 well plate and the absorbance read at 490 nm. background absorbance readings from reagent and media alone were deducted and the values expressed as a percentage of untreated cells. to quantify cellular cholesterol levels, drug treated or control macrophages were detached by pipetting with pbs containing 5 mm edta and 12 mm lidocaine (sigma), on ice. cells were lysed and the cholesterol was extracted as previously described (chung et al., 2005) . the amount of cholesterol was assayed with amplex red cholesterol assay kit (molecular probes) according to the manufacturer's instructions and the concentrations were determined from a standard curve and normalised to the number of cells. macrophages were infected in 12-well plates with 500 μl of dnase i treated hiv-1 bal by spinoculation in a sealed plate spinning centrifuge at 2000 ×g for 90 min at 37°c. the virus inoculum was removed, cells were overlaid with rpmi fcs m-csf and infections were left to proceed for 28-30 h. cells were harvested by scraping and dna was extracted using dneasy blood and tissue kit (qiagen) according to the manufacturer's instructions. qpcr reactions to detect late stage reverse transcription products were carried out as previously described (butler et al., 2001; cohen et al., 2008) . hiv-1 standards consisting of pnl4.3lucr -e -hiv-1 backbone plasmid diluted with 30 ng/μl hela dna were used in duplicate at dilutions ranging from 10 to 1 × 10 7 copies, and actin standards consisting of human xsomal dna (eurogentec) were used at 10-fold dilutions from 80 ng. to measure multiple rounds of hiv infection, macrophages were infected in 12-well plates with hiv-1 bal for 2 h at 37°c. the inoculum was removed and replaced with rpmi fcs m-csf. over a 14-16 day period, supernatant samples were taken at intervals and kept at −80°c. for analysis of hiv-1 binding, macrophages were incubated with hiv bal for 2 h on ice. the cells were then washed 5 times in ice-cold pbs to remove unbound virus, and lysed in 200 μl tes (1 × tris buffered saline [tbs], 1% empigen [fluka], 10% fcs, 0.05% tween 20) before heat inactivation. for analysis of hiv-1 production, samples from infected cells were diluted in tes and heat inactivated. to create a standard curve, recombinant p24 protein (aalto) was diluted in doubling dilutions in tes. costar eia/ria flat bottom high binding 96-well plates (corning) were pre-coated with 10 μg/ml sheep anti-hiv-1-p24 gag antibody (clone d7320, aalto) diluted in 0.05 m carbonate-bicarbonate buffer ph 9.6 (sigma) and blocked with 2% bsa (sigma) in tes. diluted samples or standards were added to the plates and after overnight incubation at room temperature, bound p24 was detected using a biotinylated mouse anti-hiv-1-p24 gag antibody (clone eh12ei, centre for aids reagents, nibsc) at 0.23 μg/ml in tt/ss (tbs, 20% fcs, 0.05% tween 20) followed by 0.625 μg/ml streptavidin-hrp (pierce). plates were developed using tmb substrate (pierce), the reaction was stopped with 1 m h 2 so 4 (sigma) and plates were then read using a molecular devices e max precision microplate reader and softmax pro software version 4.0. graphpad prism software version 4 was used to create standard curves (2-site binding equation) and to calculate unknown values. detached macrophages were washed once with facs buffer (pbs, 0.01% nan 3 , 1% fcs, 10 μg/ml human igg), and 1 × 10 5 cells were incubated with 5 μg/ml directly conjugated mouse anti-human antibodies to cd4 (clone 1180), ccr5 (clone 45531), cxcr4 (clone 44717.111) or appropriate isotype control (all r & d systems). as a control, the expression of the non-raft associated protein transferrin receptor was also measured using mouse anti-human cd71 (clone rvs10) with isotype control mouse igg1 (clone 203) (both diluted 1/ 10 and from immunotools). cells were fixed in 4% formaldehyde. flow cytometry was carried out using a becton-dickinson facscalibur flow cytometer with 10000 events collected and data analysed using flowjo software, version 7.1.3. macrophages were washed once in serum-free rpmi, and incubated with ctb-fitc (sigma) at 10 μg/ml in serum-free rpmi, for 30 min on ice or at 37°c. detached cells were then analysed by flow cytometry as described. to visualise virus and ctb, hiv-1 capable of a single round of infection nl4.3 jrfl was spinoculated (1 h at 4°c at 700 ×g) onto day 7 macrophage monolayers seeded onto glass coverslips in 24 well plates. cells were washed with ice-cold pbs and incubated in rpmi containing 10 μg/ml ctb-fitc for 20 min at 37°c. cells were fixed in 4% formaldehyde, quenched in 80 mm glycine in pbs and permeabilised in buffer containing 1% fcs, 0.1% saponin and human igg 10 μg/ml in pbs. to detect virus, cells were stained with mouse anti-hiv p17 diluted 1/500 (clone 4c9, centre for aids reagents, nibsc) followed by alexa fluor 647 conjugated goat antimouse igg2a antibody diluted 1/400 (clone a21131, invitrogen). coverslips were mounted on slides using mowiol and images collected using a zeiss pascal microscope. macrophages (1 × 10 7 ) were harvested by scraping, pelleted and suspended in 1 ml h buffer (10 mm sodium hepes, ph 7.2, 250 mm sucrose, 2 mm mgcl 2 , 10 mm naf, and 1 mm vanadate) containing protease inhibitors (roche). the cells were nitrogen-cavitated using a nitrogen cavitation bomb (model 4639; parr instrument company) equilibrated at 4°c, 50 bar for 10 min (harder and kuhn, 2000) . cells were further disrupted by adding 1 ml hne buffer (10 mm hepes, ph 7.0, 150 mm nacl, and 5 mm edta), passaged 15 times through a 23g syringe and 1% triton x-100 was added before cells were incubated for 1 h at 4°c. a 200 μl extract containing approximately 1 × 10 6 cells was adjusted to 45% optiprep™ (nycomed pharma) and transferred to an sw55 centrifuge tube (beckman coulter) and overlaid with another 200 μl 45% optiprep to make a 1 ml layer. this was overlaid with 2 ml of 35% and 0% optiprep in hne and gradients were spun at 40,000 rpm for 3 h at 4°c. nine fractions of 550 μl were collected and precipitated in an equal vol of 20% trichloroacetic acid, washed twice in ice-cold acetone and resuspended in 8 m urea and sds sample buffer. equal volumes of each fraction were run on a 10% sds-page protein gel with biorad precision plus protein standards. protein was transferred onto 0.2 μm pvdf membranes, and membranes were incubated with primary mouse anti-human antibodies to cd4 (clone 34915, 2.5 μg/ml r & d systems) ccr5 (clone ctc5, 2.5 μg/ml r & d systems), flotillin-1 (clone 18, 1.25 μg/ml, bd biosciences) and transferrin receptor (clone 2, 1.25 μg/ml, bd biosciences). to detect primary antibody binding, membranes were incubated with goat antimouse igg (fab specific) antibody conjugated to alkaline phosphatase (sigma) diluted 1/30,000. protein was detected using ecf substrate (pierce), and membranes were imaged using storm imager. statistical analysis was performed by two-tailed paired t-test (unpaired with 2 replicates) using graphpad prism version 4. stars indicate the p value: ⁎ = significant p = 0.05-0.01, ⁎⁎ = very significant p = 0.01-0.001, ⁎⁎⁎ = extremely significant p b 0.001. lipid composition and fluidity of the human immunodeficiency virus lipid composition and fluidity of the human immunodeficiency virus envelope and host cell plasma membranes bound simian virus 40 translocates to caveolin-enriched membrane domains, and its entry is inhibited by drugs that selectively disrupt caveolae specific association of glycoprotein b with lipid rafts during herpes simplex virus entry the hiv lipidome: a raft with an unusual composition human immunodeficiency virus type 1 nef protein modulates the lipid composition of virions and host cell membrane microdomains a quantitative assay for hiv dna integration in vivo virion-associated cholesterol is critical for the maintenance of hiv-1 structure and infectivity the raftpromoting property of virion-associated cholesterol, but not the presence of virionassociated brij 98 rafts, is a determinant of human immunodeficiency virus type 1 infectivity ccr5 internalisation and signalling have different dependence on membrane lipid raft integrity crosstalk between lxr and toll-like receptor signaling mediates bacterial and viral antagonism of cholesterol metabolism retroviruses human immunodeficiency virus and murine leukemia virus are enriched in phosphoinositides vaccinia virus penetration requires cholesterol and results in specific viral envelope proteins associated with lipid rafts an aptamer that neutralizes r5 strains of hiv-1 binds to core residues of gp120 in the ccr5 binding site macrophage-tropic strains of human immunodeficiency virus type 1 utilize the cd4 receptor blocking of hiv-1 infection by targeting cd4 to nonraft membrane domains statins inhibit hiv-1 infection by down-regulating rho activity duck hepatitis b virus requires cholesterol for endosomal escape during virus entry virus isolation from and identification of htlv-iii/lav-producing cells in brain tissue from a patient with aids statin compounds reduce human immunodeficiency virus type 1 replication by preventing the interaction between virion-associated host intercellular adhesion molecule 1 and its natural cell surface ligand lfa-1 role for human immunodeficiency virus type 1 membrane cholesterol in viral internalization selective accumulation of raft-associated membrane protein lat in t cell receptor signaling assemblies induction of the g{alpha}q signaling cascade by the human immunodeficiency virus envelope is required for virus entry poliovirus cell entry: common structural themes in viral cell entry pathways effects of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane variations in cd4 expression by human monocytes and macrophages and their relationships to infection with the human immunodeficiency virus segregation of cd4 and cxcr4 into distinct lipid microdomains in t lymphocytes suggests a mechanism for membrane destabilization by human immunodeficiency virus quantification of cd4, ccr5, and cxcr4 levels on lymphocyte subsets, dendritic cells, and differentially conditioned monocyte-derived macrophages lipid rafts play an important role in the early stage of severe acute respiratory syndrome-coronavirus life cycle lipid rafts and hiv pathogenesis: host membrane cholesterol is required for infection by hiv type 1 lipid rafts and hiv pathogenesis: virionassociated cholesterol is required for fusion and infection of susceptible cells human immunodeficiency virus type 1 enters brain microvascular endothelia by macropinocytosis dependent on lipid rafts and the mitogen-activated protein kinase signaling pathway ecotropic murine leukemia virus receptor is physically associated with caveolin and membrane rafts membrane raft microdomains mediate front-rear polarity in migrating cells membrane raft microdomains mediate lateral assemblies required for hiv-1 infection human immunodeficiency virus type 1 entry into macrophages mediated by macropinocytosis internalization of echovirus 1 in caveolae virus entry: open sesame human immunodeficiency virus impairs reverse cholesterol transport from macrophages statins disrupt ccr5 and rantes expression levels in cd4+ t lymphocytes in vitro and preferentially decrease infection of r5 versus x4 hiv-1 cxcr4 function requires membrane cholesterol: implications for hiv infection dynamic reorganization of chemokine receptors, cholesterol, lipid rafts, and adhesion molecules to sites of cd4 engagement lack of p56lck expression correlates with cd4 endocytosis in primary lymphoid and myeloid cells hiv-1 entry into t-cells is not dependent on cd4 and ccr5 localization to sphingolipid-enriched, detergentresistant, raft membrane domains echovirus 1 endocytosis into caveosomes requires lipid rafts, dynamin ii, and signaling events cd4 receptor localized to non-raft membrane microdomains supports hiv-1 entry. identification of a novel raft localization marker in cd4 human immunodeficiency virus type 1 uses lipid raft-colocalized cd4 and chemokine receptors for productive entry into cd4(+) t cells the molecular face of lipid rafts in model membranes syndecans serve as attachment receptors for human immunodeficiency virus type 1 on macrophages lipid microdomaindependent macropinocytosis determines compartmentation of afipia felis functional rafts in cell membranes ccr5, cxcr4, and cd4 are clustered and closely apposed on microvilli of human macrophages and t cells how viruses enter animal cells mobility of the human immunodeficiency virus (hiv) receptor cd4 and coreceptor ccr5 in living cells: implications for hiv fusion and entry events role for influenza virus envelope cholesterol in virus entry and infection human herpesvirus-6 infection induces the reorganization of membrane microdomains in target cells, which are required for virus entry differential cholesterol binding by class ii fusion proteins determines membrane fusion properties nef induces multiple genes involved in cholesterol synthesis and uptake in human immunodeficiency virus type 1-infected t cells role of cholesterol in human immunodeficiency virus type 1 envelope protein-mediated fusion with host cells modulation of brucella-induced macropinocytosis by lipid rafts mediates intracellular replication float on: lipid rafts in the lifecycle of hiv nef increases the synthesis of and transports cholesterol to lipid rafts and hiv-1 progeny virions this research was supported by grant number 9746 and a phd studentship from the uk medical research council. key: cord-276358-so390gp4 authors: nieto-torres, jose l.; verdiá-báguena, carmina; jimenez-guardeño, jose m.; regla-nava, jose a.; castaño-rodriguez, carlos; fernandez-delgado, raul; torres, jaume; aguilella, vicente m.; enjuanes, luis title: severe acute respiratory syndrome coronavirus e protein transports calcium ions and activates the nlrp3 inflammasome date: 2015-11-30 journal: virology doi: 10.1016/j.virol.2015.08.010 sha: doc_id: 276358 cord_uid: so390gp4 abstract severe acute respiratory syndrome coronavirus (sars-cov) envelope (e) protein is a viroporin involved in virulence. e protein ion channel (ic) activity is specifically correlated with enhanced pulmonary damage, edema accumulation and death. il-1β driven proinflammation is associated with those pathological signatures, however its link to ic activity remains unknown. in this report, we demonstrate that sars-cov e protein forms protein–lipid channels in ergic/golgi membranes that are permeable to calcium ions, a highly relevant feature never reported before. calcium ions together with ph modulated e protein pore charge and selectivity. interestingly, e protein ic activity boosted the activation of the nlrp3 inflammasome, leading to il-1β overproduction. calcium transport through the e protein ic was the main trigger of this process. these findings strikingly link sars-cov e protein ic induced ionic disturbances at the cell level to immunopathological consequences and disease worsening in the infected organism. coronaviruses (covs) cause respiratory diseases in humans ranging from common colds to fatal pneumonias perlman and netland, 2009) . at the end of 2002, the etiological agent of severe acute respiratory syndrome (sars-cov) emerged in guandong province, southeast china, initiating a global epidemic. approximately 8000 people were infected by the virus, whose severe disease resulted in a 10% mortality rate rota et al., 2003) . the sars-cov epidemic was controlled by the summer of 2003, and the virus has not naturally reemerged since (http://www.who.int). however, in 2012 a closely related cov appeared in saudi arabia causing the middle east respiratory syndrome (mers-cov) (zaki et al., 2012) . mers-cov induces acute pneumonia similar to that caused by sars-cov, and is sometimes accompanied with renal failure (danielsson and catchpole, 2012; zaki et al., 2012) . mers-cov is now circulating and spreading throughout the human population, and to date has infected at least 1356 people leading to death in 484 cases. initial cases were confined to countries of the arabian peninsula; however, new extensive infection clusters have been recently reported in other regions such as south korea (http:// www.who.int). furthermore, covs similar to those causing sars, mers, and many other human and animal diseases have been detected in bat species circulating all over the globe (annan et al., 2013; chu et al., 2008; drexler et al., 2010; falcon et al., 2011; muller et al., 2007; quan et al., 2010) . bats are now considered the natural host for most covs, providing a perfect scenario for recombination events among different viral species leading to the emergence of new viruses, able to cross species barriers and cause devastating illness in other animals and humans . for this reason, development and implementation of broad spectrum treatments and general therapeutic strategies against covs are a high priority. covs are enveloped viruses containing the largest positive-sense rna genomes known, around 30 kb, which encode the viral replicase and a set of structural proteins: spike (s), envelope (e) and membrane (m), present in the viral envelope, and nucleocapsid (n) located inside the viral particle . the s protein interacts with the cellular receptor to trigger viral entry into the host cell wong et al., 2004) . the e and m proteins actively contents lists available at sciencedirect journal homepage: www.elsevier.com/locate/yviro participate in viral morphogenesis (de haan et al., 1999; lim and liu, 2001; nguyen and hogue, 1997; ruch and machamer, 2012) , and the n protein coats the viral genome to form a helicoidal nucleocapsid that remains protected within the viral envelope (narayanan et al., 2000) . depending on the cov species, there are other accessory proteins, some structural, that are also encoded by the genome. sars-cov encodes the highest number of accessory genes (3a, 6, 7a 7b, 8a, 8b and 9b), which play diverse roles eventually related to pathogenesis . to analyze the mechanisms leading to the high virulence of sars-cov and mers-cov, several mutants affecting their different genes have been generated (almazan et al., 2013; dediego et al., 2007; dediego et al., 2014a; scobey et al., 2013; yount et al., 2005) . remarkably, deletion of sars-cov e gene produced a virus that was attenuated in at least three different animal models and conferred protection against sars-cov challenge (dediego et al., 2007 fett et al., 2013; lamirande et al., 2008; netland et al., 2010) . small deletions in different domains of the e protein caused similarly attenuated viruses, and are promising vaccine candidates (regla-nava et al., 2015) . in a similar approach, a mers-cov missing the e gene was generated, resulting in a replicationcompetent, propagation-defective virus which may also constitute the basis of a safe attenuated vaccine (almazan et al., 2013) . the cov e gene encodes a small transmembrane protein highly synthesized during infection (maeda et al., 2001; nieto-torres et al., 2011; raamsman et al., 2000) that mainly localizes to the golgi apparatus and the endoplasmic reticulum golgi apparatus intermediate compartment (ergic), where it facilitates virus production and morphogenesis (cohen et al., 2011; corse and machamer, 2002; maeda et al., 2001; nal et al., 2005; venkatagopalan et al., 2015) . notably, when the e protein is present, sars-cov overstimulates the nf-κb inflammatory pathway (dediego et al., 2014b) and, through its pdz-binding motif, interacts with the cellular protein syntenin, triggering p38 mapk activation . these signaling cascades result in exacerbated inflammation and immunopathology. the connection between the e protein and virulence has further encouraged the search for other e protein functions that may trigger disease worsening. one of the most striking functions displayed by the cov e protein is ion channel (ic) activity. the cov e protein self assembles in membranes forming pentameric protein-lipid pores that allow ion transport (pervushin et al., 2009; torres et al., 2007; verdia-baguena et al., 2012; wilson et al., 2004 wilson et al., , 2006 . interestingly, lipid head-groups are integral components of the pore and regulate ion conductance and selectivity (verdia-baguena et al., 2012 ) . the sars-cov e protein showed a mild preference for cations (na þ , k þ ) over anions (cl à ) when reconstituted in membranes mimicking the charge and composition of those of the ergic/golgi. in this scenario, the e protein ic showed no selectivity for any particular cation (verdia-baguena et al., 2012) , though the relevance and consequences of these ic properties in a cellular context remains unknown. alteration of ion, and specially, ca 2 þ homeostasis in favor of infection has been already demonstrated in several viral systems. to that end a wide range of viruses encode ion conductive proteins similar to e protein, named viroporins (nieva et al., 2012) . highly pathogenic rna viruses such as human immunodeficiency virus-1 (hiv-1), hepatitis c virus (hcv), influenza a virus (iav), picornaviruses and covs, encode one or more of these proteins (nieva et al., 2012) . viroporins participate in several steps of the life cycle and are usually linked with pathogenesis. whether ic properties could promote pathways leading to disease worsening has been unknown for a long time. recently, it was described that point mutations that specifically inhibited sars-cov e protein ic activity caused attenuation (nieto. mice infected with e protein ic proficient viruses presented extensive disruption of the pulmonary epithelia and edema accumulation . edema is the major determinant of acute respiratory distress syndrome (ards), the pathology induced by sars-cov, leading to death hollenhorst et al., 2011; matthay and zemans, 2011) . edema and an il-1β mediated proinflammatory response was increased in the lung parenchyma when e protein ic activity was present . il-1β is a potent proinflammatory cytokine crucial in resolving infectious processes; however, its overproduction has been correlated with diverse severe inflammatory diseases such as asthma, ards, gout, atherosclerosis and parkinson's (dinarello, 2009; dos santos et al., 2012; martinon et al., 2006; pugin et al., 1996; strowig et al., 2012) . organisms tightly control il-1β production through macromolecular complexes called inflammasomes, which are mainly expressed in macrophages and dendritic cells although other cell types, such as those of the bronchiolar epithelium, synthesize their components (ichinohe et al., 2010; triantafilou and triantafilou, 2014) . one highly studied inflammasome is the nucleotide-binding oligomerization domain (nod)-like receptor pyrin domain-containing protein 3 (nlrp3) inflammasome, relevant in the pulmonary tissue. this complex is composed of the sensing protein nlrp3, the adapter component apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (asc) and the catalytically inactive procaspase-1 (elliott and sutterwala, 2015; latz et al., 2013) . the inflammasome components are synthesized under precise danger stimuli, such as molecular patterns associated to infections. however, a second signal is sequentially needed to induce their assembly, which triggers the inflammasome. this leads to the processing of procaspase-1 into active caspase-1, which cleaves inactive pro-il-1β into its mature form il-1β that is released to the extracellular media to stimulate proinflammation (elliott and sutterwala, 2015; latz et al., 2013) . the presence of ionic imbalances within cells is a main trigger of the nlrp3 inflammasome assembly and activation . interestingly, several viroporins stimulate this pathway through alteration of cell ion homeostasis, frequently involving ca 2 þ imbalances (ichinohe et al., 2010; ito et al., 2012; triantafilou et al., 2013a; triantafilou and triantafilou, 2014) . there is clear evidence correlating nlrp3 inflammasomes and il-1β driven proinflammatory cascades with worsening of several respiratory diseases, including those caused by viruses (dos santos et al., 2012; mcauley et al., 2013; pugin et al., 1996; triantafilou and triantafilou, 2014) . however, the specific role that viral proteins with ic activity may play in this process has not been fully determined. overproduction of il-1β in the airways of the lungs of mice infected with e protein ic proficient sars-covs, strongly suggested that ion conductivity may be stimulating the inflammasome (nieto. here we demonstrate that sars-cov e protein forms a ca 2 þ permeable channel in ergic/golgi membranes. e protein ic activity alters ca 2 þ homeostasis within cells boosting the activation of the nlrp3 inflammasome, which leads to the overproduction of il-1β. this data supports that sars-cov e protein ca 2 þ channel activity may play a role in disease, through overstimulation of inflammasomes leading to immunopathology. pharmacological inhibition of this pathway may constitute the basis for combined therapeutics applicable for sars-cov and other viruses. the sars-cov e protein forms ca 2 þ permeable ion channels previously, we reported that the sars-cov e protein forms channels that are moderately selective for cations in membranes mimicking the ergic/golgi (verdia-baguena et al., , 2012, 2013). within cells, the movement of different cations through the e protein pore should be dictated by their respective gradients. ca 2þ possesses the highest asymmetrical distribution between the er-golgi lumen (hundred μm) and the cytoplasm (around 100 nm) (zhou et al., 2009) , which should allow the flow of this cation through the e protein ic channel if it is permeable. to test whether this is the case, e protein ics were first reconstituted in artificial neutral lipid membranes in order to exclude any effect coming from the lipid charge, using 100 mm cacl 2 solutions. current jumps corresponding to the assembly of channels in the lipid membrane were detected (fig. 1a) . the most frequent current jump, representing the insertion of a single e protein ic in the lipid membrane, displayed an intensity of 7.272.4 pa. interestingly, values multiple of 7.2 pa were also detected, corresponding to the insertion of two (14.4 pa) or three channels (21.6 pa), reinforcing the previous observation ( fig. 1a and b). because lipid charge can affect channel conductance (verdia-baguena et al., 2012 , single channel conductance was analyzed in ergic/golgi-like membranes that contained approximately 20% negatively charged lipids. interestingly, the e protein ic showed a slightly higher unitary current jump of 1174 pa under these conditions, and multiples of this value, evidencing the insertion of additional channels, were also detected (figs. 1c and d). the presence of the negatively charged lipid head-groups in the channel pore may facilitate ca 2þ flow through the channel enhancing current intensity under these conditions. this effect was already reported for e protein and monovalent cations (verdia-baguena et al., 2012) . additional measurements were performed in aqueous solutions with decreasing cacl 2 concentrations, to approach ca 2 þ amounts found in the ergic/golgi lumen. ic conductance (g), which is the ratio between the current intensity and the applied voltage, was calculated. single channel conductance scaled almost linearly with cacl 2 concentration in neutral membranes (fig. 1e ). this reflects that ion conduction inside the e protein ic is similar to the conduction of the solution, which increases as the electrolyte concentration rises. these data indicate that the interaction between the channel and the permeating ions is weak, and are in accordance with e protein forming a neutral pore in these conditions, as previously reported for monovalent cation salts (verdia-baguena et al., 2013) . in ergic/ golgi membranes two different regimes depending on the range of cacl 2 concentrations were observed. a linear relation between conductance and cacl 2 concentration was again detected in high concentrated solutions. however, ic conductance in ergic/golgi membranes was independent of cacl 2 concentration in low concentration solutions (below 0.2 m), and higher than in neutral membranes (fig. 1e ). this result suggests that e protein acts as a charged protein-lipid pore in ergic/golgi membranes. in this scenario, the conductance is regulated by the balance between the ions that flow into the channel to neutralize the excess of negative charges and the bulk electrolyte concentration. collectively, these data indicate that the e protein forms channels that are highly conductive in cacl 2 . furthermore, e protein ics worked very efficiently when reconstituted in ergic/golgi membranes under ca 2 þ concentrations approaching those found in the lumen of the organelles (hundred μm). to specifically test e protein selectivity for ca 2 þ , i.e. the ability of the ic to select ca 2 þ either by its charge or by its intrinsic properties, reversal potential (erev) experiments were performed. the rational of these experiments is provided next. ics were reconstituted in lipid membranes that separated two solutions, one with high and the other with low cacl 2 concentration. this concentration gradient induces the movement of ions through the channel to equilibrate their asymmetric distribution. the potential applied across the channel, which is required to counteract this ion movement leading to zero electric current is the erev. the erev can be transformed into channel permeability for ca 2 þ (pca 2 þ ) and cl à (pcl à ) by using the goldman-hodgkin-katz equation (hodgkin and katz, 1949) . to test whether lipid charge may have an effect on e protein, selectivity measurements were performed in neutral, ergic/golgi similar charged membranes, and fully negative-charged membranes. the e protein ic showed pca 2 þ /pcl à values of 0.2 (1/5) in neutral membranes. the value of this permeability ratio is barely the same expected for a non-selective neutral pore just reflecting the different diffusivities of ca 2 þ and cl à . therefore, e protein channel is equally selective for cl à and ca 2 þ in neutral membranes. interestingly, the ratio pca 2 þ /pcl à increased when the e protein ic was assembled in membranes containing negatively charged lipids, being 0.5 in ergic/ golgi membranes and 1.8 in negative membranes (fig. 2) . these data indicated that lipid charge largely influenced the channel preference for ca 2 þ showing that under conditions mimicking the ergic/golgi environment, e protein displayed a mild selectivity for ca 2 þ . multivalent ions, such as ca 2 þ , can modulate ion transport across ics by interacting with the internal charges of their pores (alcaraz et al., 2009; garcia-gimenez et al., 2012; queralt-martin et al., 2011) . therefore we tested whether e protein ion selectivity could be modified by the presence of a range of small cacl 2 concentrations. ion selectivity was measured using a ten-fold concentration gradient of kcl at ph 6 upon symmetrical addition of very small cacl 2 concentrations (mm range), at both sides of the membrane. by applying the erev, the flow of k þ and cl à ions ca 2 þ selectivity of sars-cov e protein channel. permeability ratios pca 2 þ / pcl à in neutral dphpc (magenta column), ergic/golgi (blue column) or negativelycharged dphps membranes (green column). dotted line represents the permeability ratio value for a hypothetical neutral pore. values above the line represent cation selectivity, and those below correspond to anion selectivity. error bars show standard deviations. down their electrochemical potential gradient is prevented. therefore, erev measures the relative preference of the channel for k þ cations over cl à anions, but not for ca 2 þ , because calcium concentrations are the same at both sides of the pore. to analyze the relevance of ca 2 þ on ic selectivity, this type of experiments were performed in neutral and in negatively-charged membranes (fig. 3a) . in charged membranes the addition of small amounts of ca 2 þ induced significant changes in the channel erev, whereas in neutral membranes the effect was very small, although still measurable. these results indicate that ca 2 þ interaction with e protein channel, modifying the effective pore charge, mainly occur with the lipid charges that line the pore, rather than with acidic residues of e protein tm domain. previously we reported that ph also modulates e protein channel selectivity by protonation and deprotonation of the titratable residues present in the pore (verdia-baguena et al., 2013) . the effect of ca 2 þ and ph on e protein selectivity was simultaneously tested. erev titration was studied in the presence or absence of 15 mm cacl 2 under different ph conditions (fig. 3b) . in negatively-charged lipid membranes, rising ph induced sequential deprotonation of lipid head-groups and e protein glutamic acid residues as their respective pk a (1.73 for lipid head-groups and 4.5 for glutamic acid) were overtaken, conferring an excess of negative charges to the channel and making it cation selective. the addition of ca 2 þ when e protein is reconstituted in charged membranes reduces the cationic selectivity or increases the anionic selectivity (i.e., it shifts the reversal potential towards less negative values), and reversal potential values become closer to those obtained in neutral membranes. this indicates that ca 2 þ ions interact with the acidic protein residues and the negative lipid head-groups and decrease the effective negative charge of the e protein pore. this effect was especially patent at physiological values of ph (ph 6-7.5). in addition, this interaction of ca 2 þ ions with the protein acidic residues shifts their effective pk a towards lower values. these results indicate that both ca 2 þ and h þ ions change e channel transport properties by modifying the charge of the pore, and further support that ca 2 þ enters within e protein ic. in order to assess the biological impact of sars-cov e protein with and without ca 2 þ channel activity, we evaluated previously constructed mutants containing amino acids substitutions known to affect the e protein ic. previously we demonstrated that mutations n15a and v25f in the transmembrane domain of sars-cov e protein abolished ion conductance in kcl and nacl solutions (verdia-baguena et al., 2012) . new conductance measurements were performed in ergic/golgi membranes in 100 mm cacl 2 solutions (fig. 4) . wildtype e protein transmembrane peptides showed conductance values in the range of 110 ps, whereas no conductance was observed for n15a or v25f mutants, indicating that these mutations also inhibited ca 2 þ transport, and probably prevent all ion passage as it was previously demonstrated that they also failed to transport k þ , na þ and cl à (verdia-baguena et al., 2012). ca 2 þ transport through sars-cov e protein channel activates the nlrp3 inflammasome alteration of cellular ion homeostasis by sars-cov e protein ic could have several implications. previously, we linked e protein ic with il-1β triggered proinflammation in the lungs of mice, leading to epithelial cell damage and death . higher levels of mature il-1β were detected in the airways of infected animals when e protein ic activity was present, suggesting that ion conductance could stimulate the inflammasome. to determine whether this was the case, the inflammasome complex was reconstituted in vero e6 cells by transient transfection of its components (nlrp3, asc and procaspase-1) and the inactive pro-il-1β (lo et al., 2013) , in the absence or presence of e protein, with or without ic activity. all the components of the inflammasome, besides the different versions of e protein were efficiently expressed within cells (fig. 5a) . when pro-il-1β was transfected as a control, no significant level of active il-1β was detected in the supernatant. however, when all the components were supplied to the cells, the inflammasome was stimulated, and active il-1β was detected in the media in the range of 600 pg/ml (fig. 5b) . interestingly, the production of il-1β was significantly enhanced in the presence of e protein with ic activity (ic þ ) (4800 pg/ml). this stimulation was ic activity dependent, as the e protein mutants lacking ion conductance (n15a and v25f), represented in the figure as eic à 1 and eic à 2 , respectively, did not boost il-1β levels (fig. 5b ). stimulation levels found with wildtype e protein were in the range of those previously reported for other inflammasome activating proteins, using this system (lo et al., 2013) . imbalances in ca 2 þ within cells have been described as an inflammasome inducer (ito et al., 2012; murakami et al., 2012; triantafilou et al., 2013b) . to test the specific contribution of sars-cov e protein ca 2 þ transport to inflammasome activation, the complex was reconstituted in cells in the presence or absence of e protein. cells were subsequently treated with the cell permeant calcium chelator bapta-am (fig. 6a ). this compound enters cells and binds ca 2 þ preventing inflammasome activation (ito et al., 2012) . increasing amounts of bapta-am markedly decreased the levels of secreted il-1β in the presence of e protein (fig. 6a , infl eic þ ), reaching levels close to those obtained when the inflammasome was assembled alone (fig. 6a, infl) . the ca 2 þ ionophore ionomycin stimulated active il-1β accumulation in the cell supernatant at levels similar to those seen with e protein, further confirming that ca 2 þ imbalances boost il-1β production (fig. 6b) . no significant differences in cell viability were found for any of the treatments (fig. 6c) . collectively, these results indicate that sars-cov e protein activates the nlrp3 inflammasome through its ca 2 þ transport ability. ion conductive proteins are widely distributed among viruses, being especially common in rna viruses (nieva et al., 2012) . conductance of ions facilitate diverse processes of the viral life cycle such as entry, takeover of organelles to serve as platforms for viral replication, protection of viral proteins from acidic cell compartments, and trafficking of nascent virions (nieva et al., 2012) . in addition, viroporins are often linked to pathogenesis, and in general, mutant viruses lacking them are attenuated, in many cases serving as effective vaccines (dediego et al., 2007; netland et al., 2010; watanabe et al., 2009; whitehead et al., 1999) . viroporin removal is frequently accompanied by a defect in viral production, which by itself can explain virulence attenuation. however, recent studies have indicated that ic activity may specifically trigger pathways leading to pathology. previously we demonstrated that sars-covs proficient in e protein ic activity caused increased damage of the pulmonary epithelia and edema accumulation . these disease symptoms correlated with an immunopathological response mediated by proinflammatory cytokines such as il-6, tnf and il-1β, the latter being a crucial mediator of this cascade. il-1β overproduction is linked to a wide range of inflammatory pathologies including those caused by respiratory viruses (dos santos et al., 2012; mcauley et al., 2013; pugin et al., 1996; triantafilou and triantafilou, 2014) . here, we report for the first time that cov e protein formed an ic that transported ca 2 þ in ergic/golgi membranes, where this protein locates, which may have important consequences on cell physiology. in fact, ca 2 þ leakage through e protein ic induced the activation of the nlrp3 inflammasome resulting in overproduction of il-1β. this finding together with previous in vivo observations indicates that ic activity correlates with proinflammation and pathology. generally, viroporins form poorly selective ion channels (nieva et al., 2012) . therefore, the subcellular location where viroporins assemble and the conditions of that particular environment are crucial determinants of their impact on cellular ionic homeostasis. previously, we reported that sars-cov e protein showed mild selectivity for cations (na þ and k þ ) when reconstituted in ergic/ golgi membranes, mostly conferred by the negative charges of the lipids (verdia-baguena et al., 2012 . high concentration gradients are found for na þ and k þ between the cell interior and the extracellular media (dubyak, 2004) . however, there is no known asymmetric distribution for either of these ion species between the lumen of ergic/golgi and the cell cytoplasm (chandra et al., 1991; schapiro and grinstein, 2000) . accordingly, minimal net transport of na þ and k þ through e protein pore should be expected, and therefore the biological relevance of these processes may be also limited. in contrast, the er and golgi apparatus store high amounts of calcium ions by the action of pumps such as the sarcoendoplasmic reticulum ca 2 þ atpase (serca) and the secretory pathway ca 2 þ atpase (spca) (wuytack et al., 2002) . this creates an enormous gradient of around 1000-fold between the lumen of these organelles and the cytoplasm (zhou et al., 2009) . the gradient allows controlled eventual and temporal leakages of ca 2 þ into the cytoplasm that trigger several processes relevant to cell physiology. interestingly, we have shown above that sars-cov e protein ic was also permeable to ca 2 þ in ergic/golgi membranes. furthermore, ca 2 þ interacted with the negative charges of the protein-lipid pore, modulating its properties. several other viroporins are known to transport ca 2 þ , resulting in leakage of this cation from its intracellular stores. rotavirus nsp4 protein as well as coxsackievirus, encephalomyocarditis virus, and poliovirus 2b proteins deplete er and/or golgi ca 2 þ concentrations in favor of viral proliferation (campanella et al., 2004; crawford et al., 2012; de jong et al., 2008) . alteration of protein trafficking, manipulation of apoptosis, and control of autophagy are some of the processes controlled by these ca 2 þ effluxes. whether some of these aspects are influenced in a similar manner during sars-cov infection will be explored in future experiments. protons (h þ ) are also actively confined to the lumen of the golgi apparatus and those of the organelles of the secretory pathway in a process that acidifies their interior and creates a gradient with the cytoplasm (paroutis et al., 2004) . considering that the e protein ic weakly interacts with circulating ions and that ph can modulate its net charge, it is highly likely that protons will also flow through the ic within cells. this is not an isolated case, as several viroporins such as hcv p7 and iav m2 are known to transport protons, and others such as the 2b protein of the picornaviridae family transport both h þ and ca 2 þ (de jong et al., 2006; wang et al., 1994; wozniak et al., 2010) . alkalinization of the golgi lumen is crucial to protect acid-sensitive viral progeny and prevent premature activation of viral proteins involved in entry processes (sakaguchi et al., 1996; wozniak et al., 2010) . sars-cov e protein ic mutants did not show profound growth defects, although they were outgrown in competition assays by ic proficient viruses, which suggests better proliferation when e protein ion conductance was present . whether alkalization of intracellular compartments by e protein ic may assist in sars-cov production remains to be explored. besides these considerations, it cannot be excluded that ic activity may have a greater impact in sars-cov production. inhibition of e protein ic activity may be compensated by the action of two other viroporins encoded by sars-cov, the 3a and 8a (chen et al., 2011; lu et al., 2006) ; further experiments are being performed to answer this question. disruption of ion homeostasis can have profound deleterious effects for the cells; consequently, they have evolved mechanisms to sense and control these disturbances. ion imbalances associated with infecting pathogens are recognized by inflammasomes, which are components of the innate immune system. inflammasomes orchestrate proinflammatory responses to fight viral infections, il-1β being one of the major players (strowig et al., 2012) . however, increasing evidence indicates that overstimulation of this pathway can lead to undesirable effects for the organism. in fact, immunopathology rather than direct viral destruction of infected cells is the main cause of severe disease in many viral illnesses (mcauley et al., 2013; meduri et al., 1995; triantafilou and triantafilou, 2014) . sars-covs that lack e protein ic activity induced less proinflammation and active il-1β, suggesting that ic activity could be a trigger of this pathway . indeed, wildtype e protein but not its ic à mutants boosted the production of mature il-1β through the mediation of a reconstituted inflammasome. ca 2 þ was the main trigger of this process, as chelation of this ion abolished enhanced il-1β production, and a ca 2 þ ionophore produced a similar simulation. e proteins from other human respiratory covs, such as mers-cov and hcov-229e, also display ion channel properties (surya et al., 2015; verdia-baguena et al., 2012; wilson et al., 2006) . investigating whether these proteins may favor ca 2 þ transport and activation of the inflammasome represents a relevant issue. furthermore, the identification of a possible correlation between the extent of inflammasome activation and the disease symptoms caused by these viruses, highly deleterious in the case of sars-cov and mers-cov, and mild in the case of hcov-229e, could provide key information on the impact of this pathway on the pathological outcome induced by different covs, and will be analyzed in future experiments. this study provides new insights into the molecular mechanisms governing cov viroporin activity and the consequences of this function in viral pathogenesis. what is more, il-1β overproduction has been related with the pathology induced by sars-cov and other respiratory viruses (triantafilou and triantafilou, 2014) . our results suggest that development of specific ic inhibitors and implementation of novel compounds decreasing inflammasome driven immunopathology (coll et al., 2015) may be a valuable complement to other antiviral approaches for the treatment of these infectious diseases. the african green monkey kidney-derived vero e6 cells were kindly provided by eric snijder (medical center, university of leiden, the netherlands). cells were grown at 37 1c with an atmosphere of 98% humidity and 5% co 2 , in dulbecco's modified eagle medium (dmem, gibco) supplemented with 25 mm hepes, 2 mm l-glutamine (sigma), 1% non-essential amino acids (sigma) and 10% fetal bovine serum (fbs, biowhittaker). synthetic peptides representing the full-length sars-cov e protein, or its transmembrane domain (amino acids 7-38) containing point mutations that inhibited ion channel activity (n15a and v25f), were generated by standard phase synthesis and purified by hplc, as previously described (verdia-baguena et al., 2012) . ion channels were reconstituted in lipid membranes composed of diphytanoyl phosphatidylcholine (dphpc), diphytanoyl phosphatidylserine (dphps), or a mixture of 59% dioleoyl phosphatidylcholine (dopc), 24% dioleoyl phosphatidylethanolamine (dope), and 17% dioleoyl phosphatidylserine (dops) (avanti polar lipids, inc., alabaster, al). the membranes separated two chambers filled with electrolytic solutions of cacl 2 or kcl. ion channel insertion was achieved by adding 0.5-1 μl of a 300 μg/ml solution of synthetic protein or peptides in a acetonitrile:isopropanol (40:60) buffer to one of the chambers (cis). all measurements were performed at room temperature (237 1 1c). the single-channel conductance was obtained from current measurements under an applied potential of þ 100 mv in symmetrical salt solutions of 1 m cacl 2 buffered with 5 mm hepes at ph 6. the conductance values were evaluated using the gaussian fit tool of sigma plot 10.0 (systat software, inc). for the selectivity measurements the reversal potential (erev) was obtained under a ten-fold salt concentration gradient (500 mm / 50 mm). one or several channels were inserted into the bilayer causing a net ionic current due to the concentration gradient. then the ionic current was manually set to zero by adjusting the applied potential. the potential needed to achieve zero current represents the erev; conversion of erev into the channel permeability (p þ /p à ) was by done using the goldman-hodgkin-katz (ghk) equation (hodgkin and katz, 1949) . the effect of ca 2 þ on e protein ion selectivity was defined by measuring erev in a ten-fold kcl concentration gradient (500 mm/50 mm) buffered with 5 mm hepes at ph 6, in the presence of increasing concentrations of cacl 2 . the functional interaction of ca 2 þ with e protein-lipid pore was evaluated measuring erev in dphpc and dphps membranes under different ph values, with or without 15 mm cacl 2 . sub-confluent monolayers of vero e6 cells seeded onto 24-well plates were transfected using lipofectamine 2000 (invitrogen) and a set of plasmids encoding the components of the nlrp3 inflammasome (lo et al., 2013; wu et al., 2014) : 200 ng of pcdna4-proil-1β, 25 ng of pcdna4-nlrp3-ha, 20 ng pcdna4-asc-mcherry, and 10 ng of pcdna4-procaspase-1-myc, with 100 ng of empty pcdna3, as a control, or 100 ng of either pcdna3-e, pcdna3-en15a or pcdna3-ev25f. cell lysates and culture supernatants were collected 24 h after transfection. the secreted active il-1β was measured by elisa according to the manufacturer's specifications (ebioscience). cells were lysed in a buffer containing tris/hcl 10 mm, edta 1 mm, nacl 150 mm, igepal 1%, and complete protease inhibitor (roche) at ph 8. inflammasome proteins were detected by western blot using a mouse anti-ha antibody (sigma) to detect nrlp3-ha, mouse anti-mcherry (abcam) to bind asc-mcherry, rabbit anti-myc (abcam) to label procaspase-1-myc, and rabbit anti-human il-1β (cell signaling). sars-cov e protein was detected using a polyclonal antiserum produced in rabbits . as a loading control, beta-actin was labeled using a mouse monoclonal antibody (abcam). bound antibodies were detected using hrp-conjugated goat anti-rabbit igg and rabbit anti-mouse igg and the immobilon western chemiluminicesce substrate (millipore), following manufacturer's specifications. fresh supplemented dmem containing the indicated concentration of the cell permeant calcium chelator bapta-am (life technologies) was added to cells 4 h after transfection of the inflammasome components and incubated 30 min at 37 1c. then, media was removed and a second treatment with fresh media containing bapta-am was performed. supernatants were collected 20 h post-bapta-am treatment. ionomycin (life technologies) was added at 1 μm 4 h after transfection and incubated for 20 h. the mtt colorimetric method was used to measure cell metabolic activity. cell media of transfected cells was replaced by fresh dmem supplemented with 10% fcs and 500 μg/ml of mtt. cells were incubated 2 h at 37 1c. after that, media was removed and a solution containing 0.04 m hcl 0.1% nonidet p-40 in isopropanol was added. plates were gently mixed to obtain a homogeneous solution, and optic density was measured at 570 nm. diffusion, exclusion, and specific binding in a large channel: a study of ompf selectivity inversion 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protein are attenuated and promising vaccine candidates the coronavirus e protein: assembly and beyond the ion channel activity of the influenza virus m2 protein affects transport through the golgi apparatus determinants of the ph of the golgi complex reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus inflammasomes in health and disease mers coronavirus envelope protein has a single transmembrane domain that forms pentameric ion channels conductance and amantadine binding of a pore formed by a lysine-flanked transmembrane domain of sars coronavirus envelope protein human respiratory syncytial virus viroporin sh: a viral recognition pathway used by the host to signal inflammasome activation rhinovirusinduced calcium flux triggers nlrp3 and nlrc5 activation in bronchial cells ion flux in the lung: virus-induced inflammasome activation coronavirus envelope (e) protein remains at the site of assembly analysis of sars-cov e protein ion channel activity by tuning the protein and lipid charge coronavirus e protein forms ion channels with functionally and structurally-involved membrane lipids direct measurement of the influenza a virus m2 protein ion channel activity in mammalian cells influenza a virus lacking m2 protein as a live attenuated vaccine recombinant respiratory syncytial virus bearing a deletion of either the ns2 or sh gene is attenuated in chimpanzees hexamethylene amiloride blocks e protein ion channels and inhibits coronavirus replication sars coronavirus e protein forms cationselective ion channels a 193-amino acid fragment of the sars coronavirus s protein efficiently binds angiotensinconverting enzyme 2 intracellular proton conductance of the hepatitis c virus p7 protein and its contribution to infectious virus production participation of c-flip in nlrp3 and aim2 inflammasome activation molecular physiology of the serca and spca pumps severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice isolation of a novel coronavirus from a man with pneumonia in saudi arabia viral calciomics: interplays between ca 2 þ and virus the work done by the authors was supported by grants from the government of spain (bio2013-42869-r, fis2013-40473-p), generalitat valenciana (prometeo 2012/069), fundació caixa castelló-bancaixa (p1-1b2012-03) and a u.s. national institutes of health (nih) project (5p01 ai060699). jln, jmj and jar received contracts from nih. ccr received a contract from fundacion la caixa. we thank professor ming-zong lai (institute of molecular biology, taipei) for kindly providing us plasmids encoding the inflammasome components. we thank marga gonzalez for her technical assistance. key: cord-276034-a8pixbuc authors: zhi, yan; kobinger, gary p.; jordan, heather; suchma, katie; weiss, susan r.; shen, hao; schumer, gregory; gao, guangping; boyer, julie l.; crystal, ronald g.; wilson, james m. title: identification of murine cd8 t cell epitopes in codon-optimized sars-associated coronavirus spike protein date: 2005-04-25 journal: virology doi: 10.1016/j.virol.2005.01.050 sha: doc_id: 276034 cord_uid: a8pixbuc the causative agent of severe acute respiratory syndrome (sars) has been identified as a new type of coronavirus, sars-associated coronavirus (sars-cov). cd8 t cells play an important role in controlling diseases caused by other coronaviruses and in mediating vaccine-induced protective immunity in corresponding animal models. the spike protein, a main surface antigen of sars-cov, is one of the most important antigen candidates for vaccine design. overlapping peptides were used to identify major histocompatibility complex class i-restricted epitopes in mice immunized with vectors encoding codon-optimized sars-cov spike protein. cd8 t-cell responses were mapped to two h-2(b)-restricted epitopes (s436–443 and s525–532) and one h-2(d)-restricted epitope (s366–374). the identification of these epitopes will facilitate the evaluation of vaccine strategies in murine models of sars-cov infection. furthermore, codon and promoter optimizations can greatly enhance the overall immunogenicity of spike protein in the context of replication-defective human and simian adenoviral vaccine carriers. the optimized recombinant adenoviral vaccine vectors encoding spike can generate robust antigen-specific cellular immunity in mice and may potentially be useful for control of sars-cov infection. severe acute respiratory syndrome (sars) is an emerging infectious disease associated with a novel coronavirus, sars-associated coronavirus (sars-cov), which caused worldwide outbreaks. the case fatality rate has been as high as 15% for patients younger than 60 years old and can be higher than 50% for patients 60 years or older. nearly 40% of patients developed respiratory failure that required assisted ventilation (de groot, 2003) . the severe morbidity and mortality associated with sars make it imperative that effective means to prevent and treat the disease be developed and evaluated, especially since it is not known whether the virus will exhibit a seasonal pattern or whether it will be reintroduced into the human population through animal reservoirs, or laboratory accidences, or acts of terrorism. the successful development of effective treatments and vaccines against sars-cov depends on understanding the roles of various immune effectors in protective immunity and identifying protective antigens recognized by these effector cells. like other covs, sars-cov is an enveloped plus-stranded rna virus with a¨30 kbs genome encoding replicase (rep) gene products and the structural proteins spike (s), envelope (e), membrane (m), and nucleocapsid (n) (marra et al., 2003; rota et al., 2003) . s protein is responsible for binding to specific cellular receptor , e protein plays a role in viral assembly, m is 0042-6822/$ -see front matter d 2005 elsevier inc. all rights reserved. doi:10.1016/j.virol.2005.01.050 important for virus budding, and n protein is associated with viral rna packaging (holmes, 2003) . no conclusive information is available on the immune correlates of protection to sars in patients. however, it has been reported that antibodies against sars-cov were detected in patients infected with sars . recently, using pseudotyped lentiviral particles bearing the sars-cov spike protein, it has been shown that spike-mediated infection could be inhibited by sera from sars patients, demonstrating that spike is a target for neutralizing antibodies (hofmann et al., 2004) . in addition, two identified cd8 t-cell epitopes in sars-cov spike protein have been shown to elicit specific t-cell responses in hla-a2+ sars-cov-infected patients (wang et al., 2004) . therefore, we initially focused on sars-cov spike protein as the target antigen for our vaccine development. a number of animal models have emerged for studying sars pathogenesis and evaluating therapies including macaques, ferrets, and mice hogan et al., 2004; kuiken et al., 2003; martina et al., 2003; subbarao et al., 2004) . however, for the early stage of product evaluation, a small animal model, such as mice, would be very useful. we (hogan et al., 2004) and others (subbarao et al., 2004) have shown that sars-cov replicates in mice although the infection is self-limited. to study antigen-specific immune responses in mice, mhc irestricted cd8 t-cell epitopes need to be identified. vectors based on replication-defective adenoviruses are capable of high-level gene transfer and activation of t and b cells to the transgene product (van ginkel et al., 1997; yang et al., 1996) . these properties have been exploited in the development of genetic subunit vaccines (sullivan et al., 2000; xiang et al., 1996) . therefore, several versions of replication-defective adenoviral vectors expressing spike protein were created to induce spike-specific t cell responses in mice and to screen for cd8 t-cell epitopes using an overlapping peptide library spanning the entire spike protein in ifn-g elispot and intracellular ifn-g staining assays. here, we report the detailed mapping of both h-2 b -and h-2 d -restricted cd8 t-cell epitopes from codon-optimized spike protein. these results provide critical information for analyzing cd8 t-cell responses in murine models of sars-cov infection and for developing spike-based sars-cov vaccines. more importantly, a single administration of the optimized sars-cov spike vaccine vectors based on replication-defective human and simian adenovirus can generate strong spikespecific cd8 t-cell responses in mice. to identify cd8 t-cell epitopes in sars-cov spike protein, c57bl/6 mice were injected im with adhu5. cmvspike, a replication-defective human adenovirus serotype 5-based vaccine vector encoding wild-type spike protein. splenocytes were harvested 8 days after immunization and stimulated in vitro with pools of overlapping peptides corresponding to spike protein. no pool resulted in specific stimulation of an ifn-g response in t cells using elispot assay (data not shown). we then used adhu5.cmvnspike, another replicationdefective human adenovirus serotype 5-based vaccine vector encoding codon-optimized spike protein, to immunize c57bl/6 mice im. splenocytes were harvested at 8 days after immunization and subjected to ifn-g elispot assay with pools of overlapping peptides from spike protein. of all peptide pools screened, pools 9, 11, and 18 showed specific ifn-g responses in c57bl/6. the remaining pools, as exemplified by pool 5, showed no specific response (fig. 1a) . pools 9 and 11 were selected for mapping the individual peptide(s) responsible for stimulating ifn-g expressing t cells. peptide 87 (tstgnyny-kyrylrh, corresponding to s431 -445) and peptide 88 (ynykyrylrhgklrp, corresponding to s436 -450) within pool 9 and peptide 105 (knqcvnfnfngltgt, corresponding to s521 -535) and peptide 106 (nfnfn-gltgtgvltp, corresponding to s526 -540) within pool 11 were identified as the major positive peptides responsible for specific stimulation of t cells to produce ifn-g (fig. 1b) . based on the syfpeithi algorithm (rammensee et al., 1999) , several potential cd8 t-cell epitopes for sars-cov spike protein in c57bl/6 mice were identified. table 1 showed their sequences, positions, and scores. indeed, ynykyryl (s436 -443) completely present in peptides 87 and 88 was predicted to have strong binding affinity for h2-k b ; while vnfnfngl (s525 -532) completely present in peptide 105 and partially present in peptide 106 was predicted to have weak binding affinity for h2-k b . since elispot assay cannot readily distinguish antigen-specific cd4 t-cell responses from antigenspecific cd8 t-cell responses, the immunized splenocytes were further subjected to intracellular ifn-g staining after stimulating in vitro with peptides 87 and 105 (fig. 2) . subsequently, the predicted optimal cd8 t-cell epitopes, ynykyryl resided in peptide 87 and vnfnfngl resided in peptide 105, were synthesized and used to stimulate immunized splenocytes in vitro for ifn-g secretion (fig. 2) . as predicted, ynykyryl and peptides 87 gave much stronger cd8 t-cell responses than vnfnfngl and peptides 105. furthermore, optimal epitopes, ynykyryl and vnfnfngl, were able to induce ifn-g responses in cd8 t cells either as effectively as or more effectively than their parental 15 mers, respectively. in conclusion, a single administration of a replication-defective human adenoviral vaccine vector encoding codon-optimized spike can generate strong spike-specific cd8 t-cell responses. we also identified two optimal cd8 t-cell epitopes of sars-cov spike in c57bl/6 mice. determining the dose responses of two h-2 b -restricted cd8 t-cell epitopes for t cell activation immunized c57bl/6 splenocytes were in vitro stimulated with the two identified optimal cd8 t-cell epitopes at different concentrations ranging from 5 ag/ml to 5eà7 ag/ ml, respectively. t cells activation was measured by intracellular ifn-g staining (fig. 3) . it appeared that the minimal amounts of peptides needed to fully activate t cells in vitro were similar for both epitopes, even though the magnitude of t cell response with ynykyryl was much higher than that with vnfnfngl. mapping of h-2 d -restricted cd8 t-cell epitopes in sars-cov spike protein balb/c mice were immunized in a similar fashion as c57bl/6 mice. using elispot assay, pools 7, 8, and 9 showed specific ifn-g responses in balb/c. the remaining pools, as exemplified by pool 5, showed no specific response (fig. 4a ). we focused on pools 8 and 9 because of the stronger responses observed with these pools. subsequently, peptide 73 (fstfkcygvsatkln, correspond-ing to s361 -375) and peptide 74 (cygvsatklndlcfs, corresponding to s366 -380) within pool 8 and peptide 87 (tstgnynykyrylrh, corresponding to s431 -445) and peptide 88 (ynykyrylrhgklrp, corresponding to s436 -450) within pool 9 were identified as the major positive peptides responsible for specific stimulation of t cells to produce ifn-g (fig. 4b ). based on the syfpeithi algorithm (rammensee et al., 1999) , several potential cd8 t-cell epitopes for sars-cov spike protein in balb/c mice were identified. table 2 showed their sequences, positions, and scores. indeed, cygvsatkl (s366 -374) completely present in peptides 73 and 74 was predicted to have strong binding affinity for h2-k d ; while nyny-kyryl (s435 -443) completely present in peptide 87 and partially present in peptide 88 was predicted to have weak binding affinity for h2-k d . the immunized splenocytes were also subjected to intracellular ifn-g staining after stimulating in vitro with these positive 15 mers. notably, there was no significant ifn-g production from cd8 t cells stimulated with peptides 87 and 88 by intracellular ifn-g staining (fig. 5a ), even though both peptides gave strong responses in ifn-g elispot assay (fig. 4b ). this suggested that peptides 87 and 88 may contain a cd4 ttable 1 selected candidates of cd8 t-cell epitopes for sars-cov spike protein in c57bl/6 the candidates of cd8 t-cell epitopes present in the positive 15 mers were shown in red. cell epitope that stimulated cd4 t-cell responses in ifn-g elispot assay. in contrast, peptide 74 can significantly stimulate cd8 t-cell responses (fig. 5b ). subsequently, the predicted optimal cd8 t-cell epitope, cygvsatkl resided in peptide 74, was synthesized and used to stimulate immunized splenocytes in vitro for ifn-g secretion (fig. 5b ). the results indicated that peptide 74 indeed contained an h-2 d -restricted cd8 t-cell epitope. as expected, optimal epitope was able to induce ifn-g responses in cd8 t cells more effectively than its parental 15 mer. collectively, we have identified one optimal cd8 t-cell epitope of sars-cov spike in balb/c mice. the dose responses of this fig. 2 . intracellular ifn-g staining to confirm cd8 t-cell epitopes of sars-cov spike protein in c57bl/6 mice. mice were immunized with 3 â 10 10 particles of adhu5.cmvnspike via im injection. 10 days after immunization, splenocytes were harvested and pooled from 3 mice and stimulated with either 15 mers positive peptides or optimal 8 mers cd8 t-cell epitopes, as indicated, for 5 h. the immune response was evaluated by intracellular ifn-g staining with pe-anti-ifn-g and fitc-anti-cd8 antibodies. numbers in the upper right corner of each graph represent the frequencies of ifn-g-producing cd8 t cells. experiments were done in duplicate and representative results were shown. fig. 3 . dose responses of h-2 b -restricted cd8 t-cell epitopes for t cell activation in vitro. c57bl/6 mice were immunized with 5 â 10 10 particles of adhu5.cmvnspike via im injection. 8 days after immunization, splenocytes were harvested and pooled from 3 mice and stimulated with two identified cd8 t-cell epitopes at different concentrations, as indicated, respectively, for 5 h. t cell activation was evaluated by intracellular ifn-g staining. experiments were done in duplicate and representative results were shown. cd8 t-cell epitope for t cells activation in vitro were also determined as described above (fig. 6) . the results suggested that about 100-fold more peptides were needed to fully activate t cells in vitro with this h-2 d -restricted cd8 t-cell epitope compared to those with two h-2 brestricted cd8 t-cell epitopes (fig. 3) . furthermore, peptides 87 and 88 may contain a cd4 t-cell epitope of sars-cov spike in balb/c mice. to confirm the presence of cd4 t-cell epitope(s) in peptides 87 and 88, the adhu5.cmvnspike-immunized splenocytes of balb/c mice were harvested and in vitro stimulated with three 15 mers individually, including peptides 74, 87, and 88. the cd4 t-cell response was directly evaluated by intracellular cytokine staining with fitc-anti-cd4 and pe-anti-ifn-g antibodies (fig. 7) . as expected, when stimulated with peptide 74, there was no detectable ifn-g secretion from immunized cd4 t cells, even though a significant ifn-g production from immunized non-cd4 t cells (cd8 t cells) was observed. more importantly, peptides 87 and 88 were able to stimulate immunized cd4 t cells to product ifn-g. these results indicated that peptides 87 and 88 indeed contained a cd4 t-cell epitope of spike protein in balb/c mice. increasing the immunogenicity of sars-cov spike protein in the context of replication-defective simian adenoviral vaccine carrier to circumvent the potential problem that neutralizing antibodies to human adenovirus serotype 5 vector by previous natural infections will impair its efficacy as vaccine carrier, our lab recently developed a series of novel replication-defective adenoviral vaccine carriers based on simian serotypes pinto et al., 2003) . adc7, one of those carriers, is able to induce robust transgene-specific cd8 t-cell responses in immunized mice (kobinger et al., submitted for publication) . three replication-defective simian adenovirus serotype 7-based vaccine vectors encoding either wild-type spike driven by cmv promoter (adc7.cmvspike) or codon-optimized spike driven by cmv promoter (adc7.cmvnspike) or codonoptimized spike driven by a hybrid promoter cag2 (adc7.cag2nspike) were created and used to immunize mice. cag2 promoter was created by deleting a 955-bp table 2 selected candidates of cd8 t-cell epitopes for sars-cov spike protein in balb/c the candidates of cd8 t-cell epitopes present in the positive 15 mers were shown in red. apai/aflii fragment from the original caggs promoter (niwa et al., 1991) . similar to the negative results observed when adhu5.cmvspike was used to immunize mice, there was no detectable stimulation of an ifn-g response in t cells with any of the identified positive pools using elispot assay when adc7.cmvspike was used for immunization (data not shown). in contrast, spike-specific cd8 t-cell responses were observed when adc7.cmvnspike vector was used to immunize c57bl/6 mice (fig. 8) . however, the magnitude of spike-specific cd8 t-cell responses was much lower than that observed in mice immunized with adhu5.cmvnspike vector (fig. 8) . in order to further improve the immunogenicity of spike protein delivered by the simian adenoviral vaccine carrier, adc7.cag2nspike vector was created. chicken h-actin promoter has been shown to increase expression of sars-cov spike protein in transfected cells (simmons et al., 2004) . spike-specific cd8 t-cell responses in c57bl/6 mice elicited by adc7.cag2nspike vector were examined by intracellular ifn-g staining (fig. 8) . the result indicated fig. 6 . dose responses of h-2 d -restricted cd8 t-cell epitope for t cell activation in vitro. balb/c mice were immunized with 5 â 10 10 particles of adhu5.cmvnspike via im injection. 8 days after immunization, splenocytes were harvested and pooled from 3 mice and stimulated with the identified cd8 tcell epitope at different concentrations, as indicated, for 5 h. t cell activation was evaluated by intracellular ifn-g staining. experiments were done in duplicate and representative results were shown. fig. 5 . intracellular ifn-g staining to confirm cd8 t-cell epitopes of sars-cov spike protein in balb/c mice. mice were immunized with 3 â 10 10 particles of adhu5.cmvnspike via im injection. 10 days after immunization, splenocytes were harvested and pooled from 3 mice and stimulated with either 15 mers positive peptides or optimal 8 mers cd8 t-cell epitope, as indicated, for 5 h. the immune response was evaluated by intracellular ifn-g staining with pe-anti-ifn-g and fitc-anti-cd8 antibodies. numbers in the upper right corner of each graph represent the frequencies of ifn-g-producing cd8 t cells. experiments were done in duplicate and representative results were shown. that codon and promoter optimizations can greatly enhance the overall immunogenicity of sars-cov spike protein in the context of simian adenoviral vaccine carrier. more importantly, a single administration of an optimized sars-cov spike vaccine vector based on a replication-defective simian adenovirus can generate strong spike-specific cd8 t-cell responses in mice. it has been shown that increased expression of codonoptimized hiv gag protein is responsible for its enhanced immunogenicity in mice (deml et al., 2001; gao et al., 2003a) . therefore, it was of interest to determine whether optimization of transgene expression cassette, including codon and promoter optimizations, in the context of replication-deficient adenoviral vaccine vectors also resulted in the increased expression of spike protein in vitro. expression of spike protein in ad vector-infected 293t cells was examined by western blot analysis and the bands representing spike protein were quantified by image-quant5.1. forty-three-fold increase of protein expression was achieved when cag2 promoter, instead of cmv promoter, was used in the simian adenoviral c7 vector (fig. 9 , lanes 4 and 6), while 7-fold increase of protein expression was achieved when codon optimization of spike protein was applied (fig. 9, lanes 5 and 6) . overall, the results supported that the enhanced immunogenicity of spike in mice injected with adhu5.cmvnspike and adc7.cag2nspike vaccine vectors (fig. 8) indeed correlated to high expression of spike protein in cells infected with these vectors. sars-cov represents an emerging threat. currently, no effective therapies or vaccines exist, and relatively little is known about the pathogenesis of the virus. it has been shown that 293t cells transfected with a functional receptor for sars-cov, angiotensin-converting enzyme 2 (ace2), formed multinucleated syncytia with cells expressing spike protein . more significantly, in the postmortem lung tissue samples from patients who died from sars, multinucleate giant cells of macrophage and epithelial origins have been observed within the damaged alveoli (nicholls et al., 2003) . in addition, cd8 t-cell responses are necessary for clearance of other covs, such as mouse hepatitis virus (sussman et al., 1989; williamson and stohlman, 1990) . therefore, eradication of sars-cov may not be achieved by humoral response alone and t cellmediated immunity may be also required to clear infection. to study more closely cellular immune responses in murine model, mhc i-restricted cd8 t-cell epitopes need to be identified. in this study, we have identified two octamers (s436-443 and s525 -532) as h-2 b -restricted cd8 t-cell epitopes and one nonamer (s366 -374) as an h-2 d -restricted cd8 t-cell epitope. in c57bl/6 mice, one epitope is more dominant than the other in immunized mice. the strength of these epitopes appeared to correlate with the binding affinity to h2-k b predicted by syfpeithi algorithm. in the preliminary study, splenocytes were collected from c57bl/6 mice immunized with adc7.cag2nspike vector and in vitro cultured in the presence of the dominant h-2 brestricted cd8 t-cell epitope for a week. subsequently, the cultured splenocytes were subjected to elispot assay stimulated with mc57sv cells (h-2d b , a cell line derived from b6 mice) previously infected with either adhu5.null or adhu5.cmvnspike vector. the initial results indicated that cultured splenocytes were able to induce higher level of ifn-g response after stimulated with adhu5.cmvnspike vector-infected mc57sv cells than after stimulated with adhu5.null vector-infected mc57sv cells. these data suggested that t cells raised to the epitope were able to recognize cells expressing the spike protein and that the identified cd8 epitope is naturally processed and presented by cells. currently, additional experiments are underway to address this issue more thoroughly. coronaviruses are common and worldwide pathogens that infect a variety of mammals and birds. these viruses have been classified into three groups. although sarsfig. 7 . intracellular ifn-g staining to confirm the presence of cd4 t-cell epitope of sars-cov spike protein in balb/c mice. mice were immunized with 5 â 10 10 particles of adhu5.cmvnspike via im injection. 10 days after immunization, splenocytes were harvested and pooled from 3 mice and stimulated with 15 mers, as indicated, for 5 h. the immune response was evaluated by intracellular ifn-g staining with pe-anti-ifn-g and fitc-anti-cd4 antibodies. numbers in the upper right corner of each graph represent the frequencies of ifn-g-producing cd4 t cells. experiments were done in duplicate and representative results were shown. fig. 8 . promoter optimization can further increase the immunogenicity of sars-cov spike protein delivered by the replication-defective simian adenoviral vaccine carrier. c57bl/6 mice were immunized with 5 â 10 10 particles of vaccine vectors, including adhu5.cmvnspike, adc7.cmvnspike, and adc7.cag2nspike, via im injection. 10 days after immunization, splenocytes were harvested and pooled from 3 mice in each group and stimulated in vitro with positive peptide for 5 h. the immune response was evaluated by intracellular ifn-g staining with pe-anti-ifn-g and fitc-anti-cd8 antibodies. numbers in the upper right corner of each graph represent the frequencies of ifn-g-producing cd8 t cells. experiments were done in duplicate and representative results were shown. cov exhibits a similar genome structure, it is only distantly related to known covs and fmost like_ group 2 covs, which includes bovine, murine, and human viruses (snijder et al., 2003) . the most commonly studied coronavirus is mouse hepatitis virus (mhv), in part because the natural host for this infection, the mouse, is more easily managed in the laboratory. two spike-specific cd8 t-cell epitopes (dominant s510 -518 and subdominant s598-605) were identified in mhv-infected c57bl/6 mice (bergmann et al., 1996; castro and perlman, 1995) , while no spikespecific cd8 t-cell epitope has been recognized in mhvinfected balb/c mice. the dominant s510 -518 epitope is located in a hypervariable region of spike protein that appears to be readily deleted without loss of viability of mhv (parker et al., 1989) . in contrast, it was impossible to recover infectious virus with mutations in the subdominant s598 -605 epitope (personal communication, m.m. chua and s.r. weiss). examination of sars-cov spike sequence reveals that three cd8 t-cell epitopes are present in the s1 domain of the spike protein. specifically, the dominant cd8 t-cell epitope in c57bl/6 (s436 -443) and the cd8 t-cell epitope in balb/c (s366 -374) both reside in the minimal region of s1 required for interaction with its cellular receptor (babcock et al., 2004; wong et al., 2004) . several spike-specific cd4 t-cell epitopes have also been reported in mhv-infected mice (heemskerk et al., 1995; xue and perlman, 1997) . cd4 t cells are needed to help cd8 cells for viral clearance (stohlman et al., 1998; sussman et al., 1989; williamson and stohlman, 1990) . in this study, we clearly showed that both peptide 87 and peptide 88 were able to specifically stimulate immunized cd4 t cells to produce ifn-g in vitro. however, the exact sequences of cd4 t-cell epitope in the 431 -450 region of spike protein remain to be further elucidated. the first step in designing a genetic vaccine is to enhance the intrinsic immunogenicity of the selected target gene. in this study, we demonstrated that codon and promoter optimizations can greatly enhance the overall immunogenicity of spike protein in replication-defective human and simian adenoviral vaccine vectors. we showed that the expression of spike protein in vitro was greatly increased by expression cassette optimization. this provided useful information for developing spike-based sars-cov genetic vaccines. more importantly, this may provide us a platform to quickly generate genetic vaccine vectors based on simian adenovirus. namely, cag2 but not cmv promoter should be used in simian adenoviral vaccine vector. currently, we are testing this hypothesis with other viral antigens. since the cmv promoter has been widely used in human adenoviral vectors, it is surprising to see the inferior performance of cmv promoter in simian adenoviral vector. one possibility is that certain viral sequences only present in simian adenoviral vector have negative effects on cmv promoter activity. the other possibility is that certain inflammatory cytokines induced by simian adenoviral vector may inhibit cmv promoter activity. recently, a strategy involving priming with dna vaccine and boosting with adenoviral vaccine vectors, each expressing a similar antigen, has resulted in the generation of unparalleled levels of specific immunity and afforded protection against infectious agents in animal models (shiver et al., 2002; sullivan et al., 2000) . nevertheless, dna vaccines have performed poorly in clinical trials so far (macgregor et al., 1998; wang et al., 2001) , and it is thus uncertain whether dna vaccine prime followed by adenoviral vaccine vector boost will be as efficacious in humans as in preclinical experimental animal studies. therefore, we developed an additional replication-defective adenoviral vaccine vector of chimpanzee origin to increase our repertoire of vaccine carriers that can be given sequentially pinto et al., 2003 pinto et al., , 2004 reyes-sandoval et al., 2004; roy et al., 2004; xiang et al., 2002 xiang et al., , 2003a xiang et al., , 2003b xiang et al., , 2003c . the toxicity of adenoviral vectors in vivo is directly related to the dose of injection. therefore, a major benefit of prime/boost strategy is the potential to significantly reduce the dose for immunization in order to achieve a high level of immune responses. in this study, we showed that both adhu5.cmvnspike and adc7.cag2nspike vaccine vectors could induce robust spike-specific cd8 t-cell responses in immunized mice alone. currently, we are studying whether strong cd8 t-cell responses can be achieved when mice were primed and boosted with these heterologous adenoviral vaccine vectors at a much lower dose. complementary dna (cdna) of spike gene for sars-cov was isolated by rt-pcr from the viral rna of the sars-cov (tor2 isolate). the pcr fragment was topocloned (invitrogen, ca) and characterized by sequencing at seqwright (seqwright, tx), and was found to be 100% identical to the published sequence (marra et al., 2003) . subsequently, tor2 spike protein sequence was used as a template to design a synthetic spike gene sequence with human pattern of codon usage, according to entelechon backtranslation software tool. finally, cloned tor2 spike cdna was used as a template and amplified with overlapping oligonucleotides in which human codon usage was introduced. resulting overlapping pcr fragments were fused and full-length codon-optimized spike cdna (nspike) was created. to generate the molecular clones of adhu5 vectors, wild-type or codon-optimized spike insert was cloned into a pshuttle plasmid, followed by homologous recombination in bacterial cells using padeasy system (invitrogen, ca). molecular clones of all adc7 vectors used in the study were created through a direct cloning and green-white selection procedure as described elsewhere . all of these molecular clones of replicationdefective adhu5 and adc7 vectors were transfected into 293 cells for virus rescue. the rescued vectors were expanded to large-scale infections in 293 cells and purified by the standard cscl gradient sedimentation method. genome structures of the vectors were confirmed by restriction analysis. infectivity of the vectors was determined by the standard plaque assay on 293 cells and levels of replication competent adenovirus (rca) contaminants in the vector preparations were inspected as described previously (gao et al., 2000) . 293 and 293t cells were maintained in dmem (gibco-life technologies, grand island, ny) supplemented with antibiotic and 10% fbs (hyclone, logan, ut). c57bl/6 and balb/c mice (6 -8 weeks old) were purchased from charles river laboratories (wilmington, ma) and kept at the animal facility of the wistar institute (philadelphia, pa). peptide library derived from the sars-cov spike protein sequence was synthesized as 15 mers with 10-amino-acid overlap with the preceding peptide (mimotopes, victoria, australia) and dissolved in dmso at approximately 100 mg/ ml. pools of 10 consecutive peptides were made and stored at à20 -c. peptides were used at the concentration of 5 ag/ml in all experiments except the dose response studies and dmso concentrations were kept below 0.1% (v/v) in all final assay mixtures. groups of three to five mice were immunized with recombinant adenoviral vectors diluted in 100 al phosphatebuffered saline (pbs) given im. 293t cells were infected at 1000 particles/cell with each recombinant adenoviral vector encoding spike protein. twenty-four hours later, cells were harvested and resuspended in lysis buffer and frozen at à20 -c. all samples were normalized to the lysate with the lowest total protein concentration by diluting with 4â sds sample buffer (invitrogen, carlsbad, ca) plus 5% h-mercaptoethanol. the total protein content for all lysates was determined using the bradford assay (biorad, melville, ny). diluted samples were heated to 94 -c for 4 min and loaded onto an sds -polyacrylamide gel. after electrophoresis, proteins were transferred onto pvdf membrane. blots were blocked with 8% milk diluted in tbs/tween 20 (tbs/t) for 45 min, washed three times (10 min each) with tbs/t, and probed with primary antibody at room temperature for 1 h. primary antibody consisted of whole serum isolated from rabbits inoculated with purified spike protein diluted in 5% milk/ tbs to a final concentration of 1:500. the blots were then washed three times with tbs/t before being incubated with secondary antibody for 45 min at room temperature and washed again three times with tbs/t. secondary antibody was anti-rabbit hrp diluted in 5% milk/tbs to a final concentration of 1:2000 (santa cruz biotechnology, santa cruz, ca). protein bands were developed using super-signal west pico chemiluminescent substrate (pierce, rockford, il) and exposed on kodak biomax film. the western blot image was scanned and the interested protein bands were quantified by imagequant5.1 (molecular dynamics). assay was performed using elispot mouse set (bd pharmingen, san diego, ca) following the protocol provided by the vendor. briefly, 96-well elispot plate was coated with 5.0 ag/ml anti-mouse ifn-g capture antibody overnight at 4 -c. next day, wells were washed and blocked with complete culture medium for 2 h at room temperature. splenocytes from immunized mouse were added to microwells along with spike-specific peptides. cells were incubated at 37 -c and 5% co 2 for 18 -20 h. control cells were incubated either without peptide or with nonspecific stimulator, seb (200 ng/ml). then wells were extensively washed with pbs containing 0.05% tween 20 and subsequently incubated with 2.0 ag/ml biotinylated anti-mouse ifn-g detection antibody for 2 h at room temperature. after washing, wells were incubated with 5 ag/ ml streptavidin-horseradish peroxidase antibody for 1 h at room temperature. wells were washed again, and final substrate was added to wells. color development was monitored and stopped by washing with water. after drying overnight at room temperature, wells were counted using an elispot reader. splenocytes from immunized mice were stimulated with spike-specific peptides for 5 h at 37 -c and 10% co 2 in the presence of 1 al/ml brefeldin a (golgiplug, bd pharmingen, san diego, ca). control cells were incubated without peptide. after washing, cells were stained with either a fitc-labeled anti-mouse cd8 antibody (bd pharmingen) or a fitc-labeled anti-mouse cd4 antibody (bd pharmingen). then, cells were washed and permeabilized in cytofix/ cytoperm (bd pharmingen) for 20 min on ice. subse-quently, cells were washed again and stained with a pelabeled anti-mouse ifn-g antibody (bd pharmingen). after extensively washing, cells were examined by two-color flow cytometry and data were analyzed by winmdi software. amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor the jhm strain of mouse hepatitis virus induces a spike protein-specific db-restricted cytotoxic t cell response cd8+ t-cell epitopes within the surface glycoprotein of a neurotropic coronavirus and correlation with pathogenicity how the sars vaccine effort can learn from hiv-speeding towards the future, learning from the past multiple effects of codon usage optimization on expression and immunogenicity of dna candidate vaccines encoding the human immunodeficiency virus type 1 gag protein a simian replication-defective adenoviral recombinant vaccine to hiv-1 gag aetiology: koch's postulates fulfilled for sars virus a cell line for 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the central nervous system adenoviral gene delivery elicits distinct pulmonary-associated t helper cell responses to the vector and to its transgene induction of cd4(+) t cell-dependent cd8(+) type 1 responses in humans by a malaria dna vaccine t-cell epitopes in severe acute respiratory syndrome (sars) coronavirus spike protein elicit a specific t-cell immune response in patients who recover from sars effective clearance of mouse hepatitis virus from the central nervous system requires both cd4+ and cd8+ t cells a 193-amino acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme 2 a replicationdefective human adenovirus recombinant serves as a highly efficacious vaccine carrier novel, chimpanzee serotype 68-based adenoviral vaccine carrier for induction of antibodies to a transgene product mucosally delivered e1-deleted adenoviral vaccine carriers induce transgene product-specific antibody responses in neonatal mice t helper cellindependent antibody responses to the transgene product of an e1-deleted adenoviral vaccine require nk1.1 t cells oral vaccination of mice with adenoviral vectors is not impaired by preexisting immunity to the vaccine carrier antigen specificity of cd4 t cell response in the central nervous system of mice infected with mouse hepatitis virus immunology of gene therapy with adenoviral vectors in mouse skeletal muscle this work was supported by glaxosmithkline pharmaceuticals. jmw is an inventor on patents licensed to various commercial entities. key: cord-273379-w8vy5rl8 authors: mizutani, tetsuya; repass, john f.; makino, shinji title: nascent synthesis of leader sequence-containing subgenomic mrnas in coronavirus genome-length replicative intermediate rna date: 2000-09-30 journal: virology doi: 10.1006/viro.2000.0489 sha: doc_id: 273379 cord_uid: w8vy5rl8 abstract infection with coronavirus results in the accumulation of genomic-sized mrna and six to eight subgenomic mrnas that make up a 3′ coterminal nested-set structure. genome-length negative-strand rna and subgenomic-length negative-strand rnas, each of which corresponds to each of the subgenomic mrnas, also accumulate in infected cells. the present study examined whether the genome-length negative-strand rna serves as a template for subgenomic mrna synthesis. genome-length replicative intermediate (ri) rna was purified by two-dimensional gel electrophoresis of intracellular rnas from cells infected with mouse hepatitis virus. rnase a treatment of the purified genome-length ri resulted in the production of the genome-length replicative form rna, indicating that the genome-length ri included genome-length template rna. rnase protection assays using the purified genome-length ri and two probes, which corresponded to the 5′ 300-nt region of mrna 6 and to the same region of mrna 7, showed the presence of nascent leader sequence-containing subgenomic mrnas in the genome-length ri. these data demonstrated that the genome-length negative-strand rna serves as a template for subgenomic mrna synthesis. coronavirus is an enveloped rna virus containing a large positive-strand genomic rna. coronavirus-infected cells produce a genomic-sized mrna, mrna 1, and six to eight species of successively smaller subgenomic mrnas, which form a 3ј coterminal nested-set structure (1) (2) (3) . characteristics of the unique coronavirus nestedset structure are that all the mrnas are polyadenylated at the 3ј-end; the messages are successively larger by one gene at the 5ј-end with only the 5ј-most gene on the polycistron being translated; and each has a 60-to 90nt-long leader sequence at the 5ј-end (4) (5) (6) . the leader sequences are derived from the 5ј-end of genomic rna (the only place on the genome where they are encoded) and, by an unknown type of discontinuous synthesis (7) , they join the 5ј-ends of each of the messages at the intergenic regions. infected cells also produce negativestrand genomic rna and subgenomic rnas, each of which corresponds to one mrna species (8) . these negative-strand rnas contain a poly (u) sequence at the 5ј-end and an anti-leader sequence at the 3ј-end (9) . like coronaviruses, cells infected with arteriviruses produce a 3ј coterminal nested set of mrnas with the leader sequence at the 5ј-end of the mrnas (10) . subgenomic negative-strand rnas that correspond to each of the subgenomic mrnas are also produced in arteri-virus-infected cells. coronaviruses and arteriviruses are classified under the newly established order nidovirales; both viruses probably use similar transcription strategies. one of the unsolved questions in nidovirus transcription is whether the template for subgenomic mrnas is of genome length or subgenome length. two major models have been proposed. one suggests that a genomelength negative-strand rna serves as a template for all the mrnas (11) , while the other model proposes that each negative-strand subgenomic rna is a unique template for synthesis of the correspondingly sized subgenomic mrna (8, (12) (13) (14) (15) . a study using mouse hepatitis virus (mhv), a prototypic coronavirus, demonstrated that early in infection negative-strand genomic rna, and not negative-strand subgenomic rna, is the template for subgenomic mrna synthesis (16) ; this study did not determine the template late in infection. here we describe whether mhv negative-strand genomic rna is also the template for subgenomic mrna synthesis late in infection. previous studies of replicative intermediate (ri) rnas in mhv-infected cells were based on the detection of two different types of ri rnas: subgenomic ris and genome-length ri. after a short pulse of radiolabeling, the subgenomic ris (13) appear as six to seven discrete rna bands when separated by using nondenaturing agarose gel electrophoresis. the polarity of these elongating rnas in the subgenomic ris has not been identified. the genome-length ri rna appears as a slowly migrating diffuse band on a nondenaturing electrophoretic gel (11, 13) . rnase a treatment of the genome-length ri rna results in the production of replicative-form (rf) rna 1, which comigrates with mrna 1 (11, 13) , demonstrating that the nascent rnas are elongating on genome-length template rna in the genome-length ri. rnase t1 fingerprinting analysis showed that the majority of elongating rnas in the genome-length ri are positive-sense (11) , strongly indicating that the genomelength ri rna must contain a genome-length negativestrand rna template. accordingly, if the negative-strand genome-length mhv rna is a template for subgenomic mrna synthesis, then all the subgenomic mrnas are likely to be elongating in a genome-length ri rna. to test for the possibility that subgenomic mrnas are elongating from the negative-strand genome-length mhv rna in the genome-length ri rna, we first purified genome-length ri rna. dbt cells were infected with the a59 strain of mhv at a multiplicity of infection of 20; defective interfering rnas were undetectable in this stock of mhv (13) . the mhv rnas were radiolabeled from 4 to 7 h postinfection (p.i.) with 32 pi in the presence of actinomycin d as described previously (17) . intracellular rna was extracted at 7 h p.i. using the totally rna kit (ambion), treated with dnase i, and applied onto an oligo-dt cellulose chromatography column. the rna fraction that did not bind to the oligo-dt cellulose was collected; this step lowered the amount of poly (a)-containing mature mhv mrnas in the rna preparation. then the rna sample was applied to a two-dimensional 0.8% tbe (89 mm tris, 89 mm boric acid, 2 mm edta) agarose gel containing ethidium bromide (fig. 1) . we hoped that the two-dimensional gel electrophoresis would efficiently separate genome-length ri rna from mhv subgenomic mrnas. a strong diffuse radioactive rna signal, which migrated slower than the 24-kbp-long lambda hindiii marker dna fragment, was detected after two-dimensional gel electrophoresis. the gel region that migrated more slowly than the 24-kbp-long dna fragment was excised carefully (fig. 1 ). based on a previous study of the genome-length ri (13) , this region of the gel should contain the genome-length ri. we further separated the rna in the gel slices under different electrophoretic conditions to estimate the purity and the properties of the gel-purified genome-length ri. first we looked for contamination of subgenomic mrnas in the gel-purified genome-length ri by processing the gel piece obtained after two-dimensional gel electrophoresis in a new, nondenaturing 0.8% tbe gel next to 32 p-labled mhv intracellular rnas ( fig. 2a) . electrophoresis of the purified gel piece resulted in a diffuse slowly migrating signal whose migration pattern was similar to the published mhv genome-length ri rna (11, 13) . the purified gel pieces did not contain mhv mrnas even after a long exposure of the gel (data not shown). next we examined the production of the rf1 rna after rnase a treatment of the purified gel pieces. the gel piece was treated with rnase a (0.33 g/ml) in a buffer (233 mm nacl, 3.3 mm tris-hcl, ph 7.4, 10 mm edta) for 5 min at room temperature. after rnase a treatment, the gel piece was soaked in tbe buffer for 10 min at room temperature. then the gel piece was placed into the well of a 0.8% tbe agarose gel and electrophoresed alongside labeled mhv intracellular rnas (fig. 2b) , which resulted in a single band that roughly comigrated with mrna 1. subgenomic rf rnas were not detected even after much longer exposure of the gel. the migration of this rnase-a-resistant band was very similar to that of the rf 1 rna described previously (11, 13) . we further fig. 1. two-dimensional gel electrophoresis of the poly(a)-minus fraction of mhv-intracellular rnas. 32 p-labled mhv-infected intacellular rnas that did not bind to oligo-dt cellulose were mixed with lambda hindiii marker dna fragments and separated on 0.8% nondenaturing two-dimensional gel electrophoresis. (a) ethidium bromide staining of the gel after two-dimensional gel electrophoresis. lambda hindiii marker dna fragments and ribosomal rnas (28s rrna and 18s rrna) are shown. (b) autoradiogram of the two-dimensional gel. the box region containing genome-length ri was excised from the gel and used for further study. the asterisk is an artifact of the gel electrophoresis. a low level of mrna 7, which was not completely removed by oligo-dt cellulose column chromatography, is also shown. confirmed the presence of the genome-length ri rna in the purified gel piece under denaturing gel electrophoresis. rnas were extracted from the gel-purified gel pieces using the rnaid kit (bio 101) and then separated using denaturing formaldehyde gel electrophoresis (fig. 2c) . we only detected a smeary signal, the size of which ranged from genomic in length to less than the size of mrna 7; we saw no discrete mhv mrnas. incubation of genome-length ri at 65°c for 10 min and subsequent separation in a 0.8% tbe agarose gel also produced a similar smeary rna signal (data not shown). we interpreted this absence of discrete mrnas with the presence of a smeary signal to mean that the smear represented nascent rnas that were elongating in genomelength ri. other experiments showed poor recovery of rna molecules larger than mrna 3 from a gel, while recovery of small rnas was excellent (unpublished data). this reduced efficiency of large rna recovery from the first gel appears as a drop in signal above the size of mrna 3 in the second separation (fig. 1c) . we repeated these experiments at least three times and obtained consistent results. we purified genome-length ri without contamination of any detectable level of mhv subgenomic mrnas. we used rnase protection assays to look for nascent subgenomic mrnas containing the leader sequence in the genome-length ri. for these assays, an excess of two nonradiolabeled negative-sense rna probes, one complementary to the 5ј 300-nt region of mrna 6 (probe 1) and the other to the same region in mrna 7 (probe 2), were independently mixed with heat-denatured 32 p-labeled purified genome-length ri. after hybridization and subsequent rnase a and rnase t1 treatment, rnaseresistant rna fragments were detected on electrophoretic sequence gels. if leader-sequence-containing subgenomic mrna 6 elongates on the genome-length ri, then the rnase protection assay using probe 1 and purified radiolabeled genome-length ri should produce a radiolabeled 300-nt-long rna fragment (300-nt fragment) that corresponds to the 5ј-end 300 nt of nascent mrna 6 (see fig. 3a ). in addition, two rna bands of approximately 70 and 240 nt long (240-nt fragment) should be detected, the former corresponding to the leader sequence of all the nascent mrnas other than mrna 6 and the latter to the 5ј 240-nt region of the mrna 6 body sequence, which is found in nascent mhv mrnas from 1 through 5. similar results were expected for the rnase protection assay using probe 2; in this case a 300-nt-long radiolabeled rna fragment should represent nascent leader-sequence-containing mrna 7 (fig. 3a) . if only genome-length mrna 1 elongates on genome-length ri, then the rnase protection assays using either probe should yield just a 70-nt-long band and a 240-nt-long band. two pcr products, each corresponding to the most 5ј 300 nt of mrna 6 and mrna 7, were cloned into a ta cloning vector (invitrogen). probe 1 and probe 2, the nonradiolabeled negative-strand rna transcripts, were transcribed from cloned plasmids using the t7 mmes-sage mmachine kit (ambion). in a separate experiment, 32 p-labeled genome-length ri was prepared using two-dimensional gel electrophoresis as described above. rnas were extracted from the gel pieces by using the rnaid kit, heat denatured at 65°c for 10 min, and quickly chilled on ice. labeled rnas were then applied to an oligo-dt cellulose chromatography column. we collected the rna fraction that bound to the oligo-dt cellulose, the poly(a)-containing fraction, and the fraction that did not bind to the oligo-dt cellulose, the poly(a)minus fraction. we assumed that the poly(a)-containing fraction included nascent poly(a)-containing mrnas elongating in the genome-length ri and mature mrnas that might be contaminating the purified genome-length ri preparation; and we assumed that the poly(a)-minus would include mostly nascent rna molecules and possibly be contaminated with degraded mature mhv rnas that lacked the poly(a) sequence. we separately mixed each of the rna fractions with each of the probes and performed the rnase protection using the riboquant kit (pharmingen), according to the manufacturer's instruction. after rnase a and t1 treatment, the samples were incubated with proteinase k and extracted using phenolchloroform, and the rnas were precipitated with ethanol. finally the rnas were applied to a 6% polyacrylamide-urea gel. control assays combining 32 p-labeled intracellular rnas from mhv-infected cells with probe 2 and with probe 1 both yielded the 300-and the 240-nt fragments (figs. 3b and 3c). production of these two expected fragments demonstrated that this rnase protection assay could specifically distinguish mrna 6 and mrna 7 from intracellular viral mrna species. the 240-nt fragment appeared as two closely migrating discrete bands (fig. 3b) ; a shorter exposure of fig. 3c , lane 1, also showed two closely migrating discrete bands making up the 240-nt fragment. the presence of sequence heterogeneity at the leader-body fusion site of mhv mrna 6 (18) may be the reason for the two closely migrating 240-nt fragments in the rnase protection assay using probe 2. however, the origin of two 240-nt fragments in the rnase protection assay using probe 1 is unclear, because extensive sequence heterogeneity does not exist at the leader-body fusion site of mrna 7 (18) . it is possible that the rnase protection assay did not quantitatively reflect the actual amounts of individual intracellular viral mrna species. the rnase protection assay using the poly(a)-minus fraction of the purified genome-length ri and probe 1 as well as the same rna fraction and probe 2 both showed the 300-nt fragment and the 240-nt fragment (figs. 3b and 3c ). in contrast, neither the 300-nt nor the 240-nt fragment was detected when the poly(a)-containing fraction of the purified genome-length ri was used in combination with probe 2 or with probe 1. in three repeated experiments, we consis-tently detected 300-and 240-nt fragments using the poly(a)-minus fraction, while these bands were underdetectable or detected at a very low level using the poly(a)containing fraction. if the purified genome-length ri were contaminated with a large amount of mrna 6 and mrna 7, then we should have detected the 300-nt-long fragment in the poly(a)-containing fraction. the underdetectable levels of the 300-nt-long fragment and the 240-nt fragment in the poly(a)-containing fraction indicated that contamination with mrnas was very low in the gel-purified genomelength ri. consistent with rnase protection assay data, contamination of mature mhv subgenomic mrnas in purified genome-length ri was at an underdetectable level when purified genome-length ri was separated by agarose gel electrophoresis (fig. 2) . in contrast, we clearly detected the 300-nt fragment in the poly(a)-minus fraction with either probe 1 or probe 2. as discussed above, this 300-nt fragment could be produced from nascent leader-sequence-containing mrna 6 and mrna 7 and/or from degraded mhv rnas lacking the poly(a) sequence that might be residually present in the purified genome-length ri. our preparation of mhv intracellular rna showed minimal rna degradation ( fig. 2a) ; therefore, in our rnase protection assays we think that the possibility that the 300-nt fragment was solely produced from contaminating degraded mrnas is highly unlikely. more likely, the 300-nt fragment in the poly(a)-minus fraction was produced from nascent subgenomic mrna 6 and mrna 7, both of which contain the leader sequence. theoretically, nascent leader-sequence-containing mrnas 6 and 7, which are shorter than 300 nt in length, should produce a smeared rna signal in the rnase protection assay. we could not detect the expected smeared rna signals, so we supposed that the signal was too low to be detected under the present experimental conditions; in both autoradiograms, gels were exposed to x-ray films for 3 weeks, and further exposure had little effect on signal detection. rnase a treatment of the gel-purified genome-length ri produced rf 1 (fig. 2b) , and the rnase protection assay demonstrated the presence of nascent leader-sequence-containing subgenomic mrnas in the genome-length ri. apparently, leader-sequence-containing subgenomic mrnas were produced from the genome-length negative-strand rna in the genome-length ri. these data are consistent with the model of coronavirus discontinuous transcription (7, 11) , which involves fusion of a leader sequence to every mrna body during subgenomic mrna synthesis. the characteristics of the genome-length ri that we described here agree with earlier analyses of mhv genome-length ri (11, 13) . sawicki and sawicki (13) described a slowly migrating genome-length ri during electrophoresis of intracellular rna from mhv-infected cells in a nondenaturing gel. their treatment of the gel fraction containing the genome-length ri with rnasea gave mostly rf 1 and a low level of subgenome-length rf rnas, rf 2 and rf 3; rf 2 and rf 3 may well have been due to the presence of contaminating subgenomic ris in the genome-length ri preparation. baric et al. also showed that genome-length ri rna purified by sepharose 2b-cl column chromatography migrates slowly on nondenaturing gels appearing as a smeared rna and produces rf 1 after rnase a treatment (11) . the authors compared rnase t1 fingerprinting patterns of columnpurified genome-length ris under two conditions. under one condition the genome-length ri was digested with rnase t1 without denaturation and under the other condition the genome-length ri was digested after denaturation of rna. they found that t1 spots corresponding to leader sequence and the leader-body fusion site for subgenomic mrnas appear as strong spots in the sample treated under the nondenaturing condition (11) . these data led the authors to propose that leader-sequence-containing subgenomic mrnas elongate on genome-length ri. although the previous study using the column-purified genome-length ri (11) and the present study used different experimental approaches to examine the nascent rnas in the genome-length ri, the data from these two independent studies indicated that mhv genome-length negative-strand rna is the template rna for subgenomic mrna synthesis. we showed that negative-strand genomic rna is the template for subgenomic mrna synthesis early in infection (16) . our new data strongly support the idea that negative-strand genomic rna is a template for subgenomic mrna synthesis throughout the infection. rnase a treatment of mhv genome-length ri, in which nascent subgenomic mrnas are elongating on a genome-length negative-strand rna, resulted in rf 1. in contrast, alphaviruses, which produce a subgenomic mrna that corresponds to the 3ј-region of viral genome, produce three rf rnas after rnase a treatment of alphavirus ri rna; with this virus, genome rna and subgenomic rna elongate on a genome-length negativestrand rna in its ri rna (19) . why rnase a treatment of coronavirus genome-length ri produces rf 1 but not subgenome-length rfs is unknown. although the present study and the previous study (16) established that negative-strand genomic rna is a template for mhv subgenomic mrna synthesis throughout the infection, these studies do not eliminate the possibility that mhv subgenomic mrna is also synthesized from subgenomic negative-strand template rna late in infection. a number of studies aimed to test whether positive-strand rna synthesis occurs using subgenomic negative-strand template rna in cells infected with coronaviruses (8, (12) (13) (14) and with arterivirus (15), yet did not provide direct evidence of positive-strand rna elongation from the subgenomic-length negative-strand template. sawicki and sawicki revealed the presence of subgenomic ris in mhv-infected cells (13) , and their data directly demonstrated elongation of nascent rna on subgenomic-length template in infected cells, however, the polarity of the template rna in the subgenomic ris was not described. it is interesting to note that subgenomic ris appear as discrete rna bands that comigrate with subgenomic rfs, which were produced after rnase a treatment of subgenomic ris (13); electrophoretic patterns of subgenomic ris imply that subgenomic ri exists as a completely double-stranded form. because ri rnas that are efficiently synthesizing nascent rna molecules appear as a diffuse rna signal in nondenaturing gels, multiple nascent rnas may not be produced in mhv subgenomic ris. instead, subgenomic ris may exist as complete double-stranded rnas, in which newly synthesized subgenome-length rna of either polarity hybridizes with its subgenomic-length template rna. recently, baric and yount reported the kinetic radiolabeling analysis of mhv intracellular rnas (12) . they demonstrated that the incorporation of radioisotopes into a subgenomic rf rna, rf 7, increases rapidly during 5-min pulse-labeling of mhv-infected cells; mhv rf rnas were obtained after rnase treatment of 32 plabeled mhv intracellular rnas. the authors interpreted these data that the incorporation of radioisotopes into nascent positive-strand rnas, which are elongating on subgenomic negative-strand template rna in the subgenomic ri, is saturated quickly. they also showed the gradual increase of the rf 7 radioactivity after 5-min pulse-labeling, which they interpreted to represent the synthesis of a constant and low level of subgenomic negative-strand rna, which is used as a template for subgenomic mrna synthesis. these interpretations of the data led the authors to claim that nascent positivestrand rnas are elongating on subgenomic negativestrand template rna in the subegenomic ri (12) . however, their data can be interpreted differently such that subgenomic ris are continuously produced efficiently in infected cells, whereas many of them are quickly degraded. because subgenomic ris may exist as complete double-stranded rnas, subgenomic ris can be an excellent interferon inducer; efficient accumulation of subgenomic ris in infected cells may elicit the interferon activation pathway. it is not surprising that mhv has developed a mechanism that degrades most subgenomic ris to inhibit double-strand rna accumulation in infected cells. none of the published studies demonstrated the polarity of nascent rnas elongating on subgenomic ris in nidovirus-infected cells, hence it is possible that nascent negative-strand rnas are elongating on subgenomic ris. the data presented by baric and yount (12) do not exclude the possibility that subgenomic negative-strand rna is elongating in the subgenomic ri rna, which is unstable in infected cells. mouse hepatitis virus a59: mrna structure and genetic localization of the sequence divergence from hepatotropic strain mhv-3 the virus-specific intracellular rna species of two murine coronaviruses: mhv-a59 and mhv-jhm coronavirus multiplication strategy. ii. mapping the avian infectious bronchitis virus intracellular rna species to the genome presence of leader sequences in the mrna of mouse hepatitis virus characterization of leader rna sequences on the virion and mrnas of mouse hepatitis virus, a cytoplasmic rna virus coronavirus mrna synthesis involves fusion of non-contiguous sequences evidence for coronavirus discontinuous transcription coronavirus subgenomic minus-strand rnas and the potential for mrna replicons minus-strand copies of replicating coronavirus mrnas contain antileaders the molecular biology of arteriviruses characterization of replicative intermediate rna of mouse hepatitis virus: presence of leader rna sequences on nascent chains subgenomic negative-strand rna function during mouse hepatitis virus infection coronavirus transcription: subgenomic mouse hepatitis virus replicative intermediates function in rna synthesis genetics of mouse hepatitis virus transcription: evidence that subgenomic negative strands are functional templates arterivirus discontinuous mrna transcription is guided by base pairing between sense and antisense transcription-regulating sequences coronavirus transcription early in infection analysis of genomic and intracellular viral rnas of small plaque mutants of mouse hepatitis virus discontinuous transcription generates heterogeneity at the leader fusion sites of coronavirus mrnas replication of sindbis virus. ii. multiple forms of double-stranded rna isolated from infected cells acknowledgment this work was supported by public health service grant ai29984 from the national institutes of health. key: cord-283309-ovx5fzsg authors: yang, yong-le; liang, qi-zhang; xu, shu-ya; mazing, evgeniia; xu, guo-han; peng, lei; qin, pan; wang, bin; huang, yao-wei title: characterization of a novel bat-hku2-like swine enteric alphacoronavirus (seacov) infection in cultured cells and development of a seacov infectious clone date: 2019-08-09 journal: virology doi: 10.1016/j.virol.2019.08.006 sha: doc_id: 283309 cord_uid: ovx5fzsg swine enteric alphacoronavirus (seacov), also known as swine acute diarrhea syndrome coronavirus (sads-cov), belongs to the species rhinolophus bat coronavirus hku2. herein, we report on the primary characterization of seacov in vitro. four antibodies against the seacov spike, membrane, nucleocapsid and nonstructural protein 3 capable of reacting with viral antigens in seacov-infected vero cells were generated. we established a dna-launched seacov infectious clone based on the cell adapted passage-10 virus and rescued the recombinant virus with a unique genetic marker in cultured cells. six subgenomic mrnas containing the leader-body junction sites, including a bicistronic mrna encoding the accessory ns7a and ns7b genes, were experimentally identified in seacov-infected cells. cellular ultrastructural changes induced by seacov infection were visualized by electron microscopy. the availability of the seacov infectious clone and a panel of antibodies against different viral proteins will facilitate further studies on understanding the molecular mechanisms of seacov replication and pathogenesis. swine enteric alphacoronavirus (seacov), also known as swine acute diarrhea syndrome coronavirus (sads-cov), is a novel porcine enteric coronavirus that causes acute vomiting and watery diarrhea in piglets (gong et al., 2017; pan et al., 2017; zhou et al., 2018) . this emerging virus was first isolated from clinically sick animals in commercial swine herds at guangdong province, china during february-may 2017. the mortality rate in less than 5 days old piglets was over 90%, whereas it dropped to 5% in piglets older than 8 days . the clinical samples examined by polymerase chain reaction (pcr) or reverse transcription pcr (rt-pcr) during laboratory investigation were negative for the other swine coronaviruses such as porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev), porcine deltacoronavirus (pdcov) and porcine hemagglutinating encephalomyelitis virus (phev), as well as the other known viral pathogens . isolation of the pathogen in african green monkey vero cells resulted in the discovery of seacov , which belongs to the species rhinolophus bat coronavirus hku2 identified in the same region a decade earlier (lau et al., 2007) . a retrospective study indicated that the virus had emerged in guangdong since august 2016 . the isolated virus was infectious to pigs and cause mild or severe diarrhea symptom when inoculated orally into conventional newborn piglets xu et al., 2019; zhou et al., 2018) . nevertheless, as seacov fulfilled the premises of koch's postulates, this was regarded to be the etiologic agent of the epidemic. like other covs, seacov is a single-stranded and positive-sense rna virus in the genus alphacoronavirus (α-covs) of the subfamily coronavirinae of the family coronaviridae. its genome is approximately 27.2 kb in size with the gene order of 5'-orf1a/1b (orf1ab)-spike (s)-orf3-envelope (e)-membrane (m)-nucleocapsid (n)-ns7a/ns7b-3'. seacov shared 95% nucleotide (nt) sequence identity with the bat cov hku2 strains and 96-98% nt identity with the hku2-derived bat sadsrelated coronavirus (sadsr-cov) strains at the complete genome level zhou et al., 2018) . interestingly, seacov and other hku2-related α-covs possess the unique s genes closely related to the betacoronavirus (β-cov), in a manner similar to those by rodent and asian house shrew α-covs (tsoleridis et al., 2019; wang et al., 2015 wang et al., , 2017b between α-cov and β-cov (lau et al., 2007; pan et al., 2017) . the cov genome harbors a few genus-specific accessory genes within the 3'-part genomic region encoding the four structural proteins (s-e-m-n) . it is found that seacov contains a putative open reading frame (orf), ns7a, and a downstream ns7b orf (overlapped with ns7a) after the n gene at the 3'-end genome (lau et al., 2007; pan et al., 2017) . the ns7a is shared by the hku2 and seacov strains, whereas ns7b is only present in the seacov genome . many of cov accessory proteins play some important roles in immune modulation and viral pathogenesis (liu et al., 2014) . for examples, the severe acute respiratory syndrome coronavirus (sars-cov) orf-3a was found to induce necrotic cell death, lysosomal damage and caspase-1 activation, which largely contribute to the clinical manifestations of sars-cov infection (yue et al., 2018) . in addition, sars-cov orf6 and orf7b may also be also associated with the virulence. in another newly emerged swine cov, pdcov, its accessory ns6 protein has been reported to counteract host innate antiviral immune response by inhibiting ifn-β production that interacts with rig-i/mda5 (fang et al., 2018) . whether the predicted ns7a and ns7b of seacov encode functional accessory proteins remain to be confirmed experimentally. discovery of seacov, largely dissimilar to pedv, tgev and pdcov, challenges to the prospects of detection, prevention and control of diarrheal pathogens in swine . it is pivotal to undertake comprehensive investigations on the basic genetics of this emerged enteric cov since very little is known about the molecular virology of seacov. the purpose of this study was to develop seacovspecific antibodies to distinct viral protein as the research tools used to investigate the basic characteristics of seacov infection in vitro. we also aimed to develop a dna-launched reverse genetics system for seacov that will be useful for future studies. 2.1. polyclonal antibodies against four recombinant seacov proteins can react with viral antigens in seacov-infected cells four seacov specific polyclonal antibodies (pabs) against distinct viral protein antigens were generated and validated. two viral genes, seacov n and the nonstructural protein 3 (nsp3) acidic domain (ac) of orf1a, were expressed as soluble products in the bacteria; the seacov spike subunit 1 (s1) was expressed in insect cells, secreting into the cultured medium. purified recombinant seacov proteins (n, s1 and ac) and an antigenic peptide corresponding to the last 14 amino acids (aa) at the carboxyl terminus of the m protein were used to immunize rabbits, respectively, generating four polyclonal sera that were then used to detect viral proteins on seacov-infected vero cells. immunofluorescence assay (ifa) conducted at 48 h post-infection (hpi) using respective pab showed that the four viral antigens (n, m, s1 or ac) were each expressed in the cytoplasm of the infected cells, with the anti-n and anti-m pabs displaying the higher fluorescence intensity (fig. 1a) . in contrast, mock-infected controls did not show any positive ifa signals (fig. 1a) . to determine the intracellular localization and the timing of the viral protein expression with higher magnification, time course analysis of confocal image was performed. vero cells infected with seacov were fixed at 4, 8, 12, and 24 hpi, and labeled with four pab, respectively. perinuclear and cytoplasmic foci were detected by anti-n staining at 4 and 8 hpi, and were distributed throughout the cytoplasm at 12 and 24 hpi, probably reflecting that n protein is associated with sites of viral rna replication in early infection phase and assembled into virions subsequently (fig. 1b) . anti-ac (nsp3) staining also resulted in detection of perinuclear foci at four time points, indicating localization to the viral replication-transcription complexes (fig. 1c) , which was similar to the pattern of nsp3 antibody observed in sars-cov-infected vero cells (prentice et al., 2004) . confocal microscopy detected discrete cytoplasmic fluorescence signal throughout the cytoplasm with anti-m (fig. 1d ) and anti-s1 (fig. 1e ) as early as 4 hpi. diffuse and more intense fluorescence was observed over time, demonstrating the process of virus assembly by incorporation of m and s proteins into virus particles. the anti-n pab recognized a single band of 42 kda in the lysate of seacov-infected cells but not in control cells at 48 hpi by western blot analysis (fig. 1f ). the molecular size was consistent with the deduced aa sequence of the n protein but was a little less than the purified products expressed in the bacteria (fig. 1f) . expression of the m protein with the predicted 25-kda molecular size was also detected by using anti-m pab in seacov-infected cells (fig. 1g ). the reactivity of anti-s1 or anti-ac was less distinct as seen by western blot analysis (data not shown). therefore, all the four seacov pabs can be used for specific detection of seacov infection in the cultured cell by ifa staining, and the anti-n and anti-m pabs can also be used particularly in western blot analysis. the antibodies are available to the research community upon request. genetic manipulation of viral genomes and dissection of the structural and functional relationships of viral genes depend on the development of powerful reverse genetics systems. thus far, the rna polymerases ii-based dna-launched reverse genetics system using a bacterial artificial chromosome (bac) as the backbone vector has been applied to rescue of multiple covs (almazan et al., 2014) . basically, homogenous rna transcripts are generated from transfected full-length cdna clone in permissive cells to launch virus life cycle. recently, our lab has just developed a novel and efficient method to assemble a fulllength cdna clone of measles virus (~16 kb) by using the geneart™ high-order genetic assembly system, without the need for restriction endonucleases, which was used to rescue recombinant measles virus and the derived vaccine candidates . we employed this strategy successfully to assemble the 27.2-kb seacov genomic cdna from the passage-10 virus ("seacov-p10") by a single step ligation of 15 overlapping fragments into a bac expression vector, resulting in a full-length cdna clone of seacov named psea ( fig. 2a) . the seacov genomic cdna cassette on psea was engineered with a cytomegalovirus (cmv) promoter and a hepatitis delta virus ribozyme (hdvrz) followed by a bovine growth hormone polyadenylation and termination sequences (bgh) at both termini, respectively. in addition, two silent mutations (a24222t and g24223c) in orf3 were introduced in psea as a genetic marker to distinguish the parental virus seacov-p10 ( fig. 2a) . bhk-21 cells were co-transfected with psea and a helper plasmid expressing the n protein (prk-n) in order to recover the infectious seacov. supernatants from transfected bhk-21 cells were inoculated onto fresh vero cells at 2-3 days post-transfection. seacov-induced cytopathic effects (cpe) were visualized at 48 hpi in inoculated vero cells; viral antigens were detected by ifa using anti-n, anti-m, anti-s1 or anti-ac to stain cells, confirming the successful recovery of recombinant seacov (rseacov; fig. 2b ). a region containing the marker from extracellular and intracellular samples of extracted viral rna was amplified and sequenced to determine the retention of the genetic markers in the rescued viruses. the two introduced mutations (tc) were still present in both samples, confirming that the rescued virus originated from the clone psea (fig. 2c ). there were no other mutations detected in genomic rna of rseacov by genome re-sequencing. we further assessed the morphology of the purified rseacov virions via ultracentrifugation followed by em observation. the virus particles measured 100-120 nm in diameter with surface projections (fig. 2d) , consistent with our previous report of seacov isolation in vero cells . the comparative growth kinetics of rseacov and the parental seacov-p10 were analyzed by infection of vero cells with the respective virus at the same multiplicity of infection (moi) of 0.1. the infectious virus titers were determined at different time points postinfection (2, 6, 12, 24, 36, 48, 60 and 72 hpi) . the result showed that rseacov had the growth kinetics similar to the parental seacov-p10 ( fig. 2e ). of note, the maximal rates of seacov-p10 or rseacov production were from 6 to 12 hpi, suggesting that the exponential release of virus occurred before 6 hpi, which was consistent with detection of n, m, s and ac expression as early as 4 hpi ( fig. 1b-e) . the single-cycle growth of seacov in vero cells is hence similar to those of mouse hepatitis virus (mhv), sars-cov and pdcov, taking approximately 4-6 h (prentice et al., 2004; qin et al., 2019) . these data collectively demonstrated that rseacov and its parental virus share the same virological features. to our knowledge, this is the first study describing a seacov/sads-cov infectious clone. previous studies on cov reverse genetics have shown that cov accessory genes such as orf3 [in tgev , sars-cov (yount et al., 2005) , pedv (ji et al., 2018) or human cov nl63 (donaldson et al., 2008) ] and the gene 7 [in tgev (ortego et al., 2003) ] are dispensable for propagation in vitro. the corresponding genes, orf3 and ns7a, are also present in the seacov genome; therefore, we will aim to generate reporter virus expressing luciferase or green fluorescent protein by replacement of orf3 or ns7a with the reporter gene in future studies. coronaviruses can produce multiple sgrnas are produced by discontinuous transcription. each sgrna contains a short 5' leader sequence derived from the 5'-end of the genome and a body sequence from the 3'-poly (a) stretching to a position in the upstream of each orf encoding a structural or accessory protein (sola et al., 2015) . the fusion site of the leader and body sequence in each sgrna is termed transcription regulatory sequence (trs). the seacov leader sequence of 75 nt from the 5'-end to the leader trs was proposed according to the previous report (lau et al., 2007) ; it was compared with that of another swine α-cov, pedv, indicating an identical leader trs sequence (aactaaa) shared by these two α-covs (huang et al., 2013) (fig. 3a) . the existence of all predicted subgenomic mrnas (sgrna; mrna 2 to mrna 7) for the expression of s, orf3, e, m, n and ns7a was investigated further (fig. 3b) . the leader-body junctions and surrounding regions of all of the putative sgrnas were amplified by rt-pcr. each of the combination of the forward primer (lf) and one of the six reverse primers (s1-r, sgorf3-r, sge-r, sgm-r, sgn-r and ns7a-r) amplified at least one major band of the expected size by agarose gel electrophoresis analysis (fig. 3c ). the appearance of multiple pcr bands was in line with what was expected, since except for the primers lf and s1-r, the other primer combinations could produce larger pcr fragments that seacov-infected or mock-infected vero cells with an anti-n-pab, an anti-m-pab, an anti-s1-pab and an anti-ac-pab, respectively (magnification = 200×). alexa fluor 488-conjugated goat anti-rabbit igg (green) was used as the secondary antibody in the ifa. antibody staining merged with nuclear staining using dapi (blue) is also shown. (b-e) time course analysis of n, ac, m or s1 detection using an olympus confocal microscope. vero cells infected with seacov were fixed at 4, 8, 12, and 24 hpi, and labeled with four pabs, respectively. bar = 10 μm. (f) western blot analysis using cell lysates of seacov-infected or mock-infected vero cells with an anti-n pab. the purified n protein expressed in e.coli was used as the control. (g) western blot analysis using cell lysates of seacov-infected or mock-infected vero cells with an anti-peptide pab specific to m. open arrowheads indicate the detected n or m protein. correspond to the upstream-larger sgrnas. for examples, the primer sgn-r, intended to amplify the leader-body fusion site of mrna 6, could also amplify those of mrnas 2 to 5, resulting in detection of five bands (fig. 3c) . sequencing of individual pcr fragments confirmed that the leader-body junction sequences of sgrnas are identical to the conserved core elements in the intergenic trs (fig. 3d) . we also noticed that both orfs of ns7a and ns7b are connected with a body trs in the upstream, implying a bicistronic mrna encoding ns7a and ns7b (fig. 3e ). since amplification with the reverse primer ns7a-r could not cover the entire ns7b, we next determined whether a potential ns7b sgrna is present using the leader primer lf and a new reverse primer ns7-r corresponding to the 3'-end of orf7b by rt-pcr. a single band of approximately 400-bp was amplified by optimizing the pcr condition and detected by agarose gel electrophoresis analysis; the other smaller bands were not found (fig. 3e) . sequence analysis revealed that the trs for this bicistronic sgrna ns7 was exactly aacuaaa and one nt upstream of the aug start codon of ns7a, which is consistent with the prediction (fig. 3e) . we further expressed and purified the complete ns7a or ns7b gene in the bacteria. both products were found in the inclusion bodies. however, the resulting anti-ns7a or anti-ns7b pab did not react with any antigens in seacov-infected cells by ifa and western blot analysis (data not shown) in contrast to the four working seacov pabs. this suggests that ns7a and ns7b are either, not highly antigenic or the denatured antigens used to generate pabs destroy the native protein structure. development of monoclonal antibodies against ns7a and ns7b used for experimental validation of the existence of two expression products at the protein level is underway. (e) comparison of growth kinetics between rseacov and the parental seacov-p10 in vero cells. cells were infected in triplicate with virus at a moi = 0.1. cells were harvested at 2, 6, 12, 24, 36, 48, 60 and 72 hpi, and virus titers (tcid 50 /ml) were determined in triplicate on vero cells. a number of studies on ultrastructural characterization of cov-infected cells in vitro have demonstrated the presence of altered membrane architectures such as the double-membrane vesicles (dmvs), the large virion-containing vacuoles (lvcvs) and the phagosome-like vacuoles during cov replication and morphogenesis (goldsmith et al., 2004; gosert et al., 2002; qin et al., 2019; salanueva et al., 1999; v'kovski et al., 2015) . dmvs are membrane structures where viral genomic rna is recognized by the host cell machinery and translated into non-structural proteins (orf1ab), assembling into viral replication-transcription complexes (gosert et al., 2002) , whereas lvcvs are large circular organelles that are thought to originate from golgi compartments expanding to accommodate numerous precursor virions positions of forward (lf) and reverse primers (s1-r, sgorf3-r, sge-r, sgm-r, sgn-r and ns7a-r/ns7-r) used for pcr amplification of distinct subgenomic mrnas (sgrnas) are indicated by arrows under the genome. the seven small black boxes at the 5' ends of the genomic rna (grna) and sgrnas depict the common leader sequence. genomic and subgenomic rna numbers (1 for grna and 2 to 7 for sgrnas) are also indicated. . the other type of membrane structure usually seen is phagosome-like vacuoles or lysosomes containing endoplasmic reticulum (er), small vesicles, damaged mitochondrion and other vesicles. these conserved structures were also observed directly under an electron microscope (em) in seacov-infected vero cells ( fig. 4a ; 24 hpi) but not in uninfected cells (fig. 4c) . of note, time course analysis of nsp3 detection in fig. 1c likely indicated corresponding locations of the dmvs. since infection of vero cells with either seacov or pedv resulted in indistinguishably cytopathic phenotype, i.e., syncytia formation , the ultrastructural changes in pedv-infected vero cells (at the same moi of 0.1) were examined under em for comparison of possibly morphological differences. interestingly, pedv appeared to induce a higher number of dmvs and lvcvs in large clusters surrounding the nucleus at 24 hpi and thereafter (fig. 4b) . a previous study on qualitative and quantitative ultrastructural analysis of membrane rearrangements induced by mhv proposed that cov rna synthesis is dictated by the number of dmvs, whereas an increasing production of viral particles is accommodated by lvcvs from expanding of er-golgi intermediate compartment (ergic)/golgi compartments . it will be interesting to investigate whether synthesis of pedv/seacov rna and assembly of pedv/ seacov virions are correlated with the level of ultrastructural changes in the future. in summary, we generated rabbit antisera against four of the seacov structural and nonstructural proteins and validated their reactivity and use of time course analysis of viral protein expression in seacov-infected vero cells. furthermore, we established a dna-launched reverse genetics system for seacov and rescued the recombinant virus with a unique genetic marker in cultured cells. recombinant seacov had similar growth kinetics to the parental virus. the singlecycle growth of seacov in vero cells was determined to take approximately 4-6 h. by rt-pcr analysis, we experimentally identified all proposed seacov sgrnas containing the leader-body junction sites. among six sgrnas, a bicistronic mrna 7 was utilized by the accessory ns7a and ns7b genes. finally, we characterized the cellular ultrastructural changes induced by seacov infection in vitro. our study develops essential research tools and establishes the basic characteristics of seacov that will facilitate future studies on understanding the molecular mechanisms of seacov replication and pathogenicity. a monkey kidney cell line vero (atcc ccl-81) and a baby hamster kidney fibroblast cell line, bhk-21 (atcc ccl-10) were grown in dmem supplemented with 10% fetal bovine serum (fbs) and 1% antibiotics at 37°c, respectively. the seacov isolate ch/gd-01/2017 at the passage 10 (p10) used in this study was cultured in vero cells. the virus titers were determined by endpoint dilutions as 50% tissue culture infective dose (tcid 50 ) on vero cells. the control virus pedv (zju/g2/2013 strain; genbank accession no. ku558701) was also cultured in vero cells as described earlier (ji et al., 2018; qin et al., 2017) . vero cells infected by the seacov or pedv (at 24 h postinoculation, hpi) were fixed with 2.5% glutaraldehyde in phosphate buffer (0.1 m, ph 7.0) and 1% oso4 in phosphate. ultrathin sections were prepared as described previously , stained by uranyl acetate and alkaline lead citrate for 5-10 min, and observed using a hitachi model h-7650 tem. polyclonal antibodies (pab) against the spike subunit 1 (anti-s1), membrane (anti-m), nucleocapsid (anti-n) and the nonstructural protein 3 (nsp3) acidic domain (anti-ac) of seacov were produced in rabbits. for generation of anti-m pab, prediction of transmembrane helices of the seacov m protein was first performed using the tmpred software (https://embnet.vital-it.ch/software/tmpred_form.html). the m protein antigenic peptide was predicted as "csdnltendrll-hlv", and synthesized by hua-an biotechnology co., ltd (hangzhou, china). this peptide was purified and used to immunize two new zealand white rabbits and antiserum was harvested at 55 days postimmunization (dpi). anti-s1, anti-n and anti-ac pabs of seacov were prepared in-house. briefly, full-length n (1128 nt, 379 aa,~42 kda) or ac (435 nt, 145 aa,~16 kda) of seacov were expressed with a sixhistidine tag in escherichia coli according to methods described previously (huang et al., 2011) , whereas seacov-s1 (1638 nt, 174 aa, 62 kda) with a six-histidine tag was expressed by baculovirus system in sf9 insect cells as described previously (wang et al., 2017a) . the purified proteins were used to immunize rabbits, and antisera were harvested at 55 dpi, respectively. total rna from seacov-infected vero cell was extracted using trizol reagent (invitrogen) and then reverse-transcribed with a superscript ii reverse transcriptase (invitrogen) using oligo-dt (promega) as the reverse primer according to the manufacturer's instructions. the forward primer lf (5'-atagagtccttatcttttt-3') and six gene specific reverse primers, s1-r (5'-caatggcatttctgtg tacctctc-3'), sgorf3-r (5'-agtaatctgcttacaacagc-3'), sge-r (5'-agacattaattatggggcat-3'), sgm-r (5'-gttcgcgttctgcga taaag-3'), sgn-r (5'-atctgcgtgaggaccagtac-3'), ns7a-r (5'-aatctgcaaaatctgccaac-3'), were designed for amplification of all seacov subgenomic mrnas (fig. 3a ) from the obtained cdna with a taq dna polymerase (transgen, beijing, china) in a total volume of 50 μl by pcr. the pcr condition was set at 35 cycles of 94°c for 30 s, 50°c for 30 s, 72°c for 3 min with an initial denaturing of the template dna at 94°c for 3 min and a final extension at 72°c for 5 min. the resulting pcr fragments were analyzed on a 1% agarose gel (fig. 3b) and then subcloned into a peasy-t1 vector (transgen, beijing, china) followed by sanger sequencing. for amplification of the subgenomic mrna 7 containing the entire ns7a/ns7b, the reverse primer ns7-r (5'-ttacgtgcttaccattgtgt-3') was used, and the pcr extension time was shortened to 45 s. analysis of dna sequences was performed using the lasergene package (dnastar inc., madison, wi). the expression vector, designated as psb2μ, used to construct a fulllength seacov cdna clone, was based on a bac backbone vector psmart-bac-bamhi (copyright v2.0 bac cloning kits, lucigen). this psmart-bac vector was modified to insert a yeast replication origin (2μ) from the plasmid pyes2 (invitrogen), a cytomegalovirus (cmv) promoter from the plasmid pcdna3 (invitrogen), a hepatitis delta virus ribozyme (hdvrz) sequence from a prrsv (porcine reproductive and respiratory syndrome virus) infectious clone ptri-53rz-pgxg (huang et al., 2009) , and a bovine growth hormone (bgh) polyadenylation and terminator from the plasmid pcdna3 (invitrogen) by several rounds of amplification and "in-fusion" pcr according to our previous publication . the primer sequences and approaches used in the pcr assays are available upon request. the full-length consensus sequence of seacov-p10 (27,155 nt) was determined as described previously . briefly, a total of 15 overlapping fragments covering the entire genome was amplified by rt-pcr using the q5 high-fidelity 2×master mix (new england biolabs, usa). pcr products were purified and cloned into a peasy-blunt vector (transgen, beijing, china) . for each amplicon, five individual clones were sequenced to validate the consensus sequence. to create a 2-nt genetic marker on the orf3 gene of the infectious clone, two point mutations, a to t, and g to c at nucleotide positions 24222-24223, corresponding to the seacov-p10 genome, were generated on the fragment s-2 by fusion pcr (fig. 2a) . subsequently, all 14 fragments identical to the consensus sequence together with the mutated s-2 fragment were re-amplified from each clone with primers listed in table 1 . it was then assembled into the expression vector (psb2μ) between the cmv promoter and the hdvrz+bgh element, using the geneart™ high-order genetic assembly system according to the manufacturer's manual, to create a dna-launched seacov fulllength cdna clone, psea (fig. 2) . the plasmid psea is available to the research community upon request. the sequence encoding the fulllength seacov nucleocapsid gene was amplified and inserted into a prk5 eukaryotic expression vector containing a flag-tag at its c terminus to construct prk-n-flag as a helper plasmid for rescuing the infectious clone. the plasmid psea was purified from the e. coli dh10b strain using qiaprep miniprep kit (qiagen) and quantified by a nanodrop spectrophotometry. bhk-21 cells were seeded at 2×10 5 per well of a sixwell plate and grown until 60-70% confluence before transfection. one microgram each of psea and prk-n-flag were co-transfected into the cells using lipofectamine 3000 (invitrogen) according to the manufacturer's protocol. transfected cells were cultured for 2-3 days. the supernatant was collected and passaged onto fresh vero cells on 12-well plates and cultured for 3 days before the detection of viral protein expression by ifa. the recombinant seacov rescued from the psea infectious clone was named rseacov. the rseacov titers were determined by endpoint dilutions as tcid 50 . viral particles in the supernatants from rseacov-infected cell cultures were negatively stained and examined under tem. a 1.5-kb dna fragment harboring the introduced mutations in the orf3 gene was amplified by rt-pcr using primers tf21 (5'-tactggatgttgtggcatgt-3') and tr21 (5'-ttccacttaaaatcgtcaga-3'). the amplicons were sequenced to affirm that rseacov contained the desired mutations. seacov-infected or rseacov-infected cells were washed twice with pbs, fixed with 4% paraformaldehyde in pbs for 20 min and then permeabilized with 0.5% triton x-100 for 10 min. anti-n, anti-m, anti-s1 or anti-ac pab, each at a 1:1000 dilution in pbs, was added over the cells and incubated for 1 h at 37°c. cells were then washed thrice with pbs and alexa fluor 488-labeled goat anti-rabbit igg (thermo fisher scientific) at a 1:1000 dilution was then added. after 30 min of incubation at 37°c, the cells were again washed thrice with pbs followed by 4',6-diamidino-2-phenylindole (dapi) staining, and were visualized under a fluorescence microscope (dmi3000b, leica, germany). for time course analysis of detection of n, m, s1 or ac, fluorescent images were obtained with a confocal laser scanning microscope (fluoviewver fv1000-ix81; olympus, japan). for western blot analysis, seacov-infected cells were lysed in lysis buffer (25 mm tris-hcl, 200 mm nacl, 10 mm naf, 1 mm na 3 vo 4 , 25 mm β-glycerophosphate, 1% np40, and protease cocktail [biotool, houston, tx]). samples were resolved on sds-page and transferred onto polyvinylidene difluoride (pvdf) membrane that was subsequently blocked with tris-buffered saline (tbs) containing 3% bovine serum albumin (bsa) overnight at 4°c. proteins were detected using the anti-n pab or anti-m pab at 1:1000 dilution followed by incubation with horseradish peroxidase (hrp)-conjugated anti-rabbit igg (1:5000 dilution; thermo fisher scientific). the consensus sequence of seacov-p10 used for construction of the infectious clone has been deposited in genbank under accession no. mk977618. natural science foundation of china (31872488), and the fundamental research funds for the central universities of china (2019fza6014). we thank the staff in the shared experimental platform for core instruments, college of animal science, zhejiang university for assistance with analysis of confocal microscopy. coronavirus reverse genetic systems: infectious clones and replicons systematic assembly of a full-length infectious clone of human coronavirus nl63 porcine deltacoronavirus accessory protein ns6 antagonizes interferon beta production by interfering with the binding of rig-i/mda5 to double-stranded rna a new bat-hku2-like coronavirus in swine rna replication of mouse hepatitis virus takes place at double-membrane vesicles origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states identification and characterization of a porcine monocytic cell line supporting porcine reproductive and respiratory syndrome virus (prrsv) replication and progeny virion production by using an improved dnalaunched prrsv reverse genetics system expression of the putative orf1 capsid protein of torque teno sus virus 2 (ttsuv2) and development of western blot and elisa serodiagnostic assays: correlation between ttsuv2 viral load and igg antibody level in pigs aminopeptidase-n-independent entry of porcine epidemic diarrhea virus into vero or porcine small intestine epithelial cells complete genome sequence of bat coronavirus hku2 from chinese horseshoe bats revealed a much smaller spike gene with a different evolutionary lineage from the rest of the genome accessory proteins of sars-cov and other coronaviruses transmissible gastroenteritis coronavirus gene 7 is not essential but influences in vivo virus replication and virulence discovery of a novel swine enteric alphacoronavirus (seacov) in southern china identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins characteristics of the life cycle of porcine deltacoronavirus (pdcov) in vitro: replication kinetics, cellular ultrastructure and virion morphology, and evidence of inducing autophagy genetic and pathogenic characterization of a novel reassortant mammalian orthoreovirus 3 (mrv3) from a diarrheic piglet and seroepidemiological survey of mrv3 in diarrheic pigs from east china structural maturation of the transmissible gastroenteritis coronavirus continuous and discontinuous rna synthesis in coronaviruses engineering the transmissible gastroenteritis virus genome as an expression vector inducing lactogenic immunity shared common ancestry of rodent alphacoronaviruses sampled globally qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus new insights on the role of paired membrane structures in coronavirus replication the coronavirus nucleocapsid protein is dynamically associated with the replication-transcription complexes development and application of real-time rt-pcr and s1 protein-based indirect elisa for porcine deltacoronavirus emerging and re-emerging coronaviruses in pigs discovery, diversity and evolution of novel coronaviruses sampled from rodents in china discovery of a highly divergent coronavirus in the asian house shrew from china illuminates the origin of the alphacoronaviruses enhancement of safety and immunogenicity of the chinese hu191 measles virus vaccine by alteration of the s-adenosylmethionine (sam) binding site in the large polymerase protein isolation and characterization of a highly pathogenic strain of porcine enteric alphacoronavirus causing watery diarrhoea and high mortality in newborn piglets severe acute respiratory syndrome coronavirus groupspecific open reading frames encode nonessential functions for replication in cell cultures and mice sars-coronavirus open reading frame-3a drives multimodal necrotic cell death retrospective detection and phylogenetic analysis of swine acute diarrhoea syndrome coronavirus in pigs in southern china this work was supported by the national key research and development program of china (2016yfd0500102), the national key: cord-280795-wtrt13ij authors: han, yu-tsung; tsai, chia-sheng; chen, ya-chio; lin, ming-kuem; hsu, yau-heiu; meng, menghsiao title: mutational analysis of a helicase motif-based rna 5′-triphosphatase/ntpase from bamboo mosaic virus date: 2007-10-10 journal: virology doi: 10.1016/j.virol.2007.05.013 sha: doc_id: 280795 cord_uid: wtrt13ij the helicase-like domain of bamv replicase possesses ntpase and rna 5′-triphosphatase activities. in this study, mutational effects of the helicase signature motifs and residue l543 on the two activities were investigated. either activity was inactivated by k643a-s644a, d702a, d730a, r855a, or l543p mutations. on the other hand, q826a, d858a and l543a had activities, in terms of k(cat)/k(m), reduced by 5to 15-fold. amppnp, a nonhydrolyzable atp analogue, competitively inhibited rna 5′-triphosphatase activity. analogies of mutational effects on the two activities and approximation of k(i(amppnp)) and k(m(atp)) suggest that the catalytic sites of the activities are overlapped. mutational effects on the viral accumulation in chenopodium quinoa indicated that the activities manifested by the domain are required for bamv survival. results also suggest that q826 in motif v plays an additional role in preventing tight binding to atp, which would otherwise decrease further rna 5′-triphosphatase, leading to demise of the virus in plant. bamboo mosaic virus (bamv), a member of potexvirus genus belonging to the alphavirus-like superfamily, has a positive-strand rna genome (∼ 6.4 kb) with a 5′ m 7 gpppg cap structure and a 3′ poly(a) tail . open reading frame 1 of the viral genome encodes a 155-kda replicase consisting of mrna capping enzyme, helicase-like and rnadependent rna polymerase (rdrp) domains sequentially from n to c termini. a disordered region of more than 100 amino acid residues and a proline-rich segment separate respectively the neighboring functional domains. these two flexible regions may allow one domain to interact with others dynamically during the viral replication process. the capping enzyme domain, expressed in membrane fractions of yeast, exhibited an s-adenosylmethionine-dependent guanylyltransferase activity by which gtp is methylated to form m 7 gtp before the m 7 gmp moiety is further transferred to the 5′-diphosphate end of mrna to form the m 7 gpppn cap structure (huang et al., 2005; li et al., 2001a) . such distinctive capping activity has also been demonstrated in semliki forest virus (sfv) (ahola and kääriäinen, 1995) , brome mosaic virus (ahola and ahlquist, 1999; kong et al., 1999) , tobacco mosaic virus (tmv) (merits et al., 1999) and hepatitis e virus (magden et al., 2001) ; thus, it is probably a common feature of the capping enzymes from the alphavirus-like superfamily. the e. coli-expressed helicase-like domain possessed both rna 5′-triphosphatase and ntp hydrolase activities (li et al., 2001b) . with the rna 5′triphosphatase and the aforementioned capping enzyme activities, a cap structure could be formed at the 5′ end of bamv mrna in vitro. the recombinant rdrp domain showed a template-dependent rna polymerase activity and had preferential binding activity to the 3′ noncoding region of the viral rna (huang et al., 2001; li et al., 1998) . rna 5′-triphosphatase specifically cleaves the 5′ γ-phosphate out of the nascent mrna in the first reaction step toward the cap formation. the documented rna 5′-triphosphatases can be grouped into three classes according to their primary structures and the catalytic mechanism employed. the meta-zoan and plant enzymes are of the metal-independent cysteine phosphatase type and distinguish themselves by possessing an hc(x) 5 rs/t motif, in which the cysteine residue acts as a catalytic nucleophile within the catalytic pathway (changela et al., 2001; takagi et al., 1997) . the enzymes from fungi, protozoa, and some dna viruses constitute a metal-dependent phosphohydrolases class with characteristic of possessing two essential glutamate-containing motifs (ho et al., 1998; martins and shuman, 2003) . rna triphosphatases, derived from rna helicases or helicase-like proteins, constitute another metaldependent phosphohydrolases class. this class includes the enzymes identified from rna viruses such as reovirus , alphavirus (vasiljeva et al., 2000) , dengue virus (matusan et al., 2001) , coronavirus , and bamv (li et al., 2001b) . both classes of the metal-dependent rna triphosphatases are also capable of hydrolyzing nucleoside triphosphates to nucleoside diphosphates and inorganic phosphate. according to motif-based classification, helicase-like proteins from rna viruses have been classified into three superfamilies (sf) (kadaré and haenni, 1997) . members of sf1 share similarity to nsp2 of alphavirus, while ns3-like proteins of potyvirus, flavivirus and pestivirus, and 2c-like proteins of picornavirus are representatives of sf2 and sf3, respectively. across the three sf, gks/t sequence of motif i (walker a site) is conserved, whereas the consensus sequences of motif ii (walker b site) are varied, with de/d sequences in sf1 and sf3 and dexh in sf2. based on the classification principle, the helicase-like domain of bamv replicase can be grouped into sf1. fig. 1 shows comparison of partial amino acid sequences of the bamv domain and some of the sf1 members. in relation to enzymatic activities, all three sf proteins have atpase activity. an rna helicase activity has also been broadly corroborated on proteins of sf2; however, the activity was so far demonstrated only on a couple of sf1 proteins, e.g., nsp2 of sfv (gomez de cedron et al., 1999) and the helicase domain of tmv replicase (goregaoker and culver, 2003) . like the helicase-like domain of bamv replicase, nsp2 of sfv has also been reported to have rna 5′-triphosphatase activity (vasiljeva et al., 2000) . ntp binding and hydrolysis functions of motif i and ii of helicases have been suggested by numerous mutational studies and confirmed by crystal structures of helicases such as pcra of bacillus stearothermophilus (subramanya et al., 1996; velankar et al., 1999) , rep of escherichia coli (korolev et al., 1997) and ns3 of hepatitis c virus (yao et al., 1997) . the lysine residue of motif i interacts with the phosphates of mgatp/mgadp and the threonine or serine ligates the mg 2+ ion. the first aspartic acid of motif ii also coordinates mg 2+ ion and the following glutamate or aspartate residue may act as catalytic base in atp hydrolysis. by contrast, functions of other signature motifs, particularly those of sf1, are relatively less addressed. as a step toward better understanding the relationship between activities and structures of the helicase-like domain of bamv replicase, we set out to investigate the importance of each signature motif to its ntpase and rna 5′-triphosphatase activities by mutational and kinetic analyses. results of this study strongly sug-gest a common catalytic site for the removal of 5′γ-phosphate from ntp and rna. the relation between the enzymatic activities in vitro and the viral replication in vivo is also discussed in this study. previous studies demonstrated that the helicase-like domain of bamv replicase exhibits an mg 2+ /mn 2+ -dependent γphosphohydrolase activity toward both nucleoside triphosphate and rna (li et al., 2001b) . the former (ntpase) could be a prerequisite for the putative rna helicase activity, and the latter (rna 5′-triphosphatase) could be involved in the cap structure formation. substitution of gaa for gks in motif i abolished ntpase as well as rna 5′-triphosphatase activities. in the present study, we examined the mutational effects of other conserved motifs of sf1 helicases with respect to these two activities. leucine at position 543 was also included in the mutation list because the accumulation level of the viral coat protein decreased significantly in protoplast of nicotiana benthamiana as leu543 to proline mutation was introduced into the viral replicase . in addition, a deih sequence immediately downstream motif vi was mutated because it is analogous to dexh box of motif ii of sf2. each of the targeted residues, as indicated in fig. 1 , was replaced by alanine except leu543 that was also replaced by proline. all the proteins were expressed in e. coli and purified by immobilized metal affinity and anionic exchange chromatography as described in materials and methods. the purification results are shown in fig. 2 . the importance of the mutated residues to atp hydrolysis activity was first analyzed qualitatively by tlc assay (fig. 3) . alanine substitution at k643-s644 or at d702 reduced the activity to extents similar to the background level. mutation of d730 or r855 to alanine or l543 to proline also resulted in severe damage to atp hydrolysis activity. in contrast, q826a and d858a remained active but with diminished activities. atpase activity manifested by helicases of sf2, e.g., ns3 proteins of yellow fever virus , hepatitis c virus (suzich et al., 1993) and bovine viral diarrhea pestivirus , is usually stimulated by single stranded rna. effect of rna on atpase activity of the bamv helicaselike domain was also examined in this study. inclusion of an rna transcript, corresponding to the first 200 nucleotides of bamv genome, in the reactions did not cause apparent changes in the atpase activity of any of the tested enzymes. to better characterize the ntpase activity, steady-state rates of the reaction were measured by enzyme-coupled assay, in which ntp hydrolysis was coupled to nadh oxidation. apparent k m and k cat of the wild-type enzyme toward four different mononucleotides were subsequently calculated based on ntp concentration dependences of rate (table 1 ). all four mononucleotides could be hydrolyzed by the wild-type enzyme at rates with apparent k m value ranging from 0.15 to 0.33 mm and apparent k cat from 24 to 67 s − 1 . the specificity constants (k cat /k m ) of the reactions are comparable to that of λ1 of reovirus and nsp13 of sars coronavirus and larger than that of nsp2 of sfv and ns3 of dengue virus (matusan et al., 2001; vasiljeva et al., 2000) . mutational effects of the targeted amino acid residues on the apparent kinetic parameters of atpase were subsequently analyzed by enzyme-coupled assays. consistent with tlc results, proteins with specified mutation in motif i, ii, iii or vi did not show atpase activity to any appreciable extent (not shown). l543p mutant was also inactive; however, l543a was active with an unchanged k m but a 14-fold reduced k cat (table 2) . d858a had k m and k cat reduced by factors of 2 and 10, respectively. q826a had 17-fold and 90-fold decreases in values of k m and k cat , respectively. it is worth noting that the specificity constant of q826a decreased only by a factor of 5.3; this and overtime incubation may account for the apparent atpase activity of q826a shown in the previous tlc assay. the significantly enhanced affinity to atp should account for, to some extent, the dramatic decrease of k cat in q826a. besides ntpase activity, the helicase-like domain of bamv replicase also catalyzes the removal of 5′ γ-phosphate from triphosphate-terminated rna. results of typical experiments with wild-type enzyme are shown in fig. 4 . phosphate increased steadily with time in the first 5 min under the reaction condition fig. 1 . alignment of partial amino acid sequences of the helicase-like domains of some rna viral replicases. the consensus residues within each signature motif of sf1 helicases are shown in bold. residues mutated in this study on bamv protein are indicated by asterisk. protein secondary structure was predicted at the psipred protein structure prediction server (http://bioinf.cs.ucl.ac.uk/psipred/). the rectangle and arrow symbolize the α-helix and β-strand, respectively. bamv, pvx, tmv, tymv, and hev represent bamboo mosaic virus, potato virus x, tobacco mosaic virus, turnip yellow mosaic virus and hepatitis e virus, respectively. (panel a). in addition, phosphates released were roughly proportional to the applied amounts of enzyme within a short period of reaction time (panel b). to define the mutational effects on the activity, reactions catalyzed by wild-type and various mutants were allowed to occur under conditions of enzyme excess (fig. 5) . the activities of k643a-s644a and d702a were nearly abolished. likewise, l543p, d730a and r855a had activities hardly to be observed. the apparent activity of l543a was comparable to that of wild type, while that of q826a and d858a was reduced noticeably. to characterize the effects in detail, reaction rates of l543a, q826a, d858a and the wild-type enzyme were determined at different rna concentrations, and the kinetic parameters were subsequently calculated (table 2) . considering the limitations, such as suboptimal substrate concentration, in data processing, the determined values of k m and k cat were apparent. still, they should be helpful to evaluate the mutational effects. comparing with mononucleotides, rna was hydrolyzed at much slower rates. alanine substitution at l543, q826 or d858 had unfavorable effects on both rna binding and catalysis as evidenced by the increase of apparent k m and the decrease of apparent k cat , respectively. in general, the specificity constants of the three mutants for rna hydrolysis reduced approximately 5-to 14-fold. overall, effects of the investigated mutations on atpase and rna 5′-triphosphatase were parallel, implying that the catalytic sites of the two activities are overlapped. to support the notion, effects of amppnp, a nonhydrolyzable analogue of atp, and amp on rna 5′-triphosphatase activity were examined. amppnp exerted an inhibitory effect on the activity (fig. 6a ). the higher concentration of amppnp, the greater extent of inhibition was observed. by contrast, amp did not significantly affect rna 5′-triphosphatase activity even as its concentration was up to 2 mm (fig. 6b) , suggesting that the 5′terminal γand β-phosphate groups of substrates are the major determinants for the competition. to know the inhibition mode of amppnp, the dependence of rate on rna substrate concentration was determined under different amppnp concentration (fig. 7a ). double-reciprocal plot of the data showed an approximately unchanged v max (fig. 7b) , suggesting that amppnp acted as a competitive inhibitor. the apparent k i value of amppnp in inhibiting rna 5′-triphosphatase activity the enzymatic activity was determined at 20°c by enzyme-coupled assay in 1 ml solution that contained 10 pmol enzyme, 0.1 to 3 mm ntp and other buffer components as described under materials and methods except that the amounts of pyruvate kinase were increased up to 50, 75 and 300 u for gtpase, utpase and ctpase assay, respectively, to assure the rate of ntp hydrolysis being the limiting step within the coupling reaction. was calculated to be 93 μm, which is comparable to the k m value of atp. probably, the catalytic sites for hydrolyzing 5′ γphosphate from rna and mononucleotide are identical or extensively overlapped. the competence of bamv to multiply in vivo was investigated by inoculating plasmid pcbg into leaves of c. quinoa, in which the viral replication and cell-to-cell movement could b atpase activity was determined at 20°c by enzyme-coupled assay in 1-ml solution that contained 0.1 to 3 mm atp, 10 to 600 pmol enzyme and other buffer components as described under materials and methods. data are averages of at least two independent experiments. c rna 5′-triphosphatase activity was determined by tlc analysis. reactions were carried out at 20°c in 3-μl solution that contained 1 pmol enzyme, 10 to 80 μm 5′-[γ-32 p]rna, and other buffer components as described under materials and methods. data are averages of two independent experiments. be monitored by the appearance of green fluorescence because the expression of the introduced gfp depends on the fulfillment of bamv replication (lin et al., 2004) . to see the mutational effects, each of the selected mutations was introduced into pcbg, and the resulting mutant plasmid was mechanically inoculated into plant cells. as shown in fig. 8 , spots with green fluorescence began to appear on leaves of plants infected with wild type, l543a, or d858a virus a week later after inoculation. the respective frequency and average diameter of fluorescent loci appeared on leaves were similar, suggesting that the three variants replicated equally well in c. quinoa. local lesions formed later on leaves that had shown green fluorescence spots earlier (fig. 9) . no signs of green fluorescence or disease symptoms were noted on leaves of plants infected with other mutant viruses, including that carries q826a mutation. repeated experiments with wild type, l543a, q826a and d858a confirmed the negative result of q826a mutation. disability on rna 5′-triphosphatase alone is sufficient to account for the replication incompetence of those carrying specified mutation in motif i, ii, iii, and vi and l543p since formation of 5′ cap at viral mrna is essential for substantial viral protein translation. however, the reasons for the opposite effects of the d858a and q826a mutations in plant are uncertain because the two mutations decreased rna 5′-triphosphatase activity to similar extents. would it be possible that alanine substitution at q826 affects not only the enzymatic activity but also the protein stability? protein stabilities of wild type, l543a, q826a and d858a were, therefore, investigated under 25°c by measuring their residual enzymatic activity as a function of time. the inactivation of the proteins followed first-order kinetics. the half lives (t 1/2 ) of wild type, l543a and q826a were approximately 2 h, whereas t 1/2 of d858a was 40 min (data not shown), indicating that protein stability would not be the cause. alternatively, the distinct feature of q826a on atpase activity might underlie the failure of the mutant virus to replicate in vivo. the mutant protein might not be capable of providing sufficient energy by hydrolyzing atp to the putative helicase activity, or the tight binding of atp deteriorated further the weakened rna 5′-triphosphatase activity. activity of rna 5′-triphosphatase was assayed in the presence of 0.3 mm atp, which is within physiological concentration range (hampp et al., 1982; usuda, 1988) , to explore the latter possibility (fig. 10) . under the competition conditions, rna 5′-triphosphatase activities of wild type and d858a remained approximately 30%, whereas little was left in q826a mutant. the results suggest that rna and mononucleoside triphosphate could have reciprocal inhibitory effects in vivo; and the deterioration of rna 5′-triphosphatase activity might be simply enough to disable the replication function of q826a mutant virus. helicases are defined as proteins that catalyze the separation of duplex nucleic acids into single strands in an ntp-dependent reaction. sequence comparison of the helicase-like proteins from rna viruses has disclosed the conservation of several signature motifs and grouped them into three sf (kadaré and haenni, 1997) . functions of helicase motifs have been addressed through mutational and structural analyses owing to the involvement of helicases in various important biological processes. as regards sf1, crystal structures of dna helicases pcra (subramanya et al., 1996; velankar et al., 1999) and rep (korolev et al., 1997) could provide us insights into the relationships between structures and functions of the proteins. in brief, residues on both motif i and ii are involved in the binding of atp through phosphate group recognition and mg 2+ coordination, while the second conserved acidic residue of motif ii acts as catalytic base in atp hydrolysis. other residues such as the glutamine of motif iii and the arginine of motif vi also participate in accommodating the γ-phosphate group of atp. as with other potexvirus, bamv replicase contains a helicase-like domain which can be classified into sf1. in this study, contributions of the conserved motifs of the helicase-like domain to ntpase and rna 5′-triphosphatase were investigated by mutagenesis. with respect to atpase activity, motifs i and ii are essential, consistent with the suggestions by crystal structures and mutational results reported in numerous literatures. mutations at the aspartate of motif iii and the arginine of motif vi brought on severe damages to the activity, also consistent with their involvement in atp binding. function of deih sequence immediately downstream motif vi is not analogous to that of dexh box (motif ii) of sf2 helicases because mutation of the sequence merely caused about 10-fold decrease in k cat value. q826 in motif v seems to avoid the tight binding of the protein to ground-state atp since alanine substitution at the residue reduced value of k m(atp) notably. the otherwise enhanced affinity to atp in q826a not only had adverse effect on atp hydrolysis but also caused significant interference with rna 5′-triphosphatase activity. l543, located in a predicted short β-strand outside the helicase motifs, might have a structural role supporting the active-site architecture for catalyzing atp hydrolysis. for one thing, l543 bears a hydrophobic side chain; for another, the destructive effect caused by proline substitution could be reversed to a certain extent by alanine substitution. nonetheless, this proposition needs to be verified by further structural analysis. as for rna 5′-triphosphatase activity, the effects caused by the various mutations were similar to those on atpase activity, except for q826a: the alanine substitution increased k m(rna) slightly for the rna 5′-triphosphatase activity, while decreasing k m(atp) for the ntpase activity more than 17-fold. the importance of the mutated residues in the viral accumulation in plant was also investigated. mutations of residues within the conserved motifs would disable the viral replication in vivo. by contrast, the two mutations outside the motifs, l543a and d858a, had insignificant consequence. the helicase motifs in 1a protein of brome mosaic virus have also been shown to play essential roles in rna replication in vivo by presumably being involved in viral rnas import (wang et al., 2005) . another aim of this study is to know whether the two enzymatic activities occur at a single or two independent catalytic sites. the generally parallel effects of the mutations on the two activities suggest that the protein employs the same amino acid constellation for the removal of 5′ γ-phosphate from rna and ntp. approximation between k i(amppnp) in rna 5′-triphosphatase competition assays and k m(atp) in atpase assays supports the same idea. still, we cannot rule out another possibility that the substrates might bind to two closely related sites with the atpase activity driving a conformational change that activates the rna 5′-triphosphatase activity, considering the opposite mutational effects on k m(atp) and k m(rna) of q826a. further structural evidence is needed to clarify the argument. nonetheless, employment of a common catalytic site for ntpase and rna 5′-triphosphatase activities has also been suggested on several helicase motif-base proteins such as sfv nsp2 (balistreri et al., 2007) , reovirus λ1 , sars-coronavirus nsp13 and dengue virus nsp3 proteins (bartelma and padmanabhan, 2002) . this raises a question as whether rna 5′-triphosphatase activity is an attendant function of helicases, or it is an adaptive outcome in the course of viral evolution. bamv genome contains two copies of helicase-like domains. one is the domain within viral replicase, the other is the p28 movement protein encoded by triple gene block. with the essential role to the movement of virus in the infected plant, p28 protein also possesses ntpase and rna-binding activities (wung et al., 1999) ; however, it did not show rna 5′-triphosphatase activity (li et al., 2001b) . taken together with the fact that only a few helicases (or helicase-like proteins) have been reported to have rna 5′-triphosphatase activity, we speculate that the 5′ γphosphohydrolase activity for rna molecule has evolved from ntpase activity driven by the demand for functional capping machinery, by which the mrna of rna viruses can be capped within cytoplasm of the infected cells. in spite of lacking explicit biochemical data, the helicase-like domain of bamv replicase has been thought to have rna helicase activity, which may be required for resolving intramolecular base pairing in the template rna and/or preventing the formation of extensive base pairing between template and the nascent complementary strand during rna replication process. is it possible that a protein domain can participate in rna replication while it is also responsible for the removal of 5′ γ-phosphate from the nascent rna to allow cap structure formation? with the help of the flanking flexible hinges, the helicase-like domain may be able to work with the rdrp or the capping domains at different steps during replication process. yet, we cannot rule out another possibility at this moment that the function of the helicase-like domain is for the formation of cap structure while the helicase activity required for bamv replication is provided by hosts. deferring the uncertainty of helicase activity, rna 5′-triphosphatase itself is definitely sufficient to dictate the survival of bamv in plant. mutations that abolished the enzymatic activity in vitro could result in the demise of the virus in c. quinoa. nonetheless, ∼7% of the wild-type activity, in terms of k cat /k m , seemed to be enough for the need since bamv bearing d858a mutation survived. the failure of q826a mutant in c. quinoa is intriguing because it has even higher rna 5′-triphosphatase function than d858a. further inhibition of the weaken rna 5′triphosphatase activity by tight binding of atp in vivo might account for the fatal outcome, indicating the need of a multiplefunction enzyme to coordinate all its activities for the survival of the organism. plasmid phwt, a pet32 derived-vector containing a cdna fragment encoding amino acids 514-892 of bamv replicase, was used for protein expression in e. coli as described previously (li et al., 2001b) . plasmid pcbg is an infectious clone of recombinant bamv, in which a green fluorescence protein (gfp) gene preceded by a duplicated coat protein promoter is situated between triple gene block and coat protein-coding region as described previously (lin et al., 2004) . initial transcription of the recombinant bamv genome from pcbg in plant is driven by 35s promoter of cauliflower mosaic virus. plasmid puhel is a puc18-based vector that contains an sphidigested fragment (4168 nt) isolated from pcbg. site-directed mutagenesis was done on phwt and puhel based on the protocol of quikchange site-directed mutagenesis kit (stratagene). after confirming the mutations with abi prism 3100 auto sequencer (perkinelmer), the sphi-digested fragment from the mutated puhel was put back into pcbg. the helicase-like domain of bamv replicase, fused with a thioredoxin, a poly-histidine tag, and an s-tag at the n terminus, was expressed in e. coli novablue cells (novagen) and purified as described previously with minor modifications (li et al., 2001b) . briefly, the recombinant protein, expressed as inclusion bodies, was first dissolved in urea-containing lysis buffer (50 mm tris [ph 7.5], 150 mm kcl, 0.1% brij-35, 10% glycerol, 10 mm β-mercaptoethanol, 1 mm pmsf and 8 m urea). refolding of the protein was done at 4°c by dropping 1 ml denatured protein solution (∼ 15 mg/ml) into 25 ml stirred lysis buffer. after centrifugation, the refolded protein in the supernatant was then purified by ni 2+ -nitriloacetic acid resin (qiagen) followed by q sepharose (pharmacia) using a linear gradient of nacl (20-500 mm) in equilibrium buffer (50 mm tris [ph 8.0], 0.1% brij-35, 20% glycerol, 10 mm β-mercaptoethanol, and 5 mm egta). the eluted protein was finally subjected to dialysis against equilibrium buffer containing additional 20 mm kcl. rna transcript consisting of the first 50 nucleotides of the plus-strand rna of bamv was used as substrate for rna 5′triphosphatase assay. the corresponding cdna fragment preceded by t7 promoter was first amplified from a cdna clone of bamv by pcr and purified through steps of page (10%), gel extraction and ethanol precipitation. the amplified cdna fragment was then used as template to produce 5′-[γ-32 p]rna in a 20-μl in vitro transcription reaction that contained 0.3 μg template dna, 30 mm ntp (7.5 mm each), 0.05 mci [γ-32 p] gtp (6000 ci/mmol, perkinelmer), 2 μl t7-megashortscript enzyme mix (ambion) and 1× t7 transcription buffer. after 2 h of incubation at 37°c, the reaction product was treated with 2 units of rnase-free dnase i (ambion) at 37°c for 15 min and the 5′-labeled rna was purified through 8 m urea-page (10%), gel extraction and ethanol precipitation. rna concentration was determined according to its optical density at 260 nm measured with nanodrop spectrophotometer. a 200nucleotide 5′-terminal fragment of the plus-strand rna of bamv was also prepared based on the above procedure and described before (li et al., 2001b) . ntpase activity was analyzed by detecting the liberated [α-32 p]ndp from [α-32 p]ntp on a polyethyleneimine (pei)cellulose thin-layer chromatography (tlc) plat or by the decreasing rate of od 340 nm in an enzyme-coupled assay in which ntp hydrolysis was linked to nadh oxidation through activities of pyruvate kinase and lactate dehydrogenase as described previously (li et al., 2001b) . in principle, the reactions were carried out at 20°c in solution containing 50 mm tris [ph 7.5], 5 mm mgcl 2 , 5 mm dtt and indicated amounts of substrate and protein. for enzyme-coupled assays, the solution also contained 2 mm phosphoenol pyruvate, 0.2 mm nadh, 20 u of pyruvate kinase and 20 u of lactate dehydrogenase unless otherwise indicated. rna 5′-triphosphatase activity was analyzed by measuring the γ-phosphate released from 5′-[γ-32 p]rna on pei tlc plate using a phosphorimager (typhoon 9200). standard reaction was performed at 20°c in solution containing 50 mm tris [ph 7.5], 5 mm mgcl 2 , 5 mm dtt, 5 u of rnase inhibitor (takara) and specified amounts of protein and 5′-[γ-32 p]rna. reaction was stopped by adding edta to final 25 mm. tlc was developed using 0.15 m licl-0.15 m formic acid. inhibition experiments of rna 5′-triphosphatase were conducted in the presence of indicated amounts of atp, amppnp, or amp. the amount of radiolabeled products on tlc plate was quantified according to its pixel count against a standard curve of serial diluted 5′-[γ-32 p]rna versus their respective pixels. the reaction rate was estimated from a period of reaction during which no more than 15% of the initial rna substrate was hydrolyzed. proportional ratio between the product released and the enzyme applied under this criterion (fig. 4b) suggests that the rates estimated should be close to the initial rate. the mechaelis-menten constant, k m and v max were determined from lineweaver-burk plot using grafit software. inhibition of amppnp was assessed using lineweaver-burk plot in the presence of different fixed concentrations of amp-pnp. the inhibition constant, k i , was calculated according to the dependence of the slope, k m /v max , of the lineweaver-burk plot on the inhibitor concentration. catalytic constant, k cat , was calculated based on the equation v max = k cat × [e] 0 where [e] 0 is the molar concentration of the protein used in assays. plasmid pcbg and its derivatives were inoculated mechanically into plant leaves of 3-week-old c. quinoa according to the method described previously with minor modifications . in short, 2 μg dna in 10 μl aqueous solution was rubbed gently over the surface of the selected leaf that had been dusted with a thin layer of sterile carborundum. each plasmid dna was applied to at least 9 leaves, 3-4 leaves a plantlet and total 3 plantlets. fluorescent images of inoculated leaves were obtained 7 days post-inoculation with a fluorimager with an excitation filter of 488 nm and an emission filter using calibration files. spots with green fluorescence were further observed under a fluorescent microscope. local lesions that appeared later on leaves could be observed by naked eyes. putative rna capping activities encoded by brome mosaic virus: methylation and covalent binding of guanylate by replicase protein 1a reaction in alphavirus mrna capping: formation of a covalent complex of nonstructural protein nsp1 with 7-methyl-gmp enzymatic defects of the nsp2 proteins of semliki forest virus temperature-sensitive mutants expression, purification, and characterization of the rna 5′-triphosphatase activity of dengue virus type 2 nonstructural protein 3 characterization of the reovirus λ1 protein rna 5′-triphosphatase activity characterization of the nucleoside triphosphate phosphohydrolase and helicase activities of the reovirus λ1 protein structure and mechanism of the rna triphosphatase component of mammalian mrna capping enzyme rna helicase of semliki forest virus replicase protein nsp2 oligomerization and activity of the helicase domain of the tobacco mosaic virus 126-and 183-kilodalton replicase proteins adenylate levels, energy charge, and phosphorylation potential during dark-light and light-dark transition in chloroplasts, mitochondria, and cytosol of mesophyll protoplasts from avena sativa l yeast and viral rna 5′ triphosphatases comprise a new nucleoside triphosphatase family evolution of bamboo mosaic virus in a nonsystemic host results in mutations in the helicase-like domain that cause reduced rna accumulation sequences at the 3′ untranslated region of bamboo mosaic potexvirus rna interact with the viral rna-dependent rna polymerase mrna guanylation catalyzed by the s-adenosylmethionine-dependent guanylyltransferase of bamboo mosaic virus human coronavirus 229e nonstructural protein 13: characterization of duplex-unwinding, nucleoside triphosphatase, and rna 5′-triphosphatase activities multiple enzymatic activities associated with severe acute respiratory syndrome coronavirus helicase virus-encoded rna helicases the n-terminal half of the brome mosaic virus 1a protein has rna capping-associated activities: specificity for gtp and s-adenosylmethionine major domain swiveling revealed by the crystal structures of complexes of e. coli rep helicase bound to single-stranded dna and adp identification and characterization of the escherichia coli-expressed rnadependent rna polymerase of bamboo mosaic virus characterization of the adomet-dependent guanylyltransferase activity that is associated with the n terminus of bamboo mosaic virus replicase the helicase-like domain of plant potexvirus replicase participates in formation of rna 5′ cap structure by exhibiting rna 5′-triphosphatase activity a satellite rna associated with bamboo mosaic potexvirus nucleotide sequence of the genomic rna of bamboo mosaic potexvirus arg-16 and arg-21 in the n-terminal region of the triple-gene-block protein 1 of bamboo mosaic virus are essential for virus movement virus-specific mrna capping enzyme encoded by hepatitis e virus mapping the triphosphatase active site of baculovirus mrna capping enzyme lef4 and evidence for a two-metal mechanism mutagenesis of the dengue virus type 2 ns3 protein within and outside helicase motifs: effects on enzyme activity and virus replication virus-specific capping of tobacco mosaic virus rna: methylation of gtp prior to formation of covalent complex p126-m7gtp crystal structure of a dexx box dna helicase hepatitis c virus ns3 protein polynucleotide-stimulated nucleoside triphosphatase and comparison with the related pestivirus and flavivirus enzymes an rna 5′-triphosphatase related to the protein tyrosine phosphatases rna-stimulated ntpase activity associated with the p80 protein of the pestivirus bovine viral diarrhea virus adenine nucleotide levels, the redox state of the nadp system, and assimilatory force in nonaqueously purified mesophyll chloroplasts from maize leaves under different light intensities identification of a novel function of the alphavirus capping apparatus: rna 5′-triphosphatase activity of nsp2 crystal structures of complexes of pcra dna helicase with a dna substrate indicate an inchworm mechanism brome mosaic virus 1a nucleoside triphosphatase/helicase domain plays crucial roles in recruiting rna replication templates rna-stimulated ntpase activity associated with yellow fever virus ns3 protein expressed in bacteria identification of the rna-binding sites of the 28 kda movement protein of bamboo mosaic potexvirus structure of the hepatitis c virus rna helicase domain this work was supported by grants, nsc 94-2752-b-005-012-pae, from the national science council, taiwan, republic of china. key: cord-275348-jna496x7 authors: kapadia, sagar u.; simon, ian d.; rose, john k. title: sars vaccine based on a replication-defective recombinant vesicular stomatitis virus is more potent than one based on a replication-competent vector date: 2008-06-20 journal: virology doi: 10.1016/j.virol.2008.03.002 sha: doc_id: 275348 cord_uid: jna496x7 a sars vaccine based on a live-attenuated vesicular stomatitis virus (vsv) recombinant expressing the sars-cov s protein provides long-term protection of immunized mice from sars-cov infection (kapadia, s.u., rose, j. k., lamirande, e., vogel, l., subbarao, k., roberts, a., 2005. long-term protection from sars coronavirus infection conferred by a single immunization with an attenuated vsv-based vaccine. virology 340(2), 174–82.). because it is difficult to obtain regulatory approval of vaccine based on live viruses, we constructed a replication-defective single-cycle vsv vector in which we replaced the vsv glycoprotein (g) gene with the sars-cov s gene. the virus was only able to infect cells when pseudotyped with the vsv g protein. we measured the effectiveness of immunization with the single-cycle vaccine in mice. we found that the vaccine given intramuscularly induced a neutralizing antibody response to sars-cov that was approximately ten-fold greater than that required for the protection from sars-cov infection, and significantly greater than that generated by the replication-competent vector expressing sars-cov s protein given by the same route. our results, along with earlier studies showing potent induction of t-cell responses by single-cycle vectors, indicate that these vectors are excellent alternatives to live-attenuated vsv. sars vaccine based on a replication-defective recombinant vesicular stomatitis virus is more potent than one based on a replication-competent vector introduction sars (severe acute respiratory syndrome) emerged in the fall of 2002 in china but soon caught the world's attention as it quickly spread to 28 countries. by the end of 2003 the world health organization reported over 8000 probable cases of sars, a fifth of which occurred in health care workers. the overall fatality rate was 9.6%, but in people over the age of 60, the rate exceeded 50%. (http://www.who. int/csr/sars/en/whoconsensus.pdf; peiris et al., 2004) the etiological agent was quickly identified as a coronavirus (cov) ksiazek et al., 2003) , and the 30 kb genome sequence revealed a common coronavirus genome organization (marra et al., 2003; rota et al., 2003) . six major open reading frames were identified. of those, four encoded the major structural proteins: spike (s), membrane (m), nucleocapsid (n) and envelope (e). m, n and e are involved in viral assembly and budding. s, the major glycoprotein, binds the cellular receptor, ace 2 , and mediates entry by a class i viral fusion mechanism (bosch et al., 2003) . there have been no reported cases of sars since 2004; however sources of the sars-cov still exist. animal carriers of the virus including himalayan palm civets, raccoon dogs and bats have been identified (guan et al., 2003; lau et al., 2005; li et al., 2005) . several cases of laboratory-acquired sars have also been reported. because sars-cov has not been eradicated, there is still a potential for human infections. a sars vaccine may be important in controlling future outbreaks. several experimental vaccines have been constructed and tested. these include dna vaccines, protein subunit vaccines, inactivated sars-cov vaccine and recombinant viral vaccines (gillim-ross and subbarao, 2006) . the sars-cov s glycoprotein has been used as the antigen in the development of most of these sars vaccines because it is the target of virus neutralizing antibody. we previously reported the development of an experimental vsv-based sars vaccine. vsv (vesicular stomatitis virus) is a negative strand rna virus that belongs to virus family rhabdoviridae (kapadia et al., 2005) . attenuated vectors derived from vsv have been used extensively as experimental vaccine candidates (daddario-dicaprio et al., 2006a,b; egan et al., 2004; geisbert et al., 2005; jones et al., 2005; kahn et al., 2001; natuk et al., 2006; palin et al., 2007; ramsburg et al., 2004; reuter et al., 2002; roberts et al., 1999 roberts et al., , 1998 roberts et al., , 2004 rose et al., 2001; schlereth et al., 2003 schlereth et al., , 2000 . they induce strong antibody and cellular immune responses, and with the exception of some rural populations in central and south america, there is negligible seropositivity to vsv in the human population (reif et al., 1987) making them attractive candidates for human vaccination. for populations with pre-existing immunity to vsv, nonendemic vsv serotype vectors can be used. vsv also grows to high titers in cell lines approved for vaccine production. in our initial study (kapadia et al., 2005) we showed that a vsv recombinant expressing the sars-cov s protein was capable of generating neutralizing antibodies against sars-cov in mice. furthermore, the immunized mice were protected from a sars-cov challenge. we also showed that a humoral response was sufficient for protection. in the current study we generated and tested the effectiveness of a vsv recombinant that is capable of undergoing only one round of infection because it lacks the gene encoding the vsv glycoprotein (g). use of such a replication-deficient vector would overcome the complex regulatory issues related to approval of live-virus vectors for use in humans. however, production of such vectors would require a qualification of a cell-line that expresses vsv g or some plasmid dna based complementation. furthermore, a single-cycle viral vaccine would alleviate concerns over potential risks related to the use of live viral vectors in individuals with weakened immune systems. in order to evaluate this vector as a sars vaccine candidate, we also developed a sars-cov neutralization assay using a pseudotyped vsv recombinant expressing a green fluorescent protein. in order to recover a single-cycle vsv recombinant encoding the s protein of sars-cov, the vsv glycoprotein (g) gene in a plasmid expressing the vsv anti-genome was replaced with a gene encoding sars-cov s (fig. 1a) . the resulting plasmid, pvsvδg-sars s, was used to recover a virus, vsvδg-s on bhk-21 cells expressing vsv g. because g is required for virus entry, vsv recombinants lacking the g gene must be complemented with g in order to produce infectious particles. viruses the vsvδg-egfp1 lacks the vsv g gene and has an egfp gene inserted into the first position of the genome. the rna sequences are shown in the (+) anti-genomic sense. (c) bhk-21 cells were infected with either vsvδg-s or wt vsv. cells were fixed, and sars-cov s was visualized by indirect immunofluorescence. the fluorescence images are shown on the left, and differential interference contrast (dic) images are shown on the right. (d) lysates of metabolically labeled bhk-21 cells infected with wt vsv (lanes 1 and 2), vsv-s (lanes 3 and 4) or vsvδg-s (lanes 5 and 6) were analyzed by sds-page. the lysates were also treated with pngase f to remove n-linked glycans from proteins (lanes 2, 4, and 6). complemented with g can infect cells for a single cycle, but do not propagate further in the absence of a complementing g protein. when vsvδg-s was used to infect bhk-21 cells, we observed only single infected cells and no virus spread, consistent with the absence of an encoded vsv g protein. to determine if the s protein was expressed by this recombinant, we examined cells using indirect immunofluorescence microscopy. bhk-21 cells were infected with vsvδg-s or wild type (wt) vsv, fixed, and then incubated with serum from a sars-cov-infected mouse. a secondary, alexa fluor 488-conjugated anti-mouse antibody was used for visualization by confocal microscopy (fig. 1c) . we found that the sars-cov s protein was expressed on the cell surface as indicated by the strong surface fluorescence signal visible in cells infected with vsvδg-s but not on control cells infected with wt vsv. to evaluate viral protein expression further, we infected bhk-21 cells with wt vsv, vsv-s (kapadia et al., 2005) or vsvδg-s and metabolically labeled cells with [ 35 s]-methionine. lysates of radiolabeled cells were analyzed by sds-page. because vsv infection shuts off host protein synthesis, the five vsv proteins l, g, n, p and m are readily seen without immunoprecipitation (fig. 1d , lane 1). vsv-sinfected cells expressed the sars-cov s protein in addition to the five vsv proteins (fig. 1d , lane 3). vsvδg-s-infected cells expressed s and all of the vsv proteins except g (fig. 1d , lane 5). because s is a highly glycosylated protein, we also treated the lysates with pngase f to remove glycans in order to further characterize s. after digestion, s migrated faster on the gel (fig. 1d , lanes 4 and 6), a change consistent with removal of the predicted 17 glycans (kapadia et al., 2005) . because a low level of s protein of sars-cov is incorporated into vsv particles (data not shown), it is possible that the s protein might mediate infection in the absence of g. to determine if this s protein could mediate infection of vsvδg-s, we infected vero e6 cells [cells that express the sars-cov receptor, ace2 li et al., 2003) ] with g-pseudotyped vsvδg-s. using an immunofluorescence microscopy assay for observing vsv n protein expression, we saw single infected cells after 6 h but observed no infected cells after 24 h despite a near confluent monolayer of live vero e6 cells. this result indicates that vsvδg-s is not capable of a second round of infection. additionally, we passaged vsvδg-s through bhk-21 cells to yield progeny lacking vsv g and attempted to infect vero e6 cells with this virus stock. even though we could detect s protein in these noncomplemented virions by western blot, we did not observe any infection of vero e6 cells with these particles. these results indicated that the s protein present on the virion was not capable of mediating vsv entry in tissue culture. this is consistent with reported results showing that the full-length s protein, as is encoded in vsvδg-s, is not capable of mediating infection of pseudotyped vsv (fukushi et al., 2005) . to determine if vsvδg-s is able to replicate in vivo without vsv g, we inoculated mice intramuscularly (i.m.) with non-complemented vsvδg-s. if there were any infection by this virus, we anticipated that there might be detectable immune responses to the s protein. we measured sars-cov neutralizing antibody response as a measure of replication. to control for possible immune responses to s protein on the surface of particles in the inoculum, we also administered uvinactivated, non-complemented vsvδg-s. as additional controls we immunized two groups of mice with either g-complemented vsvδg-s or uv-inactivated, g-complemented vsvδg-s. a dose of 5 × 10 5 pfu (plaque forming units) of the g-complemented vsvδg-s was used. an equivalent particle dose of the non-complemented virus was assessed from the amount of n protein in the virus preparation as determined by western blot. one month after inoculation, serum was collected from each animal and the sars-cov neutralization titers were determined. only the g-complemented vsvδg-s-inoculated animals generated any measurable neutralizing titers to sars-cov ( fig. 2 ). they averaged 1:128. the animals in the remaining groups including those inoculated with non-complemented vsvδg-s made no measurable neutralizing antibody response (even at an antibody dilution of 1:5) indicating that significant replication was not occurring. uv-inactivated g-complemented vsvδg-s did not induce a neutralizing antibody response, indicating that one round of replication is essential for a response. we also assessed the immune responses to the vsv vector in these animals. we used the serum from each animal to stain vsv-infected cells and observed vsv n expression by immunofluorescence microscopy. all animals immunized with live g-complemented vsvδg-s had an antibody response to n, while animals inoculated with noncomplemented vsvδg-s had no detectable response to vsv. this further supports the idea that vsvδg-s is not infectious in animals without vsv g. in order to test the potential of our single-cycle vsv recombinant as a sars vaccine, we conducted a study including five groups of mice. the first group included three control mice that received wt vsv intranasally (i.n.). the second group of three mice was inoculated with wt vsv i.m. the third group of six mice was immunized with vsv-s administered i.n., while the fourth group of six mice received vsv-s i.m. the last group of five mice was vaccinated with vsvδg-s i.m. a single vaccine dose of 5 × 10 5 pfu was administered. serum was collected from all mice at 5, 9 and 13 weeks post-immunization. to verify that all mice had been infected with the vectors, we measured vsv neutralizing antibody titers in the serum of individual mice at five weeks post-infection (fig. 3) . vsv g protein is the target of vsv neutralizing antibodies (kelley et al., 1972) . all mice made measurable neutralizing antibody titers to vsv consistent with successful infection. wt vsv administered i.n. produced the highest vsv neutralizing titers (mean titer of 1:8533) consistent with previous results (kapadia et al., 2005) . wt vsv given i.m. and vsv-s given either i.m. or i.n. produced mean vsv neutralizing titers between 1:2560 and 1:3760. the vsvδg-s group produced a lower vsv neutralizing titer (mean of 1:1408) consistent with the fact that this virus does not encode a vsv g protein but does carry g protein on the particles generated by complementation with vsv g. these results indicate that all mice had been effectively inoculated. because a humoral response to the sars-cov s protein is sufficient for protection against sars-cov infection, we wanted to determine the sars-cov neutralizing antibody titers in the serum of mice in this study. in a previous study, we had used direct neutralization of sars-cov to determine sars-cov neutralizing titers. in order to circumvent the level of bio-containment required for this assay, we developed and validated an assay using a vsvδg virus expressing egfp and complemented with sars-cov s protein, the target of sars-cov neutralizing antibodies. we first generated a vsv recombinant, vsvδg-egfp1. the genome of this virus (fig. 1b) has four vsv genes, n, p, l and m, and an egfp gene in the first position of the vsv genome to promote maximal egfp expression. next we inserted the gene for a tagged sars-cov s protein with its cytoplasmic tail replaced with an ha epitope tag (sδtail-ha) into a mammalian expression vector, pcaggs (niwa et al., 1991) . the deletion of the tail is required for infection in the context of pseudotyped viruses (fukushi et al., 2005; giroglou et al., 2004; moore et al., 2004) . this plasmid was transfected into bhk-21 cells. when the transfected cells were expressing sδtail-ha protein, they were in-fected with vsvδg-egfp1 complemented with vsv g. the virus was adsorbed for 1 h, and the cells were then washed three times with pbs in order to remove the input particles. the media was replaced and the infection was allowed to continue for 24 h. the resulting pseudotyped virus, vsvδg-egfp1/sδtail-ha, was present in the media collected from these cells. we next determined if the pseudotyped vsvδg-egfp1/sδtail-ha could be used to assay for sars-cov neutralizing antibodies. we incubated the pseudotyped virus with antiserum from mice inoculated with either wt vsv (which have neutralizing antibody directed to vsv g only), vsv-s (which have neutralizing antibody to vsv and sars-cov), or sars-cov (which have antibody to sars-cov only) at a dilution of 1:50 to ascertain which antibodies were capable of neutralizing the pseudotyped virus. we used vsvδg-egfp1 pseudotyped with vsv g as a control to measure neutralizing antibodies that react with vsv g. following a one-hour incubation at 37°c, the virus-serum mixtures were then transferred to a monolayer of vero e6 cells, which express the sars-cov receptor, ace 2 li et al., 2003) . the cells were incubated at 37°c for 18 h and then fixed with 3% paraformaldehyde. we determined infection by observing egfp expression using fluorescence microscopy. infection of vero e6 cells by the vsvδg-egfp1/sδtail-ha pseudotypes was not neutralized by antibodies to vsv, but was neutralized by antibodies to vsv-s (which contains antibodies to vsv and s) or sars-cov (which contains antibodies to s). in contrast vsvδg-egfp1/g was not neutralized by antibody to sars-cov, but was neutralized by antiserum to vsv or vsv-s (fig. 4) . these results show that neutralization of the s-pseudotyped virus was specific for antibody to sars-cov s. we next compared the sensitivity of our neutralization assay with the standard assay using serum standards assayed previously with the direct sars-cov neutralization assay. we used sera from mice fig. 4 . specific neutralization of vsvδg-egfp1/sδtail-ha by anti-s antibody. vsvδg-egfp1 pseudotyped with either sδtail-ha or vsv g proteins were incubated with antiserum from mice immunized with wt vsv, vsv-s or sars-cov as indicated. the pseudotypes were then transferred to vero e6 cells. infection was determined by egfp expression. both fluorescence images and differential interference contrast (dic) images are shown for each field. immunized with either wt vsv, vsv-s or sars-cov from our previous sars vaccine study (kapadia et al., 2005) . sars-cov neutralizing antibody titers of these sera were determined by incubating vsvδg-egfp/sδtail-ha virus with serial dilutions of these sera, and the virusserum mixtures were transferred to a monolayer of vero e6 cells. infection was determined by observing egfp expression by fluorescence microscopy 18 h after infection. the titer was defined as the highest dilution that completely neutralized vsvδg-egfp1/sδtail-ha. there was no detectable neutralizing activity in serum from mice vaccinated with wt vsv. the titers in serum samples from vsv-s-and sars-cov-inoculated mice were determined to be 1:40 and 1:20 respectively in pseudotype assay. these titers of these sera were 1:32 and 1:12 in the direct assay. furthermore these sera were from mice that were able to control sars-cov infection upon challenge. since an antibody response is sufficient for protecting against sars-cov (bisht et al., 2004; kapadia et al., 2005; yang et al., 2004) , a titer neutralizing titer of as low as 1:20 is indicative of protection. we then used this neutralization assay to measure the neutralizing antibody titers in the serum of the mice in our current study (fig. 5) . no sars-cov-neutralizing antibodies were detected in animals that were infected by wt vsv. there was little variability between individual mice within a group at the three time points measured (fig. 5a, b and c) . notably animals made neutralizing antibodies titers that were considerably greater than 1:20, a titer we determined previously to be protective against sars-cov challenge. animals infected by vsv-s i.n. produced the most robust response with a mean titer of 1:906 five weeks post-vaccination (fig. 5d ). this level dropped by to 1:426 by nine weeks post-vaccination. the group immunized with vsvδg-s also made a strong antibody response with average titers approximately 1:200 at all time points tested. this response was about two-fold greater than that seen in the group immunized i.m. with the replication-competent vsv-s. this difference was statistically significant at 13 weeks post-immunization (p = 0.0087, mann-whitney test). a similar trend was previously reported with a single-cycle vsv vector expressing the hiv env protein. it generally generated a better t-cell response to hiv env than the replication-competent vsv vector expressing hiv env when administered i.m., though the difference was not statistically significant . regulatory approval for the use of replication-competent vsvbased vaccine vectors in humans has been slow because of concerns about potential pathogenesis. we therefore have developed singlecycle vsv-based vectors lacking the vsv g gene that can infect cells, but cannot produce infectious particles (schnell et al., 1997) . we report here that such a replication-defective vector expressing the sars-cov s protein is highly effective at generating sars-cov neutralizing antibody in animals when given i.m. and is even better than a replication-competent vsv vector expressing sars-cov s given by the same route. we were concerned that the single-cycle vsvδg-s vaccine vector described here might be able to mediate multiple rounds of infection because some s protein is incorporated into virions. however, we did not detect any infection by non-g-complemented vsvδg-s particles in cells expressing the sars-cov receptor. furthermore, when we inoculated mice with these non-complemented pseudotyped particles, we saw no immune responses to s or to vsv n indicating that no significant infection occurred. others have also reported that fulllength sars-cov s was not able to mediate entry of vsv and found that a deletion in the carboxy-terminal tail was required for s-mediated entry (fukushi et al., 2005) . the tail of s was also inhibitory in mediating entry of retroviruses (giroglou et al., 2004; moore et al., 2004) . it is likely that the s tail sequence negatively regulates the membrane fusion activity of the s protein, and that in sars-cov virions, other proteins function to activate the s protein membrane fusion activity. consistent with these earlier reports, we found that the full-length s protein would not pseudotype vsvδg-egfp to generate infectious virions, while s protein with its cytoplasmic tail deleted and replaced with an ha tag pseudotyped effectively. taken together, all evidence indicates that vsvδg-s is a single-cycle vector. the strength of the immune response to proteins expressed by replication-competent vsv vectors given i.n. correlates positively with their ability to replicate and spread systemically simon et al., 2007) . single-cycle vectors, which do not spread systemically (simon et al., 2007) , are relatively poor vectors when given i.n., yet generate strong immune responses when given i.m. . in the studies reported here we therefore tested the single-cycle vsvδg-s vector only by the intramuscular route. we found that one dose of the vector was able to generate high levels of sars-cov neutralizing antibody titers of about 1:200. these neutralizing titers were at least ten-fold greater than what was required for complete protection against sars-cov replication in mice in our previous study (kapadia et al., 2005) , and two-fold greater than those induced by the replication-competent vsv-s given i.m.. because antibody responses are sufficient for controlling sars-cov infection (kapadia et al., 2005; yang et al., 2004) , these titers are predictive of protection in the mouse model. although the sars-cov neutralizing antibody titers obtained from mice immunized i.n. with replication-competent vsv-s were higher (average ∼1:600) than the titers obtained from animals immunized i.m., we also know that the replication-competent vectors spread systemically after vaccination by this route (simon et al., 2007) . the virus replicates in the lungs, causes a viremia, and spreads to multiple organs. such widespread dissemination of the vector could also raise safety concerns. how can we explain the greater potency of the single-cycle vectors relative to replication-competent vectors given i.m.? first, we have evidence that replication-competent and single-cycle vectors are both effectively single-cycle vectors when given i.m. (ian simon, unpublished results) . second, the single-cycle vector may be more effective because of the greater expression of s protein in the absence of the upstream g protein gene. because of transcriptional attenuation (iverson and rose, 1981) , the removal of the g gene leads to greater transcription and expression of the sars-cov s gene. in order to evaluate this possibility, we quantified the expression of s (treated with pngase f) by vsv-s and vsvδg-s relative to n/p expression in the gel shown in fig. 1d . we found that vsvδg-s expresses approximately 47% more s protein than vsv-s. lastly, it is also possible that expression of g protein from the replication-competent vector competes with the s protein for the antibody response. the results reported here, along with earlier studies showing potent induction of cellular immune responses by single-cycle vectors , indicate that these single-cycle vectors are excellent alternatives to live-attenuated vsv vaccine vectors and that they warrant further development. to construct pvsvδg-sars s, the sars-cov s gene was amplified from pvsv-sars s (kapadia et al., 2005) by pcr using the following primers: 5′-gatcgatcacgcgtaacatgtttattttcttattatttc-3′ and 5′-cgatccccccgggctagcttatgtgtaatgtaatttgacaccc-3′. the pcr product was digested with mlui and nhei (sites underlined) and ligated to the purified 12,704 bp fragment resulting from the digestion of pvsvxn2 (schnell et al., 1996) with the same enzymes. the plasmid pvsvδg-egfp1 expressing egfp from the first position in the genome was generated by digesting pvsv1xn-egfp with hpai and xbai. the ∼ 12-kb vector fragment was purified and ligated to the ∼ 1.4 kb fragment resulting from the digestion of pvsvδg (roberts et al., 1999) with the same enzymes. the resulting plasmid was designated pvsvδg-egfp1. pcaags-sars sδtail-ha was made by pcr amplification of the sars-cov s gene with primers that replaced the region encoding the last 28 residues of the carboxy-terminal tail with a sequence encoding the ha epitope tag. the following primers were used: 5′-gatc-gatcctcgagaacatgtttattttcttaattatttc-3′ and 5′-cgatc-cccccgggctagcttaggcgtaatctgggacgtcgtatgggtacttgagg-caactgcaacaactagtc-3′. the sequence encoding the ha tag is shown in bold. the resulting pcr product was digested with xhoi and nhei (sites underlined) and ligated into pcaags (niwa et al., 1991) also digested with the same enzymes. construction of pcaags-g was previously described (okuma et al., 2001) . viruses were recovered from plasmids pvsvδg-sars s and pvsvδg-egfp1 by previously described methods (schnell et al., 1997) . the recovered viral supernatants were then transferred onto bhk-21 cells that had been transfected (described below) with pcaags-g (okuma et al., 2001) . the supernatants containing vsvδg-s and vsvδg-egfp1 complemented with g were collected after 36 h. the viruses were titered on bhk-g cells (schnell et al., 1997) using a standard plaque assay. to obtain vsvδg-egfp1 pseudotyped with the sδtail-ha protein, we transfected bhk-21 cell with pcaags-sars sδtail-ha (described below). transfected cells were infected with recovered vsvδg-egfp1 complemented with vsv g. one hour after infection, the input virus was removed and the cells were washed 3 times with phosphate buffered saline (pbs). dmem containing 5% fbs was added to the cells. the media containing vsvδg-egfp1 complemented with sδtail-ha was collected after 36 h. the virus was titered on vero e6 cells by assessing the number of cells expressing egfp. vsv-sars s (vsv-s) (kapadia et al., 2005) and wt vsv (lawson et al., 1995) recovery were previously described. non-complemented vsvδg-s was obtained by infecting bhk-21 cells with vsvδg-s at an moi of 5 for 1 h. the cells were then washed 5 times with pbs to remove any input virus. dmem with 5% fbs was added to the cells and incubated overnight. the media was collected and subjected to ultracentrifugation for 1 h at 100,000 ×g in order to concentrate virus. nine micrograms of dna was diluted in 0.6 ml of optimem (invitrogen, carlsbad, ca), and 30 μl of lipofectamine reagent (invitrogen, carlsbad, ca) was also diluted in 0.6 ml of optimem. the dna and lipofectamine mixtures were combined and incubated for 30 min at room temperature. bhk-21 cells (2 × 10 6 cells plated 18 h earlier) were washed with pbs, and 4.8 ml of optimem was added. the dna/lipofectamine was added to the cells and incubated at 37°c for 5 h. then 6 ml of dulbecco's modified eagle's medium (dmem) containing 20% fetal bovine serum (fbs) was added and left overnight at 37°c. the next morning the media was replaced with dmem containing 5% fbs. the transfection was allowed to continue for 48 h after the addition of the dna/lipofectamine mixture. bhk-21 cells were infected with wt vsv, vsv-s or vsvδg-s at a multiplicity of infection (moi) of 20. after 5 h the cells were washed twice with methionine-free dmem and incubated with 100 μci of [ 35 s]-methionine in 1 ml of methionine-free dmem for 30 min at 37°c. the cells were then washed twice with pbs and solubilized with a detergent solution (1% nonidet p-40, 0.4% deoxycholate, 50 mm tris-hcl [ph 8], 62.5 mm edta). lysates were analyzed by sds-page. the protein samples were treated with peptide n-glycosidase (pngase) f (new england biolabs, beverly, ma) according to manufacturer's instructions. for indirect immunofluorescence microscopy, bhk-21 cells plated on glass coverslips were infected with either wt vsv or vsvδg-s. after 6 h the cells were washed twice with pbs and fixed with 3% paraformaldehyde. the cells were then washed twice with pbs containing 10 mm glycine and incubated with serum from a sars-cov infected mouse at a dilution of 1:200. the coverslips were washed twice with pbs-glycine and incubated with alexa fluor 488 goat anti-mouse igg (molecular probes, eugene, or) diluted 1:500. the cells were washed twice with pbs-glycine and mounted on slides. cells were imaged using a biorad μ radiance confocal scanning system on a nikon elipse te300 microscope with a 60× planapochromat objective. fluorescence microscopy to visualize egfp was performed with a nikon microphot fx microscope equipped with a 40× planapochromat objective, epifluorescence, and a spot digital camera. ten-week-old balb/c mice (charles river laboratories) were used in this study. single intranasal inoculations of 5 × 10 5 plaque forming units (pfu) of wt vsv and vsv-s were administered in a volume of 25 μl to animals lightly anesthetized with 20% isoflurane (baxter, deerfield, il) diluted in propylene glycol (v/v). single intramuscular inoculations of 5 × 10 5 pfu of wt vsv, vsv-s and vsvδg-s were administered in a volume of 50 μl in the hind leg muscle. the vsv neutralization titers are defined as the highest dilution of serum that can completely neutralize infectivity of 100 pfu of vsv on bhk-21 cells. this assay was described previously . in order to measure sars-cov neutralizing antibodies in serum, vsvδg-egfp1/ sδtail-ha was first incubated with two monoclonal antibodies, i1 and i14 (lefrancois and lyles, 1982) , at a dilution of 1:1000 per antibody for 1 h at 37°c to neutralize potential infection due to any residual vsv g that may have been incorporated into the particles pseudotyped with sδtail-ha protein. serum samples were serially diluted with dmem containing 5% fbs. approximately 75 infectious pseudotyped particles were added to each serum dilution in a final volume of 30 μl. the mixture was incubated for an hour at 37°c. 25 μl of each dilution was transferred to a monolayer of vero e6 cells grown in a 96-well plate. after 1 h at 37°c, 50 μl of dmem with 5% fbs was added to each well and the cells were incubated for 16 to 20 h at 37°c. infection was determined by visualizing egfp expression using an olympus ck40 microscope equipped for epifluorescence. each dilution was measured in duplicate. the titer was determined to be the highest dilution at which both duplicates showed no infection. severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex cross-protection against marburg virus strains by using a live, attenuated recombinant vaccine postexposure protection against marburg haemorrhagic fever with recombinant vesicular stomatitis virus vectors in non-human primates: an efficacy assessment identification of a novel coronavirus in patients with severe acute respiratory syndrome immunogenicity of attenuated vesicular stomatitis virus vectors expressing hiv type 1 env and siv gag proteins: comparison of intranasal and intramuscular vaccination routes vesicular stomatitis virus pseudotyped with severe acute respiratory syndrome coronavirus spike protein development of a new vaccine for the prevention of lassa fever emerging respiratory viruses: challenges and vaccine strategies retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus s protein isolation and characterization of viruses related to the sars coronavirus from animals in southern china localized attenuation and discontinuous synthesis during vesicular stomatitis virus transcription live attenuated recombinant vaccine protects nonhuman primates against ebola and marburg viruses replication-competent or attenuated, nonpropagating vesicular stomatitis viruses expressing respiratory syncytial virus (rsv) antigens protect mice against rsv challenge longterm protection from sars coronavirus infection conferred by a single immunization with an attenuated vsv-based vaccine the glycoprotein of vesicular stomatitis virus is the antigen that gives rise to and reacts with neutralizing antibody a novel coronavirus associated with severe acute respiratory syndrome severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats recombinant vesicular stomatitis viruses from dna the interaction of antibody with the major surface glycoprotein of vesicular stomatitis virus ii. monoclonal antibodies of nonneutralizing and cross-reactive epitopes of indiana and new jersey serotypes angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus bats are natural reservoirs of sars-like coronaviruses the genome sequence of the sars-associated coronavirus retroviruses pseudotyped with the severe acute respiratory syndrome coronavirus spike protein efficiently infect cells expressing angiotensin-converting enzyme 2 recombinant vesicular stomatitis virus vectors expressing herpes simplex virus type 2 gd elicit robust cd4+th1 immune responses and are protective in mouse and guinea pig models of vaginal challenge efficient selection for high-expression transfectants with a novel eukaryotic vector analysis of the molecules involved in human t-cell leukaemia virus type 1 entry by a vesicular stomatitis virus pseudotype bearing its envelope glycoproteins an optimized vaccine vector based on recombinant vesicular stomatitis virus gives high-level, long-term protection against yersinia pestis challenge severe acute respiratory syndrome a single-cycle vaccine vector based on vesicular stomatitis virus can induce immune responses comparable to those generated by a replication-competent vector highly effective control of an aids virus challenge in macaques by using vesicular stomatitis virus and modified vaccinia virus ankara vaccine vectors in a single-boost protocol a vesicular stomatitis virus recombinant expressing granulocyte-macrophage colony-stimulating factor induces enhanced t-cell responses and is highly attenuated for replication in animals epizootic vesicular stomatitis in colorado, 1982: infection in occupational risk groups intranasal vaccination with a recombinant vesicular stomatitis virus expressing cottontail rabbit papillomavirus l1 protein provides complete protection against papillomavirus-induced disease vaccination with a recombinant vesicular stomatitis virus expressing an influenza virus hemagglutinin provides complete protection from influenza virus challenge attenuated vesicular stomatitis viruses as vaccine vectors complete protection from papillomavirus challenge after a single vaccination with a vesicular stomatitis virus vector expressing high levels of l1 protein an effective aids vaccine based on live attenuated vesicular stomatitis virus recombinants characterization of a novel coronavirus associated with severe acute respiratory syndrome successful vaccine-induced seroconversion by single-dose immunization in the presence of measles virus-specific maternal antibodies successful mucosal immunization of cotton rats in the presence of measles virusspecific antibodies depends on degree of attenuation of vaccine vector and virus dose the minimal conserved transcription stop-start signal promotes stable expression of a foreign gene in vesicular stomatitis virus construction of a novel virus that targets hiv-1-infected cells and controls hiv-1 infection replication and propagation of attenuated vesicular stomatitis virus vectors in vivo: vector spread correlates with induction of immune responses and persistence of genomic rna a dna vaccine induces sars coronavirus neutralization and protective immunity in mice this work was supported by nih grant ai057158. key: cord-279924-09uwhxs9 authors: plaisted, warren c.; weinger, jason g.; walsh, craig m.; lane, thomas e. title: t cell mediated suppression of neurotropic coronavirus replication in neural precursor cells date: 2014-01-01 journal: virology doi: 10.1016/j.virol.2013.11.025 sha: doc_id: 279924 cord_uid: 09uwhxs9 neural precursor cells (npcs) are the subject of intense investigation for their potential to treat neurodegenerative disorders, yet the consequences of neuroinvasive virus infection of npcs remain unclear. this study demonstrates that npcs support replication following infection by the neurotropic jhm strain of mouse hepatitis virus (jhmv). jhmv infection leads to increased cell death and dampens ifn-γ-induced mhc class ii expression. importantly, cytokines secreted by cd4+ t cells inhibit jhmv replication in npcs, and cd8+ t cells specifically target viral peptide-pulsed npcs for lysis. furthermore, treatment with ifn-γ inhibits jhmv replication in a dose-dependent manner. together, these findings suggest that t cells play a critical role in controlling replication of a neurotropic virus in npcs, a finding which has important implications when considering immune modulation for npc-based therapies for treatment of human neurologic diseases. transplantation of multipotent neural precursor cells (npcs) is emerging as a feasible therapeutic strategy for the treatment of a variety of neurological disorders. recent studies have demonstrated both short and long-term clinical benefits following npc engraftment within the context of rodent models of alzheimer's disease, parkinson's disease, huntington's disease, and acute spinal cord injury (blurton-jones et al., 2009; mcbride et al., 2004; van gorp et al., 2013; yasuhara et al., 2006) . furthermore, in murine and non-human primate models of the neuroinflammatory disease multiple sclerosis (ms) the ability of human npcs to function as modulators of the immune system in addition to replacing lost or damaged neural cell populations has been suggested (aharonowiz et al., 2008; pluchino et al., 2003 pluchino et al., , 2009 . however, despite the clinical and histological benefits of npc transplantation in pre-clinical animal models of neurologic disease, there is limited evidence addressing the capacity of neural grafts to act as reservoirs for viral replication. studies using the non-polio enterovirus coxsackievirus b (cvb) demonstrate the ability of cvb to preferentially replicate in murine npcs (ruller et al., 2012) . the ensuing carrier-state infection results in increased cell death and impaired differentiation potential in vitro, as well as inflammation, microgliosis, and a variety of cns developmental defects in vivo (ruller et al., 2012; tsueng et al., 2011) . intracerebral infection of neonates with murine cytomegalovirus (mcmv) results in the loss of neural stem cells and their neuronal progeny, as well as a decrease in the production of neurotrophins imperative to normal brain development (mutnal et al., 2011) . borna disease virus (bdv) infection of human fetal human npcs results in cell death upon differentiation and impaired neurogenesis (brnic et al., 2012) . thus, the role of neural stem and progenitors as targets for a variety of neuroinvasive viruses is evident, while the consequences of infection within the context of cellular therapy remain to be elucidated. complicating npc-based therapies is the controversial issue of antigenicity of transplanted cells and immune-mediated recognition. a growing body of evidence suggests npcs are not immunoprivileged, as has previously been reported (hori et al., 2003) . indeed, we have shown that npcs derived from post-natal c57bl/6 brains express the co-stimulatory molecules cd80 and cd86 and up-regulate major histocompatibility complex (mhc) molecules in response to the pro-inflammatory cytokine interferon gamma (ifn-γ) (weinger et al., 2012) . furthermore, allogeneic npcs are rapidly rejected via a t cell mediated mechanism following intraspinal transplantation into mhc-mismatched recipients (weinger et al., 2012) . similarly, human npcs have the capacity to express mhcs i and ii and induce t cell proliferation (goya et al., 2011) . the apparent antigenicity of npcs suggests successful engraftment may require the use of immunomodulatory agents and lifelong suppression of the immune system, as with solid organ transplants. however, an unintended consequence of immune suppression is the potential for latent viruses to become activated, or for uncontrolled viral replication to occur following opportunistic infection (crough et al., 2007; jordan et al., 1977; wynn et al., 2010; young et al., 2012) . therefore, it is imperative to understand the consequences of neurotropic virus infection of npcs as cellreplacement therapies continue to move into the clinic (gupta et al., 2012; riley et al., 2013) . in this study, we demonstrate that cultured murine npcs are infected by the neurotropic jhm strain of mouse hepatitis virus (jhmv), which induces acute encephalomyelitis and chronic demyelination when injected intracranially into immunocompetent mice. jhmv-infected npcs support replication that ultimately results in increased cell death over time. importantly, cd8 þ t cells kill npcs pulsed with viral-peptides, and jhmv replication in npcs was suppressed, in part, by ifn-γ secreted from virus-specific cd4 þ t cells. npcs express the mhv receptor ceacam1a and are infected by jhmv jhmv is a neurotropic coronavirus with relatively restricted tropism for glial cells through recognition and binding to the receptor carcinoembryonic antigen-cell adhesion molecule 1a (ceacam1a) (hirai et al., 2010; thorp and gallagher, 2004) . ceacam1a expression in mouse tissues is widespread and can be detected on the surface of a variety of epithelial cells in the gastrointestinal, respiratory, and reproductive tracts, as well as on small vascular endothelia and hematopoietic cells (hemmila et al., 2004) . however, ceacam1a expression is not ubiquitous, and although it is known to be located at the surface of resident cells of the cns including glia, expression by neural stem or progenitor cells has not been evaluated. to determine if npcs derived from c57bl/6 transgenic mice engineered to express gfp (gfp-npcs) express ceacam1a, mrna was isolated from cultured npcs and receptor expression was evaluated by pcr. using ceacam1a-specific primers, pcr amplicons were detected in npcs, as well as mixed splenocytes from c57bl/6 mice acting as controls (fig. 1a) , and nucleotide sequencing confirmed homology with the specified region of the gene (data not shown). furthermore, cell surface expression of ceacam1a was confirmed with more than 90% of npcs expressing the receptor as determined via flow cytometric analysis (fig. 1b) . we next infected sox2 þ gfp-expressing npcs with jhmv to assess susceptibility to infection. infected npc cultures were fixed 72 h post-infection (p.i.) and stained with an antibody specific for the carboxyl terminus of the jhmv nucleocapsid (n) protein and subsequently imaged using fluorescence microscopy. compared to non-infected npcs that form a confluent monolayer when grown in tissue culture-treated, matrigel-coated vessels, sox2þ npcs infected at a multiplicity of infection (m.o.i.) of 0.1 displayed jhmv-specific syncytia formation by 72 h post-infection ( fig. 2a) . correspondingly, increasing viral titers were detected when plaque forming unit (pfu) assays were performed on supernatants harvested from jhmv-infected npc cultures at 24, 48, and 72 h p.i. (fig. 2b) . furthermore, determination of lactate dehydrogenase (ldh) released into the supernatants of infected cultures at defined time p.i. revealed increased npc death over time, ranging from 10.1 71.5% at 24 h p.i., increasing to 29.7 73.7% at 48 h p.i., and peaking at 35.4 7 3.6% by 72 h (fig. 2c ). as jhmv replication has been reported to occur via ceacam1a-dependent and independent mechanisms (nakagaki and taguchi, 2005) , we performed a monoclonal antibody blockade to determine the role of ceacam1a in the spread of jhmv infection in cultured npcs (fig. 2d ). by 72 h p.i., significant (p o0.05) inhibition of viral replication was observed in anti-ceacam1a-treated cells (4.4 â 10 3 7 1.4 â 10 3 pfu/ml, n¼ 3) when compared to non-treated, jhmvinfected npcs (2.6 â 10 5 75.4 â 10 4 pfu/ml). under normal culture conditions, expression of mhc classes i and ii is undetectable on npcs, yet mhc expression can be induced by treatment with ifn-γ (chen et al., 2011; weinger et al., 2012) . to investigate if jhmv infection alters mhc class i and/or ii expression on npcs, we compared surface expression levels of these molecules on non-infected and infected cells in the absence or presence of 100 u/ml ifn-γ. our findings indicated r1% of npcs were found to be positive for mhc class i ( fig. 3a fig. 3a and c). however, mhc class ii was detected on a significantly (p o0.05) lower fraction (24.572.6%, n ¼3) of infected, ifn-γ-treated npcs compared to non-infected, ifn-γ-treated npcs (32.9 72.1%, n¼ 3) (fig. 3c) . furthermore, mhc class ii could not be detected on the majority of jhmv-infected npcs as determined by dual staining for viral antigen and mhc class ii (fig. 3d ). cd8 þ and cd4 þ t cells are pivotal in controlling jhmv replication within the infected cns (sussman et al., 1989; williamson and stohlman, 1990 ). virus-specific effector cd8 þ t cells help control replication in infected astrocytes and microglia through cytolytic activity (bergmann et al., 2004) . in addition to secreting ifn-γ that limits viral replication in oligodendrocytes, cd8 þ t cells carry out perforin-dependent cytolysis of astrocytes and microglia (bergmann et al., 2004; williamson and stohlman, 1990) . we co-cultured virus-specific ctls at diminishing effectorto-target (e:t) ratios with npcs pulsed with the immunodominant cd8 peptide specific for jhmv spike (s) glycoprotein spanning amino acids 510-518 (s510-518), and treated with ifn-γ to induce mhc class i expression. subsequently, ldh released in the supernatants was evaluated to quantify ctl-mediated npc lysis; rma-s cells, a murine lymphoma cell line that presents viral peptides to ctls in an mhc class i dependent manner, were used as positive control (debruijn et al., 1991) . npcs pulsed with s510-518 peptide were specifically lysed by virus-specific ctls at an e:t ratio of 10-1 (p o0.05, n ¼ 3), indicating that virus-specific cd8 þ t cells are capable of recognizing and directly killing jhmv-infected npcs in vitro (fig. 4) . importantly, this cytolytic effect waned as the e:t to target ratio declined. cd4 þ t cells have both indirect and direct antiviral roles during acute jhmv-induced encephalomyelitis, which include inducing the effector functions of virus-specific ctls, along with ifn-γ secretion (savarin et al., 2008; stohlman et al., 2008 fig. 4 . virus-specific cd8 þ t cells target s510-518 pulsed npcs for lysis. ctls were harvested from mice immunized with the dm variant of jhmv and co-cultured at varying effector:target ratios with s510-518 pulsed, ifn-γ-treated npcs for 4 h, and lactate dehydrogenase released into the supernatant was subsequently measured. non-ifn-γtreated rma/s cells pulsed with 50 μm s510-518 were used as a positive lysis control. negative selection was performed to purify the respective t cell populations. npc media was conditioned with cd4 þ t cell cytokines for 48 h and then added to jhmv-infected npcs. supernatants from either naïve or virus-specific cd4 þ t cells suppressed viral replication in npcs at 24 and 48 h post-infection, with the most significant inhibitory effects observed in groups treated with media enriched with virus-specific cd4 þ t cell cytokines (fig. 5a) . however, while the suppressive effects of naïve t cell media appeared to wane by 72 h p.i. (4.7 â 10 5 7 2 â 10 5 pfu/ml), supernatants from npcs treated with virusspecific cd4 þ t cell conditioned media maintained low viral titers (6.8 â 10 3 76.3 â 10 3 pfu/ml) in comparison to non-treated controls (2.6 â 10 5 75.4 â 10 4 pfu/ml; fig. 5a ). t cell derived ifn-γ is critical in controlling jhmv replication in the cns (bergmann et al., 2004; smith et al., 1991) . furthermore, treatment with ifn-γ specifically inhibits jhmv replication in oligodendrocyte progenitors (opcs) derived from c57bl/6 npcs, and inhibition of ifn-γ signaling in oligodendrocytes is associated with increased viral loads and mortality (parra et al., 2010; whitman et al., 2009) . we evaluated levels of ifn-γ in naïve-versus-dm specific cd4 þ t cell conditioned media by enzyme-linked immunosorbent assay (elisa); absorbance values from media conditioned with dm-cd4 þ t cells were increased $ 60-fold when compared to naïve t cell conditioned media (p o0.0001; fig. 5b ). we subsequently treated jhmv-infected npcs with varying amounts of mouse recombinant ifn-γ for 24 h and determined its effects on viral titers. npcs treated with 1 or 10 u/ml ifn-γ maintained high jhmv titers (1.6 â 10 5 76.4 â 10 4 pfu/ml and 1.3 â 10 5 77.8 â 10 4 pfu/ml, respectively) in relation to non-treated groups (1.1 â 10 5 7 6.6 â 10 4 pfu/ml; fig. 5c ). however, jhmv replication in npcs was reduced in cultures treated with 50 or 100 u/ml ifnγ (4.3 â 10 4 72 â 10 4 pfu/ml and 4.1 â 10 4 72.3 â 10 4 pfu/ml, respectively; fig. 5c ). we next performed a 72-h time course to further probe the effects of ifn-γ (100 u/ml) on jhmv-infected npcs. a reduction from 2.8 â 10 5 7 3.4 â 10 4 pfu/ml to 9.2 â 10 4 7 4.9 â 10 4 pfu/ml was observed in ifn-γ-treated cultures by 48 h post-treatment when compared to non-treated groups (p o0.05, n ¼3), and jhmv levels were reduced to 2.3 â 10 4 73.3 â 10 3 in ifn-γ treated cultures, versus 5.2 â 10 5 71.6 â 10 5 in non-treated cultures, by 72 h post-treatment (po0.05) (fig. 5d ). we previously showed that multiple pro-inflammatory cytokines secreted by dmspecific t cells have synergistic effects with ifn-γ (weinger et al., 2012) . to confirm the role of ifn-γ as the major cytokine contributing to suppression of jhmv replication in infected npc cultures, monoclonal antibody blockade against the ifn-γ receptor was performed on npcs before and during treatment with virusspecific cd4þ t cell enriched media. as expected, by 48 h p.t. jhmv levels were significantly (po0.01) reduced in conditioned media treated cultures compared to npcs grown in non-conditioned media (1.3 â 10 5 71.8 â 10 4 and 3.1 â 10 5 72.4 â 10 4 pfu/ml, respectively; fig. 5e ). however, treatment with anti-ifn-γ receptor resulted in higher (po0.05) viral titers (5.4 â 10 5 71.4 â 10 5 ) compared to cd4þ t cell media treated cultures, thereby confirming the pivotal role of ifn-γ in cd4þ t cell mediated suppression of jhmv in npcs. we have previously shown that ifn-γ treatment of jhmvinfected opcs increases ifn-α/β secretion, and treatment with ifn-β suppresses jhmv replication (whitman et al., 2009) . type i interferon (ifn-β) levels in jhmv-infected, ifn-γ treated npc supernatants were assessed by elisa and ifn-β was not detected above background levels (data not shown). we evaluated the expression of the jhmv receptor ceacam1a on npcs following treatment with 100 u/ml ifn-γ and did not observe a change in the frequency of cecam1aþ npcs between treated and non-treated groups at 24 h p.t. (fig. 6a and b) (matthews et al., 2002) . to determine if m transcripts were decreased following ifn-γ treatment, gene-specific quantitative pcr (qpcr) was performed on total rna extracts from jhmv-infected npcs and m transcript levels were normalized to β-actin. m expression was significantly reduced in ifn-γ treated npcs compared to non-treated npcs at 48 and 72 h p.t. (po0.05; fig. 6c ). these findings suggest that the ifn-γinduced inhibitory effect on jhmv replication within npcs is related to both muted expression of ceacam1a and inhibition of viral rna synthesis. this study demonstrates that npcs derived from the brains of post-natal c57bl/6-gfp mice express the jhmv receptor, cea-cam1a, and support viral replication following ceacam1adependent infection. additionally, jhmv infection of cultured npcs induces cytopathic effects over time as evidenced by syncytia formation and elevated ldh levels. within the context of jhmv infection of the cns, these findings demonstrate that resident npcs present within defined anatomical niches may be susceptible to viral infection. moreover, we have previously shown that intraspinal transplantation of npcs into mice persistently infected with jhmv results in clinical recovery associated with remyelination (carbajal et al., 2010; totoiu et al., 2004) . data presented within this report argues that transplanted npcs may be susceptible to jhmv infection, a finding that highlights important clinical implications for emerging therapies utilizing npcs to treat human neurologic disease as engrafted cells may be susceptible to infection by persistent neurotropic viruses. jhmv infection has previously been shown to inhibit constitutive expression of mhc class i in mouse primary astrocyte cultures and to block ifn-γ-induced mhc class ii expression on murine cerebral endothelial cells (correale et al., 1995; joseph et al., 1991) . here, we show that jhmv does not significantly affect mhc class i or ii expression following infection of cultured npcs in the absence of ifn-γ. however, ifn-γ-induced expression of mhc class ii was reduced following jhmv infection. mhc expression plays an important role in immune surveillance during viral infection, and control of jhmv replication within the cns requires antigen recognition by mhc class i and mhc class ii restricted cd8þ and cd4þ t cells (bergmann et al., 2004; sussman et al., 1989; williamson and stohlman, 1990) . impaired expression of mhc class ii following ifn-γ-treatment of infected npcs may be a mechanism employed to subvert detection by infiltrating virus-specific cd4þ t cells. nonetheless, conditioned medium from virus-specific cd4þ t cells was able to suppress jhmv replication within npcs, likely due to the effects of ifn-γ. supporting this notion, treatment of infected npcs with recombinant mouse ifn-γ had a dose-dependent inhibitory effect on virus replication, and blocking ifn-γ receptor abrogated the observed suppressive effects. ifn-γ treatment resulted in fewer ceacam1a-expressing npcs with a concomitant decrease in jhmv membrane glycoprotein transcripts, suggestive of viral entry inhibition and reduced virion assembly. we also observed that npcs pulsed with the cd8-specific viral peptide s510-518 were detected and killed by virus-specific cd8þ t cells, indicating that virallyinfected npcs may be targeted for lysis by ctls infiltrating into the cns in response to infection. collectively, our findings argue that t cells are important for controlling viral replication within npcs through both cytolytic activity and ifn-γ secretion. lineage fate mapping of neural stem/precursor cells residing within the subventricular zone of lateral ventricles and subgranular zone of the hippocampus demonstrates the ability of these cells to differentiate into neurons and glia throughout development (doetsch, 2003; gage, 2000) . furthermore, endogenous npcs have been shown to proliferate, migrate, and differentiate in response to acute cns inflammatory events, such as with spinal cord injury, stroke, and experimental models of chronic inflammatory demyelinating disorders (picard-riera et al., 2002; yagita et al., 2001; zhang et al., 2004) . though viewed as a glial tropic virus, this study highlights the potential for npcs to serve as a reservoir for jhmv infection and replication. ctl-mediated lysis of jhmv-infected npcs may be detrimental to npc-mediated repair during cns inflammation, and a loss of npcs destined to become oligodendrocytes could contribute to limited remyelination observed in the jhmv-infected cns. additionally, our findings have clinical relevance, as npcs are currently being employed in clinical trials for spinal cord injury as well as for treating the pelizaeus-merzbacher disease, a genetic disorder that affects the growth of the myelin sheath (gupta et al., 2012; mayor, 2010) . as npcs used for clinical trials are unlikely to be "self-derived", they would be subject to immune recognition and potential destruction by both innate and adaptive immune responses, necessitating long-term immune suppression to prevent graft rejection (chen et al., 2011; swijnenburg et al., 2008; weinger et al., 2012) . several classes of immunosuppressive drugs used during transplantation, including calcineurin inhibitors i.e. cyclosporine and fk506, inhibit the activation and/or proliferation of t cells. such immunosuppressive drugs would foster an environment whereby opportunistic infection or reactivation of latent virus might occur. this raises the possibility that transplanted npcs may be subject to infection, and in the absence of adequate immune surveillance of the cns, could lead to damage/ death of engrafted cells. with this in mind, careful consideration should be given to potential viral infection when contemplating npc grafting for treating neurological disease. the jhm strain of mouse hepatitis virus (j2.2v-1) was added to npc cultures at a multiplicity of infection (moi) of 0.1 pfu/cell. virus was allowed to absorb overnight (16-18 h) before media were replaced. supernatants of infected cultures were collected at defined time p.i. and viral titers were determined using the dbt astrocytoma cell line as previously described (hirano et al., 1976) . npcs derived from the striatum of post-natal day 1 transgenic c57bl/6 mice expressing enhanced green fluorescent protein (gfp) were cultured as previously described (carbajal et al., 2010) . npc media consisted of dmem/f12 with glutamax (gibco), n2 supplement (1x, gibco), ciprofloxacin hydrochloride (100 μg/ ml, cellgro), gentamicin (50 μg/ml, sigma-aldrich), fungizone (2.5 μg/ml, gibco), penicillin/streptomycin (1000 u/ml, gibco), and human epidermal growth factor (20 ng/ml, sigma-aldrich). recombinant mouse ifn-γ was purchased from cell sciences. for studies involving blockade of ceacam-1a, npcs were infected overnight and monoclonal antibody cc1 (ebiosciences) was subsequently added at a concentration of 1 μg/ml. media were harvested 72 h p.i. and plaque assay performed to determine viral titers. experimental blockade of ifn-γ receptor was performed using jhmv-infected npcs incubated with 250 nm (final) antimouse cd119 (ifn gamma receptor 1; ebiosciences) or 250 nm purified rabbit igg (control; bd pharmigen) for 1 h before media were replaced with non-conditioned or cd4 þ t cell conditioned media þ/ à anti-mouse cd119 or rabbit igg. supernatants were harvested 48 h post-treatment and viral titers determined. cultured npcs were dissociated using 0.05% trypsin-edta and suspended in pbs containing 0.5% bsa and 2 mm edta (invitrogen). cells were subsequently treated with blocking antibody (purified rat igg 2b anti-mouse cd16/cd32 monoclonal antibody, 1:200; bd biosciences) for 20 min at 4 1c before being incubated with antibodies specific for ceacam1a (apc-conjugated, 0.06 μg/ test, ebioscience), mhc class i (pe-conjugated, 1:200, ebioscience), or mhc class ii (pe-conjugated, 1:200, bd biosciences), for 20-30 min. in experiments where facs analysis of jhmv was performed, npcs were fixed with 4% paraformaldhyde for 15 min before being permeabilized using bd perm/wash buffer (bd biosciences). the anti-jhmv mab j.3.3 specific for the carboxyl terminus of the viral nucleocapsid (n) protein was conjugated to alexa fluor 647 using the apex labeling system (life technologies) and used at a final concentration of 1.5 ng/ml. detection of fluorescence was performed using a lsr ii flow cytometer (bd biosciences) and analysis of facs data was performed with flowjo software (tree star). total rna was isolated from c57bl/6 splenocytes and npcs using trizol reagent (invitrogen) and purified by phenol-chloroform extraction. cdna was reverse transcribed from rna according to manufacturer's instructions using the superscript iii first-strand synthesis system (invitrogen) and random hexamers. standard pcr for ceacam1a expression was performed with an eppendorf mastercycler using the platinum taq dna polymerase kit (invitrogen) and the following primers purchased from integrated dna technologies: ttccctggggaggactactg (forward primer) and tgtatgcttgcc ccgtgaaat (reverse primer). gene products were run alongside a 1 kb plus dna ladder (invitrogen) on a 1% agarose gel containing ethidium bromide before being imaged using the bio-rad geldoc system. for quantitative rt-pcr experiments, primers specific for the jhmv membrane protein (forward: cgagccgtagcatgtttatcta; reverse: cgcatacacgcaattgaa-cata) were designed using primerquest software (integrated dna technologies, inc.). sybr green real-time pcr master mix (life technologies) was used according to manufacturer's specifications and rt-pcr was performed using the applied biosystem viia 7 real-time pcr system. c t values of m protein transcripts were normalized to β-actin c t values (forward: ggcccagagcaa-gagaggtatcc; reverse: acgcacgatttccctctcagc) and compared using the δδc t method. to evaluate jhmv infection of cultured npcs, cells were dissociated and plated on slides or cover slips coated with reduced growth factor matrigel (bd biosciences). npcs were infected with jhmv overnight and fixed 72 h p.i. with 4% paraformaldehyde for 10 min at room temperature. immunofluorescence staining was performed as previously described (whitman et al., 2009 ) using the anti-jhmv mab j.3.3 (1:20 dilution) specific for the carboxyl terminus of the viral nucleocapsid (n) protein and the alexa fluor 405 goat anti-mouse igg 1 secondary antibody (life technologies), as well as rabbit monoclonal anti-sox2 (epitomics) and alexa fluor 568 goat anti-rabbit igg 1 secondary antibody (life technologies). slides were imaged using a nikon eclipse ti inverted microscope. npc death due to jhmv infection was evaluated at 24, 48, and 72 h p.i. by measuring lactate dehydrogenase released by lysed cells according to manufacturer's recommendations using the cytotox 96 non-radioactive cytotoxicity assay (promega). briefly, spontaneous and virus-induced ldh levels were determined using the following formula: % lysis¼ (experimental ldh release)/(maximum ldh release). ldh levels from jhmv-infected cultures were then normalized to spontaneously released ldh and expressed as cell death due to infection (%). cd4 þ t cell isolation for npc media conditioning c57bl/6 mice were infected with an i.p. injection of 2.5 â 10 5 pfu of a demyelinating (dm) variant of jhmv. on day 8 p.i., cd4þ t cells were isolated from spleens by negative selection according to manufacturer's specifications using the easysep mouse cd4þ t cell isolation kit (stemcell technologies). briefly, red blood cell depleted splenocytes were suspended at a concentration of 1 â 10 8 cells/ml in pbsþ 2% fbs with 1 mm edta. normal rat serum was added at the appropriate concentration and cells were incubated with a cocktail containing a combination of biotinylated monoclonal antibodies directed against cd8a, cd11b, cd11c, cd19, cd45r/b220, cd49b, tcrγ/δ and ter119, for 10 min. subsequently, a suspension of streptavidin-coated magnetic particles in pbs was added and incubated with the cells for 2.5 min; buffer was added to the appropriate volume, and cells were incubated in the easysep magnet for 2.5 min to foster binding of magnetically-labeled unwanted cells to the tube walls before cd4þ t cells were poured off. to generate cd4þ t cell conditioned npc media, the magnetically-labeled fraction following depletion of total t cells was collected using the easysep mouse t cell isolation kit (stemcell technologies). this enriched fraction was treated with 50 μg/ml mitomycin-c (ag scientific), and 35 â 10 6 cells were co-cultured with 35 â 10 6 cd4þ t cells in 10 ml npc media containing 5 μm cd4-specific membrane (m) glycoprotein spanning amino acid residues 133-147 (m133-147, bio-synthesis) for 48 h. cd4þ t cell conditioned media were administered to jhmvinfected npcs and supernatants were harvested 24, 48, and 72 h p.i. for determination of viral titers. levels of ifn-γ in cd4þ t cell conditioned media were determined by elisa using the mouse ifn-γ duoset according to manufacturer's recommendations (r&d systems). interferon-β levels in jhmv-infected npc cultures were evaluated using the verikine mouse interferon beta elisa kit (pbl assay science). the animal protocols and procedures used for these studies were reviewed and approved by the institutional animal care and use committee of the university of california, irvine. npcs were seeded at a density of 20,000 cells/well in a flatbottom 96-well format tissue culture plate (corning life sciences) and pulsed overnight with 5 μm of the immunodominant cd8 peptide specific for mhv spike (s) glycoprotein spanning amino acids 510-518 (s510-518, bio-synthesis). npcs were simultaneously treated overnight with 100 u/ml ifn-γ to induce mhc class i expression for the presentation of s510-518. cd8 þ t cells isolated from dm-infected c57bl/6 mouse splenocytes (as mentioned for cd4 þ t cells) using the easysep mouse cd8 þ t cell isolation kit (stemcell technologies) were then plated with npcs at effector-to-target (e:t) ratios ranging from 10:1 to 0.31:1. cocultures were incubated for 4 h at 37 1c in 5% co 2 at a final volume of 200 μl/well. the amounts of lactate dehydrogenase released from lysed cells were determined using a cytotox 96 non-radioactive cytotoxicity assay (promega). the percentage of ctlmediated lysis was determined as specified by the manufacturer's protocols. rma-s cells pulsed overnight with 50 μm s510-518 were used as a positive control for cell lysis. statistical analysis was carried out using student's t test, oneway anova, or repeated measures anova and p r0.05 was considered significant. neuroprotective effect of transplanted human embryonic stem cell-derived neural precursors in an animal model of multiple sclerosis perforin and gamma interferon-mediated control of coronavirus central nervous system infection by cd8 t cells in the absence of cd4 t cells neural stem cells improve cognition via bdnf in a transgenic model of alzheimer disease borna disease virus infects human neural progenitor cells and impairs neurogenesis migration of engrafted neural stem cells is mediated by cxcl12 signaling through cxcr4 in a viral model of multiple sclerosis mhc mismatch inhibits neurogenesis and neuron maturation in stem cell allografts 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hippocampus transplantation of human neural stem cells exerts neuroprotection in a rat model of parkinson's disease resurrection of endogenous retroviruses in antibody-deficient mice stroke transiently increases subventricular zone cell division from asymmetric to symmetric and increases neuronal differentiation in the adult rat this work was supported by the national institutes of health (nih) grant r01 ns074987 to t.e.l. c.m.w. is supported by the california institute for regenerative medicine (cirm) grants rm1-01717 and tr3-05603, the national multiple sclerosis society (nmss) collaborative center research award ca1058-a-8, and the gleis family foundation. w.c.p. is supported by nih predoctoral training grant 1t32ns082174-01 and j.g.w. is supported by nmss post-doctoral fellowship fg 1960-a-1. key: cord-280287-t7uozjml authors: favier, anne-laure; burmeister, wilhelm p; chroboczek, jadwiga title: unique physicochemical properties of human enteric ad41 responsible for its survival and replication in the gastrointestinal tract date: 2004-04-25 journal: virology doi: 10.1016/j.virol.2004.01.020 sha: doc_id: 280287 cord_uid: t7uozjml human enteric adenovirus ad41 is associated with children gastroenteritis. to infect gastrointestinal cells, the invading virus must be acid-stable and resistant to inactivation by bile salts and proteases. in addition, it has to cross the mucus barrier before it infects mucosa cells. we show that ad41 infectivity is not diminished by acid exposure, a condition limiting the infectivity of the respiratory ad. this feature can be attributed to a large extent to the global basic charge of enteric ad virions and to the stability of ad41 fiber, a viral protein mediating virus attachment. upon exposure to ph shock, the respiratory ad2 loses its ability to interact with lipids while enteric ad41 still binds to the major phospholipids of gastric and intestine mucus. in addition, contrary to respiratory ad, enteric ad41 interacts with several sphingolipid components of plasma membranes. these results show that the molecular bases of the ad41 enteric tropism stem from its particular physicochemical properties. the human stomach secretes about 2.5 l of gastric juice daily. the hydrochloric acid synthesized and secreted by gastric parietal cells may reduce the intraluminal ph to below 2.0. a variety of proteases, secreted by gastric and pancreatic cells and bile salts, enter the duodenum from the biliary tract. to protect the mucosa from the gastric juice, both specialized gastric and intestine cells secrete mucus forming a gel-like layer of about 200 nm. the bicarbonate ions trapped in the mucus create a ph gradient from 6 -7 at the epithelium surface to 1 -2 in the stomach lumen. such extreme conditions encountered in the gastrointestinal tract (gi) are highly inhospitable to invading viruses. indeed, to initiate infection via the gi route, the virus must be acid-stable and resistant to inactivation by bile salts and proteolytic enzymes. it should also be able to withstand the ph jump from 1 -2 in stomach to 7.5 in the intestine. finally, it has to cross the mucus layer before it encounters and infects the cells lining the gi. enteric adenoviruses of subgroup f (ad40 and ad41) are associated with gastroenteritis in children (jacobsson et al., 1979; uhnoo et al., 1983 uhnoo et al., , 1984 and are known as an important enteric pathogen second to rotavirus. the molecular bases of their narrow and specific tropism for gi, unique among the ads, are still not understood. in contrast to enteroviruses or hepatitis a producing systemic illness, these enteric ads, similarly to rotaviruses or coronaviruses, do not cause disease outside gi, which may be related to the efficient host immune response. they can be isolated in the infectious state from the stools of infected children (gary et al., 1979) , with approximately 10 11 physical virus particles/ g of stool, which again shows their survival in the human digestive tract (de jong et al., 1983; uhnoo et al., 1984) . the adenovirus capsid is composed of three major oligomeric proteins. the trimeric hexons form 20 facettes of viral icosahedron which is sealed at each of the 12 vertices by a complex of the pentameric penton base and an outward extending trimeric fiber. these proteins are multifunctional. in addition to its structural role, the fiber protein is involved in virus attachment through binding primary cell receptor. the penton base protein is responsible for virus cell entry through binding to cellular av integrins (wickham et al., 1993) and viral release from endosomes on the pathway towards the cell nucleus (greber et al., 1993) . recently, we have shown that the hexon of respiratory ad promotes virus entry independently of car (primary) and integrin (secondary) receptors through interaction with phospholipids (balakireva et al., 2002) . this might explain the ability of the respiratory ad2 to cross the physical barrier composed of lipid surfactant before infecting alveolar epithelium. during cell attachment, the distal c-terminal globular head domain of the fiber protein interacts with the primary receptor, which for some human ad serotypes is the coxsackievirus and adenovirus receptor (car) (bergelson et al., 1997; roelvink et al., 1998) . while the majority of human ad serotypes have only one kind of fiber, the enteric serotypes 40 and 41 possess two different fibers (kidd et al., 1993; pieniazek et al., 1990; yeh et al., 1994) present in the virion in equal ratio (favier et al., 2002) . similar to fibers of respiratory ads, the ad40/41 long fiber interacts with car. however, car is not recognized by the short fiber (roelvink et al., 1998) . the narrow tropism of enteric ads cannot result alone from ad40/41 long fiber interaction with car. car mrna is preferentially expressed in the heart, testis, prostate, and pancreas, which are not tissues targeted by enteric ads infection. moreover, the internalization of the majority of ad serotypes is due to their interaction with integrins avh3 and avh5 via an rgd motif present in the virus penton base protein (bai et al., 1994; mathias et al., 1994; wickham et al., 1993) . however, ad40 and ad41 lack the rgd motif in their penton bases or fibers (albinsson and kidd, 1999) . altogether, these data imply that the entry of enteric ads is clearly different from that of respiratory serotypes. we feel that ad40/41 tropism depends to some extent on the short fiber even though studies on several cells lines showed that the short fiber is not involved in virus attachment (roelvink et al., 1998; favier, 2002, personal communication; nakamura et al., 2003) . the short fiber might be involved in virus multiplication through interaction with cellular protein partners (chroboczek et al., 2003) downstream of virus initial interaction with the cell plasma membrane. in this study, we analyzed the physicochemical properties of ad41 by distinguishing it from the respiratory serotypes. it is reasonable to assume that the tropism of ad41 is defined by a combination of features permitting to survive in the stomach and to pass through the mucus layer followed by the recognition of specific cellular plasma membrane partners leading to the successful infection of cells lining the gi. in the first attempt to understand the survival mechanism of the human enteric ad41 under acid conditions of the stomach, we compared the predicted pi values of external structural proteins of different ad serotypes (table 1) . using the primary sequences available in the databases, we found that ad40/41 fibers have very basic predicted pi values in contrast to the predicted acidic values of structural proteins of the majority of human adenoviruses. in particular, the difference of more than 3 ph units between the enteric ad41 short fiber and fibers of respiratory viruses ad2 and ad3 is very striking. ad37 and ad8 also display a high pi of the (kagnoff et al., 1987) . n.d.: sequence not known. accession numbers to the sequences used for the calculation of predicted pi values are given in table 2 (see materials and methods). fiber and surprisingly high pi of the ad37 penton base. the differences in charge of head domains of fibers of different serotypes are illustrated by the clusters of basic amino acids shown in the atomic or modeled structures of head domains of fibers of different serotypes (in blue in fig. 1 ). approximately 87% of the ad particle mass consists of proteins (green and pina, 1963) . it is thus reasonable to assume that the charge difference of some ad proteins is reflected in the charge of the whole virus particle. indeed, the particle migration varied when the mobility of different ad serotypes is compared on native gel (fig. 2) . while serotypes 2, 3, and 5 migrate towards the anode, ad41 remained in the wells. the quality of virus preparations was checked by em, and no aggregation has been noticed (results not shown). acid treatment did not seem to affect virus mobility (compare a with b in fig. 2 ), suggesting that virions were not disassembled. under non-denaturing conditions, the mobility on native gel depends on the size, the shape (form), and the charge of the migrating entity. the organization and form of ad virions are quite similar, and the serotypes differ only slightly in their dna content; for example, ad2 dna contains 35 937 bases (roberts et al., 1984) whereas ad40 dna contains 34 214 bases (davison et al., 1993) . it seems therefore that the observed mobility differences stem mainly from the charge difference of the viral proteins and in the case of ad41 can be attributed to somewhat more basic penton bases, hexons, and significantly more basic fibers (table 1) . even if it is not known which part of the digestive tract is primarily infected by enteric ads, it is clear from a mode of dissemination by oral or fecal route that these viruses have to withstand the conditions encountered in the stomach. using ph of the normal fasting human stomach, we compared the survival of infectious ads during exposure to acid. we used 293 cells as they are permissive for both ad41 and ad2 and conventionally used for ad41 production. to establish the experimental conditions for the infectivity assessment, at the beginning we obtained a curve of infection using increasing virus quantities for the same amounts of attached cells as described by favier et al. (2002) . from this we estimated the virus amount giving the maximum (saturation) of infection as well as giving 50% of maximum infection. for each virus infection, two sets of condition were used, 50% and nearly 100% of infectivity saturation, and progeny production was measured after viral inoculum has been exposed to buffered hcl. preliminary time course experiment demonstrated that for ad2 the largest drop in infectivity occurred after 5 min of acid fig. 1 . electrostatic surface potential of fiber knob domains. the molecular surface and its potential have been calculated using grasp (nicolls et al., 1991) . colors range from red (potential of à10 kt) to blue (10 kt). two views are shown, the view on top of the receptor and a side view turned by 90j around x with the top of the fiber head pointing upwards. (a) ad2 (pdb entry 1 qhv; van raaij et al., 1999) ; (b) ad5 (pdb 1 knb; xia et al., 1995) ; (c) ad3 (pdb 1 h7z; durmort et al., 2001) ; (d) theoretical model of ad41 short fiber. the model has been built using the swiss-model together with the swiss-pdb viewer graphical interface (http://www.expasy.ch/swissmod/swiss-model.html). it is based mainly on ad12 (1 nob; bewley et al., 1999) . it has been verified using o (jones and miller, 1991) and a badly constructed loop has been corrected manually. (e) theoretical model of ad41 long fiber obtained with swiss-model based mainly on the structure of ad5. treatment (results not shown). therefore, similar to studies on rotavirus acid stability (weiss and clark, 1985) , short ph exposure was performed. such exposure, in contrast with long dialysis at ph 6.4 -6.8 (pereira and wrigley, 1974; van oostrum and burnett, 1985) , does not result in ad vertex removal. because acid secretion by gastric parietal cells reduces the intraluminal ph to about 2.0 in adults and to about 4.0 in children (gryboski and walker, 1983) , both conditions were employed (fig. 3) . the scatter of results is typical for this kind of experiments; therefore, 33 experiments have been carried out after acid exposure. in about half of the experiments, the ad41 infectivity was not affected by acid exposure compared to virus diluted in culture medium. interestingly, in 17 cases out of 33, the infectivity of ad41 increased. however, at the same time the infectivity of ad2 was inhibited by acid exposure, with a negligible activation observed in three cases out of 33. on average, the ad41 infectivity was unchanged, whereas ad2 infectivity decreased by 20%. no notable difference was observed for ph 2 versus ph 4 or at room temperature (rt) versus 37 jc, in contrast with the results obtained for another enteric virus, rotavirus. for this virus, acid resistance was significantly higher at ph 4 than at ph 2 and at rt than at 37 jc (weiss and clark, 1985) . these experiments show the remarkable stability of enteric ad under ph conditions of the human fasting stomach. ad fiber is known to be an extremely sturdy protein, insensitive to different attempts to denature and de-oligomerize it (devaux et al., 1987) . as this protein mediates the initial virus attachment to infected cells, it was of interest to analyze its behavior after acid exposure. because contrary to respiratory serotypes ad2 and ad5, ad41 (as well as ad3) fibers cannot be purified from ad41-infected cells without the penton base (see favier et al., 2002) , therefore ad41 penton, complex of fiber, and penton base had to be used in these experiments. in the first experiment, the fiber interactions with the primary receptor car were compared before or after acid exposure (fig. 4) . the extracellular domain of car was tested to interact with ad41 short and long fibers contained in pentons (sp41 and lp41), with ad5 fiber (f5), and with ad3 fiber contained in ad3 penton dodecahedra, symmetrical assemblies of 12 ad3 pentons (p3) (fender et al., 1997) . in agreement with the data of roelvink et al. (1998) , neither ad3 nor short ad41 fibers did recognize car, whereas both ad5 and long ad41 fibers fig. 3 . virus infectivity after acid treatment. 293 cells were infected with ad2 or ad41 using two different moi (see materials and methods). virus portions were untreated or exposed to hcl at ph 2 or 4 for 1 min at rt or at 37 jc. exposure was stopped by dilution in culture medium. virus progeny was estimated by fluorescence as described in materials and methods. the results in percent show the activation or inhibition of infection upon hcl treatment (above or below zero line, respectively), where baseline is the infection level for the untreated virus. interacted with the receptor. these results were also found after acid exposure, showing that this treatment does not trigger any major structural change of the fiber protein, which would abolish car interaction. proteolytic analysis was employed for further analysis of the eventual changes in the fiber structure upon acid exposure. because the trimeric and well-folded fiber protein is largely resistant to chymotrypsin (it is cut at a unique nterminal location only, at the elevated amount of enzyme, devaux et al., 1987) , we used this enzyme to probe fiber protein stability. native fibers and pentons were purified from cells infected with wild-type viruses. because we had to use the pentons for ad41 (see explanation above), the penton was also used for ad5 (p5). the fibers were revealed with antibody. initial proper folding of fibers was verified by the presence of the trimeric forms. under denaturing conditions, ad5 and long ad41 fiber monomers run according to their molecular masses of about 62 000 for ad5 and long ad41 fibers and about 42 000 for short ad41 fiber (fig. 5, lanes 1) . under non-denaturing conditions, the trimeric long fibers with molecular masses of about 180 000 run above the 175-kda marker and the trimeric ad41 short fiber (molecular mass of about 130 000) runs between 82-and 175-kda markers (fig. 5, lanes 2) . as in the previous experiment, exposure to acid alone did not significantly affect the fiber trimers (lanes 3). purified ad5 fiber (f5) was destroyed by the combined action of acid exposure and chymotrypsin (fig. 5c , lane 5 for f5). contrary to that, chymotrypsin after prior acid treatment did not result in proteolysis of the ad41 fibers (figs. 5a and b, lanes 5). altogether, these results show that acid exposure followed by ph jump to 7.5 results in such subtle changes in the ad5 fiber structure that it can still interact with car (fig. 4) . however, these changes are sufficient to permit ensuing proteolysis of the respiratory serotype 5 fiber (fig. 5) . on the contrary, both fibers of enteric ad survive well acid exposure, ph jump to 7.5 and the proteolytic treatment, showing the unusual stability under gi simulating conditions encountered by enteric viruses. to understand the mechanism allowing ad41 to cross the mucus barrier separating the gi lumen from cells lining the gi, we studied ad41 interaction with lipids and compared it with the respiratory ad2. mucosa cells of the stomach contain lamellar bodies that function as lipid storage and secretory organelles, permitting synthesis, excretion, and turnover of the gastric surfactant. phospholipids exert the greatest impact on the physicochemical properties of gastric mucus. it was found initially that the major phospholipid of the lamellar bodies of the mucosa cells in the stomach is dipalmitoyl phosphatidylcholine (dppc) (schmitz and muller, 1991) . it was also thought that this phospholipid would also be predominant in the mucus (gastric surfactant) (lichtenberger, 1995) . however, recent data show that both gastric mucosa and mucus contain significant amounts of unsaturated pc together with phosphatidylethanolamine (pe) (bernhard et al., 1995; larhed et al., 1998; nardone et al., 1993) . the ads interaction with lipids was analyzed using a protein -lipid overlay assay. the assay was performed in the absence or presence of magnesium ions, following the conditions used for the investigation of lipid interaction with pleckstrin homology domains (dowler et al., 1999; thomas et al., 2001 thomas et al., , 2002 . pc, dppc, pe, and dipalmitoyl phosphatidylglycerol (dppg) were spotted onto a nitrocellulose membrane and incubated with ad2 and ad41 in comparable molar amounts. it should be noted that the assay sensitivity was ensured by the amount of the immobilized lipid being in the pmol range (thomas et al., 2001) . before acid treatment, at both binding conditions used, ad2 attached to dppc, whereas ad41 attached to dppc only at ph 6, additionally attaching strongly to dppg (fig. 6) . to simulate the abrupt ph jump induced by the passage from stomach to intestine, both viruses were exposed to ph 2 for 1 min at 37 jc, then brought up to ph 7.5 and incubated with immobilized lipids. contrary to the untreated ad2 case, no lipid interaction was observed after ad2 was exposed to a ph shock (fig. 6) . most interestingly, ph shock of enteric ad41 confirmed its interaction with dppc and dppg and improved its interaction with two other phospholipids. the loss of the lipid interaction by ad2 can be explained by the changes in virus integrity induced by acid treatment, as it has been illustrated by the decrease in infectivity and in fiber stability (figs. 3 and 5) . in the case of ad41, there is no loss of infectivity upon acid treatment, which would suggest the conservation of virion integrity. however, an increase in lipid interaction observed for ad41 upon acid shock suggests in addition some capsid transformation improving its ability to recognize different classes of phospholipids. mucosal pathogens target sites of infection through specific adherence to host glycoconjugate receptors (mahdavi et al., 2002; svensson et al., 2003) . one class of such receptors is glycoshingolipids, a highly polymorphic class of lipids, which occur in mammalian cells expressed on the . the proteins were electrophoresed on a 7.5% or 10% sds-page gel at 4 jc. lane 1, boiled fiber; lane 2, native fiber; lane 3, 1-min treatment with hcl, ph 2 at 37 jc; lane 4, fiber digested by chymotrypsin at ph 7.5 with ratio 1/5 enzyme/substrate; lane 5, fiber exposed to hcl for 1 min at ph 2 at 37 jc, neutralized, and digested by chymotrypsin at final ph 7.5 with ratio 1/5 enzyme/substrate. fibers in pentons were revealed by western blot with the appropriate antibody. ad5 fiber was visualized by staining with coomassie brilliant blue (f5, left side) or revealed by western blot (f5, right side). cell surface and are predominant in the gastric epithelium (reviewed in hakomori, 1990) . to understand the interaction of enteric ad with the surface of mucosa cells, the analysis of ad41's ability to interact with lipids was extended to sphingolipids. membrane-immobilized sphingolipids were incubated with both viruses (fig. 7) . both ad41 and ad2 interacted with sulfatide; however, only ad41 interacted specifically with four additional sphingolipids: sphingosine-1-phosphate, lysophosphatidic acid, mono-sialoganglioside gm1, and di-sialoganglioside gd3, suggesting much higher, possibly multivalent, ad41 affinity for cell surface. these experiments together show the remarkable lipophilicity of enteric ad, which most probably plays a role in virus ability to cross the protective mucus barrier and to attach to the surface of cells lining the gi. the goal of this study was to bring us closer to understanding the molecular basis of narrow tropism of enteric adenovirus serotype 41 resulting in the infection of human gi. for this we examined the behavior of ad41 virions by simulating in vitro four crucial steps leading to successful enteric infection: survival in acidic stomach environment, immunity to proteolytic attack after acid exposure, interaction with phospholipids present in the mucus barrier protecting the gi mucosa, and finally, attachment to the apical surface of gi lining cells. comparing the theoretical pi of different ad capsid proteins, we observed that enteric hexon and penton base proteins are somewhat more basic and enteric fibers are significantly more basic than the appropriate proteins of respiratory serotypes (table 1 ). the charge distribution on the head domain of fibers of different serotypes (fig. 1) shows the presence of clusters of basic amino acids for the ad41 fibers and the more positive global charge related to the higher pi of these head domains (table 1 ) this positive surface potential is particularly pronounced for the ad41 long fiber. furthermore, ad41 mobility observed during electrophoresis under native conditions indicated a much more basic charge of enteric virions than other ad serotypes (fig. 2) . this feature immediately suggested that upon encountering stomach acidic conditions, the protonation of basic ad41 particle would be much less damaging to the virion integrity than for the significantly less basic respiratory ad2 or ad5. indeed, acid treatment significantly impaired ad2 infectivity whereas ad41 viability was not affected by the acid (fig. 3) . because all mucosa possess an enzymatic barrier composed primarily of proteolytic enzymes (zhou and li wan po, 1994) , we analyzed the proteolytic resistance of ad41 fibers, viral proteins mediating cell attachment. contrary to the fiber of the respiratory serotype, ad41 fibers were not digested by chymotrypsin after acid exposure (fig. 5) . this tends to show that ad41 survival in the acid stomach environment stems from the pronounced basic character of the enteric ad particle, and in particular, from the remarkable stability of its fibers, which under acidic stomach conditions would likely retain, in the presence of proteolytic enzymes, the ability to attach to the host cells. some viruses (e.g., rotaviruses) have evolved so that proteolytic processing facilitates viral infection. our results strongly suggest that enteric adenoviruses use another adaptation mechanism, which is the prevalence of basic charge of the virions. it protects the virions against the negative effects of the protonation upon the low ph conditions encountered in the stomach and subsequently against the activity of proteolytic enzymes present in gi. the human gut harbors many microorganisms, and their acid survival can be linked to the basic character of some of their components. several types of gut-colonizing bacteria are covered by a semicrystalline layer (s-layer) composed of a single protein or a glycoprotein species. the 43-kda sprotein of lactobacillus acidophilus has a predicted pi of 9.4 (boot et al., 1993) . similarly, a vacuolating cytotoxin vaca secreted by the pathogenic strain of helicobacter pylori that is able to associate with lipid bilayers (czajkowfig. 7 . comparison of glycosphingolipid-binding properties of ad41 with ad2. the nitrocellulose membrane containing 100 pmol of the indicated sphingolipids was incubated with the purified viruses as described in materials and methods. the ligands bound to the membrane-immobilized lipids were detected with specific antibody. binding was performed at ph 6.0 without mg 2+ (*) or at ph 7.5 in presence of mg 2+ . lipid positions are indicated in the diagram: 1, sphingosine; 2, sphingosine 1-phosphate; 3, phytosphingosine; 4, ceramide; 5, sphingomyeline; 6, sphingosyl-phosphocholine; 7, lysophosphatidic acid; 8, myriocin; 9, monosialoganglioside g m1 ; 10, disialoganglioside g d3 ; 11, sulfatide; 12, sphingosylgalactoside (psychosine); 13, cholesterol; 14, phosphatidylcholine; 15, lysophosphophatidylcholine; 16, blank. sky et al., 1999) has a predicted pi of 9.44. also the antibacterial defensins, arginine-rich peptides found in the human mucus are cationic and establish electrostatic interaction with the negatively charged membranes (ganz and lehrer, 1994) . interaction of ad41 with the gi protective mucus barrier was tackled by studying its lipids affinity. one of the potentially important biophysical features of mucus relates to its ''unwettability'' linked with its hydrophobic character. the hydrophobic properties of mucus as well as its viscosity appear to depend on its lipid constituents and specifically on the presence of a phospholipid surfactant that is synthesized, stored, and secreted by gi mucus cells (reviewed in lichtenberger, 1995) . the mucus layer within the gi tract turns over continuously, and the so-called ''soluble mucus'' can be found within the gi lumen (lehr et al., 1991) . the phospholipid constituents of the mucus are mainly pc and pe, which together account for more than 60% of total phospholipid (nardone et al., 1993) . interestingly, the protective effect of the mucosa against gastric juice, damaging agents, and microorganisms can be attributed to the unsaturated phospholipid, dppc (lichtenberger, 1995; schmitz and muller, 1991 and references therein) . under close to neutral ph conditions, both ad2 and ad41 attached to dppc, with ad41 also strongly recognizing dppg (fig. 6) . of note, the assay sensitivity is ensured by the amount of the immobilized lipid in the pmol range (thomas et al., 2001) . when using isolated ads structural proteins in such an assay, we observed that the hexon, the major capsid protein, and the fiber were responsible for both ads binding to dppc (data not shown), confirming the data of balakireva et al. (2002) . in addition, ad2 penton base did not recognize any of the phospholipids used in the assay, whereas binding of enteric ad to dppg could be attributed to the penton base protein which attached strongly to dppg (data not shown). a similar experiment performed with ad3 dodecahedra gave negative results showing that neither the fiber nor the penton base of ad3 (respiratory and ocular serotype) has the ability to recognize phospholipids (results not shown). when ad2 was exposed to the acid followed by the ph jump to neutral, conditions simulating the passage from stomach to intestine, ad2 virions lost the capability of lipid interaction (fig. 6) . this can be explained by irreversible changes generated in respiratory virions upon acid exposure due to proteolytic cleavage (fig. 5) . most remarkably, acid treatment of ad41 followed by the ph jump to neutral resulted not only in the retention of the lipid interaction observed at the neutral ph but also in the improved interaction with other lipids (fig. 6) . altogether, these results suggest a scenario in which enteric ad upon encountering acidic stomach environment is still able to withstand the proteolytic attack and cross the mucus barrier through interaction with its lipid components. because the mucosa is primarily lipophilic (corbo et al., 1990) , and mucosal pathogens are known to target sites of infection through adherence to host glycoconjugate receptors (mahdavi et al., 2002; svensson et al., 2003) , the ad41 aptitude to interact with lipids was extended to a panel of glycosphingolipids, a highly polymorphic class of lipids, the constituents of plasma membrane. high amounts of sphingolipids were particularly found for the apical plasma membrane domains of intestinal cells (simons and van meer, 1988) . here again, ad41 interacted with a significantly larger group of sphingolipids than respiratory ad (fig. 7) , and these data could explain mechanistically ad41 interaction with the plasma membrane of epithelial monolayer lining the gi. this interaction is likely to be highly reinforced thanks to the car recognition by enteric ad long fiber. interestingly, several sphingolipids are involved in cell binding of enteric pathogens. h. pylori attaches to sulfatides and is able to bind phospholipid pe and gangliosides gm3 at neutral ph, but low ph pulse induces a specific recognition of sulfatides (reviewed in lingwood, 1999) . similarly, the e. coli enterotoxin b, a basic peptide of pi 9.6, recognizes sulfatide on the pig jejunum brush border epithelial cells (rousset et al., 1998) . in addition, galactosylceramide and sulfatide have attracted attention as the alternative receptors for hiv (bhat et al., 1993; cook et al., 1994; ruiz et al., 1994) , whereas glycosphingolipid asialo-gm1 is involved in rotavirus cell binding (willoughby et al., 1990) . data on the entry and infection mechanism of enteric viruses are scarce. it was observed that the proteolytic cleavage, believed to occur in the lumen of intestine, enhances rotavirus infectivity in vitro (estes et al., 1981) . in addition, observations on membrane permeabilization by rotaviruses led to the hypothesis of a direct virus penetration across the plasma membrane lipid phase (ruiz et al., 1994) . however, not much is known about the virus behavior in the gi lumen as well as about the mechanism allowing the passage through protective mucus layer. to our knowledge, our data on enteric ad41 are the first allowing the reasonable hypotheses concerning the chain of events starting from the oral route of virus entry, crossing mucus, and attaching to the surface of the epithelial monolayer lining the gi. a narrow tropism of ad41, unique among ads, is the result of a specific adaptation which rendered these viruses very efficacious pathogens of the human gi. the virulence of enteric ads against human gi has been recognized as being of value for putative applications linked to gastric gene therapy (croyle et al., 1998a) . in addition, the eventual spread of these vectors would be easy to control because their infections are confined to epithelial cells adjacent to the intestinal lumen. however, because of the lack of the recombinant ads constructed with the backbone of enteric serotypes, all gi gene therapy assays are done with respiratory ad5-derived vectors (see for example foreman et al., 1998) . alternatively, ad5-derived vectors are used in vitro in combination with cyclodextrins, rendering positively charged formulations (croyle et al., 1998b) or after manipulations allowing ablation of the native (respiratory) tropism (heideman et al., 2002) . in view of our data, it is clear that this kind of recombinant ads will not survive oral applications in vivo. the construction of gi delivery vectors based on the enteric ads backbone and further studies on the role of enteric ads capsid proteins in virus entry will bring us closer to the intelligent use of our fundamental knowledge in human health applications. the theoretical pi of hexon, penton base, and fiber proteins was obtained with the software http://www.up. univ-mrs.fr/~wabim/d_abim/compo-p.html. accession numbers to the sequences used for calculation of predicted pi values are given in table 2 . the 293 transformed human embryonic kidney 293 and a549 human lung carcinoma cells were maintained in emem supplemented with 2 mm glutamine, 20 u of penicillin -streptomycin, and 10% fbs. ad3 and ad5 were propagated in 293 cells. ad41 tak strain was propagated in 293 cells as described by favier et al. (2002) and ad2 was grown on hela cells. all viruses were purified according to kanegae et al. (1994) and tittered at 9-16 â 10 8 focus forming units (ffu)/ml, respectively. for western blot analysis, polyclonal rabbit antibodies were used at the following dilutions: anti-ad3 dodecahedron penton at 1:50 000, and anti-ad41 and anti-ad2 at 1:10 000 (all made by authors). antibodies were obtained after two intravenous and one intraperitoneal injections of the purified antigens (boiled ad3 dodecahedron or a mixture of 50% boiled -50% infectious viruses) to the rabbit (esd, france). monoclonal antibody 4d2 recognizing the fnpvypy epitope conserved in the n-terminal part of ad fibers (kind gift of j. engler; hong and engler, 1991) was used at 1:50 000. anti-mouse and anti-rabbit-horseradish peroxidase conjugates were used at 1:5000 (jackson immu-nores.). ecl detection system (amersham pharmacia) was used throughout this work. purified ads virions of serotypes 2, 3, 5, and 41 (approximately 10 ag of ad3 and ad5, 5 ag of ad41 and ad2) were electrophoresed in 0.8% agarose gel using 0.5% tbe buffer at 50 v. acid treatment was done by virus incubation with an equal volume of hcl, ph 2, at 37 jc for 1 min. the reaction mixtures were placed in ice-cold bath and 1 m tris, ph 9.6, was immediately added to reach ph 7.5. to establish conditions for hcl treatment, the curve of the maximum of infection was obtained for ad41 and ad2 on 293 cells as described by favier et al. (2002) . subsequently, conditions of about 50% or 100% saturation of infection were used as follows. confluent 293 cells were infected with ad41 at dilutions of 1/8 and 1/16 (3.37 â 10 5 and 1.68 â 10 5 ffu/well of 96-well dish) and with ad2 at 1/16 and 1/32 dilutions (1 and 0.5 â 10 5 ffu/well of 96well dish). for each condition, one mixture was prepared for the five samples containing the virus, yielding five fractions of 5 al each. incubation conditions were: 1 min at rt or at 37 jc, with 5 al of hcl at final ph 2.0 or hcl at ph 4.0 or with culture medium. the reaction was terminated by adding 150 al of emem-0.2% fbs to each sample. three portions of 293 cells in 96-well dish were infected each with 50 al of such viral solution. one hour after infection, 100 al of emem -0.2% fbs was added and the viral proteins were estimated by immunofluorescence 24 h later. native fibers of ad41 and ad5 were isolated from a cscl fraction obtained during virus purification. this fraction is localized above the virus band and contains a mixture of free viral proteins. native ad41 proteins were obtained from ad41-infected cells grown in 31 flasks of 175 cm 2 . supernatant above the virus band obtained after the first cscl gradient (kanegae et al., 1994) was dialyzed against a q2-sepharose column followed by an s2-sepharose with 20 mm mes buffer, ph 6.5. fibers were visualized by western blot with the serum recognizing ad3 penton (complex of penton base and fiber proteins) or with monoclonal antibody 4d2. dodecahedra made of 12 pentons (p3, complex of penton base and fiber) were purified as described by fender et al. (1997) . fiber samples were treated with an equal volume of hcl, ph 2, at 37 jc and after 1 min the ph was adjusted to 7.5 with 1 m tris buffer, ph 9.6. chymotrypsin digestion of fibers was performed with a 1:5 enzyme/substrate ratio for 1 h at 37 jc and ph 7.5. proteins were electrophoresed under semi-denatured conditions in a 7.5% sds-polyacrylamide gel at 4 jc as described by mitraki et al. (1999) followed by western blot. freeze-dried lipids were reconstituted in a 1:1 (v/v) mixture of chloroform/methanol at 1 mm concentration. the stock solutions were 2-fold serially diluted in a mixture of chloroform/methanol/water (1:2:0.8, by vol.) and 1 al of such dilutions (1 pmol to 1 nmol of pc, dppc, pe, dppg) was spotted onto the hybondc-extra nitrocellulose membrane (amersham) and allowed to dry at rt for 1 h. membranes were blocked for 1 h at rt in buffer a (50 mm mes -naoh ph 6.0, 150 mm nacl, and 0.1% tween 20) or buffer b (10 mm tris -hcl ph 7.5, 150 mm nacl, and 0.1% tween 20) containing 3% fatty acid-free bsa (sigma). the membrane was then incubated overnight at 4 jc in the same buffers containing 2 ag/ml of viruses. the membranes were washed five times for 10 min in the respective buffer and then incubated for 1 h with the polyclonal anti-ad41 and anti-ad2 containing 3% bsa. after three 10-min washes followed by overnight wash at 4 jc without rocking and four 15-min washes at rt, the membranes were incubated for 1 h with anti-rabbit or antimouse-horseradish peroxidase conjugate. finally, after six 30-min washes, the interactions were detected by ecl. sphingostrips membranes (molecular probes), each containing 100 pmol of 15 different lipids, were blocked for 1 h at rt in buffer a or buffer b. the membrane was then incubated overnight at 4 jc in the same buffers containing 2 ag/ml of viruses. the membranes were washed four times for 15 min in the respective buffer and then incubated for 1 h with the appropriate antibody. after four 15-min washes, membranes were incubated for 1 h with an anti-rabbit or anti-mouse-horseradish peroxidase conjugate. finally, after four 15-min washes, the interactions were detected by ecl. the overlay reactions were done either in presence or in absence of 10 mm mgcl 2 . adenovirus type 41 lacks an rgd alpha(v) integrin binding motif on the penton base and undergoes delayed uptake in a549 cells vitronectin receptor antibodies inhibit infection of hela and a549 cells by adenovirus type 12 but not by adenovirus type 2 binding of adenovirus capsid to dipalmitoylphosphatidylcholine provides a novel pathway for virus entry isolation of a common receptor for coxsackie b viruses and adenoviruses 2 and 5 composition of phospholipid classes and phosphatidylcholine molecular species of gastric mucosa and mucus structural analysis of the mechanism of adenovirus binding to its human cellular receptor the galactosyl ceramide/sulfatide receptor binding region of hiv-1 gp120 maps to amino acids 206 -275 s-layer protein of lactobacillus acidophilus atcc 4356: purification, expression in escherichia coli, and nucleotide sequence of the corresponding gene novel partner proteins of adenovirus penton binding of human immunodeficiency virus type i (hiv-1) gp120 to galactosylceramide (galcer): relationship to the v3 loop characterization of the barrier properties of mucosal membranes in vitro and in vivo assessment of 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with diarrhea in infants: a new subgroup of human adenoviruses stepwise dismantling of adenovirus 2 during entry into cells biochemical studies on adenovirus multiplication: iv. isolation, purification and chemical analysis of adenovirus gastrointestinal problems in the infant bifunctional role of glycosphingolipids. modulators for transmembrane signaling and mediators for cellular interactions selective gene transfer into primary human gastric tumors using epithelial cell adhesion molecule-targeted adenoviral vectors with ablated native tropism the amino terminus of the adenovirus fibre protein encodes the nuclear localization signal identification of an enteric adenovirus by immunoelectroosmophoresis (ieop) technique analysis of structural design features in collagen evidence for the role of a human intestinal adenovirus in the pathogenesis of coeliac disease a simple and efficient method for purification of infectious recombinant adenovirus adenovirus type 40 virions contain two distinct fibres the influence of intestinal mucus components on the diffusion of drugs an estimate of turnover time of intestinal mucus gel layer in the rat in situ loop the hydrophobic barrier properties of gastrointestinal mucus glycolipid receptors for verotoxin and helicobacter pylori: role in pathology helicobacter pylori saba adhesin in persistent infection and chronic inflammation multiple adenovirus serotypes use alpha v integrins for infection unfolding studies of human adenovirus type 2 fibre trimers. evidence for a stable domain reduction of natural adenovirus tropism to the liver by both ablation of fiber-coxsackievirus and adenovirus receptor interaction and use of replaceable short fiber phospholipid composition of human gastric mucosa: a study of endoscopic biopsy specimens protein folding and association: insights from the interfacial and thermodynamics properties in vitro reconstitution, hexon bonding and handedness of incomplete adenovirus capsid human enteric adenovirus type 41 (tak) contains a second fibre protein gene dna sequences from the adenovirus 2 genome the coxsackievirus-adenovirus receptor protein can function as protein can function as a cellular protein for adenovirus serotypes from groups a sulfatide from the pig jejunum brush border epithelial cell surface is involved in binding of escherichia coli enterotoxin b rotavirus interaction with isolated membrane vesicles structure and function of lamellar bodies, lipid -protein complexes involved in storage and secretion of cellular lipids lipid sorting in epithelial cells glycolipid depletion in antimicrobial therapy crystal structure of the phosphatidylinositol 3,4-bisphosphatebinding pleckstrin homology (ph) domain of tandem ph-domain-containing protein 1 (tapp1): molecular basis of lipid specificity highresolution structure of the pleckstrin homology domain of protein kinase b/akt bound to phosphatidylinositol (3,4,5)-trisphosphate two new serotypes of enteric adenovirus causing infantile diarrhoea importance of enteric adenovirus 40 and 41 in acute gastroenteritis in infants and young children molecular composition of the adenovirus type 2 virion structure of the human adenovirus serotype 2 fiber head domain at 1.5 a resolution rapid inactivation of rotaviruses by exposure to acid buffer or acidic gastric juice integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment rotaviruses specifically bind to the neutral glycosphingolipid asialo-gm1 structure of the receptor binding domain of adenovirus type 5 fiber protein human adenovirus type 41 contains two fibres stability and in vitro absorption of captopril, enalapril and lisinopril across the rat intestine we are indebted to mark van raaij for the gift of extracellular car domain. we acknowledge the gift of antibodies from jeff engler. key: cord-271359-dpa8zzc3 authors: sapats, s. i.; ashton, f.; wright, p. j.; ignjatovic, j. title: novel variation in the n protein of avian infectious bronchitis virus date: 1996-12-15 journal: virology doi: 10.1006/viro.1996.0670 sha: doc_id: 271359 cord_uid: dpa8zzc3 abstract the nucleocapsid protein of coronaviruses has been considered highly conserved, showing greater than 94% conservation within strains of a given species. we determined the nucleotide sequence of the n gene and the 3′ untranslated region (utr) of eight naturally occurring strains of ibv which differed in pathogenicity and tissue tropism. in pairwise comparisons, the deduced amino acid sequences of n of five strains vic s, n1/62, n9/74, n2/75, and v5/90 (group i) shared 92.3–98.8% identity. the three strains n1/88, q3/88, and v18/91 (group ii) shared 85.8–89.2% identity with each other, but only 60.0–63.3% identity with viruses of group i. amino acid substitutions, deletions, and insertions occurred throughout the n protein and involved regions previously identified as being conserved. despite the considerable variation observed between the two virus groups, all n proteins contained a high proportion of basic residues, 80% of which were conserved in position. in addition, all strains contained approximately 30 serine residues of which 10 were conserved, the majority occurring between positions 168 and 194. as for all other coronaviruses, the region between positions 92 and 103 was highly conserved. hence, a large number of amino acid changes can be tolerated within the n protein without affecting its integrity or functioning. the 3′ utr immediately downstream from the n gene was highly heterogeneous with extensive deletions occurring in the group ii strains. infectious bronchitis virus (ibv), a member of the family be highly conserved differing by only 2 to 6% at the amino acid level (4, 5) . the high level of conservation in the n coronaviridae, causes an acute highly contagious disprotein has resulted in the widely held view that the s1 ease of chickens resulting in significant economic losses glycoprotein, which may show up to 49% variation (6) , to poultry industries throughout the world. the ibv geis the only relevant structural element in assessing the nome consists of a single strand of positive sense rna genetic diversity and evolutionary direction of ibv. measuring 27.6 kb in length excluding the poly(a) tail (1) . immediately downstream of the n gene is the 3 un-the genes encoding the three major structural proteins translated region (utr) which is presumably important are situated within an 8-kb region located at the 3 end in the initiation of negative-strand rna synthesis. the of the genome. these proteins are the spike glycoprotein organization of the 3 utr differs among the coronavi-(s), the membrane glycoprotein (m), and the phosphoryruses. in porcine, canine, and feline coronaviruses this lated nucleocapsid protein (n). the n protein plays a region is conserved among strains within a species and role in viral replication, assembly, and immunity. it intercontains at least one open reading frame (orf) (7, 8) . acts with leader rna sequences facilitating viral mrna to the present time, functional orfs have not been desynthesis and also binds to the viral rna forming a helitected in the 3 utr of ibv. however, the virulent m41 cal nucleocapsid (2). the n protein of all coronaviruses virus lacks a sequence of 184-196 nucleotides that has is overall very basic and in ibv contains 409 amino acids been detected in five other ibv strains. this sequence with a predicted m r of approximately 50,000 (3); it also has been termed a hypervariable region (hvr) and was contains a high proportion of serine residues which act proposed to be exogenous in origin (3, 9) . the region as sites for phosphorylation (2). located downstream of the hvr (315 nucleotides ending the n protein of 27 strains of ibv isolated over a period at the poly(a) tail) is highly conserved in strains of ibv, of 60 years from diverse locations such as the u.s.a., the probably indicative of its role in the synthesis of negative-uk, holland, saudi arabia, and japan has been shown to strand rna. recently we reported the isolation of three ibv strains, other australian ibv strains. based on s1 sequences, the ibv strains and methods used for their propagation have been described (10, 11) . vic s is a commercial these strains formed a distant and novel genetic group of ibv (10) . in addition, the n protein of these strains vaccine (arthur webster pty ltd, castle hill, australia). strain n1/62 was isolated from unvaccinated chicks in failed to react with five monoclonal antibodies directed against different epitopes on the n protein. these epi-1962, whereas n9/74, n2/75, n1/88, q3/88, v5/90, and v18/91 were isolated from vaccinated commercial chicks topes are conserved in other australian strains of ibv, indicating unusual changes in the n genes of n1/88, q3/ between 1974 and 1991. strains were cloned either in chicken embryo kidney cells (by plaque assay) or tra-88, and v18/91 (11) . we have now sequenced the n gene and 3 utr of eight australian ibv strains isolated over cheal organ cultures (by limiting dilutions) and passaged 1-2 times in embryonated chicken eggs. all strains repli-the period of thirty years from both vaccinated and unvaccinated flocks. the results demonstrated less conserva-cated in the trachea and strains vic s, v5/90, n1/62, n9/ 74, and n2/75 also replicated in the kidneys, the latter tion in the n protein and 3 utr of ibv than previously detected. three causing 32 to 96% mortality (10). fig. 1. sequence alignment of the n protein of australian strains of ibv. the complete deduced amino acid sequence of n of vic s is shown. gaps (dashes) were introduced to align the sequences. dots indicate residues identical to vic s. asterisks indicate residues conserved in all strains. the longest region of complete conservation is boxed. the serine-rich region is underlined; the t cell epitope is double underlined. clusters of basic residues are shaded. the clustal v program was used for all sequence alignments (17) . of these five strains were also similar to those of geographically distant strains isolated in the u.s.a., europe, and japan with which they shared 91.4-92.9% identity at the amino acid level (results not shown). this confirmed the previous observation of the tendency for conservation of the n protein over a long time (ç30 years) irrespective of geographical distances and immunological pressures. contrary to this, however, the n protein of three other strains n1/88, q3/88, and v18/91 (group ii) shared only 60.0-63.3% amino acid identity with the n proteins of strains in the first group, while sharing 85.8-89.2% amino acid identity with each other. this lack of conservation of the n protein has not been reported before for any other coronavirus. an alignment of the deduced amino acid sequence of the n gene of vic s with sequences of the other australian strains is shown in fig. 1 n (3, 4, 18) . the tree was constructed using tion 337. the group ii strains also contained a number the neighbor-joining method (19) . of insertions and deletions relative to group i strains, the majority of which were at the amino and carboxy termini between positions 7 and 23 and 339 and 401, respec-viral rna was purified using methods described (10, tively. overall, only 54% (221/411) of the residues were 12). vic s, n1/88, and q3/88 cdna was synthesized using conserved in all strains, the longest region of complete random primers, and sequences obtained from the conservation occurring between positions 92 and 103, cloned cdna were used to design primers for amplificacorresponding to the part of the n protein previously tion of the n gene of all strains by reverse transcription found to have the highest degree of conservation in ibv and polymerase chain reaction. all cdnas were cloned and all other coronaviruses (13). published sequences into puc series plasmids. for each virus two or more for the n proteins of ibv also contain a region of complete independent cdna clones were sequenced using the conservation between positions 242 and 296 (4). as evi-pharmacia t7 sequencing kit. pairwise comparisons of dent from fig. 1 , the corresponding region is not conthe nucleotide and deduced amino acid sequences of served among the australian strains. the precise locathe n genes of the eight strains vic s, v5/90, n1/62, n9/ tion and role of functional domains within the n protein 74, n2/75, n1/88, q3/88, and v18/91 (excluding the first are not well understood (2, 4, 14) . however, a t cell 17 nucleotides of v5/90, n1/62, n9/74, and n2/75) are epitope has been identified in ibv corresponding to posishown in table 1 . the strains formed two distinct genotions 74-81 in fig. 1 (15) . examination of these setypic groups based on the level of nucleotide and amino quences reveals that they are completely conserved in acid identities. the first five strains in table 1 2, 3, 14) . (i) all strains possessed a high proportion of basic residues (17.2-19.6%). the majority of revealed values of identity ranging from 22.2 to 100%. within group i the identity was 92.1-100%; within group these (80%) were conserved in position and generally clustered in three regions between positions 66 and 88, ii it was 22.2-100% identity (results not shown). the hvr of group i viruses (i) contained a high u content which 181 and 234, and 334 and 373 (fig. 1) . this basic character probably facilitates protein-rna interactions. (ii) the was evenly distributed, and (ii) was similar to that of beaudette, ark99, and gray viruses (9) . little similarity to carboxy terminus contained a clustering of acidic residues located between positions 375 and 415, a region kb8523 and holl52 viruses was observed (results not shown). which otherwise showed very little conservation. however, the acidic residues were less conserved (66%) in this hvr downstream of the n gene was originally identified in the vaccine strain beaudette by comparison position than the basic residues. (iii) all strains contained approximately 30 serine residues, their precise locations with the virulent m41 strain and considered to be an insert of exogenous origin acquired through recombina-varying considerably. only 10 were totally conserved in position. the majority of these conserved serine residues tion during adaptation in eggs (3, 9) . however, the presence of the long hvr (153 to 216) in group i strains, occurred between positions 168 and 194. serine residues are potential sites for phosphorylation (2). hence it which received a low number of passages in eggs, suggests that the hvr region may have been present in appears that the n protein is able to tolerate some variability in the distribution of basic, acidic, and serine resi-an ancestral strain and subsequently deleted in some strains. in addition, in contrast with the beaudette and dues, although the basic residues were the most conserved consistent with the function of the protein as a m41 comparison, the shorter forms (10 to 38) of the hvr in the australian strains n1/88, q3/88, and v18/91 (group ribonucleocapsid protein. these residues are expected to directly influence protein charge, state of phosphoryla-ii) are associated with a decrease in virulence. thus there is no clear association between the length of the tion, and secondary structure. the phylogenetic relationship between australian and hvr and virulence. the remaining 294 nucleotides downstream of the hvr (from position 220 in fig. 3b ) showed other strains of ibv based upon the published nucleotide sequences for the n gene is shown in fig. 2 . the group considerable conservation with values of identity ranging from 84.6-100.0% in pairwise comparisons. within group i and group ii australian strains formed two clusters distinct from previously published strains. the five group i i strains, the identity was greater than 97.3%. an orf was detected within the 3 utr of n1/62 and strains formed one cluster with vic s and v5/90 being the most closely related and showing approximately the n9/74 with the potential to encode hydrophobic proteins of 8793 m r and 8711 m r , respectively. a similar orf has same degree of relatedness to n1/62 and n2/75, while n9/74 appeared to be the most divergent. the group ii been identified in four other ibv strains (9) . although the initiation codons of these orfs fit the kozak consensus strains formed another cluster having diverged markedly from all other clusters. the phylogenetic relationships sequence (16) with an a at position 03 and a g at position /4, there are no ibv mrna transcription motifs (ctt/ among the australian ibv strains based on the n nucleotide sequences were similar to those based on s1 (10). gaacaa) directly upstream. other coronaviruses such as porcine transmissible gastroenteritis virus also con-this suggests that the s1 and n genes of strains in group ii have evolved in parallel. interestingly, a comparison tain an orf within the 3 utr. the latter virus directs the synthesis of a hydrophobic and membrane-associated of the dutch strain d1466 with other european strains showed that the d1466 s1 protein had significantly di-protein of 9101 m r (7) . at present it is unknown whether the corresponding protein for ibv is synthesized in in-verged from the s1 of the other strains, similar to the distance between the australian group i and ii strains. fected cells and what its function in virus replication may be. more experiments are required to detect the protein however, this divergence was not matched by the n protein, which was conserved between d1466 and the and to examine possible mechanisms for the initiation of its translation. other european strains (5) . the 3 utr, located immediately downstream of the n gene, was sequenced for vic s and the other australian acknowledgment strains (figs. 3a and 3b) . a hvr was identified, ranging fig. 3b ) than previously reported (9) . in the references group i strains n1/62, n9/74, and n2/75 the hvr was tom-214, 214, and 216 nucleotides, respectively, while for vic ley the coronaviridae virus 12 key: cord-266861-t5h133lp authors: moller-tank, sven; maury, wendy title: phosphatidylserine receptors: enhancers of enveloped virus entry and infection date: 2014-11-01 journal: virology doi: 10.1016/j.virol.2014.09.009 sha: doc_id: 266861 cord_uid: t5h133lp a variety of both rna and dna viruses envelop their capsids in a lipid bilayer. one of the more recently appreciated benefits this envelope is incorporation of phosphatidylserine (ptdser). surface exposure of ptdser disguises viruses as apoptotic bodies; tricking cells into engulfing virions. this mechanism is termed apoptotic mimicry. several ptdser receptors have been identified to enhance virus entry and we have termed this group of proteins ptdser-mediated virus entry enhancing receptors or pveers. these receptors enhance entry of a broad range of enveloped viruses. internalization of virions by pveers provides a broad mechanism of entry with little investment by the virus itself and may allow some viruses to attach to cells, thereby making viral glycoprotein/cellular receptor interactions more probable. alternatively, other viruses may rely entirely on pveers for internalization into endosomes. this review provides an overview of ptdser receptors that serve as pveers and the biology behind virion/pveer interaction. a variety of both rna and dna viruses envelop their capsids in a lipid bilayer. this outer membrane is obtained during virus budding from either plasma or organelle membranes. while reliance on an envelope sensitizes viruses to desiccation, detergents, and heat, these envelopes provide a number of benefits for the virus, including protection of viral structural proteins from immune recognition and neutralizing antibodies, a platform for displaying viral proteins, a barrier to enclose viral and cellular proteins necessary for early steps during infection, and a mechanism for virus egress without lysing infected cells. in addition, a more recently appreciated benefit is the incorporation of phospholipids into viral envelopes. presentation of phosphatidylserine (ptdser) on the outer leaflet of these membranes disguises viruses as apoptotic bodies, thereby conning cells into engulfing virions through cell clearance mechanisms. this mechanism of enhanced virus entry is termed apoptotic mimicry. over the past three years, receptors and receptor complexes have recently been identified that enhance entry of a diverse range of enveloped viruses. this group of viral receptors shares the ability to bind to ptdser present on the viral envelope and, consequently, we have termed them ptdser-mediated virus entry enhancing receptors or pveers. the broad expression of these receptors and their ability to interact with ptdser on a wide array of enveloped viruses has huge potential implications for virus infection. most importantly, pveers enhance virus binding to cells and facilitate internalization. these receptors also contribute to immune evasion through both anti-inflammatory signaling and a mechanism of entry that does not require extracellular exposure of key receptor-binding domain and fusion epitopes. in this review we summarize what is known about the identified pveers and their role in virus entry and identify important gaps in current knowledge. apoptotic mimicry was first hypothesized to be used by hepatitis b virus (vanlandschoot and leroux-roels, 2003) , but was experimentally confirmed with vaccinia virus (mercer and helenius, 2008) . inactivation of vaccinia virus by np-40-mediated lipid depletion could be rescued by incubation with ptdser liposomes (oie, 1985) . however, the contribution of ptdser to infection was not understood. mercer et al. furthered these studies and determined that not only ptdser is present on the surface of some vaccinia infectious particles, but annexin v (anxv), a ptdser-binding protein, can bind to ptdser on viral envelopes and inhibit vaccinia infection (mercer and helenius, 2008) . these results were confirmed by another study that found substitution with a non-biologically relevant isomer of ptdser restored infectivity (laliberte and moss, 2009 ). in addition, a role for viral envelope ptdser during infection was demonstrated for pichinde virus and hiv-1 (soares et al., 2008; callahan et al., 2003) . a protein complex composed of growth-arrest-specific 6 (gas6) and the tyrosine kinase receptor, axl, was the first set of cellular proteins to be implicated in ptdser-binding enhancement of viral entry (morizono et al., 2011) . it was shown that the soluble protein gas6 binds to ptdser on the virion surface and bridges virus to the cell surface via interaction with the tyrosine kinase receptor, axl and formation of this complex is necessary for enhancement of virus entry. this study found in addition to vaccinia virus, the gas6/axl complex enhances binding and entry of lentiviruses pseudotyped with ross river gp, baculovirus gp64, or sindbis env, demonstrating for the first time that ptdser binding can enhance entry of viral particles bearing a variety of different viral glycoproteins. more recently, a variety of additional pveers were identified, including t-cell immunoglobulin and mucin domain 1 and 4 (tim-1 and 4) proteins and mfg-e8/integrin αvβ3 or αvβ5 complexes (meertens et al., 2012; jemielity et al., 2013; moller-tank et al., 2013; morizono and chen, 2014) . the details of virion interactions with different pveers are highlighted below. several recent studies have expanded our understanding of pveers. the group of viruses whose entry is enhanced by pveers now include members of the flavivirus, filovirus, new world arenavirus, baculovirus, and alphavirus families (meertens et al., 2012; jemielity et al., 2013; moller-tank et al., 2013; morizono and chen, 2014) . between viruses within these families, the extent to which pveers enhance virus entry varies, but generally viruses do not appear to utilize one pveer effectively and not another. this suggests that virion uptake mediated by the different pveers is mechanistically similar. despite the strong evidence that pveers interact with virion associated ptdser, the utilization of pveerdependent virus entry is influenced by the viral glycoprotein presence on the virion. for instance, entry of viruses bearing the envelope proteins of old world arenaviruses, coronaviruses, influenza a virus, vesicular stomatitis virus, or herpes simplex virus 1 is not enhanced by pveer expression (morizono et al., 2011; meertens et al., 2012; jemielity et al., 2013; moller-tank et al., 2013; kondratowicz et al., 2011) . thus, while pveers do not enhance entry all enveloped viruses, they do represent a potentially important class of receptors for a large and diverse collection of important human pathogenic viruses. six pveers have been identified to date: tim-1, tim-4, gas6 or protein s/axl, mer, and tyro3, and mfg-e8/integrin αvβ3 and αvβ5. a summary of characteristics of these pveers is shown in table 1 . the key property that these proteins or complexes utilize to enhance virus entry is their ability to bind ptdser and a native function of all of these receptors is to bind and clear apoptotic bodies (ravichandran, 2011) . mutation of residues involved in ptdser binding or complex formation results in inhibition of pveer function (morizono et al., 2011; meertens et al., 2012; moller-tank et al., 2013; morizono and chen, 2014) . further, competition with ptdser liposomes, but not phosphatidylcholine liposomes, inhibits entry enhancement by pveers (jemielity et al., 2013; moller-tank et al., 2013) . phosphatidylethanolamine liposomes are also somewhat inhibitory, likely due to sharing similar structure to ptdser (jemielity et al., 2013) . in some cases, prebinding virus with the ptdser-binding protein, annexin v (anxv), has been shown to inhibit pveer enhancement of entry (mercer and helenius, 2008; callahan et al., 2003; meertens et al., 2012) , but not in others (morizono and chen, 2014) . consistent with the ability of an array of ptdser receptors to serve as pveers, an artificially generated pveer containing the ptdser-binding domain from anxv was highly effective at mediating uptake of vesicular stomatitis virus (vsv) pseudovirions bearing a filovirus, alphavirus or baculovirus glycoprotein (moller-tank et al., 2014). (kobayashi et al., 2007) immune cells: filovirus: old world arenavirus: human: b cells (sizing et al., 2007) , mast cells , t h 2 cd4 þ t cells (meyers et al., 2005; umetsu et al., 2005; khademi et al., 2004) , and nkt cells kim et al., 2013) ebola• and marburg (jemielity et al., 2013; moller-tank et al., 2013; kondratowicz et al., 2011) lassa virus, lcmv (jemielity et al., 2013; moller-tank et al., 2013) huh-7, achn, a498, 786-o, caco-2, caki-1, tk-10, uo-3, vero, a549, cos-7 (meertens et al., 2012; kondratowicz et al., 2011) epithelial cells: alphavirus: new world arenavirus: kidney ichimura et al., 1998) and airway and eye mucosa ross river•, chikungunya, sindbis•, eastern equine encephalitis (jemielity et al., 2013; moller-tank et al., 2013; morizono and chen 2014) oliveros (jemielity et al., 2013) baculovirus: influenza a virus: autographa californica multicapsid nucleopolyhedrovirus• (moller-tank et al., 2013) h7n1, h1n1• (jemielity et al., 2013) rhabdovirus: coronavirus: vesicular stomatitis virus (morizono and chen, 2014) sars (jemielity et al., 2013) new world arenavirus: rhabdovirus: amapari, tacaribe•, junín, and machupo virus (jemielity et al., 2013) vesicular stomatitis virus (jemielity et al., 2013) flavivirus: herpes simplex virus: west nile virus•, dengue•, yellow fever virus• (meertens et al., 2012; jemielity et al., 2013) hsv-1• (meertens et al., 2012) tim-3 igv immune cells: flavivirus: alphavirus: mouse: t h 1 and t h 17 cd4 þ t cells (khademi et al., 2004; hastings et al., 2009; monney et al., 2002; , mast cells , dcs anderson et al., 2007) , and monocytes (anderson et al., 2007) west nile virus, dengue virus• (meertens et al., 2012; jemielity et al., 2013) sindbis (morizono and chen, 2014) raw264 igv (kobayashi et al., 2007; santiago et al., 2007; miyanishi et al., 2007) immune cells: filovirus: new world arenavirus: macrophages and mature dcs (kobayashi et al., 2007; dekruyff et al., 2010; miyanishi et al., 2007; mizui et al., 2008; meyers et al., 2005) , and b-1 cells (rodriguez-manzanet et al., 2010) ebola, marburg (jemielity et al., 2013; moller-tank et al., 2013) oliveros (jemielity et al., 2013) tissues: alphavirus: old world arenavirus: spleen, lymph node, and peritoneum (miyanishi et al., 2007; meyers et al., 2005) sindbis, ross river (morizono and chen, 2014) , eastern equine encephalitis (jemielity et al., 2013) lasv, lcmv (jemielity et al., 2013) new world arenavirus: influenza a virus: ebola, marburg (jemielity et al., 2013; shimojima et al., 2006) lasv, lcmv (jemielity et al., 2013) a549, vero, cos-7, hela, snb19, and sn12c (meertens et al., 2012; shimojima et al., 2006; brindley et al., 2011 ) platelets (angelillo-scherrer et al., 2001 rhabdovirus: new world arenavirus: immune cells: vesicular stomatitis virus (morizono and chen, 2014) machupo (jemielity et al., 2013) , (seitz et al., 2007; caraux et al., 2006; behrens et al., 2003) baculovirus: oliveros (jemielity et al., 2013) connective tissue: autographa californica multicapsid nucleopolyhedrovirus (morizono et al., 2011; morizono and chen, 2014) rhabdovirus: bone marrow stromal cells (caraux et al., 2006) alphavirus: vesicular stomatitis virus (jemielity et al., 2013) sindbis, ross river (morizono et al., 2011; morizono and chen, 2014) influenza a virus: testes (lu et al., 1999; wang et al., 2005) , cns (stitt et al., 1995; prieto et al., 2000) , retina (prasad et al., 2006) , and foreskin fibroblasts (hanayama et al., 2002; finnemann and rodriguez-boulan, 1999; antonov et al., 2004) , immature dcs albert et al., 1998) ross river and sindbis (morizono and chen, 2014) vesicular stomatitis virus (morizono and chen, 2014) hec-1a, ishikawa, heec, k562, hl60 (bocca et al., 2012; kruger et al., 2000) ; baculovirus: mouse: mammary glands (oshima et al., 1999; stubbs et al., 1990) , spleen, lymph node, brain garmy-susini et al., 2007; uehara and uehara, 2014) , and vascular system (silvestre et al., 2005; brooks et al., 1994) autographa californica multicapsid nucleopolyhedrovirus (morizono and chen, 2014) comma-1d, d1, p388d1 thery et al., 1999; oshima et al., 2002) cd300a igv (simhadri et al., 2012) immune cells: alphavirus: alphavirus: human: cd8 þ t cells (xu et al., 2012) , cd4 þ t cells (clark et al., 2007; simhadri et al., 2011; narayanan et al., 2010) , b cells (silva et al., 2011) , and nk cells (lankry et al., 2010; cantoni et al., 1999) sindbis (morizono and chen, 2014) •• ross river and sindbis (morizono and chen, 2014) thp1, u937, rec-1, sudhl5, 721.221 (silva et al., 2011; kim et al., 2012) baculovirus: autographa californica multicapsid nucleopolyhedrovirus (morizono and chen, 2014) rhabdovirus: vesicular stomatitis virus (morizono and chen, 2014) bai1 type 1 thrombospondin repeats (tsrs) (park et al., 2007) tissues: alphavirus: human: brain (shiratsuchi et al., 1997; sokolowski et al., 2011 ), muscle(hochreiter-hufford et al., 2013 , bone marrow, and spleen (park et al., 2007) ross river and sindbis (morizono and chen, 2014) j774 and raw264.7 (park et al., 2007) immune cells: baculovirus: macrophages (park et al., 2007) autographa (akirav et al., 2012; moser et al., 2007) , dcs (dumitriu et al., 2005) , macrophages sunahori et al., 2006) , and monocytes (ohashi et al., 2010) lassa (moller-tank et al., 2013) hacat , a549 (nakano et al., 2006) tissues: filovirus: smooth muscle kamioka et al., 2011) , ebola virus (moller-tank et al., 2013) cartilidge chondrocytes (loeser et al., 2005) , skin keratinocytes , and vascular system (pollreisz et al., 2010; liu et al., 2010) stabilin-1/-2 epidermal growth factor-like domain kim et al., 2010) immune cells: alphavirus: human: macrophages martens et al., 2006) ross river and sindbis (morizono and chen, 2014) pma stimulated thp1 ) tissues: baculovirus: mouse: sinusoidal endothelial cells (martens et al., 2006; falkowski et al., 2003) , spleen, lymph node, liver, bone marrow, cornea, brain, heart, and kidney (falkowski et al., 2003; goerdt et al., 1991) autographa californica multicapsid nucleopolyhedrovirus (morizono and chen, 2014) pma stimulated p388d1 cells rhabdovirus: vesicular stomatitis virus (morizono and chen, 2014) •confirmed using infectious virus. ••enhances binding only, but not infection. recent studies have demonstrated that virus internalization into hek 293t cells can occur entirely independently of the presence of a viral glycoprotein. this virion internalization is significantly enhanced by pveer overexpression and is inhibited by competition with ptdser liposomes (jemielity et al., 2013; moller-tank et al., 2013) , providing compelling evidence that virion associated ptdser/pveer interactions are responsible for virion uptake, not just virus binding. while the cellular compartment(s) into which pveers deliver virions has yet to be identified, indirect evidence indicates that uptake of cargo is into endosomes (kobayashi et al., 2007; albacker et al., 2010) . once the virion is internalized, the presence of a viral glycoprotein on the virion is necessary for fusion events with the endosomal membrane. an overview of each group of pveers is given below. tim-1 was first implicated in enveloped virus entry as a specific receptor for filoviruses . however, it was later discovered that both tim-1 and family member, tim-4, enhance virus entry as pveers through binding of ptdser (meertens et al., 2012; jemielity et al., 2013; moller-tank et al., 2013) . the human tim family members are type i, cell-surface glycoproteins that along with their murine counterparts, share a common structure. their amino terminal immunoglobulin variable (igv)-like domain extends from the plasma membrane by a heavily o-linked-glycosylated mucin-like domain (mld), which is attached to the cell surface by a transmembrane domain followed by a cytoplasmic tail ( fig. 1 ) (mcintire et al., 2004) . tim-1 signals through phosphorylation of cytoplasmic tail tyrosines (binne et al., 2007; de souza et al., 2005 de souza et al., , 2008 ; however, none of the tim family members require their cytoplasmic domains for pveer function (meertens et al., 2012; moller-tank et al., 2014) . tim-1 and tim-4 are unique among pveers as their ptdserbinding domains are a portion of a transmembrane protein rather than an independent, small protein that binds to a plasma membrane receptor. the ptdser-binding pocket is located between two loops of the igv domain ( fig. 2) (kobayashi et al., 2007; santiago et al., 2007; dekruyff et al., 2010; miyanishi et al., 2007) . conserved aspartate and asparagine residues within the upper loop are involved in coordination of a cation that, in conjunction with residues of the lower loop, form hydrogen bonds with the phosphate and serine groups of ptdser (santiago et al., 2007) . this ptdser binding is necessary for the native functions of tim-1 and tim-4: apoptotic body clearance and immune cell regulation (albacker et al., 2010; miyanishi et al., 2007; lee et al., 2010; kim et al., 2013; ichimura et al., 2008; rodriguez-manzanet et al., 2010; mizui et al., 2008; wong et al., 2010) . mutation of aspartate and/or asparagine or chelation of free cations with egta results in significant loss in ptdser binding and subsequent pveer efficacy (meertens et al., 2012; moller-tank et al., 2013) . a recent study suggests tim-4 is more sensitive than tim-1 or -3 to differences in membrane-ptdser concentrations due to additional ptdserbinding residues outside of the pocket (tietjen et al., 2014) . this altered sensitivity may have implications for virus binding. while all human tim family members can bind ptdser and enhance the uptake of apoptotic bodies (kobayashi et al., 2007; dekruyff et al., 2010) , tim-3 does not effectively enhance virus entry (o2 fold) (meertens et al., 2012; jemielity et al., 2013; kondratowicz et al., 2011) . though the igv domain of tim-3 has reduced affinity for ptdser , replacing the tim-1 or tim-4 igv domain with that of tim-3 results in a functional pveer (moller-tank et al., 2014) . thus, the reduced efficacy is not due to the tim-3 igv domain. rather, the inability of tim-3 to function effectively as a pveer is likely due to the short length of the tim-3 mld which is 2.5-fold shorter than that of tim-1 and -4 ( fig. 1 ). this is supported by studies showing that deletions within the mld of tim-1 reduce pveer efficacy (jemielity et al., 2013; moller-tank et al., 2014) . tyro3, axl, and mer (tam) are members of the tam family of receptor tyrosine kinases. these highly related proteins contain two n-terminal immunoglobulin-like domains, followed by two fibronectin type iii domains, a single transmembrane domain, and a cytoplasmic protein tyrosine kinase (ptk) domain (fig. 2) . tam n-terminal interaction with gas6 leads to activation of the tam receptors and autophosphorylation of tyrosines within the ptk domain (stitt et al., 1995) . tyro3 and mer, but not axl, are similarly activated by binding protein s (stitt et al., 1995; prasad et al., 2006) . both gas6 and protein s consist of an n-terminal domain rich in γ-carboxyglutamic acid residues (gla) that binds to ptdser ( fig. 1) (huang et al., 2003; ishimoto et al., 2000; rajotte et al., 2008) , a loop region, four epidermal growth factor-like repeats, and two c-terminal laminin g-like domains forming a sex hormone-binding globulin-like structure that binds to the ig-like domains of the tam receptors (sasaki et al., 2006 (sasaki et al., , 2002 . dimerization of tam receptors occurs after binding of their ligands, resulting in a 2:2 complex, and is necessary for signaling (sasaki et al., 2006) . all tam receptors in combination with their gas6 or protein s ligand have been shown to effectively enhance virus entry, although studies suggest that the relative effectiveness of the three tam members as pveers varies (morizono et al., 2011; meertens et al., 2012; jemielity et al., 2013; bhattacharyya et al., 2013) . this may be due to differences in binding affinities between the tam receptors and their ligands (nagata et al., 1996) or relative expression. as the ptdserbinding activity of pveers is essential for efficacy, in the case of the tam receptors, this occurs through the gla domain of gas6/protein s and removal of this domain eliminates pveer function of the tams (morizono et al., 2011; meertens et al., 2012) . similarly mutation of the gas6 binding residues of axl inhibits virus uptake by the complex (meertens et al., 2012) . thus, the pveer efficacy of the tam ligand/ receptor complexes requires both efficient binding of ptdser by ligand and binding of ligand by receptor. contrary to the tim family pveers where cytoplasmic tail signaling is not required for enhancing virus entry into cells (moller-tank et al., 2014) , signaling through the ptk domain of tam receptors is essential for enhancement of viral infection (meertens et al., 2012; bhattacharyya et al., 2013) . while the gas6/ axl complex is still able to enhance wnv binding and internalization without kinase activity, subsequent infection is significantly impaired compared to infection of cells expressing wt axl. this kinase activity is important in vivo for tam-receptor-inhibition of dc activation and inflammation (rothlin et al., 2007; tibrewal et al., 2008) . ligand binding by the receptors and assocation with the type i interferon receptor leads to activation of stat1 and induction of suppressor of cytokine signaling (socs) proteins (rothlin et al., 2007) . viruses appear to utilize this anti-inflammatory signaling pathway to dampen the immune response and promote replication (bhattacharyya et al., 2013) . while tam signaling is triggered by interaction with ligand alone, signaling is significantly enhanced by the presence of virus which helps to facilitate the interaction of tam ligands with their receptors (bhattacharyya et al., 2013) . thus, the tam receptors enhance virus infection through pveer functions by enhancing virus binding and internalization and non-pveer functions by inhibiting innate immunity, both of which require binding to ptdser on the virion surface and likely contribute to enhancing in vivo virus loads. milk fat globule-egf factor 8 (mfg-e8), the most recently identified pveer, is a secreted protein that contains two n-terminal egf repeats followed by a c1 and c2 domain, of which the latter binds to ptdser (andersen et al., 2000; ye et al., 2013) . several key positively charged residues within the c2 domain are responsible for interacting with the phosphate and serine groups, while hydrophobic residues of the domain stabilize the positioning of ptdser (fig. 1) (ye et al., 2013) . like the gas6, mfg-e8 bridges ptdser containing membranes to cells through interaction with a membrane bound protein, either integrin αvβ3 or αvβ5 (andersen et al., 2000; hanayama et al., 2002) . this occurs through an arg-gly-asp (rgd) motif present in the second egf (santiago et al., 2007) . the human cd300a igv domain crystal structure (dimasi et al., 2007) is shown with residues hypothesized for interaction (simhadri et al., 2012) . the region of the rage igv domain (3o3u) that corresponds to the binding pockets of cd300a and tim-1 igv domains is shown for comparison ; however, this domain is only hypothesized to interact with ptdser and no residues have been implicated (friggeri et al., 2011) . the thrombospondin type-1 repeats (tsrs) of bai1 are represented by tsr domain 3 of human thrombospondin-1 (3r6b) (klenotic et al., 2011) . gas6 binds ptdser via a gla domain, represented by that of bovine prothrombin (1nl2) with residues identified that interact with ptdser (red) (huang et al., 2003) . for anxv, the structure of one annexin repeat domain from rat anx v is shown (1a8a) with residues identified that interact with ptdser (red) (swairjo et al., 1995) . the c2 domain of bovine mfg-e8 binds to ptdser (3bn6) (shao et al., 2008) and while the structure was not crystallized with ptdser, key residues involved in interaction have been determined experimentally (ye et al., 2013) . an egf-like domain of stabilin-1 or -2 is represented by that from human heregulin alpha (1hre) (nagata et al., 1994). repeat of mfge-8 and mutation of this motif inhibits pveer efficacy (morizono and chen, 2014) . several aspects of mfg-e8 biology may contribute to infection. similar to the gas6/axl complex, mfg-e8/integrin complexes have been shown to induce production of anti-inflammatory cytokines such as il-10 (aziz et al., 2009; jinushi et al., 2009) , some of which are initiated by activation of stat-3 (brissette et al., 2012; jinushi et al., 2011) . interestingly, stat-3 has also been identified as an upstream regulator of tim-1 expression (ajay et al., 2014) . however, currently mfg-e8 induced signaling activity has not yet been associated with enhancement of virus entry. the presence of pveers in various tissues permissive to infection may contribute to the tropism of some viruses, particularly for flaviviruses and filoviruses, whose entries are most enhanced by pveer expression. pveers are expressed in a variety of tissues and cell types (summarized in table 1 ) that are key targets of infection by enveloped viruses. tim-1, tim-4, and axl dorfman et al., 2010; bauer et al., 2012) may enhance uptake of dengue virus into langerhans dendritic cells (dcs), one of the first targets during flavivirus infection (wu et al., 2000; marovich et al., 2001) . tim-1 is also present on the mucosal epithelia of the airway and eye , both of which may be routes of infection for filoviruses (jaax et al., 1996) . early during infection, flaviviruses and filoviruses replicate in antigen-presenting cell (apc) populations: dcs and macrophages (geisbert et al., 2003a (geisbert et al., , 2003b jessie et al., 2004) . similarly, alphaviruses infect dcs (labadie et al., 2010; gardner et al., 2000; nishimoto et al., 2007; shabman et al., 2007) and establish persistent infections in macrophages (labadie et al., 2010; linn et al., 1996) . interestingly, these apcs express a variety of pveers including tim-4 (kobayashi et al., 2007; rodriguez-manzanet et al., 2010; mizui et al., 2008; meyers et al., 2005; baghdadi et al., 2013) , tam receptors (seitz et al., 2007; caraux et al., 2006; behrens et al., 2003) , and mfg-e8/integrin αvβ3 or αvβ5 miyasaka et al., 2004; albert et al., 1998; finnemann and rodriguez-boulan, 1999; antonov et al., 2004) . however, little is known about whether these pveers play critical roles in entry of virus into apc populations. knock out of tam receptors from bone marrow-derived dcs significantly reduces infection by west nile virus or lentiviruses pseudotyped with ebola, marburg, vesicular stomatitis, or murine leukemia virus entry proteins (bhattacharyya et al., 2013) . in addition, ptdser liposomes compete for uptake of ebov virus-like particles (vlps) in mouse peritoneal macrophages (jemielity et al., 2013) . these data suggest these receptors are relevant for uptake into early infection targets. pveers are also expressed in tissues that are later targets of virus spread. after infection by flaviviruses, langerhans cells traffic to the lymph nodes (johnston et al., 2000) . once there, flaviviruses infect lymph node dcs (balsitis et al., 2009; kyle et al., 2007) , which have been shown to express tim-4 (kobayashi et al., 2007; meyers et al., 2005) . the alphavirus, chikungunya virus, and filoviruses also replicate in lymph nodes (geisbert et al., 2003a (geisbert et al., , 2003b labadie et al., 2010) , where tim-4, in conjunction with mfg-e8/integrin αvβ3 and αvβ5 garmy-susini et al., 2007) , may mediate infection. infected apcs also traffic flaviviruses and filoviruses to the liver and spleen, two major sites of replication (geisbert et al., 2003a; jessie et al., 2004; balsitis et al., 2009; baskerville et al., 1985; bhoopat et al., 1996) . expression of mfg-e8/integrin αvβ5 and tim-4 has been detected in spleen (meyers et al., 2005; uehara and uehara, 2014) . in particular, tim-4 is expressed on splenic macrophages (kobayashi et al., 2007; wong et al., 2010) , a target of flaviviruses (balsitis et al., 2009 ). tim-1 and integrin αvβ5 have been detected on hepatocellular, huh7-derived cell lines (meertens et al., 2012; kondratowicz et al., 2011) and kupffer cells (wheeler et al., 2001) , respectively, and may contribute to flavivirus and filovirus infection of the liver (geisbert et al., 2003b; balsitis et al., 2009; couvelard et al., 1999; hall et al., 1991; xiao et al., 2001; ryabchikova et al., 1999) . flaviviruses also infect bone marrow myeloid cells (jessie et al., 2004; balsitis et al., 2009) , which were recently shown to express tim-4 after stimulation (baghdadi et al., 2013) . pveer expression does not appear to affect the tropism of every virus for which they enhance entry. for instance, ross river, chikungunya, and sindbis virus infect muscle cells, which do not express any currently identified pveers (morrison et al., 2006; johnson, 1965; ozden et al., 2007) , suggesting that either other unidentified pveers exist or virus entry into muscle cells is independent of pveer expression. interestingly, the ptdserbinding receptor bai1 has been shown to be important for myoblast fusion (hochreiter-hufford et al., 2013) . although bai1 does not effectively enhance virus entry in vitro (meertens et al., 2012) , it may contribute to virus entry in vivo in its native cell type and environment. this may also be the case for other ptdser receptors that do not function as pveers in cell culture as the efficacy of these receptors was assessed after exogenous expression in cell types in which they are not endogenously expressed. interestingly, pveers may also not function effectively in every cell population in which they are endogenously expressed as tim-1 is present on some populations of t-cells (meyers et al., 2005; umetsu et al., 2005; khademi et al., 2004) , but, t cells are refractory to ebov infection (wool-lewis and bates, 1998). however, this may also be due to defects in virion internalization machinery or subsequent steps in the viral life cycle and not pveer activity. vaccinia virus does infect activated t-cells (chahroudi et al., 2005) and in this case tim-1 may contribute to entry. nonetheless, the effect of ptdser binding protein expression on virus tropism has not been thoroughly explored in tissue culture and, as discussed below, has yet to be assessed in vivo. while some ptdser-binding receptors function effectively as pveers, this is not true for all. receptor for advanced glycation end-products (rage), brain-specific angiogensis inhibitor 1 (bai1), cd300a, stabilin-1 and -2, and tim-3 have all been shown to bind to phosphatidylserine and enhance engulfment of apoptotic cells (fig. 3) hochreiter-hufford et al., 2013; park et al., 2007; he et al., 2011; friggeri et al., 2011; nakahashi-oda et al., 2012; simhadri et al., 2012; ). however, expression of these proteins does not enhance virus entry (meertens et al., 2012; moller-tank et al., 2013; morizono and chen, 2014) . of these proteins, only the mechanism responsible for reduced pveer efficacy of tim-3 has been studied. the inability of tim-3 to serve as a pveer is due to the absence of a mld stalk of sufficient length (moller-tank et al., 2014) . however, this may also explain the reduced efficacy of cd300a that, like tim-3, binds ptdser using an n-terminal igv-like domain (simhadri et al., 2012) and has a short stalk region between its igv domain and the transmembrane domain. stabilin-1/-2 and bai1 bind ptdser through epidermal growth factor-like (egf) domain repeats kim et al., 2010; and type 1 thrombospondin repeats (tsrs), respectively (park et al., 2007) (fig. 2) , although the residues within these domains mediating ptdser binding have yet to be identified. while it is unknown why these motifs are able to bind apoptotic cells, but not virions, none of the ptdser binding receptors that function as pveers bind ptdser using repeat regions. one explanation may be that the repeats require a larger or flatter surface area to bind ptdser than a virion envelope allows. this biological conundrum requires further examination and may provide insight for identification of other pveers. rage is the only ptdser receptor listed above for which the ptdser-binding-domain is unidentified. the extracellular portion of rage consists of an n terminal igv domain followed by two igc2 domains (fig. 3) . the igv domain of rage has been suggested to be responsible for ptdser binding although this has not yet been experimentally shown (fig. 2) (friggeri et al., 2011) . the lack of a similar ptdser-binding pocket to that of the cd300a and tim igvs and inability to functionally replace the igv domain of tim-1 (smt, unpublished data) would suggest at least the igv domain of rage cannot bind to ptdser on viral envelopes. additionally, the condensed structure of the rage stalk provided by the igc2 domains likely also further reduces pveer efficacy by reducing overall length (moller-tank et al., 2014) . thus, both the absence of a clear ptdser-binding pocket in the igv domain and the short stalk likely explain the absent pveer activity of rage. the key factors required for proteins to function as pveers are ptdser-binding activity and attachment to the plasma membrane (morizono et al., 2011; meertens et al., 2012; jemielity et al., 2013; moller-tank et al., 2013) . while tim-1 and tim-4 individually perform both functions, the tam receptors and αvβ3 or αvβ5 integrins provide membrane attachment and their ligands bind to ptdser. in the case of the tim family pveers, the spacer domain between the ptdser-binding pocket and plasma membrane attachment is a mld. mlds from other molecules can substitute for the tim mlds, provided the mld is of sufficient length, while the more compact structure of rage igc2 domains is unable to substitute (moller-tank et al., 2014) . these studies led us to propose that an extended structure was needed in the spacer region, perhaps to extend the ptdser binding-pocket above the extracellular matrix that surrounds the cell (moller-tank et al., 2014) . thus, sufficient distance is necessary between the two essential functions of pveers for virus binding. while we understand how pveers bind virus, much less is currently known about the pathways and/or mechanisms for pveer mediated internalization of virus. there is no evidence for direct internalization. ebola, baculo-, and vaccinia virus enter predominantly or at least partially through macropinocytosis (mercer and helenius, 2008; aleksandrowicz et al., 2011; nanbo et al., 2010; kataoka et al., 2012; saeed et al., 2010; mulherkar et al., 2011) and these viruses are amongst those whose entry is most significantly enhanced by pveer expression. however, entry of flaviviruses and alphaviruses is enhanced by pveer expression, but primarily occurs through clathrin-mediated endocytosis (chu and ng, 2004; peng et al., 2009; acosta et al., 2009; sourisseau et al., 2007; detulleo and kirchhausen, 1998; marsh et al., 1984) . thus, if pveers directly mediate virus internalization, there are currently no data to support entry via a single pathway. it is certainly possible that different pveers mediate uptake through different pathways. alternatively, in some cases, pveers may function only as attachment factors to facilitate interaction of viral proteins with other cell surface receptors that trigger internalization. this may be true for new world arenaviruses that are known to enter cells using transferrin receptor 1 (radoshitzky et al., 2007) and whose entry is only moderately enhanced by pveer expression (jemielity et al., 2013) . however, this mechanism does not account for the ability of pveers to stimulate rapid uptake of viruses lacking a glycoprotein (moller-tank et al., 2013; jemielity et al., 2013) . several viruses have been shown to expose ptdser on the outer leaflet of their membrane (soares et al., 2008; morizono et al., 2011; meertens et al., 2012; moller-tank et al., 2013) . however, it remains to be determined how ptdser, which is normally present within the inner leaflet of the cell membrane, is exposed on the outer leaflet of viral membranes. on cellular membranes the asymmetrical distribution of ptdser is maintained by the activity of flippases and floppases that transfer phospholipids unidirectionally from either the extracellular side to the cytosolic side or the reverse, respectively (leventis and grinstein, 2010) . meanwhile, scramblases disrupt asymmetry by mediating random bidirectional transfer of phospholipids. in healthy cells, exposure of ptdser can be induced by several mechanisms, including apoptosis and elevated levels of intracellular calcium (leventis and grinstein, 2010; boon and smith, 2002) . some viruses encode proteins that increase cytosolic calcium levels such as nef of hiv-1 (manninen and saksela, 2002) and p7 of hepatitis c virus (griffin et al., 2003) , reviewed in (zhou et al., 2009) , which may contribute to ptdser exposure. entry of flaviviruses into cells also increases intracellular calcium levels (nour et al., 2013) . it is also possible that cellular stresses associated with virus infection trigger apoptosis as is seen with influenza a (shiratsuchi et al., 2000) and flaviviruses (su et al., 2002; liu et al., 2014; desprès et al., 1996) . there is evidence that west nile, sindbis, and chikungunya virus actively activate apoptosis to their advantage (yang et al., 2008; krejbich-trotot et al., 2011; levine et al., 1993) . in contrast, however, apoptosis is not induced in cells that are infected by ebola viruses (geisbert et al., 2000; olejnik et al., 2013) , although internalization of ebola vlps and infection of ebola virus is still enhanced by tim-1 expression (jemielity et al., 2013; moller-tank et al., 2013; kondratowicz et al., 2011) . further, some viruses for which entry is enhanced by pveers also encode anti-apoptotic genes, such as ns1 of vaccinia virus (maluquer de motes et al., 2011; cooray et al., 2007) and p35 of baculovirus (bertin et al., 1996) . thus, there is not a clear correlation between induction of apoptosis and incorporation of ptdser on viral envelopes. viruses may not utilize cellular functions to induce ptdser flipping, but rather, concentrate the limited ptdser present on the outer leaflet. ptdser may be incorporated into the outer leaflet of viral envelopes during budding for promotion of favorable membrane curvature (graham and kozlov, 2010) . alternatively, viruses may associate with ptdser in the inner leaflet due to its anionic charge as has been suggested for the matrix proteins of ebola virus (adu-gyamfi et al., 2013) and human immunodeficiency virus-1 (chukkapalli et al., 2013; vlach and saad, 2013) . the vsv matrix has also been shown to associate with ptdser enriched domains (luan et al., 1995) . thus, the inner leaflet would be enriched with ptdser and, after budding, the absence of flippases that normally regulate ptdser asymmetry may lead to equilibration of ptdser between the outer and inner leaflets. nonetheless, the expression of the matrix protein alone results in sufficient ptdser accumulation on the outer leaflet of ebola virus vlps to allow for tim-1 enhancement of internalization (jemielity et al., 2013; moller-tank et al., 2013) . these data suggest that regardless of the mechanism, activity of the matrix protein is sufficient for ptdser incorporation into viral envelopes. assuming pveer enhancement of virus entry occurs through misrecognition of viruses as apoptotic bodies, it stands to reason that the pathways of pveer-mediated virus internalization overlap with those involved in apoptotic body uptake. initially it was believed that apoptotic bodies were internalized through macropinocytosis ogden et al., 2001) which involves the ruffling of membrane to form large cups that engulf fluid (swanson, 1989) . macropinocytosis can be stimulated by receptor signaling induced by a variety of ligands including growth factors (racoosin and swanson, 1989) or pma (swanson, 1989) . while spontaneous ruffling occurs (sallusto et al., 1995) , only signaling-induced macropinocytosis leads to complete closure of the macropinosomes (li et al., 1997) . contrary to what was previously thought, studies using sem revealed that while necrotic cells are taken up through macropinocytosis, apoptotic bodies are phagocytosed (krysko et al., 2006) . unlike macropinocystosis, which does not specifically engulf cargo, phagocytosis involves direct interaction between ligands and receptors. targets to be engulfed must be entirely covered in ligand and become enclosed as binding between receptor and ligand facilitates the binding of adjacent receptors to ligand (griffin et al., 1976) . this mechanism is appropriately compared to a zipper (griffin et al., 1975) . in theory, phagocytosis would best explain the mechanism of pveer-mediated internalization with pveers sequentially binding ptdser on the virion surface. this is particularly fitting for tim-1and tim-4-mediated internalization of apoptotic bodies, as they have been shown to form phagocytic cups around cargo (ichimura et al., 2008; wong et al., 2010) . however, both ptdser and vlps have been shown to induce macropinocytosis (aleksandrowicz et al., 2011; hoffmann et al., 2001) . additionally, amiloride and its derivative 5-(n-ethyl-n-isopropyl) amiloride (eipa), considered specific inhibitors of macropinocytosis (west et al., 1989) and not phagocytosis (fukushima et al., 1996) , inhibit ebola virus uptake into and infection of vero cells or snb19 cells that express tim-1 and axl respectively saeed et al., 2010; mulherkar et al., 2011; hunt et al., 2011) . thus, there exists disparity between mechanisms of apoptotic body and virus uptake. some of this confusion may be attributed to many components being shared between phagocytosis and macropinocytosis. both require phosphoinositide 3-kinase (pi3k) signaling (araki et al., 1996) , phospholipase cγ (amyere et al., 2000; cheeseman et al., 2006) , rac1 (caron and hall, 1998; ridley et al., 1992) , and dynamin (liu et al., 2008; cao et al., 2007; gold et al., 1999; tse et al., 2003) , making differentiation between the two processes by targeting components difficult. the only macropinocytosis specific inhibitor, eipa, functions by deregulating intracellular ph and thus may disrupt additional aspects of the viral life cycle that contribute to inhibition of virus infection (koivusalo et al., 2010) . neither phagocytosis nor macropinocytosis have been studied in the context of pveers. phagocytosis is associated with phagocytic cells such as macrophages and dendritic cells (rabinovitch, 1995) , while all cells can initiate macropinocytosis. it is possible that both mechanisms are being used and are cell-type dependent. for example ebola virus initially infects macrophages and dendritic cells, which may occur through phagocytosis, and subsequently spreads and enters a broad variety of tissues and cell types, perhaps by macropinocytosis (feldmann and geisbert, 2011) . interestingly, receptors that induce either phagocytosis or macropinocytosis require phosphorylation and recruitment of kinases and adapter proteins (sobota et al., 2005; swanson, 2008) , but several pveers have been shown not to require signaling through cytoplasmic tails for internalization of virus (meertens et al., 2012; moller-tank et al., 2014; bhattacharyya et al., 2013) . neither phagocytosis nor macropinocytosis of have been shown to be directly elicited by pveers. while axl has been associated with macropinocytosis in some cells , it is unknown whether axl triggers macropinocytosis. there are, however, several studies that show ptdser receptors coordinate to induce uptake signaling. in macrophages there is evidence that mer induces internalization of apoptotic bodies, but requires tim-4 for initial attachment due to a higher binding affinity for ptdser (nishi et al., 2014) . mer has also been shown to work synergistically with integrin αvβ5 (wu et al., 2005) . similarly, tim-4 and mfg-e8 have been implicated as partners for uptake of apoptotic bodies in which mfg-e8/integrin signaling triggers uptake (toda et al., 2012) . several details of these mechanisms remain to be elucidated, such as whether or not tim-4 actively associates with signaling partners. tim-4 has also been shown to interact with adenosine monophosphate activating kinase (ampk) (baghdadi et al., 2013) , a protein important for macropinocytosis of ebola and vaccinia virus moser et al., 2010) . however, this interaction is believed to occur after phagocytosis and does not explain how initial internalization events are triggered. this is also complicated by evidence that the cytoplasmic tail of tim-4, the only domain accessible to ampk, is unnecessary for internalization ). further, these mechanisms do not explain why entry of viruses that utilize clathrin-coated pits is enhanced by pveer expression. thus, additional studies are required to determine if and how pveers contribute directly to internalization. as mentioned above, pveer expression does not enhance entry of all enveloped viruses into every cell type. in the case of arenaviruses, there exists an interesting dichotomy between the ability of pveers to enhance entry of new world but not old world arenaviruses. unlike for filo-, baculo-, alpha-, vaccinia, and flaviviruses, definitive cellular receptors have been identified for both old world and new world arenaviruses: α-dystroglycan (cao et al., 1998) and transferrin receptor 1 (radoshitzky et al., 2007; abraham et al., 2009) , respectively. these viruses also use different entry pathways as new world arenaviruses such as junín virus enter using clathrin coated pits while old world arenaviruses such as lassa and lymphocytic choriomeningitis virus use an unknown pathway that is clathrin, caveolin, and dynamin independent (rojek et al., 2008a (rojek et al., , 2008b . pveer-mediated enhancement of some new world arenaviruses entry into tim-1 expressing hek 293t cells is more modest than that found with filoviruses and flaviviruses, but still significant (jemielity et al., 2013; moller-tank et al., 2013) . these new world arenaviruses can bind to host cells and mediate efficient entry through interaction with transferrin receptor 1, and this can clearly be supplemented by pveer expression. for old world arenaviruses, expression of and/or affinity to α-dystroglycan may be sufficiently robust that pveer expression does not notably enhance entry further. indeed, in cells lacking α-dystroglycan, other attachment factors, including axl, can enhance old world arenavirus entry (shimojima et al., 2012) . however, pveers are likely not biologically relevant for old world arenavirus entry as α-dystroglycan expression is ubiquitous (cao et al., 1998; sullivan et al., 2013) . when carrying its native glycoprotein, entry of vsv is in most cases, like the old world arenaviruses, not enhanced by pveer expression (table 1) . however, in a few cases we (unpublished data) and others (jemielity et al., 2013) have observed a slight increase in vsv g-mediated entry into cells expressing pveers. the modest effect of pveer expression on vsv entry is not due to an absence of ptdser on the viral envelope as ptdser can be readily detected on vsv pseudovirions (moller-tank et al., 2013) . instead, as we propose above for lasv, the utilization by vsv of pveers would be anticipated to be dependent upon the relative availability of its native receptor. if the native glycoprotein of vsv is sufficient for optimal virus binding to and entry into cells, pveer expression would make little to no contribution to vsv entry. entry of sars corona-, influenza a, and herpes simplex 1 viruses is not enhanced by pveer expression (meertens et al., 2012; jemielity et al., 2013) . influenza a and sars-cov have viral envelope proteins that bind effectively to sialic acid (weis et al., 1988) and ace2 (li et al., 2003) respectively. expression of tim-1 on hek 293t cells enhances internalization of pseudovirions bearing either envelope protein, but does not result in enhancement of transduction (jemielity et al., 2013) . these results indicate that pveer-mediated or -enhanced internalization results in unproductive infection by these viruses. this could occur due to virions being delivered to compartments with incompatible conditions for fusion (i.e. lacking correct ph or fusion triggers). an alternative possibility is that pveers enhance internalization of defective sars or influenza virions with low or negligible levels of glycoprotein on the viral envelope. these particles would not effectively bind cellular receptors or be able to fuse, and thus their enhanced internalization would not contribute to infection. internalization into endosomes may similarly inhibit entry of herpes simplex virus 1, which fuses at the cell surface (akhtar and shukla, 2009 ). however, as the effects of pveer expression on herpes simplex virus 1 internalization have not been tested, it is possible that, in a manner similar to that proposed for old world arenaviruses and vsv, strong association with cellular receptors masks any enhancement. interestingly in all cases, pveer overexpression on permissive cells does not appear to inhibit overall virus entry, suggesting that either a majority of these viruses either escapes these compartments or enters through natural productive routes. a major question remaining regarding pveer-mediated enhancement of virus entry is its relevancy in vivo. the efficacy of pveers on wild-type infectious virus has been demonstrated in vitro with virus harvested from both cell culture and mice (jemielity et al., 2013; moller-tank et al., 2013) . additionally, entry of ebola virus vlps into mouse peritoneal macrophages is inhibited by ptdser liposomes, providing evidence of pveer importance for filovirus entry into relevant primary cell populations (jemielity et al., 2013) . however, to date only limited studies have been done in mice to determine the effect of ptdser-binding inhibitors and none using pveer knock down or knock out. difficulty of testing in vivo relevancy of pveers using knockout mice arises from the potential for compensation by other pveers. additionally, many pveers play critical roles in regulating adaptive immunity and single or combination knockout of genes could make interpretation of results difficult and/or lead to development of autoimmunity (rodriguez-manzanet et al., 2010; miyanishi et al., 2012; xiao et al., 2012; . nonetheless, in a single study, a chimeric antibody that recognizes ptdser was shown to inhibit infection by the new world arenavirus, pichinde (soares et al., 2008) . future studies will benefit from the development of a broad ptdser-binding inhibitor that can target multiple pveers. utilization of ptdser-binding proteins by viruses to enhance internalization provides a broad mechanism of viral entry with little investment by the virus itself. this mechanism may allow some viruses to attach to cells, thereby making viral glycoprotein/cellular receptor interactions more probable. alternatively, other viruses may rely entirely on pveers for internalization into endosomes. this latter mechanism would eliminate the need of viruses to expose sensitive viral epitopes extracellularly, thereby protecting critical receptor binding or membrane fusion motifs from neutralizing antibodies. once virions are internalized into endosomes, glycoprotein structural alterations that lead to membrane fusion can occur unhindered by antibodies. additionally, the broad expression of pveers might contribute to the extensive tropism of viruses such as flaviviruses and alphaviruses that infect a broad array of insect and mammalian hosts. many questions still remain regarding pveer-mediated entry. although there is preliminary evidence as discussed above, the most important question is what is the in vivo significance of pveers and their relative contribution to infection? if pveer utilization by viruses in vivo proves to be important, a model for testing inhibitors of ptdser binding by pveers would be valuable and could lead to development of a single antiviral or cocktail capable of inhibiting multiple human viruses. however, as pveer expression does not enhance entry of all enveloped virus families, targeting of pveers as an antiviral therapeutic may be limited to those such as the filoviruses and flaviviruses. nonetheless, these viral families include highly pathogenic viruses for which limited antivirals are available. second, how is ptdser being incorporated into viral envelopes and does it matter which membrane the envelope is derived from? third, what is the mechanism mediating pveer-dependent entry? is there a mechanism of direct internalization that results in productive infection? is any cell signaling involved in these events? finally, are there additional unidentified pveers that contribute to this newly appreciated means of viral infection? this may include potential ptdser receptors that have not been tested as pveers. cd36, for example, has been shown to bind oxidized ptdser for uptake of apoptotic cells into macrophages (driscoll et al., 2013; greenberg et al., 2006) . hek 293 t cells do not express a detectable amount of the identified pveers on their surface by surface staining, yet support low but detectable levels of transduction of filoviruses, flaviviruses, and alphaviruses. certainly one possibility is that there are additional undiscovered pveers mediating entry. alternatively, while studies detecting expression of tyro3 and mer on hek 293t cells have been contradictory, low levels of expression may allow for entry (morizono et al., 2011; bhattacharyya et al., 2013; shimojima et al., 2006) . further research on these receptors will elucidate these gaps in knowledge and determine feasibility of broad-spectrum antivirals to target them. host-species transferrin receptor 1 orthologs are cellular receptors for nonpathogenic new world clade b arenaviruses alternative infectious entry pathways for dengue virus serotypes into mammalian cells the ebola virus matrix protein penetrates into the plasma membrane: a key step in viral protein 40 (vp40) oligomerization and viral egress a bioinformatics approach identifies signal transducer and activator of ranscription-3 and checkpoint kinase 1 as upstream regulators of kidney injury molecule-1 after kidney injury viral entry mechanisms: cellular and viral mediators of herpes simplex virus entry rage expression in human t cells: a link between environmental factors and adaptive immune responses tim-4, a receptor for phosphatidylserine, controls adaptive immunity by regulating the removal of antigen-specific t cells immature dendritic cells phagocytose apoptotic cells via alphavbeta5 and cd36, and cross-present antigens to cytotoxic t lymphocytes ebola virus enters host cells by macropinocytosis and clathrin-mediated endocytosis constitutive macropinocytosis in oncogene-transformed fibroblasts depends on sequential permanent activation of phosphoinositide 3-kinase and phospholipase c functional analyses of two cellular binding domains of bovine lactadherin † promotion of tissue inflammation by the immune receptor tim-3 expressed on innate immune cells deficiency or inhibition of gas6 causes platelet dysfunction and protects mice against thrombosis regulation of macrophage foam cell formation by αvβ3 integrin: potential role in human atherosclerosis a role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages mfg-e8 attenuates intestinal inflammation in murine experimental colitis by modulating osteopontindependent αvβ3 integrin signaling tim-4 glycoprotein-mediated degradation of dying tumor cells by autophagy leads to reduced antigen presentation and increased immune tolerance tropism of dengue virus in mice and humans defined by viral nonstructural protein 3-specific immunostaining ultrastructural pathology of experimental ebola haemorrhagic fever virus infection identification of axl as a downstream effector of tgf-beta1 during langerhans cell differentiation and epidermal homeostasis the mer receptor tyrosine kinase: expression and function suggest a role in innate immunity apoptotic suppression by baculovirus p35 involves cleavage by and inhibition of a virus-induced ced-3/ice-like protease enveloped viruses disable innate immune responses in dendritic cells by direct activation of tam receptors immunohistochemical characterization of a new monoclonal antibody reactive with dengue virus-infected cells in frozen tissue using immunoperoxidase technique. asian pac human tim-1 associates with the tcr complex and up-regulates t cell activation signals milk fat globule epidermal growth factor 8 (mfg-e8): a novel protein in the mammalian endometrium with putative roles in implantation and placentation chemical control of phospholipid distribution across bilayer membranes tyrosine kinase receptor axl enhances entry of zaire ebolavirus without direct interactions with the viral glycoprotein mfg-e8 released by apoptotic endothelial cells triggers antiinflammatory macrophage reprogramming requirement of vascular integrin alpha v beta 3 for angiogenesis phosphatidylserine on hiv envelope is a cofactor for infection of monocytic cells molecular and functional characterization of irp60, a member of the immunoglobulin superfamily that functions as an inhibitory receptor in human nk cells dynamin 2 mediates fluid-phase micropinocytosis in epithelial cells identification of alphadystroglycan as a receptor for lymphocytic choriomeningitis virus and lassa fever virus natural killer cell differentiation driven by tyro3 receptor tyrosine kinases identification of two distinct mechanisms of phagocytosis controlled by different rho gtpases vaccinia virus tropism for primary hematolymphoid cells is determined by restricted expression of a unique virus receptor targeting of protein kinase c-a during fcγ receptor-dependent phagocytosis requires the ϵc1b domain and phospholipase c-γ1 infectious entry of west nile virus occurs through a clathrinmediated endocytic pathway evidence in support of rnamediated inhibition of phosphatidylserine-dependent hiv-1 gag membrane binding in cells novel human cd4þ t lymphocyte subpopulations defined by cd300a/c molecule expression functional and structural studies of the vaccinia virus virulence factor n1 reveal a bcl-2-like anti-apoptotic protein report of a fatal case of dengue infection with hepatitis: demonstration of dengue antigens in hepatocytes and liver apoptosis tim-1) is expressed on in vivo-activated t cells and provides a costimulatory signal for t cell activation t cell ig and mucin domain-1-mediated t cell activation requires recruitment and activation of phosphoinositide 3-kinase t cell/transmembrane, ig, and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells the clathrin endocytic pathway in viral infection human isolates of dengue type 1 virus induce apoptosis in mouse neuroblastoma cells molecular analysis and solution structure from small-angle x-ray scattering of the human natural killer inhibitory receptor irp60 (cd300a) the phosphatidylserine receptors, t cell immunoglobulin mucin proteins 3 and 4, are markers of histiocytic sarcoma and other histiocytic and dendritic cell neoplasms macrophage adam17 deficiency augments cd36-dependent apoptotic cell uptake and the linked anti-inflammatory phenotype release of high mobility group box 1 by dendritic cells controls t cell activation via the receptor for advanced glycation end products expression of stabilin-2, a novel fasciclin-like hyaluronan receptor protein, in murine sinusoidal endothelia, avascular tissues, and at solid/liquid interfaces ebola haemorrhagic fever macrophage and retinal pigment epithelium phagocytosis: apoptotic cells and photoreceptors compete for alphavbeta3 and alphavbeta5 integrins, and protein kinase c regulates alphav-beta5 binding and cytoskeletal linkage participation of the receptor for advanced glycation end products in efferocytosis naþ /hþ exchange activity during phagocytosis in human neutrophils: role of fcgamma receptors and tyrosine kinases infection of human dendritic cells by a sindbis virus replicon vector is determined by a single amino acid substitution in the e2 glycoprotein methods to study lymphatic vessel integrins apoptosis induced in vitro and in vivo during infection by ebola and marburg viruses pathogenesis of ebola hemorrhagic fever in primate models: evidence that hemorrhage is not a direct effect of virus-induced cytolysis of endothelial cells pathogenesis of ebola hemorrhagic fever in cynomolgus macaques: evidence that dendritic cells are early and sustained targets of infection identification of a novel high molecular weight protein preferentially expressed by sinusoidal endothelial cells in normal human tissues dynamin 2 is required for phagocytosis in macrophages interplay of proteins and lipids in generating membrane curvature oxidized phosphatidylserine-cd36 interactions play an essential role in macrophage-dependent phagocytosis of apoptotic cells studies on the mechanism of phagocytosis. i. requirements for circumferential attachment of particle-bound ligands to specific receptors on the macrophage plasma membrane studies on the mechanism of phagocytosis. ii. the interaction of macrophages with anti-immunoglobulin igg-coated bone marrow-derived lymphocytes the p7 protein of hepatitis c virus forms an ion channel that is blocked by the antiviral drug, amantadine demonstration of yellow fever and dengue antigens in formalin-fixed paraffin-embedded human liver by immunohistochemical analysis identification of a factor that links apoptotic cells to phagocytes autoimmune disease and impaired uptake of apoptotic cells in mfg-e8-deficient mice expression of developmental endothelial locus-1 in a subset of macrophages for engulfment of apoptotic cells tim-3 is expressed on activated human cd4 þ t cells and regulates th1 and th17 cytokines receptor for advanced glycation end products binds to phosphatidylserine and assists in the clearance of apoptotic cells phosphatidylserine receptor bai1 and apoptotic cells as new promoters of myoblast fusion phosphatidylserine (ps) induces ps receptor-mediated macropinocytosis and promotes clearance of apoptotic cells structural basis of membrane binding by gla domains of vitamin kdependent proteins the tyro3 receptor kinase axl enhances macropinocytosis of zaire ebolavirus kidney injury molecule-1 (kim-1), a putative epithelial cell adhesion molecule containing a novel immunoglobulin domain, is up-regulated in renal cells after injury kidney injury molecule-1 is a phosphatidylserine receptor that confers a phagocytic phenotype on epithelial cells promotion of the uptake of ps liposomes and apoptotic cells by a product of growth arrest-specific gene, gas6 lethal experimental infection of rhesus monkeys with ebola-zaire (mayinga) virus by the oral and conjunctival route of exposure tim-family proteins promote infection of multiple enveloped viruses through virion-associated phosphatidylserine localization of dengue virus in naturally infected human tissues, by immunohistochemistry and in situ hybridization milk fat globule epidermal growth factor-8 blockade triggers tumor destruction through coordinated cell-autonomous and immune-mediated mechanisms tumor-associated macrophages regulate tumorigenicity and anticancer drug responses of cancer stem/initiating cells virus invasion of the central nervous system: a study of sindbis virus infection in the mouse using fluorescent antibody langerhans cells migrate to local lymph nodes following cutaneous infection with an arbovirus involvement of membrane type 1-matrix metalloproteinase (mt1-mmp) in rage activation signaling pathways baculovirus gp64-mediated entry into mammalian cells t cell ig-and mucin-domain-containing molecule-3 (tim-3) and tim-1 molecules are differentially expressed on human th1 and th2 cells and in cerebrospinal fluid-derived mononuclear cells in multiple sclerosis cd300a and cd300f differentially regulate the myd88 and trif-mediated tlr signalling pathways through activation of shp-1 and/or shp-2 in human monocytic cell lines t-cell immunoglobulin and mucin domain 1 deficiency eliminates airway hyperreactivity triggered by the recognition of airway cell death the conserved histidine in epidermal growth factor-like domains of stabilin-2 modulates ph-dependent recognition of phosphatidylserine in apoptotic cells expression, purification and structural characterization of functionally replete thrombospondin-1 type 1 repeats in a bacterial expression system tim-1 and tim-4 glycoproteins bind phosphatidylserine and mediate uptake of apoptotic cells amiloride inhibits macropinocytosis by lowering submembranous ph and preventing rac1 and cdc42 signaling t-cell immunoglobulin and mucin domain 1 (tim-1) is a receptor for zaire ebolavirus and lake victoria marburgvirus amp-activated protein kinase is required for the macropinocytic internalization of ebolavirus chikungunya virus mobilizes the apoptotic machinery to invade host cell defenses expression of human milk fat globulin proteins in cells of haemopoietic origin macrophages use different internalization mechanisms to clear apoptotic and necrotic cells dengue virus infects macrophages and dendritic cells in a mouse model of infection chikungunya disease in nonhuman primates involves long-term viral persistence in macrophages appraising the apoptotic mimicry model and the role of phospholipids for poxvirus entry expression and function of cd300 in nk cells apoptotic cells activate nkt cells through t cell ig-like mucin-like-1 resulting in airway hyperreactivity immunobiology of the tam receptors the distribution and function of phosphatidylserine in cellular membranes conversion of lytic to persistent alphavirus infection by the bcl-2 cellular oncogene uncoupling of membrane ruffling and pinocytosis during ras signal transduction expression and anatomical distribution of tim-containing molecules in langerhans cell sarcoma systemic autoimmunity in tam triple knockout mice causes inflammatory brain damage and cell death angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus antibody-dependent enhancement and persistence in macrophages of an arbovirus associated with arthritis ages increased migration and inflammatory responses of adventitial fibroblasts via rage, mapk and nf-κb pathways dengue virus subgenomic rna induces apoptosis through the bcl-2-mediated pi3k/akt signaling pathway isoform and splice-variant specific functions of dynamin-2 revealed by analysis of conditional knock-out cells articular chondrocytes express the receptor for advanced glycation end products: potential role in osteoarthritis tyro-3 family receptors are essential regulators of mammalian spermatogenesis formation of membrane domains created during the budding of vesicular stomatitis virus. a model for selective lipid and protein sorting in biological membranes inhibition of apoptosis and nf-kappab activation by vaccinia protein n1 occur via distinct binding surfaces and make different contributions to virulence hiv-1 nef interacts with inositol trisphosphate receptor to activate calcium signaling in t cells human dendritic cells as targets of dengue virus infection semliki forest virus entry and the endocytic pathway differential expression of a gene signature for scavenger/lectin receptors by endothelial cells and macrophages in human lymph node sinuses, the primary sites of regional metastasis characterization of junín arenavirus cell entry tim-1, a novel allergy and asthma susceptibility gene the tim and tam families of phosphatidylserine receptors mediate dengue virus entry vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells tim-4 is the ligand for tim-1, and the tim-1-tim-4 interaction regulates t cell proliferation identification of tim4 as a phosphatidylserine receptor synergistic effect of tim4 and mfg-e8 null mutations on the development of autoimmunity expression of milk fat globule epidermal growth factor 8 in immature dendritic cells for engulfment of apoptotic cells bimodal regulation of t cell-mediated immune responses by tim-4 role of the phosphatidylserine receptor tim-1 in enveloped-virus entry characterizing functional domains for tim-mediated enveloped virus entry th1-specific cell surface protein tim-3 regulates macrophage activation and severity of an autoimmune disease the role of phosphatidylserine receptors in enveloped virus infection the soluble serum protein gas6 bridges virion envelope phosphatidylserine to the tam receptor tyrosine kinase axl to mediate viral entry characterization of ross river virus tropism and virus-induced inflammation in a mouse model of viral arthritis and myositis receptor for advanced glycation end products expression on t cells contributes to antigen-specific cellular expansion in vivo a kinome rnai screen identified ampk as promoting poxvirus entry through the control of actin dynamics the ebola virus glycoprotein mediates entry via a non-classical dynamin-dependent macropinocytic pathway solution structure of the epidermal growth factor-like domain of heregulin-alpha, a ligand for p180erbb-4 identification of the product of growth arrest-specific gene 6 as a common ligand for axl, sky, and mer receptor tyrosine kinases phenotypic differences between th1 and th17 cells and negative regulation of th1 cell differentiation by il-17 tim-1 and tim-3 enhancement of th2 cytokine production by mast cells identification of phosphatidylserine as a ligand for the cd300a immunoreceptor association of advanced glycation end products with a549 cells, a human pulmonary epithelial cell line, is mediated by a receptor distinct from the scavenger receptor family and rage ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner human th1 cells that express cd300a are polyfunctional and after stimulation up-regulate the t-box transcription factor eomesodermin tim4-and mertk-mediated engulfment of apoptotic cells by mouse resident peritoneal macrophages restricted and selective tropism of a venezuelan equine encephalitis virus-derived replicon vector for human dendritic cells viral membrane fusion and nucleocapsid delivery into the cytoplasm are distinct events in some flaviviruses c1q and mannose binding lectin engagement of cell surface calreticulin and cd91 initiates macropinocytosis and uptake of apoptotic cells advanced glycation end products enhance monocyte activation during human mixed lymphocyte reaction reversible inactivation and reactivation of vaccinia virus by manipulation of viral lipid composition ebola virus does not block apoptotic signaling pathways lactation-dependenteexpression of an mrna splice variant with an exon for a multiplyoglycosylated domain of mouse milk fat globule glycoprotein mfg-e8 secretion of a peripheral membrane protein, mfg-e8, as a complex with membrane vesicles human muscle satellite cells as targets of chikungunya virus infection the phosphatidylserine receptor tim-4 does not mediate direct signaling the 1.5 a crystal structure of human receptor for advanced glycation endproducts (rage) ectodomains reveals unique features determining ligand binding epidermal growth factor-like domain repeat of stabilin-2 recognizes phosphatidylserine during cell corpse clearance rapid cell corpse clearance by stabilin-2, a membrane phosphatidylserine receptor stabilin-1 mediates phosphatidylserine-dependent clearance of cell corpses in alternatively activated macrophages entry of dengue virus serotype 2 into ecv304 cells depends on clathrindependent endocytosis, but not on caveolae-dependent endocytosis receptor for advanced glycation endproducts mediates proatherogenic responses to periodontal infection in vascular endothelial cells tam receptor function in the retinal pigment epithelium expression of the receptor protein-tyrosine kinases tyro-3, axl, and mer in the developing rat central nervous system professional and non-professional phagocytes: an introduction macrophage colony-stimulating factor (rm-csf) stimulates pinocytosis in bone marrow-derived macrophages transferrin receptor 1 is a cellular receptor for new world haemorrhagic fever arenaviruses gas6-mediated signaling is dependent on the engagement of its gamma-carboxyglutamic acid domain with phosphatidylserine beginnings of a good apoptotic meal: the find-me and eatme signaling pathways the small gtpbinding protein rac regulates growth factor-induced membrane ruffling t and b cell hyperactivity and autoimmunity associated with niche-specific defects in apoptotic body clearance in tim-4-deficient mice different mechanisms of cell entry by human-pathogenic old world and new world arena viruses cellular entry of lymphocytic choriomeningitis virus tam receptors are pleiotropic inhibitors of the innate immune response an analysis of features of pathogenesis in two animal models of ebola virus infection cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class ii compartment: downregulation by cytokines and bacterial products structures of t cell immunoglobulin mucin protein 4 show a metal-ion-dependent ligand binding site where phosphatidylserine binds crystal structure of a c-terminal fragment of growth arrest-specific protein gas6. receptor tyrosine kinase activation by laminin glike domains structural basis for gas6-axl signalling macrophages and dendritic cells use different axl/mertk/tyro3 receptors in clearance of apoptotic cells differential induction of type i interferon responses in myeloid dendritic cells by mosquito and mammalian-cell-derived alphaviruses crystal structure of lactadherin c2 domain at 1.7 a resolution with mutational and computational analyses of its membrane-binding motif tyro3 family-mediated cell entry of ebola and marburg viruses identification of cell surface molecules involved in dystroglycan-independent lassa virus cell entry phosphatidylserinemediated phagocytosis of influenza a virus-infected cells by mouse peritoneal macrophages cloning and characterization of bai2 and bai3, novel genes homologous to brain-specific angiogenesis inhibitor 1 (bai1) cd300a is expressed on human b cells, modulates bcr-mediated signaling, and its expression is down-regulated in hiv infection lactadherin promotes vegfdependent neovascularization differential expression of cd300a/c on human th1 and th17 cells human cd300a binds to phosphatidylethanolamine and phosphatidylserine, and modulates the phagocytosis of dead cells epitope-dependent effect of anti-murine tim-1 monoclonal antibodies on t cell activity and lung immune responses targeting inside-out phosphatidylserine as a therapeutic strategy for viral diseases binding of igg-opsonized particles to fcγr is an active stage of phagocytosis that involves receptor clustering and phosphorylation brain-specific angiogenesis inhibitor-1 expression in astrocytes and neurons: implications for its dual function as an apoptotic engulfment receptor characterization of reemerging chikungunya virus the anticoagulation factor protein s and its relative, gas6, are ligands for the tyro 3/axl family of receptor tyrosine kinases cdna cloning of a mouse mammary epithelial cell surface protein reveals the existence of epidermal growth factor-like domains linked to factor viii-like sequences japanese encephalitis virus infection initiates endoplasmic reticulum stress and an unfolded protein response is the tam receptor axl a receptor for lymphocytic choriomeningitis virus? increased expression of receptor for advanced glycation end products by synovial tissue macrophages in rheumatoid arthritis ca(2 þ )-bridging mechanism and phospholipid head group recognition in the membrane-binding protein annexin v phorbol esters stimulate macropinocytosis and solute flow through macrophages shaping cups into phagosomes and macropinosomes molecular characterization of dendritic cell-derived exosomes. selective accumulation of the heat shock protein hsc73 autophosphorylation docking site tyr-867 in mer receptor tyrosine kinase allows for dissociation of multiple signaling pathways for phagocytosis of apoptotic cells and down-modulation of lipopolysaccharide-inducible nf-κb transcriptional activation molecular mechanism for differential recognition of membrane phosphatidylserine by the immune regulatory receptor tim4 two-step engulfment of apoptotic cells differential role of actin, clathrin, and dynamin in fcγ receptormediated endocytosis and phagocytosis integrin αvβ5 in endothelial cells of rat splenic sinus: an immunohistochemical and ultrastructural study tim-1 induces t cell activation and inhibits the development of peripheral tolerance viral apoptotic mimicry: an immune evasion strategy developed by the hepatitis b virus? trio engagement via plasma membrane phospholipids and the myristoyl moiety governs hiv-1 matrix binding to bilayers immunoexpression of tyro 3 family receptors-tyro 3, axl, and mer-and their ligand gas6 in postnatal developing mouse testis structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid distinct endocytotic pathways in epidermal growth factor-stimulated human carcinoma a431 cells adenoviral gene delivery can inactivate kupffer cells: role of oxidants in nf-κb activation and cytokine production phosphatidylserine receptor tim-4 is essential for the maintenance of the homeostatic state of resident peritoneal macrophages characterization of ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines human skin langerhans cells are targets of dengue virus infection a role for mer tyrosine kinase in alphavbeta5 integrin-mediated phagocytosis of apoptotic cells defect in regulatory bcell function and development of systemic autoimmunity in t-cell ig mucin 1 (tim-1) mucin domain-mutant mice experimental yellow fever virus infection in the golden hamster (mesocricetus auratus). ii. pathology crystal structure of the extracellular segment of integrin αvβ3 in complex with an arg-gly-asp ligand peripheral cd300a þ cd8þ t lymphocytes with a distinct cytotoxic molecular signature increase in pregnant women with chronic chorioamnionitis west nile virus capsid protein induces p53-mediated apoptosis via the sequestration of hdm2 to the nucleolus nmr solution structure of c2 domain of mfg-e8 and insights into its molecular recognition with phosphatidylserine anti-receptor for advanced glycation end products therapies as novel treatment for abdominal aortic aneurysm viral calciomics: interplays between ca2 þ and virus involvement of rage, mapk and nf-κb pathways in ages-induced mmp-9 activation in hacat keratinocytes key: cord-285869-jwflooop authors: clementz, mark a.; kanjanahaluethai, amornrat; o’brien, timothy e.; baker, susan c. title: mutation in murine coronavirus replication protein nsp4 alters assembly of double membrane vesicles date: 2008-05-01 journal: virology doi: 10.1016/j.virol.2008.01.018 sha: doc_id: 285869 cord_uid: jwflooop coronaviruses are positive-strand rna viruses that replicate in the cytoplasm of infected cells by generating a membrane-associated replicase complex. the replicase complex assembles on double membrane vesicles (dmvs). here, we studied the role of a putative replicase anchor, nonstructural protein 4 (nsp4), in the assembly of murine coronavirus dmvs. we used reverse genetics to generate infectious clone viruses (icv) with an alanine substitution at nsp4 glycosylation site n176 or n237, or an asparagine to threonine substitution (nsp4-n258t), which is proposed to confer a temperature sensitive phenotype. we found that nsp4-n237a is lethal and nsp4-n258t generated a virus (designated alb ts6 icv) that is temperature sensitive for viral replication. analysis of alb ts6 icv-infected cells revealed that there was a dramatic reduction in dmvs and that both nsp4 and nsp3 partially localized to mitochondria when cells were incubated at the non-permissive temperature. these results reveal a critical role of nsp4 in directing coronavirus dmv assembly. all positive-stranded rna viruses that infect mammalian and plant hosts form membrane-associated replication complexes in the cytoplasm of infected cells (salonen et al., 2005) . coronaviruses, such as mouse hepatitis virus (mhv) and severe acute respiratory syndrome coronavirus (sars-cov) that causes severe respiratory illness in humans (peiris et al., 2004; stadler et al., 2003) , generate double membrane vesicles (dmvs), which are the sites of viral rna synthesis (baker and denison, 2008; goldsmith et al., 2004; gosert et al., 2002; snijder et al., 2006) . the dmvs are generated by the association of coronavirus nonstructural proteins (nsps) with host intracellular membranes (gosert et al., 2002; harcourt et al., 2004; prentice et al., 2004; shi et al., 1999; snijder et al., 2006) . however, the role of each of the coronavirus nonstructural proteins in the assembly of dmvs is not yet clear. the coronavirus nonstructural proteins are translated from the 5′-most gene of the genome, gene 1. gene 1 contains two open reading frames (orfs) that produce two large polyproteins, pp1a and pp1ab (fig. 1a) . polyprotein 1a is processed by viral proteinases to generate intermediates and mature products nsp1-nsp11. polyprotein 1ab is generated by a ribosomal frameshift between orf1a and orf1b and contains all 16 nsps (masters, 2006; ziebuhr and snijder, 2007) . for mhv, these large polyproteins undergo extensive proteolytic processing by three virally encoded proteinases, papain-like proteinase (plp) 1, plp2, and 3c-like proteinase (3clpro) to produce intermediate and mature replicase proteins (baker et al., 1989; bonilla et al., 1997; bost et al., 2000; denison et al., 1995; graham and denison, 2006; kanjanahaluethai and baker, 2001; kanjanahaluethai et al., 2003; lu et al., 1998 lu et al., , 1995 schiller et al., 1998) . the replicase intermediate p150 contains the mature products nsp4-nsp11 and has been proposed to be functional in the synthesis of negative strand rna and/or as a scaffold for the assembly of the transcriptase/ replicase complex (kanjanahaluethai and baker, 2000; sawicki et al., 2007 sawicki et al., , 2005 . bioinformatic analysis indicates that the replicase products nsp3, nsp4, and nsp6 have membrane spanning helices (ziebuhr et al., 2000) . biochemical fractionation studies have shown that nsp3 and nsp4 are integral membrane glycoproteins (gosert et al., 2002; kanjanahaluethai et al., 2007; oostra et al., 2007) . recently, sparks et al. (2007) showed that the amino terminal region of nsp4 is essential for viral replication as deletions in this region are lethal. furthermore, sawicki et al. (2005) sequenced a large panel of temperature sensitive (ts) mutants of mhv and identified one ts mutant, alb ts6, that contained a substitution of asparagine to threonine at amino acid position 258 within the amino terminal region of nsp4. this mutation was predicted to confer the rna minus ts phenotype, but how this mutation affects mhv rna synthesis is not yet understood. to investigate the role of specific asparagine residues within the amino terminal domain of nsp4 in mhv replication, we engineered amino acid substitutions at putative glycosylation sites nsp4-n176 and nsp4-n237 and the putative ts lesion nsp4-n258 and isolated infectious clone virus (icv). we found that nsp4-n176 and nsp4-n237 are glycosylated and that substitution of nsp4-n237 to alanine was lethal for virus replication, whereas substitution of nsp4-n176 to alanine had no effect on virus replication. our results show that the asparagine to threonine substitution at nsp4-258 was sufficient to bestow a ts phenotype, and this virus was designated alb ts6 icv. we found that proteolytic processing of p150 is unaffected in the alb ts6 icv at either temperature. however, at the non-permissive temperature, dmv assembly and mitochondria morphology are disrupted in alb ts6 icv-infected cells and viral replicase proteins partially localize with mitochondria. these data demonstrate that nsp4 is an important factor in dmv assembly and are consistent with the the first two-thirds of the mhv genome (orf 1a and orf 1b) encode the viral replicase proteins. the nonstructural proteins (nsps) are synthesized as polyproteins processed into precursors then 16 mature replicase products. (b) analysis of p150 and nsp4 after tunicamycin and endo h treatments. hela-mhvr cells were infected with mhv-jhm, untreated (u) or treated with 1 µg/ml of tunicamycin (t) for 1 h prior to and during labeling. endo h treatment (e) was performed after immunoprecipitation with α-nsp4. (c) hela-mhvr cells were infected with vtf7.3, and co-transfected with plasmids encoding plp2 and substrate encoding either wt or mutant nsp4. proteins were radiolabeled ( 35 s-trans-label) and immunoprecipitated with α-nsp4 antibodies. ip products were untreated or treated with endo h, separated on 5-10% gradient sds-page gel, and visualized by autoradiography. (d) predicted topology of nsp4 indicating the location of two glycosylation sites and a ts lesion within the luminal loop. hypothesis that nsp4 is an anchor or scaffold for the replication complex. mhv replicase precursor p150 and product nsp4 are modified by n-linked glycosylation previous studies indicated that mhv nsp4 is a glycoprotein (oostra et al., 2007) , but it was unclear if the p150 precursor was also modified. to determine if both the precursor p150 and the mature product nsp4 are modified by n-linked glycosylation, we analyzed these proteins for sensitivity to tunicamycin and endoglycosidase h (endo h). mhv-infected cells were radiolabeled with 35 s-trans-label and either mock-treated or treated with tunicamycin and lysates and were subjected to immunoprecipitation with anti-nsp4 and then treated with endo h. we found that both the precursor p150 and the mature product nsp4 were modified by n-linked glycosylation and that these modifications were sensitive to endo h, indicating that these proteins did not progress past the endoplasmic reticulum (er) (fig. 1b) . bioinformatic analysis indicated that nsp4 residues n176 and n237 are consensus sites for n-linked glycosylation (consensus sequence: nxs/t). to determine if nsp4-n176 and nsp4-n237 are specifically modified by n-linked glycosylation, a cdna clone [mhv-cen-nsp4 (kanjanahaluethai and baker, 2000) ] expressing the entire nsp4 region and the cleavage site recognized by plp2, was subjected to site-directed mutagenesis to generate alanine substitutions at these sites, and the mobility of wt and mutant forms of nsp4 was assessed by sds-page. cells were transfected with constructs expressing pplp2 (proteinase) and either wt or mutant forms of pcen-nsp4 dna (substrate) and newly synthesized proteins were radiolabeled with 35 s-trans-label. cell lysates were prepared and subjected to immunoprecipitation with an α-nsp4 antibody to detect the mature form of nsp4 generated by cleavage of cen-nsp4 by plp2. the immunoprecipitated nsp4 was either mocktreated or endo h treated and electrophoretic mobility of protein was assessed. as seen in fig. 1c , nsp4 encoding either the n176a or n237a substitutions exhibited faster migration as compared to wt nsp4. the nsp4-n176a/n237a double mutant migrated faster than either single mutant. for products treated with endo h, which cleaves n-linked oligosaccharides, the mobility of all four proteins was similar to that of the nsp4-n176a/n237a double mutant, as expected. these data indicate that nsp4-n176 and nsp4-n237 are in fact subjected to n-linked glycosylation. recent studies have suggested that an asparagine residue in the luminal domain of nsp4 is important for mhv rna synthesis. sawicki et al. (2005) analyzed a series of mhv temperature sensitive mutants that do not make viral rna at the non-permissive temperature. they identified one virus, designated as alb ts6, with an asparagine to threonine substitution at nsp4 residue 258. this residue was implicated as the site responsible for temperature sensitive defect. however, the mechanism by which this substitution in nsp4 causes the defect in rna synthesis in alb ts6 is not known. a schematic diagram of nsp4 topology indicating the position of the two asparagine residues modified by n-linked glycosylation, and an asparagine to threonine change predicted to be responsible for the temperature sensitive phenotype are depicted in fig. 1d . to determine if nsp4-n176, nsp4-n237, or nsp4-n258 is important for nsp4 function, we generated virus encoding each specific substitution. each substitution was introduced into the mhv-a59 genome using a reverse genetics approach pioneered by yount et al. (2002) as described in the materials and methods. briefly, pcr based site-directed mutagenesis was performed on the plasmid dna containing the region of nsp4 to be mutated (clone b) using specific primers (table 1) . each mutant clone b dna fragment was ligated with the remaining six wt fragments to produce full-length mhv cdna which was then in vitro transcribed using t7 rna polymerase. infectious rna was electroporated into bhk-mhvr cells and cells were laid over a semi-confluent monolayer of dbt cells. cells were incubated at 33°c and scored for cytopathic effect. supernatant from cells showing syncytia formation was collected and passaged over a fresh monolayer of dbt cells to generate a stock of infectious clone virus. rna was isolated from mock and icv-infected dbt cells and rt-pcr was performed to amplify the region containing the mutation of interest. amplicons were sequenced to verify the presence of the engineered mutation (fig. 2) . infectious viruses were successfully obtained for position n176a (referred to as nsp4-n176a icv) and n258t (designated alb ts6 icv). however, we were unable to generate the nsp4-n237a single or nsp4-n176a/n237a double mutant virus, which indicates that mutation of n237 may be lethal for nsp4 function. to determine if either alb ts6 icv or nsp4-n176a icv is temperature sensitive, we measured the amount of infectious particles produced by virus-infected cells incubated at the permissive (33°c) or non-permissive (39.5°c) temperature and titrated at the permissive temperature. two sets of dbt cells were infected with wt-a59 icv, alb ts6 icv, or nsp4-n176a icv at an moi of 0.1. one set of infected cells was maintained at the fig. 2 . sequence analysis of mutant infectious clone virus. dbt cells were infected with wt-a59 icv, nsp4-n176a icv, or alb ts6 icv and at 12 h.p.i. rna was isolated. rt-pcr was performed on viral rna using primers listed in table 1 , and pcr products were sequenced across the nsp4 region. permissive temperature of 33°c, while the other was incubated at the non-permissive temperature of 39.5°c. at 15 h.p.i., cellfree supernatant was collected. ten-fold serial dilutions (in triplicate) of isolated supernatant were used to infect dbt cells incubated at the permissive temperature. after 48 h, plaques were counted and viral titer was determined. wt-a59 icv replicated to high titers of 1.08 × 10 7 plaque forming units (pfu)/ ml at 33°c and 1.42 × 10 7 pfu/ml at 39.5°c. nsp4-n176a icv also produced a similar size of plaques and reached titers of 1.33 × 10 7 pfu/ml at 33°c and 1.75 × 10 7 pfu/ml at 39.5°c. the alb ts6 icv replicated efficiently at 33°c (1.08 × 10 7 pfu/ ml), but replication was dramatically reduced at 39.5°c (4.08 × 10 2 pfu/ml) (fig. 3a) . one-step growth curve analysis was then performed on wt-a59 icv, alb ts6 icv, and nsp4-n176a icv. dbt cells were infected with icv at a multiplicity of infection of 0.1, incubated at the permissive temperature, and production of infectious virus was monitored by plaque assay. as shown in fig. 3b , when grown at 33°c, wt-a59 icv, alb ts6 icv, and nsp4-n176a icv all replicated with indistinguishable kinetics. temperature shift experiments were then performed to further assess the ts phenotype. two sets of dbt cells were infected with wt-a59 icv, alb ts6 icv, or nsp4-n176a icv at an moi of 0.1 and incubated at 33°c. at 6 h.p.i., one set of infected cells was shifted to 39.5°c. supernatant was harvested at two hour intervals and virus production was measured by plaque assay. as depicted in fig. 4 , wt-a59 icv, nsp4-n176a icv, and alb ts6 icv at 33°c grew to similar titers and statistical analysis revealed that all icvs exhibited a common growth curve ( p = 0.9459). at 39.5°c however, alb ts6 icv titers fell over 3 logs by 12-14 h.p. i. as compared to wt-a59 icv or nsp4-n176a icv, and alb ts6 icv exhibited distinct growth kinetics ( p b 0.0001). taken together, these data indicate that the n258t mutation in nsp4 is sufficient to confer a ts phenotype to the alb ts6 icv. analysis of proteolytic processing of p150 in the alb ts6 icv and nsp4-n176a icv one possible explanation for the ts phenotype of the alb ts6 icv is that nsp4 processing is altered at the non-permissive temperature. to address this issue, dbt cells were infected with wt-a59 icv, alb ts6 icv, and nsp4-n176a icv at an moi of 1.0 and at 4 h.p.i. radiolabeled with 35 s-trans-label for 2 h. at 6 h.p.i., cell lysates were prepared and subjected to immunoprecipitation with nsp-specific antibodies. as seen in figs. 5a-d, a large precursor of 150 kda is detected from which the mature nsp4-nsp10/11 products are generated (kanjanahaluethai and baker, 2000) . the liberation of nsp4 was detected in cells infected with wt-a59 icv, alb ts6 icv, or nsp4-n176a icv virus at both the permissive and non-permissive temperature. the mobility of nsp4 in the nsp4-n176a icv was increased, which is consistent with the absence of glycosylation at amino acid 176 (fig. 5a) . the processing of nsp5, nsp8, nsp9, and nsp10 was also unaffected in cells infected with wt-a59 icv, alb ts6 icv, or nsp4-n176a icv at both temperatures (figs. 5b-d). overall, these results indicate that the ts phenotype is not due to any alteration in the processing of nsp4 or other p150-derived replicase products. we hypothesize that nsp4 is a key anchor for dmv assembly and therefore asked if the formation of dmvs was altered fig. 4 . temperature shift assay on infectious clone virus. two sets of dbt cells were infected with wt-a59 icv, alb ts6 icv, or nsp4-n176a icv at an moi of 0.1 and incubated at 33°c. at 6 h.p.i., one set of infected cells was shifted to 39.5°c. supernatant was harvested at two hour intervals and virus production was measured by plaque assay. arrow indicates time of temperature shift. in cells infected with the alb ts6 icv incubated at the nonpermissive temperature. we assessed dmv formation by transmission electron microscopy (tem) analysis to determine if the reported defect in rna synthesis is due to a defect in dmv assembly. two sets of dbt cells were infected with wt-a59 icv or alb ts6 icv at an moi of 1.0 and incubated at 33°c. at 3.5 h.p.i., one set of infected cells was shifted to 39.5°c. at 5.5 h.p.i., cells were harvested and processed for tem analysis. dmvs can be visualized by tem as darkly ringed vesicles in the cytoplasm of mhv-infected cells (gosert et al., 2002) . as shown in fig. 6 , dmv formation induced by wt-a59 icv was similar at both permissive and non-permissive temperatures. at 33°c, the alb ts6 icv induced dmv formation comparable to wt-a59 icv; however, at the non-permissive temperature of 39.5°c, the alb ts6 icv produced fewer dmvs. the most striking feature we observed was that the morphology of the mitochondria was altered in cells infected with the alb ts6 icv incubated at 39.5°c. as seen in fig. 6d , the mitochondria were larger and extensively vacuolated. the overall reduction in dmvs and the striking change in mitochondrial morphology lead us to hypothesize that the mutation in nsp4 resulted in altered localization of nsp4 and potentially other mhv replicase products, resulting in a block in viral rna synthesis. the abnormalities observed in the mitochondria of cells infected with alb ts6 icv incubated at the non-permissive temperature led us to explore whether mhv replicase proteins, which normally co-localize with er in dbt cells (shi et al., 1999) , were co-localizing with mitochondria. two sets of dbt cells were infected with wt-a59 icv or alb ts6 icv at an moi of 1.0 and incubated at 33°c. at 3.5 h.p.i., one set of infected cells was shifted to 39.5°c. at 5 h.p.i., cells were labeled with mitotracker red fluorescent dye, which is concentrated by active mitochondria. at 5.5 h.p.i., cells were harvested, fixed, and permeabilized for immunofluorescence assays. permeabilized cells were then incubated with antibodies to either nsp3 or nsp4. as shown in fig. 7a , staining for nsp4 (green) and mitochondria (red) was non-overlapping in cells infected with wt-a59 icv at either temperature. at the permissive temperature, alb ts6 icv nsp4 and mitochondria displayed a very slight increase in co-localization versus wt-a59 icv. at 39.5°c however, co-localization of nsp4 and mitochondria was substantially increased. the intensity of the red signal was also increased in cells infected with alb ts6 icv at the nonpermissive temperature, which may reflect the increased in size of the mitochondria that we observed by tem. similar results were obtained in three independent experiments, with extensive co-localization detected only in the alb ts6 icv-infected cells incubated at the non-permissive temperature. to extend these findings, we performed similar experiments using hela cells stably transfected with the mhv receptor (mhvr). two sets of hela-mhvr cells were infected with wt-a59 icv or alb ts6 icv at an moi of 1.0 and incubated at 33°c. at 3.5 h.p.i., one set of infected cells was shifted to 39.5°c. at 5 h.p.i., mitochondria were labeled with mitotracker red fluorescent dye or following fixation with an antibody to pyruvate dehydrogenase (pdh), which is a mitochondrial matrix protein. antibodies against nsp3 and nsp4 were again used to detect replicase products. as seen in fig. 8 , extensive overlap between replicase proteins nsp3 and nsp4 and mitochondria was detected only in the alb ts6 icv-infected cells incubated at the non-permissive temperature (39.5°c). similar results were obtained with mitotracker stained hela-mhvr cells and mitotracker and pdh were found to completely overlap (data not shown). these results are consistent with the tem studies and reveal that the mutant form of nsp4 is partially localized to mitochondria at the non-permissive temperature. furthermore, we also detected co-localization with mitochondria using the α-nsp3 antibody in alb ts6 icv-infected cells incubated at 39.5°c (figs. 7b and 8b) . importantly, nsp3, and perhaps other replicase products, are misdirected due to nsp4 mis-localization and are likely unable to efficiently assemble into functional dmvs in the alb ts6 icv-infected cells. this inability to generate functional dmvs and/or replication complexes would lead to an inability to synthesize viral rna, which is the reported phenotype of alb ts6 (sawicki et al., 2005) . positive-strand rna viruses express viral replicase proteins that must interact with host intracellular membranes to create an environment for optimal viral rna synthesis. coronaviruses express replicase proteins that assemble to generate dmvs in the cytoplasm of infected cells (goldsmith et al., 2004; gosert et al., 2002; snijder et al., 2006) . in this study, we investigated the role of one coronavirus replicase product, nsp4, in mhv replication. because nsp4 is a transmembrane protein, we hypothesize that nsp4 is critical for assembly of the replication complex on dmvs. to test this hypothesis, we generated viruses with specific amino acid substitutions in nsp4 and assessed the effect of these substitutions on viral replication. first, we investigated the role of two putative n-linked glycosylation sites in mhv replication. using endo h assays, we found that both the nsp4-11 precursor (p150) and the nsp4 product are modified by n-linked glycosylation (fig. 1b) . we engineered alanine substitutions (nsp4-n176a and nsp4-n237a) into wt-a59 virus and found that nsp4-n176a icv behaved identically to wt-a59 icv suggesting that glycosylation at this site is not required for mhv replication. however, we were unable to generate either nsp4-n237a icv or nsp4-n176a/n237a (double glycosylation knockout) icv suggesting that glycosylation of nsp4-n237a, or specific folding of nsp4 in this luminal domain, is required for mhv replication. next, we investigated the role of nsp4-n258 in mhv replication. sawicki et al. (2005) analyzed the sequence of 19 mhv ts mutants and reported that one of these mutant viruses, alb ts6, encoded a substitution of asparagine for threonine at nsp4-258. they hypothesized that this mutation alone was sufficient to confer the ts and rna synthesis-negative phenotype to mhv-a59. via reverse genetics, we engineered the nsp4-n258t mutation into mhv and designated this virus as alb ts6 icv. we found that the nsp4-n258t substitution was indeed sufficient to induce temperature sensitivity. alb ts6 icv titers were reduced approximately 5 orders of magnitude when incubated at the nonpermissive temperature; however, alb ts6 icv titers at 33°c were comparable to wt-a59 icv (fig. 3a) . growth kinetics, assayed by a one-step growth curve at the permissive temperature, were indistinguishable between alb ts6 icv and wt-a59 icv. however, temperature shift experiments revealed that upon incubation at the non-permissive temperature, alb ts6 icv titers fell 1000 fold (fig. 4) . using a similar reverse genetics approach, donaldson et al. (2007) found that a single amino acid substitution in nsp10 conferred temperature sensitivity to the icts-la6 virus. this analysis revealed that nsp10 is a necessary cofactor for 3clpro activity as proteolytic processing of the replicase intermediate p150 was defective in icts-la6-infected cells incubated at the non-permissive temperature. in contrast, we found that alb ts6 icv had no defects in proteolytic processing when virus-infected cells were incubated at the non-permissive temperature (fig. 5 ). an alternative explanation for the rna minus ts phenotype of alb ts6 is that a mutation in nsp4 affects assembly of dmvs. to test this hypothesis, we performed tem analysis of alb ts6 icvinfected cells. this analysis revealed that dmv assembly is severely impaired in the alb ts6 icv-infected cells incubated at the non-permissive temperature (fig. 6d) . the failure to assemble dmvs, which are necessary for viral rna synthesis, is consistent with the rna minus phenotype observed by sawicki et al. (2005) . our results demonstrate that nsp4 plays a critical role in the formation and/or maintenance of dmvs. also, tem analysis of alb ts6 icv-infected cells incubated at the non-permissive temperature showed a disruption of mitochondrial morphology; the mitochondria were enlarged and extensively vacuolated (fig. 6d ). using confocal microscopy, we assessed whether nsp4-n258t was localized to the mitochondria. we found that nsp4-n258t partially co-localized with mitochondria in virus-infected cells incubated at the nonpermissive temperature (figs. 7a and 8a) . interestingly, we found that replicase product nsp3 also co-localized with mitochondria, suggesting that nsp4-n258t may direct the localization of other replicase components (figs. 7b and 8b). currently, it is unclear if a replicase precursor or only the final replicase products are directed to specific membrane sites or if nsp4 is actually penetrating the mitochondrial membrane. since nsp4 is an integral membrane protein originally derived from the er, the co-localization detected may be due to membrane reorganization. dmvs are likely diffusible in the cytoplasm and perhaps nsp4-n258t is directing the localization of dmvs to mitochondria where they are sequestered or fused with mitochondrial membranes. further experiments will be required to address this important issue. the aberrant mitochondrial morphology and partial colocalization with nsp3 and nsp3 raises questions about the role for mitochondria in mhv replication. could nsp4-n258t be localizing to mitochondria in error resulting in reduced dmv assembly? or is there a mitochondrial phase in mhv replication whose progression is inhibited by the nsp4-n258t substitution? previous studies demonstrate that for some viruses, the replicase complex can be directed to use different membrane sources for efficient virus replication. for example, flock house virus (fhv) normally induces spherules within the outer membrane of the mitochondria providing precedence for the use of mitochondrial membranes as the site of membrane-bound replication complex assembly (kopek et al., 2007; miller and ahlquist, 2002; miller et al., 2001) . to determine if mitochondrial membranes were required for replication, miller et al. (2003) replaced the mitochondrial outer membrane targeting signal of fhv protein-a with that of an er targeting signal and measured viral replication. they found that the er-targeted replication complex functioned as efficiently, if not more efficiently, than the normal mitochondria-targeted replication complex. there-fore, a specific source of membranes for replication complex assembly is not required for fhv. for mhv, it is unclear if the replication complex could be appropriately targeted to mitochondria, or if cytoplasmic dmvs are critical for mhv replication. in addition, it will be interesting to determine if wt nsp4 or nsp4-n258t expressed in trans can direct mhv replication complexes to specific membrane sites. complementation studies are useful for identifying products which can act in trans to provide a functional protein for a defective gene product. complementation analyses have been done with a large panel of ts mutants within the mhv replicase and have provided insights into the functions of intermediate and fully processed replicase proteins donaldson et al., 2007; fu and baric, 1994; sawicki et al., 2005; schaad et al., 1990; siddell et al., 2001; younker and sawicki, 1998) . interestingly, although mhv orf1a encodes eleven mature nsps, mutants within orf1a do not complement each other. there are at least two possible explanations for these results: 1) a polyprotein precursor, such as p150, may function itself, or function in cis and therefore cannot be complemented by mature nsp products (deming et al., 2007; sawicki et al., 2005) ; and 2) mutations in one nsp may affect the production, function or localization of multiple products and therefore cannot be complemented by a trans-acting factor. for example, virus with a mutation in nsp10 (ts-la6) crossed with a nsp4 mutant (alb ts6) do not complement each other (sawicki et al., 2005) . donaldson et al. (2007) suggest that the icts-la6, which exhibits a processing defect, fails to complement due to the inability to generate mature forms of nsp4-nsp16. thus, a mutation in a single nsp (nsp10) affects the production of several nsps (nsp4-nsp16). likewise, the mutation analyzed in this study, nsp4-n258t, which results in defects in dmv assembly and localization, also affects the localization of other nsps, such as nsp3. therefore, like the icts-la6, the defect in alb ts6 icv induces an overarching defect in mhv replication. these observations highlight the complex nature of coronavirus replication complex assembly and maturation and indicate that interplay among partially and fully processed replicase products ultimately leads to competent replication complexes. the results presented in this study indicate that nsp4 is a key component in dmv assembly and are consistent with nsp4 serving as an anchor or scaffold for the replication complex. analysis of cis and trans-acting viral and host factors will further elucidate the processes required for assembly of the coronavirus transcription/replication complex. wt-a59 icv, alb ts6 icv and nsp4-n176a icv were generated using the mhv-a59 reverse genetics system developed by yount et al. (2002) . wt and mutagenized clone b plasmids were transformed into chemically competent mds (scarab genomics) cells. the remaining clones were transformed into chemically competent xl-1 blue cells. competent cells were heat shocked for 45 s at 42°c and plated on luria-bertani (lb) plates containing appropriate selection antibiotics. single colonies were picked and grown in selection media (lb + antibiotic) overnight at 25°c. subcultures of wt and mutant clone b were grown at 37°c in 350 ml of lb + antibiotic until culture density reached an o.d. of 0.8-1.0 at 590 nm. the remaining clones were treated similarly, but grown at 25°c. delayed brain tumor (dbt) and baby hamster kidney (bhk) expressing the mhv receptor (bhk-mhvr) cells were incubated at 37°c in minimal essential medium, mem, (gibco) containing 5% fetal calf serum (fcs), 10% tryptose phosphate broth, 2% penicillin/streptomycin, and 2% l-glutamine. hela-mhvr cells were propagated in dmem (gibco) containing 10% fcs, 0.001 m sodium n-2-hydroxyethylpiperazine-n′-2ethanesulfonic acid, ph 7.4, 1% penicillin/streptomycin and 1% l-glutamine. the coronavirus reverse genetics system described by yount et al. (2002) was used to generate virus encoding a single amino acid substitution compared to mhv-a59. primers with two nucleotide changes designed to generate amino acid substitutions at nsp4 positions n176 and n237 to alanine, and n258 to threonine were incorporated into the mhv b plasmid dna via pcr based site-directed mutagenesis (quikchange kit, stratagene, primers listed in table 1 ). plasmid dnas containing the specific mutations of interest were isolated and sequenced across the entire b region (sequencing primers shown in table 1 ). the b plasmid dna region of interest was excised and ligated with the mhv a, c, d, e, and f isolated dna fragments to produce full-length viral cdna, which was in vitro transcribed using mmessage mmachine t7 kit (ambion) according to the manufacturer's instructions. infectious rna was electroporated into 4 × 10 6 bhk-mhvr cells and laid over 1.5 × 10 6 dbt cells in 60 mm dishes in duplicate. cells were incubated at 33°c for 24-72 h and monitored for the characteristic cytopathic effect (cpe) of mhv, which is syncytia formation. supernatant from cultures with cpe was passaged over a fresh monolayer of confluent dbt cells to generate a stock of icv. rna was isolated from virus-infected cells and subjected to reverse transcriptase (rt)-pcr using primers that flanked the region of interest. pcr amplicons were sequenced to verify the presence of the mutation in infectious clone virus rna (fig. 2) . the region of viral rna containing the mutation of interest was rt-pcr amplified using the improm-ii rt system (promega) followed by the advantage cdna pcr kit (clonetech) according to the manufacturer's instructions. specific primers are listed in table 1 . hela-mhvr cells were infected with a recombinant vaccinia virus expressing the bacteriophage t7 polymerase (vtf7.3) at a multiplicity of infection of 10 for 1 h. then, cells were co-transfected with pplp2-cen dna and either pcen-nsp4 wild type or pcen-nsp4 mutant dna (n176a, n237a, or n176a/n237a) using lipofectamine (gibco) according to the manufacturer's instruction as previously described (fuerst et al., 1986; kanjanahaluethai and baker, 2000) . proteins were radiolabeled with 50 μci of 35 s-trans-label from 4.5 to 9.5 h. p.i. cells were harvested and lysed with lysis buffer a containing 4% sds, 3% dtt, 40% glycerol and 0.065 m tris at ph 6.8. cell lysates were subjected to immunoprecipitation assays as described previously (schiller et al., 1998) . briefly, radiolabeled cell lysates was diluted in ripa buffer (0.5% triton x-100, 0.1% sds, 300 mm nacl, 4 mm edta and 50 mm tris-hcl, ph 7.4) and immunoprecipitated with α-nsp4 rabbit antiserum and protein-a sepharose beads (amersham biosciences, piscataway, nj). for endoglycosidase h (endo h) treatment, protein-a sepharose-antibody-antigen complexes were washed once in ripa buffer. the endo h treatment was performed according to the manufacturer's instruction (roche). briefly, the complexes were resuspended in 20 µl of 50 mm sodium phosphate buffer, ph 6.0, and incubated in the presence and absence of a final concentration of 1 u/μl of endo h for 16 h at 37°c. the complex-bound sepharose beads were pelleted by centrifugation. the products were eluted from the beads by incubating with 2× laemmli sample buffer at 37°c for 30 min. protein products were separated via electrophoresis on 5-10% gradient sds-page gels and were visualized by autoradiography. in tunicamycin treatment experiments, mhv-infected hela-mhvr cells were treated with 1 µg/ml tunicamycin (boehringer mannheim) for 1 h prior to addition of 35 s-trans-label, and the drug was present during the 1 h labeling period. whole cell lysates were prepared and subjected to immunoprecipitation as described above. viral titer of the wt-a59 icv, alb ts6 icv, and nsp4-n176a icv was determined via plaque assay. two sets of dbt cells were infected with wt-a59 icv, alb ts6 icv, or nsp4-n176a icv at an moi of 0.1. one set of infected cells was maintained at the permissive temperature of 33°c, while the other was incubated at the non-permissive temperature of 39.5°c. at 15 h.p.i. cellfree supernatant was collected. ten-fold serial dilutions (in triplicate) of isolated supernatant were used to infect dbt cells seeded to 70% confluency in 12 well plates. following a 1 h absorption period, a 1 ml mixture of 0.4% noble agar (difco, detroit, mi) and mem with 1% fcs and 2% penicillin/ streptomycin was added to each well. infection was maintained for 48 h at the permissive temperature (33°c) and plates were stained with 0.1% crystal violet solution for 10 min at room temperature to visualize and count plaques. one-step growth curves were generated by infecting dbt cells at an moi of 0.1 in 6-well plates. cells were washed three times with phosphate-buffered saline (pbs) following a 1 h absorption phase. three milliliters of fresh medium were added and cells were incubated at 33°c. aliquots of supernatants were collected 2, 4, 6, 8, 10, and 24 h.p.i. and the viral titer was determined by plaque assay in dbt cells maintained at 33°c. temperature shift growth kinetics were assessed by infecting two sets of dbt cells with wt-a59 icv, alb ts6 icv, or nsp4-n176a icv at an moi of 0.1 and incubated at 33°c. at 6 h.p.i., one set of infected cells was shifted to 39.5°c. supernatant was harvested at two hour intervals from 2-14 h.p.i. and virus production was measured by plaque assay in dbt cells maintained at 33°c. the logarithm of the titer of each virus (y) was analyzed using nonlinear regression modeling and the sas® software package. since these data clearly exhibit an asymptotic growth pattern, the two-parameter exponential model, y = θ 1 (1 − exp{− θ 2 x}) + ε, was fit to the data using 6 h post infection as the baseline. separate curves were fitted to each virus and temperature combination; parameter estimates were obtained using maximum likelihood methods, and subsequent tests were performed using likelihood-based f tests (ratkowsky, 1990) . two sets of dbt cells were infected with wt-a59 icv, alb ts6 icv and nsp4-n176a icv at an moi of 1.0 and incubated at 33°c. one hour post infection, actinomycin d (sigma, st. louis, mo) was added. at 3.5 h.p.i., cells were grown in media lacking methionine for 30 min. cells were radiolabeled with 35 s-trans-label for 2 h at 4 h.p.i. at 4 h.p.i., one set of infected cells was shifted to 39.5°c. cell lysates were prepared 6 h.p.i. and subjected to immunoprecipitation with nsp-specific antibodies as described above. two sets of dbt cells were infected with wt-a59 icv or alb ts6 icv at an moi of 1.0 and incubated at 33°c. at 3.5 h.p.i., one set of infected cells was shifted to 39.5°c. at 5.5 h.p.i., cells were harvested and processed for tem analysis as previously described (gosert et al., 2002) . two sets of dbt or hela-mhvr cells were grown to semiconfluence in 8 well chamber slides coated with permanox. cells were infected with wt-a59 icv or alb ts6 icv at an moi of 1.0 and incubated at 33°c for a 1 h absorption period. at 3.5 h.p.i., one set of infected cells was shifted to 39.5°c. at 5 h.p.i., cells were labeled with 65 nm mitotracker red fluorescent dye (invitrogen). at 5.5 h.p.i., cells were washed 3 times with pbs and fixed for 30 min at room temperature with 3.7% formaldehyde in pbs. cells were then permeabilized for 10 min at room temperature with 0.1% triton x-100 in pbs. following permeabilization, cells were incubated with either α-nsp3 or α-nsp4 and/or α-pdh antibodies overnight at 4°c. cells were then washed 3 times for 30 min in pbs. after washing, cells were incubated with alexafluor 488 conjugated chicken α-rabbit igg (invitrogen) and/or alexa fluor 568 goat α-mouse igg (invitrogen) secondary antibody for 30 min at room temperature. cells were again washed 3 times for 30 min in pbs. cells were imaged on the zeiss 510 confocal microscope. identification of a domain required for autoproteolytic cleavage of murine coronavirus gene a polyprotein cell biology of nidovirus replication complexes establishing a genetic recombination map for murine coronavirus strain a59 complementation groups characterization of a second cleavage site and demonstration of activity in trans by the papain-like proteinase of the murine coronavirus mouse hepatitis virus strain a59 four proteins processed from the replicase gene polyprotein of mouse hepatitis virus colocalize in the cell periphery and adjacent to sites of virion assembly processing of open reading frame 1a replicase proteins nsp7 to nsp10 in murine hepatitis virus strain a59 replication identification and characterization of a 65-kda protein processed from the gene 1 polyprotein of the murine coronavirus mhv-a59 analysis of murine hepatitis virus strain a59 temperature-sensitive mutant ts-la6 suggests that nsp10 plays a critical role in polyprotein processing map locations of mouse hepatitis virus temperaturesensitive mutants: confirmation of variable rates of recombination eukaryotic transientexpression system based on recombinant vaccinia virus that synthesizes bacteriophage t7 rna polymerase rna replication of mouse hepatitis virus takes place at double-membrane vesicles replication of murine hepatitis virus is regulated by papain-like proteinase 1 processing of nonstructural proteins 1, 2, and 3 identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity identification of mouse hepatitis virus papain-like proteinase 2 activity processing of the replicase of murine coronavirus: papain-like proteinase 2 (plp2) acts to generate p150 and p44 identification of the murine coronavirus mp1 cleavage site recognized by papain-like proteinase 2 membrane topology of murine coronavirus replicase nonstructural protein 3 three-dimensional analysis of a viral rna replication complex reveals a virus-induced mini-organelle identification and characterization of a serine-like proteinase of the murine coronavirus mhv-a59 mouse hepatitis virus 3c-like protease cleaves a 22-kilodalton protein from the open reading frame 1a polyprotein in virus-infected cells and in vitro the molecular biology of coronaviruses flock house virus rna polymerase is a transmembrane protein with amino-terminal sequences sufficient for mitochondrial localization and membrane insertion flock house virus rna replicates on outer mitochondrial membranes in drosophila cells engineered retargeting of viral rna replication complexes to an alternative intracellular membrane localization and membrane topology of the coronavirus nonstructural protein 4: involvement of the early secretory pathway in replication severe acute respiratory syndrome coronavirus replication complex formation utilizes components of cellular autophagy handbook of nonlinear regression models viral rna replication in association with cellular membranes functional and genetic analysis of coronavirus replicasetranscriptase proteins a contemporary view of coronavirus transcription genetics of mouse hepatitis virus transcription: identification of cistrons which may function in positive and negative strand rna synthesis processing of the coronavirus mhv-jhm polymerase polyprotein: identification of precursors and proteolytic products spanning 400 kilodaltons of orf1a colocalization and membrane association of murine hepatitis virus gene 1 products and de novo-synthesized viral rna in infected cells identification of the mutations responsible for the phenotype of three mhv rna-negative ts mutants ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex genetic analysis of murine hepatitis virus nsp4 in virus replication sars-beginning to understand a new virus negative strand rna synthesis by temperature-sensitive mutants of mouse hepatitis virus systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a59 the coronavirus replicase gene: special enzymes for special viruses virus-encoded proteinases and proteolytic processing in the nidovirales we thank ralph baric for the generous donation of clones for the reverse genetics system. we also thank linda fox of the loyola core imaging facility for her help with imaging studies, and katrina sleeman, surendranath baliji, naina barretto, dalia jukneliene, and other members of the baker lab for their technical assistance and suggestions. this research was supported by public health service research grant ai 45798. key: cord-284707-72vx11aq authors: leibowitz, julian l.; devries, james r. title: synthesis of virus-specific rna in permeabilized murine coronavirus-infected cells date: 1988-09-30 journal: virology doi: 10.1016/0042-6822(88)90147-x sha: doc_id: 284707 cord_uid: 72vx11aq abstract we have developed a permeabilized cell system for assaying mouse hepatitis virus-specific rna polymerase activity. this activity was characterized as to its requirements for monoand divalent cations, requirements for an exogenous energy source, and ph optimum. this system faithfully reflects mhv-specific rna synthesis in the intact cell, with regard to both its time of appearance during the course of infection and the products synthesized. the system is efficient and the rna products were identical to those observed in intact mhv-infected cells as judged by agarose gel electrophoresis and hybridization. permeabilized cells appear to be an ideal system for studying coronavirus rna synthesis since they closely mimic in vivo conditions while allowing much of the experimental flexibility of truly cell-free systems. the coronaviruses comprise a group of large enveloped positive-strand viruses with a unique replication scheme. the genomic rna is about 27 kb in size (boursnell et al., 1987) and shares many structural features with the genomes of other positive-strand viruses, i.e., it is capped at the 5'terminus (lai et al., 1982a) , is polyadenylated at the 3' end (lomniczi, 1977; yogo et al., 1977) and can function as messenger rna in vitro (leibowitz et al,, 1982; denison and perlman, 1986) and presumably in vivo as well (denison and perlman, 1987) . the translation product(s) of the virion rna is hypothesized to include the coronavirus-specific rnadependent rna polymerase, although this has never been rigorously demonstrated. in addition to the virion rna, infected cells contain several other classes of coronavirus-specific rna (stern and kennedy, 1980a; lai et al., 1981; leibowitz et a/., 1981; spaan et a/., 1981) . lai and co-workers have demonstrated that mhv-infected cells contain a single rna species of negative polarity which is genome length (198213) . this negative-strand rna is thought to serve as the template for positive-strand mrna synthesis. for mouse hepatitis virus (mhv), one of the most extensively studied coronaviruses, there are seven species of mhv-specific mrna present in infected cells, the largest of which is indistinguishable from virion rna (leibowitz et a/., 1981; lai et a/., 1981; spaan et a/., 1981) . structural analyses of these rnas have shown them to make up a "nested set" with coterminal 3' ends (leibowitz et al., 1981; stern and kennedy, 1980a,b; lai et a/., 1981; cheley et a/., 1981; ' to whom requests for reprints should be addressed. weiss and leibowitz, 1983; spaan et al., 1982) . a unique feature of coronavirus replication is the presence of a common leader sequence of about 70 bases at the 5' end of each message which is present only once (at the 5' end) in the virion rna (spaan et a/., 1983; lai et al., 1983 lai et al., , 1984 baric et a/., 1985) . the mechanism of synthesis of all species of coronavirusspecific rnas is largely unknown. progress in studying the details of the synthesis of the mhv mrnas has been hampered somewhat by the lack of an efficient and easily reproducible in vitro transcription system which faithfully reproduces the events which occur in intact cells. several groups of workers have demonstrated actinomycin d-resistant rna polymerase activity in extracts of coronavirus-infected cells. dennis and brian (1982) have reported the presence of a membrane-associated polymerase activity in cytoplasmic extracts of tgev-infected cells. a similar activity has been demonstrated to be present in extracts of mhv-infected cells (brayton et al., 1982 (brayton et al., , 1984 mahey et a/., 1983) . recently, compton et al. (1987) have described a system based on an extract from lysolecithin-treated cells. these systems have either been difficult to work with due to their relatively low efficiencies, or they have been hard to reproduce and maintain on a daily basis, or they do not faithfully reflect the pattern of mhv rna synthesis observed in infected cells. to circumvent the short-comings of the existing in vitro mhv polymerase systems and to overcome the relative experimental limitations of intact cells, we have taken an approach similar to that used by condra and lazzarini (1980) for studying vsv replication. in this paper we report the characteristics of a permeabilized cell system and demonstrate that it incorporates ribonucle-otide triphosphates into rna molecules which appear identical to the virus-specific mrnas synthesized in mhv-infected cells. monolayer cultures of 17cl-1 cells were grown as described previously (sturman and takemoto, 1972; leibowitz et a/., 1981) . the origin and growth of the a59 (mhv-a59) and jhm (mhv-jhm) strains of mouse hepatitis virus have been described (robb and bond, 1979) . monolayer cultures of 17ci-1 cells were trypsinized and infected in suspension as described previously (robb and bond, 1979 ) at a multiplicity of infection equal to 3 pfu per cell. infected or mock-infected cells were plated in 35-mm 6-well cluster dishes (costar) at 2 x 1 o6 cells/well in dulbecco's modified eagle's medium (dme) containing 2% fetal bovine serum and incubated at 37". at 3 hr postinfection actinomycin d (sigma) was added to the cultures at 10 pg/ml to inhibit host dna-dependent rna synthesis. at the times indicated for each individual experiment, the dishes were placed on ice and washed twice with serum-free dme. buffera [50 mmtris, ph 8.0, 4.5 mm mgac,, 20 mm kci, 5 mm naci, 150 mll/l rnase-free sucrose (swartz/ mann biotech)] was then added to the cultures. for our standard permeabilization conditions, synthetic lysolecithin (l-d-lysophosphatidylcholine, palmitoyl, sigma) was added to a concentration of 150 pg/ml and the cells were held on ice for 90 sec. lysolecithin was removed from the cultures by aspirating the buffer followed by one wash with buffer a without lysolecithin. buffer b was then added to the cultures and the cells were incubated at 37". the make up of buffer b varied, as described under results, over the course of the work reported here. 1 mm dithiothreitol, 150 mm sucrose, 10 pg/ml actinomycin d, 0.24 tiu/ml aprotinin (sigma), 20 rig/ml ouabain octahydrate (sigma). [3h]ctp (icn) was added at 62.5 &i/ml yielding a final ctp concentration of 4.5 fm. following incubation at 37" for the indicated times (40 min for most experiments), the reaction was stopped by the addition of an equal volume of 2% sodium dodecyl sulfate (sigma) to the cultures. polymerase activity was assayed by precipitating labeled rna from replicate cultures by the addition of trichloroacetic acid (tca) containing 1 yo sodium pyrophosphate (sigma) to a final concentration of 5%. tcainsoluble precipitates were collected on glass fiber filters and extensively washed with 5% tca, and the tca-precipitable radioactivity was quantitated by liquid scintillation counting. extraction and electrophoresis of rna rna was extracted from cell cultures and permeabilized cells as described (wittek et al., 1984; cabirac eta/., 1986) . the cells were dissolved in 8 m guanidium hydrochloride, 0.1 m 2-mercaptoethanol, and 0.2 m sodium acetate, ph 5.0, and the dna was sheared by passage through a hypodermic needle. the rna was selectively precipitated overnight by the addition of ethanol to a concentration of 33%. the precipitated rna was collected by centrifugation and dissolved in 50 mm sodium acetate, ph 5.2, 10 mm edta, 1 yo sds, 1 mg/ml proteinase k and digested at 37" for 1 hr. after phenol extraction the rna was ethanol precipitated and collected by centrifugation prior to further analysis. rna to be analyzed by gel electrophoresis was dissolved in a buffer containing 50% formamide, 4% formaldehyde, 20 mm mops [3-(iv-morpholino)propane sulfonic acid), 5 mm sodium acetate, 1 mm edta, ph 7.0, and electrophoresed in 0.8% agarose gels containing formaldehyde (lerach et a/., 1977) . the plasmids used in this work include 9344 (kindly provided by dr. susan weiss, university of pennsylvania), a cdna clone of mhv-a59 which encompasses a portion of gene 7 all of genes 5 and 6, and the 3'pottion of gene 4 (budzilowicz et al., 1985) . previously undescribed molecular clones of mhv-jhm used in this work are 118-8a, a cdna clone which extends from the 3' poly(a) of the genome to position 119 in gene 7, a distance of 1648 bases (spaan et al., 1983) ; 370-1, a cdna clone extending from the pvull site at position 1044 of gene 7 (spaan et al. 1983 ) to the pstl site at position 2589 of gene 3 (schmidt et a/., 1987) , a distance of almost 4.05 kbp; 414-8a, which extends from nucleotide 2350 to nucleotide 199 in gene 3 (schmidt et a/., 1987) ; and 478-38a, a clone which extends from the ddel site at position 350 in gene 3 into mhv gene 2 for an additional 1.8 kbp. a molecular clone representing the barnhi fragment k (5.9 kbp) of the leporipoxvirus malignant rabbit fibroma virus (strayer et a/., 1983a,b) was used as a control for some experiments. plasmids were digested with the appropriate restriction enzyme according to the manufacturer's suggested conditions. the resulting digests were electrophoresed in a 1 o/o agarose gel and transferred to nitrocellulose as described previously (southern, 1975) . nitrocellulose filters were probed either with randomprimed cdna prepared with [32p]dctp using purified mhv-a59 virion rna as template (weiss and leibowitz, 1983) or with the permeabilized cell reaction products. hybridization was performed at 42" in 509/o formamide, 3x sspe, 5x denhardt's. the filters were washed twice in 0.1 x sspe, 1% sds at 20" and then washed four additional times in the same buffer at 50". our initial experiments were geared toward determining the optimal conditions for permeabilizing mhvinfected 17cl-1 cells. cells were infected with mhv-a59 or mock-infected and incubated until approximately 60% of the cells were involved in syncytia. for the experiments reported here this was usually between 8.5 and 9.5 hr postinfection, a time when mhvspecific rna synthesis was maximal. at this time the cells were washed as described under materials and methods and permeabilized in buffera containing lysolecithin which was varied in concentration from 20 to 250 pg/ml. after permeabilization the cells were stained with trypan blue to determine the percentage of cells which had been made permeable to the dye at each lysolecithin concentration. these preliminary experiments demonstrated that a lysolecithin concentration of 150 pg/ml permeabilized virtually all of the mhv-infected cells and greater than 95% of the uninfected cells without making the cells too fragile to withstand the subsequent incubations. higher concentrations of lysolecithin impaired our ability to subsequently maintain the cells for the polymerase reaction (data not shown). our standard permeabilization conditions were therefore set at 150 pg/ml lysolecithin. once the conditions for permeabilization were es-tablished, we investigated the ability of permeabilized cells, in the presence of actinomycin d, to incorporate labeled precursors into tca-precipitable material. these initial experiments were pet-formed at a ph of 8.0, 4.5 mh/l mg2+, 5 mm na+, 20 mm k+, and 33 mll/l nh,+. these conditions were based upon those used by brayton et a/. (1982) in a cell-free mhv polymerase system. permeabilized cells were incubated with [3h]utp or [3h]ctp in the presence of the three other unlabeled ribonucleotide triphosphafes, actinomycin d, and an energy regenerating system, in buffer b. the amount of tca-precipitable radioactivity was severalfold greater in mhv-infected cells than in mock-infected controls (data not shown). the synthesis of radioactivity labeled material from labeled ribonucleotide triphosphates required permeabilization; cells in which the lysolecithin treatment was omitted did not incorporate any radioactivity (table 1 ). the tca-precipitable material synthesized in permeabilized cells was demonstrated to be rna in subsequent experiments on the basis of it being completely sensitive to rnase digestion and completely resistant to digestion with dnase ( table 2) . the ability of permeabilized, mhv-infected cells to incorporate [c~-~~p]ctp into acid-precipitable material percentage activity present under control reaction condition@ -permeabilization -spermidine -creatine phosphokinase and creatine phosphate -mg'+ -mg*+, +mn*+ (6 mm) -mg'+, +ca*+ (5, 10, or 20 mm) -gtp -utp -utp, -gtp +50 rm pmsfb +aprotininb +40 units/ml rnasin* +ouabain, 1 rig/ml" +ouabain, 20 ng/mp was dependent upon adding an excess of atp as compared to the three other ribonucleotide triphosphates. an atp concentration of 1 .o mm achieved the best results. higher levels of atp made the permeabilized cells extremely fragile and inhibited incorporation (data not shown). previous investigators using cell-free systems had demonstrated ph optima for mhv-specific rna-dependent rna polymerase activity at 8.4, 8.0, or 7.4, depending upon the system used. to determine the optimum ph for measuring mhv-specific polymerase activity in permeabilized cells, infected and mock-infected cells were permeabilized and assayed for polymerase activity as described above, with the exception that the ph of buffers a and b was varied between 7.0 and 8.4 among replicate cultures. in this assay, incorporation of labeled substrate in the presence of actinomycin d into tca-precipitable material increased as the ph was raised from 7.0 to 8.4 (fig. 1) . the increase of polymerase activity as the ph was raised proceeded in a step-wise fashion, with the greatest increment in activity occurring as the ph was increased from 7.2 to 7.4. further increases in the ph from 7.4 to 8.4 had a relatively small effect upon the activity, with the greatest portion of that increase occurring as the ph was changed from 7.6 to 8.0. the ability of permeabilized cells to synthesize actinomycin d-resistant rna remained almost constant as the ph was varied from 8.0 to 8.4. therefore we adopted ph 8.0 for our standard reaction conditions. the magnesium requirement of mhv rna synthesis in permeabilized cells was then determined. infected and mock-infected cells were permeabilized at ph 8.0 and the magnesium concentration was varied from 0 to 9 mm in replicate cultures. as can be seen in fig. 2 and in table 1 , there was a strict requirement for mg*+ in this system. in the absence of mg*+ the polymerase activity was reduced to 19% of the amount observed at 6.0 mm mg*+. although mg*+ was needed for measuring the mhv polymerase activity in permeabilized cells the optimum was rather broad. the requirement for magnesium cannot be replaced by either mn*+ or ca*+ (table l) , both of which resulted in less activity than that obtained when magnesium was simply omitted from the reaction mix. a magnesium concentration of 6.0 mm was chosen for our standard reaction conditions. to further optimize the system we next investigated the monovalent cation requirements of the mhv polymerase/permeabilized cell system. infected and mockinfected cells were permeabilized as described above, except that the ph and magnesium concentration were held constant at 8.0 and 6.0 mm, respectively, and the naf, k+, and nh,+ concentrations were varied as described below. initial experiments determined that k+ and nh4+ seemed to be interchangeable in this sysfig. 1. determination of ph optimum for mhv rna polymerase activity in permeabilized cells. cells were infected with mhva59 at a m.o.i. of 3, or mock-infected, and incubated until 8.5 hr postinfection. the cells were permeabilized as described under materials and methods. replicate cultures were assayed for mhv rna polymerase activity using the original formulation of buffer b (33 mm nh&i, 5 mm naci, 20 mm kci, 4.5 mm mgcip, as described under materials and methods with the exception that the ph was varied between 7.0 and 8.4 among the replicate cultures. after 40 min of incubation the assay was terminated and the amount of radioactivity incorporated into tca-precipitable material was determined. all data points represent the mean of duplicate samples. the results were calculated by subtracting the amount of radioactivity in mock-infected samples from that incorporated into mhv-infected samples under identical conditions. the results are expressed in arbitrary units with the maximum activity being set at 100. the cells were permeabilized and incubated in a formulation of buffer b which contained 30 rnm tris, 33 mm nh&i, 5 mm naci, 20 mm kci, ph 8.0, as described under materials and methods with the exception that the magnesium concentration was varied between 0 and 9 mm among replicate cultures. at 40 min incubation the assay was terminated and the amount of radioactivity incorporated into tca-precipitable material was determined. all data points represent the mean of duplicate samples. the results were calculated by subtracting the amount of radioactivity in mock-infected samples from that incorporated into mhv-infected samples under identical conditions. tern, na+ was required for activity, and a total monovalent cation concentration greater than 80 rnm resulted in a decrease in polymerase activity (data not shown). the mhv polymerase activity present in permeabilized cells was relatively insensitive to monovalent cation concentrations, as long as the total monovalent cation remained below 80 mn/l. k+ was not required; the omission of k+ from buffer b decreased activity by 5-l 09/o. at concentrations above 80 mm na+ + k+ the polymerase activity decreased somewhat. we adopted final concentrations of 44 mm na+ and 20 mm k+ in buffer b for our standard reaction conditions. these concentrations were convenient to use and approximately in the center of the broad optimum concentrations of monovalent cations. the requirements of the mhv polymerase/permeabilized cell system for various cofactors were determined. as shown in table 1 , the omission of spermidine decreased the activity to 669/o of that observed with the complete system. there was a requirement for an energy regenerating system; the omission of cpk and creatine phosphate decreased activity to 5 1% of control values. the system also required all four ribonucleotide triphosphates. the omission of either gtp or utp decreased incorporation of labeled ctp by 83 and 98%, respectively. the omission of both utp and gtp decreased synthesis by 94% of that observed in the complete system. these results suggested that the activity we were detecting was not a polynucleotide terminal transferase-like activity. similarly, the ability of permeabilized cells to incorporate radiolabeled ctp as well as utp into tca-precipitable material suggests that the polymerase activity we are detecting is not due to the polyuridylate polymerase present in the cytoplasm of mammalian cells (hayashi and mcfarlane, 1979) . protease inhibitors and rnase inhibitors have both been reported to increase the rna-dependent rna polymerase activity present in extracts of west nile virus-infected cells (grun and brinton, 1986) . we therefore determined the effect of adding pmsf or aprotinin, two protease inhibitors, on the ability of permeabilized mhv-infected cells to direct the synthesis of actinomytin d-resistant rna. as shown in table 1 , 50 &i pmsf decreased incorporation of ctp into tca-precipitable rna by about 20%. however, aprotinin increased activity by about 20%. we attribute the different effects of these compounds to the much broader spectrum of activity of pmsf, a drug which inhibits most serine esterases (fahrney and gold, 1963; laskowski and sealock, 1972) . surprisingly, the addition of placental rnase inhibitor had little effect on rna synthesis by permeabilized cells. the effect of ouabain on the mhv polymerase/permeabilized cell system was investigated because of the dependence of the system on an exogenous energy source. ouabain is an inhibitor of the na+/k+-dependent atpase present in the plasma membrane (ruoho and kyte, 1974) . we reasoned that after permeabilization this enzyme might be competing with the mhv polymerase complex for atp. if this hypothesis is true we felt that the addition on an inhibitor of the atpase to the system might stimulate the mhv polymerase activity. this did appear to be the case. ouabain at concentrations of 1 and 20 rig/ml increased the polymerase activity to 125 and 131% of that observed in controls. we could not increase the concentration of atp above 1 mm to directly test the idea that ouabain exerted its stimulator-y effect on mhv polymerase activity by increasing the biologically effective atp concentration in our reaction since concentrations of atp greater than 1 mn/l caused the permeabilized cells to detach from the substrate and subsequently disintegrate. we therefore included ouabain at 1 rig/ml and aprotinin at 0.24 tiu/ml in all subsequent experiments. the tca-precipitable material synthesized in mhvinfected permeabilized cells was identified as rna by its sensitivity to rnase. it was not sensitive to dnase (table 2) . to further characterize the rna synthesized in our system, we extracted rna from permeabilized cells labeled with [a-32p]ctp and from parallel cultures of intact mhv-infected cells labeled with [3'p]orthophosphate in the presence of actinomycin d. these samples were then analyzed by electrophoresis on a formaldehyde gel. the autoradiograph shown in fig. 3 illustrates that the relative amounts and sizes of the rna species synthesized in the permeabilized cells is very similar to the mhv-specific rnas observed in intact cells. further evidence of the virus-specific nature of these rnas was obtained by southern blot hybridization. mhv-specific plasmid clones and a plasmid clone derived from the unrelated malignant rabbit fibroma virus were digested with the appropriate restriction enzyme to excise the cloned insert and resolved by agarose gel electrophoresis. the band at approximately 3.0 kbp (fig. 4a, lane a) represents the cloning vector pgem-1. the band at approximately 4.3 kbp (fig. 4a, lanes be) represents pbr322. the band at approximately 2.8 kbp (fig. 4a , lane f) represents puc19. replicate filters of molecular clones representing the most 3' 10 kb of the mhv genome were hybridized with either randomprimed cdna synthesized from a purified virion rna template (fig. 4b) , rna extracted from mhv-infected permeabilized cells labeled with [a-32p]utp after permeabilization (fig. 4c) , or rna prepared from mockinfected permeabilized cells. as expected, the randomprimed cdna probe hybridized to all of the mhv-specific clones (lanes a-e) and did not recognize the plasmid containing the malignant rabbit fibroma virus barnhi fragment k (lane f). the labeled rna synthesized after permeabilization of infected cells also hybridized specifically with the mhv inserts, although it did not give as strong a signal as random-primed cdna probe (fig. 4c) . the apparent band at about 3.7 kbp in panel c, lane e, as artifactual since no dna is present at that position in the ethidium bromide stained gel. the signal with clones 118-8a and 414-8a was considerably weaker than the signal obtained with the other mhv-specific inserts. we attribute these differences in signal, at least in part, to the lower amount of these two inserts present in the gel (fig. 4a ). this is also reflected in the relative signals obtained with the random-primed probe. the specificity of the hybridization reaction was confirmed by the lack of hybridization of mhv-infected permeabilized reaction product with an irrelevant plasmid insert (fig. 4c , lane g) and the failure of permeabilized cell reaction products from mock-infected cells to hybridize with these mhv clones (data not shown). additionally, the extent of hybridization of the reaction products from permeabilized mhv-infected cells to mhv cdna clones bound to nitrocellulose circles was similar to that of rna prepared by labeling intact mhvinfected cells with [32p]orthophosphate in the presence of actinomycin d (data not shown). the time course of incorporation of label in permecultures of mhv-infected and mock-infected cells, incubating them for 8.5 hr, permeabilizing them using the standard conditions we had developed, and labeling them for the times indicated in fig. 5 . the accumulation of radioactivity in tca-precipitable products increases, although not in a linear fashion, over the first 40 min of labeling. after that time the amount of tcaprecipitable radioactive product in the cells decreases dramatically. the kinetics of the development of the mhv-specific rna polymerase activity over the course of infection was determined in permeabilized cells. as shown in fig. 6 , the accumulation of mhv rna polymerase activity in infected cells (panel a), as detected by our assay, faithfully mirrored the kinetics of actinomycin dresistant, [3h]uridine incorporation into tca-precipitable material (panel b) during a series of 1-hr pulses. polymerase activity is first detectable at 5 hr postinfection in these experiments. subsequent experiments showed that the peak level of polymerase activity in permeabilized cells occurred at 11 hr postinfection (data not shown). similar experiments with mhv-jhm yielded similar results, although polymerase activity appeared 1 hr later than during mhv-a59 infection. it should be noted that the infection proceeded somewhat slower in the experiments reported here than in our previously reported work (leibowitz et a/., 1981) . the reasons for this discrepancy are not known at this time. in this work we report the development and characterization of a permeabilized cell system for assaying mhv-specific rna polymerase activity. this activity was characterized as to its requirements for mono-and divalent cations, requirements for an exogenous energy source, ph optimum, and its time of appearance during the course of infection. the rna products synthesized in permeabilized cells were demonstrated to be mhv-specific by agarose gel electrophoresis. the purpose of the present work was to develop and characterize a system for studying the mhv-specific rna polymerase that was more amenable to experimental manipulations than intact cells. to avoid difficulties in reproducing in vitro the intracellular environment which evolves during mhv infection we elected to pursue a path which would leave as much of the cell machinery in place as possible. the system we have developed has several advantages when compared to the cell-free systems developed by other workers. although it is difficult to compare the relative efficiencies of different systems due to the different ways in which the experimental results have been presented, we can calculate the amount of rna synthesized in our system. using the optimized reaction conditions, one 35mm well of mhv-infected permeabilized cells incorporated about 900 fmol of ump into mhv-specific rna. this is estimated to be about fivefold more rna synthesis on a per cell basis than that obtained from a cell-free extract prepared from permeabilized cells (compton et a/., 1987) . other workers using cell-free systems have reported yields on the basis of femtomoles of ump/h/ mg protein (dennis and brian, 1982; mahey et a/., 1983; brayton et a/., 1982 brayton et a/., , 1984 . these have been in the range of 150-400 fmol/hr/mg protein. one 35-mm well of mhv-infected 17cl-1 cells contains about 450 pg of protein, providing a yield on a per milligram basis which is approximately 1800 fmol of ump/mg protein/ 40 min, a figure which makes it at least four times more efficient than the previously described cell-free systems. there are no data at this time to suggest that our system, or any other mhv polymerase assay, is capable of initiating the synthesis of new strands of rna. a second advantage of permeabilized cells for studying mhv rna synthesis is that the products synthe-sized accurately reflect the rna species synthesized in intact mhv-infected cells. all seven of the mhv mrna species are made in approximately the same ratios as they are in vivo. this contrasts with truly cell-free systems in which the rna products synthesized were not characterized as to the precise molecular species of rna synthesized (brayton et al., 1982 (brayton et al., , 1984 mahey et a/., 1983) or those in which the major product was genome length (compton et al., 1987) . although the reasons for this difference in the rna products synthesized are unknown, a possible explanation for this observation is the loss of a soluble factor responsible for regulating mhv transcription during preparation of cellfree extracts. other explanations are possible as well, and additional work is needed to identify putative factors needed for the appropriate regulation of mhv rna synthesis. the kinetics of the accumulation of polymerase activitywe observed parallels the increase of actinomycin d-resistant uridine incorporation which occurs during mhv infection. no early peak of polymerase activity or uridine incorporation in intact cells was detected. in this regard our results are similar to those of compton et al. (1987) and sawicki and sawicki (1986) . these results differ from those of earlier workers (brayton et a/., 1982 (brayton et a/., , 1984 who detected a peak of polymerase activity at 2 hr postinfection followed by a fall in activity prior to a subsequent increase to maximal levels. the reasons for these differences is not known. it could relate to the different cell lines used by different laboratories 0 2 4 6 6 10 ham post hktbl ham post hfectii fig. 6. the accumulation of mhv-specific rna polymerase activity during infection. replicate cultures of 17ci-1 cells were infected with mhva59 (m) or mock-infected (a) and incubated for 2 hr. at that time, and at hourly intervals thereafter, duplicate sets of cultures were either permeabilized and assayed forthe incorporation of radioactive ctp into tca-precipitable material under standard reaction conditions (a) or exposed to actinomycin d (5 pg/ml) for 15 min and labeled with 100 &i of [3h]uridine for 1 hr and then solublized with sds and assayed for incorporation of label into tca-precipitable material (b). or be related to the vastly different reaction conditions that are employed by the different methods of assaying mhv polymerase activity. the system we have described for assaying the mhv-induced rna-dependent rna polymerase activity should prove useful for other workers. it is simple to set up, provides a system which should be amenable to pulse-chase-type experiments, and furnishes a systern where macromolecules such as rna templates or purified proteins can be added and their effect on mhv synthesis observed. it may also serve as a point of departure for developing a completely cell-free system which better reflects the intracellular synthesis of mhv rnas than the currently available systems. characterization of leader-related small rnas in coronavirus-infected cells: further evidence for leader-primed mechanism of transcription completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus characterization of two rna polymerase activities induced by mouse hepatitis virus further characterization of mouse hepatitis virus rna-dependent rna polymerases three intragenic regions of coronavirus mouse hepatitis virus strain a59 genome rna contain a common nucleotide sequence that is homologous to the b'end of the viral mrna leader sequence transcriptional mapping of early rna from regions of the genome of the shope fibroma and malignant rabbit fibroma virus genomes intracellular murine hepatitis virus virus-specific rnas contain common sequences in vitro replication of mouse hepatitis virus strain a59 replicative rna synthesis and nucleocapsid assembly in vesicular stomatitis virus-infected permeable cells. 1. l&o translation and processing of mouse hepatitis virus virion rna in a cell-free system. 1. viral replication of mouse hepatitis virus: negative stranded rna and replicative form rna are of genome length. 1. viral protein protease inhibitors-molecular aspects cell-free translation of murine coronavirus rna the virus-specific intracellular rna species of two murine coronaviruses: mhv-a59 and mhv-jhm rna molecularweight determinations of gel electrophoresis under denaturing conditions, a critical reexamination biological properties of avian coronavirus rna rna-dependent rna polymerase activity in murine coronavirusinfected cells pathogenic murine coronaviruses. i. characterization of biologic behavior in vitro and virusspecific intracellular rnaof strongly neurotropic jhmv and weakly neurotropic a59v viruses photoaffinity labeling of the ouabainbinding site on (na+ + k+) adenosine triphosphatase. proc. nat coronavirus minus-strand rna synthesis and effect of cycloheximide on coronavirus rna synthesis nucleotide sequence of the gene encoding the surface projection glycoprotein of coronavirus mhv-jhm. 1 detection of specific sequences among dna fragments separated by gel electrophoresis coronavirus mrna synthesis involves fusion of 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virus acknowledgments merase gene product in cells infected with murine coronavirus a59. virology 157, 565-568. dennis, d. e., and brian, d. a. (1982) . rna-dependent rna polymerase activity in coronavirus-infected cells. j. viral. 42, 153-l 64. fahrney, d. e., and gold, a. m. (1963) . sulfonylfluorides.i. rates of reaction with acetylcholinesterases, a-chymotrypsin and trypsin. 1. amer. chem. sot. 85,997-l 003. grun, 1. b., and brinton, m. a. (1986) key: cord-286121-ltaxmp3u authors: xu, ke; zheng, bo-jian; zeng, rong; lu, wei; lin, yong-ping; xue, liang; li, li; yang, lei-lei; xu, chen; dai, jie; wang, fei; li, qing; dong, qing-xi; yang, rui-fu; wu, jia-rui; sun, bing title: severe acute respiratory syndrome coronavirus accessory protein 9b is a virion-associated protein date: 2009-06-05 journal: virology doi: 10.1016/j.virol.2009.03.032 sha: doc_id: 286121 cord_uid: ltaxmp3u eight accessory proteins have been identified in severe acute respiratory syndrome-associated coronavirus (sars-cov). they are believed to play roles in the viral life cycle and may contribute to the pathogenesis and virulence. orf9b as one of these accessory proteins is located in subgenomic mrna9 and encodes a 98 amino acid protein. however, whether 9b protein is a structural component of sars-cov particles remains unknown. in this study, we demonstrate that 9b protein is translated from bicistronic mrna9 via leaky ribosome scanning and it is incorporated into both virus-like particles (vlps) and purified sars-cov virions. further analysis shows that sufficient incorporation of 9b protein into vlps is dependent upon the co-expression of e and m proteins, but not upon the presence of either s or n protein. our data indicate that 9b protein of sars-cov is another virion-associated accessory protein. this finding will lead to a better understanding of the properties of the sars-cov 9b protein. severe acute respiratory syndrome (sars) which broke out in 2002-2003 has caused worldwide panic due to its high mortality in humans (peiris et al., 2004) . in 2003, sars-associated coronavirus (sars-cov) was identified as the aetiological agent for this disease ksiazek et al., 2003; peiris et al., 2003) . the genome of sars-cov is a plus-stranded single rna which is 29,727 nucleotides in length excluding the polyadenylation tract at the 3′ end . fourteen orfs have been identified, which translate into two replicative polyproteins (pp1a and pp1ab), 4 structural proteins (s, e, m, n) and 8 accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b, 9b) (marra et al., 2003; rota et al., 2003) . the accessory proteins are unique to sars-cov, as they have little homology in amino acid sequence with accessory proteins of other coronaviruses. all current studies concerning accessory proteins of coronaviruses including sars-cov suggest that they are not essential for virus replication (de haan et al., 2002; yount et al., 2005) , but do affect virus release, stability, pathogenesis, and finally contribute to the virulence (weiss and navas-martin, 2005) . understanding the properties and functions of sars-cov specific accessory proteins may help to explain the differences in pathogenicity between sars-cov and other known coronaviruses. among the 8 accessory proteins of sars-cov, 3a protein was first found to be a structural component of sars-cov virions as it was incorporated into purified virions and virus-like particles (ito et al., 2005; shen et al., 2005) . our previous study also shows that 3a protein functions as an ion channel to facilitate virus release (lu et al., 2006) . in addition, orf7a protein was also characterized as a virion-associated protein of sars-cov (huang et al., 2006a) , which induces apoptosis ((kopecky-bromberg et al., 2006; tan et al., 2004) and arrests the cell cycle when over-expressed (yuan et al., 2006) . subsequently, both sars-cov orf6 and orf7b proteins were determined to be incorporated into virus particles (huang et al., 2007; schaecher et al., 2007) . orf6 protein was further reported to function as an ifn antagonist and to accelerate replication of a related mouse virus (kopecky-bromberg et al., 2007; tangudu et al., 2007) . four other accessory proteins have virology 388 (2009) [279] [280] [281] [282] [283] [284] [285] not been well investigated, and some of them may be included in virions and have important functions (tan et al., 2006) . there are nine subgenomic mrnas in sars-cov, four of which (mrna3, 7, 8 and 9) are bicistronic producing two orfs initiating at the first or additional downstream start codon weiss and navas-martin, 2005) . on bicistronic mrna9, a 98 amino acid viral accessory protein, 9b, is encoded from a complete internal orf within the n gene. the expression of orf9b has been detected in infected cells and in clinical specimens (chan et al., 2005) . anti-9b antibodies have also been found in the serum of sars patients (qiu et al., 2005) . the crystal structure additionally indicates that 9b protein is an unusual membrane binding protein with a long hydrophobic lipid-binding tunnel. it was therefore proposed to be associated with intracellular vesicles and may have function in sars-cov assembly (meier et al., 2006) . however, further analysis of 9b protein is necessary for understanding its contribution in the viral life cycle. in this study, we have examined the expression mechanism of 9b protein from mrna9. we find that 9b protein is translated via a leaky ribosomal scanning mechanism. furthermore, we provide the first evidence that 9b protein is present both in virus-like particles and in purified virions, and that the efficient incorporation of 9b protein is dependent upon the co-expression of e and m proteins. these data suggest that 9b protein is not only a viral accessory protein but also a structural component of sars-cov virions. our previous study using a proteomics approach has identified orf3a as a viral protein of sars-cov (zeng et al., 2004) . meanwhile, several peptides belonging to orf9b protein had also been detected in the cytosol of sars-cov infected vero e6 cells (data not shown). to further investigate the properties of sars-cov 9b protein, purified recombinant 9b protein was used to produce both anti-9b polyclonal and monoclonal antibodies. the expression of 9b protein in sars-cov infected cells was confirmed by western blot analysis with anti-9b monoclonal antibody. an 11 kda protein (9b protein) was recognized in infected but not in uninfected cells (fig. 1b left) . the specificity was further ensured by immunoprecipitation with anti-9b polyclonal antibody. the complex precipitated was resolved under gel electrophoresis and the corresponding band was submitted to mass spectrometry. a peptide: "afqstpivvqmtk" representing amino acids 69-81 of the sars-cov 9b protein was identified (fig. 1b right) . as none of the previous studies has illustrated the expression mechanism of 9b protein, we then examined whether 9b protein was able to be translated from subgenomic mrna9 alone and how it was translated. the 9b protein encoded by an internal orf starts at the 18th nucleotide downstream of the transcription regulatory sequence of mrna9 . the start codon for orf9b is very close to the start codon for orfn, with only 10 nucleotides in between (fig. 1a ). to confirm that 9b protein can be translated from an mrna corresponding to the sars-cov subgenomic rna9, the sequence encoding orfn which contains orf9b was cloned into the eukaryotic expression vector pcaggs and transfected into 293t cells. the results showed that 9b protein was able to be expressed in cells transfected with plasmids containing the n gene coding region ( fig. 1c orfn) , but that the expression level was relatively weaker compared with that of cells transfected with orf9b alone (fig. 1c orf9b) . when the orf9b initiation codon was mutated from atg to acg ( fig. 1c n(9b − )), 9b protein was not expressed while the expression level of n protein showed little change. when the orfn initiation codon was mutated from atg to atc ( fig. 1c n(n − ) ), n protein was not expressed while the expression level of 9b protein dramatically increased. these data not only demonstrated the expression of 9b protein from the n gene coding region, but also led us to further investigate the translation regulatory mechanism for 9b protein. the downstream orf of multicistronic mrna is mainly translated by leaky scanning ribosomes or internal ribosome entry (kozak, 1989; thiel and siddell, 1994; van vliet et al., 2002) . analysis of the sequence flanking the initiation codon of orfn and orf9b showed that the initiation of the n gene represented a suboptimal kozak context (a at −3, t at +4), while the initiation of the 9b gene indicated an optimal kozak context (a at −3, g at +4) (kozak, 1986) . in addition, a lower expression level of 9b protein had been observed already in cells transfected with orfn and then in cells transfected with orf9b or n (n − ). all of these findings make it reasonable to presume that the translation initiation of orf9b is regulated by a leaky ribosomal scanning mechanism under which the downstream gene expression is affected by the translation efficiency of the upstream gene (kozak, 1987 (kozak, , 1989 . to test this, a more ideal kozak context was induced in orfn ( fig. 1c orfn + 4t to g). we detected an obviously decreased expression level of 9b protein as expected. meanwhile, when the kozak context of orfn was changed to a much weaker one ( fig. 1c orfn −3a to c), an increased expression of 9b protein was observed. together, these data suggest that orf9b protein is translated via a leaky ribosomal scanning mechanism. in order to examine whether 9b protein is incorporated into virus particles, we established a sars-cov vlp system in vitro to study the presence of 9b protein in virus-like particles. to ensure that the release of 9b protein is associated with virus particles, we first examined whether 9b protein was able to be released from cells expressing 9b protein alone. cells were transfected with pcaggs-9b plasmid. parental pcaggs-3a or vector alone was also transfected as control. western blot analysis of the cell lysates clearly showed that both 3a and 9b proteins were expressed (fig. 2) . the clarified culture supernatants from 9b and 3a-expressing cells and control cells were applied to the top of a 20% sucrose cushion for purification. western blot analysis of the pellets demonstrated that 9b protein was not released into the culture medium of 9b-expressing cells, while 3a protein was released as previously described (huang et al., 2006b) . the abundant host protein, actin, was not detectable in the supernatants from any group of cells (fig. 2) . we then transfected pcaggs-s, pcaggs-e, pcaggs-m, pcaggs-n (9b − ) and pcaggs-9b simultaneously into 293t cells. at 48 h post transfection, the culture medium was collected and clarified. the vlps purified with 20% sucrose were then added to the top of a 20-60% continuous sucrose gradient for fractionation. twelve fractions were collected, and each was examined by western blot analysis. the results showed that 9b protein was released from the vlp system, and exhibited in fractions 5 to 9 along with s, n, and m proteins (fig. 3) . the densities of these fractions were approximately between 1.12 g/ ml and 1.20 g/ml. the greatest amount of 9b protein was detected in fraction 7 (density, 1.16 g/ml), which also contained the highest level of s, n and m proteins. the data suggest that 9b protein is incorporated into virus-like particles containing s, n, m and e proteins. e protein was not detectable in western blot due to its low abundance in sars-cov vlps (huang et al., 2006a) . to further confirm the potential association of 9b protein with sars-cov virions, purified sars-cov was prepared from the supernatant of virus-infected frhk-4 cells. the medium was collected at 36 h post infection, at which time the cells remained intact and cpe had not appeared. clarified supernatant was then subjected to sucrose gradient ultracentrifugation as described above and twelve fractions from top to bottom were collected and analyzed. the results showed that s, n, m and 9b proteins were present in fractions 7 (density, 1.16 g/ml) to 10 (density, 1.20 g/ml) at the same time (fig. 4) . the peak of the 9b protein was detected in fraction 8 along with the highest level of s, n and m proteins. the density of fraction 8 was approximately 1.18 g/ml, consistent with the density of sars-cov particles (huang et al., 2006a; ito et al., 2005; schaecher, mackenzie, and pekosz, 2007) . these data further suggest that 9b protein is incorporated into sars-cov virions. as 9b protein is present in virions, it is advantageous to characterize the role of other sars-cov structural proteins in incorporation of 9b protein into vlps. cultures of 293t cells were transfected with the indicated plasmids, and pcaggs vector was added to adjust the total amount of dna to equivalent levels. as shown in fig. 5 , in the fig. 2 . orf9b protein is not released from 9b-expressing cells. 293t cells were transfected with pcaggs-3a-ha (3a), pcaggs (vector), or pcaggs-9b (9b) independently. at 48 h post transfection, supernatants were clarified, applied to a 20% sucrose cushion, and centrifuged at 100,000 ×g for 3 h at 4°c. the pellets were suspended in 1×sds-page loading buffer (medium). cell lysates were prepared with 1×sds-page loading buffer (cell). samples were subjected to western blot analysis with anti-ha antibody to detect 3a protein (37 kda), anti-9b antibody (11 kda) to detect 9b protein and anti-actin antibody for internal control. (meier et al., 2006; thiel et al., 2003) . (b) left. 11 kda 9b protein was detected in sars infected frhk-4 cells by anti-9b monoclonal antibody, but not in uninfected cells. β-actin was detected in both infected and uninfected cells. right. 9b protein was immunoprecipitated by anti-9b polyclonal antibody from sars infected frhk-4 cell lysates in ripa buffer, the mass spectrometry analysis of the corresponding gel slices detected a specific peptide that represents 9b protein. (c) translation of orf9b by leaky ribosomal scanning. schematic diagram of cdna constructs is shown (upper panel). wild-type orf9b sequence (orf9b), wild-type n gene sequence (orfn), a point mutation eliminating 9b initiation codon in orfn sequence (n(9b − )), a point mutation eliminating n initiation codon (n(n − )), an optimal kozak context around the orfn initiation codon (orfn + 4t to g), and a weaker kozak context around the orfn initiation codon (orfn − 3a to c). 293t cells were transfected with plasmids encoding the indicated cdnas and analyzed by western blot with both anti-n and anti-9b monoclonal antibodies for each sample. β-actin was detected by actin specific polyclonal antibody. pellets of the culture medium collected from cells co-transfected with pcaggs-e and pcaggs-9b, a low level of 9b protein was detected suggesting that 9b protein is able to be released when only e protein is available. a marked increase of 9b protein in culture medium was observed when m protein was co-expressed with 9b and e proteins. the amount of 9b protein in vlps did not change significantly when s or n, or both of them were added. the results indicate that sufficient incorporation of 9b protein into sars-cov vlps is dependent upon co-expression of e and m proteins but is not influenced by either s or n protein. the effect of 9b protein on vlp production was further analyzed. vlps containing s, e, m and n proteins in 9b-expressing cells and nonexpressing cells were examined and compared. unfortunately, no obvious differences in the assembly of these proteins have been detected (fig. 5 ). as a newly emerging coronavirus in the human, sars-cov has led to acute inflammation and a lethal syndrome in patients. phylogenetic analysis showed that sars-cov had been classified as group 2b cov distantly related to known group 2 cov (gorbalenya et al., 2004; snijder et al., 2003) . the functions of viral replicase and four basic structural proteins (s, e, m, and n) of sars-cov are similar to those of other coronaviruses. however, the accessory proteins of sars-cov show little homology to those of known coronaviruses. the specific properties of these accessory proteins may contribute to the differences in pathogenicity between sars-cov and other coronaviruses. in this report, we demonstrate that sars-cov 9b protein is another viral accessory protein incorporated into virus particles. our finding makes 9b protein the fifth accessory protein of sars-cov to be found present in virions. for efficient utilization of their limited genome, viruses frequently use bicistronic rnas to produce alternative open reading frames. sars-cov orf1b, orf3b, orf7b, orf8b, and orf9b are all transcribed via alternative open reading frames. in addition to orf7b protein (schaecher et al., 2007) , orf9b protein is another accessory protein that is proven to be translated via leaky ribosomal scanning. other fig. 4 . incorporation of 9b protein into purified sars-cov virions. clarified supernatant from sars infected cells was subjected to 20% sucrose ultracentrifugation. the pellets resuspended in nte buffer were further applied on a 20-60% sucrose gradient cushion. twelve fractions were collected from top to bottom after ultracentrifugation, each fraction was condensed using 20% sucrose and subjected to western blot analysis with anti-s monoclonal antibody (s), anti-n monoclonal antibody (n), anti-m polyclonal antibody (m) and anti-9b monoclonal antibody (9b). β-actin was detected by actin specific polyclonal antibody (actin). the density of each fraction was measured and is shown. fig. 5 . release of sars-cov 9b protein in the presence of other viral structural proteins. 9b protein was co-expressed with e protein, m protein, s protein, and n protein in different combinations in 293t cells, as indicated. the quantity of each plasmid for this analysis was same, and total dna levels were adjusted by adding pcaggs. at 48 h post transfection, medium was harvested and pelleted through a 20% sucrose cushion. medium (medium) and cell lysates (cell) were analyzed for s protein (s), n protein (n), m protein (m), and 9b protein (9b) by western blotting. s, n, and 9b proteins were detected using indicated monoclonal antibodies, while m protein with ha tag was detected using anti-ha antibody. the released vlps were first pelleted by centrifugation through a 20% sucrose cushion, and then further purified over a 20-60% sucrose gradient. twelve fractions were collected from top to bottom; each fraction concentrated by 20% sucrose ultracentrifugation was analyzed using western blot analysis with anti-s monoclonal antibody (s), anti-n monoclonal antibody (n), anti-ha tag monoclonal antibody (m), and anti-9b monoclonal antibody (9b). β-actin was detected by actin specific polyclonal antibody (actin). the density of each fraction was measured and is shown. virus accessory proteins encoded by multicistronic mrnas are translated either by leaky ribosomal scanning or by internal entry of the ribosome (senanayake and brian, 1997; thiel and siddell, 1994) . the mechanisms must be identified individually. after the first sars-cov accessory protein, 3a, had been identified as a structural component of virions, accessory proteins 7a, 7b, and 6 were subsequently determined to be incorporated into virions (huang et al., 2006a (huang et al., , 2007 ito et al., 2005; schaecher et al., 2007; shen et al., 2005) . here, our data further indicate that 9b protein is the fifth virion-associated accessory protein of sars-cov to be recognized. analysis of vlps and purified sars-cov particles showed that the highest level of 9b protein was detected in the fractions also abundant in s, n, and m proteins. while this property is almost equivalent among these accessory structural proteins, they do exhibited different biochemical characteristics. some, such as 3a and 6 proteins, are released from expressing cells, while others, like 7a and 9b proteins, are not detectable in the supernatant of expressing cells. as a result, the release of the protein itself is seen not to be a determinant of incorporation. the localization of these proteins to intracellular vesicle components like golgi and er may help them to be incorporated through mechanisms bringing them close to the site of virus assembly. the mechanism of incorporating accessory proteins into virus particles is still not well understood and must be further studied. furthermore, it will be important to address the necessity of incorporation of all these accessory proteins into virions. release of 9b protein was detected from cells co-expressing e and 9b proteins, but not from cells expressing 9b protein alone. this result was similar to what was found with 7a protein; the release of these two accessory proteins with e protein may be caused by their incorporation into putative e protein-containing vesicles (maeda et al., 1999; huang et al., 2006a) . when m protein but not s or n protein was added, the release of 9b into the medium was increased markedly, indicating that e and m proteins are sufficient to allow incorporation of 9b protein into virus-like particles. this is consistent with former studies showing that e and m proteins could form smooth virus-like particles when co-expressed, while s and n were unnecessary for vlp formation (hsieh et al., 2005; mortola and roy, 2004) . as we do not have evidence for interactions between 9b and e or m proteins, we cannot determine whether the incorporation of 9b protein into vlps generated by e and m co-expressing cells is caused by the physical associations among them. however, this mechanism requires further examination. orf9b on sars-cov bicistronic mrna9 is a fully internal orf in the n gene coding region. there are also coordinate proteins similar to sars-cov 9b which are so-called "internal" or "i" proteins in other group ii coronaviruses (fischer et al., 1997; lapps et al., 1987; senanayake and brian, 1997) . in mhv, i protein is an accessory viral structural protein which can contribute to plaque morphology (fischer et al., 1997) . in our study, i protein of sars-cov (9b protein) was also shown to be a structural component of the virions. it is reasonable to propose that 9b protein of sars-cov may also play a role in the viral life cycle. however, a more thorough functional analysis of 9b protein still must be performed. although a recent study with sars-cov accessory protein deletion mutants showed little effect on pathogenicity in a mouse model (dediego et al., 2008) , these accessory proteins, including 9b protein, may still play a supportive role in the viral life cycle in patients. cell culture, transfection, and virus infection 293t, vero e6 and frhk-4 cells (atcc) were cultured in dulbecco's modified eagle's medium (dmem) containing 10% fetal bovine serum (gibco) at 37°c in a co 2 incubator. lipofectamine (invitrogen) was applied for transient transfection following the manufacturer's procedure. for sars-cov infection, frhk-4 cells were inoculated with virus (gz 50 strain) as previously described (zhong et al., 2003) at moi of 5 for 1 h in medium without fbs. after 1 h, the cells were washed with medium and cultured with complete medium for the required time. all procedures with sars-cov infection were performed in a biosafety level-3 laboratory. viral rna was extracted from filtered supernatant of virus-infected frhk-4 cells using the rnesay mini kit (qiagen) following the manufacturer's protocol. the cdna was prepared by reverse transcription using m-mlv reverse transcriptase (invitrogen) with oligonucleotide primers. s, e, m, n, 3a and 9b genes were amplified by pcr with specific primer using pyrobest polymerase (takara). s, n and 9b genes were cloned directly into pcaggs vector under a powerful chicken β-actin promoter (kindly provided by dr. jun-ichi miyazaki, osaka university). the deletion of orfn (n(n − )) was obtained by mutating the initiation codon of n gene from atg to atc, while the deletion of orf9b (n(9b − )) in the n gene coding region was achieved by mutating the initiation codon of orf9b from atg to acg. for the mutation of the kozak context, primers containing the mutated nucleotides (−3a to c, + 4t to g) were synthesized to amplify the expected orfs and then cloned into pcaggs vector. e, m and 3a genes were first cloned into pcdna-3′ha (invitrogen), and then the genes together with ha tag at the 3′ end were amplified by pcr and subcloned into the pcaggs vector. all plasmids were confirmed by sequence analysis. the monoclonal and polyclonal antibodies against 9b protein were both produced by the antibody research centre, shanghai institute of biological science. mouse anti-s and mouse anti-n antibodies were also produced by the antibody research centre, shanghai institute of biological science. rabbit anti-m antibody (ap6008b) was purchased from abgent. mouse anti-ha monoclonal antibody was obtained from covance and rabbit anti-actin from sigma. hrp-conjugated antimouse igg was purchased from sigma and hrp-conjugated antirabbit igg from southern biotech. samples were treated as previously described (zeng et al., 2004) . briefly, the infected cell lysates, which had been immunoprecipitated using 9b polyclonal antibody, were subjected to sds-page and the corresponding gel slices were excised and digested with trypsin. the digested peptides were analyzed by mass spectrometry (lcq deca xp plus, thermo finnigan). data analysis was carried out using a shot-gun approach. for sars-cov purification, the supernatant containing the virus was inactivated with 1:2000 formaldehyde at 4°c for 72 h as previously described (qu et al., 2005) . for the sars-cov virus-like particle system, the medium was collected 48 h post co-transfection for s, e, m, n and 9b. the inactivated virus and the medium from the vlp system were first purified by centrifugation at 1000 ×g for 15 min at 4°c and then by ultracentrifugation over a 20% sucrose cushion at 100,000 ×g for 3 h in an sw41 ultracentrifuge rotor (beckman). the pellets were resuspended in nte buffer (100 mm nacl, 10 mm tris-hcl [ph7.5], 1 mm edta) and loaded onto a 20-60% continuous sucrose gradient for fractionation at 100,000 ×g over 12 h at 4°c in a sw41 rotor (hsieh et al., 2005; mortola and roy, 2004) . twelve fractions from top to bottom were collected and concentrated separately by ultracentrifugation at 100,000 ×g for 3 h. each pellet was then dissolved in 1×sds-page loading buffer and analyzed by western blot assay. to determine whether 9b protein is released from cells expressing 9b protein, supernatants from cells transfected with orf3a, orf9b or empty vector were clarified by centrifugation at 1000 ×g for 15 min at 4°c. this was followed by ultracentrifugation over 20% sucrose at 100,000 ×g for 3 h in a sw41 rotor. the pellet was dissolved in 1×sds-page loading buffer and submitted to western blot analysis. frhk-4 cells were infected with sars-cov at 24 h post infection and the cell lysates were collected as described previously (lu et al., 2006) . briefly, infected cells were lysed with solution containing 40 mm tris (ph 8.3) and 0.5% np-40 at 22°c for 5 min. the supernatant was collected and subjected to immunoprecipitation and western blot assay. clarified supernatant was then incubated with anti-9b polyclonal antibody together with 5% bsa and protein a/g beads at 4°c overnight. after immunoprecipitation, beads were washed 5 times with ripa buffer; the complex was eluted and submitted to mass spectrometry analysis. expression of the sars-cov proteins in 293t cells was studied 24 h after transient transfection. the cells were lysed in sds loading buffer and subjected to sds-page electrophoresis. proteins were transferred to a nitrocellulose membrane and detected using corresponding antibodies. coronaviral hypothetical and structural proteins were found in the intestinal surface enterocytes and pneumocytes of severe 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sarsassociated coronavirus in infected vero e6 cells and sars patients epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china we thank prof. vincent deubel, prof. paul zhou, prof. jing zhong, prof. ke lan, (institute pasteur of shanghai, cas, china), prof. shi-shan yuan (shanghai institute of animal parasitology, chinese academy of agriculture science), prof. xue-liang zhu (shanghai institute of biological science), dr. sheri skinner (usa), prof. hao shen (usa) and prof. hans klenk (marburg university, germany) for reviewing the manuscript and for constructive suggestions. we thank mr. yi-min wang (freiburg university, germany) for helpful comments on this study. this work was supported by grants from the national natural science foundation of china (30325018, 30530700, 30623003 and 30421005) and cas project (kscx1-yw-r-43), grants from the technology commission of shanghai municipality (07dz22916, 064319034, 04dz14902, 04dz19108, 06dz22032 and 04dz19112), a grant from national 863 key project (2006aa02a27), a grant from the sino-germany center on sars project (gz238(202/11)), a grant from e-institutes of the shanghai universities immunology division and a grant from li kha shing foundation. key: cord-282947-3hgku2e4 authors: wong, hui hui; fung, to sing; fang, shouguo; huang, mei; le, my tra; liu, ding xiang title: accessory proteins 8b and 8ab of severe acute respiratory syndrome coronavirus suppress the interferon signaling pathway by mediating ubiquitin-dependent rapid degradation of interferon regulatory factor 3 date: 2017-12-30 journal: virology doi: 10.1016/j.virol.2017.12.028 sha: doc_id: 282947 cord_uid: 3hgku2e4 severe acute respiratory syndrome coronavirus (sars-cov) is an inefficient inducer of interferon (ifn) response. it expresses various proteins that effectively circumvent ifn production at different levels via distinct mechanisms. through the construction of recombinant ibv expressing proteins 8a, 8b and 8ab encoded by sars-cov orf8, we demonstrate that expression of 8b and 8ab enables the corresponding recombinant viruses to partially overcome the inhibitory actions of ifn activation to achieve higher replication efficiencies in cells. we also found that proteins 8b and 8ab could physically interact with irf3. overexpression of 8b and 8ab resulted in the reduction of poly (i:c)-induced irf3 dimerization and inhibition of the ifn-β signaling pathway. this counteracting effect was partially mediated by protein 8b/8ab-induced degradation of irf3 in a ubiquitin-proteasome-dependent manner. taken together, we propose that sars-cov may exploit the unique functions of proteins 8b and 8ab as novel mechanisms to overcome the effect of ifn response during virus infection. when challenged by a viral infection, the host mounts an immediate innate immune response, leading to the production of type i interferons (ifn-α and ifn-β) and the expression of hundreds of downstream ifnstimulated genes (isgs) (stetson and medzhitov, 2006; suhara et al., 2002) . central to the induction of type i ifn is interferon regulatory factor 3 (irf3) (hiscott, 2007; taniguchi et al., 2001) . the initial step of the signaling cascade leading to irf3 activation is recognition of specific viral pathogen-associated molecular patterns (pamps) by host pattern recognition receptors (prrs) in two major pathways. these include cell surface toll-like receptors (tlrs), such as tlr3, tlr7, tlr8 and tlr9, which sense viral components (kumagai et al., 2008; kumar et al., 2009a kumar et al., , 2009b , and cytosolic rna helicases such as retinoic acidinducible gene i (rig-i) and/or melanoma differentiation-associated gene 5 (mda5), which detect viral rna (loo et al., 2008; onomoto et al., 2007) . upon binding to their ligands, prrs recruit adaptor proteins to set off a series of signaling cascades to phosphorylate and dimerize irf3 (fitzgerald et al., 2003) . the activated irf3 homodimer then translocates to the nucleus, switching on ifn synthesis (lin et al., 1998; suhara et al., 2002; thanos and maniatis, 1995) . many viruses have also evolved strategies to counteract the ifn action, including mechanisms that allow virus to evade recognition by the immune surveillance system, so as to inhibit ifn induction by hijacking molecules involved in ifn activation pathways or by inhibiting downstream signal transduction (goodbourn et al., 2000; versteeg et al., 2007; weber et al., 2003) . severe acute respiratory syndrome coronavirus (sars-cov) was the etiological agent of the sars epidemic in 2003 (guan et al., 2003; marra et al., 2003) . apart from four typical structural proteins, nucleocapsid (n), envelope (e), membrane (m) and spike (s) proteins, and approximately 16 non-structural proteins (nsp1-16) involved in viral replication, sars-cov encodes an exceptionally high number of accessory proteins that bear little resemblance to accessory genes of other coronaviruses narayanan et al., 2008b) . similar to other coronaviruses, sars-cov is an inefficient inducer of ifn-β response in cell culture system (spiegel et al., 2005) and is sensitive to the antiviral state induced by ifns (spiegel et al., 2004; zheng et al., 2004) . its genome may therefore encode proteins that allow this virus to effectively circumvent ifn production in order to overcome limitations imposed by the ifn action. together with a few structural proteins and nsps, many coronavirus accessory proteins could suppress ifn production by targeting different aspects of the ifn signaling cascade (lim et al., 2016; liu et al., 2014; zhong et al., 2012) . for instance, sars-cov papain-like protease (plpro) attenuates ifn synthesis by abrogating irf3 phosphorylation and nuclear translocation by physically interacting with irf3 . ifn induction mediated by a constitutively active irf3 is also inhibited by the de-ubiquitination activity of sars-cov plpro (matthews et al., 2014) . on the other hand, nsp1 of pedv does not interfere irf3 phosphorylation and nuclear translocation, but interrupts the enhanceosome assembly of irf3 and creb-binding protein (cbp) by promoting proteasomal degradation of cbp (zhang et al., 2016) . targeting further upstream, sars-cov m protein prevents irf3 phosphorylation by inhibiting the assembly of tbk1/ikk complex (siu et al., 2009) , whereas mers-cov m protein interacts with traf3 and disrupts traf3-tbk1 association, leading to reduced irf3 phosphorylation (lui et al., 2016) . likewise, the accessory protein orf4b of mers-cov has been shown to specifically bind to tbk1 and ikkε, thereby inhibiting irf3 phosphorylation and ifn-β production . signaling molecules downstream of ifn synthesis are also targets of sars-cov proteins. for instance, sars-cov nsp1 inhibits stat1mediated transcription of isgs by inhibiting its phosphorylation (wathelet et al., 2007) , apart from inducing degradation of a wide range of host mrnas (kamitani et al., 2006; narayanan et al., 2008a) . sars-cov orf6 blocks stat1 nuclear translocation by trapping the nuclear import factors in the endoplasmic reticulum and golgi apparatus . among other ifn antagonists identified are nucleocapsid (n) protein of sars-cov and pedv ding et al., 2014) , accessory protein 4a of mers-cov (siu et al., 2014) , and plpro domain 2 (plp2) of mhv-a59 (wang et al., 2011) . ifn antagonism mediated by sars-cov n protein seemed to target a very early step of rna recognition (lu et al., 2011) . overexpression of sars-cov accessory protein 3a was found to down-regulate type i ifn receptor by promoting its ubiquitination and subsequent degradation via the lysosomal pathway (minakshi et al., 2009) . although both 8b and 8ab are encoded by sars-cov orf8, they are expressed under distinct conditions. 8ab is expressed as a single protein encoded by the single continuous orf (orf8ab) found in sars-cov isolated from animals and early stage human isolates. in contrast, as a consequence of a 29-nt deletion that results in two separate overlapping orfs (orf8a ⁄ orf8b), most human isolates obtained at the middle to later phase of the epidemic encode instead 8a and 8b as two distinct proteins (guan et al., 2003; oostra et al., 2007) . orf8 is presumably acquired from sars-related coronavirus from greater horseshoe bats through recombination (lau et al., 2015) , and the 29-nt deletion may be an evolutionary adaptation for enhancing viral pathogenesis in the human host. proteins 8a, 8b and 8ab may possess different biochemical properties and possibly cellular functions (law et al., 2006; le et al., 2007) . protein 8ab was shown to be a glycosylated er resident protein which can activate atf6 to modulate the unfolded protein response (sung et al., 2009) . protein 8a was found to enhance viral replication and induce cell death (c.y. , while 8b induces dna synthesis and down-regulates sars-cov e protein via a proteasomeindependent pathway (keng et al., 2006) . sars-cov 8b and 8ab were also shown to bind to both mono-and poly-ubiquitin when expressed in cell culture (keng et al., 2006) . whether these ubiquitin-binding properties allow them to interact with host cell proteins remains unknown. interestingly, when an in vivo attenuated recombinant sars-cov lacking the full-length e gene is passaged in mice, the orf8 sequence mutates to encode a pdz-binding motif in protein 8a, and the virus regained virulence (jimenez-guardeño et al., 2015) . in this study, we show proteins 8b and 8ab as novel ifn antagonists. evidence presented supports the direct interaction between these two proteins and irf3. the two proteins were also found to partially suppress ifn induction by limiting irf3 activation and/or promoting the proteasome-mediated degradation of irf3. african green monkey kidney cos-7 and vero cells, human nonsmall cell lung carcinoma h1299 cells and human hepatocellular carcinoma huh7 cells were cultured in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal calf serum (hyclone) and 1% penicillin/streptomycin dmem (invitrogen) and maintained at 37°c with 5% co 2 . to inhibit the proteasome activity, mg132 (sigma) at a final concentration of 10 µm was added to cells 2 h prior to harvest. to compare the growth kinetics of various recombinant virus strains, vero cells were infected with the respective recombinant ibv strains at a multiplicity of infection (moi) of 0.5, and harvested at a 4 h interval within 20 h post-infection for virus titration through plaque assay. a monolayer of vero cells seeded on 6-well plates a day prior to infection was infected with 200 µl of 10-fold serially diluted virus stock. after 1 h of incubation at 37°c with regular shaking to ensure even distribution of the virus, cells were washed with pbs and cultured in 3 ml of dmem containing 1% carboxymethyl cellulose (cmc) for 3 days. the cells were then fixed with 4% paraformaldehyde and stained with 0.1% toluidine. the number of plaques was counted in duplicates and the virus titer was calculated as plaque-forming unit (pfu) per ml. the pkto-flag-8b and pkto-flag-8ab plasmids were previously described (le et al., 2007) . the coding sequences of 8b or 8ab were also subcloned to the pxj40-flag vector, which contains the cmv promoter for expression in mammalian cell lines. for the construction of the pxj40-myc-irf3 plasmid, human irf3 gene was amplified from cdna of h1299 cells using the forward primer 5′-aacgcctcgacggaaccc caaagccacggat-3′and the reverse primer 5′-gccggtaccttattg gttgaggtggtgggg-3′ prior to ligation into pxj40-my plasmid at xhoi and kpni restriction sites. truncated mutants were then constructed based on the pxj40-myc-irf3 construct using the following primers for pcr amplification: for irf3 (1−133), forward 5′-ccgct cgagcggatgatgggaaccccaaagccacg-3′and reverse 5′-gggg tacccctcaagaagtactgcctccaccat-3′; for irf3 (399-379), forward 5′-ccgctcgagcggatggatacccaggaagacattct-3′ and reverse 5′-ggggtacccctcatccaggcagcgtcctgtctc-3′; for irf3 (241−427), forward 5′-ccgctcgagcggatgtggccagtcacactgc caga-3′ and reverse 5′-ggggtacccctcagctctccccagggccct-3′; for irf3 (134−427): forward 5′-ccgctcgagcggatggatacccagg aagacattct-3′ and reverse 5′-ggggtacccctcagctctccccagg gccct-3′. constitutively active mutant pxj40-myc-irf3-5d was generated by performing sequential site-directed mutagenesis pcr (quik-change ii site-directed mutagenesis kit; stratagene) to replace amino acids at positions 396, 398, 402, 404, and 405 with the phosphomimetic aspartic acid. cells were lysed in ripa buffer in the presence of protease inhibitors (roche diagnostics) and phosphatase inhibitors (pierce). protein lysates were separated by electrophoresis in 8% sds polyacrylamide gels and transferred to nitrocellulose membrane (amersham biosciences) via wet transfer (bio-rad). the membranes were blocked overnight at 4°c with 10% non-fat milk in pbst before probing with specific primary antibodies, followed by horse-radish peroxidase (hrp)-conjugated antimouse, anti-rabbit or anti-goat igg secondary antibodies (dako), respectively. the following commercial primary antibodies were used: βtubulin (sigma), irf3 (santa cruz biotechnology), pirf3(398) (cell signaling) and β-actin (santa cruz biotechnology). polyclonal antibodies against ibv n were raised in rabbits by this laboratory (li et al., 2005) . monolayer cells grown overnight in 6-well plates (nunc) were infected with recombinant vaccinia virus encoding the bacteriophage t7 rna polymerase before transfection of plasmids using effectene reagent (qiagen), as previously described inglis, 1991, 1992) . briefly, at 20 h post-transfection, cells were harvested with 500 µl of ripa buffer in the presence of protease (roche diagnostics) and phosphatase inhibitors (pierce). lysates were centrifuged at 13,000×g at 4°c for 15 min, and the supernatant obtained was immunoprecipitated directly with antibody-conjugated agarose beads for 2 h or with appropriate antibodies followed by incubation with protein a agarose beads (sigma) for another 2 h at room temperature. the immunoprecipitated proteins were separated on sds-page and analyzed by western blot using appropriate antibodies. the hrp-conjugated anti-myc and anti-flag antibodies were purchased from sigma, while antibodies against igg, isg56, β-actin and full-length irf3 were from santa cruz biotechnology. cells were lysed in buffer (50 mm tris-hcl (ph 7.4), 150 mm nacl, 1 mm edta, 1% np-40) containing protease inhibitors and phosphatase inhibitors for 30 min at 4°c. proteins were then separated by electrophoresis in 8% non-denaturing polyacrylamide gels, with 1% sodium deoxycholate (sigma) in the cathode buffer. irf3 monomers and dimers were detected by western blot analysis using polyclonal antibodies against full-length irf3 (santa cruz biotechnology). huh7 cells seeded on a 12-well plate were transfected with a total of 4 μg of the appropriate plasmids using lipofectamine 2000 (invitrogen) according to manufacturer's instructions. pifn-β-luc and prl-tk were purchased from promega. 10 µg of poly (i:c) complexed with 10 µl lipofectamine 2000 was then introduced into the cells 20 h later. cells were lysed 20 h post treatment in passive lysis buffer (promega) and an aliquot of the lysates was measured for firefly and renilla luciferase activities according to the manufacturer's instruction (promega). construction of recombinant ibvs (ribvs) was carried out essentially as previously described le et al., 2007; tan et al., 2006) . to generate the 8b mutant (8bm) containing lysine to arginine mutations at all three positions, standard pcr site-directed mutagenesis was performed using the construct containing orf8 insertion as a template. the genotypes of ribvs were validated by sequencing. total rna was isolated using the trizol reagent (invitrogen) as described by manufacturer's protocol. three µg total rna was reversed transcribed (roche). the relative abundance of ifn-β, isg15, isg56 and rantes mrnas in treated samples with respect to their mock treated counterparts was determined by real-time quantitative rt-pcr using the sybr green method (roche). briefly, a 20 µl pcr reaction containing cdna template, the respective primers and lightcycler fast start sybr green i dna mastermix (roche) was prepared and subjected to a qpcr program using the lightcycler (roche). pcr cycling conditions comprised of an initial denaturation step at 94°c for 10 min followed by an amplification program for 40 cycles of 30 s at 95°c, 10 s at 55°c, and 20 s at 72°c with fluorescence acquisition at the end of each extension. the relative expression of each gene is calculated using the comparative δδc t method, using the mock treated sample as calibrator and housekeeping gene gapdh as internal control. the following primer pairs were used: for gapdh forward 5′-gacaactttggtatcttggaa-3′ and reverse 5′-ccaggaaatgag cttgaca-3′; for isg56 forward 5′-tctcag aggagcctggctaag-3′ and reverse 5′-ccacactgtatttggtgtctagg-3′; for isg15 forward 5′-tggtggacaaatcgcacgaa-3′ and reverse 5′-caggcgcagattc atgaac-3′; for rantes forward 5′-ggcacgcctcgctgtcatcc tca-3′ reverse 5′-cttgatgtgggcacggggcagtg-3′; and for ifn-β forward 5′-ctctcctgttgtgcttctccac-3′ and reverse 5′-tagtct cattccagccagtgct-3′. in a previous study, we reported the construction of two recombinant ibv (ribv8b and ribv8ab) expressing sars-cov proteins 8b and 8ab, respectively (le et al., 2007) . to assess the role of sars-cov 8b and 8ab in modulating viral replication, the growth properties and kinetics of the recombinant viruses were characterized and compared to wild type ibv (wtibv). a new recombinant ibv expressing the 8a and 8b in separate orfs (ribv8a/b) was also constructed as a control. vero cells, known to lack the expression of type i ifns, were infected with wild type and recombinant ibv at an moi of~0.5 and harvested at every 4 h over a time course of 20 h for plaque assay to determine virus titers. consistent with our previous report on the impediment of virus replication by 8b expression, recombinant viruses expressing 8b, 8ab and 8a/b replicated at a slightly slower rate, compared to wild type virus during the first 12 h of infection ( fig. 1a & b) . at 16 h post-infection, however, the three recombinant ibvs were able to attain titers comparable to that of wild type virus (fig. 1b) . expression of protein 8b or 8ab could not be detected in cells infected with ribv8a/b using western blot analysis (data not shown), further supporting our previous observation that 8b is not expressed from this construct (le et al., 2007) . taken together, these results demonstrate that the inclusion of 8b and 8ab does not render detectable enhancement effects on the replication and growth of ibv in culture cells. the slightly slower growth rates observed for the recombinant viruses at early time points of the infection cycle may be due to the introduction of extra sequences into the ibv genome. this is consistent with our previous observations that such genetic manipulations may alter the replication of ibv in cells (le et al., 2007; shen et al., 2009) . as expression of proteins 8a, 8b and 8ab did not render growth advantages to ibv in normal cultured cells, these proteins may not have direct functions in viral replication, especially in a heterogeneous genome context. 3.2. expression of 8b and 8ab confers growth and replication advantages to ribv8b and ribv8ab over wtibv and ribv8a/b in the presence of poly (i:c) the ability to subvert the host innate immune response is a critical factor for establishing effective virus replication. to determine if 8b and 8ab may have a role in counteracting the action of ifn, one of the most common and potent host anti-viral defense mechanisms, we examined the relative replication efficacy of ribv8b, ribv8ab and ribv8a/b in the presence of poly (i:c). for this purpose, h1299 cells were infected with wild type or the recombinant ibvs for 8 h prior to mock or poly (i:c) transfection. cells were then further incubated for 10 h before lysates were harvested for analysis of viral protein expression (fig. 2a) . in agreement with our growth kinetics studies earlier, the recombinant viruses replicated to similar levels by 18 h (fig. 2a) in the absence of poly (i:c) treatment although the wild type virus was observed to replicate slightly faster as indicated by a higher abundance of ibv n protein (fig. 2a) . not surprisingly, replication of wild type and the three recombinant viruses were severely suppressed in cells stimulated by poly (i:c) (fig. 2a) , reflecting the sensitivity of coronavirus to interferon intervention. compared to wild type and ribv8a/b, however, ribv8b and ribv8ab were observed to replicate significantly better and express higher levels of n protein in cells stimulated by poly (i:c) (fig. 2a) . using plaque assay, viral titers attained in infected cells exposed to poly (i:c) treatment were compared to their respective mock-treated counterparts. while poly (i:c)-treatment reduced the virus titers of wtibv (from 1.1×10 6 to 2.75×10 5 ) and ribv8a/b (from 1×10 6 to 4×10 4 ) drastically by 87.5% and 96%, respectively, titers of ribv8b (from 8.2×10 5 to 4.1×10 5 ) and ribv8ab (from 7.9×10 5 to 1.98×10 5 ) were reduced by a more modest 50% and 75%, respectively (fig. 2b) . these results suggest that proteins 8b and 8ab may have a functional role in modulating the ifn pathway. as expression of protein 8a (from ribv8a/ b) did not show a similar effect, we hypothesized that this effect may be unique to the 8b region of the two proteins. in view of the central role of irf3 in regulating ifn activation during virus infection, 8b and 8ab with flag epitope-tagged to their ntermini were co-expressed with myc-tagged irf3 (fig. 3a) in cos-7 cells using the vaccinia/t7 expression system (anderson et al., 1996; lim and liu, 2001) for co-immunoprecipitation assays to determine if there is any physical interaction between the proteins. irf3 was precipitated from the cell lysates prepared from cells harvested at 20 h post-transfection using anti-myc antibody-coated agarose beads, followed by western blot analysis with antibodies against the flag epitope. as shown in fig. 3b , proteins 8b and 8ab were consistently pulled down with irf3 in samples where they were co-expressed with irf3. similarly, when the experiment was repeated using anti-flag coated agarose beads, irf3 co-precipitation was detected with both proteins. these results demonstrate that irf3 could physically interact with protein 8b and 8ab. to confirm that this interaction occurs in the context of coronavirus infection, total lysates prepared from cells infected with wtibv, ribv8b and ribv8ab together with mock infected controls were also subjected to immunoprecipitation with either polyclonal antibodies raised against sars-cov 8b or igg controls. immunoprecipitated proteins were then analyzed for the presence of endogenous irf3 using antisera against the protein. the detection of irf3 in cells infected with ribv8b and ribv8ab, but not in mock-and wtibv-infected cells (fig. 3c) indicates that protein 8b and 8ab formed complexes with irf3 during the virus replication process. it was noted that apart from the 55 kda band corresponding to the endogenous monomeric irf3, an additional band with the apparent molecular weight of approximately 85 kda was the number of infectious particles released in the supernatants was quantified using plaque assay in triplicates, and the average number of pfu for each treatment was calculated. the relative amount of virus produced after poly (i:c) treatment was expressed as a percentage of their respective control cells not treated with poly (i:c). error bars showed standard deviation from 3 independent experiments. error bars showed standard deviation from 3 independent experiments. h.h. wong et al. virology 515 (2018) [165] [166] [167] [168] [169] [170] [171] [172] [173] [174] [175] detected ( fig. 3c) in cells infected by ribv-8b. the identity of this band is not certain, but it may represent a modified form of irf3. irf3 is well documented to be heavily targeted for post-translational modifications such as phosphorylation, ubiquitination, sumoylation, and neddylation (bibeau-poirier et al., 2006; hiscott, 2007; kubota et al., 2008; ran et al., 2011) . since proteins 8b and 8ab exhibited comparable efficiency in terms of pulling down irf3, just the 8b region was then used for subsequent pull-down experiments to pinpoint the domains in irf3 essential for the interaction. four deletion constructs of irf3 either with or without a myc-tag at the n-termini were constructed (fig. 3a) and co-expressed with the flag-tagged 8b. co-immunoprecipitation experiments showed that the n-terminal region covering the first 133 residues of irf3 (myc-irf3(1-133)) failed to interact with protein 8b (fig. 3e) , suggesting that the n terminal dna-binding domain (dbd) is dispensable for the interaction. immunoprecipitation with fragments covering residues 134-240 and 134-427, respectively, resulted in effective pulldown of 8b (fig. 3e) , suggesting that the 134-240 region is likely to be important for the interaction. this region spans across several functional domains, including the nuclear export signal (nes), proline-rich region (pro), and the first 47 residues of the irf association domain (iad). interestingly, immunoprecipitation experiments repeated with just the n terminal fragment comprising of only the first 193 residues, thus excluding the 47 residues of iad, abolished the binding (fig. 3d) , indicating that just the nes and pro regions alone were insufficient for the binding. consistently, the full-length irf3 with the n-terminal myctag and the n-terminal region covering the first 375 residues could be efficiently pulled down by protein 8b (fig. 3d) . we next sought to test the effect of 8b and 8ab expression on irf3 activation. cells were transfected with either plasmid encoding flag-8b, flag-8ab or a corresponding control vector, before subjected to poly (i:c) treatment. western blot analysis coupled with native page revealed that the ectopic expression of proteins 8b and 8ab resulted in markedly decreased levels of poly (i:c)-induced irf3 dimerization (fig. 4a) . however, analysis of the same samples by sds-page showed no observable difference in the levels of hyper-phosphorylated irf3 (p-irf3) (appearing as more slowly migrating bands) in cells over-expressing proteins 8b and 8ab, compared to that in the control (fig. 4a) . it was also noted that the total amounts of irf3 were approximately comparable in these transfected cells (fig. 4a) . the concomitant decreased in irf3 dimerization with 8b and 8ab fragments that immunoprecipitated with protein 8b are denoted with (+). b. co-immunoprecipitation of myc-tagged irf3 with flag-tagged 8b and 8ab, respectively. cos7 cells were infected with the recombinant vaccinia/t7 virus at an moi of approximately 5 per cell. after incubation for 1, cells were transfected with pmyc-irf3, pflag-8b, pmyc-irf3+pflag-8b, pflag-8ab and pmyc-irf3+pflag-8ab, respectively. cells were harvested at 18 h posttransfection, lysates prepared, and subjected to immunoprecipitation with either the myc antibodyconjugated agarose beads (top two panels) or the flag-antibody-conjugated beads (bottom two panels). the precipitates were separated on sds-page and analyzed by western blot with either anti-myc (top and bottom panels) or anti-flag (2nd and 3rd panels) antibodies. c. pull down of the endogenous irf3 protein with protein 8b. h1299 cells were infected with wtibv, ribv8b and ribv8ab, respectively. cells were lysed 20 h post-infection and immunoprecipitated with either polyclonal antibodies raised against sars-cov 8b or control igg antibodies. the precipitates were probed with antibodies against the full-length irf3. d. co-immunoprecipitation analysis of the flag-tagged protein 8b co-expressed with the untagged irf3 from 1-193, 1-375 and 1-427 (full-length). h1299 cells were infected with the recombinant vaccinia/t7 virus at an moi of approximately 5 per cell. immunoprecipitation was performed as described above. total lysates and the precipitates were separated on sds-page and analyzed by western blot with either anti-myc or anti-flag antibodies. e. coimmunoprecipitation analysis of the flag-tagged protein 8b co-expressed with the myc-tagged irf3 from 1-133, 134-240 and 134-427. h1299 cells were infected with the recombinant vaccinia/t7 virus at an moi of approximately 5 per cell. immunoprecipitation was performed as described above. total lysates and the precipitates were separated on sds-page and analyzed by western blot with either anti-myc or anti-flag antibodies. expression lead us to examine the impact of such phenomenon on the activation of ifn-β and other known irf3 downstream effectors. for the study on the effect on ifn-β promoter activity, a reporter construct expressing firefly luciferase driven by the ifn-β promoter was cotransfected with either 8b, 8ab or a control plasmid prior to poly (i:c) stimulation. at 16 h post poly (i:c) treatment, lysates were assayed for luciferase activity. relative to cells transfected with control plasmid, poly (i:c)-induced ifn-β promoter activation in cells expressing 8b and 8ab were reduced to 59% and 57% respectively (fig. 4b) . this data mirrored the results obtained from the analysis of endogenous ifn-β transcript levels examined by quantitative real-time rt-pcr where relative levels of ifn-β mrna in poly (i:c)-treated cells expressing 8b and 8ab were reduced to approximately 18-20% (fig. 4c ) of control. similar to the effect on ifn-β expression, the mrna levels of other irf3 downstream target genes (grandvaux et al., 2002) , including isg56, rantes and isg15, were significantly lower in cells over-expressing proteins 8b and 8ab than those of the control. the relative levels of isg56 (fig. 4d) , rantes (fig. 4e) and isg15 (fig. 4f) were reduced to approximately 40-41, 35-40 and 25-30%, respectively, in poly (i:c)treated cells transfected with 8b and 8ab, compared to those in cells transfected with the empty vector. the negative modulation of 8b and 8ab expression on the activation of irf3 was further verified from the analysis of irf3 activation and stability in virus-infected cells shown in fig. 2a . in the absence of poly (i:c) treatment, infection by wild type and all the three recombinant viruses did not induce a detectable level of irf3 dimerization using native page analysis (fig. 5a) . while irf3 dimerization was detected in all infected cells in the presence of poly (i:c) treatment (fig. 5a) , less poly (i:c)-induced irf3 dimers could be detected in cells infected with ribv-8b, compared to those infected by wtibv (fig. 5a) . reduction in fig. 4 . suppression of poly (i:c)-induced irf3 activation by protein 8b and 8ab. a. suppression of irf3 dimerization by proteins 8b and 8ab. huh7 cells were transfected with 4 μg of empty vector, flag-8b and flag8ab, respectively, followed by stimulation with poly(i:c) for 20 h. whole cell lysates were subjected to either native page or sds-page and probed with anti-irf3. tubulin was included as a loading control. the ratio of dimeric irf3 to monomeric irf3 was calculated as the band intensity of monomer divided by the band intensity of dimer. b. suppression of poly (i:c)-induced ifn-β promoter activity by proteins 8b and 8ab. huh7 cells were transfected with control vector pcdna3.1, pcdna-8b and pcdna-8ab, respectively, together with a luciferase reporter construct under the control of ifn-β promoter. at 24 h post-transfection, cells were then further transfected with poly (i:c). at 24 h post-stimulation, cells were lysed and measured for the firefly luciferase activity. prl-tk was also co-transfected to serve as an internal control. data were represented as mean of triplicates from 3 independent experiments. c-f. huh7 cells were transfected with 4 μg of empty vector, flag-8b and flag8ab, respectively, followed by stimulation with poly(i:c) for 20 h. total rna was then extracted for quantitative real-time rt-pcr with specific primers for ifn−β (c), isg56 (d), rantes (e) and isg15 (f). the expression of each gene was expressed relative to their respective control sample transfected with empty vector. data were represented as mean of replicates from 2 independent experiments. gapdh was used as internal control. h.h. wong et al. virology 515 (2018) [165] [166] [167] [168] [169] [170] [171] [172] [173] [174] [175] irf3 dimerization, although to a lesser extent, was also observed in cells infected with ribv8ab (fig. 5a) . interestingly, analysis of the same lysates by denaturing sds-page revealed that hyper-phosphorylated irf3 was invariantly detected in the poly (i:c) treated cells regardless of whether they are mock infected, infected with wild type virus or the recombinant ibvs (fig. 5a) . these observations were in agreement with the data we described in fig. 4a . however, we did observe that the overall level of irf3 expression was significantly reduced in cells infected with ribv8b, both in the presence and absence of poly (i:c) treatment (fig. 5a) . a more moderate reduction of irf3 was also detected in cells infected with ribv8ab (fig. 5a) . when the mrna levels of ifn-β from these samples were analyzed, negligible ifn-β induction was observed in virus-infected cells in the absence of poly (i:c) stimulation (data not shown). among the samples treated with poly (i:c), ifn-β induction in cells infected with ribv8b and ribv8ab infection was suppressed (25% and 75% relative to control wtibv, respectively) (fig. 5b) . taken together, these data suggest that the observed enhanced replication of ribv8b and ribv8ab in the presence of poly (i:c) may be due to the diminished ifn activation owing to the down-regulation of irf3 levels coupled to reduced irf3 dimer formation and ifn-β induction. 6 . degradation of irf3 by 8b in a ubiquitin/proteasome-dependent manner. a. degradation of irf3 by 8b but not by a lysine-knockout mutant 8b. a lysine knockout mutant of ribv, ribv8bm, was created by substituting the three lysine residues (k4, k26 and k81) with arginine. cells were then infected up to 32 h with ribv-8b and ribv8bm, respectively, before analyzing for irf3 expression. the same membrane was also probed with anti-ibv n, sars-cov protein 8b and actin antibodies. the relative amount of irf3 was calculated as the band intensity of the protein divided by the band intensity of actin. b. ubiquitin-dependent degradation of irf3. cells transfected with either myc-tagged ubiquitin alone or co-transfected with flag-tagged 8b, in the presence or absence of mg132, were subjected to immunoprecipitation with antibodies against myc. immunoprecipitates were then probed for pull-down of endogenous irf3 with specific antibodies. the relative amount of irf3 was calculated as the band intensity of the protein divided by the band intensity of actin. h.h. wong et al. virology 515 (2018) 165-175 3.6. degradation of irf3 mediated by proteins 8b and 8ab is ubiquitindependent we had previously demonstrated that 8b binds to both poly-and mono-ubiquitin (le et al., 2007) . to address if the down-regulation of irf3 in ribv8b-infected cells is linked to its ubiquitin-binding activities, lysine knockout mutant of 8b, 8bm was generated by mutating all three lysine residues (k4, k26 and k81) in 8b to arginine. mutation of these lysine residues enhanced the stability of the 8b protein. as shown in fig. 6a , while the mutant 8bm protein was readily detected in ribv8bminfected cells at 20 and 32 h post-infection, protein 8b was not detected under the same conditions (fig. 6a) . this is consistent with previous reports of the instability of 8b protein and that protein 8b could only be detected in ribv8b-infected cells in the presence of proteasome inhibitors (le et al., 2007) . irf3 was down-regulated in cells infected with ribv8b at both 20 and 32 h post-infection (fig. 6a) . infection of cells with ribv-8bm did not result in similar reductions in irf3 protein levels, despite a similar replication efficiency of the two recombinant viruses (fig. 6a) . as mentioned previously, irf3 levels are regulated by ubiquitination during virus infection. however, some viruses exploit this mode of regulation by expressing viral proteins that promote untimely proteasomal degradation of irf3 (z. saira et al., 2009; sen et al., 2009) . to affirm the involvement of the ubiquitin-proteasome pathway in the down-regulation of irf3 observed with 8b, irf3 stability was studied in cells overexpressing protein 8b in the presence of ubiquitin. over-expression of 8b together with ubiquitin resulted in a reduction of detectable levels of irf3 compared to cells expressing ubiquitin alone (fig. 6b) . this degradation was partially suppressed in the presence of proteasome inhibitor mg132 (fig. 6b) . in a previous study, we found that proteasome-mediated rapid degradation of protein 8b could be much more efficiently inhibited by nlvs than did lactacystin (le et al., 2007) . as the supply of nlvs was discontinued, we chose to use mg132, a product shown similarly mild inhibitory effect as lactacystin in the suppression of proteasome-mediated degradation of protein 8b, in this study. finally, to address the observation of the negligible impact exerted by 8b and 8ab on irf3 phosphorylation despite their capacity to suppress irf3 dimer formation, we study the effect of 8b on activated irf3. a construct expressing the phosphomimetic form of irf3 (irf3-5d) was constructed, which contains amino acid substitutions at positions 396, 398, 402, 404, and 405 by the phosphomimetic aspartic acid. irf3-5d undergoes spontaneous dimerization leading to ifn-β induction (grandvaux et al., 2002; lin et al., 1999) . over-expression of proteins 8b and 8ab was able to reduce the irf3-5d-induced ifn-β promoter activity to approximately 58% and 49% to that of control, respectively (fig. 7a ). this suggests that 8b can act on irf3 at step(s) that is downstream of its activation. when protein 8b is co-expressed with irf3-5d, 8b reduces the expression of irf3-5d in a dose-dependent manner (fig. 7b) . for reasons yet to be known, we observed that the expression of a monomeric form of irf3-5d seemed to be more affected by the presence of 8b than its homodimeric counterpart. similar to results presented earlier with endogenous irf3, irf3-5d levels was partially rescued with the addition of mg132, indicating the role of the ubiquitin-proteasome pathway (fig. 7c) . the sars-cov genome encodes an exceptionally high number of accessory genes that bear little resemblance to other known coronavirus accessory proteins. it is believed that while these unique proteins may not participate directly in viral replication, they possess biological functions that may enhance sars-cov pathogenesis in cells. by demonstrating that recombinant ibv expressing 8b or 8ab replicates more efficiently in the background of ifn activation, data presented in this study demonstrate that expression of sars-cov orf8b and orf8ab contributes positively to viral pathogenesis. as the expression of 8b helped to overcome partially the potent inhibitory effect of ifn induction on coronavirus replication in cell culture, it suggests that 8b and 8ab are novel ifn antagonists. orf8 failed to show up in the screen for interferon antagonist in a previous study , likely due to the incomplete inhibition of ifn activation exhibited by orf8. other reasons could include the low expression of 8b in their system owing to the inherent instability of the protein (le et al., 2007) , and/or that the antagonistic activity of 8b is not apparent in their ndv model because the ifn antagonistic activity of this protein may be specific to coronavirus infection due to the requirement of other viral proteins. however, this is control vector pcdna3.1, pcdna-8b or pcdna-8ab together with a luciferase reporter construct under the control of the ifn-β promoter were transfected into huh7 cells. at 24 h post-transfection, cells were transfected with pirf3-5d and co-transfected with prl-tk to serve as an internal control. at 24 h post-stimulation, cells were lysed and measured for the firefly luciferase activity. data were represented as mean of triplicates from 3 independent experiments. b. cells were co-transfected with 200 ng of pxj40-irf3-5d and either 0, 50, 100, 200 ng of pkto-8b. the total dna transfected was made up to 400 ng using empty pkto vector. lysates harvested 20 h post-transfection were either subjected to native or sds page and probed with anti-irf3 antibodies. actin was also probed to serve as a loading control. c. irf3-5d was co-transfected pkto-8b. 20 h post transfection, co-transfected cells was either left untreated or treated with mg132 2 h prior to harvest. lysates were then probed for irf3, 8b and actin expression. the relative amount of irf3-5d was calculated as the band intensity of the protein divided by the band intensity of actin. h.h. wong et al. virology 515 (2018) [165] [166] [167] [168] [169] [170] [171] [172] [173] [174] [175] quite unlikely because the inhibitory effect could also be observed with ectopic expression of 8b and 8ab in the absence of viral replication. the other significant finding from this study is that 8b and 8ab directly physically interact with irf3 and that 8b-irf3 interaction involves part of the iad domain that is responsible for the formation of irf3 homodimers. over-expression of 8b and 8ab appears to have a more profound effect on irf3 dimerization than on its phosphorylation status. although it is still unclear whether this is a consequence of assay insensitivity or that the inhibitory actions of 8b and 8ab bypass the step of phosphorylation and targets specifically on the event of irf3 dimerization, we are inclined to believe that it is the latter owing to the ability of 8b to suppress ifn-β induced by constitutively active phosphomimetic irf3-5d. while disruption to the dimerization event as a result of direct steric interference brought about by 8b interaction at the iad domain is an attractive model, the exact mechanisms can only be ascertained through more in-depth studies such as structural analysis. we previously reported that the 8b region of sars-cov proteins 8b and 8ab consists of domains that allow for ubiquitin binding, ubiquitination and glycosylation (le et al., 2007) . based on these finding, we proposed that the 8b region may mediate the binding of 8b and 8ab to ubiquitinated cellular proteins, such as p53 and iκbα (le et al., 2007) . here we showed that irf3, another protein regulated by ubiquitination (siu et al., 2009; spiegel et al., 2004) , interacts with proteins 8b and 8ab, suggesting that the ubiquitin-binding properties of 8b region could allow them to interact with multiple cellular proteins. it would be interesting to find out what other cellular targets bind to 8b and 8ab and whether they have a regulatory role during sars-cov infection. furthermore, expression of 8b and 8ab appears to regulate the stability and function of irf3 by promoting degradation of irf3 in a ubiquitin/ proteasome-dependent manner. several viral proteins have been reported to cause proteasomal degradation of irf3 (z. saira et al., 2009; sen et al., 2009 ). irf3 degradation is typically triggered post-infection, when viral infection-induced irf3 activation leads to the ubiquitination of the protein targeting it for proteasomal degradation liu et al., 2005) . this serves to regulate type i ifn production as excessive ifn is detrimental to the cells. in this study, we observed irf3 degradation even in the absence of strong irf3 activation in cells infected with ribv8b and ribv8ab, suggesting that the 8b-and 8ab-mediated irf3 degradation is not the result of a typical negative feedback mechanism to bring the ifn level back to the physiological level at the end of viral infection as observed with other virus infection, but may be an active step undertaken by the virus to limit irf3 activation during its course of replication. at this stage, while we confirm the involvement of the proteasome, we do not know if other cellular factors are also recruited by 8b to mediate irf3 degradation. cellular factors, such as peptidyl-prolylisomerase pin1 (saitoh et al., 2006) , ro52/trim21 (pin + ro52) and e3 ubiquitin ligase rbcc protein interacting with pkc1 (rbck1) (zhang et al., 2008) , were identified to participate in the negative regulation of irf3 by targeting it for ubiquitination. additional experiments such as mass spectrometry could perhaps help elucidate if 8b interaction with irf3 also involves any of these reported proteins. considering the fact that 8b expression is unstable and that it is only expressed during the late stages of sars-cov infection, it seems counter-intuitive why the virus would express such a late stage ifn antagonist as 8b. this is especially true since sars-cov has a strong inhibitory effect on ifn induction plausibly owing to the expression of multiple viral proteins that antagonize the pathway in myriad ways during the earlier stages of infection. a possible explanation may come from a study carried out by spiegel and coworkers who reported that while nuclear translocation of irf3 remains unabated by sars-cov infection during the early stages of infection at 8 h post-infection, irf3 activation is specifically blocked during later stages of sars-cov infection at 16 h post-infection (spiegel et al., 2005) . this coincides with the late expression of 8b during sars-cov infection (keng et al., 2006) . hence, we hypothesize that the growth advantage conferred by the expression of 8b in recombinant ibv was due to the role of 8b in latestage viral pathogenesis, when the expression of 8b aids in dampening the activation of irf3 that may occur during the later phase of infection. nevertheless, we do not rule out the possibility that 8b and 8ab may regulate irf3 via mechanisms independent of its ubiquitin-binding activity as it has also been shown to down-regulate e protein via a ubiquitin-independent proteasomal pathway (keng et al., 2006 (keng et al., , 2011 . using an infectious clone system based on the urbani strain of sars-cov, the 29-nt deletion is inserted to fuse orf8a/b back into the single orf8. compared with the wild type control, this recombinant virus replicates similarly in both cell culture and in the murine model (yount et al., 2005) . theoretically, only protein 8ab is produced in cells infected with this virus, whereas both proteins 8a and 8b are produced in the wild type control. because both 8b and 8ab can antagonize ifn signaling by mediating irf3 down-regulation, it is no surprise that the recombinant virus replicates similarly as the wild type control. deletion of accessory proteins 6, 7, 7b, 8a, 8b and 9b altogether in recombinant virus rsars-cov-δ[6-9b] (dediego et al., 2008) showed that the recombinant virus replicates as well as wild type control in both cell culture and transgenic mice expressing the sars-cov receptor human angiotensin converting enzyme-2 (hace-2). since ifn antagonist function is also possessed by other sars-cov proteins (such as nsp1, plpro, m and n), it is possible that loss of functional 8b in rsars-cov-δ[6-9b] is compensated, and thus the recombinant virus is not attenuated in vivo. further studies using recombinant sars-cov with only 8b or 8ab deleted should be performed in cell culture and in appropriate animal models, to better characterize the detailed mechanisms of their involvement in modulating viral replication and pathogenesis. finally, poly (i:c) is known to be able to induce both ifn and a subset of ifn-stimulated genes through activation of 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viruses related to the sars coronavirus from animals in southern china targeting irfs by ubiquitination: regulating antiviral responses the e3 ubiquitin ligase ro52 negatively regulates ifn-beta production postpathogen recognition by polyubiquitin-mediated degradation of irf3 triggering the innate antiviral response through irf-3 activation identification of the mechanisms causing reversion to virulence in an attenuated sars-cov for the design of a genetically stable vaccine severe acute respiratory syndrome coronavirus nsp1 protein suppresses host gene expression by promoting host mrna degradation sars coronavirus 8b reduces viral replication by down-regulating e via an ubiquitin-independent proteasome pathway the human severe acute respiratory syndrome coronavirus (sars-cov) 8b protein is distinct from its counterpart in animal sars-cov and down-regulates the expression of the envelope protein in infected cells severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists virus infection triggers sumoylation of irf3 and irf7, leading to the negative regulation of type i interferon gene expression pathogen recognition by innate receptors toll-like receptors and innate immunity pathogen recognition in the innate immune response severe acute respiratory syndrome (sars) coronavirus orf8 protein is acquired from sars-related coronavirus from greater horseshoe bats through recombination expression and functional characterization of the putative protein 8b of the severe acute respiratory syndrome-associated coronavirus expression, post-translational modification and biochemical characterization of proteins encoded by subgenomic mrna8 of the severe acute respiratory syndrome coronavirus sumoylation of the nucleocapsid protein of severe acute respiratory syndrome coronavirus the missing link in coronavirus assembly retention of the avian coronavirus infectious bronchitis virus envelope protein in the pre-golgi compartments and physical interaction between the envelope and membrane proteins human coronaviruses: a review of virushost interactions virus-dependent phosphorylation of the irf-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation structural and functional analysis of interferon regulatory factor 3: localization of the transactivation and autoinhibitory domains internal entry of ribosomes on a tricistronic mrna encoded by infectious bronchitis virus association of the infectious bronchitis virus 3c protein with the virion envelope accessory proteins of sars-cov and other coronaviruses immunity by ubiquitylation: a reversible process of modification distinct rig-i and mda5 signaling by rna viruses in innate immunity sars-cov nucleocapsid protein antagonizes ifn-β response by targeting initial step of ifn-β induction pathway, and its c-terminal region is critical for the antagonism middle east respiratory syndrome coronavirus m protein suppresses type i interferon expression through the inhibition of tbk1-dependent phosphorylation of irf3 the sars coronavirus papain like protease can inhibit irf3 at a post activation step that requires deubiquitination activity the sars coronavirus 3a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type 1 interferon receptor severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type i interferon, in infected cells sars coronavirus accessory proteins regulation of antiviral innate immune responses by rig-i family of rna helicases the 29-nucleotide deletion present in human but not in animal severe acute respiratory syndrome coronaviruses disrupts the functional expression of open reading frame 8 senp2 negatively regulates cellular antiviral response by desumoylating irf3 and conditioning it for ubiquitination and degradation the infected cell protein 0 encoded by bovine herpesvirus 1 (bicp0) associates with interferon regulatory factor 7 and consequently inhibits beta interferon promoter activity negative regulation of interferon-regulatory factor 3-dependent innate antiviral response by the prolyl isomerase pin1 irf3 inhibition by rotavirus nsp1 is host cell and virus strain dependent but independent of nsp1 proteasomal degradation towards construction of viral vectors based on avian coronavirus infectious bronchitis virus for gene delivery and vaccine development severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf3.tank.tbk1/ikkepsilon complex middle east respiratory syndrome coronavirus 4a protein is a double-stranded rna-binding protein that suppresses pactinduced activation of rig-i and mda5 in the innate antiviral response inhibition of beta interferon induction by severe acute respiratory syndrome coronavirus suggests a two-step model for activation of interferon regulatory factor 3 the antiviral effect of interferon-beta against sars-coronavirus is not mediated by mxa protein type i interferons in host defense direct involvement of crebbinding protein/p300 in sequence-specific dna binding of virus-activated interferon regulatory factor-3 holocomplex the 8ab protein of sars-cov is a luminal er membrane-associated protein and induces the activation of atf6 amino acid residues critical for rna-binding in the n-terminal domain of the nucleocapsid protein are essential determinants for the infectivity of coronavirus in cultured cells irf family of transcription factors as regulators of host defense virus induction of human ifn beta gene expression requires the assembly of an enhanceosome group 2 coronaviruses prevent immediate early interferon induction by protection of viral rna from host cell recognition plp2 of mouse hepatitis virus a59 (mhv-a59) targets tbk1 to negatively regulate cellular type i interferon signaling pathway severe acute respiratory syndrome coronavirus evades antiviral signaling: role of nsp1 and rational design of an attenuated strain viral evasion of the interferon system: old viruses, new tricks middle east respiratory syndrome coronavirus orf4b protein inhibits type i interferon production through both cytoplasmic and nuclear targets severe acute respiratory syndrome coronavirus groupspecific open reading frames encode nonessential functions for replication in cell cultures and mice negative feedback regulation of cellular antiviral signaling by rbck1-mediated degradation of irf3 suppression of type i interferon production by porcine epidemic diarrhea virus and degradation of creb-binding protein by nsp1 potent inhibition of sars-associated coronavirus (scov) infection and replication by type i interferons (ifn-alpha/beta) but not by type ii interferon (ifn-gamma) recent progress in studies of arterivirus-and coronavirus-host interactions this work was partially supported by an academic research fund (acrf) tier 2 grant (acr47/14), ministry of education, singapore and by guangdong province key laboratory of microbial signals and disease control grants msdc-2017-05 and msdc-2017-06, guangdong, people's republic of china. key: cord-286060-92lazxd7 authors: stohlman, stephen a.; fleming, john o.; patton, chris d.; lai, michael m.c. title: synthesis and subcellular localization of the murine coronavirus nucleocapsid protein date: 1983-10-30 journal: virology doi: 10.1016/0042-6822(83)90106-x sha: doc_id: 286060 cord_uid: 92lazxd7 abstract the synthesis and processing of the nucleocapsid protein (pp60) of the jhm strain of murine coronaviruses were examined. pulse-chase experiments showed that pp60 was synthesized initially as a protein of approximately 57,000 in molecular weight (p57). immunoprecipitation using mouse anti-jhmv antiserum indicated that p57 was virus specific. immunoprecipitation with monoclonal antibodies specific for pp60 showed that p57 was antigenically related to pp60 and was not phosphorylated, while the intracellular protein that comigrated with the virion nucleocapsid protein, pp60, was phosphorylated. the p57 was found exclusively in the cytosol while the majority of pp60 was associated with the membrane fraction but pp60 was not an integral membrane protein. coronaviruses comprise a group of enveloped viruses which cause a variety of diseases, usually confined to their natural hosts (2.2). these viruses contain an rna genome of positive polarity that is approximately 5.4 x lo6 da in size (5). intracellular rna replication is accomplished by a virion-induced polymerase (2) which synthesizes a full-length negative strand (8) . seven distinct mrnas are then synthesized from a single species of negative-strand template and comprise a nested set with common 3' ends (7', 8, 16) . purified coronavirus particles of the a59 (mhv-a59) strain contain three structural proteins, i.e., gp90/180, gp25, and pp60, while the jhm strain (jhmv) may be composed of four, containing gp65 in addition (14, zo) . we have further shown that mhv contains only a single phosphorylated protein, the nucleocapsid protein (~~60). it is phosphorylated only on serine residues (19), presumably by a virion-associated protein kinase (13) . sturman et al. (21) have also shown that pp60 interacts with the smaller envelope glycoprotein (gp25) and have postulated that this ini author to whom correspondence should be addressed. teraction may be a regulatory mechanism operable during virus maturation. studies on the intracellular synthesis of mhv proteins by pulse-chase experiments have shown that the nucleocapsid protein, pp60, is a primary gene product (3, 12) . in vitro translation indicated that the virusspecific messenger rna no. 7 (1.8 x lo5 da) codes for a protein with a tryptic peptide map identical, or closely related, to that of virion pp60 (9, 11, 14, 15) . however, proteins of lower molecular weight are also synthesized, occasionally in excess of the virion pp60 (9, 12). it has been suggested that these proteins of lower molecular weight might be degradation products of pp60, which are generated during virus replication or during in vitro translation (11, 12) . in addition, two-dimensional nonequilibrium isoelectric focusing of infected cell lysates indicated that the nucleocapsid protein was composed of multiple heterogeneously charged species which are homogeneous in size (1). in examining the kinetics of the appearance of jhmv proteins in infected dbt cells, we noted that the region of the gel which contains the pp60 protein also contained another protein of slightly lower molecular weight (fig. 1a) . to understand the possible relationship of this protein were included as controls. lysates were analyzed on a 6-15% linear gradient gel. (designated ~57) with viral structural pro-d and 25 &i/ml of [35s]methionine (new teins, we performed kinetic studies of the england nuclear, boston). the cells were synthesis of the intracellular viral-specisolubilized in buffer consisting of 10 mm fied proteins. confluent monolayers were tris-hcl, ph 8.8, 2% sds, 4 m urea, and infected with the jhmv strain of mouse 2% 2-mercaptoethanol and heated at 56" hepatitis virus at 37" for 1 hr with an m.o.i. for 2 min. electrophoresis of equal of approximately 1.0. following removal amounts of radioactivity from infected of the inoculum, dmem containing 1 pg/ cells lysed at different time points after ml of actinomycin d was added to each infection indicated that ~57 was detectable plate. at 5 hr postinfection, the dmem throughout infection, although its ratio to was replaced with methionine-free dmem pp60 decreases as infection proceeds (fig. (mfdmem) containing 1 pg/ml actino-1a). to further examine the relationship mycin d. after 15min incubation at 37", of these two proteins, we carried out pulsethe mfdmem was replaced with 2.0 ml chase experiments early in infection (5-6 mfdmem containing 1 pg/ml actinomycin hr) to minimize possible protein degra-dation. when infected cells were pulse-labeled with [%]methionine for 2 min and then chased with excess unlabeled methionine (20 m&q for various lengths of time, only ~57 was detected within the pulse interval (fig. 1b) . within the first lo-min chase, pp60 became detectable. this is faster than the processing of p23 into gp25 (14) , since the gp25 was not detected until 20 min into the chase period (fig. 1b) . as the chase was extended, the amount of p57 decreased while pp60 increased, suggesting that ~57 is the precursor protein to pp60. the precursor-product relationship between ~57 and pp60 was further tested with hyperimmune serum and monoclonal antibodies specific for jhmv. these monoclonal antibodies recognize at least four different antigenic determinants on pp60 (fleming, j. o., in press). as shown in fig. 2a , hyperimmune serum precipitates both pp60 and ~57 suggesting that ~57 is indeed a viral protein ( fig. 2a) . also, all of the monoclonal antibodies precipitated pp60. two of these monoclonal antibodies, 53.13 and 53.14, also precipitated ~57. this result shows that pp60 shares antigenic determinants with ~57, but pp60 also contains additional antigenic determinants. to confirm that the intracellular form of pp60 is phosphorylated, and to determine whether ~57 is also phosphorylated, immunoprecipitates from [32p]orthophosphate-labeled cell lysates were analyzed. as can be seen in fig. 2b , both the monoclonal antibodies 53.13 and 53.14, which precipitated both pp60 and ~57 ( fig. 2a) , precipitated only one 32p-labeled protein which comigrated with pp60. no %p-labeled ~57 was detected. these results and those obtained by pulsechase analysis suggest that ~5'7 is not phosphorylated and that phosphate is added during the processing of ~57 into ~~60. to gain some insight into the maturation pathways of pp60, we examined the subcellular localization of ~57 and pp60. infected cells were labeled with [?s]methionine, suspended in rs buffer and lysed by dounce homogenization (18). the lysates were separated into soluble (sloo) and particulate (ploo) fractions by centrifugation at 100,000 g for 1 hr. the sloo phase contained ~5'7 and pp60, while the ploo membrane fraction contained only pp60 (fig. 3) . this result suggests that ~57 is present exclusively in cytosol while pp60 might also be associated with cellular membranes. to rule out the possibility that this apparent membranous association is due to contamination from polysomes or nonspecific association with membranes, the ploo fraction was treated with 100 mm edta to disassociate pp60 from ribosomes and then with 2 m nacl to disassociate it from membranes. neither of these treatments released any detectable pp60 from the ploo fraction (data not shown), suggesting the possibility that pp60 was an integral membrane protein, to rigorously examine this possibility, the ploo fraction was treated with alkali which removes membrane-associated proteins but not the integral membrane proteins (4, i?'). alkali treatment at ph 12, but not ph 10 removed pp60 from the ploo fraction and released it into the sloo fraction (fig. 3 ). in contrast, neither gp25 nor ~120, which are integral membrane proteins (14) , were affected by this treatment, indicating that pp60 is strongly associated with, but not integrated into, the host cell membranes. these results suggest that the nucleocapsid protein, pp60, of murine coronaviruses is synthesized first as a nonphosphorylated precursor protein, ~57, which undergoes phosphorylation immediately after synthesis. this conversion occurs rapidly, since the synthesis of ~57 in the absence of pp60 could only be detected following short (2-5 min) pulses, and large quantities of pp60 could be found after chase intervals of as short as 5 min. in fig. 1a . cells were swollen in rs buffer (10 m&f nacl, 1.5 m&f mgclz, 10 m&f tris-hcl, ph 7.4) and lysed by dounce homogenization. the lysate was centrifuged at 100,000 g for 1 br and the soluble (sloo) and particulate (ploo) fractions were analyzed. the ploo fraction was resuspended in either rs buffer, or treated for 5 min at ph 10 or 12. membranes were centrifuged again at 100,000 g for 1 hr and the supernatant and pellets examined for pp66 by electrophoresis on 6-15s linear gradient gels. lane 1, control lysate; lanes 2 and 3, rs buffer supernatant and pellet; lanes 4 and 5, supernatant and pellet following treatment at ph 10; lanes 6 and 7, supernatant and pellet following treatment at ph 12; lane 8, control jhm lysate. addition, using 9 labeling, we could detect small amounts of phosphorylated pp60 after a 5-min pulse (data not shown). the rapidity of the phosphorylation explains the previous failure to appreciate the precursor-product relationship of these two proteins. it should be noted that, in different in vitro translation systems carried out in different laboratories, the primary translation product of the mrna no. '7 of mhv obtained from infected cells was found to be either p60 or ~57 (9, 12) . it is not clear if this discrepancy is due to the possibility that p60 and ~57 were not electrophoretically separated in some studies, or whether some in vitro translation systems allowed phosphorylation of the primary gene product to take place. from the results presented here, it is more likely that p57 is the nonphosphorylated primary translation product of mrna no. 7. the conversion of ~57 to pp60 probably involves only phosphorylation, since both proteins could be immunoprecipitated by anti-jhmv serum and by two anti-jhmv monoclonal antibodies specific for the pp60 of the purified virion. the observation that most of the monoclonal antibodies did not precipitate ~57, but only pp60, suggests that phosphorylation apparently induces a drastic conformational change. this conformational change might be responsible for some regulatory functions, such as (1) controlling the rate of transcription, (2) the switching of rna transcription to replication, or (3) influencing the association of this protein with the virion genomic rna or the cellular membranes. it is not clear whether there is heterogeneity in the degree of phosphorylation of pp60. however, the finding that ~57 was not completely converted to pp60 even after prolonged chase (fig. 1b) suggests that some ~57 might not be phosphorylated under certain conditions. the most unexpected finding in these studies is the close association of pp60 with cellular membranes. this protein is synthesized on free polysomes (io), consistent with the current finding that ~57 is present exclusively in the soluble fraction of the cytoplasm (fig. 3) . these fractions also contain pp60, suggesting that phosphorylation probably takes place in the cytosol. the majority of the pp60 is associated with the cellular membranes. however, pp60 could be removed from the membrane by harsh treatment with alkali, which removes only nonintegral membrane pro-teins (4, l7), while gp23 and gp90/180, which are synthesized and processed on the cellular membranes (io), remained associated with the membranes in the ploo fraction (fig. 3) . these observations indicated that pp60 is closely associated with, but not integrated into host cell membranes. the nature of this association is very intriguing. it has been shown that pp60 is linked to gp25 in mature viral particles (21). there may be a similar interaction between these two proteins in the infected cells, which could serve as the focal points for virus maturation. it raises an exciting possibility that phosphorylation of pp60 might influence this interaction and thereby influence the virus maturation process. such an idea is presently being examined in our laboratories. top. microbiol zmmwwl we wish to thank todd kennel1 and gabriele olivka for excellent technical assistance and raymond mitchell and alisa young for editorial assistance in the manuscript preparation.this work was supported in part by grant pcm-4567 from the national science foundation, by public health service research grants ai19244 and ns18146, and grant rg 1449 from the national multiple sclerosis society. key: cord-270586-ohs8z91m authors: ballesteros, m. l.; sánchez, c. m.; enjuanes, l. title: two amino acid changes at the n-terminus of transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism date: 1997-01-20 journal: virology doi: 10.1006/viro.1996.8344 sha: doc_id: 270586 cord_uid: ohs8z91m abstract to study the molecular basis of tgev tropism, a collection of recombinants between the pur46-mad strain of transmissible gastroenteritis coronavirus (tgev) infecting the enteric and respiratory tracts and the ptv strain, which only infects the respiratory tract, was generated. the recombinant isolation frequency was about 10−9recombinants per nucleotide and was 3.7-fold higher at the 5′-end of the s gene than in other areas of the genome. thirty recombinants were plaque purified and characterized phenotypically and genetically. all recombinant viruses had a single crossover and had inherited the 5′and 3′-halves of their genome from the enteric and respiratory parents, respectively. recombinant viruses were classified into three groups, named 1 to 3, according to the location of the crossover. group 1 recombinants had the crossover in the s gene, while in groups 2 and 3 the crossovers were located in orf1b and orf1a, respectively. the tropism of the recombinants was studied. recombinants of group 1 had enteric and respiratory tropism, while group 2 recombinants infected the respiratory, but not the enteric, tract. viruses of both groups differed by two nucleotide changes at positions 214 and 655. both changes may be in principle responsible for the loss of enteric tropism but only the change in nucleotide 655 was specifically found in the respiratory isolates and most likely this single nucleotide change, which leads to a substitution in amino acid 219 of the s protein, was responsible for the loss of enteric tropism in the closely related pur-46 isolates. the available data indicate that in order to infect enteric tract cells with tgev, two different domains of the s protein, mapping between amino acids 522 and 744 and around amino acid 219, respectively, are involved. the first domain binds to porcine aminopeptidase n, the cellular receptor for tgev. in the other domain maps a second factor of undefined nature but which may be the binding site for a coreceptor essential for the enteric tropism of tgev. to amino acids 522 to 744 of the spike protein were able to efficiently recognize the papn. transmissible gastroenteritis virus (tgev) is a mem-since the s protein is responsible for the virus binding ber of the coronaviridae family (cavanagh et al., 1994, to the cell, it is expected that this protein would play an enjuanes and van der zeijst, 1995; siddell, 1995) . tgev essential role in the control of the dominant tropism of replicates in both the villus epithelial cells of the small tgev. accordingly, there are data suggesting a correlaintestine and in lung cells of newborn piglets, resulting tion between tropism and s protein structure. porcine in a mortality of nearly 100% (saif and wesley, 1992) . respiratory coronaviruses (prcvs) have been originated frequently these tgev strains are referred to as enteric, independently in europe (callebaut et al., 1988 ; pensaert as opposed to the respiratory strains which do not infect et al., 1986; sá nchez et al., 1992) and in north america the enteric tract. coronaviruses attach to host cells (vaughn et al., 1994; wesley et al., 1991 wesley et al., , 1990b ) from through the spike (s) glycoprotein (cavanagh et al., 1986; enteric isolates (enjuanes and van der zeijst, 1995; sá n-holmes et al., 1989; sturman and holmes, 1983; suñé et chez et al., 1992) . prcvs replicate to high titers only in al ., 1990) , and tgev entry into swine testis (st) cells the respiratory tract (cox et al., 1990) and have a large is mediated through interactions between the virus s deletion at the 5 end of the spike gene, in positions glycoprotein and the porcine aminopeptidase n (papn) ranging from nucleotides (nt) 45 to 745 (enjuanes and which serves as the cellular receptor (delmas et al., van der zeijst, 1995; sá nchez et al., 1992; vaughn et al., 1992) . the s glycoprotein domain recognized by the cel-1994; wesley et al., 1991) . since this deletion is present lular receptor on st cells is thought to be located spain all independently derived prcvs it may be responsible tially close to the antigenic sites a and d (suñé et al., for their loss of enteric tropism. 1990). in fact, recent studies (godet et al., 1994) showed however, it can not be excluded that other viral genes, that baculovirus-expressed polypeptides corresponding apart from the s gene, could be involved in the determination of the tropism of tgev. in fact, changes in orf3a have been associated with the loss of enteric tropism. nonical sequence cuaaac required for the leader auer et jimé nez et al., 1986) . the clone obtained was named ptv-ts-dmar1c.c12-1d.e7. the ptv-ts and primed transcription by the introduction of deletions and point mutations. these mutations lead to the lack of ptv-ts-dmar virus strains were grown at the permissive temperature (34њ). orf3a expression (britton et al., 1990; enjuanes and van der zeijst, 1995; laude et al., 1993; rasschaert et al., virus neutralization, temperature inactivation, and 1990; wesley et al., 1990a) . purification interestingly, the purdue-type virus (ptv), which displays respiratory tropism, has an s gene with an identical the procedure for virus neutralization has been desize to that of the enteric isolates. the ptv s gene was scribed (correa et al., 1988; suñé et al., 1990) . the neusequenced and compared with the homologous setralization index (ni) was defined as the log of the ratio quence of several enteric isolates. only three nucleotide of the plaque-forming units (pfu) after incubating the differences, not observed in enteric isolates, were noted virus in the presence of medium or the indicated mab. to and all introduced amino acid substitutions. two of these analyze virus inactivation by temperature, viruses were changes were located at nucleotides 214 and 655 within grown at both the permissive (34њ) and nonpermissive the area deleted in prcvs, while the other was outside, (39њ) temperatures. the temperature inactivation index at nucleotide 2098 (sá nchez et al., 1992) . the nucleotide (tii) was calculated as the log of the ratio of the pfu change at position 214 was also present in several enafter growing the virus at 34њ or 39њ. teric isolates. these data lead us to propose that alter-to partially purify tgev, st cells were grown in 500ations in the s gene around residue 655 could affect cm 2 roller bottles and infected with a multiplicity of infecenteric tropism (sá nchez et al., 1992) . tion (m.o.i.) of 10 pfu/cell. supernatant was harvested in order to analyze the role of different viral genes in 48 hr postinfection (h.p.i.) and clarified by centrifugation tropism, we have isolated recombinant tgevs by crossin a sorvall gsa rotor for 20 min at 6000 rpm. virions ing the enteric pur46 strain and the respiratory strain were concentrated by centrifugation at 25000 rpm at 4њ ptv-ts-dmar, a temperature-sensitive mutant (ts) resisin a kontron tst28.18 rotor for 2 hr through a 31% (w/v) tant to neutralization by monoclonal antibodies specific sucrose cushion. to clear the virus from the remaining for two different antigenic subsites (dmar). analysis of sucrose, the pellet was resuspended in ten (10 mm the tropism of the recombinant isolates demonstrates tris-hcl, ph 7.4, 1 mm edta, 1 m nacl) and sedimented that two changes at nucleotides 214 and 655 of the spike by centrifugation under the same conditions. the viral gene, leading to aspartic acid to asparagine and to alapellet was resuspended in 500 ml of tne (10 mm trisnine to serine amino acid changes, respectively, were hcl, ph 7.4, 100 mm nacl, 1 mm edta). associated to the loss of enteric tropism in the ptv isolate. the binding of a large panel of mabs to purified virus cells and viruses was performed by ria as previously (correa et al., 1988; sá nchez et al., 1990) using optimum amounts of virus viruses were grown in swine testis (st) cells (mcclur-(0.5 mg of protein per well). kin and norman, 1966). the virus strain pur46-mad-cc120 has been described (sá nchez et al., 1992) . the rna isolation purdue virus strain ptv was previously named neb72 (sá nchez et al., 1992) ; however, due to sequence similar-genomic rna was extracted from partially purified virus as described previously (mendez et al., 1996) . briefly, ity to the pur46 strain, its name was changed to ptv (purdue-type virus). a ts mutant derived from ptv was st cells from 10 roller bottles (500 cm 2 ) were infected with a m.o.i. of 5. medium was harvested at 22 h.p.i. kindly provided by m. welter (ambico). this ts mutant was obtained after 5-fluorouridine mutagenesis using a virions were partially purified as described above. the viral pellet was dissociated by resuspending in 500 ml previously described procedure (robb et al., 1979) and three cycles of plaque purification. the ts mutant growth of tne containing 2% sds and digested with 50 ng of proteinase k (boehringer mannheim) for 30 min at room was reduced ú10 3 -fold at the restrictive temperature (39њ) and showed a reduced capacity for rna synthesis. temperature. rna was extracted twice with phenol-chloroform and precipitated with ethanol. two neutralizing monoclonal antibodies (mabs), 1c.c12 and 1d.e7, that are specific for the antigenic subsites rna from tgev-infected st cells was obtained as described previously (mckittrick et al., 1993) . briefly, st aa and ab, respectively (correa et al., 1988) , were used to select the neutralization escape mutant (double mab-cells, grown in 8-cm 2 wells, were infected with tgev at a m.o.i. of 5. at 22 h.p.i., the cells were resuspended resistant mutant, dmar) using the ptv-ts strain. the procedure used to obtain the dmar mutant was identical to in 400 ml of phosphate-buffered saline (pbs) and were incubated for 10 min on ice with 40 ml of 2 mm vanadyl the one previously described (correa et al., 1988; gebabsence of neutralizing mabs. a significant proportion of virions resulting from this infection should have only the spike protein encoded by one virus genome. the super-m9. the origin of the nucleotides in the recombinant natant was harvested at 12 h.p.i., and neutralization with viruses, in the positions indicated by these molecular mabs 1c.c12 and 1d.e7 was performed to select recommarkers, were determined by sequencing rt-pcr-debinant viruses. potential recombinants were plaque isorived cdna fragments using the fmol dna sequencing lated at restrictive temperature in the presence of the two system (promega). the primers used to sequence the neutralizing mabs used in the selection. the surviving genetic markers m1-m9 are described in table 1 . isolates were phenotypically characterized by calculating their neutralization and temperature inactivation indices. virus tropism analysis in parallel, independent st cell cultures were infected with each of the two parental viruses and the same re-viral tropism was determined in nih miniswine (luncombinant selection procedure was attempted. ney et al., 1986; sachs et al., 1976) or in piglets derived from crossing belgium landrace and large white swine. two-to three-day-old conventional (i.e., non-colostrumdeprived) piglets were used. piglets were obtained from to identify nucleotide differences between the two pasows that were seronegative for tgev neutralizing antirental viruses, cdna fragments covering different rebodies by ria. inbred and outbred animals were oronagions of the genome were synthesized by rt-pcr. sally and intragastrically inoculated with doses of 5 1 these regions included the first 1 kb from the 5-end of 10 7 and 5 1 10 8 pfu, respectively, in a final volume of the genome, orf1 nucleotides 12208 to 20363, the first 2 ml of pbs supplemented with 2% of fetal calf serum. 5 2.3 kb of the s gene, and the most 4.3 kb 3 end of ptv-groups of piglets inoculated with the same virus were ts-dmar. these cdnas were subcloned into pbluescript grouped and housed in isolation chambers located in a (sk 0 ) (stratagene) or pgem-t (promega) vectors. plas-p3 level containment facility at 18њ to 20њ. animals were mid dna was purified using the flexiprep kit (pharmacia) fed three times per day with 30 ml of milk formula for and sequenced with sequenase 2.0 (usb). sequence newborns (nidina 1-nestlé ). virus titers at 1, 2, 3, and 4 data were compiled using the university of wisconsin days postinoculation were determined in tissue extracts genetic computer group (uwgcg) sequence analysis from jejunum and ileum and lungs. tissues were homogsoftware package and compared to previously published enized at 4њ using a tissue homogenizer pro-200 (pro-pur46 virus strains (eleouet et al., 1995; kapke and scientific) . lungs and jejunum and ileum extracts were brian, 1986; mendez et al., 1996; rasschaert et al., 1987; obtained by homogenizing the whole organs in order to sá nchez et al., 1992) . mutations were confirmed by seobtain representative samples. quencing the viral rna of the two parental viruses pur46-mad and ptv-ts-dmar. results rna was directly sequenced by oligodeoxynucleotide primer extension and dideoxynucleotide chain termina-generation and characterization of tgev mutant ptvtion procedures (sanger et al., 1977) , using a modified ts-dmar1c.c12,1d.e7 protocol previously described (fichot and girard, 1990 ). nucleotide differences between the parental viruses to generate a panel of recombinants between enteric and respiratory strains of tgev, parental viruses that were used as genetic markers and were named m1 to facilitate the selection of recombinants were generated markers, the first 1 kb from the 5 end of the genome, and characterized. a double mar mutant virus (ptv-ts-orf1 nucleotides 12208 to 20363, the first 5 end 2.3 kb dmar1c.c12-1d.e7) derived from the respiratory ptv-ts of the s gene, and the most 3 end 4.3 kb were sestrain of tgev was isolated. the growth of ptv-ts and quenced in the ptv-ts-dmar isolate. these sequences ptv-ts-dmar viruses in st cells at 39њ was at least 10 3were compared to the pur46-par strain (eleouet et al., fold lower than at 34њ, while the parental pur46 virus 1995; rasschaert et al., 1987) and to pur46-mad (menstrain replicated similarly well at both temperatures (data dez et al., 1996; sá nchez et al., 1992) . nine nucleotide not shown). the antigenic characterization of the mutant differences between the two parental viruses were iden-ptv-ts-dmar and its ancestor ptv-ts by ria and neutraltified (fig. 3a) . these markers were named m1 to m9 in ization using mabs showed (fig. 1) that the ptv-ts-dmar order from the 5-end of the genome. the ptv-ts-dmar mutant lacked the s protein antigenic subsite aa comvirus was derived from a ptv-ts isolate which originated pletely, and subsite ab partially, since some mabs spefrom ptv. these three related isolates all display respiracific for these subsites did not bind or neutralized this tory tropism. all of them differ with the enteric pur46isolate. in addition, the s protein of the escaping mutant mad by three nucleotide substitutions in the s gene (m3, was not bound by mabs 8b.f3 and 9f.c11, suggesting m4, and m6). in addition, ptv-ts-dmar differs by a fourth that the epitopes recognized by these mabs are located nucleotide change (m5), which is responsible for the in subsites aa or ab or in close association with these dmar mutation. two of the nine nucleotide differences subsites. sequencing of the s gene in the ptv-ts-dmar (m8 and m9) did not result in an amino acid change. m8 mutant showed that the loss of antigenic subsites aa was located in the intergenic region between orf3a and and ab was due to a single point mutation at nucleotide orf3b, and m9 in orf3b. neither of these two areas 1756, resulting in a change of aspartic acid to tyrosine are expressed in ptv or pur46-mad strains. at position 586. analysis of the recombinant genome sequences showed that the recombinants originated by fusing 5 sequences of the enteric parental virus to 3 sequences in order to study the molecular basis of tgev tropism of the respiratory parental virus. using these molecular recombinant viruses were obtained by coinfecting st markers, the 30 recombinants were classified into three cells with the enteric pur46-mad and respiratory ptvdifferent groups according to the position of the crossts-dmar strains (fig. 2) . st cell monolayers were infected over (fig. 3) . group 1 recombinants contained 8 clones in parallel, with either pur46-mad or ptv-ts-dmar and their crossover was located within the 1102 nucleostrains. to isolate recombinants, selective pressure tides spanning the genetic markers m4 and m5 (fig. 3 ). based on virus inactivation at high temperature and mab group 2 recombinants comprised 15 isolates that had neutralization was used. the virus titer in the st cell culture coinfected with the two parental viruses was 1.9 the crossover located between m2 and m3 markers. the 1 10 3 pfu/ml, while cells infected with the respiratory viruses included in groups 1 and 2 had the same seor enteric parental viruses contained 35 and less than 10 quence except for nucleotides 214 and 655 of the s pro-pfu/ml, respectively, indicating that recombinant viruses tein gene (genetic markers m3 and m4) that were derived resistant to the selective pressure were likely generated. from the enteric parent in group 1 isolates, and from the the supernatant from the st cell culture coinfected respiratory parent in group 2 isolates. group 3 recombiwith the enteric and respiratory strains was used to nants included 7 isolates that had recombined between plaque purify 34 putative recombinant clones and the genetic markers m1 and m2. progeny of the coinfection was phenotypically character-molecular marker m1 was sequenced in all recombiized under restrictive conditions. thirty of the 34 clones nant viruses, since it was the nucleotide difference loanalyzed showed the recombinant phenotype (table 2) . cated closest to the 5 end. all recombinants inherited most of the progeny isolates (53%) showed the expected this marker from the pur46-mad strain, indicating that selectable recombinant phenotype (sr), being resistant there was most likely only one crossover at the 5-half of to both mab neutralization and temperature inactivation. the genome. three nucleotide differences were observed among the recombinant viruses, 35% were dmar mutants from gene 3 up to the 3 end of the genome (fig. 3a) . only which were partially sensitive to the restrictive temperaone of these differences (genetic marker m7, located ture (ts intermediate phenotype recombinants, ipr). no in orf3) led to an amino acid change. however, this virus was isolated with the phenotypic characteristics nucleotide difference was not present in the respiratory against which the selection had been performed (nonse-ptv strain, strongly suggesting that it was not involved lectable recombinants, nsr) ( table 2 ). in the control of tgev tropism. the possibility of a second genotypic characterization of the recombinant crossover at the 3 half of the genome was not analyzed, isolates because if a second crossover had taken place, it would have replaced a fragment by another one with an equiva-the identification of genetic markers was required to map the recombination sites. in order to identify such lent sequence. lar for the two viruses from each group. in addition, one isolate from group 3 recombinants was studied. tropism the tropism of the three groups of recombinants was was studied in parallel in the parental viruses pur46, next studied. two isolates from group 1 and two from group 2 were evaluated. the results obtained were simi-ptv-ts-dmar, and the ptv strain. each isolate was tested at least three times. all recombinants and the parental shown) indicated that the ts mutation mapped at the orf1a of the respiratory isolate, between genetic markviruses were isolated from the lungs, but only the pur46 ers m1 and m2 (i.e., from nt 955 to nt 13272 of orf1). strain and the recombinants of group 1 could be isolated the dmar mutation was localized at position 1756 of s from the small intestine (fig. 4) . maximum virus producgene (genetic marker m5). thus, the minimum distance tion in the lungs varied from 10 4 to 10 6 pfu/g tissue (fig. for recombination with the selective pressure used (high 4). ptv-ts-dmar produced less infectious virus than the temperature and neutralization by mabs) was the interval other strains. maximum ptv-ts-dmar production was between markers m2 and m5. the recombinant isolation about 10 4 pfu/g of lung tissue, while its ancestor ptvfrequency was calculated as the ratio between the progwt could replicate to higher titers (10 6 pfu/g tissue). eny virus titer with a recombinant genotype (1.8 1 10 3 pfu) and the titer (8 1 10 7 pfu) of the parental viruses recombinant isolation frequency grown in parallel in the absence of selective pressure, the procedure used to isolate recombinant viruses divided by the distance between m2 and m5. this frefavored the selection of viruses which had recombined quency was £2.3 1 10 09 recombinants per nucleotide between the two markers used in the selection, the ts for this interval. the recombinant frequency was also calculated for recombinants of groups 1, 2, and 3 and and the dmar mutations. preliminary results (data not below this bar, the location of nine nucleotide differences (genetic markers m1 to m9) between the two parental viruses is indicated. recombinants were classified into three groups (named 1 to 3), and the origin of their genomes, whether derived from the enteric parental (dark bars) or from the respiratory parental (white bars) is indicated. in the bars corresponding to the parental viruses pur46-mad and ptv-ts-dmar, the individual nucleotide differences are indicated. (b) summary of the genetic characterization of the groups of recombinant viruses. the two markers flanking each crossover and the distance expressed in nucleotides between the two markers are indicated in columns 2 and 3, respectively. since the exact location of the ts mutation is not known, the crossover in group 3 recombinants is indicated as a maximum distance. the number of recombinants included in each group and the percentage in relationship to the total recombinant virus population are shown in column 4. the last column shows the frequency of recombinants isolated in each group, as the ratio of the number of isolates in the group relative to the number of nucleotides between the molecular markers flanking the crossover site. were 5.5 1 10 09 , 1.5 1 10 09 , 4.1 1 10 010 , respectively residues 214 and 655 of the spike gene were responsible (fig. 3b ). since the exact location of the ts mutation is not for the loss of enteric tropism. known, the interval in which the crossover takes place in group 3 recombinants can not be precisely defined and recombination among isolates of the tgev cluster the frequency provided for this group is the minimum. initial attempts to isolate tgev recombinant viruses the calculated data indicate that the frequency of recomusing selective pressure with neutralizing mabs specific binant isolation at the 5 of the s gene was 3.7-fold higher for a single epitope lead to the isolation of neutralization than that of group 2 recombinants. escape mutants instead of recombinants (data not shown). to diminish the frequency of escape mutants, discussion selective pressure with two mabs specific for different epitopes of antigenic subsites aa and ab (gebauer et in order to determine the role of different viral genes in tgev tropism, a collection of 30 recombinants was al., 1991) were used. this strategy, in fact, decreased the frequency of neutralization escape mutants, although the generated by coinfecting st cells with enteric (pur46-mad) and respiratory (ptv-ts-dmar) strains of tgev. phe-selected dmar mutant used as a parental virus in the recombination (ptv-ts-dmar) had only a single nucleotide notypic, genotypic, and biological characterizations of the recombinants showed that two nucleotide changes at change, instead of two nucleotide changes that might to study tgev tropism, 2to 3-day-old non-colostrum-deprived piglets were individually inoculated with two isolates from group 1, with two isolates from group 2, and with 1 isolate from group 3 recombinants. recombinants were isolated by crossing the enteric strain pur46 and the respiratory strain ptv-ts-dmar. top horizontal thick bars indicate the genome and the origin of each recombinant, whether enteric (dark bar) or respiratory (white bar). the thin horizontal bar indicates the s gene. triangles indicate the positions of nucleotides 655 and 2098 of the s gene and the origin of these nucleotides, whether enteric (dark triangle) or respiratory (white triangle). diagram is not at scale and the size of the s gene has been magnified. the recovery of infectious virus was determined in pfu per gram of tissue at the indicated time in h.p.i. each virus was tested at least three times. vertical thin bars indicate standard error of the mean. have been expected for a mar mutant escaping to the 3.7-fold higher than that of group 2 recombinants. this could be due to a selective advantage in their growth on simultaneous neutralization by two different mabs. isolation of tgev recombinants required the use of cell cultures or to a higher recombination frequency at the 5-end of s gene, between nucleotides 655 and 1756. selection pressure. using this procedure the frequency of recombination was estimated at £2.3 1 10 09 for tgev. in fact, extensive sequence variability has been observed in this region. during the isolation of tgev-defective in-in contrast, the isolation of the mhv recombinant does not require the use of selection pressure (makino et al., terfering viruses, a deletion is introduced at the beginning of the s gene, starting from nucleotides 6 to 74 and 1986). the recombination frequencies estimated for both coronaviruses are not directly comparable since the se-ending at orf7 (mendez et al., 1996) . in addition, during the generation of both european and american prcvs lection strategies were very different; nevertheless, from reported data it seems that the recombinant isolation in field conditions, four different deletions at the beginning of the s gene have been identified in positions rang-frequency is higher for mhv than for tgev. both recombination and the generation of defective interfering (di) ing from nucleotides 45 to 745 (sá nchez et al., 1992; vaughn et al., 1994; wesley et al., 1991) . these data genomes occurs at a lower frequency in tgev (mendez et al., 1996) than in mhv (lai, 1990) , possibly due to a suggest that the 5-half of the s gene is an area with an intrinsically high recombination frequency. although a higher accuracy in the replication of tgev rna. group 1 recombinants were isolated at a frequency selective advantage for the recombinants could not be excluded, it seems unlikely because group 1 and 2 re-tine until the fourth day postinoculation, virus was never detected in the enteric tract in any of the more than 60 combinants differ only in two nucleotide positions located at the 5-half of the s gene (nucleotides 214 and piglets inoculated with a respiratory isolate. this indicates that the virus detected in the enteric tract was not 655), and recombinants which had the same 5-half s gene as group 2 recombinants grew as efficiently as due to residual virus from the inoculation, nor swallowed virus originating in the respiratory tract, but was the result pur46. an increased recombination frequency in the s gene of mhv has also been described (fu and baric, of local virus replication in the intestine. all the isolated recombinants, including the ones lack-1994). ing enteric tropism, were temperature resistant, indicating that the ts mutation was not responsible for the loss molecular basis of tgev tropism. of enteric tropism. studies on pur46-par mar mutants also showed a only nine nucleotide differences were found between the enteric pur46-mad and the respiratory ptv-ts-dmar correlation between the n-terminal half of the s protein and viral pathogenesis (bernard and laude, 1995) . nev-strains of tgev. four of them mapped in the s protein gene at nucleotides 214, 655, 1756, and 2098. the nucle-ertheless, these results did not differentiate between virus tropism and virulence, since only parameters such otide change at position 1756 of s protein gene, which is responsible for the neutralization escape phenotype, as death, or weight loss, caused by the virus mutants were studied, but not virus replication in enteric or respi-is not responsible of the loss of enteric tropism since it was not present in the respiratory isolate ptv which ratory tissues. coronavirus spike protein is involved in virus attach-lacks enteric tropism. in order to analyze which of the other three nucleotide ment to cells (cavanagh et al., 1986; holmes et al., 1989; sturman and holmes, 1983; suñé et al., 1990) . studies changes located in the s protein gene, at positions 214, 655, and 2098, were involved in the control of the enteric on the inhibition of virus binding to cells indicated that the receptor binding site for tgev had to be located tropism, recombinant viruses containing one or the three nucleotide differences from the respiratory isolate were between antigenic sites d and a of the spike protein (suñé et al., 1990) , mapping between amino acids 385 selected. these recombinants belong to groups 1 and 2, respectively. group 2 recombinants only infected lungs, and 631. in agreement with these data, it was shown that porcine apn, the receptor for tgev (delmas et al., while group 1 replicated in the epithelial cells of both the enteric and respiratory tracts. the two nucleotide 1992), binds to s protein residues between aminoacids 522 and 744 (godet et al., 1994) . these sequences map changes between the enteric recombinants (group 1) and the respiratory ones (group 2) were at nucleotides to a distal area in relationship to amino acid 219 of s protein, which, as shown in this paper, influences tgev 214 and 655 of the s protein gene, which caused amino acid changes from aspartic acid to asparagine at residue enteric tropism. since papn is a protein present in lung epithelium and in enterocytes (kenny and maroux, 1982; 72 and from alanine to serine at residue 219. these results demonstrate that two amino acid changes at the noré n et al., 1986; semenza, 1986) , and the respiratory ptv isolate conserves the papn binding site previously n-terminus of the viral spike protein were associated to the loss of enteric tropism in the tgev cluster of viruses. described, the loss of enteric tropism in the ptv isolate should not be due to a failure in papn attachment. fur-the possibility that the loss of enteric tropism was a consequence of the addition of a nucleotide change at thermore, it has been demonstrated that prcv isolates attach to papn (delmas et al., 1992) , although they can-position 655 of s gene to a preexisting change at nt 214 cannot be completely ruled out. nevertheless, this not infect the enteric tract. this apparent discrepancy could be explained if an interaction between papn and possibility seems unlikely because most enteric viruses have the same nucleotide as ptv at position 214 (sá n-two domains of s protein located at both areas (amino acids near residue 219 and amino acids 522 to 744) are chez et al., 1992), indicating that most likely a single nucleotide change at position 655 was responsible for required to infect the enteric tract. alternatively, a putative second factor, such as coreceptor, mapping around the loss of enteric tropism. nucleotides 214 and 655 are located within the area of the s gene which is deleted amino acid 219 of the spike protein could be specifically required to infect the enteric tract and responsible for in prcvs, strongly suggesting that this deletion was responsible for the loss of enteric tropism in prcvs. in the loss of enteric tropism in ptv and prcv isolates. other explanations are also possible and the loss on human immunodeficiency and other virus systems it has also been shown that a single point mutation can alter enteric tropism could also be due to: (i) a decrease in the ph stability required to allow the virus passage tropism (takeuchi et al., 1991) . an intragastric inoculation route was employed to as-through the stomach, (ii) a decrease in virion resistance to bile salts and proteolytic enzymes in gut, and (iii) an sure that the inoculum of each isolate was introduced into the stomach, independently of their tropism. while alteration in the strength or affinity of the s/receptor interaction. recent studies also located the receptor binding viruses with enteric tropism have been found in the intescell biology of virus entry, ble gastroenteritis virus spike protein results in markedly reduced replication, and pathogenesis'' (r. w. compans, a. helenius, and pathogenicity critical epitopes in transmissible gastroenteritis virus neutralrespiratory coronaries: comparison with transmissible gastroenteriization sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsid protein gene genetic evolution and tropism of transmissible hydrolases of kidney and intestine coronavirus-organization, replication and expresgastroenteritis coronaviruses bullido, sion of genome antigenic homology among coronaviruses recoronavirus-molecular features and virus host interactions the swine major histocompatibility complex: its structure and function. in ''swine in chain-terminating inhibitors anchoring and biosynthesis of stalked brush border membrane proteins: glycosidases and peptidases of enterocytes highfrequency rna recombination of murine coronaviruses a monoclonal antibody gastroenteritis of swine. ii. selected characteristics of a cytopathogenic virus common to five isolates from transmissible gastroenteri-siddell the molecular biology of corotis efficient naviruses mechanisms of transmissible gastroenteritis coronavirus neutralization analysis of the receptor binding site of murine coronavirus spike protein elsemodification of cell tropism by a single point mutation at the neutralization epitope in the env gene three new isolates of porcine respiratory coronavirus with various pathogenicities and gastroenteritis virus in swine: old and news. 9th int porcine respiratory coronavirus differs from transmissible gastroenteritis virus by a few for the pathogenesis of transmissible gastroenteritis virus genetic analysis of porcine respiratory coronavirus, an attenuated variant of transmispartial sequence of the genomic rna, its organization and expression pathogenic murine coronaviruses. iii. biological and biochemical characterization of for a porcine respiratory coronavirus, antigenically similar to transmissible gastroenteritis virus, in the united states spike protein-dependent cellular factor other than the viral receptor is required for mouse hepatitis virus entry transmissible gastroenteritis comparison of porcine transmissible gastroenteritis virus (tgev) with site of the jhm strain of mhv in rats on the s1 subunit porcine respiratory coronavirus. viiith international congress of virolof the spike protein (suzuki and taguchi, 1996) , and in ogy, pp. p6-018. iums, berlin.mhv it was suggested that a second cellular factor, apart callebaut, p., correa, i., pensaert, m., jimé nez, g., and enjuanes, l. from the cellular receptor which interacts with the s pro(1988) . antigenic differentiation between transmissible gastroenteritis virus of swine and a related porcine respiratory coronavirus. j. tein, is involved in virus entry (yokomori et al., 1993) . gen. virol. 69, 1725 -1730 the requirement of a coreceptor to infect cells has been cavanagh, d., brian, d. a., enjuanes, l., holmes, k. v., lai, m. m. c., described in human immunodeficiency virus and in poliolaude, h., siddell, s. g., spaan, w., taguchi, f., and talbot, p. (1994). virus (deng et al., 1996; dragic et al., 1996; feng et al., revision of the taxonomy of the coronavirus, torovirus, and arteri-1996; shepley and racaniello, 1994) . orf3a is not expressed in prcvs isolates, while it is coronavirus ibv: virus retaining spike glycopolypeptide s2 but not s1 expressed in enteric strains. thus, it has been proposed is unable to induce virus-neutralizing or haemagglutination-inhibiting that orf3a plays an essential role in the control of virus antibody, or induce chicken tracheal protection. j. gen. virol. 67, enteropathogenecity (britton et al., 1991; laude et al., 1435 laude et al., -1442 laude et al., . 1993 wesley et al., 1991) . rna sequence comparison of correa, i., jimé nez, g., suñé , c., bullido, m. j., and enjuanes, l. (1988) .antigenic structure of the e2 glycoprotein from transmissible gastro-the 3-half of the respiratory virus ptv-ts-dmar, from enteritis coronavirus. virus res. 10, 77-94.orf3a to 3-utr, with that of the enteric isolate pur46 key: cord-284581-fl2nt4ak authors: kleine-weber, hannah; pöhlmann, stefan; hoffmann, markus title: spike proteins of novel mers-coronavirus isolates from northand west-african dromedary camels mediate robust viral entry into human target cells date: 2019-07-19 journal: virology doi: 10.1016/j.virol.2019.07.016 sha: doc_id: 284581 cord_uid: fl2nt4ak the highly pathogenic middle east respiratory syndrome (mers)-related coronavirus (cov) is transmitted from dromedary camels, the natural reservoir, to humans. for at present unclear reasons, mers cases have so far only been observed in the arabian peninsula, although mers-cov also circulates in african dromedary camels. a recent study showed that mers-cov found in north/west(morocco) and west-african (burkina faso and nigeria) dromedary camels are genetically distinct from arabian viruses and have reduced replicative capacity in human cells, potentially due to amino acid changes in one or more viral proteins. here, we show that the spike (s) proteins of the prototypic arabian mers-cov strain, human betacoronavirus 2c emc/2012, and the above stated african mers-cov variants do not appreciably differ in expression, dpp4 binding and ability to drive entry into target cells. thus, virus-host-interactions at the entry stage may not limit spread of northand west-african mers-cov in human cells. the middle east respiratory syndrome-related coronavirus (mers-cov) causes the severe lung disease mers (zaki et al., 2012) , which takes a fatal course in roughly~35% of infected patients (who, 2019) . mers-cov is endemic in the middle east, where the virus is transmitted from dromedary camels, the natural reservoir, to humans (perera et al., 2013; reusken et al., 2013) . human-to-human transmission is inefficient but resulted in several hospital outbreaks of mers harriman et al., 2013; memish et al., 2013) , and there is concern that the virus may adapt to humans and cause a pandemic. infection of dromedary camels with mers-cov is not limited to the middle east. african camels are frequently infected with mers-cov (ali et al., 2017a (ali et al., , 2017b chu et al., 2014 chu et al., , 2015 chu et al., , 2018 corman et al., 2014; deem et al., 2015; kiambi et al., 2018; miguel et al., 2017; ommeh et al., 2018; perera et al., 2013; reusken et al., 2013 reusken et al., , 2014 van doremalen et al., 2017) and the responsible viruses are genetically distinct from those circulating in the middle east kiambi et al., 2018; ommeh et al., 2018) . moreover, viruses isolated from animals in morocco, nigeria and burkina faso form a distinct phylogenetic subclade, c1, and exhibit reduced ability to replicate in human respiratory cells . in addition, mers-cov transmission from camels to humans has not been observed in northand west-africa (munyua et al., 2017; so et al., 2018) , although two livestock handlers in kenya were shown to harbor antibodies against mers-cov (liljander et al., 2016) , moreover, no mers cases were documented in africa. at present, the barrier(s) impeding efficient spread of african mers-cov in human cells and camel-human transmission of these viruses remain to be identified. the mers-cov spike protein (s) is incorporated into the viral envelope and facilitates viral entry into target cells (li, 2016) . for this, the s protein binds to the cellular receptor dipeptidyl peptidase 4 (dpp4, cd26) via its surface unit, s1, and fuses the viral membrane with a target cell membrane via its transmembrane unit, s2 (li, 2016) . binding of mers-s to dpp4 is essential for mers-cov infection of cells and dpp4 expression and the s protein/dpp4 interface are major determinants of mers-cov cell and species tropism van doremalen et al., 2014) . the s proteins of north-and west-african mers-cov of the c1 clade harbor 6-9 amino acid substitutions relative to mers-cov (fig. 1a , table 1 ) and these substitutions might reduce s protein-driven entry into target cells. however, this possibility has not been examined so far. we employed a previously described vesicular stomatitis virus (vsv)-based pseudotyping system to study mers-s-driven host cell entry (kleine-weber et al., 2018 known to adequately model key aspects of the coronavirus entry process. in order to study host cell entry driven by s proteins from the c1 subclade, we employed pcr-based mutagenesis to generate expression constructs for the s proteins of mers-cov from morocco (camel/morocco/cirad-hku213/2015, mo), nigeria (camel/nigeria/nv1657/ 2016, ni) and burkina faso (camel/burkina faso/cirad-hku785/ 2015, bf), using a published expression construct for mers-cov emc s protein as template (kleine-weber et al., 2018 . moreover, expression constructs for all s proteins were generated that encoded a cterminal v5 antigenic tag. western blot analysis of cells transfected to express the s proteins under study revealed that mers-s emc, mo, ni and bf were expressed and proteolytically processed to comparable levels ( fig. 1b) . moreover, these s proteins were incorporated into vsv particles with similar efficiency (fig. 1c) . these results suggest that mutations present in north-and west-african mers-s of the c1 subclade do not reduce s protein expression and proteolytic processing in human cells. we next asked whether dpp4 binding of north-and west-african mers-s was altered. for this, 293t cells transfected to express the s proteins under study were incubated with soluble dpp4 fused to the fc portion of human immunoglobulin and binding was quantified by flow cytometry, as described previously (kleine-weber et al., 2019). the results showed that mers-s emc, mo, ni, and bf bound to dpp4 robustly and with comparable efficiency while dpp4 binding to cells expressing no s protein was within the background range (fig. 2 ). finally, we tested whether the robust binding to dpp4 translated into efficient s protein-driven entry. for this, cell lines were selected that were shown to express low levels (293t), intermediate levels (vero 76) or high levels of dpp4 (caco-2, 293t + dpp4) (kleine-weber et al., 2019). mers-s mo, ni and bf mediated entry into all cell lines with at least the same efficiency as mers-s emc (fig. 3) . moreover, under conditions of low or medium dpp4 expression, entry mediated by mers-s mo and bf was even more efficient than entry mediated by mers-s emc (fig. 3 ), although these differences were not statistically significant. our results show that amino acid substitutions present in north-and west-african mers-s proteins relative to mers-s emc do not compromise s protein expression in human cells, at least when transfected cells are examined. similarly, proteolytic processing of the s proteins in the constitutive secretory pathway, which is known to be carried out by furin (gierer et al., 2015; millet and whittaker, 2014) , was not the indicated s proteins were transiently expressed in 293t cells, whole cell lysates (wcl) were prepared at 48 h posttransfection and s protein expression was analyzed via western blot, using an antibody targeting the c-terminal v5-tag. cells expressing no s protein were used as negative control and detection of β-actin (actb) served as loading control. similar results were obtained in two separate experiments. (c) rhabdoviral transduction vectors (vsvpp) harboring the indicated s proteins were concentrated by centrifugation and, following lysis, analyzed by western blot for s protein incorporation, using an antibody targeting the c-terminal v5-tag. transduction vectors harboring no s protein were used as negative controls and detection of vesicular stomatitis virus matrix protein (vsv-m) served as loading control. similar results were obtained in a separate experiment. numbers on the left side of each blot indicate the molecular weight in kilodalton (kda). further, bands representing the precursor s protein (s0, black circle) and the s2 subunit of proteolytically processed s protein (grey circle) are indicated. appreciably altered. moreover, binding of north-and west-african s proteins to dpp4 was not diminished as compared to mers-s emc, despite the presence of at least one substitution in the receptor binding domain (rbd) in each s protein tested. this finding might not be unexpected since the substituted amino acid residues do not make direct contact with residues in dpp4 (lu et al., 2013) . in keeping with these observations, all african s proteins mediated robust viral entry into non-human primate (vero 76) and human cell lines (293t, caco-2) expressing different levels of dpp4 (kleine-weber et al., 2019) . in fact, mers-s mo-and bf-driven entry into cell lines expressing low or intermediate levels of dpp4 was augmented as compared to mers-s emc, in keeping with these s proteins showing slightly enhanced dpp4 binding as compared to mers-s emc. finally, it is noteworthy that mers-s activation in caco-2 cells mainly depends on the cellular serine protease tmprss2 while activation in 293t and vero 76 cells is mediated by the cellular cysteine protease cathepsin l (kleine-weber et al., 2018, 2019). thus, north-and west-african mers-s proteins seem to be able to use both pathways available for s protein activation in human cells. confirmation of our findings with authentic viruses is pending and we cannot exclude that, for instance, the s protein modulates recognition of the virus by sensors of the interferon system, which cannot be measured with the assays available to us. moreover, we note that a recent study examining two mers-s sequences (c2 subclade) from camels in ethiopia demonstrated that these sequences, when inserted into mers-cov emc, reduced viral entry and replication and increased sensitivity to antibody-mediated neutralization (shirato et al., 2019) . the reduction in entry was observed for vero and to a lesser degree for vero-tmprss2 cells and was generally modest. nevertheless, these results suggest that s proteins from viruses circulating in ethiopia might harbor mutations that diminish entry into human cells and that are not present in the mers-s proteins studied here. amino acid residues i139, l515, e851 and s1302 in the spike protein are unique to ethiopian mers-cov and warrant further analysis. collectively, our results suggest that amino acid substitutions present in the s proteins of north-and west-african mers-cov do not compromise the ability of these viruses to enter human cells. thus, future efforts to understand why north-and west-african mers-cov isolates show reduced replicative potential in human cells should be focused on other aspects of the mers-cov lifecycle than s proteinmediated host cell entry. expression plasmids, based on the vector pcaggs, for vsv-g and mers-s emc were previously described (kleine-weber et al., 2018 . the mers-s emc plasmid was used as template for pcr-based mutagenesis to introduce the mutations found in mers-s mo (morocco, camel/morocco/cirad-hku213/2015, genbank: mg923469.1), ni (nigeria, camel/nigeria/nv1657/2016, genbank: mg923475.1) and bf (burkina faso, camel/burkina faso/cirad-hku785/2015, gen-bank: mg923471.1) ( table 1 ). in addition, pcr-based mutagenesis was used to equip the constructs with a c-terminal v5 antigenic tag. the integrity of all sequences was verified using automated sequence analysis. 293t (human embryonal kidney) and vero 76 (african green monkey kidney) cells were cultivated in dulbecco's modified eagle's medium (dmem; pan biotech). the human colorectal adenocarcinoma cell line caco-2 was grown in minimum essential media (mem, life technologies). all media were supplemented with 10% fetal bovine serum (fbs, pan biotech) and 1x penicillin and streptomycin from a 100x stock solution (pan biotech). the cells were incubated under humid conditions at 37°c and 5% co 2 . for transfection of 293t cells the calcium-phosphate precipitation method was used. genbank: mg923469.1 v26a s1 / n/a a89s s1 / n/a t424i s1 / rbd s856y s2 / n/a r884l s2 / ps(s2') a1158s s2 / n/a v1209l s2 / n/a mers-s ni camel/nigeria/nv1657/2016 genbank: mg923475.1 v26a s1 / n/a h167y s1 / n/a h194y s1 / n/a l495f s1 / rbd l588f s1 / rbd s856y s2 / n/a a1158l s2 / n/a l1200f s2 / n/a mers-s bf camel/burkina faso/cirad-hku785/ 2015 genbank: mg923471.1 v26a s1 / n/a a89s s1 / n/a h194y s1 / n/a t424i s1 / rbd s856y s2 / n/a a1158s s2 / n/a a amino acid position (numbering according to mers-s emc). b subunit / functional domain (if applicable); abbreviations: s1 = s1 subunit; s2 = s2 subunit; rbd = receptor binding domain, ps(s2') = priming site at the s2' position (884-rsar-887), n/a = not applicable. fig. 2 . s proteins of north/west-and west-african mers-cov isolates from dromedary camels efficiently bind to dpp4. 293t cells expressing the indicated s proteins or no s protein at all (control) were successively incubated with soluble dpp4 containing a c-terminal fc tag (sol-dpp4-fc) and alexafluor488conjugated anti-human antibody, before dpp4 binding to the respective s protein was analyzed by flow cytometry. presented are the combined data of three independent experiments for which sol-dpp4-fc binding to mers-s emc was set as 100%. error bars indicate the standard error of the mean (sem). statistical significance was tested by one-way analysis of variance with sidak's posttest (p > 0.05, not significant, ns; p ≤ 0.01, **). for western blot analysis, anti-v5 (mouse, 1:2,500; thermofisher scientific), anti-β-actin (mouse, 1:2,500; sigma-aldrich), anti-vsv-m (mouse, 1:2,500; kerafast) were used as primary antibodies and antimouse hrp (horse radish peroxidase) conjugated antibody (goat, 1:2,500; dianova) was used as secondary antibody. antibodies were diluted in phosphate buffered saline [pbs] containing 0.5% tween 20 [pbs-t] supplemented with 5% skim milk powder. for flow cytometry, a recombinant fusion protein of the ectodomain of dpp4 fused to the fc fragment of human immunoglobulin (sol-dpp4-fc, 1:200, acrobiosystems) and an alexaflour488-conjugated anti-human antibody (goat, 1:500; thermofisher scientific) were used (ligand and antibody were diluted in pbs containing 1% bovine serum albumin). for analysis of s protein expression, 293t cells were transfected with expression plasmid for mers-s proteins harboring a c-terminal v5 tag, as described (kleine-weber et al., 2018, 2019). to investigate mers-s incorporation into vsvpp, equal volumes of supernatants containing vsvpp bearing s proteins with v5 tag were centrifuged through a 20% sucrose cushion at 25.000 g for 120 min. subsequently, cells and vsvpp pellets were lysed and analyzed by immunoblot, following an established protocol (kleine-weber et al., 2018 . dpp4 binding was analyzed as described (kleine-weber et al., 2019) . in brief, 293t cells were transfected with expression plasmids for mers-s proteins and empty plasmid as negative control. at 48 h posttransfection, the cells were washed with pbs, pelleted and resuspended in pbs containing 1% bsa and soluble human dpp4-fc fusion protein at a final dilution of 1:200. after incubation for 1 h at 4°c, the cells were washed and incubated with alexafluor488-conjugated anti-mouse antibody at a dilution of 1:500. finally, the cells were fixed with 4% paraformaldehyde and analyzed by flow cytometry using an lsr ii flow cytometer and the facs diva software (both bd biosciences). fig. 3 . host cell entry driven by the s proteins of north/west-and west-african mers-cov isolates from dromedary camels is robust. 293t, 293t transfected to express dpp4, vero 76 and caco-2 cells were inoculated with equal volumes of rhabdoviral transduction vectors harboring the indicated s proteins or no s protein (control). at 18 h posttransduction, the activity of the virus-encoded luciferase, which served as an indicator for transduction efficiency, was measured in cell lysates. presented are the combined data of three independent experiments for which transduction mediated by mers-s emc was set as 100%. error bars indicate sem. statistical significance was tested by one-way analysis of variance (anova) with sidak's posttest (p > 0.05, ns; p ≤ 0.01, **; p ≤ 0.005, ***). transduction vectors based on a replication-deficient vsv (berger rentsch and zimmer, 2011) and pseudotyped with the indicated viral glycoproteins (vsvpp) were generated according to a published protocol (kleine-weber et al., 2018 . target cells were transduced with equal volumes of supernatants containing vsvpp and transduction efficiency was quantified at 16 h posttransduction by measuring the activity of virus-encoded firefly luciferase in cell lysates as previously described (kleine-weber et al., 2018 . cross-sectional surveillance of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels and other mammals in egypt systematic, active surveillance for middle east respiratory syndrome coronavirus in camels in egypt hospital outbreak of middle east respiratory syndrome coronavirus a vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multi-species type i interferon middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in nigeria mers coronaviruses in dromedary camels mers coronaviruses from camels in africa exhibit region-dependent genetic diversity antibodies against mers coronavirus in dromedary camels serological evidence of mers-cov antibodies in dromedary camels (camelus dromedaries) in laikipia county inhibition of proprotein convertases abrogates processing of the middle eastern respiratory syndrome coronavirus spike protein in infected cells but does not reduce viral infectivity hospital-associated middle east respiratory syndrome coronavirus infections detection of distinct mers-coronavirus strains in dromedary camels from kenya mutations in the spike protein of middle east respiratory syndrome coronavirus transmitted in korea increase resistance to antibody-mediated neutralization structure, function, and evolution of coronavirus spike proteins mers-cov antibodies in humans molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 hospital-associated middle east respiratory syndrome coronavirus infections risk factors for mers coronavirus infection in dromedary host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein no serologic evidence of middle east respiratory syndrome coronavirus infection among camel farmers exposed to highly seropositive camel herds: a household linked study genetic evidence of middle east respiratory syndrome coronavirus (mers-cov) and widespread seroprevalence among camels in kenya seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study geographic distribution of mers coronavirus among dromedary camels middle east respiratory syndrome coronavirus in dromedaries in ethiopia is antigenically different from the middle east isolate lack of serological evidence of middle east respiratory syndrome coronavirus infection in virus exposed camel abattoir workers in nigeria high prevalence of middle east respiratory coronavirus in young dromedary camels in jordan. vector borne zoonotic dis host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 middle east respiratory syndrome coronavirus (mers-cov isolation of a novel coronavirus from a man with pneumonia in saudi arabia the authors thank gert zimmer and andrea maisner for providing the replication-deficient vsv vector for pseudotyping and the vero 76 cell line, respectively. this work was supported, including the efforts of stefan pöhlmann, by the bundesministerium für bildung und forschung within the network project rapid (risikobewertung bei präpandemischen respiratorischen infektionserkrankungen; 01ki1723d). the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. key: cord-273745-mwjh5se7 authors: meng, fandan; suo, siqingaowa; zarlenga, dante s; cong, yingying; ma, xiaowei; zhao, qiong; ren, xiaofeng title: a phage-displayed peptide recognizing porcine aminopeptidase n is a potent small molecule inhibitor of pedv entry date: 2014-03-25 journal: virology doi: 10.1016/j.virol.2014.01.010 sha: doc_id: 273745 cord_uid: mwjh5se7 three phage-displayed peptides designated h, s and f that recognize porcine aminopeptidase n (papn), the cellular receptor of porcine transmissible gastroenteritis virus (tgev) were able to inhibit cell infection by tgev. these same peptides had no inhibitory effects on infection of vero cells by porcine epidemic diarrhea virus (pedv). however, when pedv, tgev and porcine pseudorabies virus were incubated with peptide h (hvtttfappppr), only infection of vero cells by pedv was inhibited. immunofluoresence assays indicated that inhibition of pedv infection by peptide h was independent of papn. western blots demonstrated that peptide h interacted with pedv spike protein and that pre-treatment of pedv with peptide h led to a higher inhibition than synchronous incubation with cells. these results indicate direct interaction with the virus is necessary to inhibit infectivity. temperature shift assays demonstrated that peptide h inhibited pre-attachment of the virus to the cells. coronaviruses belong to the family of coronaviridae and commonly cause respiratory or gastroenteric diseases (weiss and navas-martin, 2005; lai et al., 2007) . three groups of coronaviruses have been identified, based on differences in serology and genotyping (cavanagh, 1997; spaan et al., 2005) . these are enveloped viruses and consist of four major structural proteins: spike (s), membrane (m), nucleocapsid (n) and minor small envelop (e) protein (lai et al., 2007) . the host range and tissue tropism of coronaviruses depend on interactions between the viral s glycoprotein and receptors on susceptible cells (bosch et al., 2003; gallagher and buchmeier, 2001) . porcine transmissible gastroenteritis virus (tgev) and porcine epidemic diarrhea virus (pedv) are swine-specific enteric coronaviruses that are antigenically distinguishable (lai et al., 2007; pensaert and yeo, 2006) . however, they replicate in the differentiated enterocytes of the small intestine resulting in similar clinical symptoms including lethal watery diarrhea and dehydration in piglets (pensaert and yeo, 2006; sanchez et al., 1992) . two decades ago, porcine aminopeptidase n (papn) was identified as a cellular receptor for tgev (delmas et al., 1992) . since that time, several limited reports have showed that addition of exogenous papn facilitates cell infection by pedv (li et al., 2007; oh et al., 2003.) . recent evidence also indicates that increased papn receptor density on the surface of st cells contributes to cell infection by pedv (nam and lee, 2010) . the available data supports the hypothesis that blockage of papn is a good strategy for preventing cell infection by tgev or pedv. using the papn as a target protein, we identified three 12-mer peptides (designated as h, s or f) by phage display which bind to papn and competitively inhibit cell infection by tgev (ren et al., 2011a) . the initial purpose of this study was to investigate the role of papn-binding peptides h, s and f on cell infection by pedv. interestingly, although there was no surface expression of papn on vero cells, peptide h decreased the infectivity of pedv in vitro. western blots indicated that peptide h (hvtttfappppr) interacted with the s protein of pedv. altering incubation temperatures further demonstrated that peptide h affected pre-attachment of pedv to cells. it is important to identify small molecules such as peptides that prevent infection by pedv, inasmuch as highly effective pedv vaccines which are currently not available. the peptide h identified herein may be one such candidate. concentration of compound that decreased the percentage of formazan produced in uninfected, peptide-treated cells to 50% of that produced in uninfected, peptide-free cells. the cc 50 values were greater than 1000 μg/ml. all subsequent antiviral experiments were performed at peptide concentrations below the experimentally-determined cc 50 value. in order to test the abilities of the three peptides to prevent attachment of pedv to cells, all combinations of peptide, virus and cell treatment were performed. cell post-treatment assays (fig. 1a) were performed to evaluate whether the three peptides were able to inhibit replication of pedv after infecting vero cells. plaque assays indicated that none of the three peptides inhibited pedv infection; however, rabbit anti-pedv decreased vial infectivity by more than 50% when the dilutions were reduced from 1:64 to 1:4. when vero cells were pre-treated with peptide (cell pretreatment assay) prior to virus infection (fig. 1b) , little changes in virus titers were observed between the control and peptide treatment groups; some small effects were observed with the rabbit anti-pedv neutralizing antibodies. finally, in the virus pretreatment assays where pedv was incubated with peptides prior to cell infection (fig. 1c) , the results indicated that both peptides h and s inhibited pedv infectivity where ec 50 values were approximately 1 μg/ml and 62.5 μg/ml, respectively. the antiviral activity of peptide h was dose-dependent and at 250 μg/ml it exhibited greater than 95% anti-pedv activity which is significantly higher than peptides s or f (p o0.01); at 15.6 μg/ml, inhibition was greater than 70%. the selectivity indices si of peptides h and s were 1000 and 16, respectively. peptide f showed little inhibitory activity against pedv infection even at concentrations z 1000 μg/ml. inasmuch as papn may be involved in cell infection by pedv, the existence of papn on st, vero and mdck cells was analyzed by ifa. as shown in fig. 2 , the endogenous papn expressed only on the surface of st cells, a porcine cell line. no expression was found on the surfaces of vero cells or mdck cells suggesting that the inhibitory activity of peptide h on pedv infection in vitro did not involve papn. the specificity of the inhibition of peptide h on pedv infection was assessed by comparing antiviral activities of peptide h on tgev and prv. further, peptide-induced cytotoxicity in st cells was also evaluated. results clearly show that peptide h had no demonstrable effects on tgev or prv even at very high peptide concentrations (1 mm/ml) (fig. 3) suggesting that a non-specific reactivity with virus envelopes is unlikely to be the cause for attenuating pedv infectivity. the effect of peptide h on the level of virus rna was quantified by real-time rt-pcr. the results demonstrated a dose-dependent decrease of viral rna synthesized in pedv-infected cells (fig. 4) . at 31.25 μg/ml and 15.625 μg/ml, peptide h showed reduction of viral rna synthesis (po 0.05). however, at 250 μg/ml, 125 μg/ml and 62.5 μg/ml it significantly decreased viral rna synthesis (p o0.01) when compared to the no peptide treatment group. the inhibitory activity of peptide h against pedv infection was confirmed by conventional rt-pcr. analysis of the pedv-rnas indicated that the density of the amplified sequences decreased with increasing concentrations of peptide h (data not shown). binding characteristics of the phage h (phage encoding peptide h) to pedv was analyzed by western blot. as shown in fig. 5 , the phage h reacted with a protein with an approximate molecular mass of 220 kda which is coincident with the molecular mass of the pedv s protein. antibody against the pedv s protein was used as a positive control. other controls including the m13 phage library and two phages bearing non-selected peptides did not react with the pedv s protein (fig. 5) . these results indicate that the peptide h binds to the s protein of pedv. to further examine the mechanism of action of peptide h on cell infection by pedv, we investigated the effects of incubation temperature on cell infectivity. plaque assays showed that peptide infected with pedv at an pfu of 5 â 10 3 /ml. (c) peptides h, s or f were first incubated with pedv at 37 1c for 1 h, and then the peptide treated viruses (pfu ¼ 5 â 10 3 /ml) were used to infect vero cells at 37 1c. plaque assays were performed at the end of each experiment. serially-diluted polyclonal antibody against pedv and pbs were used as positive and negative controls, respectively. peptide concentrations 1, 2, 3, 4, and 5 are 250 μg/ml, 125 μg/ml, 62.5 μg/ml, 31.25 μg/ml, and 15.625 μg/ml, respectively. anti-pedv antibody dilutions 1, 2, 3, 4, and 5 are 1:4, 1:8; 1:16; 1:32; and 1:64, respectively. bars show the standard deviation from three independent assays. h exhibited significantly higher inhibitory effects (po 0.01) than peptide f or pbs on the pre-attachment of pedv to vero cells ( fig. 6a ) when virus was incubated with peptide h at 4 1c for 1 h prior to incubation with the cells. when peptide h and pedv were co-incubated with vero cell at 4 1c for 1 h before shifting to 37 1c (binding only), this allowed us to measure peptide h effects on early-and pre-attachment of the virus. results showed that pedv pre-treated with peptide h exhibited slightly higher inhibition (p 40.05) than when peptide h and pedv were co-incubated with cells absent a pre-incubation step (fig. 6b) suggesting that there is a direct effect of peptide h on pre-attachment. little to no inhibition of infection was observed once the virus became attached to the cell surface. furthermore, when the peptides and pedv were co-incubated with vero cells at 37 1c absent any preincubation step, no effective inhibition of cell infection was observed (fig. 6c ). to characterize the temperature effect on interactions between peptide h and pedv as well as cell infection, various concentrations of the peptides were incubated with pedv (pfu ¼5 â 10 3 /ml) at 4 1c or 37 1c for 1 h, then the peptide-treated viruses were used to infect cells at 37 1c. the results showed that the inhibition rate at 37 1c was significantly higher (p o0.01) than that at 4 1c when the lower concentrations of peptide h were applied. the inhibition ratio reached 61.86% at 37 1c but only 1.25% at 4 1c at the lowest concentration (15.625 μg/ml). in contrast, high concentrations of peptide h gave rise to a similar inhibition of pedv infectivity (fig. 6d ). infection with tgev and pedv can cause high mortality in piglets and therefore enormous economic loss in the pig industry. the prevalence of pedv and tgev in asian countries such as china and korea has been documented (ren et al., 2011b; li and ren, 2011) . at present, live vaccines against the both viruses are extensively used in china which in turn decreases the occurrence of diseases to some extent. however, small molecule inhibitors to tgev or pedv are alternative approaches to controlling swine viral diarrhea diseases. using combinatorial phage-display peptide libraries can be a powerful tool for selecting ligands that bind target proteins. phage display techniques have been used to generate diagnostic and therapeutic peptides for bacteria (bishop-hurley et al., 2005 carnazza et al., 2008) , fungi (bishop-hurley et al., 2002; fang et al., 2006) and viruses (ren et al., 2011a ferrer and harrison, 1999; welch et al., 2007; wu et al., 2011; yang et al., 2003) . the papn is a member of a membrane-bound metalloprotease family and predominantly expressed on the surface of epithelial cells of the kidney, small intestine, and respiratory tract (nam and lee, 2010; kenny and maroux, 1982; lendeckel et al., 2000) . it is known that papn is a cellular receptor for tgev and that anti-papn antibody efficiently decreases cell infection by this virus (delmas et al., 1992; liu et al., 2009) . recently, three papn-binding peptides h, s, and f were identified using papn as an immobilized target for panning a 12-mer phage display peptide library. these peptides exhibited high affinity binding to papn and inhibited cell infection by tgev completely . as a member of group i coronaviruses, tgev and pedv have similar infection characterizations and as such it is difficult to differentiate these pathogens based only upon clinical symptoms. recent evidence indicates that pedv may also bind papn, a type ii glycoprotein, as a functional receptor (li et al., 2007; oh et al., 2003) . interestingly, tgev can be easily propagated in swine-originated cells such as st cells (delmas et al., 1992; hofmann and wyler, 1988) whereas pedv is adapted and cultivated in african green monkey kidney (vero) cells rather swine cells. given the stark similarities as well as differences between tgev and pedv, we were interested in evaluating the antiviral effects of the h, s and f peptides on cell infection by pedv. we first analyzed potential blockage of the papn-binding peptides on vero cell infection by pedv. plaque assays indicated no significant decrease in the infectivity of pdev even though prior studies showed that both anti-papn antibody and peptides h, s or f were capable of inhibiting tgev infection in vitro (ren et al., 2011a; liu et al., 2009) if pre-incubated with tgev permissive cells. there were limited reports indicating that papn may serve as a functional receptor of pedv; however, studies herein clearly demonstrated that papn is only expressed on the surface of st cells and is not present on vero or mdck cells. as such, the inhibitory activities of peptide h were not due to binding papn. further, we showed that only pre-treatment with peptide h inhibited infection by pedv. the prv is a swine neurotropic herpesvirus with a dna genome that can be propagated in many cell lines including vero cells. therefore, we used prv as an additional control to further confirm and evaluate the inhibition specificity of the h peptide. peptide h did not prevent infection of pretreated tgev or prv suggesting its inhibitory activity was specific and not due to virucidal effects of amphipathic peptides. clearly, peptide h was able to interact with pedv; however, it was unclear as to the mechanism of its antiviral activity. as such, the binding characteristics between peptide h (using the phage bearing the h peptide) and pedv were further examined by western blot. results clearly showed that phage h reacted with a protein with a molecular mass of 220 kda closely approximating the molecular mass of the pedv s protein. this supposition was corroborated using antibody against the pedv s protein. in contrast, control phages bearing other peptides did not show such reactivity. these results demonstrate that the peptide h abrogates infectivity in part by binding to the pedv s protein. this is consistent with the function of the coronavirus s protein that mediates cell infection. further, cell post-treatment assays evaluating the effects of each peptide on the replication of pedv in vitro relative amplification of the pedv x-n gene in the infected cells was normalized to that of beta-actin and calculated using the 2 à δδct method. peptide concentrations 1, 2, 3, 4, 5, and 6 are 0 μg/ml, 15.625 μg/ml, 31.25 μg/ml, 62.5 μg/ml, 125 μg/ml, 250 μg/ml, respectively; line 7 is cell control group. displayed results are averages of three independent experiments. clearly demonstrated that the peptides do not interfere with the intracellular replication of pedv. our results showed that only peptide h and not peptides s or f exhibited very high, dose-dependent inhibitory activity against pedv where as little as 1 μg/ml needed to achieve ec 50 . this was confirmed by real-time rt-pcr which showed decreasing amounts of viral rna in pedv-infected cells. this corroborated the relationship between the antiviral activity of peptide h and either blockage of the viral attachment or entry into vero cells. the impact of peptide h on the entry of pedv was first investigated by performing the cell post-treatment and co-incubation assays. when pedv was incubated with cells prior to treatment with peptide h no significant effects on pedv infection were observed. similar results were seen when peptides, pedv and the cells were combined at the same time and co-incubated at 37 1c suggesting that peptide h did not affect pedv entry into the cell postadsorption. however, when pedv was pre-incubated with peptide h prior to incubation with vero cells, peptide h blocked the attachment of pedv as determined from plaque assays and rt-pcr analysis. it became clear that peptide h did not interact with vero cells directly. rather, it exhibits its inhibitory activity via the interplay between the peptide h and pedv. it is accepted that virus adsorption occurs at 4 1c and internalization does not happen until the temperature is raised to 37 1c (baldick et al., 2010) . our results clearly showed that the effects of peptide h occurred during the incubation step at 4 1c rather than the 37 1c internalization step again targeting a specific interaction between peptide h and pedv that affects binding to the cell surface. both pedv and tgev are group i coronaviruses (bridgen et al., 1993) and propagate in vero and st cells, respectively; prv is a dna virus that also propagates in vero cells. as such, we selected tgev and prv as control viruses to examine any specificity in the inhibitory activity of peptide h on cell infection by these viruses. as expected, the results showed no significant inhibitory activity of peptide h on either tgev or prv infection. further, peptide h was not cytotoxic to either st or vero cells. these results corroborate our hypothesis that peptide h functions in part, by interacting with the s protein of pedv and affecting the ability of the virus to bind to the cell surface. future studies will focus on identifying the specific site of interaction of peptide h and whether or not such a peptide can be used in vivo to abrogate or attenuate pedv infections. swine testis (st), monkey kidney cell lines (vero) and madin-darby canine kidney (mdck) cells were maintained in dulbecco's modified eagle medium (dmem) (invitrogen, us) supplemented with 10% fetal bovine serum, (fbs, gibco, us), 100 units/ml of penicillin and 100 units/ml of streptomycin. all cells were purchased from american type culture collection (atcc) and kept in our laboratory. pedv isolate hljby was propagated in vero cells in the presence of 10 μg/ml trypsin (gibco, us) (ren et al., 2011b) . tgev strain pur46-mad was propagated in st cells (ren et al., 2008; yin et al., 2010) . porcine pseudorabies virus (prv) strain kaplan was propagated in vero cells (ren et al., 2011c) . st or vero cells were seeded in 96-well plates at a density of 5 â 10 4 cells/well and cultured in dmem containing 10% fbs at 37 1c under 5% co 2 for 24 h followed by addition of serial dilutions (62.5-1000 μg/ml) of the tested peptides. the cells were allowed to grow for 48 h at 37 1c and proliferation was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) method. briefly, the medium was removed and 10 μl of mtt solution (0.5 mg/ml, invitrogen, us) was added and incubated at 37 1c for 4 h. then 100 μl of dimethyl sulfoxide (dmso) was added and incubated for 15 min to solubilize the formazan crystals. cell survival rate was calculated as (od 490 treatment)/(od 490 control) (paeshuyse et al., 2006) . the 50% cytostatic concentration (cc 50 ) was defined as the concentration inhibiting the proliferation of (b) pedv (pfu ¼ 5 â 10 3 /ml), peptides h or f, and vero cells were co-incubated at 4 1c for 1 h, washed, then shifted to 37 1c. (c) pedv (pfu ¼ 5 â 10 3 /ml), peptides, and vero cells were co-incubated at 37 1c for 1 h then washed prior to assaying. (d) pedv was treated with various concentrations of peptides h or f at 4 1c or 37 1c for 1 h then used to infect vero cells (pfu ¼5 â 10 3 /ml) at 37 1c. peptide concentrations 1, 2, 3, 4, and 5 are 250 μg/ml, 125 μg/ml, 62.5 μg/ml, 31.25 μg/ml, and 15.625 μg/ml, respectively. anti-pedv antibody dilutions 1, 2, 3, 4, and 5 are 1:4, 1:8, 1:16, 1:32 and 1:64, respectively. plaque assays were further performed at 72 h post-infection. bars show the standard deviation from three independent assays. exponentially growing cells by 50%. non-cytotoxic concentrations of peptides (r cc 50 ) were used for antiviral assays. three different treatment approaches were used to analyze the antiviral action of the selected peptides h (hvtttfappppr), s (svvpskatwgfa) and f (fkpssppsitlw) as previously defined (kwon et al., 2010) . in the first method, i.e., cell post-treatment assay, vero cells were grown in 24-well plates at a density of 5 â 10 5 cells/well for 24 h. pedv at a pfu (plaque-forming unit) of 5 â 10 3 /ml was inoculated onto confluent cells for 1 h followed by removal of the medium and incubation of the infected cells with various noncytotoxic concentrations (r cc 50 ) of peptides for 1 h at 37 1c. the cells were then overlaid with 1% methylcellulose in dmem and incubated for 72 h at 37 1c followed by crystal violet staining and plaque assays as previously described with modifications (ren et al., 2011a (ren et al., , 2011d . briefly, after the medium was removed, the cells were fixed with 4% formaldehyde in pbs for 1 h at room temperature followed by staining with 1% crystal violet solution for 20 min. the staining solution was removed, the cells were washed with pbs and the plaques were counted. decreases in virus infectivity were calculated from the plaque assay as follows: 100 â [1 à (treatment wells/control wells)]. average ec 50 values (concentration inducing 50% inhibition of virus replication) were calculated and the effectiveness of each peptide were expressed using the selectivity index (si) (si¼ cc 50 /ec 50 ) (paeshuyse et al., 2006; müller et al., 2007) . in the second method, i.e., cell pre-treatment assay, vero cells were first grown in 24-well plates at a density of 5 â 10 5 cells/well for 24 h then treated with non-cytotoxic concentrations of peptide for 1 h prior to incubation with virus. the peptides were removed and the cells were washed twice with pbs. pedv at a pfu of 5 â 10 3 /ml was inoculated onto the pre-treated vero cells for 1 h. after the virus was removed, the cells were overlaid with 1% methylcellulose in dmem and incubated for 72 h at 37 1c followed by plaque assays. in the third experiment i.e., virus pre-treatment, various concentrations of the peptides were mixed with pedv (pfu ¼ 5 â 10 3 /ml) at 37 1c for 1 h prior to incubation with cells. vero cells were grown in 24-well plates at 5 â 10 5 cells/well for 24 h then the peptide/virus mixture was added to the cultured cells for 1 h. after the mixture was removed and the cells washed with pbs, the cells were overlaid with 1% methylcellulose in dmem and cultured for an additional 72 h at 37 1c followed by plaque assays as described above. in parallel, pedv-neutralizing, rabbit antiserum serially-diluted 1:2 and pbs were used as positive and negative controls, respectively, for the above-mentioned experiments. each concentration of the peptide and antibody was assayed in triplicate. immunofluorescence assays to identify papn on cell lines from different species st, vero and mdck cells were seed into the 24-well plates and incubated at 37 1c for 24 h. indirect immunofluorescence assays (ifa) were performed (ren et al., 2011d; baldick et al., 2010) with modification. the cells were washed twice with pre-chilled pbs then fixed with 4% paraformaldehyde in pbs for 30 min at room temperature. following two washes with pbs, they were quenched with 0.1 m glycine for 5 min then blocked with 2% bsa (sigma, us) in pbs for 30 min. samples were incubated for 1 h with anti-papn polyclonal antibody (1:1500 in pbs) (liu et al., 2009) , washed three times with pbs, and incubated with fluorescein isothiocyanate (fitc) conjugated goat anti-rabbit igg (1:1500 in pbs, zhongshan, china) for 1 h in the dark. the samples were washed three times with pbs and the fluorescence signals and phase contrast images were detected by fluorescence microscopy (leica, wetzlar, germany). various concentrations of peptide h were incubated with the tgev, pedv and prv (pfu¼5 â 10 3 /ml) at 37 1c for 1 h then added to confluent vero or st cell monolayers for 1 h. the mixtures were removed and the cultured cells washed twice with pbs then incubated with 1% methylcellulose in dmem for 72 h at 37 1c. the cells were then stained with crystal violet staining and plaque assays were performed as described above. the effect of peptide h on pedv infection of vero cells was confirmed by semi-quantitative real-time reverse transcription (rt-pcr) (ren et al., 2011d) . vero cells in 6-well plates were infected with pedv (pfu ¼5 â 10 3 /ml) pretreated with different concentrations of peptide h at 37 1c for 1 h. the culture was replaced with dmem at 37 1c for 24 h then washed 3 times with pbs. the virus-containing cultures were frozen and thawed three times followed by addition of an equal volume of 20% polyethylene glycol (pge) 8000 at room temperature and incubation for 30 min. the samples were centrifuged at 12,000 rpm for 5 min and the pellets were re-suspended in rnase-free water. total rna was extracted with a commercial kit (fastgene, china) according to the manufacturer's instructions. reverse transcription was performed in a total of 20 μl consisting of 5 μl total rna (2.5 μg), 1 μl oligo dt, 1 μl dntp (10 mm), 0.5 μl rnase inhibitor, 7.5 μl sterile water, 1 μl mlv, and 4 μl 5 â rt-pcr buffer. the mixture was incubated at 30 1c for 10 min, 42 1c for 60 min and 95 1c for 5 min. real-time pcr was performed using abi prism 7500 real-time pcr machine (applied biosystems, usa). the information regarding primers and rt-pcr products is provided in table 1 . the real-time pcr mixture included 0.5 μl (0.5 μg) of cdna template, 10 μl of sybr taq polymerase, 0.4 μl of rox pge 2, 0.5 μl (10 pmol) of each primer, and 8.1 μl of sterile water. the reactions were incubated at 95 1c for 10 s followed by 40 cycles of 95 1c for 5 s and 60 1c for 34 s. we examined virus rna levels using primers that specifically amplify a 244 bp fragment encompassing the 3' end of a small, non-structural gene (x) and the 5' end of the pedv-n gene ( table 1 ). the expression of pedv x-n in pedv-infected vero cells was normalized to that of beta-actin and taken as 100% compared to expression of the peptide h treatment group. data analysis is based on the measurement of the cycle threshold (ct). the difference in δct (ct sample fragment mean ct value-beta-actin fragment mean ct value) was used as a measure of the relative fragment with the 2 à δδct method in correlation to the amplification size of pedv x-n fragment. for each experimental condition, real-time pcr was conducted in quadruplicate and the resulting average ct values for the pedv x-n fragment was used to quantify viral load. the experiment was performed in triplicate. information on primers and real-time rt-pcr products. sense 5' cactggttgggctttctatgtc pedvx-n antisense 5'tgttagtgggtacagcgttgtt 244 sense 5' ggctcagagcaagagaggtatcc β-actin antisense 5' ggtctcaaacatgatctgagtcatct 208 western blot analysis of peptide h binding to pedv the pedv (1 â 10 6 pfu/ml) was harvested from vero cells and clarified by centrifugation at 1300g for 15 min followed by ultracentrifugation at 150,000g for 1.5 h to collect the virus. the pellets were suspended in pbs, subjected to 10% sds-page then blotted to a nitrocellulose (nc) membrane. the nc membranes were blocked overnight at 4 1c with 5% non-fat dry milk in pbs. after three washes with pbs, membranes were sliced and incubated with phage h (1 â 10 11 pfu), anti-pedv s polyclonal antibody (1:1000 in pbs), m13 phage library (1 â 10 11 pfu), and control phage bearing either the peptides svsvgmkpsprp or mscndtlcllpn. the membranes were then washed with pbs and successively incubated with anti-m13 polyclonal antibody (abcam, 1:600 in pbs) and horseradish peroxidase hrp-conjugated goat anti-rabbit igg (1:1500 in pbs) at room temperature for 1 h. protein bands were visualized using 3,3'-diaminodbenzidine (dab, the thermo scientific) to examine the effect of temperature on the binding of virus to the cells, four experiments were performed. first, various concentrations of peptide h and control peptide f were incubated with pedv (pfu¼ 5 â 10 3 /ml) at 4 1c for 1 h then added to confluent vero cells seeded in 24-well plates at 4 1c for 1 h followed by infection at 37 1c for 72 h. second, the peptides, pedv and vero cells were co-incubated at 4 1c for 1 h, after which the incubation temperature was raised to 37 1c for 72 h. third, the peptides, pedv and vero cells were co-incubated at 37 1c for 1 h then assayed without prior incubation at 4 1c. finally, peptides were pre-incubated with pedv at 4 1c or 37 1c for 1 h followed by cell infection at 37 1c for 72 h. all experiments were terminated by extensive washing of the cells and plaque assays to quantify the infection. statistical analysis of the data was performed using spss 11.5 software; p o0.05 and p o0.01 were defined as statistically significant and highly statistically significant, respectively. a novel small molecule inhibitor of hepatitis c virus entry phage-displayed peptides as developmental agonists for phytophthora capsici zoospores phage-displayed peptides selected for binding to campylobacter jejuni are antimicrobial peptides selected for binding to a virulent strain of haemophilus influenzae by phage display are bactericidal the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex 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transmissible gastroenteritis coronavirus infection in vitro action mechanisms of lithium chloride on cell infection by transmissible gastroenteritis coronavirus development of a porcine epidemic diarrhea virus m protein-based elisa for virus detection phages harboring specific peptides that recognize the n protein of the porcine reproductive and respiratory syndrome virus distinguish the virus from other viruses cholesterol dependence of pseudorabies herpesvirus entry genetic evolution and tropism of transmissible gastroenteritis coronavirus virus taxonomy, eighth report of the international committee on taxonomy of viruses coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. microbiol potent dpeptide inhibitors of hiv-1 entry phage displayed peptides to avian h5n1 virus distinguished the virus from other viruses potent suppression of viral infectivity by the peptides that inhibit multimerization of human immunodeficiency virus type i (hiv-1) vif proteins cholesterol is important for a post-adsorption step in the entry process of transmissible gastroenteritis virus this work was supported by national natural science foundation of china (31340003 and 31372438), sponsored by chang jiang scholar candidates program for provincial universities in heilongjiang (2013cjhb002), the program for new century excellent talents in university of ministry of education of p.r. china (ncet-10-0144). key: cord-274122-n9jnu2ah authors: mielech, anna m.; kilianski, andy; baez-santos, yahira m.; mesecar, andrew d.; baker, susan c. title: mers-cov papain-like protease has deisgylating and deubiquitinating activities date: 2014-02-01 journal: virology doi: 10.1016/j.virol.2013.11.040 sha: doc_id: 274122 cord_uid: n9jnu2ah coronaviruses encode papain-like proteases (plpro) that are often multifunctional enzymes with protease activity to process the viral replicase polyprotein and deubiquitinating (dub)/deisgylating activity, which is hypothesized to modify the innate immune response to infection. here, we investigate the predicted dub activity of the plpro domain of the recently described middle east respiratory syndrome coronavirus (mers-cov). we found that expression of mers-cov plpro reduces the levels of ubiquitinated and isgylated host cell proteins; consistent with multifunctional plpro activity. further, we compared the ability of mers-cov plpro and severe acute respiratory syndrome coronavirus (sars-cov) plpro to block innate immune signaling of proinflammatory cytokines. we show that expression of sars-cov and mers-cov plpros blocks upregulation of cytokines ccl5, ifn-β and cxcl10 in stimulated cells. overall these results indicate that the plpro domains of mers-cov and sars-cov have the potential to modify the innate immune response to viral infection and contribute to viral pathogenesis. middle east respiratory syndrome coronavirus (mers-cov) is a recently described coronavirus with high mortality. as of november 18, 2013, there have been 157 confirmed cases and 66 deaths (http://www.who.int/csr/don/2013_11_18/en/index.html). mers disease is characterized primarily by respiratory symptoms but several patients also developed renal failure drosten et al., 2013) . in most cases reported thus far, immunosuppression or other types of medical disorders have been associated with more severe disease (assiri et al., 2013) . the sequence of the rna genome of mers-cov is most similar to bat coronaviruses hku4 and hku5 ; however, the origin of mers-cov is not known. a recent report showed that dromedary camels have high levels of neutralizing serum antibodies against mers-cov, suggesting a possible zoonotic source (reusken et al., 2013) . in addition, analysis of fecal samples from bats identified an egyptian tomb bat as a potential source of infection (memish et al., 2013) , but more work is needed to identify the animal reservoir(s) for mers-cov. limited humanto-human transmission of mers-cov has been reported, which considering the high mortality, raises a concern that the virus has a potential to become a threat to public health (assiri et al., 2013; guery et al., 2013) similar to severe acute respiratory syndrome coronavirus (sars-cov). the sars-cov pandemic from 2002-2003 was controlled by public health measures of identification and isolation of infected, symptomatic individuals and their contacts which broke the chain of human-to-human transmission. a sars-cov-like virus is endemic in chinese horseshoe bats, but changes in the sequence of the spike glycoprotein are required for this virus to efficiently infect humans (lau et al., 2005; rockx et al., 2007) . for mers-cov, it is unclear if the virus can jump directly from bats to humans, if there are any mutations in the viral genome that facilitate infection or disease in humans, and if there are both symptomatic and asymptomatic cases, which would make any potential epidemic more difficult to control by public health measures alone. our goal was to apply the knowledge gained from the study of sars-cov to identify and characterize the mers-cov papain-like protease domain as an innate immune antagonist and as a potential target for therapeutics. mers-cov, similar to other coronaviruses, is a positive-strand rna virus that upon entry into cells is translated to produce a viral replicase polyprotein. the replicase polyprotein is processed by viral proteases to generate a membrane-associated viral replication complex (snijder et al., 2006; perlman and netland, 2009 ). sequence analysis of mers-cov indicates that the canonical papain-like protease (plpro) and 3c-like proteinase (3clpro) are likely responsible for processing the polyprotein to generate 16 nonstructural proteins that assemble to form the replication complex. the majority of coronavirus papain-like proteases (plps), including sars-cov plpro, have been shown thus far to act as deubiquitinases and interferon antagonists (clementz et al., 2010; frieman et al., 2009; ratia et al., 2006; zheng et al., 2008; xing et al., 2013; barretto et al., 2005; sulea et al., 2005; chen et al., 2007) . the ubiquitin pathway is important for regulating a number of innate immune pathways and the ability of a viral protein to cleave ubiquitin from host cell proteins can contribute to virus pathogenesis. in addition to ubiquitination, modification of cellular proteins with interferon-stimulated gene 15 (isg15) is known to have a broad-spectrum antiviral activity. isg15 is ubiquitin-like protein that can be conjugated to cellular targets via a mechanism called isgylation, regulating innate immune responses. coronavirus plps are known to have the ability to remove isg15 conjugates from cellular substrates (clementz et al., 2010; lindner et al., 2005 lindner et al., , 2007 . in this study, we demonstrate the deisgylating and deubiquitinating (dub) activities of the papain-like protease from mers-cov, and provide new information on the potential role of coronavirus protease/dubs to inhibit the innate immune response. mers-cov plpro is encoded within nonstructural protein 3 (nsp3) of the replicase polyprotein (fig. 1a) . to gain insight into the potential of mers-cov plpro to recognize and cleave ubiquitin and isg15 from proteins, we used the high-resolution x-ray structure of sars-cov plpro in apo-enzyme form (pdb:2fe8, chain c) to generate a homology model of mers-cov plpro. we threaded the mers-cov primary amino acid sequence into the sars-cov structure and then energy minimized the structure. the homology model displays several conserved structural features between mers-cov and sars-cov plpro; including the ubiquitinlike domain (ubl), a catalytic triad consisting of c1594-h1761-d1776 and the ubiquitin-binding domain at the zinc finger. to model ubiquitin (ub) into the zinc finger and palm domains of mers-cov plpro, we used the x-ray structure and associated electron density of sars-cov plpro in complex with ub aldehyde (ubal) (pdb:4mm3) for refinement and energy minimization of the model in complex with ub. the resulting mers-cov-ubal model displays a nearly ideal fit of the ub moiety within the palm and the zinc finger regions of the enzyme with the c-terminal extension of ubiquitin oriented properly towards the mers-cov substrate subsites and catalytic triad (fig. 1b) . from this model, we hypothesize that the plpro domain from mers-cov, like sars-cov is a multifunctional enzyme with protease, deubiquitinating and likely deisgylating activity. we recently described expression and protease activity of mers-cov plpro in cell culture (kilianski et al., 2013) . to determine the deisgylating activity of mers-cov plpro, we transfected hek293t cells with c-myc-isg15 plasmid, isg15 conjugation machinery, and increasing amounts of plasmids expressing mers-cov plpro wild-type and catalytic mutant c1594a (plproca). cysteine 1594 is predicted to be the active site cysteine nucleophile that attacks the substrate peptide bond and mutation to alanine should significantly reduce or abolish enzymatic activity (fig. 1 ). in addition, we transfected cells with plasmids expressing sars-cov plpro wild-type or catalytic mutant (c1651a). we harvested cell lysates at 20 h posttransfection to evaluate the presence of isgylated proteins. we found that both mers-cov and sars-cov plpro can deconjugate isg15 from multiple cellular substrates in a dose-dependent manner. in contrast, plpro catalytic mutants did not deconjugate isg15, indicating that catalytic activity of plpro is required for its deisgylating activity ( fig. 2a) . thus, mers-cov plpro like sars-cov plpro (lindner et al., 2007) has deisgylating activity. to assess the dub activity of mers-cov plpro, we transfected hek293t cells with plasmid expressing flag-ub and increasing amounts of wild-type plpro or plproca. we determined that plpro can deubiquitinate multiple cellular substrates, and that plpro catalytic activity is required for dub activity (fig. 2b ). this dub activity is also observed with expression of sars-cov plpro, consistent with previous reports (frieman et al., 2009; ratia et al., 2006; barretto et al., 2005; lindner et al., 2005 lindner et al., , 2007 . in these experiments, we noticed the difference in the expression levels of sars-cov and mers-cov plpros in transfected cells, which may be due to differences in codon optimization in the mers-cov plpro construct. further in vitro studies using purified enzymes are needed to determine the relative kinetics of sars-cov and mers-cov plpro dub and deisgylating activities. taken together, our data indicate that mers-cov plpro is a potent deisgylating enzyme that also exhibits dub activity and that both activities require cysteine 1594 for catalysis, likely in the context of the predicted catalytic triad (fig. 1b) . coronavirus plps have been shown to block interferon β (ifnβ) induction in transfected cells (clementz et al., 2010; frieman et al., 2009; devaraj et al., 2007) . in addition, the deubiquitinase function of an arterivirus papain-like protease has been shown to have a role in interferon antagonism during virus infection (van kasteren et al., 2013) . therefore, we assessed the ability of mers-cov plpro to antagonize interferon production. first, we addressed if mers-cov plpro can inhibit mda5 induced ifnβ reporter, since mda5 has been implicated in recognition of coronaviruses during virus infection (zust et al., 2011) . we transfected hek293t cells with plasmids expressing ifn-β-luciferase, renilla luciferase, pef-bos-mda5 (rothenfusser et al., 2005) and increasing amounts of wild-type plpro or plproca. at 16 h posttransfection we assessed luciferase reporter activity. we determined that mers-cov plpro can potently inhibit mda5 mediated induction of ifnβ in a dose-dependent manner and that catalytic activity of mers-cov plpro is required for ifnβ antagonism (fig. 3a ). using overexpression of an active form of rig-i, we determined that mers-cov plpro can also inhibit n-rig-i induced ifnβ reporter. similarly to the experiment with mda5 stimulation, the catalytic activity of mers-cov plpro is necessary for ifnβ antagonism upon n-rig-i stimulation (fig. 3b) . upon recognition of viral rna by pattern recognition receptors (prrs) such as mda5 or rig-i the signal is transmitted downstream via mitochondrial antiviral signaling protein (mavs). thus, we tested if plpro is able to inhibit mavs induced ifnβ reporter. to stimulate the ifnβ reporter, we overexpressed pef-bos-mavs (rothenfusser et al., 2005) in hek293t cells, co-expressed reporters, and either the wild-type plpro or plproca. we found that plpro, but not plproca inhibits mavs induced ifnβ reporter (fig. 3c ). finally, we tested the ability of mers-cov plpro to inhibit nf-κb reporter activity as observed with sars-cov plpro. we transfected cells with plasmids expressing nf-κb luciferase, renilla luciferase, and mers-cov wild-type plpro or plproca, treated cells with tnfα to activate the nf-κb pathway, and harvested cell lysates at 4 h post-treatment to assess luciferase activity. we determined that wild-type plpro can reduce induction of nf-κb reporter in a dose-dependent manner and that the catalytic cysteine residue is required for this activity (fig. 3d) . taken together these results indicate that mers-cov plpro is an interferon antagonist and that catalytic activity is required for the antagonism. in addition, plpro can reduce tnfα-mediated induction of nf-κb reporter activity and catalytic activity is also required. to further investigate the role of coronavirus plpros in inhibiting innate immune responses we tested the effect of mers-cov plpro on the expression of endogenous cytokines. first, using the human innate and adaptive immune responses pcr array (sabiosciences) we determined that in hek293t cells ccl5 (rantes), ifnβ, and cxcl10 (ip-10) mrna levels are upregulated more than 20-fold upon mda5 stimulation (data not shown) and therefore selected these genes for further analysis. to determine the effect mers-cov plpro and sars-cov plpro on cytokine expression, we performed qrt-pcr to measure mrna encoding ccl5, ifnβ, and cxcl10 levels in the presence of cov plpros. hek293t cells were transfected with pef-bos-mda5, and wild-type or catalytic mutants of mers-cov or sars-cov plpros. at 18 h posttransfection the total rna was extracted and qrt-pcr was performed. we found that both mers-cov and sars-cov plpro can potently inhibit (over 3-fold reduction) expression of ccl5 upon mda5 stimulation and that catalytic activity is required for this inhibition (fig. 4a ). in agreement with the results from luciferase reporter assays, we observed that expression of ifnβ in mda5 stimulated cells is inhibited in the presence of wild-type mers-cov plpro and sars-cov plpro (fig. 4b ). cxcl10 mrna levels were also significantly reduced (po0.0005) when wild-type, but not catalytic mutant versions of mers-cov plpro and sars-cov plpro were expressed (fig. 4c) . to our knowledge, this is the first report showing that both mers-cov plpro and sars-cov plpro can reduce induction of endogenous proinflammatory cytokines in cells, and that the mechanism requires catalytic activity. viruses must "do more with less" because of the compact nature of their genomes. one example of this is the multifunctional plp domain encoded in all members of the order nidovirales. nidoviruses, including those in the coronavirus and arterivirus families, encode one or more plp domain. these plps are critical for proteolytic processing of the viral replicase polyprotein. in addition to protease activity, many of these plps have also been shown to act as viral deubiquitinating enzymes (dubs), able to deconjugate ubiquitin and isg15 from cellular substrates. coronavirus dub activity was first proposed by molecular modeling of the sars-cov plpro domain which predicted that the protease may be multifunctional . indeed, analysis of the dub activity of purified cov plps and the x-ray crystal structure of sars-cov plpro fully support the initial prediction of viral dub activity (ratia et al., 2006; barretto et al., 2005; lindner et al., 2005; ratia et al., 2008) . analysis of plps from coronaviruses and arteriviruses have revealed conserved dub activity; although the enzymes in the coronavirus family fall into the ubiquitin specific protease (usp) family whereas the arterivirus plps are in the ovarian tumor (otu) domain family of enzymes. the identification of a newly emerged coronavirus mers-cov provides an opportunity to evaluate plpro enzymatic activity and develop new hypotheses about how this protease/dub may contribute to viral pathogenesis. our modeling of the mers-cov plpro domain onto the structure of sars-cov led to the prediction of viral dub/deisgylating activity. although the enzymes are only $ 30% identical to sars-cov plpro at the amino acid level, we show that deisgylating, dub and interferon antagonism activities are conserved. importantly, we show that coronavirus plpro activity can modulate the innate immune response. coronaviruses have been shown to modulate immune responses upon infection, however the mechanisms involved in the regulation are not yet clear (totura and baric, 2012) . cytokine and chemokine responses to sars-cov in non-lymphatic cells and in infected patients results in low levels of several cytokines, including ccl5 and ifnβ (wong et al., 2004; spiegel and weber, 2006) . in addition, cxcl10, ccl5, and ifnβ among others, are not induced in cloned bronchial epithelial cell line and human alveolar type ii cells infected with sars-cov early post infection (yoshikawa et al., 2010; qian et al., 2013) . the innate immune response to mers-cov is also intriguing. microarray analysis of mers-cov infection of calu-3 cells results in distinct immune response compared to sars-cov infection. expression of multiple genes involved in activation of adaptive immune responses, such as mhc class i and ii, are downregulated in mers-cov infected cells (josset et al., 2013) . the ability of sars-cov and mers-cov to modulate early immune responses is likely due to multiple proteins encoded within virus genomes that may act as interferon antagonists. previous reports showed that several coronavirus proteins can block the activation of innate immune responses, particularly production and induction of ifnβ response (reviewed in (totura and baric, 2012) ). structural proteins, such as sars-cov nucleocapsid (n) and membrane protein (m), in addition to being critical elements of the viral particles, have been shown to block the ifn response. several accessory proteins (sars-cov orf3b, orf6, and mouse hepatitis virus ns2) are known to act as antagonists of innate immunity (kopecky-bromberg et al., 2007; zhao et al., 2012) . indeed, mers-cov accessory protein 4a has been reported to block induction of ifn (niemeyer et al., 2013) . in addition, nonstructural proteins including nsp1, nsp7, nsp15 have been implicated as ifn antagonists (frieman et al., 2009; kamitani et al., 2009) . importantly, plps encoded within nsp3 have been shown to block ifnβ induction. sars-cov plpro and hcov-nl63 plp2 are interferon antagonists and catalytic activity is important for plpro antagonism (clementz et al., 2010; frieman et al., 2009; devaraj et al., 2007) . sars-cov plpro is the only plp in the sars-cov genome and it is both a deisgylating and a deubiquitinating enzyme (ratia et al., 2006; lindner et al., 2005; lindner et al., 2007) . on the other hand, hcov-nl63 encodes two papain-like proteases, plp1 and plp2, in the genome but only plp2, which has 22% homology to sars-cov plpro, is an ifn antagonist with deisgylating and dub activities (clementz et al., 2010) . hcov-nl63 plp1 is devoid of these activities . in addition, mouse hepatitis virus plp2 and porcine epidemic diarrhea virus plp have dub activity and act as ifn antagonists (zheng et al., 2008; xing et al., 2013) . plps from arteriviruses are also known to block ifn responses. the n terminal region of nsp2 encodes the papain-like protease in porcine reproductive and respiratory syndrome virus (prrsv). this plp has been characterized as an otu with deubiquitinating and deisgylating ability (frias-staheli et al., 2007) . in addition, sun et al. (2010) , showed that prrsv plp domain can block sendai virus induced ifnβ, and can also inhibit nf-κb by preventing iκbα degradation by its deubiquitination. a more recent report showed that prrsv plp has also deisgylating activity which suggests multiple roles of prrsv papain-like protease in antagonism of innate immunity (sun et al., 2012) . nsp2 of another member of the arteriviridae, equine arteritis virus (eav), has deubiquitinating and deisgylating activities as well (frias-staheli et al., 2007) . the deubiquitinating ability of eav plp can block rig-i induced ifn by inhibiting ubiquitination of rig-i, which is required for its activation . co-crystal structure of eav plp with ubiquitin reveled potential interaction sites between those molecules, and mutagenesis studies showed that plp dub activity is required for inhibition of innate immunity in infected cells (van kasteren et al., 2013) . specific deubiquitinating and deisgylating activities have been shown for crimean-congo hemorrhagic fever virus (cchfv) which is a highly pathogenic, negative-strand rna virus belonging to the family bunyaviridae. the l protease of cchfv contains otu domain with the ability to cleave isg15 modification. l protease can remove isg15-meidated immune protection in type i ifn receptor knock-out mice and make them highly susceptible to sindbis virus infection (frias-staheli et al., 2007) . overall, the results from multiple laboratories studying a variety of coronavirus, arterivirus, and bunyavirus proteases indicate that deubiquitinating and deisgylating activity of viral proteases have an important role for inhibition of innate immune responses and possibly virus pathogenesis. here, we characterized the papain-like protease from mers-cov revealing the deisgylating and deubiquitinating activities, and that it can act as an interferon antagonist. further, we showed for the first time that sars-cov plpro and mers-cov plpro can block induction of several endogenous proinflammatory cytokines. our data suggest that antagonism of innate immune responses mediated by mers-cov and sars-cov plpros is not limited to ifnβ, but may affect expression of many cellular cytokines. our results suggest that plpro might contribute to the modulation of innate immune responses upon sars-cov and mers-cov infection, however, the exact mechanism and the role of coronavirus plps and their associated dub and deisgylating activities in these processes remains to be determined. the crystal structure sars-cov plpro (pdb:2fe8) was used as the template structure to generate a homology model of mers-cov plpro using the automated web-based homology modeling server 3d-jigsaw (bimolecular modeling laboratory, cancer research uk, england). ccp4 program suite 6.2.0 and coot version 0.6.2 were used for final refinement, energy minimization and modeling of ub into the zinc finger and palm regions of mers-cov plpro by using the electron density of sars-cov plpro in complex with ubiquitin-aldehyde (pdb:4mm3). hek293t cells were cultured in dulbecco's modified eagle medium (dmem) with 10% fetal calf serum (fcs) and 2% glutamine. transfections were performed with 70% confluent hek293t cells in cell bind plates (corning) using transit-lt1 reagent (mirus) according to manufacturer's protocol. the mers-cov plpro (pcdna-mers-plpro) expression plasmid and generation of catalytic mutant were described previously (kilianski et al., 2013) . pcdna-sars-plpro wild-type and catalytic mutant expression plasmids were described elsewhere (barretto et al., 2005) . for the luciferase assay experiments we used ifnβ-luc deisgylating activity assay hek293t cells in 12-well plates were transfected with 10, 25, 50, 100 ng of pcdna-mers-plpro wild-type or catalytic mutant, and 250 ng pisg15-myc, 125 ng pubch8, 125 ng pube1l, and 125 ng pherc5. at 20 h post-transfection, cells were lysed with lysis buffer (20 mm tris (ph 7.5), 150 mm nacl, 1 mm egta, 1 mm edta, 1% triton x-100, 2.5 mm na pyrophosphate, 1 mm betaglycerophosphate, 1 mm na ortho-vanadate, 1 mg/ml leupeptin). proteins were separated by sds-page, and transferred to pvdf membrane using a semi-dry transfer apparatus (biorad). following transfer, the membrane was blocked using 5% dried skim milk in tbst buffer (0.9% nacl, 10 mm tris-hcl, ph ¼7.5, 0.1% tween 20) overnight at 4 1c. the membrane was incubated with mouse anti-myc antibody (mbl) at the dilution of 1:2500. the membrane was washed 3 times for 15 min in tbst buffer. following the membrane was incubated with secondary goat-anti-mouse-hrp antibody at the dilution 1:5000 (amersham). then the membrane was washed 3 times for 15 min in tbst buffer. the detection was performed using western lighting chemiluminescence reagent plus (perkinelmer) and visualized using fluorocheme imager (protein simple). to verify expression of the plpro the membrane was probed with mouse anti-v5 antibody (invitrogen) at the dilution 1:5000. mouse anti-calnexin antibody (cell signal) at the dilution 1:2000 was used to determine loading standard. to assess dub activity, hek293t cells in 12-well plates were transfected with 400 ng pcdna3.1-flag-ub and 0.25, 0.5, or 1 mg pcdna-mers-plpro wild-type or catalytic mutant. at 18 h posttransfection, cells were lysed with 100 ml of lysis buffer. proteins were separated by sds-page and transferred to pvdf membrane as described above. membrane probing was performed using mouse anti-flag m2 antibody (sigma) at the dilution of 1:2000. hek293t cells in 24-well plates were transfected with 50 ng renilla-luciferase, 100 ng ifn-β-luc, and 25, 50, and 100 ng pcdna-mers-plpro wild-type or catalytic mutant expression plasmids. as a stimulation 150 ng pef-bos mda5, or 50 ng pef-bos mavs, or 50 ng n-rig-i per well was transfected. empty pcdna3.1-v5/his-b vector plasmid was used to standardize the total amount of dna used for transfection. at 16 h post-transfection cells were lysed using 1x passive lysis buffer (promega). alternatively, the cells were transfected with 50 ng pgl4 32 [luc2 nf-κb-re hyrgo], 100 ng ifnβ-luc and pcdna-mers-plprov5 wild-type or catalytic mutant for 12 h and then treated with 10 ng/ml tnfα (roche) for 4 h, and lysed. for all experiments firefly and renilla luciferase were measured using dual luciferase reporter assay system (promega) and luminometer (veritas). results were normalized to renilla luciferase expression control. experiments were performed in triplicate. remaining lysates were incubated with lysis buffer a (0.9% nacl, 10 mm tris-hcl, ph 7.5, 0.1% tween-20) and analyzed by sds-page as described above. hek293t cells in 12-well plates were transfected with 300 ng pef-bos mda5 and 200 ng pcdna-mers-plpro wild-type or catalytic mutant expression plasmids, or 200 ng pcdna-sars-plpro wild-type or catalytic mutant expression plasmids. empty vector plasmid pcdna3.1-v5/his-b vector was used to standardize the total amount of dna in each sample. the cells were lysed 18 h posttransfection with buffer rlt (qiagen) and rna was extracted using rneasy mini (qiagen). reverse transcription was performed using 1 mg of total rna and the rt 2 first strand kit (qiagen) according to manufacturer's protocol. 1 ml of cdna was used to set up qrt-pcr reaction according to the manufacturer's protocol using single primer assay for ifnβ, cxcl10, and ccl5 (sabiosciences). c t values were normalized to housekeeping gene (rpl13). hospital outbreak of middle east respiratory syndrome coronavirus the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity deubiquitinating and interferon antagonism activities of coronavirus papainlike proteases proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus nl63 clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection regulation of irf-3-dependent innate immunity by the papain-like protease 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rna helicase lgp2 inhibits tlr-independent sensing of viral replication by retinoic acid-inducible gene-i a noncovalent class of papain-like protease/deubiquitinase inhibitors blocks sars virus replication ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex deubiquitination, a new function of the severe acute respiratory syndrome coronavirus papain-like protease? inhibition of cytokine gene expression and induction of chemokine genes in non-lymphatic cells infected with sars coronavirus the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein 2 possesses deubiquitinating and interferon antagonism functions nonstructural protein 2 of porcine reproductive and respiratory syndrome virus inhibits the antiviral function of interferon-stimulated gene 15 sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon deubiquitinase function of arterivirus papain-like protease 2 suppresses the innate immune response in infected host cells arterivirus and nairovirus ovarian tumor domaincontaining deubiquitinases target activated rig-i to control innate immune signaling plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase dynamic innate immune responses of human bronchial epithelial cells to severe acute respiratory syndrome-associated coronavirus infection isolation of a novel coronavirus from a man with pneumonia in saudi arabia plp2, a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production ribose 2′-o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda5 antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns2 protein is required for virus replication and liver pathology we thank dr. xufang deng for helpful discussions and review of the manuscript. this work was supported by the nih grant r01 ai085089 (to scb and adm). amm was supported by arthur j. schmitt dissertation fellowship from loyola university chicago. ak was supported by the nih training grant in experimental immunology (nih t32 ai512795). key: cord-283998-whwksoxt authors: tannock, gregory a.; hierholzer, john c. title: the rna of human coronavirus oc-43 date: 1977-12-31 journal: virology doi: 10.1016/0042-6822(77)90126-x sha: doc_id: 283998 cord_uid: whwksoxt abstract a homogeneous rna complex with a sedimentation coefficient of 70 s and an apparent molecular weight of approximately 6.1 × 106 was released from purified 32p-labeled, mouse-brain-derived oc-43 virus after treatment with 1% sodium dodecyl sulfate (sds) for 15 min at 23°. the complex was highly susceptible to heat, releasing 4 s rna fragments at 37° and breaking down to fragments of 4–70 s at 60°; it was also degraded by centrifugation through dimethyl sulfoxide gradients. unlike tobacco mosaic virus or rous sarcoma virus rna, oc-43 rna prepared by extraction with phenol-sds or phenol-chloroform degraded into a range of fragments with coefficients of 15–55 s; 4 s rna was also present as a minor component. this suggests that (a) extensive nicking of a large rna molecule has occurred during viral growth, due to ribonucleases which are inactivated during phenol extractions; (b) heterogeneity for oc-43 rna is not due to internal ribonuclease activity and fragments are held together by noncovalent linkages much weaker than those present in the 70 s retroviral rna complex, or by small proteins; or, most probably, (c) a combination of extensive nicking and weak noncovalent linkages results in the heterogeneous denaturation products. or, most probably, (cl a combination of extensive nicking and weak noncovalent linkages results in the heterogeneous denaturation products. the coronaviridae were described as a separate taxonomic group by almeida et al. (1968) but, despite their ubiquity and economic importance, few biophysical characterization studies have been reported. the rna of avian infectious bronchitis virus (ibv) was first described by tannock (1973) , who obtained from purified virions a highly heterogeneous array of rna fragments using a phenol-sodium dodecyl sulfate (sds) extraction procedure. fragments ranged in molecular weight from 0.5 x lo6 to more than 3.0 x 106; a smaller, more homogeneous class of a size similar to ribosomal 4 s rna was also present. more recently, watkins et al. (1975) scribed ibv rna, after its release from virions by sds lysis, as a single species having a molecular weight of 9.0 x lo6 and a sedimentation coefficient of 50 s. they reported, but without supporting evidence, that by phenol extraction a range of rna fragments could be obtained which was similar to that tannock (1973) described. garwes et al. (1975) examined the rnas of two porcine coronaviruses, transmissible gastroenteritis virus (tgev) and hemagglutinating encephalomyelitis virus (hev), after preparation by sds lysis. they obtained a single large rna peak with electrophoretic mobility similar to that of 70 s rous sarcoma virus (rsv) rna. like rsv rna (duesberg, 19681 , the rnas of tgev and hev could be readily dissociated to 35 s and 4 s subunits, if prepared by sds lysis at temperatures above 60". in the present work, we attempt to resolve apparent differences in coronaviral rna profiles obtained by different methods of preparation, using human coronavir-us oc-43 as the model for study. copyright 0 1977 by academic press, inc. all rights of reproduction in any form reserved. issn 00426622 was first isolated in human tracheal organ culture, but it has been adapted to grow to high titer in suckling mouse brain (mc-intosh et al., 1967) . unlike 2293, a human coronavirus strain which can be propagated only in cell culture but is extremely labile (hierholzer, 1976) , oc-43 is a relatively stable virus and possesses a hemagglutinin (kaye et al., 1970; hierholzer et al., 1972) . it is, therefore, a more convenient model for study. solutions and reagents. the following buffers were used: net (0. virus was released from cells in the suspension by freezing and thawing four times, and particulate matter was removed by centrifuging at 5,000 g in a spinco 40 rotor for 20 min. the supernatant was retained in an ice bath while the pellet was resuspended in pbs to 20% of the original volume and recentrifuged; both supernatants were then combined for purification. virus was partially purified by adsorption to and elution from human "0" erythrocytes according to the method of kaye et al. (1970) . virus-adsorbed erythrocytes were washed three times with ice-cold pbs, and virus was finally eluted in two steps at 37" into approximately 20% of the original volume of pbs. in some early purifications, the eluate was then treated with ribonuclease and deoxyribonuclease (each at 50 pg/ml final concentration) in order to degrade any cellular nucleic acids adherent to the virus. when later work revealed that the profile of rna extracted from virus treated in this manner was similar to that from untreated virus, we omitted the step. virus in the eluate was then concentrated by centrifuging through a 15% sucrose interface to a 0.75-ml cushion of 65% sucrose in net at 43,200 g for 30 min in a spinco sw 36 rotor. the concentrates were pooled, diluted with net, and centrifuged to equilibrium for 15 hr at 97,loog through a lo-ml 25-65% sucrose gradient in net at 7" in an sw 36 rotor. thirtydrop fractions were collected, and aliquots of each were assayed for total radioactivity, hemagglutinin, density (from refractive index), and absorbance at 280 nm. a typical profile is seen in fig. 1a . two visible bands are present: (1) a lower opalescent band in the density range 1.168-1.202 g/ml containing all hemagglutinin activity and (2) an upper, more flocculent band of membranous material, having a density of 1.074-1.133 g/ml and no hemagglutinin. fractions 6-10 containing the lower band were then pooled, diluted with net, and centrifuged in a velocity gradient in the sw 36 rotor for 1 hr at 43,200 g in a 9.0ml 15-65% sucrose gradient in net. fractions were collected and assayed for hemagglutinin and radioactivity. the profile in fig. 1b equilibrium centrifugation may be further resolved into (1) a fast-sedimenting peak with radioactivity but little hemagglutinin, and (2) an upper heterogeneous peak of coincident hemagglutinin and radioactivity. the lower peak, which is not usually visible, probably consists of internal cell membranes and has been observed by pocock and garwes (1975) in the purification of tgev. heterogeneity in the upper peak is consistent with the high degree of pleomorphism observed for coronaviruses (almeida et al., 1968) . fractions from the upper peak, which were shown by electron microscopy to contain highly purified virus, were pooled, diluted to 5% sucrose with net, and held at 4" for rna analysis. final virus preparations contained ap proximately 11 cpm/ha unit and purification resulted in a 1cfold enrichment of hemagglutinin per unit of protein. rna extraction. the following methods were used. (1) in the warm phenol-sds (wps) method (tannock, 19731 , the viral suspension was shaken for 10 min at 37" with phenol containing 1% sds. the two phases were separated by centrifugation, and the extraction was repeated. after a suitable marker rna, 100 pg of carrier rna, and sodium acetate to 0.15 it4 were added, 2 vol of ethanol were added and all rnas were allowed to precipitate for 16 hr at -15". the precipitate was collected by centrifuging at 43,200 g for 30 min in an sw 36 rotor and then dissolved in a suitable volume of net. all rnas were stored -70" for analysis. (2) in the sds-lysis (sl) method , sds was added to 1% (w/v) to a purified virion suspension, and the mixture was held at 23" for 15 min. the rnas released were mixed with suitable marker rna and immediately analyzed in preformed sucrose gradients or in polyacrylamide gels. (3) in the phenol-chloroform (pc) method for influenza modified from pons (1975) by using the ribonuclease inhibitor diethyl pyrocarbonate (dep) in place of bentonite, the virus sample was mixed with 5% dep, 0.1 vol2-mercaptoethanol, 1 vol pc extraction buffer, and 1 vol of phenol:chloroform (1:l) and shaken for 3 min at 23". the phases were separated by centrifugation, and the extraction of the aqueous phase was repeated. precipitation in the presence of marker and carrier rnas and centrifugation were as described for the wps method. rna sucrose gradient analysis. prepared by phenol extraction were centrifuged through 4.4 ml of 15-30% sucrose gradients in net in spinco sw-50.1 rotor in a model l-2 for 2.25 hr at 189,000 g and 7". those prepared by sds lysis were centrifuged in a beckman model l-4 ultracentrifuge for 2 hr at 189,000 g and 7" (tannock and griffith, 1975) . methods used for other coronaviruses rna was prepared from two samples of 32p-labeled oc-43 virus by the wps and sl methods. profiles of oc-43 and marker rnas from each gradient after centrifugation are shown in fig. 2 . with the wps method ( fig. 2a) uted broadly in the 15-50 s region of the gradient, and a smaller quantity of 4 s rna was also present. with the sl method (fig. 2b) , a single large homoge-oc-43 rna, prepared by the sl method, neous rna was obtained with a sedimenmigrates as a homogeneous complex with tation coefficient identical to the major an electrophoretic mobility similar to that (70 s) component of rsv rna. the pc of both 45 s ribosomal precursor rna and method was used for all subsequent ex-(not shown in this figure) 70 s rsv rna. tractions involving phenol because of the semilogarithmic plots of molecular weight greater rna yields obtained (tannock versus electrophoretic mobility from repliand hierholzer, unpublished observation) cate experiments gave a mean apparent and the observation (molloy et al., 1974) molecular weight of 6.1 x lo6 for this undethat phenol-chloroform mixtures congraded complex (fig. 4) . acrylamide gels served polyadenosine sequences in rna. electrophoresed at 23" revealed considera-page of oc-43rna ble breakdown of the complex; this thermolability of oc-43 rna was confirmed in the profiles obtained for oc-43 rna by later experiments (see fig. 5 ). no discrete sucrose-gradient centrifugation were com-viral 4 s rna can be seen in fig. 3a and pared with those obtained by page. figthe firm this with oc-43 rna, preparations of oc-43 virus were treated with 1% sds by the sl method at 23", 37", and 60". the distributions of acid-insoluble radioactivity for each gradient suggest that at 37 there was some breakdown of the 70 s complex into smaller 4 s fragments, and at 60" a more generalized breakdown to a range of intermediate species occurred (fig. 5) (repik and bishop, 1973) ; 28 and 18 s ribosomal rna, 1.75 and 0.70 x 106, respectively (loening, 1968) ; 4 s ribosomal rna, 0.025 x 10" (staynov et al., 1972) . the position of the oc-43 rna complex released by the sl method is indicated. rna components found at the top of gradients for total radioactivity (see fig. 7a ). the electrophoretic profiles of oc-43 rna, after preparation by the pc method, were determined at 5". clearly, the pc method resulted in considerable breakdown of the large complex into a range of rna fragments with electrophoretic mobilities between 45 s and 18 s (fig. 3b) . a minor 4 s component was also present; it was similar to that found for ibv rna after extraction by the wps method (tannock, 1973 garwes et al. (1975) reported that rna complexes from the porcine coronaviruses tgev and hev were degraded if extracted by sds lysis at temperatures above 60", as has been noted for the rnas of retroviridae (duesberg, 1968 ). to con(duesberg, 1968; bishop et al., 1970a, b; baltimore, 1974; king, 1976) . rnas in each mixture, after preparation, were precipitated with ethanol in the presence of carrier rna and analyzed in sucrose gradients . the profiles of acid-insoluble radioactivity for each gradient are shown in fig. 6 was mixed with 25 ~1 of net containing 5 pg of ribonuclease for 30 min at 25"; the acid-insoluble radioactivity remaining was then determined after drying on paper strips. figures 6a and 6b suggest the same heterogeneity for the major class of oc-43 rna fragments already noted by the phenol extraction method (figs. 2, 31 , and again confirm the presence of a minor 4 s component. both tmv and the major 70 s rna of rsv remain undegraded after extraction. this suggests either (a) that the heterogeneity of the major rna class is caused not by the activation of ribonucleases but by disruption of noncovalent linkages between rna fragments which are much weaker than similar linkages within 70 s rsv rna, or (b) a large rna genome is extensively nicked by virion ribonucleases during viral growth and these enzymes are inactivated by phenol extraction. sedimentation coefficients for the major rnas released by phenol (15-55 s; fig. 6b ) were less than those for the homogeneous 70 s peak obtained by the sl method (fig. 2b) . all 32p-labeled structures throughout the gradient in fig. 6a are clearly rna, as judged by their uniform susceptibility to ribonuclease. a similar experiment was then carried out to extend this finding for 32p-labeled material released by the sl method. sl-extracted virus was centrifuged to release the rna as a 70 s complex (fig. 2b) , and half of each gradient fraction was treated with ribonuclease, as above. the profiles for acid-insoluble ragradient fraction were assayed for total radioactivity, and the profile is shown in a. fractions 3-7, containing 70 s rna, were pooled and reextracted in the presence of 0.1 ml of wps-extracted 13hluridine tmv rna. all rnas present were then reprecipitated in ethanol after 100 pg of carrier rna was added. the precipitate was dissolved in 0.5 ml of net and analyzed in 15-30% sucrose gradients. the profile of acid-insoluble radioactivity for and tmv (0---0) rnas is shown in b. fifty microliters of a preparation of 3zp-labeled oc-43 was treated to release 70 s rna by the sl method. the preparation was then centrifuged through 5-20% sucrose gradients in dmso containing 10 pm licl for 5 hr at 189,000 g and 22"; the profile of total radioactivity is shown in c. tannock and hierholzer dioactivity (fig. 6c) indicate that the major 70 s complex released by sds lysis is fully susceptible to ribonuclease, whereas the 32p-labeled material occurring at the top of the gradient remains fully resistant. the latter probably represents structural phospholipids which remain at the top of the gradient after sds lysis and are distinct from ribonuclease-sensitive 4 s rna obtained by the pc method (fig. 6a ). of oc-43 70 s rna in the presence of phenol and dmso further evidence that oc-43 70 s rna is a complex of fragments held together by weak, noncovalent bonds was obtained by (1) isolation of 70 s oc-43 rna by sds lysis and centrifugation, followed by extraction by the pc method in the presence of tmv rna, and (2) isolation of rna by the sl method and centrifugation in dmso gradients. oc-43 70 s rna was lysed with sds and centrifuged to isolate the rna complex as described (see fig. 7 legend). the profile for total (acid-soluble and -insoluble) radioactivity from aliquots of each gradient fraction reveals a sharp peak of 32p radioactivity in the 70 s region (the rna complex) and a much larger peak of low density material at the top of the gradient (fig. 7a) . similarly located but much smaller peaks for acid-insoluble, ribonuclease-resistant radioactivity are seen in figs. 2b, 5a, and 6c. it therefore seems likely that sds lysis separates rna from large amounts of acid-soluble components, some acid-insoluble phospholipids, and perhaps other components in the outer virion coat. fractions 2-10 were extracted by the pc method in the presence of 13hluridine tmv rna, precipitated with ethanol in the presence of carrier rna, and analyzed. the profile for acid-insoluble radioactivity reveals that such treatment completely degraded oc-43 70 s rna, whereas tmv rna remained intact (fig. 7b ). this experiment thus confirms earlier evidence that the 70 s complex is held together by weak noncovalent bonds which are destroyed by phenol extraction (figs. 2, 3) or gentle heating (fig. 5) . the breakdown of isolated 70 s rna by the pc extraction method is more complete than by similar extraction of purified virions. it does not appear to be due to ribonuclease activity because tmv rna within the same mixture remains intact, although phenol extraction could denature oc-43 70 s rna and so reveal breaks in an otherwise intact large rna molecule which were produced by such activity. since dmso has been used as a critical test for noncovalent linkages in rsv 70 s rna (duesberg, 1968) , we investigated its effect on 70 s oc-43 rna prepared by sds lysis. the profile for total radioactivity (fig. 7c) shows a complete breakdown of the 70 s rna complex to smaller fragments which sediment slightly faster than structural phospholipids at the top of the gradient (fig. 7a ). duesberg (1968) noted similar instability for rsv 70 s rna, whose constituent rna fragments are held together by noncovalent bonds less susceptible to phenol extraction than oc-43 rna (fig. 2b ). the oc-43 rna complex released by sds lysis and 70 s rsv rna have identical sedimentation coefficients. the latter is a complex of several noncovalently linked subunits (mw 2.5-3.3 x lo'? which are freed by heating or treatment with dmso (duesberg, 1968; king, 1976) . in addition to 70 s rna, a range of smaller fragments, including a 4 s species, is present in phenol extracts of rsv (robinson et al., 1965; duesberg, 1968; bishop et al., 1970a, b; dahlberg et al., 1974) . the 70 s oc-43 complex may also consist of noncovalently linked rna subunits which are disrupted by dmso, but unlike 70 s rsv, are highly susceptible to phenol. the complex is more readily broken down by heating than are similar complexes isolated from rsv (duesberg, 1968) and the tgev and hev coronaviruses . its apparent molecular weight, determined comparatively by coelectrophoresis with other rnas, is 6.1 x 106; this value is only considered to be approximate, however, since 2.2% gels do not adequately resolve high molecular-weight rnas (loening, 1969; king, 1976) . native oc-43 rna is readily denatured into fragments by organic solvents and heat, much like that observed with b77 sarcoma virus rna by stoltzfus and snyder (1975) . however, the sizes of the oc-43 rna fragments from the disrupted complex seem to vary considerably. phenol extraction of purified virions produces fragments ranging between 15 and 50 s, and a small amount of a homogeneous 4 s rna is also present (figs. 2, 3, 6) . a range of fragments of molecular weight 0.7-5 x lo6 and a smaller species was obtained after page, similar to that described by tanneck (1973) for ibv rna. coextraction experiments with tmv and rsv (fig. 6 ) did not implicate internal ribonuclease activity as a cause of this heterogeneity. when the 70 s complex released by sds lysis was isolated and reextracted with phenolchloroform, only 5-10 s rna fragments were obtained (fig. 7) . why fragments extracted in this manner should be smaller is unknown. again, however, ribonucleases could not be detected, because a preparation of tmv rna, when included in the extraction mixture, remained undegraded (fig. 7b) . possibly, rna extracted by phenol from purified virions contains more secondary structure. alternatively ribonucleases could have been present throughout viral growth, producing nicks in an otherwise intact rna molecule and subsequently being destroyed by phenol-chloroform extraction. the release of 4 s rna from the 70 s complex by heating has also been observed for tgev and hev rnas . recent studies have shown that polyadenylate residues are present in 15-55 s but not in the 4 s rnas obtained by pc extraction (tannock and hierholzer, in preparation) , indicating a functional difference between these rna classes. detailed studies are needed to determine what these functions are. although sds is recognized as an effective rna deproteinizing agent (boatman and kaper, 1973) , the susceptibility of the 70 s oc-43 complex to phenol suggests that small protein linkers could be required to hold the rna fragments together and that they are not removable by sds lysis. evidence against this comes from cesium sulfate gradient experiments in which the buoyant densities of the oc-43 complex prepared by sds lysis and phenol-extracted tmv rna were identical (tannock and hierholzer, unpublished observations) . additionally, lowry protein determinations and 8% acrylamide gels (protein-stained) on purified rna samples containing 120 pg rna did not detect protein, implying that any protein linkers present must constitute less than 2% of the rna structure. the complexing of a few small proteins to a large rna molecule, however, may produce no detectable difference in buoyant density, as sen et az. (1976) have noted for rauscher murine leukemia viral rna and phosphoprotein p12, with a type-specific rna binding affinity. extensive nicking of retroviral 70 s rna due to ribonuclease activity has been observed after extended viral growth in cell culture (bader and steck, 1969; king and wells, 1976) and during virus purifcation (scheele and hanafusa, 1972) . the integrity of the nicked 70 s rna is still maintained after extraction from virions by phenol, being only apparent after treatment with rna denaturants. extensive changes in oc-43 rna conformation produced by phenol suggest that, even if extensive nicking of the 70 s complex occurs during viral growth, there are marked differences in its secondary structure compared to that of 70 s retroviral rna. to determine whether a genuine subunit structure is present in the coronaviral complex, it would be highly desirable to examine the virion rna present at different times after maturation in a more strictly defined milieu than that of the mouse brain. however, for human coronaviruses, the lack of convenient model systems is a severe impediment to this work. coronaviruses analysis of the ribonucleic acid of murine leukemia virus tumor viruses: 1974. cold spring harbor symp the low molecular weight rnas of rous sarcoma virus. i. the 4s rna forces responsible for the generation of virus structures: the use of sds to probe protein-rna interaction structure and properties of an rna primer for initiation of rous sarcoma virus dna synthesis in vitro physical properties of rous sarcoma virus rna identification of heat-dissociable rna complexes in two porcine coronaviruses purification and biophysical properties of human coronavirus 2293 protein composition of coronavirus oc-43 standardized viral hemagglutination and hemagglutination-inhibition tests. ii. description and statistical evaluation purification and further characterization of an "ibv-like" virus (coronavirus) high molecular weight rnas from rous sarcoma virus and moloney murine leukemia virus contain two subunits all intact subunit rnas from rous sarcoma virus contain poly(a) molecular weights of ribosomal rna in relation to evolution the determination of the molecular weight of ribonucleic acid by polyacrylamide-gel electrophoresis. the effects of changes in conformation molecular weights of some hela ribosomal rnas growth in suckling-mouse brain of "ibvlike" viruses from patients with upper respiratory tract disease arrangement of specific oligonucleotides within poly(a) terminated hn rna molecules the influence of ph on the growth and stability of transmissable gastroenteritis virus in vitro intbrenza virus messenger ribonucleoprotein determination of the molecular weight of animal rna viral genomes by nuclease digestions. i. vesicular stomatitis virus and its defective t particle the nucleic acid of the bryan strain cf rous sarcoma virus: purification of the virus and isolation of the nucleic acid electrophoretie analysis of the rna of avian tumor viruses specific binding of the type c viral core protein p12 with purified viral rna molecular weight determination of nucleic acids by gel electrophoresis in non-aqueous solution structure of b77 sarcoma virus rna: stabilization of rna after packaging the nucleic acid ofinfectious bronchitis virus a re-examination of the molecular weight of poliovirus rna degradation of myxovirus virion rna by periodate the ribonucleic acid of infectious bronchitis virus key: cord-271763-cual2qv4 authors: abraham, sushma; kienzle, thomas e.; lapps, william; brian, david a. title: deduced sequence of the bovine coronavirus spike protein and identification of the internal proteolytic cleavage site date: 1990-05-31 journal: virology doi: 10.1016/0042-6822(90)90257-r sha: doc_id: 271763 cord_uid: cual2qv4 abstract the sequence of the spike (also called peplomer or e2) protein gene of the mebus strain of bovine coronavirus (bcv) was obtained from cdna clones of genomic rna. the gene sequence predicts a 150,825 mol wt apoprotein of 1363 amino acids having an n-terminal hydrophobic signal sequence of 17 amino acids, 19 potential n-linked glycosylation sites, a hydrophobic anchor sequence of approximately 17 amino acids near the c terminus, and a hydrophilic cysteinerich c terminus of 35 amino acids. an internal lysargargserargarg sequence predicts a protease cleavage site between amino acids 768 and 769 that would separate the s apoprotein into s1 and s2 segments of 85690 and 65153 mol wt, respectively. amino terminal amino acid sequencing of the virion-derived gp100 spike subunit confirmed the location of the predicted cleavage site, and established that gp120 and gp100 are the glycosylated virion forms of the s1 and s2 subunits, respectively. sequence comparisons between bcv and the antigenically related mouse hepatitis coronavirus revealed more sequence divergence in the putative knob region of the spike protein (s1) than in the stem region (s2). the bovine coronavirus (bcv) is an important cause of neonatal calf diarrhea (14, 26) and may also be the cause of winter dysentery in adult cattle (30). the mechanisms by which bcv causes disease and persistent infection are not understood, nor are current vaccines universally regarded as effective. toward these ends, we have begun a detailed study of the bcv protein and genome structure. bcv is comprised of four major structural proteins (17). these are (i) a 200-kda spike (peplomer) glycoprotein (s), that exists on the virion as cleaved subunits of approximately 120 and 100 kda, (ii) a 140-kda glycoprotein (he) that has both hemagglutinating (18) and esterase (37) activities, and which is comprised of two identical, disulfide-linked 65-kda subunits (10, 12, 16, 28) , (iii) a 26-kda integral membrane glycoprotein (m) (21), and , (iv) an internal phosphorylated nucleocapsid protein (n) (21). of these, the s protein is presumed to ' sequence data from this article have been deposited with the embugenbank data libraries under accession no. m31053. ' present address: department of molecular biology and microbiology, case western reserve university, cleveland, oh 44106. 3 present address: dnx corporation, one president street, athens, oh 45701-2979. 4 to whom requests for reprints should be addressed. be the major structure by which coronaviruses attach to cells and initiate infection (reviewed by spaan et a/. (34)). the he protein, however, may also bind to cells to initiate infection, and for bcv, the relative importance of these two proteins in initiating infection is not known. both s and he are probably important in inducing immunity since antibodies to each are known to neutralize virus infectivity in cell culture and in calves (8, 9) . s and he, therefore, may both be useful in developing effective engineered vaccines against bcv. cdna cloning of bcv genomic rna was accomplished essentially as previously described (11, 21) except that random 5mer oligodeoxynucleotides (pharmacia) and 17-mer oligodeoxynucleotides of specific sequences were used as primers for first-strand synthesis. clones were mapped relative to one another and to the 3' end of the genome using a matrix spot hybridization technique. some clones were sequenced by the chemical method of maxam and gilbert (25) and some by the dideoxynucleotide-induced chain termination method of sanger (3 1) as described by kraft et a/. (19) using sequenase enzyme (united states biochemicals). for much of the sequencing, restriction endonuclease fragments were subcloned into the pgem4z vector (promega) and forward and reverse sequencing kilobases 20 9 8 7 6 5 4 3 2 1 4, ' i 1 i 1 i i 1 i i 3 primers for the pgem vectors were used. sequencespecific oligodeoxynucleotides were also synthesized and used for sequencing within certain regions of the large clones. the amino-terminal ends of purified gp120 and gplo0 subunits were subjected to sequencing by the method of matsudaira (24) . unlabeled bcv was purified by isopycnic sedimentation in sucrose gradients and the proteins were electrophoretically separated after reduction in 2-mercaptoethanol (17) and electroblotted (13) onto polyvinylidene difluoride membrane (24). proteins were visualized by staining with coomassie brilliant blue and the gpl20 and gplo0 bands were excised and shipped to dr. matsudaira for analysis. complete sequencing of clone ma7 which extends 4.2 kilobases from the 3' end of the genome (fig. 1) revealed a continuous open reading frame located on the 5' side of the orf for a potential 4.9-kda protein (abraham et al., to be published elsewhere). the deduced amino acid sequence of the extended orf demonstrated high sequence similarity to the c-terminal end of the antigenically related mhv-a59 (22) and mhv-jhm (32) s proteins, both antigenic homologs of the bcv s protein (13). these data suggested that the s protein gene of bcv lies in the same relative position on the genome as does the spike protein gene of mhv. to complete the sequencing of the s gene, both strands of three clones, ii, hpa2, and g6, generated by random priming, and three clones, lk5, lp6 and 29, generated by specific priming, were sequenced ( fig. 1) . the total sequence for the putative s orf extended to a position 7.4 kb from the 3' end of the genome and contained 4089 bases (fig. 2) . we conclude this orf to be the s gene since it potentially encodes a 1363 amino acid protein of 150,825 da, the approximate size of the unglycosylated spike precursor (io), and be-cause its deduced amino acid sequence shows extensive sequence similarity throughout with the s proteins of both strains of mhv. five other open reading frames ranging in size from 34 to 66 amino acids were also found within the s gene sequence in the plus one reading frame, but their significance is not known at this time. the putative s orf is preceded immediately upstream (beginning at base 12 in fig. 2 ) by the consensus cyaaac sequence thought to play a role in leader priming of coronavirus transcription. the sequence is also found three times within the s orf, beginning at positions 817, 1667, and 3776, but it is not established that transcripts initiate at any of these sites. five features of the deduced bcv s protein reflect the properties of four other coronavirus spike proteins that have been characterized to date from nucleotide sequence data (i, 2, 15, 20, 22, 27, 29, 32) . (i) there is an n-terminal hydrophobic stretch of amino acids which predicts a signal peptide with a cleavage site between amino acids 17 and 18 (38). (ii) there are 19 potential asparagine-linked glycosylation sites that could give rise to the only kind of glycosylation demonstrated for this protein (hogue and brian, unpublished data; 10) . (iii) there is a hydrophobic stretch of 17 amino acids near the c terminus that could serve as a stoptransfer and anchor sequence. (iv) there is a stretch of 8 amino acids on the immediate n-terminal side of the predicted anchor sequence (-k-w-p-w-y-v-w-l-, beginning with amino acid 1305) that is identical in all coronavirus s proteins sequenced to date. (v) there is a cysteine-rich hydrophilic c-terminus of 35 amino acids that is probably the intravirion domain. in common with mhv-(22,32) and ibv (1, 2, 20, 27) , but not in common with tgev (15, 29; tung and brian, unpublished) and fipv (i!?), is also an internal sequence of basic amino acids that, in the case of mhv and ibv, lies on the immediate n-terminal side of the protease cleavage site (6, 22) . in bcv the sequence is k-r-r-sr-r beginning 10 20 30 40 50 60 70 80 90 100 110 120 tagaccataa~tgtmtgatact~~~tccttaccaatggct~gctg~ataggaga~~gtgtactacgg~cca~mtgatg~acaccggtgctcc~ -mflillislpmafavigdlkcttvsindvdtgap 130 140 150 160 170 180 190 200 210 220 230 240 ctattagcactgatattgtcgatgttactaatgg~aggtac~attatg~agatcgtgtgta~~tactacg~g~gc~~t~~a~accctac~cagg~ctacat sistdivdvtnglgtyyvldrvylbttlll ngyyptsgst 4b 250 260 270 280 290 300 310 320 330 340 350 360 atcgtaatatggcactgaagggaacmactattgagcagactattgagcagactatggttt~ccacc~~ct~ctga~a~~tggta~gct~~tc~taccaagg~a~~ yrnmalkgtlllsrlwfkppflsdfingifakvkntkvik 8b 370 380 390 400 410 420 430 440 450 460 470 i advyrripnlpdcnieawlndksvpsplnwerktfs 326 1090 1100 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200 attgtaattttaatatgag~agcctgatgtcttttattcaggcagactca~acttgt~taata~gatgctgct~gatatatggtatgtg~ccagcat~ctatagat~gt ncnfmmsslmsfiqad sftcnnidaakiygmcfssitidk 366 1210 1220 1230 12q0 1250 1260 1270 1280 1290 1300 1310 1320 ttgctatacccaatggtaggaaggttgacctacaattgggcaat~gggctatttgcagtc~tt~ctatag~ttgatactactgctacaagttgtcag~gta~ataat~acctg faipngrkvdlqlgnlgylqs fnyridttatscqlyynlp 406 1330 1340 1350 1360 1370 1380 1390 1400 1410 1420 1430 1440 ctgctaatgmctgttagcaggtttaatccttctacttggaataggagat~ggt~acag~caa~gt~~aagcctc~cctgtaggtg~actcatcatgatg~g~ aabvsvsrfnpstwnrrfgfteq fvfkpqpvgvfthhdvv 446 1450 1460 1470 1480 1490 1500 1510 ls20 1530 1540 1550 1560 atgcacaacattgttttaaagctccctc~at~ctgtccgtgtaaa~ggatgggtct~gtgtgtaggtaatggtcctggtatagatgctgg~at~~tagt~tataggcac~ yaqhcfkapsnfcpckldgslc vgngpgidagyknsgigt ---t;t 1570 1580 1590 1600 1610 1620 1630 1640 1650 1660 1670 1680 52b 1690 1700 1710 1720 1730 1740 1750 1760 1770 1780 1790 1800 tagttggcataggtgagcactgttcggggtc~gcta~aa~gtga~attgtggaggtaatcc~gtac~gcc~ccacaagca~~gggctggtctg~gactc~g~ac~g lvgigehcsclaiksdyc~cnpctcppqaflgwsvdselq -_--_-_------56b 1810 1820 1830 1840 1850 1860 1870 1880 1890 1900 1910 1920 gggataggtgtaatamttgctaatmatmgcatgatgatgttaatagtggtactacttg~ctactga~ac~~atc~acacagacat~~c~ggtg~gtg~aattatg gdrcnifanfilhdvnsgttcstdlqksntdi ilgvcvny 606 1930 1940 1950 1960 1970 1980 1990 2000 2010 2020 2030 2040 atctttatggta~acaggcc~ggta~~g~gaffi~aatgcgac~attat~tag~ggcagaacc~tatatga~ct~tggt~tctctatgg~tagagactac~~ dlygitgq gifvevbatyynswqnllyd sngnlygfrdyl 640 2050 2060 2070 2080 2090 2100 2110 2120 2130 2140 2150 2160 caaacagaactmatgattcgtagttgctatagcggtcgtgtttcagcggcctttcatgct~ctc~ccg~ccagcattgcta~cggaata~~tgc~~acg~~~ta tbrtfmirscysgrvsaafhaes 68b sepallfrnircnyvfb 2170 2180 2190 2200 2210 2220 2230 2240 2250 2260 2270 2280 atactcmcacgacagctgcaacctattaactamtgata~gatagttatc~gg~gtg~gtc~tgctgat~tagtac~ctagtg~g~c~catgtgat~cacagtaggtagtg ntlsrqlqpinyfd sylgcvvnadbs tssvvqtcdltvgs 2290 2300 2310 2320 2330 2340 2350 2360 2370 2380 2390 2400 gttactgtgtggattactctacaaaaagacgaagtagcctgtag gyc"dystkrrsrraittgyrfttfepftvnsv,?jdslepv 760 35 2410 2420 2430 2440 2450 2460 2470 2480 2490 2500 2510 2520 gtggtttgtatgaaattcaaataccttcagagag~actataggt~tatggaggag~a~c~acmgctctcct~g~acta~ga~g~ctgc~tctgtggtga~atg gglyeiqi pseftignmeefiqts spkvtidcsafvcgdy 800 2530 2540 2550 2560 2570 2580 2590 2600 2610 2620 2630 2640 cagcatgtaaatcacagttgg~g~tatggtagcttctgtgacaatattaatgctatactcacagaagtaaatgaactacttgacactacacagttgcaagtagct~ta~t~~~ga aacksqlvey gsfcdninailtevne lldttqlqvan 846 2650 2660 2670 2680 2690 2700 2710 2720 2730 2740 2750 2760 atggtgtcactcttagcactaagcttaaagatggcgtt~~tc~tgtagacgacatcaatt~cccctgta~aggttg~aggaagcgattgtaat~gt~ccagcagatctg ngvtlstklkdgvnfnvddiwfs pvlgclgsdcnkvssrs sei 2770 2780 2790 2800 2810 2820 2830 2840 2850 2860 2870 2880 ctatagaggatttacttmtctaaagtapiagtaaag~atctgatgtcggt~cgttgaggcttataat~ttgtactggaggtgccgaaattagggacctca~gtgtgc~gttataatg aiedllfskvklsdvgfveaynac tggaeirdlicvqsyn 926 2890 2900 2910 2920 2930 2940 2950 2960 2970 2980 2990 3780 3790 3800 3810 3820 3830 3840 ttpcaacacccaacctccatgattttaaggaaggaagagttggatcaatggttt~aa pictcagtggcaccagatptgtcacttgattatatp~cttggacctacaag istpnlhdfkeeldqwfk&svapdlsldyi,ii,vtfldlq 1246 3850 3860 3870 3880 3890 3900 3910 3920 3930 3940 3950 3960 atgaaatgaataggttacaggaggcaataaaagttttaag~tt~atcagagctacatcaatctcaaggaca~ggtacatatgagta~atgt~aatggccttggtatgtatggc~aa~g demnrlqeaikvloq syinlkdigtyeyyvkwpwyvwlli 12sb 3970 3980 3990 4000 4010 4020 4030 4040 4050 4060 4070 4080 gctttgctggtgtagctatgcttgmpactattctattc~catatgctg~gtacaggatgtgggactag~gt~taagatatgtggtgg~gttgtgatgattatactggacaccaggagt gfagvamlvllfficcctgcgtscfkicggccddytghqe 1326 4090 4100 tagtaattaaaacatcacatgacgactaa lviktshdd m t t 1366 fig. 2. nucleotide sequence of the s gene and its deduced amino acid sequence. the nucleotide sequence shown begins with the tag termination codon of the he gene (underlined) 17 bases upstream of the presumed s start site (7407 bases from the poly(a) tail), and ends with the taa termination codon of the s protern. the first three amino acids of the putative 4.9.kda protein are shown beginning at base position 4099. consensus cyaaac sequences are boxed. the presumed amino-terminal signal peptide and carboxy-terminal anchor sequences are underlined. potential n-linked glycosylation sites (nxs or nxt. where x # p) are boxed. the proteolytic cleavage site separating sl and 52 is identified with an arrow. the extended sequence of amino acids missing in mhv jhm is identified by individually underlined amino acids, and that missing in mhv a59, by asterisks. with amino acid 763, and, on the basis of the pattern in mhv and ibv, predicts a cleavage between amino acids 768 and 769 (note arrow in fig. 2 ). cleavage at this point would divide the unglycosylated s protein into an n-terminal segment of 85,690 da (sl) and a cterminal segment of 65,153 da (s2), from amino acid sequencing studies, no n-terminal sequence could be obtained from the virion-derived 120-kda subunit, possibly because of n-terminal blockage. the n-terminal sequence of the loo-kda subunit could be obtained, however, and was determined to be x-l-t-t-g-y-x-f-, identifying the first amino acids downstream from the predicted internal cleavage site. these results confirmed the predicted internal cleavage site and established that the 120-kda subunit is sl and the 1 00-kda subunit is s2. the bcv and mhv s proteins show remarkable sequence homology suggesting that these viruses are re-a599 ' i i 1111 i ii iii1 i bcvq ' j ' i i illll i i i i iiiii ii i i ii i ill i ii i ii ii i i i i i ii* iii i ii ii ii i i i 4 cently diverged. after aligning sequences for maximal homology, the following points emerge. (i) relative to bcv, a large deletion appears in the mhv sl subunits. for jhm it is a contiguous gap of 138 amino acids, and for a59 it is a discontiguous gap of 50 amino acids (figs. 2 and 3 ). the function of the additional sequence in the bcv sl subunit is not known, but it is possibly a structure that interacts in some way with the he glycoprotein, a structural protein not found on mhv (13, 34) except under certain rare conditions (33). no electron micrographic or chemical data exist, however, to suggest that s and he do physically interact (3, 17, 18) . it is interesting to note that the entire region in the bcv s protein corresponding to the gap region of the jhm s protein is especially rich in cysteine residues and contains 15 (26%) of the 56 total cysteines in the bcv s protein (figs. 2 and 3 ). this suggests that this part of the molecule may be important for intramolecular or intermolecular disulfide linkages. (ii) exclusive of the large gap in the mhv sequences, the sl subunits of jhm and a59 show 62 and 60% identity, respectively, with bcv, and the s2 subunits show 75 and 74% respectively. throughout the s protein, 41 of 56 cysteine positions and 13 of 19 potential n-linked glycosylation sites are conserved. the internal proteolytic cleavage position (not yet confirmed for jhm) is also conserved. the pattern of greater amino acid sequence divergence in the sl subunit is consistent with the model of cavanagh (4) and de groot et a/. (7) which proposes that the sl subunit comprises the exposed bulbous structure of the spike and probably contains most (5) but not all (23, 36) of the neutralizable antigenic sites. it is the structure most likely to undergo changes as a result of immunologic selective pressures. fusion of cells in culture is one biological activity associated with cleavage of the mhv s protein (35). despite its extensive sequence similarity with the mhv s protein, however, the bcv s protein shows little fusion activity. in fact, fusion is a behavior we have not observed with the mebus strain of bcv even though the s protein is primarily in the cleaved form on the virion (13, 17). it is not clear why bcv and mhv behave so differently in their fusogenic properties, but functional evaluation of sequence differences near the cleavage sites of these two viruses may aid in clarifying the mechanisms of fusion by mhv. this is especially interesting since hydrophobic regions, common at the cleavage sites on fusion proteins of paramyxoviruses and myxoviruses, are absent in the mhv s protein (22) and different mechanisms of fusion may be employed. biotech-niques6 proc. /vat/. acad. sc;. usa 84 proc. nat/. acad. sc;. usa 74 this work was supported by grant al-14367 from the national institute of allergy and infectious diseases, and by grant 82-crsr-z-1090 from the united states department of agriculture. t.e.k. and w.l. were predoctoral trainees on grant t32-al07 123 from the national institutes of health. key: cord-284968-eymvj6k3 authors: namazue, junko; campo-vera, harvey; kitamura, kenji; okuno, toshiomi; yamanishi, koichi title: processing of virus-specific glycoproteins of varicella zoster virus date: 1985-05-31 journal: virology doi: 10.1016/0042-6822(85)90112-6 sha: doc_id: 284968 cord_uid: eymvj6k3 abstract monoclonal antibodies to varicella zoster virus (vzv) glycoproteins were used to study the processing of three glycoproteins with molecular weights of 83k–94k (gp 2), 64k (gp 3), and 55k (gp 5). immunoprecipitation experiments performed with vzv-infected cells, pulse labeled with [3h]glucosamine in the presence of tunicamycin, suggest that o-linked oligosaccharide is present on the glycoprotein of gp 2. use of the enzyme endo-β-n-acetylglucosaminidase h revealed that the fully processed form of gp 3 had high-mannose type and that of gp 5 had only complex type of n-linked oligosaccharides. experiments with monensin suggest that the precursor form (116k) of gp 3 is cleaved during the processing from golgi apparatus to cell surface membrane. the extension of o-linked oligosaccharide chain and the complex type of n-linked oligosaccharide chains also occurs during this processing. varicella zoster virus (vzv) specifies several glycoproteins which are expressed on both virion and infected cell surfaces (grose, 1980; shemer et a& 1980; zweerink and neff, 1981; shiraki et al, 1982) . monoclonal antibodies of vzv have been produced in several laboratories and antigenic analysis of vzv has been attempted (grose et al, 1983; okuno et cd., 1983) . in our laboratory, three groups of monoclonal antibodies against vzv were isolated and the processing of vzv glycoproteins using these antibodies has been investigated (okuno et a& 1983) . recently the glycosylation of viral proteins using inhibitors of glycosylation such as tunicamycin and monensin has been studied in detail for several enveloped viruses including influenza, paramyxo viruses, vesicular stomatitis virus, sindbis virus, retroviruses, semliki forest virus, and herpes simplex virus (schwarz et d, 1976; leavitt et al, 1977; nakamura and compans, 1978; diggelmann, 1979; witte and wirth, 1979; johnson and schlesinger, 1980; pizer et ' to whom reprint requests should be addressed. stallcup and fields, 1981; johnson and spear, 1982; nakamura et al., 1982; wenske and courtney, 1983) . since the antiviral drug tunicamycin (tm) inhibits the formation of lipid-linked n-acetylglucosamine compound (takatsuki et al, 1971 (takatsuki et al, , 1975 and another drug monensin inhibits the glycoprotein pathway from golgi apparatus to cell surface membrane (tartakoff and vassalli, 1978; uchida et cd, 1979) , they are useful in identifying and studying the polypeptide moieties of glycoprotein. in this paper, the processing of three vzv glycoproteins (gp 2, gp 3, and gp 5) in the infected cells was investigated using tm and monensin. the kawaguchi strain of vzv was used throughout these studies. the preparation of cell-free vzv was described previously (yamanishi et al, 1980) . human embryonic fibroblast (huef) cells, which were passaged 10 to 15 times, were propagated in a mixture of eagle's mem and medium 199 containing 10% fetal calf serum and 0.025% nahc03 for growth medium and glycosidase treatment of immunoprecipi-preparation of radiolabeled antigen ex-tates tract prior to enzyme treatment, the immu-labeled cells were washed three times noprecipitates were washed with washing with pbs, harvested by scraping from the buffer and incubated in 0.8% sds for 20 min at 37" followed by boiling for 30 sec. the protein a-sepharose-bound antibodies were removed by centrifugation. supernatants were diluted to 0.2% sds and made 50 mm with respect to sodium citrate buffer (ph 5.5). endo+n-acetylglucosaminidase h (10 ~1 of 1 iu/ml, miles laboratories, elkhard, ind.) was added to samples. the reaction mixtures were incubated with constant rotation at 3'7" for 20 hr and the reactions were terminated by the addition of cold acetone and centrifugation. the pellets were resuspended in sample buffer and analyzed by sds-page. in order to investigate the effect of tm on the synthesis of vzv glycoprotein, infected cell cultures were labeled with rhlglucosamine for 18 hr, and cell extracts were immunoprecipitated with three kinds of monoclonal antibodies (cl 9, cl 8, and cl 12) which react with glycoproteins gp 2, gp 3, and gp 5, respectively (okuno et a,?., 1983) . when [3hlglucosamine was employed as a specific carbohydrate label, three bands with molecular weight of approximately '75k, 83k, and 94k which reacted with cl 9, two bands with molecular weight of 116k (faint) and 64k (major) which reacted with cl 8, and two bands with molecular weight of 94k (faint) and 55k (major) which reacted with cl 12, were observed in vzv-infected cultures (fig. 1, lanes a, c, e) . when [3hlglucosamine was used to label vzvinfected cells, only one diffuse band with molecular weight of 82-85k which reacted with cl 9 was observed in tm-treated cell extract (fig. 1, lane b) . however, no polypeptide was detected in cultures reacted with cl 8 and 12 monoclonal antibodies (fig. 1, lanes d, f) radiolabeled cells were immediately harvested or chased for 4 hr. when cl 9 antibody was used for immunoprecipitation test, radioactive bands at 65k (minor) and '75k (major) were observed in immunoprecipitates from cells labeled for 20 min (fig. 2, lane a) . as shown in fig. 2 lane c, these polypeptides were mainly replaced during chase by 83k and 94k molecules. in contrast, the immunoprecipitates formed with extracts of tm-treated cells gave prominent band of 73k at pulse labeling and additional 85k polypeptide during chase (fig. 2, lanes b, d) . next, when cell extracts were reacted with antibodies of cl 8, a band at 106k was seen at pulse labeling, and additional 116k and 64k (prominent) polypeptides were detected during chase (fig. 2, lanes e, g) . in tm-treated cell extracts, the 102k polypeptide (faint) at pulse labeling and 56k band (major) during chase were observed (fig. 2, lanes f, h) . finally, when cell extracts in the absence of tm were reacted with antibody from cl 12, 49k (major) and 43k (minor) polypeptides from cell cultures of pulse labeling and 55k (major) and 94k (minor) polypeptides were observed during chase (fig. 2, lanes i, k) . in contrast, a 39k (major) band from cell extract of pulse labeling in the presence of tm and 45k (major) and 83k (faint) polypeptides were observed during chase (fig. 2, lanes j, 1) . the oligosaccharides of vzv glycoproteins were characterized by use of endo h. endo h cleaves n-linked oligosaccharides of the high-mannose type, but not the complex type (koide and muramatsu, 1974) . vzv-infected cells were pulse labeled with [%!l]methionine for 20 min or pulsed and chased for 4 hr as described above. cell extracts were immunoprecipitated with three monoclonal antibodies and immunoprecipitates were treated or not treated with endo h. the 75k, 106k, and 49k polypeptides, which presumed to be precursor proteins of gp 2, gp 3, and gp 5, respectively, were detected in pulse-labeled cultures (fig. 3 , lanes a, e, i) and they were replaced by the 73k, 103k, and 46k polypeptides after endo h treatment (fig. 3, lanes b, f, j) . this evidence suggests that these precursor polypeptides were sensitive to endo h and contain high-mannose n-linked oligosaccharides. in contrast, the 83k and 94k polypeptides (gp 2) and 55k polypeptide (gp 5) which appeared during chase and are presumed to be final products were insensitive to endo h (fig. 3, lanes d, 1) . on the other hand, the 64k polypeptide which reacted with antibodies from cl 8 and corresponds to gp 3 was sensitive to this enzyme (fig. 3, lane h) . this shows that among three vzv glycoproteins only gp 3 contains high-mannose type oligosaccharides. monensin is an ionophore and blocks transport of glycoprotein from the golgi to the plasma membrane (tartakoff and vassalli, 1978; uchida et al, 1979) . figure 4 illustrates the results of experiment in which vzv-infected cells were labeled with [%s]methionine for 20 min or pulsed and chased in the presence or absence of monensin. cell extracts were reacted with the three kinds of monoclonal antibodies described above. the 75k, 106k, and 49k polypeptides which correspond to the precursor proteins of gp 2, gp 3, and gp 5, respectively, were detected in extracts from monensintreated or nontreated cells (fig. 4, lanes a, b, e, f, i, j) . when cell extracts from cultures treated or nontreated with monensin were reacted with these monoclonal antibodies, the 94k (gp 2) and 55k (gp 5) polypeptides, predominantly labeled in the absence of monensin, were replaced by the 83k and 52k polypeptides, respectively (fig. 4, lane d, 1) . finally the 64k polypeptide (gp 3) was predominantly observed during chase in nontreated cell culture, whereas this polypeptide was not detected in monensin-treated cells but the 106k to 116k bands were observed (fig. 4 , lane h). the purpose of this report was to extend our previous work (okuno et ak, 1983) in which we demonstrated the synthesis and processing of three vzv glycoproteins (gp 2, gp 3, and gp 5) using monoclonal antibodies. this report demonstrates that three species of vzv glycoproteins differ in several important structural and biovzv-infected cells not treated (-) or treated (+) with monensin were labeled with fsjmethionine as described in the legend to fig. 2 . cell extracts were precipitated with monoclonal antibodies cl 9 (lanes a-d), cl 8 (lanes e-h), and cl 12 (lanes i-l). (0) polypeptides related with gp 2, (m) polypeptides related with gp 3, (k) polypeptides related with gp 5. synthetic properties. these include the carbohydrate structure and the processing steps at the post-translational level (scheme 1). tunicamycin is known to block addition of n-linked oligosaccharides (takatsuki et az., 1975) . our experiments (fig. 2, lanes d, h, i) showed that the formation of the product proteins (gp 2, gp 3, and gp 5) were inhibited by this drug, suggesting that these vzv glycoproteins contain nlinked oligosaccharides. next, the vzvinfected cells were labeled with rh]glucosamine in the presence or absence of tm. if glycoprotein contains only n-linked oligosaccharides, no polypeptide should be labeled with [3h]glucosamine in the presence of tm. on the contrary, glycoproteins containing o-linked oligosaccharides would be labeled with [3h]glucosamine in the presence of tm. only the 82-85k polypeptide which reacted with cl 9 monoclonal antibody was labeled with ['hlglucosamine in the presence of tm (fig. 1) . from these data, it could be supposed that vzv gp 2 contains o-glycoside oligosaccharides. recently the presence of 0-glycosidically linked oligosaccharides has been reported for virusspecific glycoproteins of corona virus (nieman and klenk, 1981) , vaccinia hemagglutinin (shida and dales, 1981) , and herpes simplex virus (olofsson et ab, 1981a (olofsson et ab, , b, 1983 johnson and spear, 1983) . next, of particular interest is the appearance of newly detected proteins in the presence of tm. the clone 9 mono-clonal antibodies reacted with the 75k polypeptide (major) in the pulse labeling, and with the 83k and 94k polypeptides during chase (fig. 2, lanes a, c) . the 73k polypeptide was detected in the tmtreated and pulse-labeled cell culture, and the additional 85k polypeptide appeared during chase (fig. 2, lane d) . this suggests that the nascent polypeptide chain elongates and 0-glycoside oligosaccharides would be added to the polypeptides. next, cl 8 monoclonal antibodies, which react with gp 3, immunoprecipitated the 102k polypeptide in the pulse labeling and the 56k polypeptide during chase (fig. 2 , lane h). since precursor polypeptide (116k) will be considered to cleave to 64k product protein and 64k polypeptide appear in the virions and on the membrane of cells (okuno et al, 1983) , the 56k polypeptide found in tm-treated cells could be postulated to be a product protein cleaved as the case of 64k polypeptide in the absence of tm. it is reported that the cleavage of semliki forest virus polypeptide occurs in the presence of tm (garoff and schwarz, 1978) . this supports that the glycosylation of gp 3 is not necessary for the cleavage of polypeptide. finally, in the ease of gp 5, clone 12 monoclonal antibodies reacted with the 45k polypeptide (major) in the pulse labeling, and with the 55k (major) and 94k (minor) polypeptides. the 94k band was always observed in the long labeled or pulse-chased cultures . it was previously supposed that gp 5 and gp 2 glycoproteins would share some (okuno et ah, 1983 ). the 39k polypeptide was mainly detected in the pulse labeling and the 45k polypeptide was observed during chase. because of the data from vesicular stomatitis virus (rothman and lodish, 1977) , which indicate the glycosylation of polypeptide proceeds and the nascent chains are elongating, we believe that the 39k and 45k polypeptides are not normally present as precursor proteins, but the antigenic determinants are similar to the viral glycoproteins. the enzyme endo h have been shown to cleave selectively n-linked oligosaccharide of high-mannose type but not of the complex type (koide and muramatsu, 1974) . the precursor polypeptides of 75k (pm 2), 106k (pgp 3), and 49k km 5) were all sensitive to endo h as shown in fig. 3 . while product proteins, 83k and 94k (gp 2) and 55k (gp 5) were insensitive to endo h, the 64k (gp 3) was sensitive to this enzyme (fig. 3, lane h) . this indicates that the n-linked oligosaccharide chain is present in gp 3, and gp 5 contains complex type n-linked oligosaccharide, and that o-linked and n-linked complex type of oligosaccharides exist in gp 2. vzv-infected cells were treated with monensin and labeled for a short time or pulse labeled and chased. when cell extracts from monensin-treated cells were reacted with monoclonal antibodies, no effect was observed on the synthesis of polypeptide at the pulse labeling. however, the product proteins were not detected in monensin-treated cells but fast-migrating polypeptides were observed in gp 2 and gp 5. the monensin blocks the addition of o-linked oligosaccharides to glycoprotein and also it inhibits the conversion of nlinked high-mannose type oligosaccharide to the complex type (johnson and spear, 1983 ). considering the data described above, it can be confirmed that gp 2 contains n-linked and o-linked oligosaccharides and gp 5 contains complex type nlinked oligosaccharides. finally, the diffuse band between 106k and 116k was observed in monensin-treated cell extracts and no major polypeptides like 64k were detected in nontreated cells. since the 116k (pgp 3) would be cleaved to 64k product protein (okuno et al, 1983) , this result implies that the cleavage occurs between golgi apparatus and membrane. recently it was reported that gp 3 would be the cleaved-product form of gp 140 which has a disulfide-linked structural form (grose et ah, 1984) . we also confirmed that a 140k polypeptide was detected besides the 116k one, when immunoprecipitate was heated in sample buffer lacking 2-mercaptoethanol (unpublished data). however, the relationship between the 116k and 140k glycoproteins is not clear now. sindbis and vesicular stomatitis viruses effects of glucosamine,2-deoxyglucose, and tunicamycin on glycosylation, sulfation, and assembly of influenza viral proteins effect of tunicamycin on the replication of sendai virus coronavirus glycoprotein el, a new type of viral glycoprotein 0-glycosidic carbohydrate-peptide linkages of herpes simplex virus glycoproteins unusual lectin-binding properties of a herpes simplex virus type-l-specific glycoprotein glycoprotein c of herpes simplex virus type 1: characterization of o-linked oligosaccharides synthesis and processing of glycoproteins of varicella-zoster virus (vzv) as studied with monoclonal antibodies to vzv antigens effect of tunicamycin on herpes simplex virus glycoproteins and infectious virus production synchronized transmembrane insertion and glycosylation of a nascent membrane protein suppression of glycoprotein formation of semliki forest, influenza, and avian sarcoma virus by tunicamycin isolation and polypeptide characterization of varicella-zoster virus biogenesis of vaccinia: carbohydrate of the hemagglutinin molecule polypeptides of varicella-zoster virus (vzv) and immunological relationship of vzv and herpes simplex virus (hsv) the replication of measles virus in the presence of tunicamycin inhibition of biosynthesis of polyisoprenol sugars in chick embryo microsomes by tunicamycin comparative studies of intracellular transport of secretory proteins monovalent ionophores inhibit secretion of procollagen and fibronectin from cultured human fibroblasts glycosylation of herpes simplex virus type 1 gc in the presence of tunicamycin structure of the murine leukemia virus envelope glycoprotein precursor virus replication and localization of varicella-zoster virus antigens in human embryonic fibroblast cells infected with cell-free virus immune response after exposure to varicella zoster virus: characterization of virus-specific antibodies and their corresponding antigens diggelmann, h. (1979) key: cord-273246-4s54jrww authors: niu, shengniao; cao, shishu; wong, sek-man title: an infectious rna with a hepta-adenosine stretch responsible for programmed −1 ribosomal frameshift derived from a full-length cdna clone of hibiscus latent singapore virus date: 2014-01-20 journal: virology doi: 10.1016/j.virol.2013.11.021 sha: doc_id: 273246 cord_uid: 4s54jrww hibiscus latent singapore virus (hlsv) is a member of tobamovirus and its full-length cdna clones were constructed. the in vitro transcripts from two hlsv full-length cdna clones, which contain a hepta-adenosine stretch (phlsv-7a) and an octo-adenosine stretch (phlsv-8a), are both infectious. the replication level of hlsv-7a in nicotiana benthamiana protoplasts was 5-fold lower, as compared to that of hlsv-8a. the replicase proteins of hlsv-7a were produced through programmed −1 ribosomal frameshift (−1 prf) and the 7a stretch was a slippery sequence for −1 prf. mutations to the downstream pseudoknot of 7a stretch showed that the pseudoknot was not required for the frameshift in vitro. the stretch was found to be extended to 8a after subsequent replication cycles in vivo. it is envisaged that hlsv employs the monotonous runs of a and −1 prf to convert its 7a to 8a to reach higher replication for its survival in plants. the genomic rnas of viruses are often polycistronic and need to use strategies to express their downstream open reading frames (orfs). production of subgenomic rnas, readthrough of the stop codon, ribosome leaky scanning and programmed à 1, à 2 or þ 1 ribosomal frameshift (prf) are strategies used by viruses (plant, 2012) . prf has been reported in some plant virus groups, including luteovirus, dianthovirus, umbravirus, polerovirus and enamovirus, and most proteins translated via ribosomal frameshifting are the rna-dependent rna polymerase (rdrp) (miller and giedroc, 2010) . typically, à 1 prf requires two cis-acting rna elements. the first element is a hepta-nucleotide sequence where the reading frame shifts. this sequence usually fits the consensus xxxyyyn (x is any identical base, y is a or u, and n is not g; dreher and miller, 2006; jacks et al., 1988) . a second element is an rna secondary structure, a pseudoknot or a very stable rna structure (giedroc and cornish, 2009) , immediately downstream from the shift site (brierley et al., 1989; brierley and pennell, 2001) . this rna structure is regarded as a physical barrier to stop translating ribosomes and to shift the reading frame (namy et al., 2006) . there are examples in which slippery sequence could trigger frameshift, independent of the adjacent downstream rna secondary structure (wang et al., 2006; wilson et al., 1988) . in addition, far downstream rna elements of the slippery sequences in barley yellow dwarf virus (bydv) and red clover necrotic mosaic virus (rcnmv) required for à 1 prf through base pairing with the immediate downstream rna sequence were also reported (barry and miller, 2002; paul et al., 2001; tajima et al., 2011) . the genus tobamovirus consists of 33 species reported by the international committee on taxonomy of viruses in 2012 (http:// ictvonline.org/virustaxonomy.asp?version=2012). hibiscus latent singapore virus (hlsv), discovered in singapore (srinivasan et al., 2002 (srinivasan et al., , 2005 , is one of two members of tobamovirus infecting hibiscus (kamenova and adkins, 2004) . it has a similar genome structure to that of other tobamoviruses with a 5′ untranslated region (utr), four open reading frames (orfs) encoding viral replicase components (128 kda and 184 kda), movement protein (mp, 31 kda), coat protein (cp, 18 kda) and a 3′ utr (ishikawa and okada, 2004; zaitlin, 1999) . different from other tobamoviruses, only hibiscus latent fort pierce virus (hlfpv) and hlsv possess a unique internal poly(a) tract (personal communication, wong and adkins). hlsv contains a variable length ranging from 77 to 96 nucleotides (nt) upstream of the trna-like structure (tls) in the 3′ utr (srinivasan et al., 2005) . there is no report about viral protein expression by à 1 prf in tobamoviruses. in this study, hlsv full-length cdna clones were constructed and were found to be infectious. a full-length cdna clone of hlsv with a 7a stretch was located in its replicase gene replicated with a lower level, compared to another hlsv full-length cdna clone with an 8a stretch. mutational analyses showed that the 7a stretch is the slippery sequence required for à 1 prf and the downstream pseudoknot is dispensable. an extension from 7a to 8a stretch was observed during subsequent hlsv replication. in vitro transcripts of hlsv full-length cdna clones are infectious in nicotiana benthamiana and the replication level of hlsv-7a is lower than that of hlsv-8a to study the replication of hlsv, a biologically active full-length cdna clone has to be constructed. according to the hlsv complete genome sequence (genbank accession no. gi 113460144), primers were designated for construction of hlsv cdna full-length clone. during cloning, we found that one of the clones, designated as phlsv-7a, possessed a hepta-adenosine stretch, in contrast to an octoadenosine stretch of another clone at the position nt 2078, designated as phlsv-8a (fig. 1a) . the in vitro transcripts of hlsv-7a were able to replicate in nicotiana benthamiana protoplasts and infect n. benthamiana systemically by northern blot analysis (fig. 1b ), although its rna accumulation level was $ 5-fold lower than that of hlsv-8a, as determined by quantitative real-time rt-pcr (qrt-pcr) analysis in transfected protoplasts (fig. 1c) . hepta-adenosine stretch in hlsv is the slippery sequence for à 1 prf according to the deduced protein sequence of hlsv-7a, only a premature 78 kda protein was translated from the 5′ replicase gene sequence. since both replicase proteins of tobamoviruses are essential for efficient virus replication (bao et al., 1996; mizumoto et al., 2010; ogawa et al., 1991) , we wondered how hlsv-7a was able to replicate without both functional replicases. surprisingly, two additional bands, a clear 128 and a faint 184 kda replicase proteins, were detected, in addition to the prominent 78 kda protein in in vitro translation products using wheat germ extract. the level of the two additional proteins was much lower as compared to those generated from hlsv wild-type virions rna (fig. 2b ) and in vitro transcripts of hlsv-8a (fig. 2c , first lane from the right). these two proteins must have been produced by à 1 prf due to the absence of an adenosine residue at nt 2078 in hlsv-7a. to determine if the 7a stretch is the slippery sequence for à 1 prf, two other hlsv mutated subclones phlsv-aagaaga and phlsv-aagaagaa, in which 7a and 8a stretches were mutated to nonslippery sequences aagaaga and aagaagaa, respectively, were constructed ( fig. 2a) and confirmed by dna sequencing. the deduced à 1 prf product 108 kda protein was expected when phlsv-7a was digested with bsphi (at nt 2940 of hlsv genome) (fig. 2c , fourth lane from the right), as compared to the full-length 128 kda protein produced in phlsv-7a when digested with xhoi ( fig. 2c , second lane from the right). if the 7a stretch is a slippery sequence, only a 78 kda or a 108 kda target protein will be produced from in vitro transcripts of hlsv-aagaaga or hlsv-aagaagaa. in vitro translation experiment showed that 78 kda (fig. 2c , second lane from the left) and 108 kda proteins (fig. 2c , third lane from the left) were detected from hlsv-aagaagaa and hlsv-aagaaga, respectively (fig. 2c) . therefore, we conclude that the 7a stretch is the slippery sequence for à 1 prf in hlsv-7a. immediate downstream pseudoknot of the 7a stretch in hlsv-7a is dispensable for à 1 prf in most cases, slippery sequence and its immediate downstream rna secondary structure are needed for efficient à 1 prf (brierley et al., 1989; brierley and pennell, 2001 ; tsai et al., 1999). the immediate downstream 80 nt rna sequence of the hepta-adenosine stretch of hlsv was analyzed with the online software knotinframe (http://bibiserv.techfak.uni-bielefeld.de/kno tinframe) and a predicted 60 nt pseudoknot composed of 3 stems was obtained (fig. 3a , the first panel from left). to investigate the function of the pseudoknot on à 1 prf, the 3 stems were mutated partially or completely in phlsv-7a (fig. 3a ) and the pseudoknot structure was not detected using the software, when all three stems were mutated (data not shown). similar level of à 1 prf product, 128 kda protein, was detected in each mutant, as compared to that in hlsv-7a, which indicates that the pseudoknot is not required for the à1 prf in hlsv-7a (fig. 3b , left panel). as expected, the 128 kda protein was not detected in hlsv-7a-ms1 þms2 þ ms3stop in which a stop codon was introduced into the third stem of the pseudoknot (fig. 3b, right panel) . in bydv and rcnmv, the far downstream rna elements of the slippery sequences are required for à1 prf through base pairing with the immediate downstream rna sequence (barry and miller, 2002; paul et al., 2001; tajima et al., 2011) . in hlsv, a à 1 prf product, 108 kda protein, was still detected using transcripts derived from bsphi digested phlsv-7a, in which the far downstream hlsv sequence was cut off (fig. 2c , fourth lane from the right). therefore, the far downstream rna sequence is not required for the à 1 prf in hlsv-7a in vitro. 7a stretch could extend to 8a stretch in hlsv during its replication to determine whether the 7a stretch in hlsv could extend to 8a stretch during its replication, n. benthamiana leaves inoculated with hlsv-7a were collected at 2 and 6 days post-inoculation (dpi) and total rnas were extracted from these inoculated leaves. rt-pcr to amplify the region containing the stretch was carried out, followed by insertion of these pcr products into pgem s -t easy vector. sequence analyses of the transformants showed that only 3 out of 30 clones were added with 1a at 2 dpi. however, a large number of clones were found to have 1a added (24 out of 30) at 6 dpi (table 1 ). this result indicates that the 7a stretch can extend to 8a stretch by 1a insertion during its replication. we report here the construction of hlsv biologically active full-length cdna clones and the identification of a heptaadenosine stretch in hlsv genome as a slippery sequence responsible for à 1 prf, independent of its downstream pseudoknot structure and the far downstream rna sequence in vitro. the virus is able to generate functional viral replicases through the à 1 prf and extension from 7a to 8a stretch during replication in plants. according to earlier studies on à 1 prf, most of the frameshifting signals consist of a slippery sequence and a downstream rna secondary structure (pseudoknot or stem-loop) (biswas et al., 2004; brierley et al., 1992; jacks et al., 1988; liao et al., 2011; ten dam et al., 1990) . in this study, a downstream 60 nt rna pseudoknot sequence of the 7a stretch in hlsv was predicted by using the online software knotinframe (theis et al., 2008) . mutation analyses of the three stems within the pseudoknot showed that they were not required for à 1 prf (fig. 3b ) in vitro. translation analysis of bsphi digested phlsv-7a showed that the far downstream rna sequences are also not required for the à1 prf (fig. 2c , fourth lane from the right). these results indicate that the 7a stretch can function solely for the à 1 prf in vitro. similarly, a 7u stretch has been reported to be a sole signal for efficient à1 prf in coronavirus (wang et al., 2006) . earlier study on hiv also showed that frameshift was independent of the downstream pseudoknot structure (wilson et al., 1988) . the slippery sequences in the two cases mentioned above are all monotonous runs of u. taken together, we believe that, compared to other slippery sequences, the monotonous runs of a or u are the sole signals for à1 prf. since monotonous runs of nucleotides can also cause à 2 or þ 1 prf (brierley et al., 1992) , we noticed that in the in vitro translation products using hlsv-8a transcripts, a 78 kda protein was detected (fig. 2c , first lane from the right). one possible explanation is that þ1 prf has occurred in the 8a stretch during translation. however, in the translation products from purified viral rna from infected n. benthamiana leaves, there was no detectable 78 kda protein band (fig. 2b , first lane from the right). it has been reported that capping in the 5′ end of mrna reduces the efficiency of þ1 prf (charbonneau et al., 2012; gendron et al., 2008) . all the viral rna molecules extracted from purified hlsv virions should be capped. however, only $ 70% of the in vitro transcripts derived from phlsv-8a/xhoi were capped (according to the transcription kit manual). we also noticed larger size of translation products in hlsv-aagaagaa and hlsv digested with bsphi (fig. 2c , third and fifth lanes from the left, respectively). it may have resulted from incomplete digestion of these two plasmids, although dna bands may appear completely digested after gel electrophoresis (data not shown). among plant viruses, reversions of mutated sites to wild-type sequences are not uncommon (arguello-astorga et al., 2007; shepherd et al., 2005; wang et al., 2004) . in hlsv, the 7a stretch was extended to 8a during its replication. we believe that hlsv replicases can slide backward or forward on the slippery sequence of the positive or negative strand rna such that one additional a or u is added on the respective strands. once an a is added, the virus will replicate faster through more replicase production. therefore, hlsv with 8a becomes dominant in hlsv-7a inoculated leaves at 6 dpi. for tobamoviruses such as tobacco mosaic virus and odontoglossum ringspot virus, their replicase proteins produced by a replication-competent mutant are able to complement replication-defective mutants in trans but the efficiency is low (ogawa et al., 1991; wang et al., 2004) . thus, the dominance of hlsv with 8a is due to its faster replication through production of higher amount of replicase proteins. fig. 3 . a downstream pseudoknot structure of hlsv hepta-adenosine stretch is dispensable for à 1 prf. (a) schematic representation of the pseudoknot mutants. s1, s2 and s3 (boxed) are the first, second and third stems within the pseudoknot, respectively. the mutated nucleotides are circled and shadowed. (b) in vitro translation from transcripts of the pseudoknot mutants in wheat germ extract. the frame shift (fs in %) efficiency was calculated as the mean value of p128 to p78 ratio from three independent experiments, analyzed by the imagej software (national institutes of health). the standard deviation (sd) and t-test p (comparison of fs% between each pseudoknot mutant and hlsv-7a) are indicated. the in vitro translation products were labeled with biotinylated lysine. length of adenosine stretch 7a 8a 7a 8a number of clones randomly selected and sequenced (total ¼30) 27 3 6 24 a in vitro transcripts derived from hlsv-7a were mechanically inoculated onto n. benthamiana leaves and total rna was extracted from inoculated leaves at 2 and 6 days post-inoculation (dpi), respectively, followed by rt-pcr, cdna cloning and sequencing. to detect the distribution of 7a and 8a stretch in wild-type hlsv infected plants, in vitro transcripts derived from xhoi digested phlsv-8a were inoculated onto n. benthamiana leaves. the upper leaves were collected at 21 dpi for virions purification, followed by viral rna extraction, rt-pcr, cdna cloning and sequencing. in randomly selected 61 clones, 60 clones contained 8a stretch and one clone contained 11a stretch, but no 7a stretch was detected (data not shown). this may be due to limited number of colonies sequenced. according to this result, the possibility of the 7a stretch in phlsv-7a resulting from pcr error is very low. while we cannot rule out the possibility of slippery sequence occurring during in vitro transcription, it will be logistically difficult to check the rare occurrence. since we have observed an 11a sequence in the 8a inoculated plants, it is evident that the slippery event does occur in vivo. the error rate (mismatch) for t7 rna polymerase during transcription is 6 â 10 à 6 and no insertion or deletion event was detected (brakmann and grzeszik, 2001; rong et al., 1998) . the existence of 11a stretch in hlsv infected plant reveals that virus replicase may have been slided backward 3a in the 8a stretch during replication. earlier study on the prf and programmed transcriptional realignment events in bacterial genomes showed that the most frequent hepta nucleotide stretch for à 1 prf is aaaaaac, followed by aaaaaag and aaaaaaa. these results indicate that à1 prf tends to be evolutionarily conserved, and also some genes tend to select monotonous runs of nucleotides to ensure the right protein production once deletion or insertion happens (sharma et al., 2011) . we believe that hlsv also uses the a stretch to ensure its replicase proteins production once deletion happens. in conclusion, the hlsv-7a uses monotonous runs of 7a, à 1 prf and 1a insertion to reach its optimal replication through the functional replicase production in plants. to construct hlsv full length cdna clone, reverse transcription was carried out using purified hlsv rna as template by superscript iii reverse transcriptase (life technologies, invitrogen), by using primer hl-r1. all the primers used in this study are listed in supplementary table s1 . the synthesized cdna was used to amplify the hlsv 5′-end (nt 1-3333) and 3′-end (nt 3334-6474) fragments using primer pairs hl-f1/hl-r2 and hl-f2/hl-r1, respectively. the 3′-end fragment was first digested with xbai and mlui and inserted into modified pbluescript ii ks(þ ), resulting in p3′hlsv. the 5′-end fragment was inserted into pgem s -t vector (promega) to generate p5′hlsv which was cut with kpni and xbai and inserted into the p3′hlsv, resulting in hlsv fulllength clones phlsv containing 87 nt internal poly(a) tract. to construct the slippery sequence mutants, a subclone phlsv2960 was constructed by inserting the pcr product amplified by primers hl-f22 and hl-r18 using phlsv as template into pgem s -t easy vector (promega). the following two plasmids were constructed using phlsv2960 as a template by quick change mutagenesis (stratagene) and verified by sequencing. primers hl-f25 and hl-r20 were used for 7a stretch mutants, designated as phlsv-aagaaga. primers hl-f19 and hl-r16 were used for 8a stretch mutant, designated as phlsv-aagaagaa. to construct the downstream pseudoknot mutants, in which partial or all three stems within the pseudoknot were disrupted by nucleotide substitution, primer pairs were designed to amplify phlsv-7a. the designated plasmid names and their corresponding primers were as follows: hl-f39 and hl-r33, hl-f40 and hl-r34, hl-f41 and hl-r35, hl-f44 and hl-r38, hl-f45 and hl-r39 and hl-f43 and hl-r37 were for phlsv-7ams1þ s2þs3, phlsv-7a-ms1þ ms2þ s3, phlsv-7a-ms1þms2þms3, phlsv-7a-ms1þ s2þms3, phlsv-7a-s1þ ms2þms3 and phlsv-7a-s1þs2þ ms3, respectively. to introduce a stop codon in the third stem in phlsv-7a, primers hl-f46 and hl-r40 were used to amplify phlsv-7a and this mutant was designated as phlsv-7a-ms1þ ms2þ ms3stop. in vitro transcription and in vitro translation assay hlsv full-length cdna clones phlsv-7a and phlsv-8a were linearized with either xhoi or bsphi, followed by phenol/chloroform extraction and ethanol precipitation. phlsv-aagaaga and phlsv-aagaagaa were digested with bsphi, and the downstream pseudoknot mutants were digested with xhoi, followed by phenol/chloroform extraction and ethanol precipitation. the purified dnas were used for in vitro transcription by using mmessage mmachine in vitro transcription kit (life technologies, ambion). transcripts purified by licl precipitation were used for in vitro translation. in vitro translation was carried out by using a wheat germ extract system (promega) and products were labeled with easytag ™ l-[ 35 s]-methionine (neg709a001mc, perkin-elmer) or biotinylated lysine (transcend ™ trna, promega). protoplast transfection, total rna extraction and qrt-pcr n. benthamiana protoplasts were isolated and transfected with in vitro transcripts derived from phlsv-7a and phlsv-8a, respectively (qiao et al., 2009) . total rnas were extracted by the sdsphenol method (hans et al., 1992) from harvested protoplasts at 48 hpt, followed by cdna synthesis using primers hl-r8 and actin-r. synthesized cdna was used for qrt-pcr in triplicates with kapa sybr s on a cfx384 real-time pcr system (bio-rad) by using actin as an internal control. gene specific primers hl-f9 and hl-r9 were used for qrt-pcr and primers actin-f and actin-r were used for actin gene amplification. in vitro transcripts (0.5 mg for each leaf) derived from xhoi digested phlsv-7a were inoculated onto n. benthamiana leaves and total rnas were extracted from inoculated and upper leaves at 15 dpi. rna (5 mg each) was separated in 1.2% agarose gel and transferred onto positively charged nylon membranes, followed by hybridization with probe (covering hlsv cp region) labeled with a pcr dig probe synthesis kit (roche) with primers hl-cp-f and hl-cp-r, using phlsv-7a as template. total rnas were extracted from n. benthamiana leaves inoculated with hlsv-7a at 2 and 6 dpi, respectively; and viral rna was extracted from wild-type virions purified from hlsv-8a infected plants at 21 dpi. the rnas were used for cdna synthesis with primer hl-r6, followed by pcr with primers hl-f12 and hl-r18. the rt-pcr products were cloned into pgem s -t easy vector. selected colonies were cultured and purified plasmids were sequenced with primer hl-f12. high-frequency reversion of geminivirus replication protein mutants during infection the 126-and 183-kilodalton proteins of tobacco mosaic virus, and not their common nucleotide sequence, control mosaic symptom formation in tobacco a à 1 ribosomal frameshift element that requires base pairing across four kilobases suggests a mechanism of regulating ribosome and replicase traffic on a viral rna the human immunodeficiency virus type 1 ribosomal frameshifting site is an invariant sequence determinant and an important target for antiviral therapy an error-prone t7 rna polymerase mutant generated by directed evolution characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot mutational analysis of the "slipperysequence" component of a coronavirus ribosomal frameshifting signal structure and function of the stimulatory rnas involved in programmed eukaryotic-1 ribosomal frameshifting. cold spring harbor symp the 5′ utr of hiv-1 full-length mrna and the tat viral protein modulate the programmed à 1 ribosomal frameshift that generates hiv-1 enzymes translational control in positive strand rna plant viruses the presence of the tar rna structure alters the programmed à 1 ribosomal frameshift efficiency of the human immunodeficiency virus type 1 (hiv-1) by modifying the rate of translation initiation frameshifting rna pseudoknots: structure and mechanism replication of grapevine fanleaf virus satellite rna transcripts in chenopodium quinoa protoplasts replication of tobamovirus rna signals for ribosomal frameshifting in the rous sarcoma virus gag-pol region transmission, in planta distribution, and management of hibiscus latent fort pierce virus, a novel tobamovirus isolated from florida hibiscus the many paths to frameshifting: kinetic modelling and analysis of the effects of different elongation steps on programmed à 1 ribosomal frameshifting recoding: expansion of decoding rules enriches gene expression the 126-and/or 183-kda replicases or their coding regions are responsible both for inefficient local and for systemic movements of paprika mild mottle virus japanese strain in tomato plants a mechanical explanation of rna pseudoknot function in programmed ribosomal frameshifting trans complementation of virusencoded replicase components of tobacco mosaic virus a sequence required for à 1 ribosomal frameshifting located four kilobases downstream of the frameshift site ribosomal frameshift signals in viral genomes plastocyanin transit peptide interacts with potato virus x coat protein, while silencing of plastocyanin reduces coat protein accumulation in chloroplasts and symptom severity in host plants template strand switching by t7 rna polymerase a pilot study of bacterial genes with disrupted orfs reveals a surprising profusion of protein sequence recoding mediated by ribosomal frameshifting and transcriptional realignment a three-nucleotide mutation altering the maize streak virus rep prbrinteraction motif reduces symptom severity in maize and partially reverts at high frequency without restoring prbr-rep binding determination of complete nucleotide sequence of hibiscus latent singapore virus: evidence for the presence of an internal poly(a) tract hibiscus virus s is a new subgroup ii tobamovirus: evidence from its unique coat protein and movement protein sequences a long-distance rna-rna interaction plays an important role in programmed à 1 ribosomal frameshifting in the translation of p88 replicase protein of red clover necrotic mosaic virus rna pseudoknots: translational frameshifting and readthrough on viral rnas knotinframe: prediction of à 1 ribosomal frameshift events sufficient length of a poly(a) tail for the formation of a potential pseudoknot is required for efficient replication of bamboo mosaic potexvirus rna mutation of phe50 to ser50 in the 126/183-kda proteins of odontoglossum ringspot virus abolishes virus replication but can be complemented and restored by exact reversion identification of hepta-and octo-uridine stretches as sole signals for programmed þ1 and à 1 ribosomal frameshifting during translation of sars-cov orf 3a variants hiv expression strategies: ribosomal frameshifting is directed by a short sequence in both mammalian and yeast systems elucidation of the genome organization of tobacco mosaic virus this work was supported by national university of singapore research grant r-154-000-552-112. supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2013.11.021. key: cord-275664-qbafkxtr authors: wei, li; liu, jue title: porcine circovirus type 2 replication is impaired by inhibition of the extracellular signal-regulated kinase (erk) signaling pathway date: 2009-03-30 journal: virology doi: 10.1016/j.virol.2009.01.010 sha: doc_id: 275664 cord_uid: qbafkxtr postweaning multisystemic wasting syndrome, which is primarily caused by porcine circovirus type 2 (pcv2), is an emerging and important swine disease. we have recently shown that pcv2 induces nuclear factor kappa b activation and its activation is required for active replication, but the other cellular factors involved in pcv2 replication are not well defined. the extracellular signal-regulated kinase (erk) which served as an important component of cellular signal transduction pathways has been shown to regulate many viral infections. in this report, we show that pcv2 activates erk1/2 in pcv2-infected pk15 cells dependent on viral replication. the pcv2-induced erk1/2 leads to phosphorylation of the ternary complex factor elk-1, which kinetically paralleled erk1/2 activation. inhibition of erk activation with u0126, a specific mek1/2 inhibitor, significantly reduced viral progeny release. investigations into the mechanism of erk1/2 regulation revealed that inhibition of erk activation leads to decreased viral transcription and lower virus protein expression. these data indicate that the erk signaling pathway is involved in pcv2 infection and beneficial to pcv2 replication in the cultured cells. porcine circovirus (pcv) is classified in the genus circovirus of the family circoviridae (todd et al., 2005) . two genotypes of pcv have been identified. pcv type 1 (pcv1), which was first recognized in 1974 as a contaminant of a continuous porcine kidney cell line (pk15) (tischer et al., 1982) , is known to be non-pathogenic to pigs (allan et al., 1995) . infection with pcv type 2 (pcv2) has been associated with postweaning multisystemic wasting syndrome (pmws) in young weaned pigs, which was first recognized in canada in 1991 (clark, 1997) . nowadays, this disease and related pcv2-associated diseases are occurring in all swine-producing areas of the world and have become increasingly serious threats to global pig production (allan et al., 1998; allan and ellis, 2000; choi et al., 2000; edwards and sands, 1994; fenaux et al., 2000; mankertz et al., 2000; onuki et al., 1999; segalés and domingo, 2002) . usually pmws appears in pigs aged 5 to 18 weeks, and affected pigs show fever, wasting or unthriftiness, respiratory distress, enlarged lymph nodes and, occasionally, jaundice and diarrhea harding, 1996; segalés and domingo, 2002) . mortality rates may vary from 1 to 2% up to 30% in complicated cases when co-infections with porcine reproductive and respiratory syndrome virus, porcine parvovirus, or mycoplasma hyopneumoniae. other risk factors described in the madec principles have been developed to correlate with pmws (rose et al., 2003) . microscopic lesions are characterized by lymphocyte depletion of follicular and interfollicular areas together with macrophage infiltration of lymphoid tissues in pmws-affected pigs. several lines of field and experimental evidence have suggested that severely pmws affected pigs may develop immunosuppression (segalés et al., 2004) . pcv genome is a closed circular, single-stranded dna molecule of about 1.7 kb. two major open reading frames (orfs) have been recognized for pcv, orf1, called rep gene, which encodes a protein of 35.7 kda involved in virus replication (mankertz et al., 1998) , and orf2, called cap gene, which encodes the major immunogenic capsid protein of 27.8 kda (cheung, 2003; nawagitgul et al., 2000) . in addition, a third open reading frame (orf3) coding for an apoptosisassociated protein has been reported for pcv2 and it is involved in viral pathogenesis in vitro and in vivo (liu et al., 2005 (liu et al., , 2006 . a recent report has shown that pcv2 induces nuclear factor kappa b (nf-κb) activation in cultured cells, and further revealed the role of nf-κb activation in viral replication and pcv2-mediated apoptotic change (wei et al., 2008) . however, whether the other signaling pathways may also contribute to pcv2 infection in the cultured cells is still unclear. the extracellular signal-regulated kinase (erk) signaling pathway is one of the three mitogen-activated protein kinase (mapk) cascades that play important roles in the regulation of cell proliferation and virology 386 (2009) 203-209 differentiation, cytokine production and apoptosis (garrington and johnson, 1999; roux and blenis, 2004) . the erk activation is initiated by receptor tyrosine kinases that signal through the small gtpbinding protein ras. activation of ras leads to phosphorylation of raf kinase and in turn phosphorylates mek1/2 followed by activating erk1/2 via phosphorylation on tyrosine and threonine residues (rubinfeld and seger, 2005) . the activated erk1/2 translocates into the nucleus and phosphorylates numerous downstream substrates such as transcription factors c-myc, ets, elk-1, and egr-1, which ultimately regulate gene expression (luttrell, 2003) . therefore, the erk signaling pathway involves in a wide range of cellular functions including cell proliferation, transformatiom, differentiation, and cell survival and death (sebolt-leopold et al., 1999; luttrell, 2003) . research data have shown that many viruses such as human cytomegalovirus (johnson et al., 2001) , human immunodeficiency virus type 1 (yang and gabuzda, 1999) , influenza virus , herpes virus (perkins et al., 2002) , coxsackievirus b3 (luo et al., 2002) , vaccinia virus (andrade et al., 2004; de magalhães et al., 2001) , respiratory syncytial virus (kong et al., 2004) , borna disease virus , and coronavirus (cai et al., 2007) manipulate the erk signaling pathway to regulate viral replication and gene expression. however, the role of the erk signaling pathway during pcv2 replication is not clear. here, we reported that the erk signaling pathway was activated in the cultured cells by pcv2 infection and found that activation of the host cellular pathway is essential for efficient pcv2 infection. further experiments revealed that inhibition of this pathway reduces viral transcription, protein synthesis, and pcv2 viral progeny release. these results illustrate a mechanism by which pcv2 manipulates the erk signaling pathway to facilitate its infection and replication during viral infection. it was reported that erk is phosphorylated during varieties of virus replication. in order to assess whether pcv2 infection activated erk1/2, the phosphorylation status of erk1/2 was monitored on pk15 cells at different time points after infection by western blot analysis. the pk15 cells were infected with pcv2 strain bjw at a moi of 1 tcid 50 , and whole cell lysates were prepared at the indicated times after virus infection. inoculation of pbs into pk15 cells served as mock-infected controls. as shown in fig. 1a , infection with pcv2 led to progressive accumulation of p-erk1/2 signals over time, and the maximal induction of erk1/2 was seen at 72 h postinfection. the increased levels of erk1/2 phosphorylation were concurrent with expression of viral capsid protein orf2 in the infected cells, but not due to the enhanced production of these molecules or the difference in protein extracts loaded, as the protein levels of total amounts of erk2, as well as β-actin, in each sample were comparable. to further determine activated erk1/2 quantitatively in the infected cells, we used a fast activated cell-based elisa (face) assay to investigate the levels of ability of erk1/2 phosphorylation at different time points after pcv2 infection. consistent with the results shown in fig. 1a , there was a time-dependent increase in the erk1/2 phosphorylation in the pcv2-infected cells at 72 h postinfection which decreased thereafter (fig. 1b) . at 72 h after infection, the activation of phosphorylated erk1/2 showed approximately 3.1-fold higher than that in the mock-infected cells. in the mock-infected cells, the level of erk1/2 phosphorylation fell to its basal level. in addition, the levels of total erk1/2 remain unchanged in the pcv2-infected cells at various time points after infection when compared to that in the mock-infected cells. thus, these data indicate that pcv2 infection induces the activation of the cellular signaling pathway mediated by erk1/2. to determine whether pcv2 replication was required for erk1/2 phosphorylation, we used the face assay to examine erk1/2 phosphorylation in the cultured cells when infected with a uv lightirradiated virus sample. pk15 cells were infected an unirradiated or irradiated virus samples for 72 h postinfection. as shown in fig. 2 , no significant increase in activation of phosphorylated erk1/2 was observed in the uv-irradiated pcv2-infected cells as compared to that in the mock-infected cells. in contrast, the levels of erk1/2 phosphorylation increased by 3.5-fold when infected with the unirradiated pcv2 at 72 h postinfection. the levels of total erk1/2 remain unchanged in the pcv2-infected cells when compared to that pcv2-infected cells 24, 48, 72, and 96 h were harvested, whole cell lysates were prepared and resolved by sds-page, transferred to nitrocellulose membranes, and immunoblotted by using antibody specific for erk2, phosphorylated erk1/2, as well as pcv2 viral capsid protein orf2. the amounts of β-actin were also assessed to monitor the equal loadings of protein extracts. (b) erk1/2 activation induced by pcv2 infection was quantitatively determined using facs assay. pk15 cells were fixed at the indicated time points with 4% formaldehyde and incubated with normal or anti-phospho-specific antibodies directed against erk1/2 followed by hrp-conjugated igg antibodies. total and phosphorylated erk were each assayed in triplicate. cell numbers were normalized using crystal violet. these results are representative of three independent experiments. values are shown as the mean ± sd from triplicate wells. p-, phospho-. in the mock-infected cells. the result demonstrated that pcv2 replication was required for erk1/2 phosphorylation. to confirm that phosphorylation of erk1/2 following pcv2 infection truly activated its downstream substrate elk-1, the phosphorylation of the transcription factor was measured in the infected cells by western blotting. no activation of elk-1 was detected in the mock-infected pk15 cells. sustained erk1/2 activation after pcv2 infection leads to prolonged phosphorylation of elk-1 (fig. 3a) , with kinetics that paralleled those observed for erk1/2. cells exposed to the uv-irradiated virus sample failed to phosphorylate elk-1 (fig. 3b ). in addition, elk-1 phosphorylation was specifically impaired by preincubation with mek1/2 inhibitor u0126 at 20 μm (fig. 3b ). the result demonstrated that activation of erk1/2 induced by pcv2 infection is possibly through the activation of its downstream components such as elk-1. to determine whether activated erk plays any role in the replication of pcv2, we examined the effect of the kinase on progeny virus production in the pcv2-infected pk15 cells by blocking erk activation. we infected pk15 cells with pcv2 in the presence of the inhibitor u0126 (5-20 μm) or control (dmso), and determined the viral concentration in the cell culture supernatant 72 h after infection. the kinase inhibitor was also present during infection and in subsequent incubation periods. the selected concentrations of the kinase inhibitor were tested to show their effectiveness in inhibiting virus-induced erk1/2 activity by the facs assay, which demonstrated that activation of erk1/2 reduced dose-dependently (data not shown). seventy-two hours postinfection supernatants were collected and viral production was determined by the ifa method. the inhibitor u0126 at 5 μm inhibited pcv2 production in the cultured cells by 29%, compared with controls (fig. 4) . treatment with the inhibitor u0126 at 10 μm and 20 μm reduced pcv2 growth by 73% and 92%, respectively. as expected, the uv-irradiated pcv2 failed to grow comparable to those seen in the mock-infected cells (fig. 4) . the result suggested that erk signal regulates the replication of pcv2 in pk15 cells. to more specifically identify the stage of pcv2 infection that was targeted by the inhibition of erk activation, we quantified pcv2 production after applying the inhibitor u0126 at different time points postinfection. the cells were incubated until 72 h postinfection, and the supernatants were collected for virus production assay. as shown in fig. 5 , addition of u0126 at the time of infection led to a 91% inhibition in virus production as compared to the dmso-treated control. treatment between 2 and 8 h postinfection resulted in significant reduction of virus production (73% to 28% reduction compared to the dmso-treated control). in contrast, no significant differences in viral production were observed when the inhibitor was added at 12 or 24 h postinfection. the result indicates that inhibition of pcv2 infectivity by the inhibitor indeed occurs at the early stage in pcv2 infection. pcv2 replication is required for erk phosphorylation. monolayer pk15 cells were infected with pcv2 strain bjw at a moi of 1 tcid 50 . at 72 h postinfection, pcv2-infected cells and uv-irradiated pcv2-infected cells were fixed with 4% formaldehyde. the face assay was performed using normal or anti-phospho-specific antibodies directed against erk followed by hrp-conjugated igg antibodies. total and phosphorylated erk were each assayed in triplicate. cell numbers were normalized using crystal violet. these results are representative of three independent experiments. values are shown as the mean ± sd from triplicate wells. to help delineate the mechanism of erk1/2 regulation of pcv2 infection, we examined the effect of erk inhibition on viral protein expression. pk15 cells were infected with pcv2 (moi of 1) in the presence or absence of u0126 (20 μm), and orf2 protein expression was monitored by a fluorescence microscopy. the orf2 protein expression was significantly reduced when cells were treated with u0126, as demonstrated by the increased number of pcv2-positive cells observed in dmso-treated infected cells (fig. 6a) . no significant differences were seen in the orf2 protein expression between dmsotreated infected cells and untreated infected cells (data not shown). no orf2 protein expression was detected in the uv-irradiated pcv2infected as well as mock-infected cells (fig. 6a) . we then examined the effect of erk1/2 inhibition on pcv2 viral mrna synthesis, the real-time rt-pcr analysis was performed with rna extracted from pk15 cells 48 h after treatment with u0126 at 20 μm. the amount of each viral mrna was normalized to that of βactin mrna in the same sample. the abundance of pcv2 mrna was significantly decreased in the pcv2-infected cells with u0126 treatment (fig. 6b ). at the indicated time point, the amount of pcv2-specific mrna in u0126-treated cells was approximately 2.6% that of untreated cells. in contrast, the mrna was detected in the dmso-treated pcv2-infected cells as seen in that in the pcv2-alonedinfected cells (data not shown). no viral mrna accumulation was detected in the uv-irradiated pcv2-infected as well as mock-infected cells (fig. 6b) . the results indicate that inhibition of erk1/2 activation did have an inhibitory effect on viral protein synthesis and the accumulation of viral-specific mrna in the pcv2-infected cells. infection with a variety of viruses lead to the perturbation of host cell signaling pathways including erk mapk cascade, which can affect cellular function and virus replication. in the present study, we show that erk1/2 mapk pathway was activated during the course of pcv2 infection in pk15 cells (figs. 1a and b) , and its activation was required for viral replication (fig. 2) . also, we show that pcv2 infection induced the transcription factor elk-1, which is a downstream substrate of erk1/2, with kinetics that paralleled those observed for erk1/2 (fig. 3) . pcv2 replication was inhibited with the mek1/2 u0126 (fig. 4) . u0126 inhibited viral replication by 28% when added up to 8 h postinfection (fig. 5) , but subsequent addition had little effect, confirming that erk is important at early steps of the viral cycle. in addition, pcv2 viral transcription (fig. 6b ) and virus protein synthesis (fig. 6a) were reduced in the presence of u0126. together, these data suggest that the erk1/2 signaling pathway is manipulated by pcv2 and that the pathway plays a beneficial role in pcv2 replication. activation of the erk pathway seems to be an essential requirement to regulate signals associated with biological functions during virus infections. many viruses utilize the erk pathway for maximal viral replication. activated erk1/2 was reported to enhance the infectivity of human immunodeficiency virus (hiv), whereas treatment of cells with the mek1/2 inhibitor significantly inhibited hiv infectivity gabuzda, 1998, 1999) . erk mapk is required for visna virus replication and virus-induced neuropathology (barber et al., 2002) . treating cells with the mek1/2 inhibitor also significantly inhibited the propagation of influenza a virus , borna disease virus , coxsackievirus b3 (luo et al., 2002) , human cytomegalovirus (johnson et al., 2001) , vaccinia virus (andrade et al., 2004) , coronavirus (cai et al., 2007) , and astrovirus (moser and schultz-cherry, 2008 ). in the present study, we determined whether pcv2-induced erk1/2 activation is involved in viral replication by examining the effect of the inhibitor u0126 on pcv2 production and found that inhibition of erk1/2 activation significantly reduced the production of progeny virus (fig. 4) . this shows that optimal pcv2 replication requires the erk1/2 activity, and raises the possibility that pcv2 has acquired the ability to activate the kinase to aid its replication. therefore, our findings indicate that pcv2-induced activation of erk is truly involved in the replication of pcv2 in pk15 cells and add a new member to the growing list of viruses whose replication is modulated by the erk signaling pathway. erk1/2 regulation of viral replication can act at specific steps of the replication cycle, such as attachment, entry, gene transcription, protein expression, and assembly. borna disease virus requires active erk for entry . hiv-1 depends on the activation of mek erk pathway to deliver its genomes into the cultured cells (liu et al., 2002) . inhibition of erk during coronavirus infection specifically decreases genomic and sgrna production but has no effect on protein synthesis (cai et al., 2007) . erk activates adenovirus gene transactivators, modulating protein expression (schümann and dobbelstein, 2006; whalen et al., 1997) , with little effect on rna levels (schümann and dobbelstein, 2006) . inhibition of mek/erk pathway reduces kaposi's sarcoma-associated herpesvirus infectivity but has no effect on virus binding to the cell surface receptor (naranatt et al., 2003) . vaccinia virus-induced erk activation was significantly blocked by inhibition of erk, which was simultaneously paralleled by both delayed viral early-gene expression and a decrease in viral dna synthesis (andrade et al., 2004) . inhibition of erk activation mediated by astrovirus reduces all steps of the viral life cycle, including early and late protein expression as well as subgenomic and genomic rna transcription (moser and schultz-cherry, 2008) . the function of activated erk during pcv2 infection was investigated in the present study. our data showed that treatment of cells with u0126 suppressed virus propagation at the step of viral rna transcription as well as viral protein synthesis. pcv replicates via rolling circle replication involving an intermediate double-stranded replicative form of dna, and the rep proteins nick and join the nucleotide segments at the initiation and termination of the replication cycle (cheung, 2006; steinfeldt et al., 2006) . thus, it was not surprising that treatment of pcv2-infected cells with inhibition of erk reduced viral transcripts as well as protein expression, because viral transcription and protein synthesis are closely related in the replication of pcv2. however, a detailed mechanistic understanding of the inhibition of erk activation on pcv2 replication requires the identification of particular cellular factors which are the components of the erk signaling pathway. in conclusion, we have shown that pcv2 infection induces the activation of erk and its involvement in elk-1 activation in the cultured cells and demonstrate that the activation of erk is required for efficient pcv2 replication. inhibition of erk activation significantly reduces viral protein expression and viral rna transcription. the role of erk activation in pcv2 replication will contribute important information about the molecular mechanism of pcv2 infection. the permanent pk15 cell line, which was free of pcv, was maintained in minimal essential medium (mem) supplemented with 5% heat-inactivated fetal bovine serum (fbs), 5% l-glutamine, 100 u of penicillin g/ml, and 100 μl of streptomycin/ml at 37°c in a humidified 5% co 2 incubator. the pcv2 virus used in the study was originally isolated from a kidney tissue sample of a pig with naturally occurring pmws (strain bjw) (liu et al., 2005) . for pcv2 infection, pk15 cells seeded the day before were infected with pcv2 strain bjw at a multiplicity of infection (moi) of 1 tcid 50 . cells were additionally treated with 300 mm d-glucosamine at 24 h after infection as described previously (tischer et al., 1987) . u0126 was purchased from calbiochem (la jolla, calif.). pk15 cells were treated with either dmso which is the solvent for u0126 or various concentrations (5-20 μm) for 1 h prior to infection. after 1 h of virus adsorption, the virus inoculum was removed and fresh basal medium containing fresh inhibitor was added to the culture. the cytotoxicity of the inhibitors on pk15 cells was determined by trypan blue exclusion dye staining. it was noted that throughout all doses of the inhibitor used in this study, cell viability assay showed no detectable cell death in pk15 cells. rabbit antibodies against erk2, β-actin, as well as phosphorylated erk1/2 (p-erk1/2) were purchased from santa cruz biotechnology (hercules, ca). antibody specific for phosphorylated forms of elk-1 (p-elk-1) was obtained from cell signaling technology. horseradish peroxidise (hrp)-linked secondary antibodies were purchased from sigma. face kit to monitor the levels of erk1/2 mapk activation was obtained from active motif. procedure was performed strictly according to the manufacturer's instructions. briefly, pk15 cells were seeded in 96-well plates 1 day prior to infection. after treatment and/or infection, cells were fixed with 4% formaldehyde in pbs. after washing and blocking, cells were reacted overnight with an anti-erk1/2 or anti-phospho-erk1/2 antibody. following incubation with a hrp-conjugated secondary antibody, colorimetric analysis was performed. a450 was determined using a plate spectrophotometer. total cell rnas were prepared from pcv2-infected pk15 cell 48 h after being treated with various concentrations of the inhibitor u0126 by using trizol rna extract reagent (invitrogen). the following primers were used: orf2 (sense) (5′-atcaagcgaaccacag-3′) and orf2 (antisense) (5′-ggtcataggtgaggggc-3′) for pcv2 orf2 and sense (5′-cacgccatcctgcgtctgga-3′) and antisense (5′-agcacc-gtgttggcgtagag) for β-actin. the rna samples were incubated with dnase i for 60 min at 37°c to remove any contaminating viral dna. cdnas were reverse transcribed from total rnas by the use of antisense primers and the first-strand synthesis system (avian myeloblastosis virus reverse transcriptase kit; roche). quantitative real-time pcr was performed on a lightcycler (roche) instrument according to the instructions of the lightcycler fast start dna master plus sybr green i kit (roche). the pcr parameters consisted of an initial denaturation at 94°c for 5 min, followed by 40 cycles of 94°c for 10 s, 52°c for 5 s and 72°c for 10 s. subsequent melting curve analysis and c t value determination were performed using roche lightcycler software version 3.5. each sample was run in triplicate. the relative amount of target viral mrna was normalized to that of β-actin mrna in the same sample. pk15 monolayer cells seeded in 24-well culture plates were infected with pcv2 strain bjw. at 72 h, the cells were washed with phosphate-buffered saline (pbs) and fixed in 4% paraformaldehyde (pfa). after three washes, the cells were incubated with mouse anti-orf2 antibody diluted in 3% bovine serum albumin (bsa)-pbs at room temperature (rt) for 1 h. after three further washes, cells were incubated with fluorescein isothiocyanate (fitc)-conjugated antimouse immunoglobulin g (sigma) at rt for 1 h and washed with pbs three times. the cells were examined under a fluorescence microscopy and cells positive for pcv2 viral antigens were counted in six fields of view. whole cell lysate extracts from pk15 cells after infection at various time points were prepared with the nuclear extract kit (active motif) according to the manufacturer's protocol. protein concentration was determined by the bio-rad protein assay (bio-rad, hercules, ca) with bsa as a standard. the whole cell lysate extracts prepared as described above were diluted in 2× sample buffer and boiled for 5 min. twenty micrograms of each extract was resolved on 10% sodium dodecyl sulphatepolyacrylamide gel electrophoresis (sds-page) and blotted onto nitrocellulose (nc) membranes (stratagene) with a semidry transfer cell (bio-rad trans-blot sd). the membranes were blocked for 2 h at rt in blocking buffer tbst (20 mm tris-hcl [ph 7.4], 150 mm nacl, 0.1% tween-20) containing 5% skim milk powder to prevent nonspecific binding, and then incubated with specific primary antibodies raised against orf2, erk2, phosphorylated (p)-erk1/2, and (p)-elk-1, as well as β-actin at rt for 2 h. the membranes were washed three times with tbst buffer, and incubated for 2 h at rt with hrp-conjugated secondary antibodies diluted in blocking buffer (1:2 000). immunoreactive bands were visualized by enhanced chemiluminescence system (amersham biosciences). results are presented as averages ± the standard deviations or standard errors of the means, as indicated. statistical comparisons are made by using student's t test, and differences between groups were considered significant if the p value was b 0.05. porcine circoviruses: a review pathogenesis of porcine circovirus: experimental infections of colostrum deprived piglets and examination of pig foetal material isolation of porcine circovirus-like viruses from pigs with a wasting disease in the usa and europe the vaccinia virus-stimulated mitogen-activated protein kinase (mapk) pathway is required for virus multiplication visna virus-induced activation of mapks is required for virus replication and correlates with virus-induced neuropathology suppression of coronavirus replication by inhibition of the mek signaling pathway transcriptional analysis of porcine circovirus type 2 rolling-circle replication of an animal circovirus genome in a thetareplicating bacterial plasmid in escherichia coli porcine postweaning multisystemic 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target cells early during infection: implications for infectivity open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein detection of porcine circovirus from lesions of a pig with wasting disease in japan the herpes simplex virus type 2 r1 protein kinase (icp10 pk) blocks apoptosis in hippocampal neurons, involving activation of the mek/mapk survival pathway mek-specific inhibitor u0126 blocks spread of borna disease virus in cultured cells influenza virus propagation is impaired by inhibition of the raf/mek/erk signalling cascade risk factors for porcine post-weaning multisystemic wasting syndrome (pmws) in 149 french farrow-to-finish herds erk and p38 mapk-activated protein kinases: a family of protein kinases with diverse biological functions. microbiol the erk cascade: a prototype of mapk signaling adenovirus-induced extracellular signalregulated kinase phosphorylation during the late phase of infection enhances viral protein levels and virus progeny blockade of the map kinase pathway suppresses growth of colon tumors in vivo postweaning multisystemic wasting syndrome (pmws) in pigs: a review immunosuppression in postweaning multisystemic wasting syndrome affected pigs demonstration of nicking/joining activity at the origin of dna replication associated with the rep and rep' proteins of porcine circovirus type 1 a very small porcine virus with circular single-stranded dna replication of porcine circovirus: induction by glucosamine and cell cycle dependence circoviridae porcine circovirus type 2 induces the activation of nuclear factor kappa b by iκbα degradation phosphorylation within the transactivation domain of adenovirus e1a protein by mitogen-activated protein kinase regulates expression of early region 4 mitogen-activated protein kinase phosphorylates and regulates the hiv-1 vif protein regulation of human immunodeficiency virus type 1 infectivity by the erk mitogen-activated protein kinase signaling pathway this work was supported by grants from national natural science foundation (30871866) and beijing municipal science and technology contract project (z07010501780701), the people's republic of china. key: cord-275993-isff6lp2 authors: han, dong p; kim, hyung g; kim, young b; poon, leo l.m; cho, michael w title: development of a safe neutralization assay for sars-cov and characterization of s-glycoprotein date: 2004-08-15 journal: virology doi: 10.1016/j.virol.2004.05.017 sha: doc_id: 275993 cord_uid: isff6lp2 the etiological agent of severe acute respiratory syndrome (sars) has been identified as a novel coronavirus sars-cov. similar to other coronaviruses, spike (s)-glycoprotein of the virus interacts with a cellular receptor and mediates membrane fusion to allow viral entry into susceptible target cells. accordingly, s-protein plays an important role in virus infection cycle and is the primary target of neutralizing antibodies. to begin to understand its biochemical and immunological properties, we expressed both full-length and ectodomain of the protein in various primate cells. our results show that the protein has an electrophoretic mobility of about 160–170 kda. the protein is glycosylated with high mannose and/or hybrid oligosaccharides, which account for approximately 30 kda of the apparent protein mass. the detection of s-protein by immunoassays was difficult using human convalescent sera, suggesting that the protein may not elicit strong humoral immune response in virus-infected patients. we were able to pseudotype murine leukemia virus particles with s-protein and produce sars pseudoviruses. pseudoviruses infected vero e6 cells in a ph-independent manner and the infection could be specifically inhibited by convalescent sera. consistent with low levels of antibodies against s-protein, neutralizing activity was weak with 50% neutralization titers ranging between 1:15 to 1:25. to facilitate quantifying pseudovirus-infected cells, which are stained blue with x-gal, we devised an automated procedure using an elispot analyzer. the high-throughput capacity of this procedure and the safety of using sars pseudoviruses should make possible large-scale analyses of neutralizing antibody responses against sars-cov. during the first epidemic of severe acute respiratory syndrome (sars), which began in november of 2002 in guandong province of the people's republic of china, and lasted for about 7 months, close to 8100 people were infected worldwide, among which 774 people died (who, 2003) . the etiological agent of this atypical respiratory disease has been identified as a novel coronavirus (designated as sars-cov) fouchier et al., 2003; ksiazek et al., 2003; peiris et al., 2003; poutanen et al., 2003) . with a mortality rate of over 9%, sars-cov had a major health and socioeconomic impact. fortunately, there have been very few incidences of sars infections during the winter season of [2003] [2004] . however, with multiple modes of virus transmission and a wide range of potential nonhuman reservoirs including wild animals commonly found in markets (e.g., civet cats and raccoon dogs; guan et al., 2003) as well as domestic cats (martina et al., 2003) , it is highly likely that a virus of this nature will most certainly resurface in the future. currently, there are no antiviral drugs, immunotherapeutic agents, or vaccines available against the virus. to better control or prevent future epidemics, anti-sars-cov drugs and/or vaccines need to be developed. sars-cov belongs to coronaviridae family. the genomic organization of the virus is similar to that of other coronaviruses with a general order of replicase (rep; orfs-1a and 1b), spike (s)-glycoprotein, envelope (e), membrane protein (m), and nucleocapsid (n) from 5v to 3v direction (marra et al., 2003; rota et al., 2003) (fig. 1) . several openreading frames have also been identified, which may encode additional proteins (marra et al., 2003; rota et al., 2003; snijder et al., 2003) . their functions, however, are not known at the present time. the protein of a major interest as a target of antiviral drug development efforts as well as for developing vaccines is s-glycoprotein. s-protein of coronaviruses, which is thought to function as a trimer (delmas and laude, 1990) , is responsible for both binding to cellular receptors and inducing membrane fusion for virus entry into target cells (collins et al., 1982; godet et al., 1994; kubo et al., 1994) . mutations in the protein have been shown to alter virulence and cellular tropism (fazakerley et al., 1992; leparc-goffart et al., 1998; sanchez et al., 1999) . taken together, the s-protein plays a critical role in the biology and pathogenesis of coronaviruses. not surprisingly, it is an important target of virus-neutralizing antibodies (chang et al., 2002; collins et al., 1982; fleming et al., 1983; godet et al., 1994; kant et al., 1992; kubo et al., 1993 kubo et al., , 1994 takase-yoden et al., 1991) . moreover, mice immunized with a recombinant s-protein, or a peptide derived from it, are protected from lethal challenges with murine hepatitis virus (mhv) (daniel and talbot, 1990; koo et al., 1999) . s-protein is a type i membrane glycoprotein, which is translated on membrane-bound polysomes, inserted into rough endoplasmic reticulum (rer), cotranslationally glycosylated, and transported to the golgi complex. during the transport, s-proteins are incorporated onto maturing virus particles, which assemble and bud into a compartment that lies between the rer and golgi (lai and holmes, 2001) . virions are carried from golgi to plasma membrane in secretory vesicles. virions are released from cells when virion-containing vesicles fuse with plasma membrane. excess s-proteins not incorporated onto virus particles are transported to the surface of plasma membrane (lai and holmes, 2001; tsai et al., 1999; yamada et al., 1998) . s-protein of sars-cov is 1255 amino acids long (fig. 1) . it is predicted to have a 13 amino acid signal peptide at the amino-terminus, a single ectodomain (1182 amino acids) and a transmembrane region followed by a short cytoplasmic tail (28 residues) at the carboxy-terminus (marra et al., 2003; rota et al., 2003) . due to low sequence homology between the s-protein of sars-cov and that of the other coronaviruses (marra et al., 2003; rota et al., 2003) , the structural and immunogenic properties of sars-cov sprotein must be ascertained experimentally. the cellular receptor for sars-cov has recently been identified to be angiotensin-converting enzyme 2 (ace2; li et al., 2003) . the molecular interactions between the s-protein and ace2 are not yet known. better understanding of the interactions could lead to development of virus entry inhibitors. neutralizing antibodies (nabs) play a critical role in protection against a variety of viral diseases. an accurate assessment of nab responses in virus-infected patients is needed to determine immune correlates of protection. it is also an essential and integral part of a vaccine development process. conventional virus-neutralization assays require the use of replication-competent, infectious viruses. evaluating virus-neutralizing activity of a large number of antisera with these assays is undesirable due to safety concerns, especially for a biosafety level 3 (bsl3) pathogen like sars-cov. the same safety concerns have prompted our laboratory to utilize replication-defective pseudoviruses for hiv-1 neutralization assay (kim et al., 2001) . in this assay, nonreplicating moloney murine leukemia virus (mulv) particles pseudotyped with hiv-1 envelope glycoproteins are used (schnierle et al., 1997) . these pseudoviruses encode a h-galactosidase gene, which allows detection of individual infected cells when stained with x-gal (5-bromo-4-chloro-3-indolyl-h-d-galactopyranoside). in this study, we report development of a sars-cov pseudovirus neutralization assay, which should be particularly valuable for researchers who may not have easy access to bsl3 containment facility. additionally, we describe a high-throughput system for quantitative analyses of x-gal stained cells. this assay system should facilitate rapid evaluation of antibody responses to vaccine candidates and/or entry inhibitors against sars-cov. to express sars-cov s-glycoprotein, we initially cloned a dna fragment encoding the protein into pcdna-3 vector (pcdna-s; fig. 1b ). to detect s-protein, western blot was performed with convalescent sera from sars-cov-infected patients. however, no clear protein band was detected despite number of attempts. we reasoned that one of the possibilities for the inability to detect the protein is a low level of s-protein expressed from pcdna-s. to increase the amount of sprotein expressed, we subcloned the s gene into phcmv-g vector (burns et al., 1993) , which expresses high level of vesicular stomatitis virus (vsv) g glycoprotein. although we were able to express higher amount of s-protein (see below), this was not sufficient to detect a clear band on western blots. an alternative explanation is that antibodies against the protein in convalescent sera cannot recognize s-protein subjected to denaturing conditions of sds-page (viz. linear epitopes). however, because results from radioimmunoprecipitation and indirect immunofluorescence assays were also ambiguous, it is most likely that the antibody titer against s-protein is very low in convalescent sera. to further increase protein expression level, s gene was subcloned into a ptm vector (moss et al., 1990) . with this vector, a protein of interest is under the control of a strong t7 rna polymerase (t7rnap) promoter and the protein is expressed when cells transfected with the plasmid are infected with a recombinant vaccinia virus expressing t7rnap (vtf7-3; fuerst et al., 1986) . the presence of encephalomyocarditis virus internal ribosome entry site (ires) at the 5v end of rna transcripts allows efficient translation of mrna transcribed in cytoplasm. using ptm-s, we were able to detect a faint, but distinct band of approximately 160 -170 kda by western blot (fig. 2a , lane 3). we also reevaluated pcdna-s as this vector has a dual promoter system (cmv and t7 promoter). using t7 promoter, we were able to detect a protein band of a similar size, albeit less clear than using ptm-s (lane 2). the lower expression of the protein is likely due to the lack of ires in the pcdna vector. because the calculated molecular weight of s-protein without 13 amino acid signal peptide is about 138 kda, the result suggested posttranslational modification (e.g., glycosylation). to better demonstrate this, we generated another clone (ptm-eshis) that expresses the entire ectodomain of s-protein (amino acids 1 -1190) with a six-histidine tag at the carboxy terminus. the ectodomain of s-protein migrated with an approximate molecular weight of 163 kda while its calculated molecular weight is only 131.2 kda (fig. 2b , lane 2). to demonstrate that this difference is due to glycosylation, eshis protein was treated with endoglycosidase h (endo-h) or peptide: n-glycosidase f (pngase f). as shown in fig. 2b (lanes 3 and 4), treatment with either glycosidase increased the mobility of the protein to approximately 133 kda. because the mobility of the protein treated with either glycosidases was the same, s-protein is most likely modified with high mannose and/or hybrid, rather than complex, oligosaccharides. while s-glycoprotein of some coronaviruses is cleaved into two subdomains, s1 and s2, the fact that we observed only a single band suggests that sars-cov s-protein functions as a single unit. despite difficulties in detecting s-protein directly by immunoassays, proteins expressed from both pcdna-s and phcmv-s constructs were able to pseudotype mulv particles to produce sars pseudoviruses that could readily infect vero e6 cells (fig. 3a) . none of the other cell lines we tested, including hela, a549, 293t, and bs-c-1, were susceptible. this is in contrast to vsv-g pseudotyped viruses, which could infect all cell lines (data not shown). the fact that bs-c-1 cells, which, like vero e6 cells, are african green monkey kidney cells, were not susceptible was somewhat unexpected. however, when we performed infections with a high multiplicity of infection, we were able to detect infected bs-c-1, albeit at a significantly reduced titer (7 -8-fold compared to vero e6 cells; data not shown). this result was not too surprising because it has been shown that even 293t cells, which express small amounts of ace2, support some basal level of sars-cov replication . many pseudovirus-infected cells appeared as a doublet, which is the result of a cell division following integration of mulv pseudovirus genome encoding h-galactosidase. these doublets are counted as a single infectious unit. a typical yield of sars pseudoviruses was about 2 â 10 4 infectious units per milliliter of culture supernatant using phcmv-s, which was about fivefold greater than using pcdna-3. this yield is comparable to what we have been able to achieve for hiv-1 pseudoviruses (between 2 â 10 3 and 2 â 10 4 depending on envelopes; kim et al., 2001) , but lower than vsv-g pseudovirus yield (between 3 â 10 4 and 9 â 10 4 depending on target cell lines used). interestingly, sars pseudovirus production was about 20-fold less efficient when plasmid transfection was performed by calcium phosphate method compared with using cationic lipids (lipofection). this difference, however, was not observed for vsv-g pseudovirus production. an additional difference was that a longer incubation time was needed to achieve peak pseudovirus production for sars-s compared to vsv-g (3 vs. 2 days posttransfection, respectively). the reasons for these discordant results are unknown at the present time. cellular entry of coronaviruses can occur either by acidic ph-dependent or -independent pathway (gallagher et al., 1991; lai and holmes, 2001; cavanagh, 1990, 1992; nash and buchmeier, 1997; payne et al., 1990) . to investigate whether sars-cov infection requires low ph, we examined sensitivity of pseudovirus infections to lysosomotropic agents chloroquine and nh 4 cl. as expected, infectivity of viruses pseudotyped with vsv-g was reduced by chloroquine and nh 4 cl in a dose-dependent manner 3 and 4, respectively) . the protein was detected by western blot with anti-6âhis antibody. no band was detected from cells transfected with an empty vector (lane 1). acrylamide gradient gel (4 -12%) was used. ( fig. 3b and c, respectively) . in contrast, sars pseudovirus infection was virtually unaffected, suggesting that sars-cov infection proceeds in an acidic ph-independent manner. to assess whether sars pseudoviruses we generated could be used to quantify virus-neutralizing antibodies, we examined their susceptibility to convalescent sera from sars-cov-infected patients. as shown in fig. 4a , sera from two patients were able to specifically neutralize sars pseudoviruses; the same convalescent sera could not neu-tralize hiv-1 or vsv-g pseudoviruses and no neutralizing activity was observed with a normal serum. to determine neutralizing antibody titers in virus-infected patients, we performed the assay with serially diluted sera from seven patients. as shown in fig. 4b , antibody levels were quite similar in all patients with 50% neutralization titer between 1:15 and 1:25. although the pseudovirus neutralization assay is sensitive, quantitative, and safe, it has one disadvantage of having to count individual x-gal-stained cells through a microscope. to overcome this problem, we looked into a possibility of automating the data collection procedure using an elispot reader (immunospot analyzer, cellular technology ltd.). although this instrument is commonly used to quantify antigen-specific t cell cytokine responses by counting chromogenic immunospots (e.g., ifn-g), we rationalized that it might be able detect x-gal-stained blue cells. as shown in fig. 5 , there was no problem with using the instrument to count spots at a single-cell resolution and the analysis was highly efficient as the entire 96-well plate could be processed in less than 20 min. virus-infected cells appearing as doublets did not pose a problem because parameters on the analysis software could be adjusted to count two stained cells adjacent to each other as one. the number of infectious foci counted was quite linear as a function of virus inoculum (fig. 5c ), validating the methodology. this procedure could be used to quantify other assays based on x-gal staining of cells (e.g., recombinant vaccinia viruses that express h-galactosidase). in this study, we expressed sars-cov s-glycoprotein, which was able to pseudotype mulv particles. sars pseudoviruses were able to efficiently infect vero e6 cells, which have been shown to support sars-cov infection. the infection did not require low ph, suggesting viral entry is mediated by a direct fusion event between viral and plasma membranes. this result is consistent with a previous report that cell-to-cell fusion mediated by sprotein and its cellular receptor ace2 occurred at neutral ph (xiao et al., 2003) . however, our result is in direct disagreement with recently published article by simmons et al. (2004) . there are three major differences in exper-imental procedures between the two studies. first, we pseudotyped mulv particles whereas simmons et al. used hiv-1. second, we used an authentic s-glycoprotein whereas they used a c-terminal fusion protein that included a v5 epitope and polyhistidine tag, which totaled, by our estimation, 27 extra amino acids. whether the discrepant result is due to the use of different s-glycoproteins and/or different virus cores needs to be further investigated. the third difference between the studies is the concentrations of lysosomotropic agents used. while we used nh 4 cl at 0-50 am amounts, which are sufficient to inhibit vsv-g-mediated fusion ( fig. 3 ; picard-maureau et al., 2003) , they used millimolar (mm) amounts. at these concentrations, nh 4 cl could have a secondary effect on sglycoprotein. we were unable to find concentrations of chloroquine used in their study. it is interesting to note that while simmons et al. observed that pseudovirus infections required low ph, s-protein-mediated cell-to-cell fusion did not. the sars pseudoviruses we generated could be specifically inhibited by convalescent sera from sars-cov infected patients, indicating that s-glycoprotein of sars-cov is a target of neutralizing antibodies as it is for other coronaviruses. the major purpose of generating sars pseudoviruses was to devise an assay system to assess virus-neutralizing antibodies safely and rapidly without having to use infectious, replication-competent sars-cov. the results of our study indicate that sars pseudoviruses could be used to evaluate efficacy of various s-glycoprotein-based vaccine candidates to elicit virus-neutralizing antibodies. they could also be used to perform structurefunction analyses of s-glycoprotein. due to a large size of sars-cov genome, it would be difficult to perform such analyses directly in the context of the virus, not to mention potential safety hazards from working with it. in contrast, mutational analyses of the protein could be performed readily using pseudoviruses. our attempt to characterize biochemical and immunological properties of the s-protein was hampered by the fact that antibody titers against the protein in convalescent sera were extremely low; we were able to identify only a faint band on a western blot (with high background) and attempts to detect the protein by immunofluorescence and radioimmunoprecipitation assays were less than successful. in contrast, convalescent sera have been successfully used to detect sars-cov-infected cells by an immunofluorescence assay (hsueh et al., 2003; peiris et al., 2003) . together, the available data seem to suggest that s-protein might not be immunogenic, at least compared to other viral proteins. in fact, immunoreactivity analyses of a panel of synthetic peptides derived from s, membrane (m), and nucleocapsid (n) proteins suggested that n protein might be the most immunogenic protein . the nonimmunogenic nature of s-protein might present potential problems in developing a vaccine that can elicit potent neutralizing antibodies against sars-cov. in this regard, it is interesting to note that s-protein is highly glycosylated with 23 potential asparagine-linked glycosylation sites. based on our analyses of the ectodomain of the protein, carbohydrate residues account for approximately 30 kda (based on mobility in sds-page). the glycans were primarily high mannose and/or hybrid type. this, however, needs to be verified using proteins produced from nonvaccinia virus expression system, because the virus infection could possibly affect cellular glycosylation machinery. extensive glycosylation of hiv-1 envelope glycoprotein has been one of the major obstacles in eliciting good humoral responses against the protein and in developing an effective vaccine against the virus (cho, 2003) . it remains to be seen whether and to what extent glycans on s-protein affect immunogenic properties of the protein. interestingly, potential glycosylation sites are clustered into three regions of the protein (fig. 1a) : n-terminal, middle, and c-terminal. it has been shown that individual glycosylation sites on hiv-1 surface glycoprotein gp120 may have different functions; while some are important for evading immune responses, others are critical for maintaining proper protein structure necessary to interact with cellular receptors and mediate membrane fusion (ogert et al., 2001; reitter et al., 1998) . additional studies are needed to determine whether glycosylation sites in different clusters of s-protein serve different functions. in the absence of an effective vaccine and/or antiviral drugs against sars-cov, early detection of virus-infected patients would be critical for effective containment of future epidemics. quantitative rt-pcr-based diagnostic assays have been described for sars-cov (grant et al., 2003; lau et al., 2003; ng et al., 2003; poon et al., 2003a poon et al., , 2003b tang et al., 2004; yam et al., 2003) . despite high sensitivity, their utility has some limitations: (i) the detection rate varies widely between 20% and 80% depending on clinical sam-ples and protocols used for the assay; (ii) the window of detectability is limited to early stages of infection; and (iii) the assay is not suitable for routine surveillance. antibodies against sars proteins have been shown to appear as early as 9 days after the onset of illness (hsueh et al., 2003) . therefore, development of a high-throughput serologybased diagnostics could complement pcr-based assays. in this regard, a virus-neutralization assay could be used as a confirmatory test, which would enhance the accuracy of early diagnosis of sars-cov. because neutralizing antibodies are important for virus clearance, the assay could also be used to assess disease prognosis. in either case, the availability of sars pseudoviruses allows avoiding the use of infectious sars-cov. the overall cloning strategy is shown in fig. 1b . two parental plasmids encoding a sars-cov s gene (urbani strain), pentr-s and pcr-s, were obtained from the u.s. centers for disease control and prevention. two s-proteinexpressing plasmids (pcdna-s* and pcdna-s) were generated using pcdna-3 (invitrogen). the s gene in pcdna-s*, which was transferred from pentr-s (bamhi -ecori fragment), lacks the original translation stop codon taa because it was changed to aat of ecori restriction site (gaattc). pcdna-s with a stop codon was constructed by replacing a swai-ecori fragment of pcdna-s* with the same fragment from pcr-s. to generate phcmv-s, a bamhi -ecori fragment from pcdna-s was inserted into a bamhi site of phcmv-g following blunting ends with klenow. to construct ptm-s, a bamhi -xhoi fragment from pcdna-s was cloned into the corresponding sites of ptm-ndei (cho et al., 1994) . despite the fact that ptm-s has a small open-reading frame that encodes eight amino acids between the internal ribosome entry site of ptm-ndei vector and the s gene, s-protein was efficiently expressed and the plasmid was used as is without further modification. to generate ptm-eshis, 3v end of the ectodomain was pcr amplified using a sense primer 5v-gtc gtc aac att caa aaa gaa-3v (nts 3472 -3492 of s gene) and an antisense primer 5v-aat gaa gcg gat cccggg tta gtg atg gtg gtg atg atg ttg ctc ata ttt tcc caa-3v. base-pairing region (nts 3553-3570) is shown in bold and the six histidine residues are italicized. the amplified fragment was digested with swai (nt 3521) and smai (underlined) and subsequently cloned into ptm-s digested with swai and stui. cell culture, protein expression, and western blots all cell lines, except for vero e6, were maintained in dmem supplemented with 10% fetal bovine serum (fbs), 2 mm l-glutamine, and penicillin -streptomycin antibiotics. vero e6 cells were maintained in emem with the same supplements plus 0.1 mm nonessential amino acids. cells were cultured at 37 jc in 5% co 2 incubators. to express sprotein, cells were transfected with plasmids by a calcium phosphate precipitation method. briefly, 0.5 ml of 0.25 m cacl 2 solution containing 30 ag of plasmids was slowly mixed with 2â hbs (50 mm hepes, 1.5 mm na 2 hpo 4 , 280 mm nacl, ph 7.1) and the mixture was added to cells. after an overnight incubation, culture medium was replaced and cells were further incubated for two additional days. for expression from ptm-s and ptm-eshis, transfected cells were infected with vtf7-3 (fuerst et al., 1986 ) at a multiplicity of infection of 5. following 2 days of infection, cells were lysed with a hypotonic cell lysis buffer (10 mm tris, ph 8.0, 10 mm nacl, 1.5 mm mgcl 2 , 1% np-40). insoluble cell debris and nuclei were removed by a brief centrifugation in a microfuge. cell lysates were subjected to sds-page and western blot. s-proteins were detected with either a pool of convalescent sera (1:100 dilution) or anti-his (c-terminal) monoclonal antibody (invitrogen; 1:3000 dilution) followed by horseradish peroxidase-conjugated goat anti-human or anti-mouse igg antibody (pierce), respectively. protein bands were visualized using supersignal west pico chemiluminescence detection system (pierce). molecular weights of the protein bands were approximated by the mobility of standard molecular weight markers. pseudoviruses were generated as previously described (kim et al., 2001) . briefly, mulv packaging cell line telceb6 (schnierle et al., 1997) was transfected with pcdna-s, phcmv-s, phcmv-g (burns et al., 1993) , or pltr-gp140 (hiv-1 dh12 ; kim et al., 2001) using either calcium phosphate precipitation or lipofection (lipofectin; invitrogen) method. two days posttransfection (3 days for sars pseudovirus), cell culture medium was harvested and subjected to centrifugation (1700 â g, 10 min) to remove cell debris. supernatant was aliquoted, stored at à80 jc and used as a virus stock. virus titer was determined in vero e6 cells for sars-s and vsv-g or in hos-cd4-ccr5 (cheng-mayer et al., 1997; deng et al., 1996) for hiv-1 gp140 pseudotyped viruses. typically, cells were infected with 60-80 infectious units for 36 h. cells were washed with pbs and incubated with a fixative (1% formaldehyde, 0.05% glutaraldehyde in pbs) for 10 min at room temperature. the cells were washed twice with pbs and incubated with a freshly prepared staining solution (pbs containing 5 mm potassium ferricyanide, 5 mm potassium ferrocyanide, 2 mm magnesium chloride, and 1 mg/ml of x-gal) for >2 h at 37 jc. for routine analyses, x-gal-stained blue cells were manually counted using an inverted microscope. to determine ph-dependency of viral entry, vero e6 cells were incubated in culture medium containing 0 -100 am chloroquine for 1 h at 37 jc before adding viruses. vsv-g or sars-s pseudoviruses were allowed to adsorb to cells for 1 h at 37 jc in the absence of chloroquine. following adsorption, virus inoculum was removed, cells were washed, and infection was allowed to proceed for about 36 h in the absence of chloroquine. for nh 4 cl, cells were incubated with 0 -50 am. due to minimal cytotoxicity, nh 4 cl was present throughout the infection period including 1 h incubation before virus addition. all infections were done in duplicates. neutralization assay was performed as previously described (kim et al., 2001) using convalescent sera (13 -50 days post-onset of symptoms) obtained from cdc or from patients hospitalized in queen mary hospital, hong kong. approximately 60 -80 infectious units of pseudoviruses were incubated with serially diluted, heat-inactivated (56 jc, 30 min) convalescent or normal sera for 1 h at 37 jc. the mixture was subsequently added to vero e6 (for sars-s or vsv-g) or hos-cd4/ccr5 (for hiv-1 gp140) cells. virus infection was allowed to proceed for another 36 h. virus-neutralizing activity was determined relative to no serum control. the general pseudovirus infection procedure is the same as described above. the major difference was that 96-well plates with a white membrane bottom normally used for elispot assays (plate m200; bd biosciences) were utilized rather than regular tissue culture plates. immunospot analyzer from cellular technology ltd. was used as per manufacturer's recommendations. vesicular stomatitis virus g glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus macrophage tropism of human immunodeficiency virus type 1 and utilization of the cc-ckr5 coreceptor subunit protein vaccines: theoretical and practical considerations for hiv-1 membrane rearrangement and vesicle induction by recombinant poliovirus 2c and 2bc in human cells monoclonal antibodies to murine hepatitis virus-4 (strain jhm) define the viral glycoprotein responsible for attachment and cell -cell fusion protection from lethal 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1 the sars-cov s glycoprotein: expression and functional characterization evaluation of reverse transcription-pcr assays for rapid diagnosis of severe acute respiratory syndrome associated with a novel coronavirus requirement of proteolytic cleavage of the murine coronavirus mhv-2 spike protein for fusion activity we are grateful to cdc for providing plasmids encoding sars-cov s gene and convalescent sera, to dr. bernard moss for vtf7-3, to dr. franc ßois-loïc cosset for telceb6 cell line, and to drs. jonathan silver and mario skiadopoulos for vero e6 cells. hos-cd4-ccr5 cell line was obtained from dr. nathaniel landau through the aids research and reference reagent program, division of aids, niaid, nih. we specially thank dr. magdalena tary-lehman for providing assistance with using immuno-spot analyzer. key: cord-281237-asnpuami authors: garten, wolfgang; stieneke, andrea; shaw, elliott; wikstrom, peter; klenk, hans-dieter title: inhibition of proteolytic activation of influenza virus hemagglutinin by specific peptidyl chloroalkyl ketones date: 1989-09-30 journal: virology doi: 10.1016/0042-6822(89)90103-7 sha: doc_id: 281237 cord_uid: asnpuami abstract lysates of cultured cells have been analyzed for arginine-specific endoproteases using peptidyl-p-n itroanil ides as chromogenic substrates. the enzymes present in mdbk, mdck, vero, bhk, and chick embryo cells required lysinearginine or arginine-arginine pairs as cleavage sites, whereas chorioallantoic membrane cells contained, in addition, an activity that could cleave at a single arginine. the effect of peptidyl chloroalkyl ketones on the activation of the fowl plague virus hemagglutinin by the proteases specific for paired basic residues has been investigated. when virions containing uncleaved hemagglutinin were incubated with lysates of uninfected cells, cleavage was completely inhibited by peptidyl chloroalkyl ketones containing paired basic residues at a concentration of 1 mm. in contrast a compound containing a single arginine had no inhibitory activity. when dibasic peptidyl chloroalkyl ketones were added to infected cell cultures, cleavage of hemagglutinin and multiple cycles of virus replication were inhibited at 10 mm. however, a 100-to 200-fold increase of the inhibitory activity in intact cells could be achieved by n-terminal acylation. these studies suggest a potential role of peptidyl chloroalkyl ketones as antiviral agents biosynthesis of the hemagglutinin of influenza virus involves post-translational proteolytic cleavage of the precursor ha into the subunits ha, and ha;!. cleavage is necessary for the expression of the fusion capacity of the hemagglutinin and, thus, for initiation of infection (klenk et a/., 1975; lazarowitz and choppin, 1975) . two host enzymes are involved in cleavage: first a "ttypsin-like" endoprotease acts on a distinct arginine-glycine bond. subsequently, a carboxypeptidase eliminates the basic amino acids at the cleavage site. proteolytic activation proved to be of high importance for the spread of infection and for pathogenicity. the hemagglutinins of mammalian and of apathogenic avian influenza viruses which cause local infections are cleaved only in a restricted number of cell types. in contrast, the hemagglutinins of pathogenic avian influenza viruses which cause systemic infection are activated in a broad range of different host cells. important determinants for these differences in proteolytic activation are the structure of the cleavage site on the hemagglutinin and the specificity of the endoproteases present in individual cells. the hemagglutinin of most apathogenic avian and of mammalian influenza viruses have a single arginine residue at the cleavage site which is recognized by enzymes present only in few ceils, whereas ' to whom requests for reprints should be addressed. the hemagglutinins of the pathogenic avian influenza viruses have a cleavage site consisting of several basic amino acids which can be cleaved by endoproteases present in many cells (for review see klenk and rott, 1988) . post-translational proteolytic cleavage at multiple basic residues appears to be a general principle by which many hormones, enzymes, and other biologically important peptides or proteins are activated (docherty et a/., 1982; herbert and uhler, 1982) . this type of cleavage has been also observed with a whole series of other viral glycoproteins. these include the f protein of paramyxoviruses (scheid and choppin, 1974; paterson et al., 1984) , the precursor to the e2 protein of coronaviruses (binns et al., 1985) , the surface glycoprotein of arenaviruses (romanowski et al., 1985) , and the env protein of retroviruses (shinnick et al., 1981) . the minimal consensus sequence that can serve as a recognition signal for the converting enzymes appears to be a pair of basic amino acids, most frequently lysine-arginine or arginine-arginine. however, the length of the basic peptide at the cleavage site often exceeds these 2 amino acids, and the available evidence indicates that the additional basic residues may increase cleavability (kawaoka et al., 1988) and pathogenicity (ohuchi et al., 1989) . peptidyl chloroalkyl ketones are potent protease inhibitors, that covalently label the substrate binding sites of proteolytic enzymes. considerable specificity for inactivating "trypsin-like" enzymes involved in coagulation and fibrinolysis has been obtained by modifying the peptidyl moiety of the reagents (kettner and shaw, 1981) . in the present study we have analyzed the effect of various peptidyl chloroalkyl ketones on hemagglutinin cleavage and replication of a pathogenic avian influenza virus, fowl plague virus. we show that compounds containing paired basic amino acids are effective inhibitors. the influenza virus strains a/fpv/rostock/34 (h7n 1) and a/fpv/dutch/27 (h7n7) (dobson variant, adapted to growth in bhk cells) were used. seed stocks were propagated in the allantoic cavity of 1 1 -day-old embryonated eggs. the dutch strain was grown in bhk21 -f cells in the presence of ionophore a23187 (calbiothem, frankfurt) added to reinforced eagle's medium (rem) lacking calcium chloride, when virus with uncleaved hemagglutinin was needed (klenk eta/., 1984) . the restock strain was grown in confluent monolayers of chicken embryo cells (cec). to obtain radioactive virus particles, [35s]methionine (20 &i/ml) or [3h]glucosamine (5 &i/ml) was added to methionine-free medium or to medium with glucose replaced by 10 mlvi fructose, respectively. before metabolic labeling with [35s]methionine, cells were depleted of methionine for 30 min. virus released into the supernatant of the cell cultures was collected by centrifugation and purified on sucrose gradients (klenk et al., 1977) . when virus was propagated in the presence of peptidyl chloroalkyl ketones, inhibitors were solubilized in 1 mm hci, except for the palmitoylated one, which was dissolved in dimethyl sulfoxide. the stock solutions of the peptidyl chloroalkyl ketones, which were stored at -2o", where diluted 1 o-fold with pbs or rem just before use. when the palmitoylated inhibitor was added to medium it was dispersed by ultrasonification in a branson sonifier (10 set, 50 w). hemagglutination and plaque assays were carried out for quantification of the virus according to standard procedures. samples containing radioactively labeled viral glycoproteins were analyzed by sds-page (laemmli, 1970) directly or after immunoprecipitation. gels contained 12% acrylamide and 0.32% bisacrylamide. after treating gels with enhance (new england nuclear), fluoro-graphies were carried out on kodak x-omat ar films at -70". nonradioactive viral proteins were analyzed by immunoblotting (kuroda et a/., 1986) . for immunoblotting and immunoprecipitation a rabbit antiserum against fpv (restock strain) was used. monolayer cultures of cec, chorioallantoic membrane (cam) cells (klenketal., 1975) and various other cell types were scraped from petri dishes (5-cm diameter) and resuspended in 1 ml pbs, containing 10 mm caci, and 2% n-octylglucoside. the cells were sonicated with a branson sonifier for 3 set at 0" and immediately used for assays (klenk et a/., 1984) . the following chromogenic substrates, which were purchased from bachem (bubendorf, switzerland), were used: /v-cbz-l-tyrosyl-l-lysyl-l-arginine-p-nitroanilide. 2tfa (cbz-ykr-pna), n-cbz-r--arginyl-l-argininep-nitroanilide. 2hci (cbz-rr-pna), and iv-cbz-l-arginine-p-nitroanilide (cbz-r-pna). aqueous solutions of these substrates were added to 50 ~1 cell lysates in a total volume of 500 ~1, containing 10 mm caci,. after incubation at 37" for 2 hr the reaction mixture was centrifuged at 10,000 g for 5 min, and the optical density of the supernatant was measured at 405 nm. when hemagglutinin was used as substrate, about 200 hau of radioactively labeled fpv (dutch strain) containing uncleaved hemagglutinin were incubated in 25 ~1 pbs with 10 ~1 cell lysates in the presence of 10 mlli caci, (klenk et al., 1984) . proteolytic cleavage was stopped by adding 35 ~1 sample buffer, containing 1% sds and 1% dithiothreitol. for electrophoresis, samples were boiled for 2 min and applied to slab gels. proportions of uncleaved and proteolytically cleaved hemagglutinin before and after protease treatment were estimated by scanning fluorographies on a bio-rad model 620 videodensitometer. alanyl-lysyl-arginyl chloromethyl ketone (akr-cmk), tyrosyl-alanyl-lysyl-arginyl chloromethyl ketone (yakr-cmk), phenylalanyl-alanyl-lysyl-arginyl chloromethyl ketone (fakr-cmk), and alanyl-phenylalanyl-arginyl chloromethyl ketone (afr-cmk) were synthesized as described by kettner and shaw (1981) . phenylalanylalanyl-lysyl-arginyl chloroethyl ketone (fakr-cek) and palmitoyl-phenylalanyl-alanyl-lysyl-arginyl chloroethyl ketone (palfakr-cek) were prepared by related procedures (wikstrom et a/., 1989). a peptic&-p-nitroanilides (2 mn/l) were incubated with cell lysates in pbs containing 10 m m caci, and 2% n-octylglucoside at 37" for 2 hr. extinction ofp-nitroanrlrne was measured at 405 nm. the values indicate the percentage of cleavage relative to total cleavage as obtarned after incubation of substrate with trypsin (1 pg/ml). to characterize the proteases responsible for the activation of the fpv hemagglutinin we have used virus with the uncleaved glycoprotein as substrate in previous work. such virus, which is grown in the presence of the calcium-specific ionophore a 23 187 (klenk et a/., 1984) , can only be obtained in small amounts. furthermore, the ceil lysates employed in these studies are likely to contain a wide spectrum of different proteases, and it is known that the influenza virus hemagglutinin can be cleaved into fragments similar in size to ha, and ha, by several nonactivating enzymes that differ in their substrate specificities from the trypsin-like proteases (garten et al., 198 1) . for these reasons we have used, in addition to virus grown in the presence of a 23 187, chromogenic peptides mimicking the cleavage site as an alternate substrate in the present study. as shown in table 1 , peptidylp-nitroanilides containing two basic amino acids are cleaved by lysates obtained from all cells analyzed. there are differences in the relative cleaving activities of the individual cell lines with mdbk and bhk cells on the extreme ends. variation in the sequence of the two basic residues (arginine-arginine versus lysine-arginine) ( table 1) or n-terminal elongation of the peptide by adding several uncharged amino acids (alanine or tyrosine) (data not shown) had no significant effect on the substrate specificity. however, when the basic sequence was reduced to a single arginine, the peptide had lost its function as a substrate with most cells, except for cam cells. thus, the chromogenic peptides resemble very closely in their differential substrate specificities the influenza virus hemagglutinins among which those containing a single arginine are also cleaved only in cam cells, whereas those containing several basic residues are usually cleaved in many cells. the chromogenic peptides are therefore suitable substrates for the proteases activating the hemagglutinin. it has been shown that peptidyl chloroalkyl ketones with amino acid sequences analogous to physiological substrates specifically inactivate the corresponding serine proteases (kettner and shaw, 1981) . we have therefore used such substances to inhibit the proteases activating the hemagglutinin. table 2 shows an experiment in which a cell lysate has been first incubated with akr-cmk as an inhibitor and subsequently with chromogenic substrates containing also two basic residues at the cleavage site. the data indicate, that at inhibitor concentrations of 0.1 and 1 mm the proteolytic activity is inhibited by 50 and 1 oo%, respectively. we have then used hemagglutinin as substrate to analyze the effect of various peptidyl chloroalkyl ketones on proteases in cell lysates. table 3 shows that all compounds including a palmitoylated derivative had inhibitory activity if they contained a pair of basic residues in contrast, the ketone with a single arginine was ineffective. this observation indicates that the specificity of these inhibitors varies depending on the number of basic residues, as is the case with the substrate specificity of the proteases they interfere with. ' enzymatic activity in lysates preincubated with the inhibitor was assayed as described under materials and methods, substrates were used at a concentration of 2 m m. note. purified fpv (dobson variant) with uncleaved hemagglutinin which has been grown in bhk cells in the presence of a231 87 (0.25 pm) and [3h]glucosamine (2 &i/ml) was incubated with mdbk cell lysates as described under materials and methods. before incubation with the virus, cell lysates have been pretreated at room temperature for 60 min with various concentrations of the inhibitors indicated. the water-soluble inhibitors were dissolved in 1 m m hci, the acylated inhibitor in dimethyl sulfoxide. thereafter the virions were disintegrated in sample buffer under reducing conditions. viral proteins were analyzed by sds-page and fluorography. cleaved and uncleaved hemagglutinin were quantitated by densitometry. the difference in the amount of uncleaved hemagglutinin found in untreated virus and in virus exposed to cell lysate in the absence of inhibitors was considered 100% cleavage. it was now of interest to find out whether peptidyl chloroalkyl ketones are able to interfere with hemagglutinin processing in the course of virus replication in the intact cell. after pretreatment with fakr-cmk for 24 hr, monolayers of ce cells were infected with fpv under single replication cycle conditions. virus released from the cultures was purified and analyzed for its polypeptide composition (fig. 1) . essentially the same results have been obtained, when the restock strain, as shown here, and the dutch strain (data not shown) have been used. when the inhibitor was present before and after infection, virus with uncleaved hemagglutinin was produced. to obtain complete inhibition of cleavage, inhibitor concentrations of 10 mm were needed (fig. 1 b) . when the inhibitor was removed from the cell culture after the pretreatment, virus was produced with cleaved hemagglutinin (fig. 1 a) as was the case in the control experiment performed without inhibitor (fig. lc) . incubation with inhibitor did not result in a significant reduction of virus release as measured by hemagglutination titers in the media. even extended inhibitor pretreatment for as much as 3 days had little effect on the cells. these observations indicate that fakr-cmk prevents cleavage not only in a cell lysate, but also in the infected intact cell. furthermore, fakr-cmk showed little toxicity in cell culture. the monobasic inhibitor far-cmk, in contrast, proved to be quite toxic. already at a concentration of 1 mm, cells were severely damaged and did no longer allow virus replication. the intracellular compartmentalization and the membrane anchorage of the cleavage enzyme (klenk et al., 1984) may explain the relatively high concentrations of the water soluble inhibitors (10 mm) that are needed to prevent hemagglutinin cleavage in cell culture. because of its amphophilic character, the acylated compound palfakr-cek should have better enzyme access in the intact cell. as has been pointed out above (table 3) this inhibitor had to be used in similar amounts as the water soluble compounds to be effective in cell lysates. however, when added to cells infected with the restock strain, palfakr-cek caused total inhibition of cleavage already at a concentration of 50 pm (fig. 2a) , whereas 10 mm were required with fakr-cmk (fig. 1) . even at the 5-p/\/1 concentration of palfakr-cek, partial inhibition was still observed (fig. 2b) . similar results have been obtained with the dutch strain (data not shown). thus, it is clear that acylation enhanced the inhibitory effect in intact cells. (1 o6 pfu/ml) and incubated in dulbecco's medium containing 50 (a) or 5 pm (b) pal-fakr-cmk. after 4 hr the medium was replaced by rem without methionine but still containing the inhibitor at the respective concentrations. after further 30 min, 20 &i/ml of [35s]methionine was added for 15 hr. viral proteins were immunoprecipitated from cell lysates and analyzed by sds-page. a control experiment carried out in the absence of inhibitor is also shown (c). since inhibition of hemagglutinin cleavage has been accomplished, it was of interest to find out whether the compounds interfere with the spread of virus infection. virus was therefore propagated in ce cell cultures under conditions of multiple cycle replication, and the effect of the inhibitors on virus growth was analyzed. again, essentially the same results have been obtained with the restock and the dutch strain. as shown in fig. 3a , fakr-cmk had to be added to the culture at a concentration of 10 mm to obtain a significant effect. again, the efficiency of the inhibitor could not be altered by replacing the methyl by an ethyl group, or by adding uncharged amino acids to the amino-terminal end of the peptide (data not shown). however, acylation of the peptide resulted in a distinct increase in inhibitory activity (fig. 3b) . the observation that virus production was only retarded, but not completely suppressed, even under the most effective inhibitory conditions, is most likely explained by gradual degradation of the peptidyl chloroalkyl ketones in cell culture. we demonstrate here that dibasic peptidyl chloroalkyl ketones inhibit cleavage activation of the fpv hemagglutinin. these results have been obtained by analyzing hemagglutinin processing, first in ce cell cultures infected with the restock and the dutch strain of this virus, and second, in an in vitro system using lysates of mdbk cells and virions of the dutch strain con-taining the uncleaved hemagglutinin precursor (klenk et al., 1984) . since the hemagglutinin has essentially the same cleavage site in the restock and in the dutch strain (klenk and rott, 1988) , it is legitimate to compare the results obtained in both systems. specific peptidyl chloroalkyl ketones have been shown in previous studies to be useful agents for the selective inactivation and characterization of viral (korant et al., 1979) and cellular cysteine and serine proteinases. the latter group of proteinases includes trypsin, plasmin, acrosin, and several bacterial proteinases, which under certain conditions may also serve as activating enzymes (lazarowitz et al., 1973; klenk et a/., 1977; garten et al., 1981; tashiro et a/., 1987a,b) . it has been reported that tamino caproic acid and aprotinin interfere with activation of the influenza virus hemagglutinin by plasmin present in the alveolar fluid (zhirnov et a/., 1982a,b) , and leupeptin inhibits proteinases secreted by coinfecting bacteria (tashiro et al., 1987a,b) . in contrast to these inhibitors that act in the extracellular space, the peptidyl chloroalkyl ketones, as used in the present study, block intracellular enzymes. the observation that the palmitoyl derivative of the inhibitor is loo-fold more effective than the free form supports our previous finding that the enzyme is bound to cellular membranes. we have also reported that the protease is calcium-dependent and acts at neutral ph (klenk et a/., 1984) . cleavage of the fpv hemagglutinin is a late event in intracellular transport and is assumed to occur shortly before the glycoprotein arrives at the plasma membrane (klenk et al., 1981) . the enzyme responsible for activation of the fpv hemagglutinin shows a number of striking similarities in subcellular location and catalytic properties to enzymes believed to process prohormones at pairs of basic residues in endocrine tissues. proteases cleaving the insulin precursor, that have been isolated from secretory granula of pancreatic p cells, have been found to be under the control of intraorganellar calcium and ph (davidson et al., 1987 (davidson et al., , 1988 . a protease has been isolated from secretory vesicles of the bovine pituitary neural lobe that cleaves proteins and peptides at neutral ph (parish et al., 1986) . the a-factor matrng pheromone, a peptide hormone from yeast, is activated at pairs of basic residues by a membrane-bound serine protease that is strictly calcium-dependent and has a neutral ph optimum (thomas et a/., 1988) , exactly as has been observed for the activating enzyme of the fpv hemagglutinin. it is therefore reasonable to assume, but remains to be proven, that the enzymes activating viral glycoproteins and peptide hormones belong to the same family of proteinases. our data show that the enzymes cleave peptidyl-p-nitroanilides with a similar substrate specificity as influenza virus hemagglutinin. thus, these chromogenes may substitute for the hemagglutinin, when large amounts of substrate are required, as may be the case, for instance, in studies aimed at the characterization of the activating proteinases. as has been pointed out above, a large number of viral glycoproteins are cleaved by cellular proteases at arginine-arginine and lysine-arginine sites. it will be interesting to see whether cleavage of these glycoproteins can also be inhibited by the compounds analyzed here. with many of these viruses, e.g., the togaviruses, the coronaviruses, the arenaviruses, the herpes viruses, and most of the retroviruses, the biological significance of the cleavage reaction is not understood. studies with inhibitors of the type analyzed here should throw light on this problem. in cases, where cleavage of a protein at an arginine-containing site has been suspected, but not definitely proven, the use of peptidyl chloroalkyl ketones might result in the accumulation of polypeptides which so far have not been identified as precursors. the inhibitors might, thus, also help to elucidate new precursor-product relationships. the list of viruses that depend on proteolytic activation of a glycoprotein to spread in the organism in-cludes hiv 1 (mccune et al., 1988 ) and many other important pathogens. the application of peptidyl chloroalkyl ketones as antiviral agents is therefore worth considering. as has already been pointed out above, the available evidence indicates that cleavage activation at arginine residues is a mechanism by which also essential cellular functions are regulated. peptidyl chloroalkyl ketones are therefore usually considered toxic and their application for therapeutic purposes may not be feasible. however, we have shown here that activation of the fpv hemagglutinin is blocked by fakr-cmk, but not by fak-cmk. thus, our data provide evidence for some sequence-specificity. as has been pointed out above, with most viral and cellular proteins the cleavage site consists not only of a pair but of a whole series of basic residues, which all may contribute to its specificity as a recognition signal for the activating proteinases. furthermore, among different proteins there is considerable sequence variation in the connecting peptides. it may therefore be desirable to construct chloroalkyl ketones with an extended peptide showing exact homology to a specific cleavage site. such inhibitors may be more selective among the proteases they interact with and, thus, be less toxic. cloning and sequencing of the gene encoding the spike protein of the coronavirus ibv proteolytic converslon of proinsulin into insulin intraorganellarcalcium and ph control proinsulin cleavage in the pancreaticp cell wa two distinct site-specific endopeptidases conversion of prolnsulin to insulin: involvement of 31,500 molecular weight thiol protease proteolytic activation of the influenza hemagglutinin: the structure of the cleavage site and the enzymes involved in cleavage biosynthesis of polyprotein precursors to regulatory peptides sequence requirements for cleavage activation of influenza virus hemagglutinin expressed in mammalian cells inactivation of trypsln-like enzymes with peptides of arginine chloromethyl ketone processing of hemagglutlnin. ln "genetic variation among influenza viruses inhibition of proteolytic cleavage of the hemagglutlnin of influenza virus by calciumspecific ionophore a23187 the molecular basis of influenza virus pathogenicity further studies on the activation of influenza virus by proteolytic cleavage of the hemagglutinin activation of influenza a virus by trypsin treatment virus speclfied protease in pollovirus-infected hela cells expression of the influenza virus hemagglutinin in insect cells by a baculovlrus vector cleavage of structural proteins during the assembly of the head of the bacteriophage t 4 enhancement of the infectivity of influenza a and b viruses by proteolytic cleavage of the hemagglutinin polypeptide proteo-lyt~c cleavage of the hemagglutlnin polypeptide of influenza virus. function of the uncleaved polypeptlde ha endoproteolytic cleavage of gpl60 is required for the activation of human immunodeficiency virus mutations at the cleavage site of the hemagglutinin alter the pathogenicity of influenza virus a/ chick/penn/83 (h5n2) purification and characterization of a paired basic reslduespecific prohormone-converting enzyme from bovine pituitary neural lobe secretory vesicles complete sequence of the srna of lymphocytlc choriomeningitls virus (we strain) compared to that of plchlnde arenavirus ldentlfication of biological activities of paramyxovirus glycoproterns. activation of cell fusion, hemolysis, and lnfectlvlty by proteolytlc cleavage of an inactive precursor protein of sendai virus nucleotide sequence of moloney murine leukaemia virus synergistic role of staphylococcal proteases in the induction of influenza virus pathogenicity role of staphylococcus protease in development of influenza pneumonia yeast kex2 endopeptidase cleaves a neuroendocrine prohormone in mammalian cells. sc/-ence 241 the properties of peptldyl diazoethanes and chloroethanes as protease inactivators protective effect of protease inhibitors in influenza virus infected animals proteolytic activation of influenza wsn virus in cultured cells is performed by homologous plasma enzymes we thank 6. trier for excellent technical assistance. this work was supported by the deutsche forschungsgemeinschaft (sfb 47) and by the fonds der chemischen industrie. key: cord-290640-kh2t0kfz authors: o'connor, jennifer black; brian, david a. title: downstream ribosomal entry for translation of coronavirus tgev gene 3b date: 2000-03-30 journal: virology doi: 10.1006/viro.2000.0218 sha: doc_id: 290640 cord_uid: kh2t0kfz abstract gene 3b (orf 3b) in porcine transmissible gastroenteritis coronavirus (tgev) encodes a putative nonstructural polypeptide of 27.7 kda with unknown function that during translation in vitro is capable of becoming a glycosylated integral membrane protein of 31 kda. in the virulent miller strain of tgev, orf 3b is 5′-terminal on mrna 3–1 and is presumably translated following 5′ cap-dependent ribosomal entry. for three other strains of tgev, the virulent british fs772/70 and taiwanese tfi and avirulent purdue-116, mrna species 3–1 is not made and orf 3b is present as a non-overlapping second orf on mrna 3. orf 3b begins at base 432 on mrna 3 in purdue strain. in vitro expression of orf 3b from purdue mrna 3-like transcripts did not fully conform to a predicted leaky scanning pattern, suggesting ribosomes might also be entering internally. with mrna 3-like transcripts modified to carry large orfs upstream of orf 3a, it was demonstrated that ribosomes can reach orf 3b by entering at a distant downstream site in a manner resembling ribosomal shunting. deletion analysis failed to identify a postulated internal ribosomal entry structure (ires) within orf 3a. the results indicate that an internal entry mechanism, possibly in conjunction with leaky scanning, is used for the expression of orf 3b from tgev mrna 3. one possible consequence of this feature is that orf 3b might also be expressed from mrnas 1 and 2. expression of coronavirus genes occurs through the synthesis of a 3ј coterminal nested set of mrnas. although coronavirus mrnas are structurally polycistronic (the 3ј-most mrna in many but not all viral species is monocistronic), evidence from studies of translation both in vitro and in vivo has suggested that most function as monocistronic messages. that is, despite their polycistronic configuration, usually only the 5ј-terminal orf on each is abundantly translated (reviewed in lai and cavanagh, 1997, and luytjes, 1995) . the location of some coronavirus genes, however, is not 5ј-terminal on any mrna, which would require that the gene, if expressed, be translated by a mechanism allowing translation reinitiation, leaky scanning, frameshifting, or a downstream entry of ribosomes. examples of such genes include (1) orf 1b on mrna 1, from which a polyprotein is synthesized following ϫ1 ribosomal frameshifting (brierly et al., 1987; eleouet et al., 1995) ; (2) orfs 3b and 3c on avian infectious bronchitis virus mrna 3 (boursnell et al., 1985) , from which 7.4-and 12.4-kda proteins are synthesized following leaky scanning and internal ribosomal entry, respectively (liu et al., 1991; liu and inglis, 1992; le et al., 1995) ; (3) orf 5b on mouse hepatitis virus mrna 5 (skinner et al., 1985) , from which a 9.6-kda protein(the e protein) is synthesized (budzilowicz and weiss, 1987; leibowitz et al., 1988) , by an apparent internal ribosomal entry mechanism (thiel and siddell, 1994) ; and (4) the i orf in mrna 7 of the bovine and mouse hepatitis coronaviruses, from which the i protein is made in the ϩ1 reading frame relative to n (senanayake et al., 1992; fischer et al., 1997) following ribosomal scanning (senanayake and brian, 1997) . in this study, we examine the mechanism by which gene 3b is expressed from mrna 3 in the purdue strain of tgev and demonstrate that, surprisingly, it may be approached by ribosomes entering internally and not necessarily through a leaky scanning step as would be predicted from mrna 3 sequence. gene 3b in tgev is unusual in that for one strain of virus, the virulent miller strain (wesley et al., 1989) , it is expressed as the 5јterminal orf on mrna 3-1, whereas in three other strains, the virulent british fs772/70 and taiwanese tfi strains and the avirulent purdue-116 strain, it is expressed as the second orf on mrna 3 (britton et al., 1989; chen et al., 1995; this study) (note mrna structures in fig. 1a ). the differences in transcription patterns appear to be a function of the canonical tgev ucuaaac intergenic sequence positioned 18 nt upstream of gene 3b in the genome, which in the miller virus totally conforms to the canonical sequence but in the british fs772/70 and purdue-116 strains is ucuaaau and in the taiwanese tfi strain is acaaaac. the nonconforming intergenic sequences apparently fail to promote synthesis of a subgenomic mrna. translation from orf 3b when it occurs as the second orf on mrna 3 must, therefore, require either a reinitiation of translation after translation of the upstream orf, a leaky scanning by ribosomes over a long distance (431 nt; fig. 1b ), or a downstream entry of ribosomes. because gene 3b is not intact in some strains of tgev (i.e., it is either severely truncated by frameshift mutations as in the purdue-115 strain [rasschaert et al., 1987] or by deletions as in the avirulent miller strain [wesley et al., 1990 [wesley et al., , 1991 ) and cannot produce a full-length product, it has been suggested that its product fulfills a specialized function, perhaps during animal infection (reviewed in enjuanes et al., 1995) , and is not required for virus replication. similar conclusions were reached after revelations of a truncated gene 3b in strains of the closely related porcine epidemic diarrhea virus (vaughn et al., 1995) . it might therefore be assumed that gene 3b is not translated when it occurs as a downstream orf as in mrna 3. however, a product from gene 3b is made in cells infected with the purdue-116 strain of virus (o'connor and brian, 1999) , indicating a mechanism must exist for its synthesis from mrna 3. here we report that, whereas mrna 3 has a sequence predicting leaky scanning for the translation of orf 3b by the model of kozak (1989) , experimental results with mutant constructs suggested downstream entry of ribosomes might also be used. furthermore, deletion analysis indicated that the internal entry of ribosomes did not depend on an immediate upstream internal ribosomal entry structure (ires) and suggested ribosomes are entering very close to the orf 3b start site by a mechanism resembling shunting. mrna 3, but not mrna 3-1, is made in cells infected with the purdue-116 strain of tgev northern analyses of purdue-116 virus-infected cells carried out previously in our laboratory with oligonucleotide probes specific for the 3ј end of the genome (i.e., a sequence from within the 3ј-proximal hp orf) had identified eight species of mrna, leading us to conclude that orfs encoding the 7.7-kda (gene 3a) and 27.7-kda (gene 3b) proteins are each 5ј-terminal on separate mrna species (sethna et al., 1989) . to test this conclusion, separate northern analyses were done with probes specific for the 90-nt leader and for genes 3a and 3b. our rationale was that mrna species 3 and 3-1 would be distinguishable with probes binding within orfs 3a and 3b because transcripts of 3875 and 3561 nt (or even 4075 and 3761 nt if they included poly a tails of 200 nt in length) are resolvable on a gel of 1% agarose. northern analyses with the separate probes revealed bands with identical mobilities, indicating the presence of mrna 3, but not mrna 3-1, in rna from purdue-116 virus-infected cells ( fig. 2a, lanes 4-9) . to test this conclusion by a second method, rt-pcr analysis was done with oligonucleotide primers specific to gene 3b and the minus strand of the leader. amplified products of 463 and 876 nt would be expected from mrnas 3-1 and 3, respectively. a product of 5248 nt northern blot analysis using gene-specific probes. (b) rt-pcr analysis of rna from purdue or miller virus-infected cells using a plus-strand-detecting oligonucleotide from within orf 3b as the first primer (for rt and subsequent amplification) and a minus-strand-detecting oligonucleotide from within the leader sequence for amplification. products were electrophoresed on a gel of 1% agarose and stained with etbr. might also be found from mrna 2, the mrna encoding the spike protein. from rt-pcr analysis only a single product of 876 nt with the proper sequence as determined by cloning and sequencing was obtained (fig. 2b , lane 3; sequencing data not shown), indicating the presence of mrna 3 but not mrna 3-1. to establish that the experimental protocol would have detected mrna 3-1 if present, rna was extracted from miller virus-infected cells and used in parallel. from this, the expected 463-nt mrna 3-1-derived product was obtained (fig. 2b , lane 4). no product was obtained with rna from uninfected cells (fig. 2b, lane 2) . genes 3a and 3b, but not gene 4, are translated in vitro from synthetic mrna 3-like transcripts containing all three orfs to test by in vitro translation whether the 27.7-and 20-kda gene 3b products (o'connor and brian, 1999) are synthesized when orf 3b is positioned downstream of orf 3a (beginning at base 337) on synthetic transcripts, uncapped transcripts of porf3a-3b-4 dna linearized at the bamhi site 50 nt downstream from the stop codon of gene 4 (fig. 1c) were translated in either wheat germ extract or rabbit reticulocyte lysate. in both, products from genes 3a (the 7.7-kda protein containing one methionine) and 3b (the 27.7-kda form of the protein containing eight methionines and the 20-kda form presumably containing seven methionines), but not gene 4 (the 9.2-kda e protein containing four methionines), were obtained (results for wheat germ extract are shown in fig. 3a , lane 5; note the marker positions in lanes 2-4 and the absence of endogenous product in lane 1). because a nearly 27-kda protein was also synthesized from an endogenous transcript in (some) rabbit reticulocyte lysates and the presence of abundant globin protein in the lysate interfered with the resolution of small proteins (data not shown), all subsequent studies described were carried out in wheat germ extract. thus, from uncapped transcripts bearing similarity to mrna 3 the first methionine codon in gene 3b (for synthesis of the 27.7-kda protein) and also the second (assuming synthesis of the 20-kda protein initiates at the second methionine codon [o'connor and brian, 1999] ) are accessed for translation. on a molar basis, the amount of 27.7-kda gene 3b product is approximately one-fifth of that from gene 3a (fig. 3b ). there was no evidence of a 9.2-kda e protein from orf 4 in this (fig. 3a , lane 5) or in subsequent experiments, indicating ribosomal accessibility of orf 3b was probably not the result of template fragmentation. an upstream leader-containing sequence in transcripts of plorf3a-3b-4 ( fig. 1c) , although containing an additional 14 nts not found on mrna 3, had only a small effect on the rate of translation from orf 3b relative to 3a (fig. 3a , lane 7, and fig. 3c ), indicating the leader se-quence may not strongly influence translation from the downstream orf; however, this needs confirmation with transcripts precisely mimicking the 5ј end of mrna 3. as with transcripts of porf3a-3b-4, no product was evident from orf 4. translation of gene 3b from mrna 3-like transcripts shows a pattern not fully consistent with a leaky scanning model from precedents in eukaryotic mrnas, it is unlikely that ribosomes would approach orf 3b on mrna 3 by a mechanism of translation reinitiation, since three inframe strong stop codons follow orf 3a (fig. 1b) . however, an approach by leaky scanning according to the model of kozak (1989 kozak ( , 1991a ,b) might be expected, since the initiator codon for orf 3a (ugua123ugg, in the quantitation of products from the indicated constructs as determined from ambis radioanalytic imager scans of the gel shown in (a). note that only detected proteins are represented in the bar graph. * denotes a putative aggregate of the orf 3b product; † indicates plasmid dna was linearized at the scai site within orf 3b before transcription. same reading frame as orf 3b) and for three other potential small orfs within gene 3a (uaga167ugc, caua283ugc, and ucca424ugc, all in the ϩ1 reading frame relative to orf 3b) are within contexts considered weak for initiation, whereas that for orf 3b (aaaa432uga) is considered relatively strong. to test for ribosomal scanning on mrna 3-like transcripts, three approaches were taken. in the first, the effect of a 5ј cap on the synthesis of 3a and 3b gene products was measured. increased synthesis from both would be expected if 5ј cap-dependent entry followed by leaky scanning were used (kozak, 1989 (kozak, , 1991a . increased synthesis from 3b might also be expected if a cap-dependent shunting mechanism were used (jackson, 1996; mathews, 1996) . as can be observed in fig. 3a , lanes 6 and 8, and figs. 3b and 3c, enhanced translation of both orfs 3a and 3b resulted when capped transcripts of porf3a-3b-4 and plorf3a-3b-4 were translated. these results are therefore consistent with the mechanisms of leaky scanning and cap-enhanced ribosomal shunting. in the second approach, the competitive effect of a soluble cap analog on the translation of orfs 3a and 3b from capped transcripts of porf3a-3b-4 was measured. with either leaky scanning or cap-dependent shunting, but not with cap-independent internal entry, competitive inhibition of translation from both orfs would be expected (iizuka et al., 1994; jackson, 1996; mathews, 1996) . nearly the same rate of inhibition was found, 70-75% with 0.1 mm and 85-90% with 2 mm cap analog (figs. 4a and 4c), indicating either mechanism of capdependent entry could be functioning in the translation for orf 3b. in the third approach, the sequence context surrounding the 3a start codon in transcripts of porf3a-3b-4 was modified to become strongly favorable for translation (gccgccatgg) (kozak, 1991b) and the relative amounts of 3a and 3b gene products were measured. with leaky scanning, a diminished synthesis from 3b relative to 3a would be expected regardless of the capped status of the transcripts (kozak, 1991b) , whereas with shunting a change in the relative amounts would not necessarily be expected. as can be noted in figs. 5a and 5b, whereas the accumulation of 3a product increased almost 20% relative to 3b with the improved kozak consensus for capped transcripts, no increase was observed with uncapped transcripts. intriguingly, the absolute amount of gene 3b product appeared nearly identical under all conditions of translation. these results, therefore, are not fully consistent with the leaky scanning model for orf 3b translation of orf 3b is not blocked by the upstream insertion of an 884-nt-long sequence containing three sequential orfs to test for an internal entry of ribosomes onto orf 3b, porf3a-3b-4 was modified to pscatorf3a-3b-4 by the placement of an 884-nt-long sequence containing three sequential orfs upstream of orf 3a (fig. 1c ) and the products of translation were quantitated. an internal entry of ribosomes, either directed by an ires element or by a shunting mechanism, would typically not be blocked by the presence of upstream orfs of this dimension (reviewed in jackson et al., 1995; jackson, 1996; mathews, 1996) . transcripts of pscatorf3a-3b-4 possessed a 5ј utr of 59 nt; a five-methionine-containing 450-nt scat orf beginning within an excellent kozak context (aaaatgg) at base 60; a five-methionine-con-taining 4.4-kda protein-encoding orf beginning within a fair kozak context (atcatgc) at base 534; a one-methionine-containing 4.5-kda protein-encoding orf beginning within a fair kozak context (cggatga) at base 685; and orf 3a beginning at base 944, orf 3b beginning at base 1253, and orf 4 beginning at base 1974. in addition, there is a 66-kda protein-encoding orf beginning within a fair kozak context (tccatga) at base 346 within the scat orf (in the ϩ1 reading frame relative to scat). all in all, 29 aug codons exist upstream of orf 3b. when transcripts of pscatorf3a-3b-4 were translated the following features were noted: 1. from uncapped transcripts, only products from the scat and 3b orfs, in a molar ratio of approximately 1:0.15, were obtained (fig. 3a, lane 9; fig. 3d ), indicating that among the downstream orfs, translation from orf 3b had been a selective one and was probably not the result of initiation on fragmented transcripts. 2. from capped transcripts, an enhanced accumulation from the scat orf was observed but not from the 3b orf (fig. 3a, lane 10; fig. 3d ), unless an inexplicable putative aggregated form of orf 3b (identified by an asterisk in fig. 3a , lane 10, and noted earlier [o'connor and brian, 1999] ) was included in the total. (the aggregate was not included in the bar in fig. 3d.) 3. from capped transcripts in the presence of soluble cap, a similar rate of inhibition was observed for products of both the scat and 3b orfs (50-70% with 0.1 mm and 80-90% with 0.2 mm cap analog; figs. 4b and 4d), mirroring the results with mrna 3-like transcripts of porf3a-3b-4 (figs. 4a and 4c). 4. no translation of the remaining five orfs was observed from either uncapped or capped transcripts. these results show that translation of orf 3b positioned 1263 nt downstream from the 5ј terminus in the synthetic construct is initiated by some form of internal entry of ribosomes and not by scanning, is influenced by a cap, and is not the result of a fragmented template. although no universally identifying primary or secondary structural features of ires elements are known, certain secondary structural features do appear necessary for ires function (reviewed in jackson, 1996) . within tgev gene 3a, secondary structures can be predicted (figs. 6a and 6b) that share features with the putative ires element in ibv mrna 3 (liu and inglis, 1992; le et al., 1995) , leading us to postulate that gene 3a might contain an ires. the predicted structures are five stemloops (i-v), four of which can be drawn as components of pseudoknots. the free energies of these are calculated to be, respectively, ϫ6.8, ϩ6.0, ϫ3.8, ϫ0.8, and ϩ5. are relatively unstable and suggest a low probability for their existence in viral rna. nevertheless, to test whether gene 3a might function as an ires for translation of orf 3b, deletions within it were prepared and tested. these were (mostly) bidirectional for distances of 16, 86, 74, 192, 314, 337, 334, and 523 nt, and represented 3ј-ward deletions of 11, 41, 44, 120, 140, 149, 289 , and 534 nt from the first nucleotide in gene 3a, respectively, for which the mutants were named. results shown in fig. 6c and summarized in fig. 6d indicate that the relative molar amounts of scat and the 27.7-kda gene 3b products (ϳ1:0.12) remained essentially unchanged between wild-type and ⌬149. the only exception was for ⌬41, for which the molar ratio was 1:0.56 along with an inexplicable enhancement of an uncharacterized band with an approximate molecular weight of 6 kda. for ⌬289, which leaves only 17 nt upstream of the orf 3b start codon, the relative amounts were surprisingly 1:0.07 and not 1:0, as expected. for ⌬534, there was abundant synthesis of the scat protein but no synthesis of the 27.7-kda protein; however, there was synthesis of the 20-kda gene 3b product (o'connor and brian, 1999) (fig. 6d) . these results are not consistent with a mechanism of ribosomal entry within gene 3a but rather with one in which ribosomes enter within 50 nt from the start codon of gene 3b. because the 20-kda gene 3b product is found with mutant ⌬534, the intriguing possibility exists that ribosomes are entering at or downstream of the gene 3b start site and are scanning in the upstream direction to reach the start codon. based on precedents in eukaryotes (reviewed in mathews, 1996) , four mechanistic possibilities should be considered as explanations for how ribosomes approach the downstream orf 3b on tgev mrna 3 for translation: ribosomes could (1) translate the upstream orf and then reinitiate synthesis on the downstream orf, (2) scan through the upstream orf(s) without the act of translation in a manner known as leaky scanning, (3) bypass the upstream orf(s) by using an internal ribosomal entry site similar to that used by picornaviruses and flaviviruses on genomic rna, or (4) bypass the upstream orf(s) after first binding to the mrna in a cap-dependent manner and then undergo shunting to a downstream site on the mrna. among these, shunting is the most recently recognized and is exemplified by translation on pregenomic rna of the cauliflower mosaic virus (futterer et al., 1993) and rice tungro bacilliform virus (futterer et al., 1996) , both pararetroviruses, on adenovirus mrna (yueh and schneider, 1996) , and on sendai paramyxovirus mrna kolakovsky, 1988, 1989; latorre et al., 1998) . we conclude that orf 3b is translated from mrna 3, and that the likelihood is high that an internal entry of ribosomes is used, possibly one with shuntlike features, and perhaps in conjunction with leaky ribosomal scanning through orf 3a. the relative contribution of each mechanism on mrna 3 could not be established by the experiments performed here. however, an internal entry of ribosomes was demonstrated by the use of constructs, in which four extensive orfs within an 884-nt sequence were placed upstream of orf 3a and synthesis from orf 3b was shown to remain approximately one-eighth to one-fifth of that from the 5ј-terminal orf. the internal entry showed some properties of shunting in that (1) no ires element of the type directing internal entry in picornaviruses and togaviruses could be demonstrated within sequence upstream of gene 3b and (2) translation of orf 3b in capped transcripts from the synthetic multicistronic pscatorf3a-3b-4 showed some inhibition by a competing soluble cap in the translation mix. that is, internal entry in the multicistronic transcript may follow a cap-dependent step as described for shunting in the adenoviruses, pararetroviruses, and paramyxoviruses. in general, the mechanistic features of ribosomal shunting, so far described for only viral mrnas (jackson, 1996; mathews, 1996) , remain to be clarified. in the case of adenovirus and pararetrovirus mrnas, an upstream donor structure appears necessary for the shunting step. in pararetrovirus, this appears to be a stable hairpin preceded by a short open reading frame (hemmings-mieszczak and hohn, 1999) . in the case of tgev orf 3b shunting reported here, a requirement for an upstream structure seems unlikely, since shunting took place in the presence of foreign sequence (pscatorf3a-3b-4) as well as (relatively) native sequence (porf3a-3b-4) at the 5ј terminus. in this respect, the tgev orf 3b shunting pathway bears similarity to that in paramyxovirus mrna, for which no apparent requirement for a donor structure was found (latorre et al., 1998) . likewise, it is not clear what determines the landing site in a ribosomal shunt. certainly in the experiments reported here it is not apparent how ribosomes might have been directed to land so close to the 3b initiation codon in tgev mrna 3. it is clearly not the postulated secondary structures within gene 3a, because internal entry took place after these had been removed or disrupted (⌬149, figs. 6c and 6d ). one possibility is that ribosomes are directed to land at or near the start site of gene 3b by specific sequences or by higher-order structures situated very near the landing site. precedents for this are found in sendai virus, wherein sequences both upstream and downstream of the y1 orf are required for shunt landing (latorre et al., 1998) , and in hepatitis c virus, wherein sequences extending 28 nt into the orf are required for ires-directed landing (reynolds et al., 1995) . curiously, such a landing site might require that ribosomes backscan to find the gene 3b start codon, a process postulated to explain the translation of certain sv40 and influenza virus transcripts (peabody et al., 1986; williams and lamb, 1989) . our findings were particularly intriguing because some evidence had suggested the existence of iresdirected, cap-independent translation for the third orf in tricistronic ibv mrna 3 (liu and inglis, 1992; le et al., 1995) and for the second orf in the bicistronic mhv mrna 5 (thiel and siddell, 1994) . the influence of the cap, however, was not examined in the mhv studies, and the possibility remains that a form of shunting might also be exhibited during the translation of these mrnas. the consequences of an internal ribosomal entry onto gene 3b for virus replication are not immediately apparent, but one might be that it enables a constitutive synthesis of 3b protein because, in principle, any of the viral mrnas containing gene 3b (mrnas 1, 2, and 3) could serve as templates. cells and virus. the purdue-116 and miller strains of tgev were obtained from e. bohl, ohio state university. purdue-116 virus was plaque-purified from infectious genomic rna, grown on swine testicle (st) cells in medium containing 10% fetal calf serum (atlanta biologicals), and used within eight passages of plaque purification (brian et al., 1980; kapke and brian, 1986) . miller virus was similarly grown but was plaque-purified twice from infectious virus on st cells and used within 10 passages of plaque purification. northern analysis of tgev mrnas. northern analyses were performed as described (sethna et al., 1989) and quantitation was done with the ambis photoanalytic imaging system (ambis, san diego, ca). cells were infected with tgev at a multiplicity of infection (m.o.i.) of 10 and total rna was extracted at 6 and 9 h postinfection (hpi). blots were probed with radiolabeled synthetic oligodeoxynucleotide specific for the leader (oligonucleotide l3ϩ, 5јcgggatcctcgggtttagttcgagttggtg-tccgaagacaaaatctagcacaaggctagttaaagt-aaaagaagagatat3ј), gene 3a (oligonucleotide 7.7, 5јgttcgtcaagtacagcatctacgg3ј), or gene 3b (oligonucleotide 4, 5јcttctcataaacggtgcagctct-gcc3ј). probes were radiolabeled to a specific activity of 1.5 to 3.5 ϫ 10 6 cpm/pmol by the forward reaction. construction of plasmids. tgev purdue sequences used in this study have been published (kapke, et al., 1988a; sethna et al., 1991) . the construction of porf3b-4 ( fig. 1c) , formerly called porf2, has been described (o'connor and brian, 1999) . porf3b-4 carries genes 3b and 4 and 308 nt of gene 5 in vector pgem-4z (promega biotech) (a sequence obtained from cdna clone pft44 [ fig. 1a] ). porf4 (fig. 1c) was made from porf3b-4 by first removing the 291-nt m-containing sphi fragment, religating, and then removing the 716-nt hindiii-bbsi fragment and religating after blunt-ending with mung bean nuclease. porf3a-3b-4 (fig. 1c) , which carries genes 3a, 3b, and 4 downstream of the t7 rna polymerase promoter in pgem-3z (promega biotech), was made in three steps. first, psp6orf3a, from which gene 3a and 591 nt of gene 3b can be transcribed with rna polymerase sp6, was created by ligating the 1111 bp nsii-psti fragment from clone pft39 (a clone containing nucleotides 3968 to 5449 from the genome 3ј end [ fig. 1a ; tung et al., 1992] , prepared as described in kapke et al., 1988a,b) into the psti site of pgem-3z. second, psp6orf3a-3b-4 was created by ligating the 925-bp bsrgi-ecori fragment from porf3b-4 into the 4048-nt vector-containing ecori-bsrgi-linearized fragment of psp6orf3a. third, the entire 1340-nt sphi-hinfi insert from psp6orf3a-3b-4, after blunt-ending with t4 dna polymerase, was ligated in the reverse orientation into similarly blunt-ended ecori-saci-linearized pgem-3z. to place the viral leader upstream of orf 3a, plorf3a-3b-4 ( fig. 1c ) was constructed by a previously published procedure (sethna et al., 1991; hofmann et al., 1993) . briefly, cdna was made from the 5ј end of mrna 3 with a primer specific to gene 3b (oligo 4(ϩ) [5јcttct-cataaacggtgcagctctgcc3ј]), and amplified by pcr using oligo 4(ϩ) and oligo leadergac(ϫ) (5јgcgggc-ccgggacttttaaagtaaag3, which binds to the minus-strand of the leader), to create a leader-containing fragment. the product was digested with smai and bsrgi, and the large fragment was ligated in a three-way ligation reaction with the 590-nt bsrgi-sphi fragment of porf3a-3b, and with pgem-4z that had been linearized with hindiii, blunt-ended with t4 dna polymerase, and digested with sphi. to create a strong kozak context for the 3a start codon, thus creating porf3a(k)-3b-4, an overlap pcr mutagenesis procedure (horton et al., 1990) was used. for this, complementary mutagenesis primers 7.7kozak(ϩ) (5јcaa-tgtcaatggtggccctgtaatgac3ј) and 7.7kozak(ϫ) (5јgtcattacagggccaccatggacattg3ј), and primers oligo 5(ϩ) (5јtgccaccatacaatgtgaca3ј, which binds to bases 465-484 within orf 3b) and pgem3zf(ϫ)ndei(ϫ) (5јgagagtgcaccatatgcggtgt3ј, which binds to bases 2498-2519 within pgem-3z), were used together in the overlap procedure to amplify a 1066-nt product from porf3a-3b-4 dna. after digestion with restriction enzymes nari and bsrgi, the 932-nt product was cloned into nari-bsrgi-linearized porf3a-3b-4. to create pcatorf3a-3b-4 (fig. 1c) , the orf3a-3b-4containing 1534-nt sphi fragment from porf3a-3b-4 was placed into the sphi site of pcat (fig. 1c) , which was made by cloning the bamhi fragment from pcm4 (pharmacia) into the bamhi site of pgem-3z. for better size resolution of the large proteins, the cat gene in pcatorf3a-3b-4 was truncated by 91 nt on its 3ј end by digestions with ncoi and nuclease bal 31, thus forming pscatorf3a-3b-4 (fig. 1c) . this, along with a frameshift, resulted in a total shortening of the cat protein (now called scat) by 46 aa. the junctions of all constructs were confirmed by sequencing plasmid dna. preparation of nested deletions within gene 3a, the postulated internal ribosomal entry region. to obtain deletions within gene 3a, pscatorf3a-3b-4 dna was linearized at base 7 of orf 3a with tth111 i, treated with bal 31 and mung bean nucleases, purified by electrophoresis, and religated. transformants were screened for deletions by pcr and the sequenced constructs were named for the number of bases deleted downstream of the gene 3a start site (the total number of deleted bases is also noted). in vitro translation and analysis of products. in vitro transcription with t7 rna polymerase was carried out on linearized plasmid dnas as recommended (promega biotech). porf3b-4 was linearized with ecori, porf3a-3b-4 with scai or bamhi, as indicated, and pscatorf3a-3b-4 with asei. the 1274-nt dna fragment from scailinearized porf3a-3b-4 was purified by affinity chromatography (geneclean; bio 101) to ensure transcription of only orf 3a. for preparation of capped rna transcripts, 0.5 mm m7g(5ј)ppp(5ј)g and 0.25 mm gtp replaced 2.5 mm gtp in the transcription mix (promega biotech). each preparation of rna was purified by biospin column chromatography (bio-rad), quantitated by spectrophotometry, and monitored for degradation by agarose gel electrophoresis. in vitro translation was carried out in methionine-depleted wheat germ extracts or rabbit reticulocyte lysates as recommended by the manufacturers (promega biotech and ambion, inc.). in some preparations, translation products were treated with rnase a before electrophoresis as recommended by ambion, inc., to remove a 27-kda endogenous band caused by the binding of charged trna to proteins. fifty-microliter reaction volumes contained 50 ci 35 s-methionine (1000 ci/mmol; icn) and 1.0 g of rna transcript. to test for inhibition of translation by exogenous methylated cap analog, m7g(5ј)ppp(5ј)g (new england biolabs) was added to the translation mix to final concentrations of 0.1, 0.2, 0.4, and 0.8 mm. radioactivity in the separated products was quantitated by scanning dried gels with the ambis photoanalytic imaging system (san diego, ca) or by scanning autoradiograms of the gels with the bio-rad imaging spectrophotometer (bio-rad). each experiment depicted was done minimally on three separate preparations of transcript rna. standard deviation measurements were made from the results of three separate experiments. sequencing of coronavirus ibv genomic rna: three open reading frames in the 5ј "unique" region of mrna d genome of porcine transmissible gastroenteritis virus an efficient ribosomal frame-shifting signal in the polymerase-encoding region of the coronavirus ibv sequence of the coding regions from the 3.0 kb and 3.9 kb mrna subgenomic species from a virulent isolate of transmissible gastroenteritis virus in vitro synthesis of two polypeptides from a nonstructural gene of coronavirus mouse hepatitis virus strain a59 cloning and sequencing of an 8.4 kb region from the 3ј end of a taiwanese virulent field isolated of the coronavirus transmissible gastroenteritis virus (tgev) scanning independent ribosomal initiation of the sendai virus x protein scanning independent ribosomal initiation of the sendai virus y proteins in vitro and in vivo complete sequence (20 kilobases) of the polyproteinencoding gene 1 of transmissible gastroenteritis virus molecular basis of transmissible gastroenteritis coronavirus (tgev) epidemiology. in "the coronaviridae the internal open reading frame within the nucleocapsid gene of mouse hepatitis virus encodes a structural protein that is not essential for viral replication nonlinear ribosome migration on cauliflower mosaic virus 35s rna position-dependent att initiation during plant pararetrovirus rice tungro bacilliform virus translation a stable hairpin preceded by a short open reading frame promotes nonlinear ribosome migration on a synthetic mrna leader leader-mrna junction sequences are unique for each subgenomic mrna species in the bovine coronavirus and remain so throughout persistent infection gene splicing by overlap extension: tailor-made genes using polymerase chain reaction cap-dependent and cap-independent translation by internal initiation of mrnas in cell extracts prepared from saccharomyces cerevisiae a comparative view of initiation site selection mechanisms cap-dependent and cap independent translation: operational distinctions and mechanistic interpretations sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsid protein gene nucleotide sequence between the peplomer and matrix protein genes of the porcine transmissible gastroenteritis coronavirus identifies three large open reading frames the amino-terminal signal peptide on the porcine transmissible gastroenteritis coronavirus matrix protein is not an absolute requirement for membrane translocation and glycosylation the scanning model for translation: an update an analysis of vertebrate mrna sequences: intimations of translational control structural features in eukaryotic mrnas that modulate the initiation of translation the molecular biology of coronaviruses sendai virus y proteins are initiated by a ribosomal shunt distinct structural elements and internal entry of ribosomes in mrna 3 encode by infectious bronchitis virus detection of a murine coronavirus nonstructural protein encoded in a downstream open reading frame a polycistronic mrna specified by the coronavirus infectious bronchitis virus internal entry of ribosomes on a tricistronic mrna encoded by infectious bronchitis virus coronavirus gene expression: genome organization and protein synthesis interactions between viruses and the cellular machinery for protein synthesis the major product of porcine transmissible gastroenteritis coronavirus gene 3b is an integral membrane glycoprotein of 31 kda effect of upstream reading frames on translation efficiency in simian virus 40 recombinants enteric coronavirus tgev: partial sequence of the genomic rna, its organization and expression unique features of internal initiation of hepatitis c virus rna translation bovine coronavirus i protein synthesis follows ribosomal scanning on the bicistronic n mrna the nucleocapsid gene of bovine coronavirus is bicistronic minus-strand copies of replicating coronavirus mrnas contain antileaders coronavirus subgenomic minus-strand rna and the potential for mrna replicons coronavirus mhv-jhm mrna 5 has a sequence arrangement which potentially allows translation of a second downstream open reading frame internal ribosomal entry in the coding region of murine hepatitis virus mrna 5 improved estimation of secondary structure in ribonucleic acids the 9.1 kilodalton hydrophobic protein encoded at the 3ј end of the porcine transmissible gastroenteritis coronavirus genome is membrane associated sequence comparison of porcine respiratory coronavirus isolates reveals heterogeneity in the s, 3, and 3-1 genes nucleotide sequence of coronavirus tgev genomic rna: evidence for 3 mrna species between the peplomer and matrix protein genes genetic basis for the pathogenesis of transmissible gastroenteritis virus genetic analysis of porcine respiratory coronavirus, an attenuated variant of transmissible gastroenteritis virus effect of mutations and deletions in a bicistronic mrna on the synthesis of influenza b virus nb and na glycoproteins selective translation initiation by ribosome jumping in adenovirus-infected and heat-shocked cells we thank seulah ku and gwyn williams for the construction of pcat. this work was supported by public health service grant ai 14367 from the national institutes of health and grant 92-37204-8046 from the u.s. department of agriculture, and in part with funds from the university of tennessee college of veterinary medicine center of excellence program for livestock diseases and human health. key: cord-270929-utn21ce1 authors: wise, annabel g.; kiupel, matti; maes, roger k. title: molecular characterization of a novel coronavirus associated with epizootic catarrhal enteritis (ece) in ferrets date: 2006-05-25 journal: virology doi: 10.1016/j.virol.2006.01.031 sha: doc_id: 270929 cord_uid: utn21ce1 a novel coronavirus, designated as ferret enteric coronavirus (fecv), was identified in feces of domestic ferrets clinically diagnosed with epizootic catarrhal enteritis (ece). initially, partial sequences of the polymerase, spike, membrane protein, and nucleocapsid genes were generated using coronavirus consensus pcr assays. subsequently, the complete sequences of the nucleocapsid gene and the last two open reading frames at the 3′ terminus of the fecv genome were obtained. phylogenetic analyses based on predicted partial amino acid sequences of the polymerase, spike, and membrane proteins, and full sequence of the nucleocapsid protein showed that fecv is genetically most closely related to group 1 coronaviruses. fecv is more similar to feline coronavirus, porcine transmissible gastroenteritis virus, and canine coronavirus than to porcine epidemic diarrhea virus and human coronavirus 229e. molecular data presented in this study provide the first genetic evidence for a new coronavirus associated with clinical cases of ece. epizootic catarrhal enteritis (ece), a relatively new enteric disease of domestic ferrets (mustelo putorius furo), was first described in the spring of 1993 on the east coast of the united states (williams et al., 2000) . since then, the disease has spread throughout the u.s. and other countries. clinically, ece is characterized by a foul-smelling bright green diarrhea with high mucus content and is commonly associated with lethargy, anorexia, and vomiting. the morbidity for ece approaches 100%, but the overall mortality rate is low (b5%), with juvenile ferrets often developing only mild and even subclinical disease. clinical signs are more severe in older ferrets and the mortality rate is often higher. earlier investigations into the etiology of ece implicated a coronavirus (williams et al., 2000) . the microscopic lesions observed in affected ferrets were consistent with those described for intestinal coronavirus infections in other species. transmission electron microscopy revealed coronavirus-like particles in the feces and in affected jejunal enterocytes of ferrets with ece. moreover, positive immunohistochemical staining, using a feline coronavirus-specific monoclonal antibody, confirmed the presence of coronaviral antigen in sections of affected intestines. there have been no reports confirming these previous findings at the genetic level. coronaviruses are enveloped, positive-strand rna viruses classified under the genus coronavirus within the family coronaviridae, order nidovirales (enjuanes et al., 2000; holmes, 1999; murphy et al., 1999) . virions are pleomorphic, with a diameter of 60-220 nm. the coronavirus genome consists of a nonsegmented, positive-sense, single-stranded rna that is approximately 27-31 kb in length. the capsid has a helical confirmation and is made up of monomers of the nucleocapsid (n) protein. inserted in the envelope is the spike virology 349 (2006) 164 -174 www.elsevier.com/locate/yviro (s) protein the membrane (m) protein and the small envelope (e) protein. coronaviruses are subdivided into three groups, based originally upon serologic properties and more recently upon sequence homology (chouljenko et al., 2001; enjuanes et al., 2000; gonzalez et al., 2003; hegyi et al., 2002; holmes, 1999; murphy et al., 1999; siddell, 1995; stephensen et al., 1999; wege et al., 1982) . group 1 includes human coronavirus strain 229e, porcine transmissible gastroenteritis virus, canine coronavirus, feline coronavirus, and porcine epidemic diarrhea virus. group 2 includes human coronavirus strain oc43, murine hepatitis virus, rat coronavirus, bovine coronavirus, porcine hemagglutinating encephalomyelitis virus, and equine coronavirus. the avian viruses, infectious bronchitis virus of chickens, turkey coronavirus, and pheasant coronavirus, belong to group 3. based upon sequencing, the recently identified sars virus appears to be the prototype of a fourth group (marra et al., 2003; rota et al., 2003; snijder et al., 2003) . using consensus pcr assays for the genus coronavirus, followed by additional genomic sequencing and phylogenetic analyses, we provide the first molecular evidence that the virus associated with ece in ferrets is a new coronavirus, tentatively designated as "ferret enteric coronavirus" (fecv). amplicons of the expected sizes were derived for each consensus coronavirus rt-pcr assay performed on rna extracted from the diarrheic feces of ferrets clinically diagnosed with ece. the respective product sizes obtained were 628 bp, 735 bp, and 251 bp, for the s, m-n and polymerase gene regions. after cloning and sequencing the products, similarity searches with blast (altschul et al., 1990) showed the sequences to be authentic coronavirus sequences, with the most significant matches to porcine transmissible gastroenteritis virus, feline, and canine coronaviruses. percentage nucleotide sequence identities of the ferret coronavirus sequences (excluding primer sequences) with the corresponding sequences of porcine transmissible gastroenteritis virus (genbank accession nos. ay35549, for the s and the m genes, and aj011482, for the polymerase gene) were as follows: 60.8% for the 586-bp partial s gene sequence, 66.7% for the 334-bp partial m gene sequence, and 76.4% for the 208-bp partial polymerase sequence. these initial findings provided the first nucleotide sequence-based evidence that an enteric coronavirus is shed in the feces of ferrets clinically affected with ece. the entire n gene of the ferret enteric coronavirus (fecv), hereafter referred to as strain fecv-msu1, was derived by the 3′ race method. the n gene of fecv-msu1 is 1125 nucleotides in length and translates into a 374-amino-acid protein (fig. 1) . it is flanked upstream by a 16-base intergenic sequence, followed by the open reading frame corresponding to the sequenced 3′-end portion of the m protein gene. the recognized transcription-regulating sequence (trs), 5′-ctaaac-3′, of coronaviruses (budzilowicz et al., 1985; horsburgh et al., 1992; kapke and brian, 1986; snijder et al., 2003; spaan et al., 1988) is conserved in fecv-msu1, and found within the intergenic sequence preceding the n gene. blast analysis of the entire fecv-msu1 n gene sequence consistently demonstrated significant similarities to reported coronavirus n gene sequences in the genbank database. as was previously observed with the partial s, m and polymerase nucleotide sequences analyzed, group 1 coronavirus sequences produced the most significant alignments with the ferret sequence. the fecv-msu1 n gene shared nucleotide identities of 51.6, 48.7, and 48.2% with canine coronavirus (genbank accession no. ay342160), porcine transmissible gastroenteritis virus (genbank accession no. af104420), feline coronavirus (genbank accession no. ab086902), respectively. the deduced amino acid sequence of the fecv-msu1 n protein was aligned with corresponding coronavirus sequences from genbank, representing each of the coronavirus antigenic groupings. table 1 shows the sequence identities between the n protein of fecv-msu1 and those of porcine transmissible gastroenteritis virus (tgev), canine coronavirus (ccv), feline coronavirus (fcov), porcine epidemic diarrhea virus (pedv), human coronavirus (hcov) 229e, bovine coronavirus (bcv), mouse hepatitis virus (mhv), hcov oc43, sars virus, avian infectious bronchitis virus (ibv), and turkey coronavirus (tcov). the genbank accession numbers for these coronavirus strains are given in fig. 2 . group 1 coronaviruses, ccv, fcov, tgev, pedv, and hcov 229e, shared the highest sequence identities with fecv-msu1 at 57.1, 56, 57.3, 31.2, and 35.7%, respectively. group 2 (bcv, mhv, hcov oc43), sars virus, and group 3 (ibv, tcov) coronaviruses, showed much lower identities with fecv-msu1 at 22.9 to 26.9%. the n protein sequence alignment data was used to generate a phylogenetic tree ( fig. 2 ) which clearly shows that the ferret coronavirus, fecv-msu1, is a novel coronavirus that groups within the group 1 coronaviruses. within this group, fecv-msu1 is more closely related to fcov, ccv, and tgev than to pedv and hcov 229e. fig. 3 shows the alignment of the deduced n protein sequences of fecv-msu1, tgev, ccv, fcov, and pedv. the amino acid substitutions that occurred in the fecv sequence in relation to the consensus (majority) sequence appear randomly throughout, with stretches of up to 6 substitutions. insertions and deletions appear to be random as well, with the largest deletion of 6 residues, with respect to the consensus, observed to have occurred between amino acid positions 178 and 179. a highly conserved region between the n proteins of fecv-msu1, tgev, ccv, and fcov was noted at amino acid positions 88-109 (22 total residues), with only 3 substitutions occurring within the fecv-msu1 sequence. pedv stands out as the most distantly related among this group of animal coronaviruses, possessing extra sequence strings, not found in the consensus, of up to 35 amino acid residues at a stretch. phylogenetic analyses based upon partial sequences of the polymerase, s and m proteins the predicted partial amino acid sequences of the fecv-msu1 polymerase, s and m proteins were also aligned with corresponding sequences of other known coronaviruses and phylogenetic trees were derived. as was evident from the n protein sequence analysis, the data presented in figs. 4-6 show that fecv-msu1 is most similar to group 1 coronaviruses and more specifically to fcov, ccv, and tgev. amplification of the 3′-end of fecv-msu1 by the 3′ race method enabled determination of the entire n gene sequence and a 994 nucleotide sequence downstream from the n gene (fig. 1) . analysis of this sequence revealed the presence of two orfs. the first orf is 225 nucleotides in length with its start codon located 9 nucleotides downstream of the n gene. the second orf is 615 nucleotides in length and overlaps the first orf by 62 bases. the deduced amino acid sequences of the two orfs were subjected to blast analyses. the first orf codes for a putative 74-amino-acid protein (8.5 k polypeptide) which is 23.9% similar to the hypothetical 3x protein of the insavc strain of ccv strain from the u.k. (genbank accession no. baa02410) (horsburgh et al., 1992) . the second orf codes for a putative 204-amino-acid protein which is most similar to the nonstructural protein 7b of enteric canine coronaviruses and feline coronaviruses. this putative 7b protein sequence shares 37.3% homology with the corresponding protein of the ccv insavc-1 strain, and 35.8% similarity to the 7b protein of feline coronavirus (genbank accession no. caa62193). the conserved trs, 5′-ctaaac-3′, was found upstream of the 3x-like orf, but not of the 7blike orf (fig. 1) . the 10-base sequence highly conserved in the 3′ noncoding region of all coronaviruses, 5′-gggaa-gagct-3′ (horsburgh et al., 1992; kapke and brian, 1986) , was observed 169 bases downstream of the 7b-like orf ( fig. 1) , with 2 base mismatches, a t instead of a g in the first base, and a g instead of an a at the seventh base. cytopathic changes were not observed in any of the inoculated cultures. fecv-specific rt-pcr on inoculated crfk rna extracts yielded negative results. an fecv n gene-specific rt-pcr assay was used to analyze fecal, saliva, and serum samples from 13 ferrets naturally affected with ece. fecv was detected in feces and saliva, but not in serum, of all ferrets examined on days 0, 7, 12, and 26 (fig. 7 , panels a and b). the pcr products derived from 2 fecal samples on day 0 were sequenced and confirmed as fecv. amplification products were not observed using tgev, ccv, fcov, and bcv rna as templates. fecv rna and antigen were detected in the cytoplasm of enterocytes at the villi tips in the jejunum of affected ferrets on day 17 (n = 10) and 26 (n = 3). the ish signal (fig. 8 , panel a) co-localized with viral protein detected by ihc (fig. 9 , panel a). fecv rna or antigen was not found in the large intestine, lymph nodes, spleen, esophagus, stomach, and parotid salivary glands. fecv-msu-1 sequences have been assigned genbank accession numbers dq340560, for the partial polymerase sequence; dq340561, for the partial s sequence; and dq340562, for the partial m and downstream orf sequences (n, 3x and 7b) up to the partial 3′ utr. epizootic catarrhal enteritis (ece), an enteric disease of ferrets, was first described in 1993. previous work (williams et al., 2000) utilizing electron microscopy and immunohistochemistry implicated a coronavirus as the causative agent of ece. based upon these previous data, consensus pcr protocols using primers that target conserved areas within the genomes of coronaviruses were utilized in this study to confirm the association of a coronavirus with ece. sequencing of the pcr products and blast analyses confirmed the coronavirus origin of the amplification products and provided the first genetic evidence for the detection of a novel coronavirus in ferrets, tentatively designated ferret enteric coronavirus (fecv). subsequently, the sequences of the entire n gene and downstream orfs were derived. sequence alignment and phylogenetic analysis based upon the predicted n protein sequences of fecv and other coronaviruses clearly indicated that fecv clusters with group 1 coronaviruses. fecv appears to be more closely related to the group 1 coronaviruses fcov, ccv, and tgev, than to pedv and hcov 229e. yet, fcov, ccv, and tgev together form a tighter cluster with pairwise amino acid sequence identities from 74.9 to 89.5%, compared to only 56.8% average identity shared by fecv with these three viruses. the close serologic relationship between ccv, fcov, tgev, and the related prcv is well established (sanchez et al., 1990; siddell et al., 1983; spaan et al., 1988) . likewise, the close genetic relationship between these viruses, based upon sequence analysis, has been well documented (enjuanes et al., 2000; gonzalez et al., 2003; horsburgh et al., 1992) . pedv, although a group 1 coronavirus of animal origin, has been shown to be most closely related to hcov 229e (bridgen et al., 1993; kocherhans et al., 2001) . these two viruses were observed to clearly form their own cluster separate from the other group 1 coronavirus members in the phylogenetic trees shown in figs. 2 and 6. the predicted partial polymerase, s and m protein sequences of fecv were also compared to corresponding sequences of other known coronaviruses. as with the analysis of the n protein sequence, phylogenetic analyses based upon these three other regions of the genome gave similar results, further supporting the classification of fecv as a member of group 1 coronaviruses with highest similarities to ccv, fcov, and tgev and more distantly related to pedv and hcov 229e. the extent of genetic relatedness of fecv to other coronaviruses appears to be consistent, at least for the four regions analyzed so far. group 1 feline and canine coronaviruses are known to contain two additional orfs, namely orfs 7a and 7b, downstream from the n gene at the 3′-end of their genomes (herrewegh et al., 1995; vennema et al., 1992a) . tgev was found to have an orf 7a but not an orf 7b in its genome (herrewegh et al., 1995; vennema et al., 1992a) . these orfs code for accessory nonstructural proteins, the functions of which have not yet been defined. orf 7a codes for a small hydrophobic protein that is membrane-associated (tung et al., 1992) , while orf 7b appears to be a secretory glycoprotein not stably associated with viral particles (vennema et al., 1992b) . analyses of the 3′-end of fecv-msu1 downstream from the n gene showed the presence of 2 orfs. the gene corresponding to orf 7a of other group 1 animal coronaviruses was not found in the fecv genome. instead, an orf with 23.9% homology to the 3x pseudogene of ccv strain insavc-1 (horsburgh et al., 1992) was found in the genomic location of orf 7a. within the ccv genome, orf 3x is located between the s and m genes. tgev has a counterpart to the ccv pseudogene in a similar location but with a deletion of 92 nucleotides (horsburgh et al., 1992) . fig. 10 shows the gene arrangements of the 3′ termini of the genomes of fecv, fcov, ccv, and tgev. interestingly, the 3x gene is clearly in a different genomic location in the strain of fecv being analyzed, but this could be a strain-specific feature. it is possible that the 3x pseudogene homologue in fecv-msu1 is the result of an insertional event. it is well known that recombination, insertions, and deletions are typical features of coronavirus biology (lai, 1992 (lai, , 1996 sawicki and sawicki, 1998; spaan et al., 1984; zhang et al., 2005) . even though the 3x gene of ccv insavc-1 could conceivably encode a 71amino-acid 10 k polypeptide, this was considered unlikely, based upon codon usage and base preference programs (horsburgh et al., 1992; staden, 1982) . it was suggested that orf 3x is an evolutionary redundant sequence which is no longer required. of note, the significant similarity of the fecv 3x-like gene to that of ccv was only apparent after blast analysis without the default filtering of low complexity fig. 7 . fecv-specific rt-pcr on clinical samples from ferrets with ece. (a) lane 1, 100-bp dna ladder. lanes 2-5, amplicons from fecal specimens collected from one of the ferrets on days 0, 7, 12, and 26, respectively. lane 6, fecv positive control (113-bp product). lane 7, negative reagent control (rnase-free water as the template). (b) lane 1, 100-bp dna ladder. lanes 2-5, amplicons from saliva specimens collected from one of the ferrets on days 0, 7, 12, and 26, respectively. lane 6, fecv positive control (113-bp product). lane 7, negative reagent control (rnase-free water as the template). sequences. examination of the deduced amino acid sequence of the fecv 3x-like gene showed multiple regions of biased composition including homopolymeric runs and overrepresentation of some residues characteristic of low complexity sequences (data not shown) (wootton and federhen, 1996) . therefore, the 3x-like orf in fecv-msu1, as in insavc-1, does not appear to encode a functional viral protein. fecv-msu1 is missing a counterpart to orf 7a of feline and canine group 1 coronaviruses. it is known that the accessory nonstructural protein genes of coronaviruses can be acquired or lost easily in their evolution (snijder et al., 2003) . the orf 7a in tgev was found to have a deletion of 69 nucleotides (de groot et al., 1988) . the genomic region downstream of the n gene is known to be a "deletion hot spot" in coronaviruses (collison et al., 1990; de groot et al., 1988; horsburgh et al., 1992) . hence, it is not unusual for fecv to have a missing orf 7a homologue, most likely due to a deletion event. there is a need to sequence and analyze more strains to ascertain whether this is a common feature among fecv isolates. the putative orf 7b protein of fecv appears to be intact and has identities of 37.3% and 35.8% to ccv and fcov, respectively. this orf was observed to be the least conserved between feline and canine coronaviruses, with only 57% identity between the two (horsburgh et al., 1992) . a number of feline enteric coronavirus strains with deletions in orf 7b have been identified (herrewegh et al., 1995) . in feline coronaviruses, the orf 7b glycoprotein was found dispensable for viral replication in tissue culture (vennema et al., 1992b) and for infection of the natural host (pedersen et al., 1984) . it has been presumed that the 7b glycoprotein functions as a virus-encoded mediator of the host immune response (herrewegh et al., 1995) . the minimal conserved transcription regulatory sequence (trs), ctaaac, found in group 1 animal coronaviruses, is essential in the synthesis of subgenomic mrnas during viral replication (budzilowicz et al., 1985; snijder et al., 2003; spaan et al., 1988) . in the genus coronavirus, the nonreplicase orfs are expressed from a nested set of subgenomic mrnas that are both 5′ and 3′ coterminal with the viral genome (lai and cavanagh, 1997) . it has been postulated that trss guide the discontinuous synthesis of negative-stranded subgenomic mrnas that serve as templates for subgenomic mrna synthesis (pasternak et al., 2000; sawicki and sawicki, 1995) . the trs, ctaaac, was found to be conserved in fecv-msu1 upstream of the n gene and the 3x-like orf, but not upstream of orf 7b. in the ccv strain insavc-1, the conserved trs was present upstream of orf 7a but was absent upstream of orf 7b (horsburgh et al., 1992) . it is presumed that orfs 7a and 7b are likely to be expressed from a single mrna species, since polycistronic coronavirus mrnas have been previously identified (liu and inglis, 1992; liu et al., 1991) . hence, the fecv 7b protein may very well be expressed from a bicistronic mrna, containing the nonfunctional 3x-like orf at its 5′-end. the 10-base sequence, 5′-gggaagagct-3′, highly conserved in the 3′ noncoding region of all coronaviruses (horsburgh et al., 1992; kapke and brian, 1986) , was also observed 169 bases downstream of orf 7b of fecv. it was conserved, except for 2 base mismatches. this sequence is believed to be a recognition site for the attachment of the polymerase, as synthesis of the negative strand rna is initiated (kapke and brian, 1986) . our attempt to isolate fecv in crfk cells was negative. virus isolation was previously attempted by williams et al. (2000) in mv-1-lu, hrt-18, dk-5966, crsk, and a72-163 cells but was unsuccessful. other enteric coronaviruses, e.g., type 1 feline enteric coronavirus, have been shown to be difficult to propagate in cell culture. ece is a common clinical diagnosis, and the clinical signs associated with ece are fairly distinct (green slimy diarrhea). however, some of the clinical signs such as vomiting and hypoproteinemia may be observed with other enteric diseases of ferrets including inflammatory bowel disease, gastric helicobacteriosis, and rotavirus infections in juveniles. the availability of fecv genomic sequence data will facilitate the development of molecular assays to confirm clinical diagnosis. in this study, fecv was detected in feces and saliva of naturally infected ferrets using an n gene-specific rt-pcr. this pcr assay has potential as a standard diagnostic assay, but further validation is required. the tissue localization of fecv may be similar to that of fcov. both antigen and nucleic acid were detected in the cytoplasm of enterocytes at the tip of villi in the jejunum of affected ferrets by ihc and ish, respectively. viral antigen or nucleic acid was not present in the large intestine, lymph nodes, spleen, esophagus, stomach, or parotid salivary glands. in a recent field case of ece, we detected viral proteins both in the small and large intestines (data not shown). herrewegh et al. (1997) , using nested rt-pcr, detected fcov-specific rna in kidney, lung, brain, tonsils, salivary gland, and bone marrow. viral rna was most abundant in duodenum, jejunum, ileum, colon, and rectum. interestingly, when evidence of replicating virus was evaluated with an rt-pcr targeting n gene-specific mrna, evidence of viral replication could be obtained only in homogenates of ileum, colon, and rectum. immunohistochemistry confirmed that fcov-specific antigens were present only in the ileum, colon, and rectum. in conclusion, this study provides the first molecular evidence indicating that a novel coronavirus, herein designated as ferret enteric coronavirus, is associated with enteric catarrhal enteritis in ferrets. further definition of the pathogenesis of fecv will be dependent upon the development of an in vitro culture system. fecal specimens from a ferret clinically diagnosed with ece were obtained from a veterinary clinic. total rna was extracted from fecal specimens using the qiagen rneasy mini kit (valencia, ca). degenerate consensus primers were used to amplify portions of the s (spike), m (membrane) glycoprotein, and n (nucleocapsid) genes of any coronavirus (tobler and ackermann, 1996) . primer 55: 5′-ggakaaggtkaatgartgygt-3′ and primer 56: 5′-ccakacvtaccawggccaytt-3′ delineate an approximately 700-bp region of the coronavirus s gene. primer 25: 5′-gactagttggtggagwtttaa yccwga-3′ and primer 24: 5′-ctcgagcgacccagamgacw-ccktc-3′, bracket an approximately 700-bp region of the coronavirus genome, beginning from the 3′ terminus of the m gene to the 5′ terminus of the n gene. rt-pcr was performed using the qiagen onestep rt-pcr kit with 0.6 μm of each primer. cycling conditions for both s and m-n regions amplifications were as follows: cdna synthesis at 45°c for 45 min; predenaturation at 95°c for 15 min; 50 cycles, each consisting of denaturation at 94°c for 30 s, annealing at 45°c for 1 min, and extension at 72°c for 2 min and 30 s. a final extension at 72°c for 7 min was added after the last pcr cycle. a pair of degenerate primers, 2bp: 5′-actcarwtraa-tytnaaataygc-3′ and 4bm: 5′-tcacayttwgga-tartccca3′, was used to amplify a 251-bp region of the coronavirus polymerase gene within orf 1b (stephensen et al., 1999) . rt-pcr was carried out with the qiagen onestep rt-pcr kit with 0.6 μm of each primer. cycling conditions were as follows: cdna synthesis at 40°c for 45 min and predenaturation at 95°c for 15 min; 5 cycles of pcr at 94°c for 1 min, 40°c for 2 min, and 72°c for 1 min; then 40 cycles of pcr at 94°c for 1 min, 50°c for 1.5 min, and 72°c for 1 min; with a final extension of 72°c for 10 min. pcr products were analyzed by agarose gel electrophoresis and visualized by uv transillumination of ethidium bromidestained gels. the pcr products were extracted from the gel using the qiaquick gel extraction kit (qiagen). the purified products were ta-cloned into a plasmid vector using the qiagen pcr cloning kit. the inserts were amplified with m13 forward and reverse primers, which prime the cloning vector at positions just outside the multiple cloning site. pcr products were sent to the genomic technology support facility of michigan state university for automated bi-directional sequencing. a 683-nucleotide sequence (excluding primer sequences) of the ferret coronavirus, which spans the last 334 nucleotides of the m gene, 16 nucleotides of intergenic sequence, and the first 333 nucleotides of the n gene, was obtained by sequencing of amplicons generated with consensus primers. based upon this sequence, the entire n gene sequence was derived by the 3′ race method, using a commercial kit (gibco brl, life technologies, rockville, md). complementary dna was synthesized from 5 μl of fecal rna template with an oligo-(dt)-containing adapter primer according to the kit manufacturer's protocol. the gene-specific primer (5′-acccat-gaaggtaagaagccc-3′) used for pcr amplification of the target cdna was based on the initially derived 5′ sequence of the n gene. the abridged universal primer (auap) provided in the kit was used as reverse primer. pcr was carried out with the qiagen hotstar taq dna polymerase kit, using 2 μl of cdna template, 0.5 μm of the gene-specific primer, 2 μl of auap and the following cycling conditions: predenaturation at 95°c for 15 min, followed by 50 cycles of denaturation at 94°c for 1 min, annealing at 55°c for 1 min, and extension at 72°c for 2 min and 30 s, and a final extension at 72°c for 7 min. pcr products of the expected size were purified for direct sequencing. sequence data for the approximately 2 kb product were obtained by initially using the gene-specific primer described above and a poly-t sequencing primer, followed by primer-walking with newly designed 5′ and 3′ primers derived with the oligo 6 primer analysis software (molecular biology insights, cascade, co). blast (altschul et al., 1990 ) searches against the genbank database were performed to confirm that the sequence data obtained were authentic coronavirus sequences. sequence assembly and analyses, including multiple alignments of nucleotide and predicted amino acid sequences using the clustal w method (thompson et al., 1994) , were done with the lasergene biocomputing software (dnastar, inc., madison, wi). phylogenetic trees were constructed with the treecon software package ( van de peer and de wachter, 1994) , using the neighbor-joining method (saitou and nei, 1987) and 100 bootstrap analyses (efron and gong, 1983; felsenstein, 1985) . sequence alignment data generated with lasergene were converted to the treecon format using the forcon software (raes and van de peer, 1999) . virus isolation was attempted in the crandell reese feline kidney (crfk) cell line, obtained from the american type culture collection (ccl 94). the crfk cells were grown in eagle's minimum essential medium (emem), supplemented with 10% heat-inactivated fetal bovine serum, 100 u/ml of penicillin, and 100 μg/ml of streptomycin (brl-life sciences, gaithersburg, md). confluent monolayers were inoculated with 0.45 μm filtrates of fecal homogenates. the inoculated cells were observed during a 7-day period for the appearance of cytopathic changes. rna was extracted from inoculated and mock-inoculated cell homogenates and tested by rt-pcr, using the fecv n gene-specific primers described above. a group of 13 ferrets, naturally affected with ece, was housed at the msu laboratory animal resources (ular) facility. ferrets were kept in individual cages and fed a commercial ferret diet. ferrets were monitored clinically on a daily basis. fecal samples, rectal and oral swabs were collected daily from each ferret. ferrets were bled on a weekly basis to obtain serum samples. the 13 ferrets were exsanguinated and euthanized on days 17 (n = 10) or 26 (n = 3). necropsies were performed immediately thereafter, and samples of small and large intestine, lymph nodes, spleen, esophagus, stomach, and salivary glands were collected. sections of tissues were fixed in 10% neutral buffered formalin and routinely processed for histopathology, in situ hybridization, and immunohistochemistry. a pair of primers was designed from the nucleocapsid gene sequence of fecv-msu1 using the oligo 6 software. the selected primer pair, 5′-aca ggt ggt tct ttt act acc-3′ (forward primer) and 5′-tgt agg cac agt ttt agc ac-3′ (reverse primer) targets a 113-bp region of the n gene. total rna from fecal, oral and serum samples were extracted using the qiagen rneasy mini kit (valencia, ca). the qiagen onestep rt-pcr kit was used with an optimal primer concentration of 0.6 μm for each primer in a final reaction volume of 50 μl. five microliters of the extracted rna was used as the template. the optimized cycling conditions were as follows: cdna synthesis at 50°c for 30 min; predenaturation at 95°c for 15 min; 40 pcr cycles of 94°c for 30 s, 53°c for 30 s, and 72°c for 30 s; and a final extension step of 72°c for 7 min. pcr products were run on a 2% agarose gel stained with ethidium bromide and were visualized with a uv transilluminator. amplicons of the expected size from two fecal samples, collected on day 0, were purified using the qiaquick pcr purification kit (qiagen, inc., valencia, ca) and were directly sequenced at the genomics technology support facility at michigan state university. fecv-positive control rna used was the fecal rna extract previously amplified with consensus coronavirus pcr primers. specificity of the assay was evaluated with rna extracted from tgev, ccv, fcov, and bcv. a 25-mer fecv-specific probe (5′-tcggtaacgt-tgctgtctattcca-3′) was designed based upon the fecv nucleocapsid gene sequence using the oligo 6 software. the probe was synthesized commercially, 3′-end labeled with digoxigenin and hplc-purified (idt dna, coralville, ia). tissue sections were deparaffinized then subjected to proteolytic digestion with 0.2n hcl for 20 min and with proteinase k at 37°c for 15 min. subsequently, the slides were post-fixed in 4% paraformaldehyde for 5 min. prehybridization and hybridization were performed according to a previously described protocol (kiupel et al., 2001) . briefly, prehybridization was performed at 50°c for 1 h in 100% formamide. this was followed by hybridization at 105°c for 5 min and at 37°c for 1 h, using fischer's microprobe workstation (fischer scientific, hampton, nh). high stringency washes were performed in saline sodium citrate buffers to ensure specific binding of the probe to the target. the detection system consisted of anti-digoxigenin antibody (boehringer mannheim biochemica, indianapolis, in) conjugated with alkaline phosphatase (1:500) applied at 37°c for 45 min, and the substrate, nbt/x phos (nitro-blue tetrazolium/5 bromo-4-chloro-3-indoylphosphate, boehringer mannheim biochemica, indianapolis, in). dye reduction to insoluble blue formazan was allowed for 120 min. slides were counterstained with light green manually to best highlight the signal. negative reagent control slides were incubated with hybridization buffer from which the fecv probe was omitted. immunohistochemical staining of tissues with monoclonal antibody fcv3-70 (custom monoclonals international, west sacramento, ca), known to react with feline, canine, and porcine coronaviruses (kipar et al., 1998a (kipar et al., , 1998b , and ece-associated coronavirus in ferrets (williams et al., 2000) , was performed as described previously by williams et al. (2000) . basic local alignment search tool sequence determination of the nucleocapsid protein gene of the porcine epidemic diarrhea virus confirms that this is a coronavirus related to human coronavirus 229e and porcine transmissible gastroenteritis virus three intergenic regions of coronavirus mouse hepatitis virus strain a59 genome rna contain a common nucleotide sequence that is homologous to the 3′ end of the viral mrna leader sequence comparison of genomic and predicted amino acid sequences of respiratory and enteric bovine coronaviruses isolated from the same animal with fatal shipping pneumonia sequence comparisons of the 3′ end of the genomes of five strains of avian infectious bronchitis virus sequence analysis of the 3′ end of the feline coronavirus fipv 79-1146 genome: comparison with the genome of porcine coronavirus tgev reveals large insertions a leisurely look at the bootstrap, the jackknife, and cross-validation virus taxonomy. classification and nomenclature of viruses 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the biology of coronaviruses unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage transcription strategy of coronaviruses: fusion of non-contiguous sequences during mrna synthesis coronaviruses: structure and genome expression automation of the computer handling of the gel reading data produced by the shotgun method of dna sequencing phylogenetic analysis of a highly conserved region of the polymerase gene from 11 coronaviruses and development of a consensus polymerase chain reaction assay clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice identifikation und charakterisierung von neuen und unbekannten coronaviren mit hilfe von rt-pcr und degenerierten primern the 9-kda hydrophobic protein encoded at the 3′ end of the porcine transmissible gastroenteritis coronavirus genome is membraneassociated treecon for windows: a software package for the construction and drawing of evolutionary trees for the microsoft windows environment genomic organization and expression of the 3′ end of the canine and feline enteric coronaviruses a novel glycoprotein of feline infectious peritonitis coronavirus contains a kdel-like endoplasmic reticulum retention signal the biology and pathogenesis of coronaviruses coronavirus-associated epizootic catarrhal enteritis in ferrets analysis of compositionally biased regions in sequence databases testing the hypothesis of a recombinant origin of the sars-associated coronavirus we thank dr. cheryl isenhour (college of veterinary medicine class of 2005) for the excellent technical assistance. the authors would like to thank marshall farms group, ltd. for their generous sponsorship of this research project. this study was also supported by grants from the office of intellectual property of michigan state university and from the companion animal fund of the college of veterinary medicine, michigan state university. key: cord-279813-mrei5kih authors: temeeyasen, g.; sinha, a.; gimenez-lirola, l.g.; zhang, j.q.; piñeyro, p.e. title: differential gene modulation of pattern-recognition receptor tlr and rig-i-like and downstream mediators on intestinal mucosa of pigs infected with pedv non s-indel and pedv s-indel strains date: 2017-12-14 journal: virology doi: 10.1016/j.virol.2017.11.024 sha: doc_id: 279813 cord_uid: mrei5kih porcine epidemic diarrhea virus (pedv) strains can be divided into non-s-indel and s-indel strains. pedv pathogenesis is strain-specific, and studies in neonatal pigs have demonstrated that the pedv non-s-indel strains are more pathogenic than the pedv s-indel strains. rna viruses, including pedv, can interact with a large number of pattern recognition receptors (prrs) in the intestinal mucosa, including toll-like receptors (tlrs) and rig-i-like receptors (rlrs). we investigated the differential gene modulation of tlrs, rig-i, and downstream mediators on the intestinal mucosa of neonatal pigs infected with pedv s-indel and non-s-indel strains. ten five-day-old piglets were inoculated orally with 10 ml of 10(4) tcdi(50)/ml of either pedv non-s-indel or s-indel strains. pedv s-indel infection induced pro-inflammatory cytokines through the non-canonical nf-κb signaling pathway by activating rig-i. in contrast, pedv non-s-indel infection suppressed the induction of pro-inflammatory cytokines and type 1 interferon production by down-regulation of tlrs and downstream signaling molecules. porcine epidemic diarrhea virus (pedv) belongs to the order nidovirales, family coronaviridae, and genus alphacoronavirus, and is an enveloped virus with a single-stranded positive rna genome. it contains four structural proteins-spike (s), membrane (m), nucleocapsid (n), and envelop (e)-responsible for viral infectivity and the induction of immune response kocherhans et al., 2001) . pedv causes enteric disease, resulting in significant morbidity and mortality in neonatal pigs, and has been reported as a major source of substantial economic losses in most swine producer countries (j. chen et al., 2010; q. chen et al., 2013; x. chen et al., 2012; cima, 2014; song and park, 2012; takahashi et al., 1983) . in 2014, a less pathogenic pedv strain was reported in the united states and several other swine producer countries vlasova et al., 2014; yamamoto et al., 2015) . this new strain presented insertions and deletions on the amino terminal region of the s protein. on the basis of differences in the s gene and virulence, emerging pedv strains can be divided into non-s-indel (s gene insertions and deletions) and s-indel strains (vlasova et al., 2014) . pedv pathogenesis is strain-specific, and pathogenesis studies in neonatal pigs have demonstrated that the pedv non-s-indel strain is more pathogenic than the pedv s-indel strain wang et al., 2016; yamamoto et al., 2015) . pedv pathogenesis is also inversely correlated with the age of the animals. in adult pigs, pedv infection is self-resolving regardless of previous pedv immune status. moreover, pedv s-indel was shown to be clinically relevant in neonates, but clinical disease could not be reproduced in pigs older than three weeks (annamalai et al., 2015; chen et al., 2016) . host pattern recognition receptors (prrs), such as toll-like receptors (tlrs) and the cytosolic retinoic acid-inducible gene-1 (rig-1)-like receptors, recognize pathogen-associated molecular patterns (pamps) during viral infection alexopoulou et al., 2001b; takeuchi and akira, 2010; uematsu and akira, 2008) . this interaction triggers the interferon regulatory transcription factor (irf3/7) and activates nf-κb, which modulates the expression of several pro-inflammatory cytokines and chemokines. type 1 interferon alpha/beta (ifnα/ifnβ) are the two essential cytokines that can control viral infections (kawai et al., 2005; seth, 2005; xu et al., 2005) . within nidoviruses, acute respiratory syndrome coronavirus (sars-cov) and middle-east respiratory syndrome coronavirus (mers-cov) can evade the host immune system by interfering with the nf-κb signaling pathway (dediego et al., 2014; matthews et al., 2014) , and the infectious bronchitis virus (ibv) inhibits the phosphorylation of kinases that are necessary to activate downstream signaling cascades (chen et al., 2013; devaraj et al., 2007; kint et al., 2015a; sun et al., 2012; zhou, 2007) . two signaling pathways, known as the classical (canonical) pathway and the alternative (non-canonical) pathway (kawai and akira, 2010; loo and gale, 2011) , lead to the activation of nf-κb. the canonical pathway includes the recruitment of the myeloid differentiation primary response gene 88 (myd88), containing the toll/interleukin-1 receptor (tir) domain for eventual activation of nf-κb and induction of type i interferons akira, 2007, 2010 ; thompson and locarnini, 2007) . in addition, endosomal receptor tlr3 contains exclusively tir-domain-containing adapter-inducing interferon-β (trif) adapter proteins that interact with traf6, which induces irf3/7 similar to the myd88 pathway (yamamoto et al., 2003; zhengfan, 2004; ) . finally, both the myd88 and trif pathways activate nf-κb and induce expression of the antiviral type i interferons (thompson and locarnini, 2007) . the non-canonical pathway is tlrindependent, and nf-κb can be modulated by rig-i-traf3 mediated through irf3 (devaraj et al., 2007) . several in vitro studies have tried to elucidate the role of pedv in innate immune response at the cellular level. it has been demonstrated that the nucleocapsid (n) protein of pedv, during infection of hek-293t cells, inhibited ifn-β production by annexing the vital interaction between irf3 and tbk1 (ding et al., 2014b) . other in vitro studies in porcine intestinal epithelial cells (iecs) determined that pedv infection impeded the production of ifn-β by inhibiting the rig-i pathway and hampering the activation of irf3 (cao et al., 2015a) . studies in vero cells showed that pedv infection degraded stat-1 and disrupted the ifn response . hence, it has been shown that pedv can regulate different immunological pathways in vitro; the main regulatory effect of pedv on mucosal innate immunity and its strain-dependence on viral pathogenicity is still unknown. the aim of this study was to investigate the differential gene modulation of pattern recognition tlr and rig-i-like receptors and downstream mediators on the intestinal mucosa of neonatal pigs infected with pedv non-s-indel and pedv s-indel strains. thirty 5-days-old conventional piglets were selected for the molecular evaluation of the gene modulation of pattern recognition tlr and rig-i-like receptors and downstream mediators on intestinal mucosa. the experimental design as well as information of the virus strains used in this study, kinetic of virus shedding, virus distribution in tissues, and pathogenicity was previously described in a pedv clinical pathogenesis study . in brief, pigs were injected intramuscularly with a dose of excede (zoetis, kalamazoo, mi) at time of delivery to iowa state university, laboratory animal resources facilities (ames, ia). all pigs were confirmed negative for pedv, pdcov, tgev, and porcine rotaviruses (groups a, b, and c) by virus-specific pcrs on rectal swabs, and seronegatives by pedv indirect immunofluorescent assay. after one day of acclimation, six-day-old pigs were inoculated orogastrically with 10 ml of 10 4 tcid 50 / ml of pedv non-s-indel (usa/ in19338/2013) or pedv s-indel (usa/il20697/2014), or 10 ml of virus-negative culture medium. five pigs from each group were euthanized at three and seven days post-inoculation (dpi), respectively. sections of distal small intestine of approximately 0.5 cm in length were snap frozen and saved at −80°c. frozen intestine tissue samples were aliquoted, placed in an rnalater™ stabilization solution (life technologies, carlsbad, ca), and kept at −80°c until further use. 2.2. rna extraction from porcine intestine tissue rna was extracted from 5 mg of porcine intestinal tissue using the ambion® magmax™ total rna isolation kit (life technologies) and a kingfisher® 96 magnetic particle processor (thermo fisher scientific, waltham, ma) following the manufacturer's instructions. rna was eluted into 70 µl of elution buffer and stored at −80°c. 2.3. expression of mrna for tlrs, inflammatory signaling pathways, and cytokines on porcine intestinal mucosa the relative quantification of gene expression of toll-like receptors tlr2, tlr3, tlr4, tlr7, tlr8, and tlr9 was evaluated on total rna extracted from porcine intestinal tissues. gene expression of inflammatory signaling pathway molecules, including rig-1, trif, myd88a, myd88b, irf7, traf6, nf-κb1 (p105) nf-κb1 (p50), and rela (p65), was also conducted by sybr-green rt-pcr on the rna extracted from intestinal pig mucosa. gene expression of cytokines ifnα, il-6, il-12, and tnf-α in intestinal mucosa was also investigated following the same protocol described herein for the rest of the genes. all reactions were performed in triplicate and the relative gene expression of each target gene was evaluated in reference to the expression of housekeeping genes gapdh and beta-actin. all primers were custom-synthesized (integrated dna technologies, inc., coralville, ia) to target amplicons, with sizes ranging between 95 and 120 nt base pairs according to the cdna sequence of each target gene, collected from the national center for biotechnology information (ncbi) database (table 1) . the mrna expression levels were quantified according to the δδct method (livak and schmittgen, 2001) . briefly, the difference in cycle times, δct, was determined as the difference between the tested gene and the reference housekeeping genes. the δδct was obtained by finding the difference between groups. the fold change was calculated as 2 -δδct . real-time rt-pcr was performed with eluted rna and primers, mixed with the commercial power sybr green rna-to-c t ™ 1-step kit (applied biosystems, foster city, ca), following the manufacturer's recommendations. the reverse transcription reaction was conducted at standard mode for 30 min at 48°c, enzyme activation was conducted at 95°c for 10 min using an applied biosystems™ 7500 realtime pcr instrument. the strands were denatured at 95°c for 15 s, then annealed and extended at 60°c for 1 min (40 cycles). a dissociation curve was obtained for each quantitative pcr run to assess its target specificity. the real-time rt-pcr was analyzed with a threshold fixed at a 0.1 setting. cycle threshold (ct) values ≤ 35 were considered positive for the housekeeping and tlr gene expressions. all samples were tested in triplicate and the results were expressed as fold changes relative to the control animals. data were analyzed for normality by kolmogorov-smirnov test. the statistical significance between the two treatment groups was determined by non-parametric statistical analysis using the mann-whitney test. significance was assessed at p < 0.05. data analysis was performed using graphpad prism ® (graphpad software inc., la jolla, ca). rna viruses, including pedv, can interact with a large number of pattern recognition receptors (prrs) in the intestinal mucosa, such us toll-like receptors (tlrs) and rig-i-like receptors (rlrs). this interaction plays a critical role in the activation of the innate immune response. tlrs are normally classified based on their anatomical location in membranes or cytoplasmic receptors. numerous tlrs are expressed in porcine enterocytes with different functions in molecule recognition. in addition, other intracytoplasmic prrs, such as retinoic acid-inducible gene (rig)-i-like receptors (rlrs) can sense and recognize double-stranded or single-stranded rna from a variety of pathogens. in this study, we evaluated gene modulatory effects on a group of prrs, including tlrs and rlrs, in the intestinal mucosa during early infection by pedv non-s-indel and s-indel strains. pigs infected with pedv s-indel showed a significant up-regulatory effect (p < 0.05) on tlr3, tlr4, and tlr7 gene expression by 3 dpi compared to non-infected control pigs and pigs infected with the pedv non-s-indel strain ( fig. 1b-d) . the regulatory effect observed in the pedv s-indel-infected group was transient, returning to basal control levels by 7 dpi. pedv s-indel infection had a significant downregulatory effect (p < 0.05) on tlr2 gene expression at 3 and 7 dpi (fig. 1a) . however, pedv s-indel infection did not affect gene modulation of tlr8 and tlr9 compared to the control group (fig. 1e , f). in contrast, infection with pedv non-s-indel was shown to have a downregulatory effect on the expression of tlr4, tlr7, tlr8, and tlr9 (p < 0.05) compared to the non-infected control and pedv s-indelinfected groups consistently at 3dpi (fig. 1c-f) . however, no statistically significant differences in tlr2 and tlr3 gene modulation levels (p > 0.05) were observed between the pedv non-s-indel and control groups at either 3 or 7 dpi (fig. 1a, b) . the modulation of rig-i was only affected in response to pedv s-indel infection at 3 dpi, with a significant increment in gene expression compared to the control and non-s-indel groups (p < 0.05). however, this modulatory effect was transient, with expression levels returning to the same levels observed for the control and pedv non-s-indel groups by 7 dpi (fig. 2) . fig. 7 summarize the differential modulatory effect on prrs genes induced by pedv s-indel vs non s-indel at dpi 3. prrs of the innate immune system initiate signal transduction cascades in response to ligation by microbial-associated molecular patterns (mamps), which leads to the transcriptional modulation of downstream signaling molecules. with the exception of tlr3, the myeloid differentiation primary response gene 88 (myd88) product is the most generally utilized tlr adapter, either by direct interaction (tlr5, 7-9) or via an intermediary interaction (tlr4) with the tir table 1 primers used for real-time pcr analysis of genes expression of pattern-recognition receptor tlr and rig-i-like and downstream mediators on pig intestinal mucosa. primer name primer sequence (5'−3") amplicon length accession number glyceraldehyde 3-phosphate dehydrogenase (gapdh); β-actin; toll-like receptor (tlr); retinoic acid-inducible gene-1 (rig-1)-like receptor; tir-domain-containing adapter-inducing interferon-β (trif); myeloid differentiation primary response gene 88 (myd88); nuclear factor (nf)-κb; nuclear factor nf-kappa-b p65 subunit (rela); interferon regulatory transcription factor (irf); tnf receptor associated factor (traf); interleukin (il); tumor necrosis factor (tnf); interferons (ifn). domain-containing adapter protein (tirap)/mal. engagement of myd88 leads to recruitment and assembly of the cytoplasmic il-1 receptor-associated kinases (irak) and the tnf receptor-associated factor (traf) 6 to form the irak-traf6 complex, which leads to the activation of the nuclear factor (nf)-κb pathway. the nf-κb transcriptional factor family is composed of nf-κb1 (p105), nf-κb1 (p50) and rela (p65), subunits that translocate into nuclei and regulate nf-κb. tram and trif mediate a signal transduction cascade downstream of both tlr3 and tlr4 (kawai and akira, 2010; thompson and locarnini, 2007) . the myd88-and trif-dependent pathways lead to the activation of interferon regulatory factors (irfs) and the secretion of type-i interferon (ifn) and pro-inflammatory cytokines by activated nf-κb signaling. despite the pedv s-indel strain inducing a positive modulatory effect on tlr3, tlr4, and tlr7 gene expression, no significant differences (p > 0.05) were observed in gene expression levels of trif, myd88 (subunits a and b) (fig. 3a, c, d) . moreover, the pedv s-indel strain appears to have a down-regulatory effect on traf6 and irf7 genes at 3 dpi (p < 0.05) (fig. 4a, b) . pedv s-indel-infected animals showed a differential gene modulation characterized by a significant up-regulatory effect in expression levels of nf-κb1 and (p50) genes (p < 0.05) at 7 dpi, and a down-modulatory effect on the expression of the rela/p65 gene compared to both the control and pedv non-s-indel-infected group at 3 and 7 dpi (fig. 5a, b) . contrary to the pedv s-indel strain, a down-regulatory effect of tlr4, tlr7, tlr8 and tlr9 genes was observed in response to infection with the pedv non-s-indel strain (fig. 1) , which also negatively affected the gene expression of downstream signaling molecules (p < 0.05), including trif, myd88 (subunits a and b), and traf6 (figs. 3a, c, d, 4a). although the expression levels of both p50 and p65 were down-regulated after infection with the pedv non-s-indel strain (fig. 5) , the down-regulatory effect on myd88 and trif gene pathways did not negatively affect the expression of nf-κb (p105) (fig. 3b) . fig. 7 summarize the differential modulatory effect of nf-κb through tlr downstream adapters induced by both pedv strains at dpi 3. pro-inflammatory cytokines and type i interferons (ifns) are produced at the local intestinal mucosal level as part of the innate immune response during the infection process. the modulatory effect of proinflammatory interleukins and ifn genes is the result of a long-elaborated pathway that includes the activation of prrs and downstream mediators, including myd88 and trif, via nf-κb activation. we evaluated whether the differential modulatory effect observed in prrs and downstream mediators in response to infection with the pedv s-indel versus the non-s-indel strain was also translated into a differential modulation in the expression of gene coding for pro-inflammatory cytokines and type i interferons. neither infection with pedv s-indel nor non-s-indel was demonstrated to have a modulatory effect on local production of il-6 (fig. 6a) . the local expression of il-12 was not affected at 3 dpi regardless of the pedv strain. however, a positive modulatory effect on il-12 gene expression was exerted by pedv s-indel at 7 dpi (fig. 6b) . the pedv non s-indel strain showed a transient negative modulatory effect of the tnf-α gene at 3 dpi, returning to basal control levels by 7 dpi. however, the pedv s-indel strain consistently up-regulated the expression of the tnf-α gene during the study (fig. 6c ). the expression of the ifn-α gene was negatively affected only in response to infection with the pedv non-s-indel strain at 3 dpi (p < 0.05) (fig. 6d ). fig. 7 summarize the effect of pro-inflammatory cytokine gene regulation by pedv s-indel and pedv non-s-indel strain at dpi 3. pathogen recognition by tlrs and rlrs activates the innate immune response through signaling pathways, resulting in the production of pro-inflammatory cytokines, type i interferons, and chemokines. intestinal mucosa are composed of a variety of specialized cells that play specific functions during the disease process (kawai and akira, 2006; kumar et al., 2009) . collectively, intestinal epithelium, dendritic cells, m cells, immune cells in the lamina propria, and peyer patches (e.g., lymphocytes and macrophages) play a role in the intestinal mucosa immune response against pathogens. prrs are constitutively expressed in all cell populations referred to above and may have differential roles in each cell type and component of the intestinal mucosa. initial prr-induced responses are critical in controlling infectious agents, but are also tightly regulated through time-, location-, and cell type-dependent mechanisms. positive and negative modulation interaction of signaling pathways is the main mechanism for maintaining inmate immune homeostasis. interferon production, especially type 1 interferon and anti-viral cytokines, is important for host protection against viral invasion. many viruses can evade the host immune system by regulating signaling pathways, resulting in the blockage of cytokine production (mccartney and colonna, 2009). porcine epidemic diarrhea virus (pedv) causes enteric diseases, resulting in significant economic losses. pedv pathogenesis is strainspecific, and pathogenesis studies in neonatal pigs have demonstrated that the pedv non-s-indel strain is more pathogenic than the pedv s-indel strain wang et al., 2016; yamamoto et al., 2015) . pedv s-indel was shown to be clinically relevant in neonates, but clinical disease could not be reproduced in pigs older than three weeks (annamalai et al., 2015; chen et al., 2016) . the molecular mechanism of innate immune modulation has only been evaluated in vitro, and thus available information is scarce (cao et al., 2015a (cao et al., , 2015b ding et al., 2014a; gao et al., 2015; xu et al., 2013) . however, it has been demonstrated that in vitro studies are highly dependent on virus strain and cell type, and are not valid for the evaluation of innate immune response against low-virulent strains (kint et al., 2015b) . therefore, in this study, we evaluated the differential modulation of intestinal mucosa prr signaling mounted against pedv non-s-idel and s-indel in there is limited information about the role of antiviral innate immunity in the pathogenesis of pedv infection. however, it is well known that viral infections are usually detected by cell membranes and endosomal-associated tlrs (e.g., tlr4 and tlr3, tlr7/8) and cytosolic rig-i-like receptors (rlrs), such as rig-i and mda5. activated tlr and rig-i/mda-5 signaling pathways initiate effective antiviral innate immune responses, in particular inducing type i infs (thompson and locarnini, 2007) . previous in vitro studies demonstrated that pedv strain cv777 infection of intestinal epithelial cells (iecs) modulates the nf-κb signaling pathway through up-regulation of tlr2, tlr3, and tlr9, but not rig-i (cao et al., 2015a) . however, our results showed that in vivo, pedv s-indel infection up-regulated the nf-κb signaling pathway through tlr3, tlr4, tlr7, and rig-i, resulting in increased expression levels of tnf-α. although no statistical significance was found, increased levels of ifn-α gene expression were also observed. interestingly, both pedv cv777 and pedv s-indel strains belong to genogroup 1, and both are associated with mild clinical disease. therefore, the differential modulation observed in the nf-κb signaling pathway could be more associated with in vivo-related conditions than genotypic characteristics of the virus strain used in this study. conversely, pedv non-s-indel down-regulated the nf-κb signaling pathway through a negative modulatory effect of tlr4, tlr7, tlr8, and tlr9, resulting in final attenuation of pro-inflammatory tnf-α and ifn-α gene expression. moreover, rig-i gene modulation after pedv non-s-indel remained unaffected. in a previous study, it was demonstrated that pedv non-s-indel had a significantly higher replication rate compared to the less virulent pedv s-indel strain . our study demonstrated that the ability of pedv to induce type i ifns is strain-specific. thus, type i ifns may play an important role in pedv replication and pathogenesis. the signaling pathway during ligand binding of single-stranded and double-stranded viral rna involves the cytoplasmic membrane tlr4 receptor and endosomal tlr7/8 and tlr-9, all of which are used as signaling pathway mediators, (myd88 and trif) for eventual activation of nf-kb akira, 2007, 2010 ; thompson and locarnini, fig. 3 . changes in toll/interleukin-1 receptor (tir), myeloid differentiation primary response gene 88 (myd88), and nuclear factor (nf)-κb genes mrna expression induced by porcine epidemic diarrhea virus (pedv) non s-indel and pedv s-indel strains in intestinal mucosa (a-d). ten pigs in each group were infected with pedv non s-indel, pedv s-indel or media (negative control) and five pigs from each group were necropsied at 3 and 7 days post-infection (dpi). the mrna levels of trif (a), nf-κb (b), myd88a (c), and myd88b (d) at the intestinal mucosa was determined individually in each animal by sybr-green qrt-pcr. all samples were tested in triplicate and the results are expressed as fold changes relative to the control animals data are presented as means ± standard errors. significant difference between pedv s-indel pedv non s-indel and control group are expressed with their p values. *p < 0.05; **p < 0.01, ***p < 0.001. 2007). however, endosomal receptor tlr3 (alexopoulou et al., 2001a) contains exclusively trif-adapter proteins that interact with traf6, which induces phosphorylation of irf3/7 similar to the myd88 pathway (yamamoto et al., 2003; zhengfan, 2004) . finally, both myd88 and trif pathways activate nf-kb and induce expression of the antiviral type i interferons (thompson and locarnini, 2007) . in this study, pedv non-s-indel infection had a negative gene modulation on membranes and endosomal tlrs. this negative modulatory effect was translated into a down-regulation of cytoplasmic mediator myd88 and trif genes. in vitro studies demonstrated that silencing trif and myd88 but not rig-i inhibited pedv-induced nf-kb activation, suggesting that the tlr signaling pathway is involved in pedv-induced nf-kb activation (cao et al., 2015b) . in addition, myd88 is a required component of the innate immune response to mouse-adapted sars-cov infection in vivo (totura et al., 2015) . although pedv s-indel induces nf-kb activation, tlr pathway mediators trif and myd88 were not significantly affected by the positive modulation of the cytoplasmic membrane and endosomal tlr genes in response to infection with pedv s-indel. these contradictory results suggest that trif-and myd88-independent pathways might be involved in nf-κb activation after pedv s-indel infection. traf6 is crucial for both rig-i-and tlr-mediated antiviral responses. the absence of traf6 resulted in enhanced viral replication and a significant reduction in the production of type i ifns after infection with the rna virus (konno et al., 2009 ). activation of nf-kb and irf7, but not irf3, is normally traf6-mediated. in this study, we fig. 4 . changes in tnf receptor associated factor (traf) 6 and interferon regulatory factor 7 (irf7) genes mrna expression induced by porcine epidemic diarrhea virus (pedv) non s-indel and pedv s-indel strains in intestinal mucosa (a-b). ten pigs in each group were infected with pedv non s-indel, pedv s-indel or media (negative control) and five pigs from each group were necropsied at 3 and 7 days post-infection (dpi). the mrna levels of traf 6 (a), and irf7 (b) at the intestinal mucosa was determined individually in each animal by sybr-green qrt-pcr. all samples were tested in triplicate and the results are expressed as fold changes relative to the control animals data are presented as means ± standard errors. significant difference between pedv s-indel pedv non s-indel and control group are expressed with their p values. *p < 0.05; **p < 0.01, ***p < 0.001. observed that both pedv s-indel and non-s-indel showed a negative modulatory effect on traf6, which was translated into a significant down-regulation of the irf7 gene only in pedv s-indel-infected animals. traf6 induced activation of irf7, while traf3 is thought to activate both irf3 and irf7 (konno et al., 2009 ). however, due to the differential modulation of irf7 observed in this study, the role of the traf6-dependent pathway cannot be fully elucidated, and perhaps irf3 plays a more important role in type i ifn production in pedv infection. the role of traf3 was not explored in this study but may likely play a role in irf7 and ifn-α gene modulation. in addition, the down-regulatory effect observed in traf6 gene expression for both pedv strains did not have the same modulatory effect on nf-kb gene expression. this study showed that nf-kb gene expression was pedv strain-dependent. although it may need further confirmation, the upregulation of nf-kb gene expression observed in response to pedv s-indel infection could be linked to the up-regulation of rig-i gene expression induced after infection with the pedv s-indel strain. signaling pathways (canonical and non-canonical) lead to the activation of nf-κb (kawai and akira, 2010; loo and gale, 2011) . modulation of nf-kb family members (hetero-dimer p50-p65) is affected through th tlrs, which unbound the inhibitory ikb proteins and allowed activation of the nf-kb canonical pathway (wietek and o'neill, 2007) . we observed that pedv non-s-indel exerted a down-regulatory effect on the p50-p65 hetero-dimer. the result observed during pedv non-s-indel infection is in agreement with a previous in vitro study that demonstrated that pedv-encoded nucleocapsid (n) protein can impede pro-inflammatory cytokine and type i interferon production by direct interaction with tank-binding kinase 1 (tbk1), resulting in inhibition of transcription factors, such as irf3 and irf7, causing final nf-κb interference (ding et al., 2014b) . moreover, it has been reported in vitro that pedv non s-indel nsp1 protein induced nf-κb suppression (zhang et al., 2017) . inhibition of the p50-p65 hetero-dimer blocks activation of the nf-κb canonical pathway and inhibits early tnf-α response (wietek and o'neill, 2007) . our results demonstrate that pedv non-s-indel could use this strategy to evade the host immune system in addition to exhibiting an ifn-α down regulatory effect. although in this study we observed that pedv s-indel down-regulated the p65 gene, the nf-κb pathway was up-regulated. it has been fig. 6 . changes in proinflammatory cytokines il-6, il-12, and tnf-α, and ifn-α genes mrna expression induced by porcine epidemic diarrhea virus (pedv) non s-indel and pedv s-indel strains in intestinal mucosa (a-d). ten pigs in each group were infected with pedv non s-indel, pedv s-indel or media (negative control) and five pigs from each group were necropsied at 3 and 7 days post-infection (dpi). the mrna levels of il-6 (a), il-12 (b), tnf-α (c), and ifn-α (d) at the intestinal mucosa was determined individually in each animal by sybr-green qrt-pcr. all samples were tested in triplicate and the results are expressed as fold changes relative to the control animals data are presented as means ± standard errors. significant difference between pedv s-indel pedv non s-indel and control group are expressed with their p values. *p < 0.05; **p < 0.01, ***p < 0.001. demonstrated that inhibition of the p50-p65 hetero-dimer or the p50 homo-dimer can be compensated for by the p52-relb hetero-dimer, resulting in the activation of the nf-κb non-canonical pathway (wietek and o'neill, 2007) . in vitro studies demonstrated that pedv protein e is capable of inducing p65 in intestinal epithelial cells and the activation of the nf-κb non-canonical pathway (xu et al., 2013) . moreover, further studies demonstrated that during pedv cv777 infection, nf-kb p65 was found to be translocated from the cytoplasm to the nucleus, and pedv-dependent nf-kb activity was associated with viral dose and active replication. in addition, the same study corroborated that upon pedv n protein overexpression in transfected iecs, p65 was detected in the nucleus (cao et al., 2015b) . the production of pro-inflammatory cytokines and type i interferon (ifn) at the local intestinal mucosal level is part of the innate immune response during viral infection. interleukin-6 (il-6) is associated with improving humoral and mucosal immune response (meng et al., 2013) . in this study, il-6 mucosal gene expression was not affected by pedv infection. il-6 expression levels are not necessarily correlated with the positive gene modulation of other pro-inflammatory cytokines. il-12 is the major type i cytokine produced by macrophages and dendritic cells. il-12 is believed to be responsible for enhancing th1 and s-iga response at the mucosal level (boyaka et al., 1999) . in this study, we observed that il-12 was up-regulated at 7 dpi by pedv s-indel. this modulatory effect is in agreement with a previous in vitro study, which also evaluated a low-virulent strain of cv777 (gao et al., 2015) . in this study, we observed a positive tnf-α gene regulatory effect likely associated with the up-regulation of nf-kb in response to pedv s-indel infection. we speculate that this might be the result of the rig-i noncanonical pathway of nf-κb, perhaps through the mitochondrial antiviral-signaling protein (mavs) and traf3 (not evaluated in this study) (bowie and unterholzner, 2008) . other coronaviruses, such as sars-cov, also showed a positive modulatory effect of tnf-α through the activation of nf-kb. conversely, pedv infection with the highvirulent non-s-indel strain induced a down-regulation effect on tnf-α gene expression. this negative modulatory effect on tnf-α could be associated with the severe pathological response characteristic of pedv non-s-indel strains. modulation of type i ifn response seems to be a common evasion strategy of viruses in the order nidovirales . sars-cov and mers-cov, both within the genus betacoronavirus, do not induce significant ifn response in respiratory cells in vitro. however, transmissible gastroenteritis epidemic virus (tgev) within the alphacoronavirus genus induces a high level of ifn-α in newborn pigs. pedv also exists within the alphacoronavirus genus; however, we found virus strain-related differences in ifn-α gene modulation. although no significant ifn-α gene modulation was observed after pedv s-indel infection, there was an increment consistent with previous reports on tgev. the down-regulation observed in pedv non-s-indel is more consistent with the effect observed with other members of the betacoronavirus genus. the differential modulatory effect in the tnf gene between pedv strains observed in this study is coincident with the severity in pathogenicity between pedv s-indel and non-s-indel. in summary, the aim of this study was to investigate the differential gene modulation of pattern recognition tlr and rig-i-like receptors and downstream mediators on the intestinal mucosa of neonatal pigs infected with pedv non-s-indel and pedv s-indel strains. our results suggest that pedv s-indel infection induces pro-inflammatory cytokines through the non-canonical nf-κb signaling pathway by activating fig. 7 . differential gene modulation of pattern-recognition receptor tlr and rig-i-like, and downstream mediators on intestinal mucosa of pigs infected with pedv non s-indel and pedv s-indel strains. this figure present differential gene modulation at day post-infection (dpi) 3. pedv non-s-indel infection suppressed the induction of the pro-inflammatory cytokine tumor necrosis factor alpha (tnf-α), and type 1 interferon production (ifn-α) through the down regulation of the cytoplasmic membrane and endosomal tlrs (tlr4, tlr7/8, tlr9), and tlr-downstream signaling molecules (myd88/trif and traf6). although the expression levels of both p50 and p65 were down-regulated after infection with the pedv non-s-indel strain, the down-regulatory effect on myd88 and trif gene pathways did not negatively affect the expression of nf-κb ( ). contrary, pedv s-indel infection induced a positive modulatory effect on tlr3, tlr4, and tlr7 gene expression. however, no significant modulatory effect was observed in the levels of trif, and myd88, genes. pedv s-indel infection induced the pro-inflammatory cytokines tnf-α, and interleukin (il)−12 through the non-canonical nf-κb signaling pathway by the activation of the intracytoplasmic rig-i receptor ( ). the rig-i receptor (fig. 7) . meanwhile, pedv non-s-indel infection suppresses the induction of pro-inflammatory cytokines and type 1 interferon production by down-regulation of the cytoplasmic membrane and endosomal tlrs, as well as tlr downstream-signaling molecules (canonical nf-κb pathway) (fig. 7) . these data present novel in vivo 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7 , which was modified for use in this manuscript. we sincerely apologize to many scientists whose works were not cited in the reference list because of the space limitation. key: cord-281820-oltqsd6n authors: watanabe, rie; sawicki, stanley g.; taguchi, fumihiro title: heparan sulfate is a binding molecule but not a receptor for ceacam1-independent infection of murine coronavirus date: 2007-09-15 journal: virology doi: 10.1016/j.virol.2007.06.034 sha: doc_id: 281820 cord_uid: oltqsd6n a highly neurovirulent mouse hepatitis virus (mhv) jhmv strain (wt) with receptor (mhvr)-independent infection activity and its low-virulent mutant srr7 without such activity were found to attach to mhvr-negative, non-permissive bhk cells. to identify the molecule that interacts with jhmv, we focused on heparan sulfate (hs) since it works as a receptor of a mutant mhv-rec1 that infects in an mhvr-independent fashion. the present study indicates that hs interacts with both wt jhmv and srr7 but it does not function as an entry receptor as it apparently does for mhv-rec1. furthermore, hs failed to serve as an entry receptor in the mhvr-independent infection of wt jhmv, indicating that hs is not a host factor that wt jhmv utilizes in an mhvr-independent infection. the highly neurovirulent jhmv strain of mhv is able to spread from cells infected via the receptor for mhv (mhvr), a carcinoembryonic cell adhesion molecule 1 (dveksler et al., 1991) , to cells without mhvr (mhvr-independent infection) (gallagher et al., 1992; taguchi and matsuyama, 2002) , while a mutant srr7 (soluble-receptor-resistant mutant 7) isolated from the jhmv cl-2 strain (wt jhmv) because of its resistance to inactivation by soluble form of mhvr (somhvr) lacks this ability . the mutation responsible for the srr7 phenotype was mapped to s2 (saeki et al., 1997) . mhvr-independent infection is attributed to a unique feature of the s protein of wt jhmv, namely the labile association of s1 with s2. dissociation of s1 from s2 triggers a conformational changes in s2 and facilitate virus-cell membrane fusion (gallagher, 1997; krueger et al., 2001; matsuyama and taguchi, 2002) . a key condition for this infection may be that the dissociation of s1 takes place in close proximity to mhvr-negative cells, so that the fusion peptide is exposed and penetrates into the adjacent cell membrane. we found that both wt jhmv and srr7 attached to mhvr-negative cells (watanabe et al., 2006) and infection could be activated by the addition of somhvr, indicating that wt jhmv and srr7 may bind to molecules other than mhvr. mutants of the a59 strain of mhv that arose during persistent infection of cell expressing mhvr (sawicki et al., 1995) were reportedly able to infect mhvr-negative cells (baric et al., 1999 (baric et al., , 1997 schickli et al., 1997) . one of the mutants, mhv-bhk, utilized heparan sulfate (hs) as a receptor (de haan et al., 2005) . this virus has three copies of the putative hs-binding motif in its s protein: one in the s1 as a 7-amino acid insertion that is not present in the original mhv-a59, one in the cleavage site and one in the s2 subunit (de haan et al., 2005) , as illustrated in fig. 1 . these binding motifs are thought to make it possible to use hs as an attachment/entry receptor. there is one copy of the hs-binding motif adjacent to the cleavage site in the s protein of both wt jhmvand srr7 (fig. 1 ). this suggests that hs might also interact virology 366 (2007) we also addressed whether or not hs is responsible for the mhvr-independent infection by the wt jhmv. we have previously reported that highly neurovirulent wt jhmv could infect cells lacking mhvr if it was forced to attach to cells by spinoculation, i.e. infection by centrifugation at 3000 rpm for 2 h at 4°c. we also found that both wt jhmv and srr7 attached to mhvr-negative bhk cells during a standard infection protocol, i.e. without spinoculation (watanabe et al., 2006) . to further confirm these findings, we inoculated 10 5 pfu, corresponding to ca. 10 7 copies of genome of those viruses onto mhvr-negative bhk cells and bhk-r1 cells, which express mhvr, without spinoculation. we then evaluated the copy number of the attached viruses by real-time pcr analysis. as shown in fig. 2a , about 10 5.5 and 10 5 copies of wt jhmv and srr7, respectively, attached to the bhk cells, which was about 50% of the binding to bhk-r1 cells. this finding clearly indicated that wt jhmv and srr7 attached, even onto mhvr-negative cells. to evaluate the infectivity of the attached virus, 50 nm of somhvr was added to the culture of bhk cells inoculated with wt jhmv and srr7 and those cells were further incubated for 14 h at 37°c. as shown in fig. 2b , srr7 efficiently infected bhk cells in the presence of 50 nm of somhvr but not at all without somhvr. infection of mhvrnegative cells with wt jhmv was greatly enhanced by somhvr, although a very low level of infection was found without somhvr (fig. 2b) , which was presumably due to an extremely inefficient mhvr-independent infection after ordinary protocol of infection. these results are in good agreement with our previous findings that somhvr facilitated the infection of both wt jhmvand srr7 after adsorption onto mhvr-negative cells (watanabe et al., 2006) , suggesting that some molecule(s) on the cell surface other than mhvr allow the attachment of both wt jhmv and srr7. binding of jhmv to hs on the cell surface hs is the major glycosaminoglycan (gag) found on most cells and was recently reported as an entry receptor for mhv-bhk, a strain that has an extended host range and infects mhvr-negative cells (de haan et al., 2005) . because jhmv also has one potential hs-binding site, we evaluated the contribution of hs to wt jhmv and srr7 attachment to the cell surface by treating the cells with heparinase. as shown in fig. 3a , heparinases reduced cell surface hs effectively as shown by facs analysis, but had little effect on the level of mhvr of bhk-r1 cells. to determine the effect of removing hs on wt jhmvand srr7 attachment of bhk and bhk-r1 cells, the cells, either treated with heparinases or left untreated, were then inoculated with 10 7 copies of viruses and incubated for 1 h at 4°c. after removal of unattached virus by washing with pbs, cell-associated total rna was extracted, and the number of viral genomes was measured by real-time pcr. the data in fig. 3b show that heparinases reduced viral attachment by approximately one-half on both bhk and bhk-r1 cells, suggesting that about 50% of jhmv attached to target cells via hs, irrespective of the presence or absence of mhvr on the cell surface. we further examined whether jhmv bound to cells via hs is infectious or not. bhk and bhk-r1 cells treated with heparinase i or iii were inoculated with 2 × 10 4 and 200 pfu of srr7, respectively, and incubated for 15 h in the presence of somhvr for bhk and in its absence for bhk-r1 cells to evaluate infectivity. as shown in fig. 3c , virus infection of bhk cells was reduced by heparinase treatment in a heparinase concentration-dependent manner. at the highest concentration, a 70-90% reduction was observed, when compared to untreated cells. together with the data shown in fig. 3b that the binding of srr7 is reduced by heparinase treatment, the data in fig. 3c suggest that srr7 bound hs in a physiologically active form since addition of somhvr facilitated the infection to cells to which srr7 bound. the infection was not reduced in mhvrpositive bhk-r1 cells by heparinase treatment (fig. 3c ), indicating that hs does not influence the infection by srr7 via mhvr. we have additionally examined the binding of srr7 with hs using heparin. the pretreatment of viruses with heparin with the same disaccharide-repeating units as hs can generally block virus infection when cell surface hs contributes to their attachment/infection (liu and thorp, 2002) . we mixed srr7 with heparin and incubated the sample at 4°c for 1 h before inoculation of bhk or bhk-r1 cells. infection of bhk cells was examined in the presence of somhvr. heparin reduced somhvr-mediated infection of bhk cells by srr7 at a concentration of 5 μg/ml or higher (fig. 3d) , suggesting that hs is the molecule that interacts with srr7 to attach it to the cell surface. there was no effect of heparin on virus infection of bhk-r1 cells. this suggested that the region of s protein responsible for heparin binding is different from that required for mhvr binding. in combination with the observation that normal bhk cells, untreated with heparinase, are not at all permissive to srr7 infection, the above data collectively suggest that hs is a binding molecule but does not function as a receptor for infection nor enhance mhvr-mediated infection. hs as a functional receptor for mhv-rec1 but not for jhmv mhv-rec1 contains the same s protein as the virus isolated from mhvr-positive 17cl-1 cells persistently infected with mhv-a59 (schickli et al., 1997; ) and it utilizes hs as a receptor (de haan et al., 2005) . we evaluated the requirement of hs for infection by mhv-rec1 and jhmv. although both viruses could infect and form a large syncytium on mhvr-positive dbt cells ( fig. 4a) , there was a clear difference between mhv-rec1 and wt jhmv in the infection of mhvr-negative bhk cells. mhv-rec1 could infect bhk cells when they were inoculated by the ordinary infection method, whereas wt jhmv required spinoculation to infect efficiently (fig. 4a) . the major difference among mhv-rec1, wt jhmv and srr7 in the use of hs for infection became apparent with the use of heparinase-treated dbt cells. dbt cells treated with heparinase had reduced amounts of hs by facs analysis as described above (data not shown). heparinase-treated or untreated dbt cells were infected with mhv-rec1, wt jhmv and srr7. then, their infectivity was evaluated by counting the number of plaque that was formed 15 h after infection. as shown in fig. 4b , wt jhmv and srr7 infection of dbt cells was not affected by the heparinase pretreatment, whereas mhv-rec1 infection was ca. 90% suppressed, confirming that hs serves as a fully functional receptor for mhv-rec1 infection but it does not for either wt jhmv or srr7. to support the result obtained above, the infection-interference assay with heparin was performed. two hundred plaqueforming units of wt jhmv, srr7 and mhv-rec1 was mixed with various concentrations of heparin and incubated at 4°c for 1 h. the mixture was inoculated onto dbt cells and the number of plaque was counted after incubation for 15 h. as shown in fig. 4c , almost 90% of mhv-rec1 infectivity was blocked by the heparin at a concentration of 0.01 mg/ml, whereas the infectivity of both wt jhmv and srr7 was not inhibited by heparin at a concentration of 1 mg/ml. these results clearly show the major difference between jhmv and mhv-rec1 on the usage of hs as a receptor for entry into cells. we also addressed whether hs works as an entry receptor for mhvr-independent infection by wt jhmv. bhk cells were treated with heparinase i and spinoculated with 10 4 pfu of wt jhmv. then, the infection was monitored by plaque formation. as shown in fig. 4d , no reduction of mhvr-independent infection was observed. we also confirmed this result by using heparin interference assay. 10 4 pfu of wt jhmv was treated with heparin at 4°c for 1 h, and the infectivity was measured by spinoculation. the results also show no reduction in mhvrindependent infection by wt jhmv after its treatment with heparin. these two different approaches clearly indicated that hs is not involved in the mhvr-independent infection of wt jhmv, which is highly neurovirulent. in the present study, we showed that wt jhmv, and a mutant derived from this strain by selection for being resistant to inactivation by somhvr, interacts with hs on the cell surface, but fails to utilize this molecule as an entry receptor. it was further shown that hs does not work as a receptor for mhvrindependent infection by wt jhmv, namely infection by spinoculation. we have also confirmed the previous observation that mhv-rec1, another mhv with mhvr-independent infection activity, is able to use hs as a functional receptor. the remarkable biological difference between wt jhmv and mhv-rec1 (schickli et al., 1997) is that the latter infects cells without mhvr by the standard protocol of infection; however, the former fails to infect under such conditions. wt jhmv must be forced to attach to cells by spinoculation when it executes the infection of mhvr-negative cells. this difference could be attributed to the nature of s protein in terms of its process of binding to hs; mhv-rec1 has three copies of the hs-binding motif, while wt jhmv s contains only one copy (fig. 1) . this difference could affect the strength of binding between hs and the s protein. it is possible that tight binding of mhv-rec1 with hs could trigger the conformational changes of the s protein and facilitate its infection, while weak binding of the wt jhmv s protein with hs fails to trigger the conformational changes and also, therefore, entry. in fact, de haan et al. (2006) reported that cooperative involvement of two regions containing hs consensus sequences is important for the utilization of hs as an entry receptor by mhv-rec1. wt jhmvas well as the mhv-rec1, both of which infect in an mhvr-independent fashion, must have been selected under an environment with strong selection pressure. before permissive cell lines become available for virus propagation, jhm strains of mhv have been maintained over the years by passage through mouse brains, where only microglia cells are positive for mhvr among the various types of cells (nakagaki et al., 2005; ramakrishna et al., 2004) . to survive in the brain, jhmv would have to have been selected during passage through the mouse brains because of its unique ability to spread to a variety of mhvr-negative cells. thus, the original virus may have been more like srr7 that infects truly in an mhvr-dependent fashion. also, mhv-a59 mutant viruses that have a wide range of hosts were selected from unusual infection environments: some were forced to infect cells without mhvr (baric et al., 1999; baric et al., 1997) and the others were isolated from persistently infected cells with profoundly reduced mhvr expression (sawicki et al., 1995; schickli et al., 1997 schickli et al., , 2004 . thus, the viruses that survived to grow in an environment of reduced or no receptor expression could have adapted to use another molecule as a receptor, as did mhv-rec1, while some others, like wt jhmv, established unique features to allow them to survive in the environment. comparative studies on the s proteins of these viruses that infect in an mhvr-independent fashion will be of interest in terms of the molecular mechanism of receptor independence in viral infection. the present study showed that hs works as a functional receptor for mhv-rec1, but not for wt jhmv, both of which infect in an mhvr-independent fashion. these findings suggest that hs does not play a role to make mice susceptible to mhvr-independent infection by wt jhmv. however, pathogenic studies on mhv-rec1 are very limited and little information is available on the participation of hs in mhv pathogenesis. such studies will possibly provide new insights into mhv pathogenicity. bhk cells, bhk-r1 cells stably expressing mhvr (matsuyama and taguchi, 2000) and dbt cells were maintained in dulbecco's minimum essential medium (dmem: nissui, tokyo, japan) supplemented with 5% fetal bovine serum (fbs, sigma, st. louis, mo) as previously reported . a highly neurotropic jhmv cl-2 (defined as wt jhmv) (taguchi et al., 1985) , and a solublereceptor-resistant mutant derived from wt jhmv, srr7 (saeki et al., 1997) , as well as mhv-rec1 derived from mhv-a59 (schickli et al., 2004) , were propagated and assayed on dbt cells. viral infectivity is shown as plaque-forming units (pfu). srr7 has a single amino acid mutation at position 1114 (leu to phe) of the s2 subunit of the s protein relative to wt jhmv (saeki et al., 1997) . heparinase treatment was performed mostly as described previously (klimstra et al., 1998) . bhk and bhk-r1 cells were prepared in a 24-well culture plate (falcon, franklin lakes, nj) and were treated with heparinase i (sigma) and iii (sigma) dissolved in a buffer (10 mm phosphate buffer (ph 7.4) containing 0.15 m nacl, 3 mm kcl, 0.5 mm mgcl 2 , 1 mm cacl 2 , 0.1% glucose, 1% fbs and 0.5% bovine serum albumin) for 1 h at 37°c. then bhk and bhk-r1 cells were chilled on ice and inoculated with 2 × 10 4 and 200 pfu of viruses, respectively, and further incubated for 1 h at 4°c. after washing three times with phosphate buffered saline, ph 7.2 (pbs), cells were incubated with dmem containing 1% fbs for 14 h at 37°c. cells were fixed and stained with crystal violet, and the number of plaque was counted under light microscopy. to infect the bhk cells, the culture was supplemented with somhvr (50 nm in final concentration). the somhvr used for this purpose consisted of only the n domain from the mhvr (miura et al., 2004) , which was expressed by recombinant baculovirus and purified by using its tag as described previously . the heparin competition assay was performed as described previously (klimstra et al., 1998) . viruses at 2 × 10 4 pfu (for bhk) or 200 pfu (for bhk-r1) in 50 μl were mixed with an equal volume of heparin (sigma) and incubated for 1 h at 4°c. bhk or bhk-r1 cells prepared as described above were inoculated with those mixtures and incubated for 1 h at 4°c. cells were washed in ice-cold pbs and incubated with dmem supplemented with 1% fbs for a further 14 h at 37°c. the number of plaque was obtained as described above. to confirm the infection of the bhk cells, somhvr was added at 50 nm in a final concentration. the level of hs or mhvr on the cell surface was evaluated by flow cytometry analysis as previously described (de parseval and elder, 2001) . cells were incubated with anti-hs mab f58-10e4 (seikagaku corporation, tokyo, japan) alone or in combination with anti-mhvr mab cc1, a gift of dr. k. holmes. fitc-conjugated anti-mouse igm (bd pharmingen, san diego, ca) and phycoerythrin (pe)-conjugated antimouse igg1 (jackson immunoresearch, west grove, pa) were used to detect f58-10e4 and cc1, respectively. the fluorescence intensity was measured using a facscalibur (becton dickinson, san jose, ca, usa) and analyzed by cellquest software. attachment of inoculated viruses onto cells treated with heparinase i, heparinase iii or untreated cells was estimated by real-time pcr as described previously using a lightcycler rna master mix (loche diagnostics, mannheim, germany) (watanabe et al., 2006) . spinoculation was done as described previously (watanabe et al., 2006) . cells prepared in a 24-well plate were inoculated with viruses in 300 μl dmem, centrifuged at 3000 rpm (1750×g) for 2 h at 4°c and incubated with dmem supplemented with 1% fbs for an additional 14 h at 37°c. somhvr was added onto the cells infected with srr7. the number of plaque was counted after staining with crystal violet as described above. episodic evolution mediates interspecies transfer of a murine coronavirus persistent infection promotes cross-species transmissibility of mouse hepatitis virus murine coronavirus with an extended host range uses heparan sulfate as an entry receptor cooperative involvement of the s1 and s2 subunits of the murine coronavirus spike protein in receptor binding and extended host range binding of recombinant feline immunodeficiency virus surface glycoprotein to feline cells: role of cxcr4, cellsurface heparans, and an unidentified non-cxcr4 receptor cloning of the mouse hepatitis virus (mhv) receptor: expression in human and hamster cell lines confers susceptibility to mhv a role for naturally occurring variation of the murine coronavirus spike protein in stabilizing association with the cellular receptor cell receptor-independent infection by a neurotropic murine coronavirus adaptation of sindbis virus to bhk cells selects for use of heparan sulfate as an attachment receptor variations in disparate regions of the murine coronavirus spike protein impact the initiation of membrane fusion cell surface heparan sulfate and its roles in assisting viral infections impaired entry of soluble receptor-resistant mutants of mouse hepatitis virus into cells expressing mhvr2 receptor receptor-induced conformational changes of murine coronavirus spike protein n-terminal domain of the murine coronavirus receptor ceacam1 is responsible for fusogenic activation and conformational changes of the spike protein receptor-independent spread of a highly neurotropic murine coronavirus jhmv strain from initially infected microglial cells in mixed neural cultures expression of the mouse hepatitis virus receptor by central nervous system microglia identification of spike protein residues of murine coronavirus responsible for receptor-binding activity by use of soluble receptor-resistant mutants persistent infection of cultured cells with mouse hepatitis virus (mhv) results from the epigenetic expression of the mhv receptor the murine coronavirus mouse hepatitis virus strain a59 from persistently infected murine cells exhibits an extended host range the nterminal region of the murine coronavirus spike glycoprotein is associated with the extended host range of viruses from persistently infected murine cells soluble receptor potentiates receptorindependent infection by murine coronavirus characterization of a variant virus selected in rat brains after infection by coronavirus mouse hepatitis virus jhm receptor-independent infection of murine coronavirus: analysis by spinoculation we are grateful to miyuki kawase for the excellent technical assistance and dr. sarah connolly for the editing manuscripts and valuable comments. we also thank dr. kathyrin holmes for mab cc1 specific for mhvr. this work was financially supported by grants from the ministry of education, culture, sports, science and technology (16017308, 17390138) and a grant from human science foundation (kh51050). key: cord-289991-wx4rsr4g authors: bhowmick, rahul; banik, george; chanda, shampa; chattopadhyay, shiladitya; chawla-sarkar, mamta title: rotavirus infection induces g1 to s phase transition in ma104 cells via ca(+2)/calmodulin pathway date: 2014-03-21 journal: virology doi: 10.1016/j.virol.2014.03.001 sha: doc_id: 289991 cord_uid: wx4rsr4g viruses, obligate cellular parasites rely on host cellular functions and target the host cell cycle for their own benefit. in this study, effect of rotavirus infection on cell cycle machinery was explored. we found that rotavirus (rv) infection in ma104 cells induces the expression of cyclins and cyclin dependent kinases and down-regulates expression of cdk inhibitors, resulting in g1 to s phase transition. the rotavirus induced s phase accumulation was found to be concurrent with induction in expression of calmodulin and activation of camki which is reported as inducer of g1–s phase transition. this cell cycle manipulation was found to be ca(+2)/calmodulin pathway dependent. the physiological relevance of g1 to s phase transition was established when viral gene expressions as well as viral titers were found to be increased in s phase synchronized cells and decreased in g0/g1 phase synchronized cells compared to unsynchronized cells during rotavirus infection. manipulating the cell cycle machinery in order to sustain their own replication is a common mechanism employed by different viruses (emmett et al., 2005) . among the three check points of cell cycle at g 1 /s, g 2 /m and the metaphase/anaphase boundary, g 1 /s is the time window where cell decides for growth or quiescence depending on environmental conditions (bartek and lukas, 2001) . during the hours preceding the g 1 phase, retinoblastoma (rb) remains in hypo phosphorylated form, which can bind and inhibit a group of transcription factors (like e2f) responsible for transcribing genes required for replication as well as cell cycle progression (giacinti and giordano, 2006) . when cell gets stimuli essential for the g 1 to s phase transition, rb becomes hyperphosphorylated and loses ability to bind and inhibit e2f, which then translocates to nucleus and induces expression of genes like thymidine kinase, thymidine synthetase, dihydrofolate reductase, cyclins and cdks required for g1 to s phase transition (harbour and dean, 2000; fan and bertino, 1997) . ca þ 2 /cam-stimulated proteins, including the family of multifunctional ca þ 2 /cam-stimulated protein kinases (camk), have been identified as one of the important mediators among several stimuli that induce cell cycle progression (kahl and means, 2003) . camki induces rb phosphorylation and cyclin d1 upregulation and cdk 4 activation for overall g1 to s phase transition (skelding et al., 2011) . viruses are obligate parasites which lack many of the proteins required for genome replication, so they rely on the host cell for resources and target their cell cycle check points to create a favorable environment for their own replication (schang, 2003) . dna viruses are extensively studied for cell cycle manipulation as primary site of their replication is in the nucleus. to support viral replication small dna viruses lacking their own polymerase, such as simian virus 40 (decaprio et al., 1988; fanning and knippers, 1992) , human papillomavirus (werness et al., 1990) and adenovirus (eckner et al., 1994; howe et al., 1990) promote cell cycle progression to s phase. to avoid competition with host for replication machinery large dna viruses (herpes viruses) arrest cell cycle in the g 0 /g 1 phase (flemington, 2001) . retroviruses such as hiv type 1 arrests cell cycle in g 2 /m phase by employing vpr protein for proper viral gene expression (goh et al., 1998; he et al., 1995) . single stranded rna viruses, whose primary site of replication is cytoplasm were also been found to affect host cell cycle either inducing g 2 /m phase arrest (infectious bronchitis virus) (dove et al., 2006) or g 0 /g 1 arrest (murine corona virus, influenza virus) (chen and makino, 2004; he et al., 2010) , however whether self-limiting double stranded rna viruses like rv manipulates cell cycle remains largely unknown. rv, a nonenveloped double stranded rna virus of reoviridae family is an important threat to mankind as it causes over 5 million deaths each year with 485% of these deaths occurring in children aged below five years in developing countries (rossen et al., 2004; estes and kapikian, 2007) . in addition it also infects live stocks, thus it has huge economic importance. the virus with its 11 segmented ds rna genome encodes six structural proteins (vp1 to vp4, vp6 and vp7) which form the structure of the virus particle and six nonstructural proteins (nsp1 to nsp6), which like non-structural proteins of other viruses confirm viral replication and establish infection in host cells (autret et al., 2008; bitko et al., 2007; bollati et al., 2009; ehrhardt et al., 2007; foy et al., 2003; holloway et al., 2009; spann et al., 2004; talon et al., 2000) . previous studies have identified mechanisms by which rv activates pi3k/akt pathway (bagchi et al., 2010) or degrades p53 (bhowmick et al., 2013) to subvert host innate immune system that tries to induce programmed cell death in response to viral infection (roulston et al., 1999) . many observations such as sharing of same morphological characters (cell shrinkage, chromatin condensation and membrane blebbing) and regulatory proteins (p53, rb, e2f) suggests link between apoptosis and cell cycle regulation, which prompted us to explore the effect of rv infection on cell cycle (alenzi, 2004) . in this study we attempted to understand effect of rv infection on host cell cycle machinery and its implication on virus infectivity. we found that rv induces higher proportion of cells to accumulate in s phase during infection by activating ca þ 2 /cam pathway. accumulation of cells in s phase during initial stage of infection may benefit rv in executing proper life cycle. based on the observation that rv inhibits apoptosis and induces survival pathways during early infection (bagchi et al., 2010; bhowmick et al., 2012) , we examined whether rv can also regulate cell cycle especially during early infection by infecting ma104 cells with sa11 (g3p2), a5-13 (g8p1) and osu (g5p7) strains of rv at 3 moi (multiplicity of infection) or keeping them mock infected for increasing time points. cells were then treated with propidium iodide (50 mg/ml) and cell cycle status was analyzed using flowcytometry as described in material and methods. as shown in fig. 1a significant accumulation (20-32%) of infected cells in s phase was observed during early time point of infection (2-6 hpi), compared to mock infected cells (10-11%) in a strain independent manner. whereas at 8 hpi number of cells in s phase was not significantly higher (14-16%) in case of all the three strains, compared to uninfected controls (10-11%) and at later time points (10-12 hpi) no of cells started to increase in sub g 0 phase compared to uninfected control (fig. 1a) . to confirm the direct effect of rv on host cell cycle machinery, ma104 cells were synchronized in g 1 phase by serum starvation and were infected with sa11 at 3 moi or kept mock infected followed by cell cycle analysis using flowcytometry. cell cycle analysis revealed an increased accumulation of cells (20-30%) in s phase in sa11 infected cells compared to mock infected cells (9-10%) (fig. 1b) . to know whether this cell cycle modulation is viral replication dependent or not, sa11 was inactivated with uv irradiation (li et al., 2009 ) and ma104 cells were infected with uv inactivated virus or live virus. cells were collected at indicated time points and subjected to flowcytometry analysis. it was found that viral replication is necessary for cell cycle manipulation as no significant accumulation of infected cells in s phase was observed in uv inactivated virus infected cells whereas active live virus induced s phase accumulation (fig. 1a) . thus overall results suggest rv manipulates cell cycle machinery to drive cells from g 1 to s phase. fig. 1 . rv induces g 1 to s phase transition in a strain independent but viral replication dependent manner. (a) ma104 cells were infected with sa11 or a513 or osu or uv inactivated sa11 strain for 2-12 h or kept mock infected followed by propidium iodide (50 mg/ml) staining and cell cycle analysis using bd facsaria ii flow cytometer. each bar represent % of cell present in specific phase of cell cycle. results are representative (mean 7 sd) of three independent experiments. (b) ma104 cells were serum starved for 48 h followed by sa11 infection at 3 moi for indicated time points or kept mock infected followed by cell cycle analysis. each bar represents % of cell present in specific phase of cell cycle. results are representative (mean 7 sd) of three independent experiments. the retinoblastoma (rb) protein is the central molecule which controls the g 1 /s transition by connecting all the signals important for the decision of transition with transcription machinery (weinberg, 1995) . to know the phosphorylation status of rb during rv infection, ma104 cells were infected with the rv sa11 strain (at a moi of 3) and incubated for 0-8 hpi. cell extracts were immunoblotted with phospho rb specific antibody. compared to mock infected cells rv infection induced rb hyper phosphorylation during early infection (2-6 hpi) but from 8 hpi onwards the rb phosphorylation was reduced ( fig. 2a lower panel) . furthermore, to monitor the downstream effect of rb phosphorylation, nuclear fractions of same set of experiments was immunoblotted with e2f specific antibody to analyze level of activated e2f. results revealed increased translocation of e2f into nucleus during early infection (2-6 hpi), which is concurrent with rb hyper phosphorylation ( fig. 2a upper panel) confirming the g1 to s phase stimulation during the early hours of rv infection. in addition to nuclear translocation, expression of e2f gene was also induced during rv infection as assessed by immunoblotting and real time pcr (fig. 2b ). functionality of increased nuclear translocation of e2f was further validated by assessing expression of different e2f regulated genes such as thymidine kinase, thymidine synthase and dihydrofolate reductase in sa11 infected ma104 cells by quantitative rt-pcr. as shown in fig. 2c , all three e2f regulated genes were significantly upregulated during 2-4 hpi consistent with nuclear translocation of e2f. since rb hyper phosphorylation was observed during rv infection, the expression levels of cyclin and cdk's following sa11 (3 moi) infection were assessed by either immunoblotting or quantitative rt-pcr. compared to mock infected cells expression of both transcript and protein levels of cyclin d1, cyclin d3 and cdk4, cdk6 was induced in sa11 infected cells during 2-6 hpi followed by decrease at 8 hpi. cyclin e1 and cdk2 transcripts were induced as early as 2 hpi but significant increase in protein level were observed at 4-6 hpi ( fig. 3a and b ). as expression of both cdk and cyclin increased during infection, kinase activity of cdk4, cdk6 and cdk2 was evaluated by immunoprecipitating cyclin-cdk complexes from sa11 (3 moi) infected and mock infected cells. the kinase activity of cyclin-cdk complex was measured by incubating (30 min at 37 1c) them with specific substrates such as rb for cdk4, cdk6 and histone h1 for cdk2 in vitro in specific kinase buffer followed by immunoblotting with prb and phistone h1 specific antibody. increases in activity were observed for cdk4, cdk6 during early infection (2-6 hpi) whereas cdk2 was transiently activated only at 4-6 hpi ( fig. 3c ). activation of cdks depends on the level of cdk inhibitors. to assess whether rv modulates expression of cdk inhibitors to regulate cell cycle, whole cell lysates or total rna of ma104 cells infected with either sa11 (3 moi) or mock infected were subjected to either immunoblotting or real time pcr with p15, p21, p27 specific antibodies or primers, respectively. results revealed that representative cdk fig. 2 . rv infection induces e2f translocation by activating cdks involved with g1 to s phase transition. (a) whole cell lysates or nuclear fraction isolated by differential centrifugation of ma104 cells infected with sa11 were subjected to western blot analysis using prb (lower panel) or e2f specific antibody (upper panel) respectively and compared with mock infected controls at corresponding time points. gapdh and tbp were used as loading control for cellular or nuclear protein respectively. results are representative of three independent experiments. (b) whole cell lysates or total rna from ma104 cells infected with sa11 for indicated time points were subjected to either western blot analysis using e2f and gapdh (loading control) specific antibody or quantitative rt-pcr with e2f specific primers using sybr green dye and compared with mock infected controls. fold changes of transcripts were obtained by normalizing relative gene expression (with respect to mock infected corresponding controls) to gapdh using the formula 2 à δδct (δδct¼ δc t sample à δc t untreated control ). results are representative (mean7 sd) of three independent experiments. (c) total rna from ma104 cells infected with sa11 for indicated time points were isolated using trizol and subjected to real time pcr with thymidine kinase, thymidine synthase, dihydrofolate reductase specific primer using sybr green dye. fold changes of mrna level were obtained by normalizing relative gene expression (with respect to mock infected corresponding controls) to gapdh using the formula 2 à δδct (δδct¼ δc t sample à δc t untreated control ). results are representative (mean 7 sd) of three independent experiments. inhibitors of both ink4 and cip/kip family were significantly down regulated during early sa11 infection (2-6 hpi) ( fig. 3d and e) . camki is a cam activated kinase which regulates g 1 to s phase progression of cell (skelding et al., 2011) . in a previous study from our group, cam level was found to be modulated during rv infection (weinberg, 1995) . to know the activation level of camki during rv infection, ma104 cells were infected with the rv sa11 strain (at a moi of 3) and incubated for 0-8 hpi. cell extracts were immunoblotted with phospho camki and cam specific antibody. results indicated increased phosphorylation (activation) of camki along with upregulation of cam expression during initial time points of infection (2-6 h), followed by decrease at 8 hpi (fig. 4a ). to delineate relation between camki activation and cell cycle progression, ma104 cells were either infected with rv sa11 strain at 3 moi or kept mock infected in presence or absence of either calcium chelator bapta-am which chelates ca þ 2 ions and inhibits cam activation or cam inhibitor w7 which bind selectively to cam and inhibit its downstream functions (dhillon et al., 2003) , for indicated time points followed by cell cycle analysis using flowcytometry (treatments were done post viral absorption). quantitive analysis revealed that both bapta-am and w7 inhibit cell cycle progression from g1 to s phase as found in only sa11 infected ma104 cells (fig. 4b) . inhibition of camki activation by using bapta-am and w7 was proved by immunoblotting the cell extracts of sa11 infected or mock infected ma104 cells treated with bapta-am and w7 with phospho camki specific antibody (fig. 4c) . to define the mechanism behind ca þ 2 /cam activated camki mediated cell cycle manipulation, we assessed the levels of rb phosphorylation and e2f translocation to nucleus during sa11 infection in presence or absence of bapta-am or w7 treatment. both bapta-am and w7 significantly minimized rb phosphorylation and nuclear translocation of e2f compared to only virus infected cells (fig. 5a) . to know the effect of ca þ 2 chelation and cam inhibition during sa11 infection on cyclins, cdks and ckis associated with g1 to s phase transition, ma104 cells were either infected with (c) ma104 cells were either infected with sa11 or kept mock infected for indicated time points and subjected to immunoprecipitation with either cdk4 or cdk6 or cdk2 specific antibody. immunoprecipitates were incubated with either rb (for cdk4, cdk6) or histone h1 (cdk2) followed by immunoblot analysis using prb and phistone h1 specific antibody. results are representative of three independent experiments. (b, e) total rna from ma104 cells infected with sa11 for 2-8 hpi were isolated using trizol (invitrogen) and subjected to quantitative rt-pcr with cyclin d1 (b), cyclin d3 (b), cyclin e1 (b), cdk4 (b), cdk6 (b), cdk2 (b), p15 (e), p21 (e), p27 (e) specific primers using sybr green dye. fold changes of transcripts were obtained by normalizing relative gene expression (with respect to mock infected corresponding controls) to gapdh using the formula 2 à δδct (δδct¼ δc t sample à δc t untreated control ). results are representative (mean 7sd) of three independent experiments. sa11 or kept mock infected in presence or absence of either bapta-am or w7 followed by immunoblotting with cdk2, cdk6, cyclin e1, cyclin d1, p27 and p21 specific antibodies. no significant change in expression level of cyclins and cdks was observed in sa11 infected cells in presence of w7 or bapta-am compared to mock infected control (fig. 5b) confirming ca þ 2 /cam dependent camki mediated upregulation of cyclin and cdk expression (fig. 3a) during rv infection. but in case of ckis, following rv infection, expression of p21 and p27 was downregulated both in presence (fig. 5b) or absence (fig. 3d ) of bapta-am and w7 suggesting that cki regulation is ca þ 2 /cam independent. previous experiments showed rv infection drives cells from g 1 to s phase during early infection. to know the physiological relevance of this phenomenon during virus infection ma104 cells synchronized at g 0 /g 1 phase (terfenadine treated) or at s phase (azt treated) were infected with sa11 (moi 3) and expression of viral gene was assessed by either realtime pcr (fig. 6a) or immunoblotting (fig. 6b ) with nsp3 specific primers or antibodies and compared with viral gene expression in infected unsynchronized cell. result revealed in g 0 /g 1 synchronized cells, viral gene expression was significantly low compared to unsynchronized cells but it was significantly higher in s phase synchronized cells ( fig. 6a and b) . furthermore, viral titers were measured by plaque assay in unsynchronized ma104 cells or cells synchronized at g 0 /g 1 phase or at s phase following infection with sa11 strains at 1 moi. compared to cells synchronized at g 0 /g 1 phase significantly higher viral titers were observed in cells synchronized at s phase or unsynchronized cells (fig. 6c) . this is consistent with nsp3 expression pattern suggesting increased rotaviral replication during s phase. entry into and progression through the cell cycle is considered as a key event in maintaining cellular homeostasis, anomalies in which can cause either cell death or can lead to uncontrolled cell division (vermeulen et al., 2003) . to ensure correct cell division in higher eukaryotes, cell cycle is intricately controlled by numerous complex mechanisms (schafer, 1998) , which in turn are manipulated by many viruses to favor their replication (emmett et al., 2005) . in our current study we found rv also influences cell cycle status of infected cells by propelling them from g 1 to s phase in a strain independent manner (fig. 1a) . replication of rv is important for cell cycle manipulation as uv inactivated viruses could not drive g 1 to s phase transition (fig. 1a) as well as level and activity manipulation of cell cycle machinery proteins (data not shown). phosphorylation status of rb controls the transition from g 1 to s phase which was found to be hyper phosphorylated during initial stages of rv infection ( fig. 2a) . this results in its inability to bind to and prevent nuclear translocation of e2f ( fig. 2a) , since e2f in nucleus can activate transcription of several downstream genes like thymidine kinase, thymidine synthase and dihydrofolate reductase (fig. 2c) , which promote cell cycle progression and dna replication, nuclear accumulation of e2f by rv during early infection facilitates g 1 /s restriction point modulation in favor of rv replication. up-regulation of in vivo thymidine kinase activity in mice infected with rv has also been previously reported (collins et al., 1988) . sequential regulation of rb phosphorylation and e2f activation during interphase is controlled by specific cyclin-cdk complexes such as cdk4, cdk6, cdk2 and cyclins (cyclin d1, d3 and e1) (bloom and cross, 2007) . d type cyclins (cyclin d1, cyclin d3) preferably binds and activates cdk4 and cdk6 to hyper phosphorylate rb and free the e2f protein to induce the expression of cyclin e1 and cdk2 which after forming the holoenzyme retains the hyper phosphorylated state of the rb molecule (malumbres and barbacid, 2009 ). concurrent to rb phosphorylation pattern, all the cdks involved in g 1 to s phase transition were activated during early hours of infection (fig. 3c) . formation of active cyclin-cdk complex depends on expression level of cyclins and cdks (malumbres and barbacid, 2009) , manipulation of which might be one of the mechanisms employed by viruses to activate cdk complex. as speculated up-regulation of all the cyclins and cdks was observed at both transcript or protein levels during early infections ( fig. 3a and b ). other than the expression level of cyclins and cdks which is essential for formation of the holo enzyme, ckis play an important regulatory role to control cdk activation. there are two families of ckis, cip/kip and ink4 family. the cip/kip family of proteins includes p21, p27 and p57, which are specific inhibitors of kinase activity of all type of cdks; by contrast compounds of the ink4 family (p15, p16, p18, p19) prevent the association as well as function of only cdk4, cdk6 and d type cyclins (besson et al., 2008) . during rv infection, in addition to up-regulation of cyclins and cdks, members of ckis were significantly down-regulated at both transcript and protein levels, which may have additive effect on cdk activation ( fig. 3d and e) . the level of change in transcript and protein levels of cyclins, cdks or ckis did not correlate exactly at all time points (fig. 3a, b , d, and e) which could be due to inhibition of translation of cellular proteins by different mechanisms (like inhibition of nucleocytoplasmic rna transport) (rubio et al., 2013; padilla-noriega et al., 2002) . during time course study, progression of cells from g 1 to s phase during early rv infection but no further progression to m phase was observed. blockage in s to m phase transition is employed by different viruses (luo et al., 2013a,b) to recruit fig. 5 . ca þ 2 /cam pathway inhibition during rv infection prevents modulation of cell cycle regulatory proteins except ckis. (a) ma104 cells were kept mock infected or infected with sa11 strain in presence or absence of w7 and bapta-am for 2-8 h or kept mock treated and whole cell lysates or nuclear fraction isolated by differential centrifugation were subjected to western blot analysis using prb (lower panel) or e2f specific antibody (upper panel) respectively and compared with mock infected controls of corresponding time points. gapdh and tbp were used as loading control for whole cell lysate or nuclear lysate respectively. results are representative of three independent experiments. (b) ma104 cells were kept mock infected or infected with sa11 strain in presence or absence of w7 and bapta-am for 2-8 h or kept mock treated followed by western blot analysis using cyclin d1, cyclin e1, cdk6, cdk2, p21, p27 specific antibodies. gapdh was used as loading control. results are representative of three independent experiments. cellular replication factors for viral replication. in case of rotavirus this could be due to stabilization of microtubules (eichwald et al., 2012) . in rv infection after accumulation of cells in s phase till 6 hpi, no of cells in g 0 /g 1 phase started to increase (6 hpi and onwards) keeping consistency with the level of cyclins, cdks, which started to decrease and ckis, which were upregulated during 8 hpi. after that (10-12 hpi) activation of apoptotic pathways during late infection as observed in early studies by upregulation of p53 (bhowmick et al., 2013) , a modulator of cell cycle (agarwal et al., 1995) was observed in cell cycle analysis of sa11 infected ma104 cells with increase in sub g 0 (apoptotic) population of cells (fig. 1a) . modulation of ca þ 2 signals at various stages of the cell cycle and its role in cell cycle progression has been reported (berridge et al., 2000) and ca þ 2 concentrations was found to be increased during rv infection (brunet et al., 2000) . versatile and complex ca þ 2 signaling activates different ca þ 2 binding proteins which then further modulate distinct cellular responses (machaca, 2010) . cam is one of the important downstream effectors of ca þ 2 which gets activated after binding ca þ 2 (chin and means, 2000) and its expression levels have been linked to cell cycle progression both experimentally and physiologically (choi and husain, 2006) . cam expression is significantly increased during the g1-s transition (chafouleas et al., 1982; chafouleas et al., 1984) . increased expression of cam during early hours (2-6 hpi) of rv infection has been observed in differential proteomics study by our group (weinberg, 1995) . cam has been shown to activate several family of proteins namely serine/threonine phosphatase, calcineurin, multifunctional ca 2 þ /cam-dependent protein kinases, adenyl cyclises, ion channels, phosphodiesterases, myosin light chain kinases and protein phosphatises (chin and means, 2000) of which ca 2 þ /cam-dependent protein kinases have been shown to modulate cell cycle (skelding et al., 2011) . camki has been shown to induce g 1 to s phase transition (skelding et al., 2011) , concurrently activation of camki (phospho camki) was also observed during early rv infection (2-6 hpi) which correlated with increased cam expression and accumulation of cells in s phase (fig. 4a) . significance of ca þ 2 / cam signaling in modulation of cell cycle during rv infection was fig. 6 . synchronization of cells at s phase assist rv replication while synchronization of cells at g0/g1 phase hampers it. (a) ma104 cells either synchronized in s (treated with azt) phase or in g 0 /g 1 phase (treated with terfenadine) or left unsynchronised were infected with sa11 for 2-8 hpi and total rna was isolated and subjected to quantitative rt-pcr for viral gene expression and compared with sa11 infected unsynchronized cells. fold changes of transcripts were obtained by normalizing relative gene expression (with respect to sa11 infected untreated cells) to gapdh using the formula 2 à δδct (δδct ¼δc t sample à δc t untreated control ). results are representative (mean7 sd) of three independent experiments. (b) ma104 cells either synchronized in s (treated with azt) phase or in g0/g1 phase (treated with terfenadine) or left unsynchronized were infected with sa11 for 28 hpi. total lysates were subjected to westernblot analysis for viral gene expression (nsp3). (c) ma104 cells either synchronized either in s (treated with azt) phase or in g0/g1 phase (treated with terfenadine) or left unsynchronized were infected for 12 hpi, 18 hpi, 24 hpi followed by plaque assay. viral titers were measured as plaque forming units [log (pfu/ml)]. results are representative (mean 7 sd) of three independent experiments. confirmed when ca þ 2 chelator (bapta-am) and cam inhibitor (w7) inhibited g1 to s phase transition (fig. 4b) . rb phosphorylation and nuclear translocation of e2f was also inhibited in presence of bapta-am or w7 (fig. 5a ). similar to rb phosphorylation, expression levels of cyclins, cdks were not modulated in presence of bapta-am or w7 during rv infection (fig. 5b) , however the ckis namely p21 and p27 were down regulated both in presence (fig. 5b) or absence (fig. 3d ) of bapta-am or w7. down regulation of p21 and p27 is probably regulated by p53 which has been shown to be downregulated during early hours of rv infection (bhowmick et al., 2013) . overall results suggest role of ca þ 2 /cam signaling in rv induced g1 to s phase transition. ca þ 2 /cam signaling is also important for rv replication as in presence of bapta-am or w7 reduced viral titers were observed pérez et al., 1998) . to further study relevance of g 1 to s phase transitions, during rv infection, specific inhibitors which can synchronize cells in s phase (azt) or g 0 /g 1 phase (terfenadine) were used. azt inhibits dna replication by inhibiting thymidine kinase and preventing deoxythymidine triphosphate formation as a result it synchronizes cells at s phase (chandrasekaran et al., 1995) whereas terfenadine prevents rb phosphorylation and induce the level of ckis to prevent cell cycle progression and synchronizes cells at g 0 /g 1 phase (liu et al., 2003) . in rv infected s phase synchronized cells, increased viral gene expression and viral titers were observed whereas cells blocked in g 0 /g 1 phase had reduced viral gene expression and titers compared to unsynchronized cells infected with sa11 confirming that rv induces g 1 to s phase transition for enhanced viral replication. overall the study focused on mechanism by which rv modulates the host cell cycle machinery. the results suggests a novel mechanism i.e. ca þ 2 /cam activation, employed by rves to drive cells to accumulate in s phase by manipulating expression and activity of cell cycle regulatory proteins involved in g1 to s transition similar to hepatitis b virus (yang and cho, 2013; benn and schneider, 1995) . increase of cells in s phase during initial stages of infection was found to be beneficial for viral life cycle, which may be due to either stabilization of mt network during s phase which is helpful for viroplasm translocation (eichwald et al., 2012) or increase in host replication proteins such as topo-isomerase during s phase which may assist in rotaviral replication as reported in case of ebola virus infection (takahashi et al., 2013) . it is also possible that s phase accumulation provides an anti-apoptotic environment for proper completion of viral replication cycle. the rhesus monkey epithelial cell line ma104 cells were cultured in minimal essential medium (mem) supplemented with 10% heat-inactivated fetal bovine serum (fbs) and 1x psf (penicillin, streptomycin and fungizone) at 37 1c humifiied incubator with 5% co 2 . sa11, a513 and and osu strains of rv were used in this study (gifted by prof. n. kobayashi, japan) . for infection, viruses were activated with 0.1% acetylated trypsin (10 g/ml) (gibco, life technologies, carlsbad, ca) at 37 1c for 45 min (min) and added to the cells at 3 multiplicity of infection (moi, infectious virus particles per cell) for 60 min at 37 1c. unbound virus was removed by media wash and infection was continued in fresh mem supplemented with 0.01% acetylated trypsin and antibiotic. the time of virus removal was taken as 0 h post infection for all experiments. extracted and purified viral preparations were titrated by plaque assay (smith et al., 1979) . antibodies against e2f, gapdh, cam, phospho cam kinase i, cdk2, cyclin e1, tbp were from santa cruz biotechnology (santa cruz, ca). antibody against phospho-rb (ser807/811), cdk4, cdk6, cyclin d1, cyclin d3, p27, p21, p15 were from cell signaling technologies (danvers, ma, usa). phospho histone h1 antibody was from millipore (billerica, ma, usa). all antibodies were used at dilutions recommended by the manufacture. mouse polyclonal antibody against nsp3 was raised against full length recombinant protein according to standard protocols at the department of virology and parasitology, fujita health university school of medicine, aichi, japan and used in 1:3000 dilutions. recombinant retinoblastoma and histone h1 proteins were purchased from sigma aldrich (st. louis, mo, usa). bapta-am, azt, terfenadine were purchased from sigma aldrich and w7 was purchased from santa cruz biotechnology. to determine cytotoxicity of azt, terfenadine, bapta-am, w-7 in ma104 cells, cell viability assays were performed in 96-well plates (e 5 â 104 cells/well). cells were treated with the drugs as mentioned in supplementary fig. 1 for 24 h followed by an mtt assay (sigma-aldrich). briefly, 10 ml of mtt solution (5 mg/ml in pbs) was added and incubated at 378 1c for 4 h. the formazan was dissolved in 200 ml dmso and the optical density (od) of the solution was measured at 570 nm and 630 nm to obtain the sample signal (od570-od630). the cytotoxicity was measured as cell viability compared with dmso treated cells. whole cell lysates [extracted by incubating in ice for 15 min with totex buffer (20 mm hepes at ph 7.9, 0.35 m nacl, 20% glycerol, 1% np-40, 1 mm mgcl 2 , 0.5 mm edta, 0.1 mm egta, 50 mm naf and 0.3 mm na 3 vo 4 ) containing mixture of protease and phosphatase inhibitors (sigma, st. louis, mo), cytoplasmic or nuclear extracts or immunoprecipitated products were prepared. samples were incubated in protein sample buffer (final concentration: 50 mm tris, ph 6.8, 1% sds, 10% glycerol, 1% β-mercaptoethanol, and 0.01% bromphenol blue) for 30 min at either 4 1c or, alternatively, boiled for 5 min before sds-page at room temperature followed by immunoblotting with specific antibodies as described previously (chawla-sarkar et al., 2004) . primary antibodies were identified with hrp conjugated secondary antibody (pierce, rockford, il) and chemiluminescent substrate (millipore, billerica, ma). where necessary, to confirm protein loading blots were reprobed with gapdh or tbp. the immunoblots shown are representative of three independent experiments. total rna was isolated using trizol (invitrogen, grand island, usa) according to the manufacturer's instructions. cdna was prepared from 1 to 2 μg of rna using the superscript ii reverse transcriptase (invitrogen) with random hexamer primers. realtime pcr reactions (50 1c for 2 min, 95 1c for 10 min, followed by 40 cycles of 95 1c for 15 s and 60 1c for 30 s and 72 1c for 10 min) were performed in triplicate using sybr green (applied biosystems, foster city, ca, usa) in step one plus (applied biosystems, life technologies, carlsbad, ca) with primers specific for thymidine kinase, thymidine synthase and dihydrofolate reductase, cdk4, cdk6, cdk2, cyclin d1, cyclin d3, p27, p21, p15, cyclin e1 [primer sequences are available on request]. the relative gene expressions were normalized to gapdh using the formula 2 à δδct (δδct¼ δctsampleàδct untreated control), where ct is the threshold cycle. for preparation of cytosolic and nuclear fraction cells were washed in ice-cold phosphate buffered saline (pbs), ph 7.2, then in hypotonic extraction buffer (heb: 50 mm pipes ph 7.4,50 mm kcl, 5 mm egta, 2 mm mgcl 2 , 1 mm dithiothreitol and 0.1 mm phenyl methyl sulphonyl fluoride (pmsf)) and centrifuged. the pellet was re suspended in heb and lysed in a dounce homogenizer. this cell lysate was centrifuged for 10 min at 1000g at 4 1c to pellet nuclei and the clarified supernatant (cytosolic fraction) was stored at à80 1c. nuclear fractions were prepared by re suspending the pellet in ice cold buffer c (10 mm hepes ph 7.9, 500 mm nacl, 0.1 mm edta, 0.1 mm egta, 0.1% np-40, 1 mm dtt, 1 mm pmsf, 8 mg/ml aprotinin, 2 mg/ml leupeptin (ph 7.4)), and kept for 30 min on ice with intermittent vortexing. resuspended fraction was then spun at 14,000g for 30 min at 4 1c and the supernatant (nuclear fraction) was stored at à80 1c. for cell cycle analysis nuclear dna content was measured using propidium iodide (pi) staining. briefly, adherent ma104 cells were collected by treatment with trypsin-edta and were then washed with ice cold pbs. the cells were fixed in 1 ml of cold 70% ethanol overnight at 4 1c and resuspended in staining buffer (50 mg/ml pi [sigma], 20 mg/ml rnase in pbs) for 20 min at 37 1c. pi-stained cells were then analyzed using facs (facsaria; bd), and at least 20,000 cells were counted for each sample. data analysis was performed by using modfit lt, version 2.0 (verity software house). in this study cells were either synchronized at s phase or g 0 /g 1 phase as described previously with some modifications (chandrasekaran et al., 1995; liu et al., 2003) . for s phase synchronization ma104 cells (50-60% confluent) were treated with 3'-azido-3'-deoxythymidine (azt) (200 mm) in complete media (mem, 10%fbs) for 24 h. for g 0 /g 1 phase synchronization ma104 cells (80-90% confluent) were serum starved (mem, 0.05% fbs) for 24 h followed by incubation with 5 μm terfenadine in complete media (mem, 10%fbs) for 8 h. after that the cells were washed with pbs and subjected to infection in absence of drug as described previously. to test kinase activity of cdk4, cdk6, cdk2 in mock and sa11 infected ma104 cells at increasing time points, cells were lysed in 50 mm tris (ph 8.0), 0.5% np-40, 2 mm edta, 137 mm nacl, 10% glycerol, 2 mm sodium orthovanadate, 100 m m leupeptin, and 1 mm pmsf by incubating in ice for 30 min. cell debris was removed by centrifugation and the supernatants were precleared with protein a-coupled agarose beads (invitrogen) for 2 h. then lysates (500 mg of protein each) were incubated at 4 1c with 1 mg of either anti-cdk2, or cdk4 or cdk6 specific antibody (santa cruz biotechnology) in 0.5 ml of immunoprecipitation buffer (50 mm tris, ph 8.0; 0.5% np-40, 2 mm edta, 137 mm nacl, 10% glycerol), collected on 25 ml of protein a agarose beads, and washed twice with immunoprecipitation buffer and two times with kinase buffer (50 mm tris, ph 7.4; 10 mm mgcl 2 , 1 mm dithiothreitol). kinase reactions were done in 25 ml of kinase buffer with 10 mg of substrate (histone h1 protein for cdk2 and retinoblastoma for cdk4, cdk6), 100 mm atp. reaction mixtures were incubated for 30 min at 37 1c and analyzed by immunoblotting with prb and phistone h1 specific antibodies after separation by sds-page. data are expressed as mean 7the standard deviations of at least three independent experiments (n z3). in all tests, p ¼0.05 was considered statistically significant. p53 controls both the g2/ m and the g1 cell cycle checkpoints and mediates reversible growth arrest in human fibroblasts links between apoptosis, proliferation and the cell cycle early phosphatidylinositol 3-kinase/akt pathway activation limits poliovirus-induced jnk-mediated cell death rotavirus nonstructural protein 1 suppresses virus-induced cellular apoptosis to facilitate viral growth by activating the cell survival pathways during early stages of infection pathways governing g1/s transition and their response to dna damage hepatitis b virus hbx protein deregulates cell cycle checkpoint controls the versatility and universality of calcium signaling cdk inhibitors: cell cycle regulators and beyond rv-encoded nonstructural protein 1 modulates cellular apoptotic machinery by targeting tumor suppressor protein p53 rotaviral enterotoxin nonstructural protein 4 targets mitochondria for activation of apoptosis during infection nonstructural 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dynamic processes requiring stabilized microtubules the cell cycle and virus infection fields virology functional roles of e2f in cell cycle regulation structure and function of simian virus 40 large tumor antigen herpesvirus lytic replication and the cell cycle: arresting new developments regulation of interferon regulatory factor-3 by the hepatitis c virus serine protease rb and cell cycle progression hiv-1 vpr increases viral expression by manipulation of the cell cycle: a mechanism for selection of vpr in vivo the rb/e2f pathway: expanding roles and emerging paradigms human immunodeficiency virus type 1 viral protein r (vpr) arrests cells in the g2 phase of the cell cycle by inhibiting p34cdc2 activity influenza a virus replication induces cell cycle arrest in g0/g1 phase rotavirus antagonizes cellular antiviral responses by inhibiting the nuclear accumulation of stat1, stat2, and nf-kappab retinoblastoma growth suppressor and a 300-kda protein appear to regulate cellular dna synthesis regulation of cell cycle progression by calcium/ calmodulin dependent pathways uv inactivation and resistance of rotavirus evaluated by integrated cell culture and real-time rt-pcr assay molecular mechanisms of g0/g1 cell-cycle arrest and apoptosis induced by terfenadine in human cancer cells human parvovirus b19 infection causes cell cycle arrest of human erythroid progenitors at late s phase that favors viral dna replication smc1-mediated intra-s-phase arrest facilitates bocavirus dna replication ca (2 þ ) signaling, genes and the cell cycle cell cycle, cdks and cancer: a changing paradigm rotavirus protein nsp3 shuts off host cell protein synthesis oncosis in ma104 cells is induced by rotavirus infection through an increase in intracellular ca 2 þ concentration inhibition of cyclooxygenase activity reduces rotavirus infection at a postbinding step viruses and apoptosis rotavirus prevents the expression of host responses by blocking the nucleocytoplasmic transport of polyadenylated mrnas the cell cycle: a review the cell cycle, cyclin-dependent kinases, and viral infections: new horizons and unexpected connections controlling the cell cycle: the role of calcium/calmodulin-stimulated protein kinases i and ii a plaque assay for the simian rotavirus saii suppression of the induction of alpha, beta, and lambda interferons by the ns1 and ns2 proteins of human respiratory syncytial virus in human epithelial cells and macrophages dna topoisomerase 1 facilitates the transcription and replication of the ebola virus genome activation of interferon regulatory factor 3 is inhibited by the influenza a virus ns1 protein the cell cycle: a review of regulation, deregulation and therapeutic targets in cancer the retinoblastoma protein and cell cycle control association of human papillomavirus types 16 and 18 e6 proteins with p53 hepatitis b virus x gene differentially modulates cell cycle progression and apoptotic protein expression in hepatocyte versus hepatoma cell lines supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2014.03.001. key: cord-293375-qcy56ui7 authors: strauss, ellen g.; de groot, raoul j.; levinson, randy; strauss, james h. title: identification of the active site residues in the nsp2 proteinase of sindbis virus date: 1992-12-31 journal: virology doi: 10.1016/0042-6822(92)90268-t sha: doc_id: 293375 cord_uid: qcy56ui7 abstract the nonstructural polyproteins of sindbis virus are processed by a virus-encoded proteinase which is located in the c-terminal domain of nsp2. here we have performed a mutagenic analysis to identify the active site residues of this proteinase. substitution of other amino acids for either cys-481 or his-558 completely abolished proteolytic processing of sindbis virus polyproteins in vitro. substitutions within this domain for a second cysteine conserved among alphaviruses, for four other conserved histidines, or for a conserved serine did not affect the activity of the enzyme. these results suggest that nsp2 is a papain-like proteinase whose catalytic dyad is composed of cys-481 and his-558. since an asparagine residue has been implicated in the active site of papain, we changed the four conserved asparagine residues in the c-terminal half of nsp2 and found that all could be substituted without total loss of activity. among papain-like proteinases, the residue following the catalytic histidine is alanine or glycine in the plant and animal enzymes, and the presence of trp-559 in alphaviruses is unusual. a mutant enzyme containing ala-559 was completely inactive, implying that trp-559 is essential for a functional proteinase. all of these mutations were introduced into a full-length clone of sindbis virus from which infectious rna could be transcribed in vitro, and the effects of these changes on viability were tested. in all cases it was found that mutations which abolished proteolytic activity were lethal, whether or not these mutations were in the catalytic residues, indicating that proteolysis of the nonstructural polyprotein is essential for sindbis replication. proteins encoded by plus-stranded rna viruses are commonly produced as polyproteins which are posttranslationally processed by one or more virally encoded proteolytic enzymes (reviewed in ). viral proteinases have been described which resemble cellular proteinases belonging to three distinct families: the aspartate proteases that resemble pepsin, the serine/cysteine proteinases that resemble ttypsin or chymotrypsin, and the thiol proteases that resemble papain. the best characterized viral proteases are the aspartate proteases encoded by retroviruses. the residues making up the catalytic triad (asp, thr, gly) of the hiv proteinase have been confirmed by site-specific mutagenesis (loeb et a/., 1989) , and the structure of the proteinase has been determined to high resolution by x-ray crystallography (navia et al,, 1989) . the three-dimensional folding of the viral proteinase resembles pepsin, suggesting a common evolutionary origin. proteinases similar to ttypsin or chymotrypsin have been identified in picornaviruses; alphaviruses; plant ' to whom reprint requests should be addressed. * present address: academic hospital leiden. p.o. box 320, nl-2300 ah leiden, the netherlands. 3 present address: department of biochemistry and biophysics, university of california, san francisco, ca 94143.0448. coma-, nepo-, and potyviruses; coronaviruses; and flaviviruses (and their proposed relatives pestiviruses and hepatitis c virus.) originally these domains were predicted to have proteolytic activity based on the presence of certain conserved amino acid residues and on the basis of protein-modeling studies (bazan and fletterick, 1989; boege et a/., 1981; gorbalenya et al., 1989; hahn eta/., 1985) . the catalytic triad of trypsin is composed of a serine, a histidine, and an aspartic acid; in the flavivirus ns3 proteinase (chambers et a/., 1990) , the pestivirus p80 proteinase (wiskerchen eta/., 1991) , and the alphavirus capsid proteinase , such a triad has been confirmed by mutagenesis. furthermore, the crystal structure of the sindbis alphavirus capsid protein has been determined to 3 a, and the c-terminal 151 amino acids are folded into a structure remarkably similar to that of chymotrypsin, with his-141, asp-163, and ser-215 forming the catalytic site (choi et al., 1991 b) . the similarity in the folding of this capsid protein to that of chymotrypsin suggests that the virus acquired a proteinase from a host at some time in the past, and that all of the viral chymotrypsin-like proteinases will in fact be similar in structure. the poliovirus 3c proteinase and the comparable 3c-like proteinases found in plant viruses and coronaviruses are also believed to be related to chymotrypsin, but in these cases the catalytic serine has been replaced by cysteine (reviewed in bazan and fletterick, 1990; goldbach, 1990; harris eta/., 1990) . mutagenesis experiments and x-ray structure experiments should shortly confirm or disprove this prediction. recently, we proposed that the nonstructural proteinase of sindbis alphavirus was related to papain, thus representing a third family of viral proteinases (hardy and strauss, 1989; strauss and strauss, 1990) , and gorbalenya et al. (199 1) proposed that papain-like proteinases were present in several rna viruses. in papain, a cysteine and a histidine form a catalytic dyad, and there is conflicting evidence as to whether an asparagine residue is also an essential component of the active site (higaki et al., 1987; kamphuis et al., 1985) . other proposed papain-like proteinases in rna viruses include two domains within orfl a of coronaviruses identified by protein modeling studies (baker et al., 1991; lee et al., 1991) and the hc-pro protein of potyviruses, in which the catalytic cys and his residues have been identified by mutagenesis (oh and carrington, 1989) . interestingly, in a number of cases viruses encode proteinases of more than one type. coronaviruses, for example, encode one or two proteins with sequences characteristic of a papain-like proteinase as well as one with similarity to a 3c-like proteinase within orfl a (boursnell eta/., 1987; lee et al., 1991) . similarly, in potyviruses there is both a 3clike proteinase (the 49-kda protein) as well as a papainlike proteinase (the hc or helper component protein) (oh and carrington, 1989 ). finally, as described above, the alphaviruses possess a chymotrypsin-like structural proteinase and a papain-like nonstructural proteinase. in this paper we report that cys-481 and his-558 have been identified as the catalytic dyad of the sindbis virus nsp2 nonstructural proteinase. furthermore, we present data to show that none of the asparagine residues in the proteinase domain of sindbis nsp2 that are conserved among alphaviruses are absolutely required for proteolytic activity, but that trp-559, adjacent to his-558, is essential for function. for mutagenesis, the bglll (nt 2288) to pstl (nt 3949) fragment from toto50, a cdna clone containing the entire sequence of sindbis virus from which infectious rna can be transcribed with sp6 polymerase (rice et a/., 1987) was inserted into the replicative form of m 13mpl8, which had been digested with barnhi and wi, to form m 13mpl8t2.155. uracil-containing single-stranded dna was prepared from phage by the method of kunkel (1985) and used as the template for oligonucleotide-directed mutagenesis (zoller and smith, 1984) after removal of low-molecular-weight material using nacs prepac cartridges (bethesda research laboratories). both degenerate oligonucleotides capable of producing multiple mutations and oligonucleotides containing a single altered codon were used. single-stranded dnas from mutated bacteriophages were sequenced using appropriate sequencing primers and sequenase (united states biochemical) according to the manufacturer's instructions. mutated fragments 1236 nt in length were excised from m 13mpl8t2.155 replicative form with c/al and pstl and inserted into the full-length clone by a three-piece ligation with the f'stl(3948) + spel(5262) and spel(5262) + cial(2712) fragments of toto50. fulllength clones were checked for the presence of the mutations by direct sequencing of the double stranded plasmid dna as previously described (shirako et al., 1991) . miniprep dna (sambrook et al., 1989) was treated with rnase a, linearized with xhol, and treated with proteinase k followed by repeated phenol extraction and ethanol precipitation. linearized dna was resuspended in rnase-free tne and frozen at -70" until use. rna transcripts were synthesized with sp6 polymerase as previously described (rice et al., 1987) . approximately 10 to 50 ng of rna were translated in 9 pi of nuclease-treated, methionine-depleted reticulocyte lysate (promega) supplemented with 20 pii/ of an amino acid mixture minus methionine and 1 o-l 5 pc [35s]methionine, for 90 min at 30". translation mixes were diluted 1: 1 with 2x laemmli sample buffer containing 10% p-mercaptoethanol, and analyzed on 1 oq/o sds-containing polyactylamide gels (laemmli, 1970) . gels were fluorographed using en3hance (nen research products) according to the manufacturer's instructions. infectivity assays rna transcribed in vitro was transfected onto confluent monolayers of bhk cells in six-well cluster dishes (costar) using the deae-dextran method described in rice et al. (1987) . for plaque assays the monolayers were overlayered with eagle's medium containing 10% fetal bovine serum and 19/o agarose. plates were incubated for 2 days at 30" and stained for at least 8 hr at 30" with neutral red. during infection by sindbis virus, the viral genome of 11.7 kb serves as a messenger for the translation of rna-mutants previously mapped (hahn er al., 1989) . solid-stalked symbols indicate reduced or absent proteolytic activity; open symbols have wild type enzymatic activity as determined in w'fro. the shape of the symbol indicates the parental wild type amino acid; circles for cys, squares for his, and triangles for asn. checkered diamonds indicate that the mutants are temperature-sensitive for proteolysis in viva . these substitutions are phe-509 + leu, ala-51 7 + thr, asp-522 + asn. and gly-736 -, ser. the nonstructural proteins that form the viral replicase (strauss and strauss, 1986) . two polyproteins are produced upon translation. the first terminates at an inframe opal codon at nucleotides 5748-5750 and this polyprotein of 1896 amino acids is subsequently cleaved into nonstructural proteins nsp1, nsp2, and nsp3, numbered in order from 5' to 3' along the viral genome. the second polyprotein of 25 13 amino acids is produced by readthrough of the opal codon and contains in addition the sequences of nsp4; cleavage of this longer polyprotein produces nsp1, nsp2, nsp3, and nsp4, as well as nsp34, which is also thought to be functionally important . the genome organization and cleavage sites in the polyproteins are schematically illustrated in fig. 1 . when sindbis rna, whether derived from the virion or from sp6 transcripts, is translated in a cell-free system, cleavage occurs at all three sites in the polyproteins to produce mature nsp1, nsp2, nsp3, and nsp4 and various intermediates (de groot eta/., 1990 (de groot eta/., , 1991 hardy and strauss, 1989) . in addition some unprocessed p123 and p1234 usually remain. the polypeptides produced in such a translation are shown in fig. 2a (labeled "wild type"). the proteinase that cleaves all three sites in the polyproteins has been mapped by deletion studies to the c-terminal half of nsp2; only deletions that invaded the domain between amino acids 475 and 728 abolished all activity of the enzyme, and both nsp2 and polyproteins containing nsp2 were found to be proteolytically active (de groot eta/., 1990; hardy and strauss, 1989) . hardy and strauss (1989) proposed that the enzyme is a papain-like proteinase in which cys-481 and one of the five conserved histidines in this region form the active site of the enzyme. to confirm that cys-481 is required for activity of the nsp2 proteinase, we changed this residue to serine, glycine, or arginine. we also examined the effect of changing cys-525, one of the two remaining conserved cysteine residues in the c-terminal half of nsp2, to serine or arginine, as well as changing ser-535, which is found in a domain of limited similarity to the active site serine of serine proteinases, to threonine. the mutations were introduced into a full-length cdna construct, and rna was transcribed with sp6 rna polymerase and translated in rabbit reticulocyte lysates. representative results with the substitutions for cys-481 and cys-525 are shown in fig. 2 , results for ser-535 are shown in fig. 3 , and a summary of these results is given in table 1 . in the case of every mutant tested, multiple independent clones were examined to guard against the possibility that extraneous changes other than the mutation of interest might have affected the results. change of cys-481 to serine, glycine, or arginine resulted in abolishment of proteolytic activity. however, at least one substitution for cys-525 and for ser-535 resulted in an active enzyme. taken together, these results are consistent with the hypothesis that cys-481 is in fact a component of the active site of the nsp2 proteinase. his-558 is also required for cleavage all five conserved histidines in the proteinase region, his-558, his-61 9, his-701, his-702, and his-709, were site specifically changed to alanine. in addition, his-558 was also changed to glutamine and tyrosine, and his-709 was also changed to tyrosine and arginine. results for a representative sample of these mutants are shown in fig. 3 , and the results for all of the his mutants are summarized in table 1 . all three changes in his-558 totally abolished proteolytic activity. in contrast, change of any of the other four histidine residues to alanine, or change of his-709 to tyrosine, resulted in an active protease. thus his-558 is uniquely required for proteolytic activity, and we postulate that this residue forms part of the active site of the nsp2 proteinase. as a word of caution on this method of determining catalytic site residues of a proteinase, note that two other substitutions, presumably not in the catalytic ' "lethal" rndrcates that no plaques were found after transfection. wild type constructs in this assay give "large" plaques. "not detn." means that the viability was not tested. site, also abolished the enzymatic activity: his-709 + arg and cys-525 + arg. it appears that the large, positively charged arginine residue perturbs the structure such that the protein is no longer proteolytically active. to rule out such structural alterations, in most cases we have tested more than one substitution for the wild type residue. it has been suggested that in addition a cysteine and a histidine an asparagine residue is also important for catalysis by papain (garavito et a/., 1977; higaki et a/., 1987) . to investigate the possible role of an asparagine residue in the catalytic activity of the nsp2 proteinase, we mutagenized all four asparagine residues within the catalytic domain that are conserved among alphaviruses, namely asn-561, asn-609, asn-614, and asn-693. in each case we changed the asparagine to both aspartic acid and to serine, and the results are summarized in table 2 . substitution of either serine or aspartic acid for asn-56 1 and asn-6 14 produced enzymes with near wild type activity. substitutions at asn-693 gave variable amounts of processing, which depended upon the particular preparation of reticulocyte lysate used. some experiments showed almost wild type processing (data not shown), while in the translation shown in fig. 4 asp-693 and ser-693 both show markedly reduced processing. similar variability in processing during in vitro translation in reticulocyte lysates has been noted for constructs containing the ns3 proteinase of flaviviruses (preugschat et a/., 1991) . the reason for such variability is not clear, but may be due to variation in the translation efficiency or in the fidelity of folding of the translated proteins. asn-609 + ser gave reduced and variable processing; in the translation shown in fig. 4 normal products are made but in very reduced amounts. for asn-609 + asp certain constructs seemed completely inactive, while others gave very incomplete processing, the extent of which was again dependent upon the reticulocyte preparation used. the severely reduced activity of the enzyme containing the asn-609 + asp mutation was not due to some additional lesion elsewhere in the protein. as a control a restriction fragment of 375 nucleotides containing this change from the mutant construct was excised, sequenced in its entirety, and reinserted into the toto background, and the resulting construct was also nearly inactive. presumably the presence of a charged residue at this position alters the overall conformation of the protein, leading to reduced activity. multiple independent isolates of this mutation all had similar phenotypes. we conclude that none of the conserved asparagines is absolutely required for proteolysis by the nsp2 proteinase, although substitutions for asn-609 markedly reduce the activity of the enzyme, apparently without affecting the specificity for the cleavage sites. these results suggest that an asparagine residue is not a component of the active site of the enzyme. in the case of most papain-family proteinases, the amino acid c-terminal to the catalytic histidine is small. in plant and animal proteinases, glycine or alanine is present at this position, whereas in other viral proteinases cysteine, serine, orvaline is found. however, all of the alphaviruses have tryptophan in this position. to determine whether trp-559 was essential for proteolysis, we examined the mutant trp-559 + ala. when this construct was transcribed and translated in vitro, it was totally inactive. we conclude that trp-559 is required for activity of the nsp2 protease. we have tested a number of these constructs for viability, since the mutations had been inserted into a full-length clone of sindbis virus from which infectious rna can be transcribed in vitro. bhk cells were transfected with this rna to look for the formation of plaques and the results of these experiments are shown in the last columns of tables 1 and 2. in all cases in which proteolysis was abolished, no plaques could be detected after transfection, indicating that proteolytic processing of the nonstructural polyprotein is essential for sindbis replication. these lethal mutations include all substitutions tested for the catalytic cys-481 and his-558, as well as for arg-709, arg-525, and ala-559. the effects of the asparagine substitutions are not as straightforward. asp-561 and ser-561 gave normal processing during in vitro translation but produced small plaques and mixed plaque sizes respectively after transfection (table 2) . on the other hand, the ser-609 virus, which showed very little processing, gave wild type large plaques and the asp-609 variant, which scarcely processed at all, gave minute plaques. still more interesting are the results with the substitutions at asn-614. ser-614 gave processing similar to wild type and made slightly smaller plaques than the wild type. asp-6 14 gave normal amounts of the fully processed proteins nsp1, nsp2, and nsp3 and seemed to process more effectively, since no uncleaved p123 was seen after in vitro translation (fig. 4) but surprisingly, asp-61 4 is lethal. ser-693, which produced variable processing results, is also lethal. we have shown that cys-481 and his-558 are required for proteolytic activity by the sindbis virus nsp2 proteinase. any change in either of these residues abolished all activity by the enzyme and we propose that these two residues form the catalytic dyad of a papain-like proteinase. we also found that trp-559 is required for proteolytic activity. all of the mutations which we made in this study, as well as temperaturesensitive mutations mapped in the proteinase domain of nsp2 (hahn et al., 1989) are shown diagramatically in fig. 1 . it is notable that in some cases substitution with an arginine residue killed the enzymatic activity, whereas substitution of other residues did not. a great deal has been learned about the thiol papain-like proteinases over the last few years. these proteinases include the prototype papain from papaya and other plant enzymes such as actinidin from kiwifruit and bromelain from pineapple as well as animal lysosomal enzymes such as the various cathepsins and calpain (reviewed in baker and drenth, 1987; brocklehurst, 1987) . the crystal structure of papain has been determined to 1.65a resolution (kamphuis et al., 1984) , but structural information is lacking for other enzymes. because the amino acid similarities around the active site residues or throughout the rest of the proteins are limited and even the spacing between the active cysteine and histidine is quite variable, it remains to be determined what the relationships are between the plant, animal, and viral enzymes. in particular, it is not clear whether the folding of the viral proteins resembles that of the plant or animal proteins; similar folding would be strongly indicative of the descent of animal, viral, and plant proteins from a common ancestral enzyme. in addition to the catalytic cys-25 and his-l 59 residues of papain, other amino acid residues have been suggested to be important for catalysis. one such residue is asn-175 which occupies precisely the same position in a possible catalytic triad of papain as asp-l 02 in the catalytic triad of trypsin (garavito et a/., 1977; higaki et a/., 1987) . furthermore, there is considerable sequence identity among papain-like enzymes around this amino acid. in order to examine whether an asparagine might be required for activity of the sindbis nsp2 proteinase, all four asparagine residues within the proteinase domain that are conserved among alphaviruses were subjected to mutagenesis. in every case at least one substitution was partially tolerated, and we conclude that no asparagine residue is absolutely essential for proteolysis. this is not to say that changes at various asparagine residues were without effect: changes at both asn-609 and asn-693 reduced the extent of proteolysis, and asp-614 appeared to enhance proteolysis. if the role of asn-175 in papain were to stabilize the active site histidine in a tautomerically favorable conformation through its hydrogen bonding to a nitrogen in the imadozole ring of his-l 59, it is conceivable that trp-559 adjacent to his-558 could fulfill a related role in alphaviruses. as described above, the presence of a tryptophan following the active site histidine is exceptional among all of the papain-like enzymes described to date. in this model trp-559 might interact with trp-482 adjacent to the catalytic cysteine to maintain his-558 and cys-481 in an active conformation. in any event, an important role for this tryptophan residue is fig. 5. aligned amino acid sequences of papain-like proteinases in the vicinity of the conserved cys and his catalytic residues, alignment of the sequences in according to gorbalenya et al. (1991) . sequences are from the following sources: papain (mitchel eta/., 1970) ; actinidin (carne and moore, 1978) ; bromelain. stem bromelain (ritonja et al., 1989) ; cpl and cp2, cysteine proteinases i and ii from dicryostelium discoideum (pears eta/., 1985) ; cathepsin h and cathepsin l, human cathepsins (wiederanders et a/., 1991) ; cathepsin b, human cathepsin b (ritonja eta/., 1985) ; calpain, chicken calpain (ohno eta/., 1984) ; ibv, avian infectious bronchitis virus (boursnell eta/., 1987) ; mhvl and mhv2, two copies of a papain-like proteinase in the long orf 1 of mouse hepatitis virus (lee er al., 1991) ; rub, rubella virus (dominguez et al., 1990) ; sin, sindbis virus (strauss et a/., 1984) ; sf, semliki forest virus (takkinen, 1986) ; onn, o'nyong-nyong virus (levinson et al., 1990) ; rr, ross river virus (faragher et a/., 1988) ; tev, tobacco etch virus, ppv, plum pox virus, tvmv, tobacco vein mosaic virus, and pvy. potato virus y (oh and carrington, 1989) ; baymv, barley yellow mosaic virus (kashiwazaki et al., 1991) ; hav, hypovirulence-associated virus of chestnut blight fungus (choi et a/., 1991a) . numbers preceeding the first amino acid are either the number of residues to the known n-terminus of the protein (parentheses), or the number of residues to the beginning of the orf encoding the proteinase (square brackets). residues which are the same as those of papain are indicated with a dot. within the alphaviruses and the potyviruses, residues which are conserved among four of five and three of four viruses, respectively, are boxed suggested by the fact that change of this residue to alanine resulted in loss of enzymatic activity. other residues that have been proposed to be important for catalysis in papain-like enzymes include gly-23, the two residues n-terminal to the catalytic cys-25, and gln-19, all of which are invariant in the plant and animal enzymes. it was suggested that these residues were involved in substrate binding and that a longer side chain at position 23 might block substrate binding by approaching too closely the side chain of gln-19 (baker and drenth, 1987) . in alphaviruses the residue corresponding to position 23 of papain is asparagine, whereas that at position 19 is ser in sindbis virus and gln in other alphaviruses (fig. 5) . no firm conclusions can be drawn in the absence of crystal structures, because the substrate specificity of alphavirus nsp2 is very different from that of papain or cathepsin. papain has a strong preference for bulky hydrophobic amino acids, phenylalanine, tyrosine, valine, or leucine in the p2 position (the penultimate amino acid relative to the site of cleavage), while the animal cathepsins require nonpolar side chains at this position. the p2 position of alphavirus cleavage sites is always glycine. when this glycine is changed to valine or to glutamic acid, the site becomes noncleavable . comparisons of this nature assume that the overall folding of the viral proteinases is similar to that of papain, but confirmation of this will require three-dimensional crystal structures of the viral counterparts. finally, there is the question of the viability of the various mutants. it is not surprising that mutations which kill the proteinase are lethal, such as the substitutions at cys-481, his-558 and trp-559, or that some substitutions which gave severely reduced processing were lethal or gave minute plaques. it was more surprising to discover that at position 614 the change from asn to asp-614 gave enhanced cleavage and apparently normal cleavage products but was lethal for viral replication, while the change to ser-6 14 gave normal processing and normal virus production. note that in fig. 4 that the most obvious difference in the translation/processing pattern of asp-61 4 was the lack of any residual p123. with the wild type, the amount of residual p123 varied with the batch of reticulocyte lysate, and there was a significant amount in the experiment shown in fig. 4 . the lethality of this particular substitution may indicate a separate and necessary role for p123 (or for some other processing intermediate) in alphavirus replication. similarly, it is of interest to compare the results for the two substitutions at asn-693. both gave variable and somewhat reduced proteolysis, but in one case (ser-693) the change was also lethal. this may mean that slight changes in the rate of proteolysis, which may not be detectable in the cell-free translation system used, can still interfere with efficient virus replication. it is also possible that some of these changes affect the overall conformation of nsp2 and polyproteins containing nsp2. such an altered conformation could affect the helicase activity or impair some as yet unknown function of nsp2 other than proteolysis. the fact that many changes are poorly tolerated illustrates the fact that the alphaviruses, as isolated in nature, appear to be optimized for replication. primary structure of the 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rule pathway sequence of the genome rna of rubella virus: evidence for genetic rearrangement during togavirus evolution genome sequences of a mouse-avirulent and a mouse-virulent strain of ross river virus convergence of active site geometries plant viral protelnases cysteine proteases of positive strand rna viruses and chymotrypsln-like serine proteases: a distinct protein superfamily with a common structural fold putative papain-related thiol proteases of positive-strand rna viruses the thiol proteases: structure and mechanism. ln "biological macromolecules and assemblies: the active sites of enzymes identification of a domaln required for autoproteolytic cleavage of murine coronavlrus gene a polyprotein sequence analysis of three sindbls virus mutants temperature-sensitive in the capsld autoprotease detection of a trypsin-like serine protease domain in flavlvlruses and pestiviruses site-directed mutagenesis of the proposed catalytic amino acids of the slndbis virus capsld protein 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complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and rna polymerase determination of the complete nucleotide sequence of the genomic rna of o'nyong-nyong virus and its use in the construction of phylogenetic trees mutational analysis of human immunodeficiency virus type i protease suggests functional homology with aspartic proteinases the complete amino acid sequence of papain three-dimensional structure of aspartyl protease from human immunodeficiency virus hiv-l identification of essential residues in potyvirus proteinase hc-pro by site-directed mutagenesis evolutionary origin of a calcium-dependent protease by fusion of genes for a thiol protease and a calcium-binding protein characterization of two highly diverged but developmentally co-regulated cysteine proteinase genes in dictyostelium discoideum flavivirus enzyme-substrate interactions studied with chimeric proteins: identification of an intragenic locus important for substrate recognition production of infectious rna transcripts from sindbis virus cdna clones: mapping of lethal mutations, rescue of a temperature sensitive marker, and in vitro mutagenesis to generate defined mutants amino acid sequence of human liver cathepsin b. fe&s lett stem bromelain: amino acid sequence and implications for weak binding of cystatin molecular cloning: a laboratory manual structure of the ockelbo virus genome and its relationship to other sindbis viruses cleavage between nsp1 and nsp2 initiates the processing pathway of sindbis virus nonstructural polyprotein p123 complete nucleotide sequence of the genomic rna of sindbis virus structure and replication of the alphavirus genome. ln "the togaviridae and flaviviridae viral proteinases alphavirus proteinases complete nucleotide sequence of the nonstructural protein genes of semliki forest virus primary structure of bovine cathepsin s pestivirus gene expression: the first protein product of the bovine viral diarrhea virus large open reading frame, p20, possesses proteolytic activity laboratory methods: oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded dna template this work was supported by grants al 10793 and al 20612 from the national institutes of health. r.j. de g. was supported by a fellowship from european molecular biology organization (altf 280. 1988 ). key: cord-286232-jo24ia4s authors: hasebe, rie; sasaki, michihito; sawa, hirofumi; wada, ryuichi; umemura, takashi; kimura, takashi title: infectious entry of equine herpesvirus-1 into host cells through different endocytic pathways date: 2009-10-25 journal: virology doi: 10.1016/j.virol.2009.07.032 sha: doc_id: 286232 cord_uid: jo24ia4s we investigated the mechanism by which equine herpesvirus-1 (ehv-1) enters primary cultured equine brain microvascular endothelial cells (ebmecs) and equine dermis (e. derm) cells. ehv-1 colocalized with caveolin in ebmecs and the infection was greatly reduced by the expression of a dominant negative form of equine caveolin-1 (ecavy14f), suggesting that ehv-1 enters ebmecs via caveolar endocytosis. ehv-1 entry into e. derm cells was significantly reduced by atp depletion and treatments with lysosomotropic agents. enveloped virions were detected from e. derm cells by infectious virus recovery assay after viral internalization, suggesting that ehv-1 enters e. derm cells via energyand ph-dependent endocytosis. these results suggest that ehv-1 utilizes multiple endocytic pathways in different cell types to establish productive infection. viruses deliver their genomes and accessory proteins into host cells in order to initiate their replication. certain enveloped viruses, including retroviruses (stein et al., 1987) , enter cells through direct fusion of the virion envelope with the plasma membrane, a process that is followed by the release of the viral capsid or genome into the cytoplasm. other enveloped viruses, such as influenza virus (matlin et al., 1981) and semliki forest virus (helenius et al., 1980) , as well as most nonenveloped viruses rely on the cellular endocytic machinery for their entry into host cells. productive infection with alphaherpesviruses had been thought to be established only by direct fusion of the viral envelope with the plasma membrane, as demonstrated by electron microscopic analysis and the effects of treatment with neutralizing antibodies (fuller and spear, 1987; fuller et al., 1989; fuller and lee, 1992) . agents that perturb endocytosis were found to have little or no effect on herpes simplex virus (hsv) infection in hep-2 and vero cells (wittels and spear, 1991) . furthermore, entry of hsv-1 via endocytic vesicles was shown to result in degradation of the virus particles (campadelli-fiume et al., 1988) . however, it has recently become clear that hsv successfully infects hela, receptor-expressing cho, and c10 murine melanoma cells as well as primary and transformed human epidermal keratinocytes via endocytosis (nicola et al., 2003 nicola and straus, 2004; milne et al., 2005) . the cellular and viral requirements for the endocytic entry of hsv into these cells have been characterized. in hela and receptor-expressing cho cells, infectious entry of hsv requires trafficking of the virus to an acidic intracellular compartment, phosphatidylinositol 3-kinase activity, glycoprotein d (gd) receptors, as well as viral gb, gd, and gh-gl (nicola et al., 2003; nicola and straus, 2004) . the pathway into c10 murine melanoma cells is gd receptor dependent but independent of vesicles with a low ph (milne et al., 2005) . hsv enters primary and transformed human epidermal keratinocytes, an important target cell population in vivo, by a ph-and tyrosine phosphorylation-dependent mechanism . equine herpesvirus-1 (ehv-1), an alphaherpesvirus of the family herpesviridae, is distributed worldwide and causes rhinopneumonitis, abortion, and encephalomyelitis in horses (storts and montgomery, 2001) . with the use of ultrastructural analysis, we have previously suggested that ehv-1 enters equine brain microvascular endothelial cells (ebmecs) via endocytosis (hasebe et al., 2006) . similar ultrastructural observations were described for ehv-1 endocytosis in mouse fibroblast l-m cells (abodeely et al., 1970) . frampton et al. (2007) demonstrated that ehv-1 strain l11δgiδge, which lacks gi and ge, enters cho-k1 cells by endocytosis, while entry pathway into equine dermis (e. derm) cells and rabbit kidney (rk13) cells is direct fusion of viral envelope with the plasma membrane. van de walle et al. (2008) reported that integrin on the surface of the host cells is involved in the endocytosis of ehv-1. it has remained unclear, however, whether the other strains of ehv-1 utilize endocytosis to enter the susceptible cells. here, we have investigated the entry mechanism of ehv-1 into ebmecs and e. derm cells. with the use of confocal immunofluorescence microscopy, we examined the localization both of ehv-1 and of the endocytic markers clathrin and caveolin during viral internalization. moreover, we evaluated the involvement of caveolar endocytosis in ehv-1 entry with the cells expressing a dominant negative form of caveolin-1. we also assessed the role of tyrosine kinase activity and of low ph in the endosomal compartment in ehv-1 entry with the use of pharmacological approaches. we also performed energy depletion experiments and infectious virus recovery assay for direct indications of endocytosis. our data identify caveolar endocytosis as an entry pathway for alphaherpesviruses. moreover, our results demonstrate the existence of multiple endocytic pathways for ehv-1 entry. ebmecs and e. derm cells were examined for their ability to support ehv-1 replication. kinetics of viral growth in e. derm cells was similar to those in ebmecs ( fig. 1) , which were typical of a fully productive infection (hasebe et al., 2006) . these results demonstrated that both ebmecs and e. derm cells were susceptible to ehv-1 infection and that there was no significant difference in viral replication between ebmecs and e. derm cells. we have previously suggested that the entry of ehv-1 in ebmecs occurs via endocytosis by using electron microscopy (hasebe et al., 2006) . here, we further examined the mode of ehv-1 entry into ebmecs and e. derm cells using electron microscopy. at 10 min post infection (p.i.), enveloped virions were detected in noncoated vesicles within the cytoplasm of ebmecs ( fig. 2a , hasebe et al., 2006) and e. derm cells (figs. 2b, c). these observations imply that ehv-1 enters e. derm cells as well as ebmecs via endocytosis. we were unable to quantify the numbers of enveloped viral particles in the endosomes, because we could not catch enough particles having the complete recognizable structure (i.e. core, capsid and envelope) for the quantification. clathrin-dependent endocytosis plays a major role in the entry of many viruses, having been classically described for semliki forest virus, vesicular stomatitis virus, and influenza virus (helenius et al., 1980; matlin et al., 1981 matlin et al., , 1982 . although the vesicles containing the virions appeared not to possess clathrin coats by electron microscopy ( fig. 2 ; hasebe et al., 2006) , these observations did not exclude the possible involvement of clathrin-dependent endocytosis in ehv-1 entry. we therefore examined the ehv-1-infected cells by two-color immunofluorescence staining for ehv-1 and clathrin-heavy chain at 10 or 30 min p.i. both in infected ebmecs (figs. 3a-f) and e. derm cells (figs. 3g-l), ehv-1 immunoreactivity did not colocalize with clathrin at any time points. lack of cross reactivity of anti-ehv-1 antibodies with anti-clathrin antibody was confirmed by the double staining of uninfected ebmecs and e. derm cells (data not shown). the specificity of the mouse monoclonal antibody to clathrin-heavy chain in ebmecs and e. derm cells was examined by western blotting, in which the antibody detected the specific band at 180 kda (data not shown). localization of ehv-1 and caveolae during viral internalization caveolar endocytosis has emerged as a route of entry for several viruses including simian virus 40 (sv40) (anderson et al., 1996) , mouse polyomavirus (richterová et al., 2001; gilbert et al., 2003; gilbert and benjamin, 2004) , echovirus (marjomäki et al., 2002) , human papillomavirus type 31 (bousarghin et al., 2003; smith et al., 2007) , human polyomavirus bk (bkv) (eash et al., 2004) , and species c human adenovirus (colin et al., 2005) . so we next examined the possible association of caveolae with ehv-1 during viral internalization. two-color immunostaining revealed that ehv-1 gb and caveolin, the major component of caveolae, were colocalized in ebmecs at 10 (figs. 4a-c) and 30 min p.i. (figs. 4d-f). three fields were chosen at random and more than 50 signals for ehv-1 immunoreactivity were counted. approximately 40% of the ehv-1 signal in ebmecs was colocalized with caveolin at 10 min p.i. the percentage of colocalization was reduced to 19% at 30 min p.i. such colocalization was not detected in infected e. derm cells at any time points (figs. 4g-l). lack of cross reactivity of anti-ehv-1 antibody with anti-caveolin antibodies was confirmed by the double staining of uninfected ebmecs and e. derm cells (data not shown). the specificity of the rabbit polyclonal antibodies to caveolin in ebmecs and e. derm cells was examined by western blotting, in which the antibodies detected the specific bands at 24 kda for alpha isoform and/or 21 kda for beta isoform (data not shown). previous studies have reported that the tyrosine phosphorylation of caveolin-1 at residue 14 mediates the release of caveolae from the plasma membrane and is an integral part of certain signaling pathways (parton et al., 1994; aoki et al., 1999; orlichenko et al., 2006) . to confirm the involvement of caveolae vesicles in ehv-1 entry into ebmecs, we constructed equine caveolin-1 mutant. this mutant encodes a protein in which tyrosine 14 is mutated to phenylalanine (ecavy14f). this mutant has been known to act as a dominant negative inhibitor of caveolin-1 (orlichenko et al., 2006) . the ecavy14f construct and wild type equine caveolin-1 (wt ecav) were expressed in ebmecs and e. derm cells using a lentivirus vector. cells expressing either wt ecav or ecavy14f were infected with ehv-1 strain ab4-gfp (ab4-gfp). the ab4-gfp harbors a gfp expression cassette between gene 62 and gene 63 (ibrahim et al., 2004) . therefore, the viral infected cells show gfp signal. fewer ehv-1infected ebmecs were observed in cells expressing ecavy14f than in cells expressing wt ecav (p b 0.01). however, there was no difference in the number of the ehv-1-infected e. derm cells expressing ecavy14f or wt ecav (figs. 5a, b). expression of wt ecav and ecavy14f was confirmed by western blotting with rabbit anticaveolin polyclonal antibodies (fig. 5c ). the effects of endocytosis inhibitors on ehv-1 infection were assessed by quantification of icp0 rna, the product of an early gene of ehv-1. the production of icp0 rna is indicative of successful entry of the viral genome into the nucleus and is maximal at 3 h p.i. in rk13 cells (kimura et al., 2004) . given that the onset of ehv-1 dna synthesis in rk13 and l-m cells was detected at 4 h p.i. (caughman et al., 1985; o'callaghan et al., 1968) , the abundance of icp0 rna at 3 h p.i. is thought to reflect the number of virions that have infected the cells. the icp0 rna could be detected in ehv-1-infected rk13 cells at a multiplicity of infection (m.o.i.) of 0.004 plaque forming unit (p.f.u.) per cell (data not shown). previous studies have suggested that cellular tyrosine kinase activity aids ehv-1 infection (frampton et al., 2007) . cellular tyrosine kinase activity is important for receptor-mediated endocytosis (greenberg et al., 1993; lamaze et al., 1993; mcpherson et al., 2001) . furthermore, tyrosine phosphorylation of caveolin-1 at residue 14 is important in signaling pathways mediating release of caveolae from the plasma membrane since caveolar fission is decreased by kinase inhibition (parton et al., 1994; aoki et al., 1999) . we therefore examined the effects of genistein, a tyrosine kinase inhibitor (akiyama et al., 1987) , on the endocytosis of ehv-1. the amount of icp0 rna in ebmecs at 3 h p.i. was greatly reduced by treatment with genistein at a concentration of 50 μg/ml (fig. 6a ). in contrast, genistein had no effect on the abundance of icp0 rna in e. derm cells. to eliminate the possibility that the results of e. derm cells were due to the inefficient uptake of genistein, we assessed the effect of genistein on the tyrosine phosphorylation of caveolin-1 in e. derm cells (fig. 6b ). tyrosine phosphorylation of caveolin-1 at residue 14 was diminished by the treatment of genistein at 100 μg/ml, suggesting that the concentration of genistein used in this study was effective to down-regulate the tyrosine phosphorylation of caveolin-1. neither the morphology of both cell types nor the level of expression of the cellular housekeeping gene for horse gapdh was affected by genistein in both cell types at 50 μg/ml and 100 μg/ml (data not shown). low ph in endosome is important for many viruses to enter the host cells either via clathrin-dependent endocytosis or clathrin-and caveolae-independent pathway yoshimura and ohnishi, 1984; blumenthal et al., 1987; nicola et al., 2003) . intracellular low ph is involved in bkv entry by caveolae-dependent endocytosis (eash et al., 2004) although sv40 infection by caveolar endocytosis is ph independent (ashok and atwood, 2003) . to determine whether an acidic compartment is required for ehv-1 infectivity, we examined the effects of lysosomotropic agents on ehv-1 icp0 rna production. cells were treated with bafilomycin a1 and ammonium chloride to neutralize the ph of acidic organelles (tsiang and superti, 1984; van weert et al., 1995; dröse and altendorf, 1997) and infected with ehv-1 in the continued presence of the reagent. the abundance of icp0 rna in e. derm cells was reduced in a concentration-dependent manner by treatment with bafilomycin a1 (fig. 7a ) and ammonium chloride (fig. 7b ). on the other hand, ehv-1 entry into ebmecs was not inhibited by bafilomycin a1 (fig. 7a) and ammonium chloride at 10 mm (fig. 7b ). the effect of ammonium chloride at 20 mm in ebmecs was not determined, because the expression of horse gapdh was significantly reduced (p b 0.01; data not shown) suggesting that 20 mm of ammonium chloride is toxic in ebmecs. a fluorescent ph indicator probe, lysosensor™ yellow/blue dnd-160, was used to confirm that the ph of the organelles was neutralized. this probe exhibits a ph-dependent increase in fluorescence intensity upon acidification when the cells are excited at 405 nm and the fluorescence is emitted at 490 nm. untreated ebmecs and e. derm cells exhibited punctuate staining. the staining of lysosensor was diminished in ebmecs by the treatment with 0.2 μm of bafilomycin a1 and with 10 mm of ammonium chloride (figs. 7c, d) . in e. derm cells, signals of lysosensor were diminished by the treatment with 1 μm of bafilomycin a1 and 20 mm of ammonium chloride (figs. 7c, d). the concentration of bafilomycin a1 and ammonium chloride used here seemed to be non-toxic, because neither the morphology of both cell types nor the level of expression of gapdh was affected (data not shown). effects of atp depletion on ehv-1 entry into e. derm cells em studies and the effects of lysosomotropic agents suggest the possibility that ehv-1 enters e. derm cells via endocytosis. however, these data are insufficient to demonstrate that endocytosis is involved in ehv-1 entry into e. derm cells. to assess this possibility, we examined the effect of atp depletion on ehv-1 infection to e. derm cells. atp depletion is known to inhibit endocytosis, but has no effect on herpesvirus entry by direct fusion of viral envelopes with plasma membranes (nicola et al., 2003) . e. derm cells were pretreated with glucose-free media containing 2-deoxy-d-glucose for 1 h, and then infected with ab4-gfp for 1 h in the continued presence of glucosefree media. after virus infection, the media were replaced with complete growth media. to confirm that atp depletion affects the viral entry step, the cells were infected with ehv-1 with media including glucose for 1 h, and then cultured with glucose-free media for 2 h. the number of infected cells was later evaluated by counting gfp-positive cells at 12 h p.i. atp depletion during viral entry greatly reduced ehv-1 infection (p b 0.001) whereas atp depletion post viral entry did not significantly reduce ehv-1 infection (fig. 8) . frampton et al. (2007) performed infectious virus recovery assays to demonstrate that ehv-1 l11δgiδge strain enters cho-k1 cells via endocytosis. since endocytosed virions possess envelopes in the early phase of entry, infectivity can be detected by titration on rk13 cells. on the other hand, when virus penetrates by direct fusion of envelope with plasma membrane, infectious virions cannot be detected due to the loss of the viral envelope. to confirm that ehv-1 enters e. derm cells via endocytosis, we performed infectious virus recovery assay. after incubation at 4°c for 5 min, the cells were infected with ehv-1 at an m.o.i. of 10 p.f.u. per cell for 2 h at 4°c. the temperature was then shifted to 37°c to allow the virus internalization. at 0, 7.5, 15, 30 and 45 min after the temperature shift, the viruses on the cell surface were inactivated by washing with acidic buffer. the internalized infectious viruses were titrated on rk13 cells. for a control to eliminate the possibility of detecting virions remaining on the cell surface, we used nih3t3 cells, which seem to be resistant to ehv-1 entry because we could not detect viral rna at 12 h p.i. by rt-pcr (data not shown). at 0 and 7.5 min, no infectious virus was detected from e. derm and nih3t3 cells. at 15 min, virus was recovered from e. derm cells. the virus titer from e. derm cells reached a peak at 30 min and slightly declined at 45 min. in contrast, no virus was recovered from nih3t3 cells at any time point (fig. 9 ). the colocalization of ehv-1 with caveolin at the early stage of infection and the significant effect of a dominant negative form of caveolin-1 on the ehv-1 infection suggests that the virus enters ebmecs via caveolar endocytosis. the results of double immunolabeling indicated that clathrin-dependent endocytosis plays a relatively minor role in ehv-1 entry into ebmecs. as far as we are aware, our study is the first to demonstrate alphaherpesvirus entry into cells via caveolar endocytosis. the production of ehv-1 icp0 rna in ebmecs was blocked by the tyrosine kinase inhibitor genistein, indicating a requirement for tyrosine phosphorylation in the entry of ehv-1 into these cells although we cannot exclude the possibility that genistein might affect icp0 gene expression after viral entry. tyrosine phosphorylation initiates signal transduction events that lead to receptor-mediated endocytosis (mcpherson et al., 2001) . in caveolar endocytosis, tyrosine kinase activity is required for phosphorylation of caveolin at residue 14, which induces caveolar vesiculation and enclosure of ligands within caveolae (aoki et al., 1999; chen and norkin, 1999) . our result indicated that ehv-1 may induce caveolin phosphorylation, which activates the subsequent signal transduction. caveolae have traditionally been described as smooth invaginations of the plasma membrane with a diameter of 50 to 80 nm (palade, 1953; yamada, 1955) . most viruses that have been shown to enter host cells via caveolae are nonenveloped and therefore smaller than typical caveolar invaginations. however, recent studies have shown that this traditional description of caveolar morphology is inadequate, as caveolae with flat or tubular forms have also been detected (anderson, 1998) . caveolae-dependent endocytosis has also been found to contribute to the entry of enveloped viruses, such as filoviruses and human coronavirus (empig and goldsmith, 2002; nomura et al., 2004) . our data now provide support for the notion that caveolar vesicles mediate the delivery of large enveloped viruses. infection of endothelial cells in the horse central nervous system (cns) is required for establishment of ehv-1-induced encephalomyelitis, which is characterized by vasculitis, thrombosis, and secondary ischemia of neuronal tissue (edington et al., 1986) . we have previously proposed that ebmecs are an appropriate in vitro model for studies of the endotheliotropism of ehv-1 (hasebe et al., 2006) . primary cultured brain microvascular endothelial cells (bmecs) retain several characteristics of cns endothelial cells in vivo (joó, 1996) . infection of human bmecs with escherichia coli k1 results in the formation of abundant caveolae that mediate bacterial uptake (sukumaran et al., 2002) . immunohistochemical studies of normal brain tissue have also shown the caveolar compartment to be pronounced in endothelial cells, suggesting an important physiological role for caveolar mediated endocytosis in vivo (virgintino et al., 2002) . it is therefore reasonable to propose that ehv-1 makes use of caveolar endocytosis to infect endothelial cells in the horse cns. viral entry through caveolae has traditionally been considered to occur in a ph-neutral setting, bypassing the acidic endosome (ashok and atwood, 2003) . recently, caveolin-1-positive endosomes have been shown to deliver caveolae-internalized cargo to the golgi complex (nichols, 2003) and bkv enters vero cells via this pathway (eash et al., 2004) . the effects of the lysosomotropic agent used in this study suggest that acidic intracellular organelles do not facilitate ehv-1 infection in ebmecs. therefore, ehv-1 may be transported to phneutral organelles after internalization via caveolae. immunofluorescence microscopic analysis and the lack of an effect of the dominant negative form of caveolin-1 on ehv-1 infection in e. derm cells suggested that ehv-1 entry occurred by a caveolaeindependent route. such a route might be operative in vivo for ehv-1 infection of certain cell types, such as lymphocytes, that do not appear to form caveolae. the principal pathway of ehv-1 entry into e. derm cells also appears to be clathrin independent, given that ehv-1 immunoreactivity did not colocalize with clathrin-heavy chain in these cells. despite the lack of colocalization of ehv-1 with endocytic markers, our data from electron microscopy, lysosomotropic agent treatment, energy depletions and infectious virus recovery assays suggest that ehv-1 enters e. derm cells via energy-and ph-dependent endocytosis. in contrast, frampton et al. (2007) suggested that ehv-1 strain l11δgiδge entry into e. derm cells occurs by direct fusion at the cell surface. the difference between frampton's study and our own could be explained by the difference in virus strain. the strain l11δgiδge is a mutant which lacks gi and ge, resulting in attenuated virulence in mice and reduction of viral growth on rk13 cells compared to the parental virus strain racl11 (frampton et al., 2002) . it has been thought that ge of varicella zoster virus (vzv), another varicellovirus, is associated with viral entry. notably, li et al. (2006) reported that ge interacts with insulin-degrading enzyme, which acts as a cellular receptor mediating cell-free vzv infection and cell-to-cell spread. therefore, the lack of ge might influence the ehv-1 entry mechanism. the dependence of entry mechanisms on virus strain has been reported for human papillomavirus by bousarghin et al. (2003) , who demonstrated that although they are very closely related viruses human papillomavirus types 16, 31 and 58 use different pathways to enter cells. in conclusion, our results suggest that ehv-1 entry pathways are cell type dependent. furthermore, they show that ehv-1 enters certain cell types via caveolar endocytosis, a pathway that has not previously been known to mediate the entry of alphaherpesviruses. the ebmecs were isolated from the brain of a 6-month-old horse as described previously (hasebe et al., 2006) and were cultured in medium 199 earl's (invitrogen, carlsbad, ca, usa) supplemented with 10% fetal bovine serum (fbs; sigma, st. louis, mo, usa) and both penicillin (100 u/ml) and streptomycin (100 μg/ml) (invitrogen). the e. derm cells were obtained from american type culture collection (manassas, va, usa) and cultured in dulbecco's modified eagle's medium (dmem) supplemented with 0.1 mm nonessential amino acids (invitrogen) and 10% fbs. the rk13 cells were cultured in minimum essential medium (mem) supplemented with 10% fbs. the 293t cells were cultured in dmem supplemented with 10% fbs. the ehv-1 strain hh1 was isolated from an aborted equine fetus in japan (kawakami et al., 1970 ). an ehv-1 mutant, ab4-gfp, was generously provided by dr. h. fukushi. (gifu university, gifu, japan). the ab4-gfp was constructed by inserting a gfp expression cassette into the intergenic region between orf62 and orf63 of ehv-1 ab4 strain (ibrahim et al., 2004) . stock viruses were grown in confluent monolayers of rk13 cells. in preparation for rna dot-blot analysis, viruses were treated with rnase (20 ng/ml) for 1 h at 37°c to remove contaminating rna in the stock virus. viral titer was determined by a plaque formation assay using rk13 cells. genistein was obtained from sigma, and bafilomycin a1 and ammonium chloride were from wako (osaka, japan). bafilomycin a1 and genistein were dissolved in dimethyl sulfoxide at 1 mm and 100 mg/ml, respectively. ammonium chloride was dissolved in distilled water at 5 m. the final concentration of dimethyl sulfoxide in culture medium was ≤0.1%, and the same concentration was also added to control incubations. rabbit polyclonal antibodies to ehv-1 were kindly provided by dr. r. kirisawa (rakuno gakuen university, hokkaido, japan), and a mouse monoclonal antibody to ehv-1 gb protein was kindly provided by dr. t. matsumura (japan racing association, tochigi, japan). rabbit polyclonal antibodies to caveolin, a mouse monoclonal antibody to caveolin (py14) and a mouse monoclonal antibody to clathrin-heavy chain were obtained from bd transduction laboratories (san jose, ca, usa). alexa fluor 488-conjugated goat antibodies to mouse immunoglobulin g, alexa fluor 594-conjugated goat antibodies to rabbit immunoglobulin g, 4′,6-diamidino-2-phenylindole (dapi) and lyso-sensor™ yellow/blue dnd-160 were from molecular probes (leiden, the netherlands). horse radish peroxidase (hrp)-conjugated goat antibodies to mouse immunoglobulin and hrp-conjugated goat antibodies to rabbit immunoglobulin were obtained from biosource (camarillo, ca, usa). confluent monolayers of ebmecs or e. derm cells seeded into 24well plates were infected with ehv-1 at an m.o.i. of 5 p.f.u. per cell. the infected cells were incubated at 37°c for 1 h to allow attachment of the virus, then washed three times with phosphate-buffered saline (pbs), provided with fresh growth media, and incubated further at 37°c. at 0 h (immediately after seeding the virus), 8 h, 16 h or 24 h p.i., the supernatants were removed and the cells were collected. the cells were suspended in 1 ml of mem and lysed by three cycles of freezing and thawing. viral titer was determined by the plaque formation on rk13 cells. ebmecs or e. derm cells cultured in six-well plates were exposed to ehv-1 at an m.o.i. of 150 p.f.u. per cell and incubated for 2 h at 4°c to allow attachment of the virus to the cell surface. after subsequent incubation for 10 min at 37°c, the cells were collected and fixed overnight at 4°c with 2.5% glutaraldehyde. the cells were subsequently exposed to 2% osmic acid, dehydrated, and embedded in epon 812 (shell chemical company, new york, ny, usa). sections were cut at a thickness of 70-80 nm, mounted on coated grids, stained with uranyl acetate and lead citrate, and examined with an electron microscope (jem-1210; japan electron optics laboratory, tokyo, japan). confluent monolayers of ebmecs or e. derm cells in eight-well chamber slides (bd falcon, san jose, ca, usa) were infected with ehv-1 at an m.o.i. of 10 p.f.u. per cell. after incubation at 37°c for 10 or 30 min, the cells were fixed with 3.7% paraformaldehyde for 5 min and permeabilized with 0.1% triton x-100 for 5 min. the cells were washed with pbs and incubated for 1 h at room temperature first with 2% bovine serum albumin (sigma) and then with primary antibodies. for simultaneous detection of ehv-1 and clathrin, cells were stained with rabbit anti-ehv-1 polyclonal antibodies and mouse anticlathrin-heavy chain monoclonal antibody. for simultaneous detection of ehv-1 and caveolae, cells were stained with mouse anti-ehv-1 gb monoclonal antibody and rabbit anti-caveolin polyclonal antibodies. then the cells were incubated for 1 h at room temperature with secondary antibodies, mounted with the use of fluorescence mounting medium (dako cytomation, carpinteria, ca, usa), and examined with a laser-scanning confocal microscope (olympus, tokyo, japan). for all primary antibodies, control images were evaluated to ensure nonoverlapping binding of secondary antibodies and specific detection for each excitation channel. the images were processed with fv10-asv 1.4 viewer (olympus). coincidence of the immunoreactivity between rabbit anti-ehv-1 polyclonal antibodies and mouse anti-ehv-1 gb monoclonal antibody was confirmed (data not shown). the lentiviral vector system was provided by dr. hiroyuki miyoshi, riken bioresource center, ibaraki, japan. full-length equine caveolin-1 cdna amplified by pcr from cdna of ebmecs was cloned into pcdna 3.1(−) (invitrogen) and csii-cmv-mcs-ires2-bsd (riken bioresource center). these constructs were designated as pcdna 3.1 (−)-ecav and csii-cmv-ecav-ires2-bsd, respectively. a mutant form of equine caveolin-1 was generated from pcdna 3.1(−)-ecav via pcrbased mutagenesis and subcloned into csii-cmv-mcs-ires2-bsd, which was designated as csii-cmv-ecavy14f-ires2-bsd. lentiviral vectors pseudotyped with vesicular stomatitis virus g glycoprotein (vsv-g) were generated according to the instruction provided by the riken bioresource center. briefly, 293t cells were transfected with the package construct (pcag-hivgp), the vsv-g-expressing construct (pcmv-vsv-g-rsv-rev) and the self-inactivating vector construct (csii-cmv-ecav-ires2-bsd or csii-cmv-ecavy14f-ires2-bsd). after 48 h, the supernatant was concentrated by ultracentrifugation at 50,000 g for 2 h. the pellet was resuspended in hanks' balanced salt solutions (invitrogen). vector titer was determined using hiv p24 elisa kit (perkinelmer life sciences, inc., wellesley, ma, usa). the ebmecs and e. derm cells were infected with lentiviral vectors at an m.o.i. of 0.01 infectious unit per cell and incubated for 24 h. then the cells were infected with ehv-1 at an m.o.i. of 1 p.f.u. per cell. after 16 h, the cells were fixed with 3.7% paraformaldehyde, permeabilized with 0.1% triton x-100 and counterstained with dapi, a nuclear stain. the gfp and dapi signal was evaluated with fluorescent microscopy (olympus). the number of gfp expressing cells and the number of nuclei were counted by image-j (nih, bethesda, md, usa). the relative proportion of ehv-1-infected cells was calculated by dividing the number of the gfp-positive cells by the number of the nuclei. the number of the gfp-positive cells in wild type ecav expressing ebmecs or e. derm cells was defined as 100%. expression of the transgene products was confirmed by western blotting. the cells infected with lentiviral vector at 24 h p.i. were lysed in ripa buffer [10 mm tris-hcl (ph 7.5), 150 mm nacl, 5 mm edta, 50 mm naf, 10% glycerol, 1% triton x-100, 1% sodium deoxycholate, 0.1% sds, 5 mm phenylmethylsulfonyl fluoride], and mixed with complete protease inhibitor cocktail (roche diagnostics, mannheim, germany). the cell lysates were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page) and transferred to pvdf membrane (millipore, billerica, ma, usa). the membrane was blocked with 2% low-fat milk in tris-buffered saline containing 0.05% tween 20 (tbs-t) at 4°c overnight, incubated with a rabbit polyclonal antibodies to caveolin (1:1,000 dilution in 2% low-fat milk in tbs-t) for primary antibody, and then with hrp-conjugated goat antibodies to rabbit immunoglobulin (1:5000 dilution in 2% lowfat milk in tbs-t) for secondary antibody. the immunocomplex was visualized with immobilon™ western chemiluminescent hrp substrate (millipore) and las-1000mini (fujifilm, tokyo, japan). complementary dna fragments corresponding to ehv-1 icp0 rna and horse glyceraldehydes-3-phosphate dehydrogenase (gapdh) were cloned by reverse transcription and pcr from total rna extracted either from ehv-1-infected rk13 cells or from uninfected equine placenta, respectively, using pcr primers 5′-tttttggccgt-ggattctgg-3′ and 5′-agttctgcttggacgatgag-3′ for icp0 rna and 5′-agttccatggcacagtcaag-3′ and 5′-acaaacattggggcat-cagc-3′ for gapdh. the pcr products were cloned into the pgem-t vector (promega, madison, wi, usa), and the resultant plasmids were sequenced to verify their identity. antisense rna probes were prepared with a digoxigenin-based rna labeling kit (sp6/t7; roche diagnostics). plasmids containing cloned cdna were linearized with not i for synthesis of rna in the presence of digoxigenin-11-utp. the labeled probes generated from 1 μg of plasmid dna were precipitated with ethanol, dissolved in 50 μl of rnase-free water, and stored at −80°c. confluent monolayers of ebmecs or e. derm cells in six-well plates were treated with inhibitor at 37°c for 1 h and then infected with rnase-treated ehv-1 at an m.o.i. of 5 p.f.u. per cell for 1 h at 37°c in the continued presence of an inhibitor. after additional 2 h incubation in the presence of inhibitor, total rna was extracted with using trizol reagent (invitrogen), treated with dnase with the use of a kit (ambion, austin, tx, usa), and diluted to a concentration of 2 mg/ml. hybridization was performed as previously described (kimura et al., 2004) . in brief, 1 μl of each sample was spotted onto a dry positively charged nylon membrane (roche) and allowed to dry in air. the rna was fixed to the membrane with a uv cross-linker (xl-1000; spectronics, lincoln, ne) and baked for 30 min at 80°c. the membrane was then incubated for 3 h at 68°c in a solution containing 0.25 m sodium phosphate buffer (ph 7.2), 10% sds, 1 mm edta, and 2% blocking reagent. hybridization was performed for 12 h at 68°c in the same solution containing the digoxigenin-labeled crna probe (20 ng/ml), after which the membrane was washed three times (each for 20 min) with 25 mm sodium phosphate buffer (ph 7.2) containing 10% sds and 1 mm edta. hybridization complexes were detected with alkaline phosphatase-conjugated antibodies to digoxigenin and disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo [3.3.1.1. 3,7 ]decan}-4-yl) phenyl phosphate (cspd) as the chemiluminescent substrate (roche). quantitative analysis of autoradiograms was performed with scion image software. the relative amount of ehv-1 icp0 rna was calculated by dividing the intensity of the signal for icp0 rna by that of the signal for gapdh mrna. the adjusted signal intensity for infected but mock-treated cells was defined as 100%. confluent monolayers of e. derm cells in 60 mm dishes were treated with genistein at 37°c for 1 h, washed once with pbs and lysed in ripa buffer with complete protease inhibitor cocktail. the cell lysates were mixed at 4°c with rabbit polyclonal antibodies to caveolin for 1 h and collected on protein a-sepharose beads (ge healthcare bio-science corp, nj, usa). the immunoprecipitates were subjected to sds-page and western blotting with a mouse monoclonal antibody to caveolin (py14) (1:1000 dilution in 2% low-fat milk in tbs-t) or rabbit polyclonal antibodies to caveolin for primary antibodies and hrp-conjugated goat antibodies to mouse immunoglobulin (1:5000 dilution in 2% low-fat milk in tbs-t) or hrpconjugated goat antibodies to rabbit immunoglobulin for secondary antibodies. cells seeded on 35 mm glass bottom dishes were incubated with or without bafilomycin a1 or ammonium chloride at 37°c for 1 h, and then exposed to 2 μm of a ph sensitive fluorescence dye, lysosensor yellow/blue dnd-160, in pre-warmed growth medium. after incubation at 37°c for 5 min, the medium was replaced with fresh medium. the fluorescence was observed using a confocal laserscanning microscope with excitation at 405 nm and emission was measured at 480-510 nm. cells were incubated in atp depletion media composed of glucosefree, fbs-free dmem (invitrogen) with 10 mm 2-deoxyglucose (sigma) for 1 h and infected with ab4-gfp at an m.o.i. of 5 per cell for 1 h in atp depletion media. after ehv-1 infection, the cells were treated with citrate buffer ph 3.0 to inactivate remaining viruses on cell surface. the media were replaced with regular culture media and the cells were cultured at 37°c. for untreated controls, the cells were incubated with fbs-free dmem for 1 h, infected with ab4-gfp in fbsfree dmem for 1 h and the media were replaced with regular culture media. for samples depleted of atp after post entry, the cells were infected with ab4-gfp in fbs-free dmem. after viral infection for 1 h, the cells were incubated in atp depletion media for 2 h and the media were replaced with regular culture media. at 12 h p.i., the cells were harvested, fixed with 4% paraformaldehyde and gfp-positive cells were counted by facs canto (bd biosciences, sun jose, ca, usa). infectious virus recovery assay was performed as previously described in frampton et al. (2007) with some modifications. e. derm and nih3t3 cells seeded in 24 well plates were washed with ice-cold dmem supplemented with 25 mm hepes and 1% fbs and incubated on ice for 5 min. the cells were infected with ehv-1 hh1 strain at an m.o.i. of 10 p.f.u. per cell for 2 h at 4°c. the media were replaced with pre-warmed fresh dmem containing 25 mm hepes and 1% fbs at 37°c. at 0, 7.5, 15, 30 and 45 min after incubation at 37°c, the cells were washed with glycine ph 3.0 for 1 min at room temperature, washed with dmem containing 25 mm hepes and harvested. the cells were freeze-thawed once and sonicated three times for 15 s each. the infectious virus was detected by titration on rk13 cells. triplicate samples were measured for each time point. quantitative data are expressed as means ± sd and were compared with student's t test. a p value of b0.05 was considered statistically significant. morphology and entry of enveloped and deenveloped equine abortion (herpes) virus genistein, a specific inhibitor of tyrosine-specific protein kinases the caveolae membrane system bound simian virus 40 translocates to caveolin-enriched membrane domains, and its entry is inhibited by drugs that selectively disrupt caveolae tyrosine phosphorylation of caveolin-1 in the endothelium contrasting roles of endosomal ph and the cytoskeleton in infection of human glial cells by jc virus and simian virus 40 ph-dependent fusion of vesicular stomatitis virus with vero cells. measurement by dequenching of octadecyl rhodamine fluorescence human papillomavirus types 16, 31, and 58 use different endocytosis pathways to enter cells entry of herpes simplex virus 1 in bj cells that constitutively express viral glycoprotein d is by endocytosis and results in degradation of the virus equine herpesvirus type 1 infected cell polypeptides: evidence for immediate early/early/late regulation of viral gene expression extracellular simian virus 40 transmits a signal that promotes virus enclosure within caveolae efficient species c hadv infectivity in plasmocytic cell lines using a clathrin-independent lipid raft/caveola endocytic route bafilomycins and concanamycins as inhibitors of v-atpases and p-atpases infection of vero cells by bk virus is dependent on caveolae endothelial cell infection and thrombosis in paralysis caused by equid herpesvirus-1: equine stroke association of the caveola vesicular system with cellular entry by filoviruses contribution of gene products encoded within the unique short segment of equine herpesvirus 1 to virulence in a murine model equine herpesvirus 1 enters cells by two different pathways, and infection requires the activation of the cellular kinase rock1 anti-glycoprotein d antibodies that permit adsorption but block infection by herpes simplex virus 1 prevent virion-cell fusion at the cell surface herpes simplex virus type 1 entry through a cascade of virus-cell interactions requires different roles of gd and gh in penetration neutralizing antibodies specific for glycoprotein h of herpes simplex virus permit viral attachment to cells but prevent penetration uptake pathway of polyomavirus via ganglioside gd1a cell penetration and trafficking of polyomavirus tyrosine phosphorylation is required for fc receptor-mediated phagocytosis in mouse macrophages differential susceptibility of equine and mouse brain microvascular endothelial cells to equine herpesvirus 1 infection on the entry of semliki forest virus into bhk-21 cells inhibition of semliki forest virus penetration by lysosomotropic weak bases growth and virulence alterations of equine herpesvirus 1 by insertion of a green fluorescent protein gene in the intergenic region between orfs 62 and 63 endothelial cells of the brain and other organ systems: some similarities and differences an outbreak of abortion due to equine rhinopneumonitis virus among mares in the hidaka district, hokkaido. i. epizootiological survey and virus isolation decreased expression of equine herpesvirus-1 early and late genes in the placenta of naturally aborted equine fetuses recruitment of epidermal growth factor and transferring receptors into coated pits in vitro: differing biochemical requirements insulin degrading enzyme is a cellular receptor mediating varicella-zoster virus infection and cell-to-cell spread internalization of echovirus 1 in caveolae infectious entry pathway of influenza virus in a canine kidney cell line pathway of vesicular stomatitis virus entry leading to infection signaling on the endocytic pathway glycoprotein d receptor-dependent, low-ph-independent endocytic entry of herpes simplex virus type 1 caveosomes and endocytosis of lipid rafts cellular and viral requirements for rapid endocytic entry of herpes simplex virus roles for endocytosis and low ph in herpes simplex virus entry into hela and chinese hamster ovary cells herpes simplex virus type 1 enters human epidermal keratinocytes, but not neurons, via a ph-dependent endocytic pathway human coronavirus 229e binds to cd13 in rafts and enters the cell through caveolae kinetics of cellular and viral dna synthesis in equine abortion (herpes) virus infection of l-m cells epithelial growth factorinduced phosphorylation of caveolin 1 at tyrosine 14 stimulates caveolae formation in epithelial cells fine structure of blood capillaries regulated internalization of caveolae caveolae are involved in the trafficking of mouse polyomavirus virions and artificial vp1 pseudocapsids toward cell nuclei human papillomavirus type 31 uses a caveolin-1 and dynamin 2-mediated entry pathway for infection of human keratinocytes ph-independent hiv entry into cd4-positive t cells via virus envelope fusion to the plasma membrane tomson's special veterinary pathology escherichia coli k1 internalization via caveolae requires caveolin-1 and protein kinase calpha interaction in human brain microvascular endothelial cells ammonium chloride and chloroquine inhibit rabies virus infection in neuroblastoma cells equine herpesvirus 1 entry via endocytosis is facilitated by αv integrins and an rsd motif in glycoprotein d transport from late endosomes to lysosomes, but not sorting of integral membrane proteins in endosomes, depends on the vacuolar proton pump expression of caveolin-1 in human brain microvessels penetration of cells by herpes simplex virus does not require a low ph-dependent endocytic pathway the fine structure of the gall bladder epithelium of the mouse uncoating of influenza virus in endosomes the authors gratefully acknowledge the invaluable suggestions by key: cord-279432-aik5bo6o authors: digard, paul; blok, vivian c.; inglis, stephen c. title: complex formation between influenza virus polymerase proteins expressed in xenopus oocytes date: 1989-07-31 journal: virology doi: 10.1016/0042-6822(89)90523-0 sha: doc_id: 279432 cord_uid: aik5bo6o abstract all three influenza virus polymerase (p) proteins were expressed in xenopus oocytes from microinjected in vitro transcribed mrna analogs, with yields of up to 100 ng per oocyte. to examine the functional state of the xenopus-expressed p proteins, the polypeptides were tested for their ability to form stable complexes with each other. as seen in virus-infected cells, all three p proteins associated into an immunoprecipitable complex, suggesting that the system has considerable promise for the reconstruction of an active influenza rna polymerase. examination of the ability of paired combinations of the p proteins to associate indicated that pb1 contained independent binding sites for pb2 and pa, and so probably formed the backbone of the complex. sedimentation analysis of free and complexed p proteins indicated that pb1 and pb2 did not exist as free monomers, and that similarly, complexes of all three p proteins did not simply consist of one copy of each protein. the heterodisperse sedimentation rate seen for complexes of all three p proteins did not appear to result from their binding to rna, suggesting the incorporation of additional polypeptides polymerase complex. influenza virus gene expression depends on the coordinate transcription and replication of eight segments of negative-sense single-stranded rna. this is mediated by the three viral polymerase proteins pbl , pb2, and pa in association with the nucleoprotein (lnglis et a/., 1976) and possibly with other viral proteins or unidentified host cell factors. on infection of a permissive cell the ribonucleoprotein-polymerase complexes migrate to the nucleus where viral mrnas are transcribed (her-z et a/., 1981) . these mrnas are initiated by, and contain, 5'-capped rna fragments generated by a viral cap-dependent endonuclease from host cell mrnas (plotch eta/., 1979) . they are also polyadenylated, but lack sequences complementary to the extreme 5'-end of their corresponding genome rna template (hay et a/., 1977a) . at later times, dependent on the production of virus specific proteins, full-length, nonpolyadenylated, noncapped copies of the genomic rnas are made, which then serve as templates for production of more vrna (hay et a/., 197713) . the polymerase (p) proteins have been shown to exist and probably function as a complex both in virions (braam et al., 1983) and in the infected cell (detjen et a/., 1987; ak-' to whom requests for reprints should be addressed. kina et a/., 1987), but little is known about the role of the individual proteins, or the precise composition of the (presumably differing) complexes which catalyze the synthesis of the three types of virus rna. to address the structure-function relationship of the polymerase proteins a system is needed whereby individual virus proteins can be expressed in an appropriate environment in sufficient quantity to facilitate reconstitution of activity. it should then be possible to dissect the system to reveal the individual functions of particular proteins. our approach has been to seek expression of the polymerase-associated polypeptides through transcription of cloned dna into artificial mrna analogs using the bacteriophage sp6 rna polymerase, and translation of these mrnas in xenopus oocytes. such a system offers the advantage that proteins may be produced individually, or in any desired combination, simply by translating the appropriate mrna mixture. in addition, since the polypeptides can be produced simultaneously in an environment similar to that in the infected cell, the likelihood is that they will display appropriate physiological interactions. thus, the system offers considerable promise for the reconstitution of enzyme activity. in this report we describe the establishment of such a system and its preliminary characterization. in addition, as a first step toward reconstitution of enzyme ac-tivity, we show here that in the absence of any other virus specific polypeptides all three p proteins can interact to form complexes, and report preliminary characterization of these complexes. all enzymes were obtained from boehringer-mannheim, and all radiochemicals from amersham (england). nuclease-treated rabbit reticulocyte lysate was obtained from dr. t. hunt (cambridge). influenza strain a/pr8/34 was propagated in, and purified from, embryonated eggs as previously described (inglis et al., 1976) . infected cell lysates were prepared at 6 hr postinfection from confluent monolayers of chick embryo fibroblasts infected at a multiplicity of infection of around 10. transcription vectors for the three polymerase genes were constructed by the insertion of cdna copies of the relevant influenza a/pr8/34 segments (the generous gift of dr. p. palese) into a plasmid containing an sp6 rna polymerase promoter. the sp6 vector used, psp64-t (krieg and melton, 1984 ; obtained from dr. a. colman), also provides flanking 5'-and 3'-noncoding sequences from the xenopus ,&globin gene, which enhance the translation of foreign genes in oocytes (drummond et al., 1985) . copies of segments 1, 2, and 3 were excised from the plasmids paprlo1, papr206, and papr303 (young et a/., 1983) by digestion with the restriction enzymes barnhi, hindill, and ecori, respectively. segment 1 was inserted directly into the bg/ll site separating the 5'-and 3'-globin noncoding sequences in psp64-t, while segments 2 and 3 were end-filled by klenow fragment dna polymerase (boehringer) and blunt-end-ligated into a similarly endfilled psp64-t. the resulting plasmids containing the pbl , pb2, and pa genes, in positive orientation relative to the sp6 promoter, were designated pstl+, pst2+, and pst3+, respectively. all manipulations were carried out according to standard procedures (maniatis et a/., 1982) . in vitro transcription and translation was carried out as previously described (brierley et a/., 1987) . briefly, the three transcription plasmids were linearized downstream to the globin 3'-noncoding sequence by digestion with smal, and sp6 rna polymerase run-off transcripts were synthesized under conditions which re-sulted in the incorporation of a synthetic 5'-m'gpppg cap structure (new england bio-labs). the product rna was then phenol-extracted and checked for structural integrity by agarose gel electrophoresis (maniatis eta/., 1982) . for in vitro translation, messenger-dependent reticulocyte lysate (mdl; pelham and jackson, 1976) was programmed with mrna to a final concentration of around 0.1 pg/pl and incubated at 30" for 1 hr. oocytes were taken from the frog, maintained, and injected essentially according to standard procedures (colman, 1984) . each oocyte received a maximum of 50 ng of rna in a constant injection volume of 50 nl. at 2 hr postinjection, groups of eight oocytes per rna were transferred to modified barth's saline (mbs) containing [35s]methionine (sp act 1 150 cvmmol; amersham, england) at 1 .o mci/ml (10 &i/oocyte) and subsequently harvested by mechanical disruption into tkm buffer (20 mm tris-hci, ph 7.6, 100 mni kci, 5 mm mgcl>, 1 mm phenylmethylsulfonyl fluoride (pmsf), 50% glycerol; 10 plloocyte) at 5 hr postinjection. the resulting lysates were then clarified by microcentrifugation and stored at -20" prior to analysis. the production of antisera directed against the pb2 protein has already been described (brierley et a/., 1987) and a similar strategy was used to prepare antisera against pbl and pa. briefly, portions of p protein coding sequence were expressed in bacteria as c-terminal fusions with ,&galactosidase, using the pex series of plasmids (stanley and luzio, 1984) . these fusion proteins were then purified by gel elution and used to immunize rabbits. in this study, antibodies raised to amino acids 50-370 of pbl, 342-463 of pa, and 585-759 of pb2 (f5) were used. lmmunoprecipitation from oocyte lysates for immunoprecipitation, 10 ~1 of [35s]methionine-labeled oocyte lysate (containing the equivalent of one oocyte) was diluted to 100 ~1 with oocyte immunoprecipitation buffer, (oipb; 50 mm tris-hci, ph 7.6, 100 mm kci, 5 ml\/l mgci,, 1% triton x-l 00, 1% sodium deoxycholate, 0.1% sds, 1 mll/l pmsf) and left on ice for 30 min before the addition of 4 ~1 of rabbit antiserum. after a further 30 min on ice, 50 ~1 of a 50% suspension of protein a-sepharose (sigma) in oipb was added, and the tubes were rotated at 4" for 30 min. finally, the sepharose-bound material was collected by centrifugation, washed once with 1 ml of oipb, and pbid pb2d ipa abcdefghij fig. 1. expression of the influenza virus p proteins in xenopus oocytes, and their reactivity with the monospecific antipolymerase sera. lanes a, d. g, j-unprecipitated lysates: oocytes microinjected with pstl+. pst2+, pst3+, h20, respectively. lanes c, f, i-precipitated with a-pbl , or-pb2, a-pa, respectively. lanes b, e. h-precipitated with the corresponding preimmune bleeds. eluted in laemmli sample buffer (laemmli, 1970) . proteins contained in the supernatant were separated on a 10% polyacrylamide gel and detected by autoradiowwb immunological detection of nitrocellulose-bound proteins samples were subjected to polyacrylamide gel electrophoresis (page) (laemmli, 1970) and transferred to nitrocellulose according to standard procedures (towbin et a/., 1979). the nitrocellulose was blocked by incubation with a solution of 4% bsa in pbs for 1 hr at 37", followed by a 1 hr incubation at room temperature with the antiserum-diluted 1 in 200 in 4% bsa, 2% newborn calf serum in pbs. the blot was then rinsed with lo/o np-40 in pbs, before incubation with 1 &i of lz51labeled protein a for 1 hr. a final wash with 1% np-40 in pbs was carried out before the blot was air-dried and exposed to film. oocyte lysates (from 10 oocytes) were layered on top of linear 5-20% (w/v) sucrose gradients in 20 ml\/l tris-cl, ph 7.6, 100 mm kci, 5 mm mgc&, 0.1% np-40, 1 mm pmsf and centrifuged at 100,000 g,, for 12 hr. fractions were then collected, adjusted to 1% triton x-l 00, 1% sodium deoxycholate, 0.1% sds, and analyzed for their p protein content by immunoprecipitation and page. all gradients had bovine serum albumin (bsa; m, 68,000) and apoferritin (rji, 440,000) included as internal standards. to test the capacity of xenopus oocytes for expression of the influenza p proteins, artificial mrnas corresponding to each gene were transcribed in vitro using the sp6 rna polymerase and microinjected into the oocytes. after a 2 hr recovery period, the cells were labeled by incubation with [35s]methioninefor 3 hr, harvested, and analyzed by gel electrophoresis before and after immunoprecipitation with specific anti-p protein sera (fig. 1) . in the unprecipitated tracks, strong bands of the expected mobility could be seen [lane a, pstl+ transcript (pbl); lane d, pst2+ transcript (pb2); lane g, pst3+ transcript (pa)] and were not seen in control water-injected oocytes (lane j). in each case these polypeptides were specifically precipitated by the homologous antiserum (lanes c, f, i), but not by the corresponding preimmune sera (lanes b, e, h). the extent of radiolabeling of the p proteins in the above experiment suggested that their production was quite efficient. to obtain a quantitative estimate, the accumulation of the p proteins was examined over a period of time after microinjection of mrna, by western blotting with the directed antisera. the result of such an experiment for pbl is shown in fig. 2a . the antiserum specifically detected a single band in lysates from pst1 +-injected oocytes which comigrated both with [35s]methionine-labeled pbl (synthesized in mdl using pstl+ transcript) and with pbl from purified virions. the results indicated that the total amount of pbl increased up to 40 hr postinjection and then declined slightly. known amounts of purified virus were also included in the western blot as standards to quantify the amount of pbl produced. since the equivalent of half an oocyte was loaded in each track, it was estimated from densitometry scans that after 40 hr each oocyte had accumulated on average 100 ng of pbl [assuming that pbl comprises 1% w/w purified influenza virions (inglis et al., 1976) ]. similar analyses were carried out for the other p proteins (data not shown). the accumulation of pa was similar to that of pbl, but pb2 was produced less efficiently (approximately 10 ng per oocyte). this appears to be the result of protein turnover, since microinjected [35s]methionine-labeled pb2 (synthesized prior to injection in mdl programmed with pst2f transcript) was found to have a half-life of 3 hr, and since the accumulation of pb2 was similar to that of the other p proteins up to about 3 hr postinjection (data not shown). a further experiment was carried out to assess p protein production in oocytes relative to virus-infected cells; fig. 2b shows a western blot comparing the amount of pbl accumulated per half oocyte 36 hr after microinjection, with that present in an equivalent amount (in terms of total protein) of chick embryo fibroblast (cef) cells at 6 hr postinfection (approximately 10" cells). a more intense signal was observed from the pst1 +-injected oocyte track than that from the infected cell track. however, the rate of production in the infected cell must be higher given the difference in incubation times. nevertheless, the overall quantities seem comparable. an important initial step in the biogenesis of the influenza polymerase is likely to be the formation of a complex of the three p proteins; accordingly, the oocyte-expressed p proteins were examined for their ability to associate with each other. groups of a dozen oocytes were injected with solutions containing one, two, or all three of the mrna analogs. for these experiments, the rnas were mixed in equal quantities and single rnas were diluted in hz0 to keep the concentration of any one transcript constant throughout. the oocytes were then metabolically labeled and harvested 3 . analysis of the ability of oocyte-expressed p proteins to form complexes with each other. oocytes were injected wrth the combinations of pst transcripts shown, and radiolabeled lysates prepared as described under materials and methods. the lysates were then precipitated with lu-pbl (lanes l), a-pb2 (lanes 2) or a-pa (lanes 3). see text for full description, as before. a 3-hr labeling period was employed to avoid the problem of the instability of pb2, and allow the production of approximately equal amount of all three proteins. following harvest, the oocyte lysates were analyzed by immunoprecipitation and page (fig. 3) . the antisera can be seen to be truly monospecific in that none of the p proteins were significantly precipitated by the heterologous antisera when expressed in isolation (lanes a-i). however, each antiserum precipitated all three p proteins from oocytes injected with a mixture of all three transcripts (lanes j-l). this specific coprecipitation provides evidence that when cotranslated in an oocyte, the p proteins are present as a complex. given that all three p proteins would associate into a complex, it was of interest to determine which of the polypeptides were interacting with which. therefore, paired combinations of the mrna analogs were microinjected, and their products assayed for association as before by immunoprecipitation with specific antisera (fig. 3, lanes m-r) . pbl and pb2 (lanes m and n), and pbl and pa (lanes q and r) were found to coprecipitate, but not pb2 and pa (lanes o and p), thus indicating that pbl may act as the backbone of the complex, and also implying discrete binding sites on pbl and pb2 and pa. in order to characterize further the association of the three p proteins in oocytes, individual and complexed the absence of pb2 in the fourth fraction from the top of the gradient is the result of an error during the immunoprecipitation of that sample. p proteins were analyzed by velocity gradient sedimentation. the aims of this were twofold; first to confirm the physical existence of complexes, by showing an increased sedimentation rate for the coexpressed p proteins, and second, to examine the size of complex formed. lysates from oocytes microinjected with single or mixed p protein mrnas were fractionated on sucrose gradients, and then each fraction was assayed for its p protein content by immunoprecipitation. figure 4 shows the result of this experiment for individually expressed p proteins. pa migrated slightly faster than the bsa marker (as expected for an m, 82,000 protein), but surprisingly pbl and pb2, which are similar in size, sedimented as much larger and more heterogeneous bodies. the majority of the proteins sedimented in fractions corresponding to a size of around m, 250,000, but significant amounts of material were also present in fractions corresponding to much larger sizes. for pbl and pb2 then, it seemed likely that demonstration of a convincing mobility difference between the individual and complexed form would be difficult. however, such an experiment remained possible for pa, given its lower and more discrete sedimentation rate. accordingly, oocyte lysates containing either pa alone or pa in combination with pbl and pb2 were fractionated on a gradient, and the fractions immunoprecipitated with anti-pa serum. the results of this experiment are shown in fig. 5 . again, paalone migrated as a reasonably defined band near the top of the gradient (top panel). however, in the presence of the other two p proteins, it sedimented throughout the whole of the gradient (middle panel), suggesting its inclusion in complexes. in addition, the coprecipitation of pbl and pb2 with pa could also be seen, further confirming the existence of an interaction between the polypeptides. it is interesting to note, however, that the ratio of the three polypeptides present in the complexes was not constant throughout the gradient. in particular, the ratio of pbl and pb2 to pa increased in the faster sedimenting species, suggesting either that pa has more than one binding site for each of the basic p proteins, or perhaps more likely, that the former proteins can form complex structures linked through self-association. from the migration pattern of pa seen in complexes, the marked heterogeneity in sedimentation rate of pbl and pb2 appears to be extended to a complex of all three p proteins. in view of the fact that the p proteins must interact with rna, it seemed possible that the high sedimentation values obtained for individually expressed pbl and pb2 (fig. 4) and for complexes containing pbl and pb2 could have arisen from the polypeptides binding to rna present in the lysate. if this was the case, rnase treatment of the lysate prior to gradient fractionation should significantly decrease the sedimentation rate of the complexes. the bottom panel in fig. 5 shows the result of such rnase treatment; no difference in mobility between treated and untreated complexes could be seen. furthermore, rnase treatment of lysates containing individually expressed pb2 also failed to affect its sedimentation pattern (not shown), suggesting that the heterogeneous size distribution of the p protein complexes is not the result of association with rna. a major goal in the study of the influenza polymerase is the reconstitution of an active enzyme from cloned components. this would then allow detailed analysis of the biochemical reactions catalysed by the enzyme, while manipulation of the dna templates would facilitate structural and functional analysis of individual components. here, we have demonstrated the feasibility of producing all three influenza virus polymerase proteins for functional studies by the translation of in vitro transcribed mrna analogs in xenopus oocytes. high levels of expression were achieved for pbl and pa after prolonged incubation, reaching 100 ng per oocyte. however, pb2 appeared to be unstable, with a half-life of about 3 hr, which meant maximal expression was around 10 ng per oocyte. other groups have noted the instability of pb2 when expressed in isolation; pb2 expressed in nih 3t3 cells using an inducible bovine papilloma virus vector has a similar half-life of around 3 hr (braam-markson el al., 1985) . nevertheless, 10 ng of each individual polymerase protein is equivalent to the amount present in about 1 pg of purified virus, and virion transcriptase activity can easily be detected in reactions containing only 5-10 pg of purified virus (bishop et al., 1971) . therefore, we believe that the system offers considerable promise for the reconstitution of enzymatic activity. our results indicate that all three p proteins expressed in oocytes formed a complex. this parallels the situation seen in the infected cell, where similarly, all three proteins associate, and also confirms the observations of other workers that a complex of the p proteins can exist in virus-infected cells independently of virus rnps (detjen et al., 1987; akkina et al., 1987) . it is also the first direct demonstration that all three artficially expressed p proteins can reassociate into a complex, probably a vital preliminary step toward reconstitution of the influenza polymerase. in a previous report, where the influenza p proteins were expressed using baculovirus vectors (st. angelo eta/., 1987) only pbl and pb2 formed an immunoprecipitable complex. the authors suggested from this that the presence of another influenza gene product was needed for the incorporation of pa into a stable complex. in the light of the results presented here this seems unlikely; an alternative explanation is that amphibian cells provide a more suitable environment for the complex formation than do insect cells. for example some kind of posttranslational modification of the p proteins might be necessary for formation of a full complex and this could be more faithfully carried out in xenopus oocytes. the factors involved are likely to be quite subtle, because when the p proteins are cotranslated in mdl, no complex formation can be detected (not shown). akkina et al. (1987) reported that most of the p proteins found in the cytoplasm of virus-infected cells were not in the form of complexes. however, our work would suggest that a functional nucleus is unlikely to be necessary for complex formation, as the p proteins do not localize to the nucleus in xenopus oocytes, and furthermore, all three proteins associate normally in enucleated oocytes (not shown). expression of pairs of p proteins indicated that pb2 and pa can associate independently with pb 1, yet cannot form a complex directly with each other. thus pbl can be considered the "backbone" of the complex. this idea is consistent with its suggested central role as the protein responsible for elongation (braam et a/., 1983) , with the other two polypeptides as adjuncts fulfilling more peripheral functions such as substrate selection or cap-binding. gradient analysis of individually expressed p proteins indicated that pbl and pb2 did not exist as free monomers, but rather as heterogeneous populations of high-molecular-weight aggregates. it is not clear at present whether these structures represent self-aggregation, or the proteins binding to cellular components. this property of a large and heterogeneous sedimentation rate was also a feature of complexes of all three p proteins, as seen in the markedly different migration of pa when expressed alone or in the presence of the other two p proteins. most of the complexed material sedimented with an apparent molecular weight much greater than 250,000, the expected size for a simple trimolecular complex. again, this could reflect some kind of association with a cellular component, but it is unlikely to be the result of the complexes binding to rna present in the lysate, as rnase treatment prior to gradient analysis does not reduce their rate of sedimentation (fig. 5) or that of pb2 expressed alone (not shown). a second possibility is that the larger complexes contain extra copies of the p proteins. support for this idea comes from the observation that the faster sedimenting forms of the complex appear to contain an increased ratio of pbl and pb2 to pa, suggesting the existence of different stoichiometric forms of complex, varying in their relative content of pbl and pb2. these might arise from pa binding directly to more than one copy of each protein, but given the heterogeneous size distribution of individually expressed pbl and pb2 (and the lack of any observable direct interaction between pa and pb2), the existence of pa associated multimers of the basic p proteins seems a more likely explanation. it has also been suggested previously that the influenza polymerase is a multimeric structure (krystal et a/., 1986) . it is possible that pbl and pb2 behave in a manner analogous to that of sv-40 large t antigen, a large multifunctional protein which exists in different oligomeric forms with discrete biochemical activities (reviewed in rigby and lane, 1983) . in respect of the varying ratios of the p proteins found in the xenopus-expressed complexes, it is interesting to note that complexes detected in infected cells do not necessarily seem to consist of equimolar quantities of all three proteins. akkina et al. (1987) reported that complexes deficient in pb2 were present in the cytoplasm, and showed data to suggest the existence of rnp-associated complexes with a less than equimolar ratio of pa (our own unpublished observations support the latter observation). it is therefore possible that different functions of the influenza rna polymerase may be attributable to different forms of the complex. the successful reconstruction of the influenza polymerase complex provides an indication of the potential of the xenopus system for reconstruction of an active polymerase. as yet we have been unable to observe reassociation of the xenopus-expressed polymerase complex with vrna, the next step toward reassembly of the transcription complex, because vrna is rapidly degraded in oocytes (data not shown). however, it may be possible to circumvent this problem by partially purifying the complexes from an oocyte lysate before incubating them with the rna substrate, or alternatively, by providing natural or artificially assembled rnp structures. various other expression systems have been used to study the influenza polymerase. krystal et a/. (1986) constructed cell lines expressing all three p proteins and showed that these were able to functionally complement viruses bearing ts lesions in the p protein genes. however, no biochemical data were reported on this system, possibly because of the low levels of expression obtained. st. angelo eta/. (1987) expressed the p proteins in recombinant baculoviruses, but although the system offered considerable promise in terms of the levels of protein expressed, as discussed above, only an incomplete polymerase complex was formed. recently, it was demonstrated that an active influenza polymerase could be reassociated from the purified polypeptide components of disrupted virion rnps by renaturation with escherichia co/i thioredoxin (szewczyk et al., 1988) . although this is a significant result and provides an exciting system for the study of the polymerase, it is ultimately limited to the examination of wild-type proteins. our approach has the significant advantage that mutant polypeptides can be generated easily by manipulation of the dna protein coding sequence. characterization of such altered polypeptides will allow a more detailed analysis of the structure and function of the polymerase proteins. intracellular localisation of the viral polymerase proteins in cells infected with influenza virus and cells expressing pbl protein from cloned cdna transcription of the influenza ribonucleic acid genome by a virion polymerase. 1. optimal conditions for in vitro activity of the ribonucleic acid-dependent ribonucleic acid polymerase activity molecular model of a eukaryotic transcription complex: functions and movements of influenza p proteins during capped rna-primed transcription expression of a functional influenza viral cap-recognising protein by using a bovine papilloma virus vector an efficient ribosomal frame-shifting signal in the polymerase encoding region of the coronavirus ibv translation of eukaryotic messenger rna inxenopus oocytes the three influenza virus polymerase (p) proteins not associated with viral nucleocapsids in the infected cell are in the form of a complex the effect of capping and polyadenylation on the stability, movement and translation of synthetic messenger rnas in xenopus oocytes influenza virus messenger rnas are incomplete transcripts of the genome rnas transcription of the influenza virus genome influenza virus, an rna virus, synthesises its messenger rna in the nucleus of infected cells polypeptides specified by the influenza virus genome. 1. evidence for eight distinct gene products specified by fowl plague virus functional messenger rnas are produced by sp6 in vitro transcription of cloned cdnas expression of the three influenza virus polymerase proteins in a single cell allows growth complementation of viral mutants cleavage of structural proteins during the assembly of the head of bacteriophage t4 molecular cloning: a laboratory manual messenger rna translation in reticulocyte lysate transfer of 5-terminal cap of globin mrna to influenza viral complementary rna during transcription in vitro structure and function of simian virus 40 large t-antigen two of the three influenza viral polymerase proteins expressed by using baculovirus vectors form a complex in insect cells construction of a newfamilyof high efficiency expression vectors: identification of cdna clones coding for human liver proteins purification, thioredoxin renaturation, and enzymatic activity of the three subunits of the influenza a virus rna polymerase electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications cloning and expression of influenza virus genes we thank dr. p. palese for the gift of the influenza cdna clones, dr. a. colman for the plasmid psp64-t, and dr. c. dingwall for advice and oocytes. this work was supported in part by mrc grant ge62397 1 ca and a grant from the wellcome foundation. p. digard thanks the states of guernsey education authority for support. key: cord-294056-7e477y1x authors: la monica, nicola; yokomori, kyoko; lai, michael m.c. title: coronavirus mrna synthesis: identification of novel transcription initiation signals which are differentially regulated by different leader sequences date: 1992-05-31 journal: virology doi: 10.1016/0042-6822(92)90774-j sha: doc_id: 294056 cord_uid: 7e477y1x abstract the mrna synthesis of mouse hepatitis virus (mhv) has been proposed to be the result of interaction between the leader rna and the intergenic sites. previously, we have identified a transcription initiation site (for mrna 2-1), which is more efficiently transcribed by viruses containing two copies of ucuaa sequence in the leader rna than by those with three copies. in this study, we have identified several sites which are regulated in the opposite way, namely, they are efficiently transcribed by the leader rna with three ucuaa copies but not by those with two copies. these sites were characterized by primer extension and amplification by polymerase chain reaction. one of these sites is in the gene 3 region of a recombinant virus between a59 and jhm strains of mhv. another is in the gene 2 region of mhv-1 strain. both of these sites have a sequence similar to but different from the consensus transcription initiation signal (ucuaaucuauc and uuuaaucuu, as opposed to ucuaaac). these two novel intergenic sequences are not present in the genome of the jhm strain, consistent with the absence of these mrnas in the jhm-infected cells. the discovery of this type of transcription initiation site provides additional evidence for the importance of the leader rna in the transcription initiation of mhv mrnas. mouse hepatitis virus (mhv), a member of the coronaviridae, is an enveloped virus containing a nonsegmented positive-sensed rna genome of 31 kb (i, 2). mhv synthesizes seven to eight virus-specific mrnas that have a 3'-coterminal nested-set structure (1, 3). the 5' unique portion of each mrna is utilized for translation (4, 5). these mrnas encode four structural proteins, including s (spike), translated from mrna 3, n (nucleocapsid) from mrna 7, m (membrane) from mrna 6, and he (hemagglutinin-esterase) from mrna 2-l. other mrnas encode nonstructural proteins, which have not been well characterized [for review, see ref. (i) ]. mrna 2-1 is unusual in that it is synthesized by only some, but not all, of the mhv strains (6); its presence or absence is apparently regulated by a stretch of leader sequence located at the 5'-end of the genomic rna (7). this leader rna sequence (approximately 72 nucleotides) is also found at the 5'-end of every mhv mrna species (8). the leader rna makes only a small quantity of mrna 2-1, while the same virus with two copies makes a large quantity of this transcript (7, 10). the amounts of some other mrnas, e.g., mrna 2 and 4, are also affected, but to a much smaller extent (7, 10). at the 3'-end of the leader sequence, there are several copies of a repeat pentanucleotide sequence ucuaa, whose copy number varies from two to four, depending on the virus strain (9) and decreases upon serial passage of the virus in vitro (70). significantly, a jhm virus which contains three copies of ucuaa in ' present address: lstituto di ricerche di biologia molecolare, pomezia, rome, italy. the level of mrna 2-l expression can also be affected by the sequence at the transcription initiation site for this mrna. for example, the a59 strain of mhv has a single base substitution within this transcription initiation site and does not synthesize mrna 2-1, despite the presence of two ucuaa copies (7, 11). these results suggest that mhv mrna synthesis is determined by the interaction between the leader rna and the intergenic sites. thus, the intergenic sequence for gene 2-1 may represent a unique class of sequence which is differentially regulated by leader rnas with different ucuaa copy numbers. the basis for this differential regulation of transcription is not clear. to understand the mechanism of mhv mrna transcription, we have attempted to determine whether the differential regulation of transcription initiation by leader rna containing different ucuaa copy numbers is unique to mrna 2-l. in this report, we have identified several additional transcription initiation sites which are regulated in different manners by leader rna with different copy numbers of the ucuaa sequence. these studies provide further insight into the mechanism of mhv mrna transcription. ' has a genome structure in which the 5'-end of the genome, including the leader rna and part of gene 1, was derived from jhm, while the remaining sequence was from a59 (12). this virus synthesized a novel mrna 3-1 (previously named 3a), which was initiated within the gene 3 (12). by passaging bl virus in tissue culture, a new virus isolate was obtained, which differed from the original bl virus in that it had two copies of the ucuaa sequence in the leader, in contrast to the three copies in the original virus (10). this virus isolate synthesized only a trace amount of mrna 3-l (10). the analysis of several other recombinant viruses with different genome structures suggested that mrna 3-l was synthesized only by viruses with a leader rna containing three copies of ucuaa and gene 3 sequence derived from a59 but not jhm (10, 13). these observations suggested a new type of optional transcription initiation site, which was transcribed by the leader rna with three ucuaa copies but not those with two copies, opposite to the transcription pattern of mrna 2-l. we, therefore, attempted to further characterize this site. figure 1 shows that mrna 3-l was synthesized in large quantity in cells infected with bl(3) but not with bl(2) viruses, which contained three and two copies of ucuaa, respectively. however, neither jhm(2) nor jhm(3) synthesized this mrna, even though the latter contained three copies of ucuaa. thus, other se-quences, probably transcription initiation sequence, also accounted for the presence or absence of mrna 3-1 transcription. in contrast, mrna 2-l was synthesized only by jhm(2) but not by bl(2) viruses, although both had two ucuaa copies. this was consistent with our previous conclusions (7, io), since bl(2) virus had an a59-derived gene 2-1 sequence (12), which had a defective transcription initiation sequence for this gene (7, 11) . similar to the previous observation (7) jhm(2) synthesized a smaller amount of mrna 2 than jhm(3). however, the relative amount of this mrna was variable from virus preparation to preparation. an additional minor rna species, l-1 , was noted in b1(3)-and jhm(3)-but not in b1(2)-or jhm(2)-infected cells, suggesting that it was also a differentially regulated transcription initiation site, which was transcribed by the leader rna with three ucuaa copies, but not with two copies. several of these viruses also had additional primer extension analysis of the 5'.end of mrna 3-1 from bl(3). the synthetic oligonucleotide 348 was y-end-labeled with [y-32p]atp by polynucleotide kinase and extended with reverse transcriptase as previously described (9). briefly, poly(a)+ rna from bl(3))infected dbt cells was denatured with 10 mm methylmercury and incubated at 45" for 30 min in 50 ~1 reaction buffercontaining 60 u of rnasin (promega biotech), 10 mm mgci,, 100 mm kci, 50 mm tris-hydrochloride, ph 8.3, 10 mm dithiothreitol, 12.5 mm each of datp, dctp, dgtp, ttp, and 35 u avian myeloblastosis virus reverse transcriptase (seikagaku). reaction products were analyzed by electrophoresis on a 6% polyacrylamide gel containing 8.3 m urea. a sequencing ladder was used as size markers. the primer extended product is indicated by an arrow. note. the genomic sequence at the intergenic site for mrna 3-l was obtained by direct sequencing of cdna spanning this region, which had been amplified by pcr using oligomers 197 and 198 as specific primers. cdna synthesis and pcr amplification were carried out as previously described (6). oligomer 198 (5'.g-a-caagcttgaccaccggtgcaatta-3') was complementary to nucleotides 2676-2695 of gene 3, and oligomer 197 (b-gactcgaa ttcagtrgataagmgctgtaccccg-3') was identical to nucleotides 1166-l 191 of the same gene (14). the mhv-specific cdna products representing nucleotides 1166-2695 of gene 3 were excised from low melting agarose (seaplaque, fmc bioproducts), extracted with phenol/chloroform, and sequenced by dideoxyribonucleotide chain-termination method (16). sequence determination for the transcription initiation site of mrna 2-2 was described previously (6). underscored letters indicate diverged sequences. a sequence obtained from luytjes et al. (14). b not determined. minor rna species between mrnas 3 and 4. as discussed previously (6), these rnas most likely represented ribosomal rnas, which could be removed by purification on an oligo(dt) column. these results suggested further that mrna transcription depends not only on the leader sequence but also on the transcription initiation sequence. therefore, we determined the sequences of the transcription initiation sites for these optional rnas. from the nested-set structure of mhv mrnas and on the basis of the migration pattern of the rnas, the transcriptional start site of mrna 3-l was predicted to be located within gene 3 of bl(3) virus at a site approximately 2 kb downstream from the initiation site of mrna 3. to determine its exact location, we performed primer extension studies on poly(a)+ rna from b1(3)-infected dbt cells. a synthetic oligonucleotide (oligo 348, 5'-attaggaagcgcctcatccg-3!) complementary to nucleotides 2072-2092 of the published a59 gene 3 sequence (14) was used as a primer. the 5'-end-labeled primer was hybridized to poly(a)+ rna and extended with reverse transcriptase. primer extension products were then analyzed by electrophoresis on a 6%~ polyacrylamide gel containing 8 m urea (10). as shown in fig. 2 , an extension product of 204 nucleotides was detected. since the leader sequence of bl(3) was approximately 75 nucleotides long (8, 9) , this result suggested that the transcription initiation site of mrna 3-l was located around nucleotide 1955 of gene 3. this conclusion was also supported by the primer extension reaction using a different primer, oligo 42 (5'-gatacccagtagllxtg-3'), which was complementary to nucleotides 2151-2171 of gene 3 and was located 80 bases downstream of the oligo 348 binding site. the extension product was 284 nucleotides long (data not shown), consistent with the transcription initiation site determined using oligo 348 as a primer. to precisely define the transcription initiation site of mrna 3-1, the 5'-unique portion of mrna 3-l was cloned after amplification by polymerase chain reaction (pcr). for this purpose, intracellular rna from b1(3)-infected cells was reverse transcribed using oligo 42 as a primer and then amplified with oligo 78 (5'-agctttacgtaccctctctactctaaaactctt-tgtacttt-3') as a second primer. the latter primer was homologous to the middle portion of the leader rna and contained seven additional nucleotides representing the snabl site (15). the 280-nucleotide-long pcr product thus represented the 5'-end (including the leader sequence) of mrna 3-l and was cloned into the smal site of the ptz18u vector (u.s. biochemicals) by blunt-end ligation as previously described (6). nucleotide sequence analysis of cdna clones showed that the sequence of mrna 3-l diverged from that of the genomic rna immediately upstream of the sequence ucuaaucuauc, which was located at nucleotides 1950-l 961 of gene 3 (14), indicating that this region was the transcription initiation site of mrna 3-l (data not shown) (table 1) . significantly, the copy number of ucuaa sequence in the leader region of different cdna clones varied from two to four (data not shown). this observation was in agreement with the previously described heterogeneity of mhv mrna, possibly resulting from the imprecise interaction between the ucuaa sequence at the 3'-end of the leader rna and the transcription initiation sequence (9). this finding further established that this sequence represented a bona fide transcription initiation site of mhv mrna. however, the sequence determined here was different from the corresponding sequence of the published a59 gene 3 (14) (table l), even though bl(3) virus derived its gene 3 from the parental a59 virus (12). this result suggested that the parental a59 virus used in our laboratory may have a different sequence in this region. we therefore used the same procedures to sequence the a59 used in our laboratory. the results showed that its sequence was identical to that of bl(3) rnas were analyzed by electrophoresis as described in fig. 1 . mrna 2-l and 2-2 are indicated by asterisks. the di rnas are indicated by arrows. (b) the virion rna of p5-h, pl o-h, or p15-h virus was used for primer extension studies using a primer complementary to nucleotides 172-l 88 from the 5'.end of jhm genomic rna according to the procedures as described in fig. 2 . as demonstrated previously (9). the extended product was 188 nucleotides long if the leader rna contained three ucuaa copies (indicated by an arrow) and 183 nucleotides long if it contained two copies (indicated by an arrowhead). a dna sequencing ladder was run in parallel to serve as size markers. virus in this region, confirming that this was the authentic sequence of the parental and recombinant viruses (table 1) . this result also explained why mrna 3-1 was not synthesized by jhm (3), inasmuch as the sequence of jhm in this region was the same as that of the published a59, which did not make mrna 3-l (table 1). additionally, we sequenced bl(2) virus, which failed to make mrna 3-1, and found that its sequence was identical to that of bl(3) and the parental a59 within this region. the sequence identity between bl(2) and bl(3) extended for at least 100 nucleotides on either side of the transcription initiation site for mrna 3-1. therefore, we concluded that the difference in mrna 3-l synthesis by b1(2), b1(3), and a59 was most likely due to the difference in the copy number of the ucuaa pentanucleotide in the leader rna. we have recently identified another novel mrna species (mrna 2-2) in cells infected with another strain of mhv, mhv-1 (6). the transcription initiation sequence of this mrna was similar to but slightly different from the consensus sequence for mhv mrnas (table 1) . to determine whether this novel rna was differentially regulated by the copy number of ucuaa in the leader rna, we performed undiluted serial passages of mhv-1 in dbt cells. the 32p-labeled intracellular viral rna isolated from virus-infected cells at different passage levels were examined by electrophoresis on agarose gel. to rule out the effects of defective-interfering (di) rna, we have also examined intracellular viral rnas after a low multiplicity of infection (m.o.i.) passage of the different virus isolates to remove di rnas. figure 3a shows that the original mhv-1 synthesized mrna 2-2 in addition to the seven standard mrnas. after 10 passages in dbt cells, the virus did not synthesize mrna 2-2, but instead synthesized mrna 2-1, which had previously been characterized as being transcribed by the leader rna with two ucuaa copies (7). at both passages 5 and 8, both of these mrnas were synthesized. no difference in the intracellular viral rna pattern could be detected in the presence or absence of di rna at every passage level. to determine whether the change of mrna 2-1 and 2-2 expression correlated with the ucuaa copy number in the leader rna, we examined the structure of note. oligonucleotide primers complementary to different regions of gene 1 (around nucleotides 5942 and 7999, respectively) (2) were used separately as primers for reverse transcription of the intracellular rna from jhm(3)infected cells. a second primer (78) representing leader rna sequence (nucleotides 22 to 56) was then used for pcr amplification by ta9 polymerase. the pcr products were cloned into ptz18u vector and sequenced. the sequences were aligned with the published gene 1 sequence (2). the regions where the mrna sequences diverged from the published genomic sequences were identified, which represented the transcription initiation sites for these mrnas. the ucuaa copy numbers within the leader rna of each mrna are indicated. the nucleotide numbers indicated in the genomic sequences represent nucleotide positions from the y-end of the genome (2). the leader rna of mhv-1 at different passage levels by primer extension and sequencing. the results showed that both virus isolates from passage levels 10 and 15 had two ucuaa copies (fig. 38 ) in contrast to the original mhv-1, which had three ucuaa copies (6). interestingly, the virus at passage level 5 contained a mixture of two and three ucuaa copies, corresponding to the detection of both mrna 2-l and 2-2 (fig. 3a) . this result suggested that the leader sequence of mhv-1 underwent evolution from three to two ucuaa copies during passage in culture, similar to that of jhm virus (io), and that the synthesis of mrnas 2-2 and 2-l correlated well with the presence of three and two ucuaa copies in the leader, respectively. the entire mhv-1 leader sequence was identical to that of jhm (data not shown). this result indicated that the initiation site of mrna 2-2 of mhv-1 was utilized by the leader sequence with three but not two ucuaa copies. additionally, rna l-l was also found to be transcribed only by mhv-1 with three ucuaa copies, but not by those with two copies (fig. 3a) , consistent with the findings with jhm and bl viruses (fig. 1) . we attempted to determine the transcription initiation site of rna l-l by pcr cloning. using various primers representing sequences of different regions of gene 1 of the jhm strain of mhv (2) and a second primer representing the 5'-end of the leader rna, we detected several pcr products which represented mrnas initiated from various sites. these transcription initiation sequences appeared to bear some resemblance to the consensus transcription initiation signal (table 2) . we have also attempted to perform primer extension studies to determine which of these was the transcription initiation signal for rna l-l. however, since the transcription initiation site of rna l-l was located within a gene of 22 kb (2) and the molar amount of this rna was much less abundant than the other mrnas, the results were not conclusive. nevertheless, the detection of these pcr products with the correct leader sequence indicated that these sequences, which were similar to the consensus intergenie sequence, could be utilized as transcription initiation sites, although at a very low efficiency. to determine whether these optional and differentially regulated rnas encode any truncated products of the viral proteins, we examined the virus-specific protein synthesis in the cells infected with bl(3) and other viruses by labeling with [35s]methionine and precipitating with antiserum specific for mhv. no proteins other than the s, n, m and, in some cases, he, were detected (data not shown). thus, mrnas 2-2 and 3-l are most likely not functional. however, other possibilities, such as rapid turnover of the protein or the inefficiency of immunoprecipitation because of the change of epitopes on the protein, cannot be ruled out. since the gene product of mrna 1 is a nonstructural protein and has not been identified, we cannot determine whether mrna l-l, which represents a truncated version of mrna 1, is functional. these studies have identified, thus far, at least four transcription initiation sites which were regulated in a different manner by the leader rna with different copy numbers of ucuaa sequence. several of these (mrnas 2-2, 3-1, and l-1) were transcribed efficiently by the leader sequence with three ucuaa copies, while the other (mrna 2-l) was transcribed efficiently by those with two copies. it is not clear how the interaction of these intergenic sequences with the leader rna containing different copy numbers of ucuaa can affect transcription initiation. it is possible that this interaction requires rna-protein interactions involving rna polymerase and transcription factors, but not simply base-pairing between the leader rna and the intergenie sequences. the three-dimensional structure of rna may be affected by the copy number of ucuaa. since these intergenic sequences are identical in the same pair of viruses studied here, these results strongly suggest that the leader rna plays a regulatory role for transcription. this interpretation is most consistent with the leader-primed transcription mechanism (1) proposed for mhv mrna synthesis. proc. nat/. acad. sci. usa key: cord-280957-cdd6ngf1 authors: narkpuk, jaraspim; jongkaewwattana, anan; teeravechyan, samaporn title: the avian influenza virus pa segment mediates strain-specific antagonism of bst-2/tetherin date: 2018-10-02 journal: virology doi: 10.1016/j.virol.2018.09.016 sha: doc_id: 280957 cord_uid: cdd6ngf1 bst-2 is an antiviral protein described as a powerful cross-species transmission barrier for simian immunodeficiency viruses. influenza viruses appear to interact with bst-2, raising the possibility that bst-2 may be a barrier for cross-species transmission. an mdck-based cell line expressing human bst-2 was generated to study human-derived a/puerto rico/8/36 (h1n1; pr8) as well as two low pathogenic avian influenza viruses (subtypes h4n6 and h6n1). the h4n6 and h6n1 viruses were less affected by bst-2 expression than pr8, due to their ability to decrease bst-2 levels, a function localized to the pa segment of both avian viruses. experiments with pa-mutant and -chimeric viruses confirmed that the avian pa segment conferred bst-2 downregulation and antagonism. these results indicate a species-specific ability of pa from low pathogenic avian viruses to mitigate human bst-2 antiviral activity, suggesting that bst-2 is unlikely to be a general cross-species barrier to transmission of such viruses to humans. bst-2 (bone marrow stromal cell antigen 2), also known as tetherin, cd317 or hm1.24, is an interferon (ifn)-induced cellular protein that was initially described in 2008 as a restriction factor for human immunodeficiency virus type 1 (hiv-1) (neil et al., 2008; van damme et al., 2008) . while bst-2 has been almost exclusively studied as a mammalian antiviral protein, an in silico study identified a bst-2 ortholog as far back in vertebrate evolution as the elephant shark, dating the appearance of this gene to over 450 million years ago (heusinger et al., 2015) . other than in fish, this study also identified orthologs in marsupials, reptiles, and birds, with alligator bst-2 being tested and found to possess antiviral function against hiv-1 release. among birds, bst-2 was found in turkeys and chickens, but appears to have been lost through gene erosion among many bird species. as a type ii membrane protein, bst-2 possesses a c-terminal gpi (glycophosphatidylinositol) modification and an n-terminal transmembrane domain flanking an extracellular coiled coil central region, and is present on the cell surface as a homodimer (kupzig et al., 2003) . the resulting four-membrane-anchor conformation is considered central to the ability of bst-2 to restrict hiv-1 virion release, where it acts as a "tether" linking the membranes of budded virions to the host cell membrane (perez-caballero et al., 2009) . as bst-2 acts directly upon the host cell membrane rather than viral components, bst-2 does not target a specific virus but rather has been found capable of restricting virion release and spread for a range of enveloped viruses beyond the retroviruses, such as filoviruses (jouvenet et al., 2009; kaletsky et al., 2009; sakuma et al., 2009) , arenaviruses (radoshitzky et al., 2010; sakuma et al., 2009) , and various coronaviruses (taylor et al., 2015; wang et al., 2014) . many of the viruses described to be sensitive to bst-2 restriction are zoonotic. most notably, bst-2 has been described as a cross-species transmission barrier that shaped the evolution of the simian immunodeficiency virus (siv) and hiv (evans et al., 2010) . a recent publication also demonstrated the possible role of bst-2 as a crossspecies transmission barrier for various orthobunyaviruses (varela 2017), with human viruses being restricted by sheep bst-2 but not the human ortholog, and vice versa. also, equine bst-2 was observed to restrict the growth of both equine and human influenza viruses more effectively than human bst-2 (wang et al., 2018) . early reports examining the interplay between influenza viruses and bst-2 suggested that virus-like particles (vlps) but not wild-type viruses were susceptible to human bst-2 restriction (watanabe et al., 2011 , bruce et al., 2012 . these observations lent credence to the possibility that influenza viruses universally encode an antagonist to bst-2. contradictory reports soon emerged, however, of viruses inherently sensitive to bst-2 restriction (gnirss et al., 2015; hu et al., 2017; mangeat et al., 2012) . differential abilities of various influenza virus neuraminidases (na) in circumventing bst-2 activity (leyva-grado et al., 2014; yondola et al., 2011) made it apparent that influenza virus sensitivity to bst-2 is likely to be strain-specific. further studies supported the possibility that influenza virus na acts a strainspecific antagonist to bst-2 (leyva-grado et al., 2014; mangeat et al., 2012) . mangeat et al. also reported a reduction in bst-2 protein expression, which may be associated with hemagglutinin (ha) and na together (gnirss et al., 2015) or the m2 protein (hu et al., 2017) , but their observations of decreased bst-2 mrna levels during influenza virus infection remained unexplained. given the variety of influenza strains, host cells, and methodologies used to study the bst-2-influenza interplay, the conflicting data, at the very least, appear to suggest that a variety of influenza virus strains interact with and counteract bst-2 in some fashion. given that influenza is and remains an important zoonotic disease, the possibility of bst-2 being a host restriction factor that acts as a cross-species transmission barrier for avian influenza viruses is intriguing. thus far, however, studies examining the intersection between bst-2 and influenza viruses have mostly focused on human viruses, whether laboratory-adapted, seasonal, or pandemic. these viruses have been successful in maintaining themselves in the human population, suggesting that they possess an inherent capacity to circumvent or antagonize the antiviral activity of bst-2. therefore, we were interested in comparing human and low pathogenic avian influenza virus strains, which are generally not deemed a direct threat to human health, with the goal of identifying differences in their response to bst-2. to study the impact of bst-2 on human and avian influenza viruses, we first generated an mdck cell line stably expressing human bst-2 cloned from hela cells (narkpuk et al., 2014) (supplementary fig. 1 ). the mdck-bst-2 cell line generated by lentiviral transduction was examined by immunofluorescence staining and bst-2 was observed as punctate clusters on cell surface membranes (fig. 1a ), in accordance with expected bst-2 localization. cells were also probed for bst-2 expression by western blotting and revealed the characteristic pattern of monomeric and dimeric bst-2 in its various glycosylated forms (fig. 1b) . to test for strain-specific differences in sensitivity to human bst-2, we compared the low pathogenic avian viruses a/duck/hong kong/ 365/78 (h4n6) and a/duck/suphanburi/ai157/2005 (h6n1) against a/puerto rico/8/34 (h1n1) (pr8 for short). these viruses were used to infect mdck and mdck-bst-2 cells at a low multiplicity of infection (moi), and their supernatants were harvested at 48 h post-infection for plaque titration on mdck cells. as low pathogenic avian viruses have not been reported to cause disease in humans, we expected that the h4n6 and h6n1 viruses would be more susceptible to human bst-2 antiviral activity than pr8. surprisingly, the pr8 virus exhibited greater sensitivity to bst-2, with titers decreasing by around 1 log in mdck-bst-2 cells ( fig. 2a) . neither the h4n6 nor h6n1 virus appeared to be negatively affected by the presence of bst-2, with h4n6 showing a slight trend towards increased replication in mdck-bst-2 cells (fig. 2b, c) . when infected cells were probed to confirm bst-2 expression, western blotting revealed a dramatic disappearance of bst-2 protein in mdck-bst-2 cells infected by h4n6 and h6n1 viruses (fig. 2d ). similar levels of bst-2 protein down-regulation were not observed with pr8 infection prior to complete cell death. analysis of bst-2 mrna demonstrated that this reduction was accompanied by reduced mrna levels as well (fig. 2e ). pr8 infection also resulted in decreased bst-2 mrna levels, albeit not as drastically as infection with the avian viruses. while this result reflects the ability of influenza a viruses in general to mediate host shut-off (rivas et al., 2016) , it also suggests a difference in mrna down-regulation that may be associated with decreased levels of bst-2. alternatively, it is also possible that mrna and protein down-regulation effects occur through distinct mechanisms. these avian viruses should not have been under selection pressure to develop a specific antagonistic mechanism against human bst-2, due to the nature of their host range. these observations therefore pointed to a general host response inhibition factor present in the virus. the influenza virus nonstructural protein 1 (ns1), in particular, is known for its ability to inhibit an ifn-induced antiviral state through a variety of activities such as blocking host sensors of viral rna and signal transduction pathways leading to induction of ifn expression (marc, 2014) . as bst-2 expression in our cell line is driven by a constitutive promoter, however, we do not expect that these particular activities will be involved in bst-2 down-regulation. to determine which viral factors of h4n6 and h6n1 are involved in decreasing bst-2 levels, we studied the impact of each of their genomic segments on bst-2 expression in the context of transfection. we amplified each segment from viral rna for cloning into the reverse genetics vector phw2000. individual genomic segments of pr8, h4n6, and h6n1 were then co-transfected with a bst-2 expression plasmid into hek293t cells for western blot analysis. while none of the pr8 genomic segments affected bst-2 expression (fig. 3a) , the pa of h4n6 and h6n1 notably reduced bst-2 protein levels ( fig. 3b, c) . additionally, the h6n1 ns segment also exhibited the capacity to reduce protein expression, which is likely due to ns1-associated host shut-off capabilities (marc, 2014) . as the effect of the pa segment appeared common to both low pathogenic avian viruses, we further tested the effect of pa on bst-2 among all the pa genes tested, the low pathogenic avian influenza viruses h4n6 and h6n1 resulted in the most significant decrease in bst-2 protein levels ( fig. 3d , supplementary fig. 2a ). the pandemic h1n1 strain (non), which has an avian-derived pa, along with the highly pathogenic h5n1 pa, had a noticeable, albeit very mild, effect on bst-2 levels. the pr8 and h3n2 viruses had little observable impact on bst-2 levels. pa protein levels roughly corresponded to their effect on bst-2 levels; h4n6 and h6n1 had the greatest amounts of pa protein, followed by the highly pathogenic h5n1 and the pandemic h1n1. pr8 and h3n2 pa levels were notably lower, likely due to the presence of bst-2 itself, which has been previously shown to decrease expression of co-transfected genes (narkpuk et al., 2014) . indeed, in the absence of bst-2, pa of pr8 and the h3n2 and h5n1 viruses were expressed at higher levels, whereas h4n6 and h6n1 pa were expressed at lower levels (fig. 3e ). these results suggest that the h4n6 and h6n1 pa-mediated down-regulation of bst-2 may pre-empt bst-2 activity, preventing bst-2 from decreasing pa expression. the main product of the pa segment is the pa protein, which functions as an endonuclease (fodor et al., 2002; hara et al., 2006) . interestingly, this segment also encodes an alternative product, pa-x, containing the n-terminal endonuclease domain of pa fused to a frameshifted c-terminal tail (jagger et al., 2012) . pa-x has been demonstrated to actively target host rna polymerase ii-derived transcripts for cleavage (khaperskyy et al., 2016) and is thought to play a role in decreasing host competition for resources as well as down-regulating host responses (hayashi et al., 2015; jagger et al., 2012) . in addition, it displays host-and strain-specific impact on influenza virus pathogenicity and transmissibility (hu et al., 2018) . to dissect the roles of both pa segment products in mediating bst-2 down-regulation, we mutated the pa segment based on work by jagger et al., which had identified the frameshift motif (ucc uuu cgu c; fig. 4a ) required for pa-x expression as well as narrowed down the essential residues to 3 positions that resulted in under 0.1% frameshifting efficiency when mutated (ucc uuu cgu c to ucc uuc aga c) (jagger et al., 2012) . they also generated a single c nucleotide deletion mutant (ucc uuu cgu c to ucc uuu -gu c) which shifted the pa-x orf (open reading frame) into a position in-frame with the start codon, resulting in expression of pa-x but not pa. we emulated their mutagenesis approach for pr8 and the h6n1 virus ( fig. 4b ), mutating the frameshift motif to either reduce frameshifting into the pa-x orf (fs) or to force expression solely in the pa-x orf (δc), and assessed which orf is responsible for the observed decreases in bst-2 levels. wild-type or mutant pa was co-transfected with a bst-2 expression plasmid into hek293t cells, and cell lysates were harvested for western blot analysis (fig. 4c, supplementary fig. 2b ). as seen previously, the h6n1 wild-type pa decreased bst-2 levels to a much greater extent than the pr8 wild-type pa. the h6n1 δc pa, which is frameshifted to express only the pa-x orf, resulted in undetectable levels of bst-2 while the fs pa lost its activity. these results indicate that the effect of the h6n1 pa segment on bst-2 levels may be attributed to the pa-x orf. similarly, the pr8 δc mutant decreased bst-2 levels while the fs mutant had no effect, further demonstrating that the pr8 pa-x orf also has the ability to decrease bst-2 protein levels in transfected cells. however, because pr8 wild-type and δc pa both exhibit less down-regulation of bst-2 protein compared to its h6n1 counterparts, we surmise that pr8 pa-x expression levels or activity may be lower. since our observations demonstrated impact of the avian pa segment on bst-2 down-regulation in the context of transfection, we were interested in examining whether or not such impact translated to viral growth in the context of infection. first, we generated chimeric pr8 and h6n1 viruses where the pa, ns or both pa and ns segments were swapped, as well as pr8 and h6n1 viruses carrying the fs mutation in their pa segments. mdck-bst-2 cells were infected with these viruses at an moi of 0.001, and cell lysates were assessed for bst-2 and viral np (nucleoprotein) expression (fig. 5a, supplementary fig. 2c ). the pr8-based chimeras carrying h6n1 ns and/or wild-type pa were capable of decreasing bst-2 levels, with the strongest impact being seen with the wild-type pa segment alone. the pr8 viruses with fs pa, either its own or that of h6n1, did not exhibit bst-2 down-regulation. on the other hand, the h6n1 chimeric viruses carrying either the pr8 wild-type pa or both pr8 ns and wild-type pa completely lost their ability to decrease bst-2 levels. the h6n1 fs mutant lost most of their activity, while the virus carrying pr8 ns lost some activity but still yielded noticeably decreased bst-2 levels. these results suggest that h6n1 pa plays the primary role in down-regulating bst-2, with some contribution from its ns segment. interestingly, the presence of both h6n1 ns and wild-type pa in pr8 did not result in an additive effect on bst-2 levels, but rather resulting in a weaker effect on bst-2 than h6n1 wild-type pa alone. this suggests that viral context also plays a role in how pa and ns manifest their bst-2 antagonism. to examine the role of the pa segment further, we infected mdck-bst-2 cells at an moi of 5 and incubated the cells for 18 h in the absence of tpck-treated trypsin to limit the viruses to a single round of growth. this would allow us to examine the impact of viral infection on bst-2 levels without being confounded by differences in viral spread due to variable bst-2 sensitivity. western blotting results reflect our transfection observations, although to a less dramatic extent (fig. 5b , supplementary fig. 2d) , likely due to the single-round nature of these infections. the h6n1 virus resulted in the lowest bst-2 levels, followed by pr8 (h6n1 pa), the pr8 virus carrying the h6n1 pa segment. bst-2 mrna levels were also examined (fig. 5c) . loss of frameshifting into the pa-x orf resulted in notable losses in the ability to down-regulate bst-2 mrna, strongly suggesting a role for pa-x in controlling bst-2 transcription. however, swapping the pa segment as a whole did not affect bst-2 mrna levels significantly. considering the impact of the pa segment on bst-2 protein levels ( fig. 5a and b ), this appears to indicate a distinct role for the pa segment as a whole in bst-2 protein down-regulation. to assess the role of the pa segment in rescuing viral replication in the presence of bst-2, we assessed the growth kinetics of pr8 (h6n1 pa) and h6n1 (pr8 pa) alongside the wild-type viruses (fig. 5 d-g) . mdck or mdck-bst-2 cells were infected and their supernatants were harvested at various time points for plaque titration on mdck cells. the growth kinetics data for the wild-type viruses reflect those observed previously, with pr8 exhibiting sensitivity to bst-2 while h6n1 replicated similarly in both mdck and mdck-bst-2 cells. with the chimeric viruses, the h6n1 pa was able to confer bst-2 resistance to pr8, while h6n1 (pr8 pa) was rendered sensitive to bst-2. all together, our data suggest that the pa segment carries the determinants for bst-2 resistance in our study system, with the avian h6n1 pa antagonizing bst-2 by down-regulating its expression at both mrna and protein levels. several studies have examined the impact of bst-2 on influenza virus replication, and a handful of viral proteins have been proposed as possible antagonists of human bst-2. given the zoonotic nature of influenza viruses, we were interested in exploring this question from the perspective of human bst-2 acting as a cross-species transmission barrier for avian influenza viruses. we report here that, contrary to our initial hypothesis that low pathogenic avian influenza viruses incapable of transmission to human hosts would be strongly restricted by human bst-2, h4n6 and h6n1 viruses grew to robust titers in mdck cells constitutively expressing bst-2 and appeared completely resistant to its antiviral activity. mechanistically, our data suggest that this resistance is likely conferred by the drastic down-regulation of bst-2 levels by the avian pa segment through the reported ability of pa-x to mediate general host shut-off (jagger et al., 2012) . before discussing the implications of our pa-associated findings, it is important to note that the h6n1 ns segment also exhibited the ability to decrease bst-2 protein levels. unlike pr8, this h6n1 virus contains the f103 and m106 residues in its ns1 protein that are associated with host cpsf30 (30-kda subunit of the cleavage and polyadenylation specificity factor) interaction and ns1-mediated host-shut-off (das et al., 2008) . cpsf30 interaction may not be directly related to bst-2 down-regulation or the sole contributor to this effect, however, as the h4n6 virus also carries this motif despite having an ns segment that did not decrease bst-2 protein levels. the h4n6 ns1 protein is divergent from h6n1 ns1 at five positions, carrying k20, i129, i137, a155, and s165 instead of r20, t129, n137, v155, and f165. interestingly, three of these distinct h4n6 ns1 residues are shared with pr8, namely those at positions 129, 155, and 165. residue 165, in particular, is located in the sh3 binding motif 1 that is involved in interaction with the pi3k (phosphatidylinositol 3-kinase) p85β subunit (shin et al., 2007) . while we did not extensively examine the role of ns1 nor these residues in the down-regulation of bst-2, ns1 of a/wsn/33 (h1n1) and a/texas/36/91 were previously reported to prevent the induction of bst-2 upon ifn-α treatment (mangeat et al., 2012) . this effect is not evident in most influenza virus-bst-2 studies which have focused on engineered cell lines constitutively expressing bst-2 (bruce et al., 2012; winkler et al., 2012) . even so, our data revealed a strain-specific ability of ns1 to decrease bst-2 levels during infection in such a cell line, supporting the diversity of ns1 functionality in modulating the host innate immune response beyond its role in ifn suppression. the other products of the ns segment, nep (nuclear export protein) and ns3 are unlikely to be associated with the observed impact of h6n1 ns on bst-2, however. the ns3 splicing motif, an a to g mutation at nucleotide position 374 (selman et al., 2012) , is not present in any of the three viruses, while the nep protein sequence is perfectly conserved between the h4n6 and h6n1 viruses. additionally, while we focused primarily on the pa segment, our results do not rule out the possibility that other viral proteins encoded on two or more segments may work in concert in order to effect bst-2 decrease, as previously seen with na and ha (gnirss et al., 2015; hu et al., 2015) . neither do these results discount the presence of other anti-bst-2 mechanisms involved in sequestering or altering bst-2 localization, which would thereby decrease the impact of bst-2 on influenza virus replication through means other than direct down-regulation of bst-2 expression as assessed by mrna and protein levels. early on during this work, we hypothesized that pa-x, first described in 2012 by jagger et al. (2012) as a frameshift product of the pa gene with endonuclease and host shut-off activity, might be responsible for bst-2 down-regulation as both protein and mrna levels were affected. indeed, in the transfection studies, the pa-x orf appears to be strongly involved in mediating the decrease in bst-2 expression levels. this effect was likely due to endonuclease-mediated cleavage of rna, preventing protein translation. likewise, decreases in both bst-2 mrna and protein were observed in infected cells. suppression of bst-2 mrna during infection that has previously been reported (mangeat et al., 2012) could likely also be explained by the effect of the pa segment as well. however, the interpretation of pa and pa-x orf involvement in bst-2 down-regulation changes slightly in the context of infection, as bst-2 is pre-expressed in the mdck-bst-2 cell line. the ability of h4n6 and h6n1 infection to reduce both bst-2 mrna and protein levels suggest that there is either post-translational down-regulation or extremely rapid turnover of bst-2, or both. if the former effect is indeed the case, it would be interesting to map out the mechanism by which pa-x may have exerted such a dramatic effect on protein stability. nevertheless, our infection studies (figs. 2 and 5) show a distinct difference between bst-2 mrna and protein levels in pr8-infected cells, where mrna levels are decreased but protein levels are not. this suggests that the endonuclease and protein down-regulation activities may be distinct. alternatively, the impact of mrna degradation may be amplified at a certain threshold due to high bst-2 turnover. in our transfection experiments with mutant pa, we found that the h6n1 wild-type pa exhibited a bst-2 restriction phenotype similar to the δc pa mutant which expresses only the pa-x orf, whereas the pr8 wild-type pa phenotype was reflected in the fs pa mutant. these observations suggest that the pa-x orf may be fundamentally different between the two viruses. it is possible that the h6n1 pa-x protein is expressed at very high levels from the wild-type pa gene, similar to that of the δc mutant. this in turn implies that expression of the full-length pa protein itself may suffer as a result, due to higher rates of ribosomal frameshifting during translation. in other words, h6n1 and pr8 pa may differ in frameshifting rates as determined by factors outside the identified, highly-conserved frameshift motif. alternatively, activity of their pa-x may be different despite neither having a truncated pa-x associated with changes in protein activity (bavagnoli et al., 2015; gao et al., 2015) . the significance of the pa-x orf was further highlighted in our attempts to rescue the h4n6 virus. this virus was not used in the later viral mutagenesis studies because we could not rescue it through reverse genetics in mdck cells. intriguingly, we were able to successfully rescue one virus: the h4n6 fs mutant carrying the frameshift motif mutation in the pa gene, shown previously to decrease pa-x expression (jagger et al., 2012) . that this mutation was able to render the recombinant h4n6 virus rescuable in cell culture suggests a role for the pa-x orf in modulating viral growth. indeed, pa-x has been described to suppress viral polymerase activity in certain contexts (gong et al., 2017; hu et al., 2015) , and its loss was accordingly associated with increased viral replication in both tissue culture and infected animals. it is tempting to speculate that this shifting of balance between polymerase activity and host shut-off may play some role in host tropism and cross-species transmission. a recent publication by hu et al. describes a very similar study to the work we have reported here (hu et al., 2017) . from the observation that infection by a/wsn/33 (h1n1) reduced bst-2 levels, they assessed the major viral proteins individually for their effect on bst-2 expression and identified m2 as responsible for bst-2 down-regulation. while we did not specifically separate m1 and m2 expression in our study, the m segments of pr8, h4n6, and h6n1 as a whole did not confer bst-2 down-regulation. similarly, bruce et al. tested m2-deficient pr8 and found that loss of m2 did not notably affect alter its sensitivity to bst-2 (bruce et al., 2012) . on the other hand, hu et al. did not observe any impact of pa on bst-2 levels; this indicates that a/ wsn/33 pa may be similar to that of pr8, whose pa also lacks the activity seen with h4n6 and h6n1 viruses. these disparate, seemingly contradictory results actually point towards a single conclusion-that influenza a resistance to and antagonism of bst-2 is strain-specific, and that the role of the pa segment may possibly be associated with low pathogenic avian strains. overall, our data further elaborate upon the interaction between the antiviral protein bst-2 and influenza viruses. we identified the pa segment as a species-specific determinant of sensitivity to bst-2, with those of low pathogenic avian influenza viruses exhibiting the ability to down-regulate human bst-2 in both transfection and infection contexts. that such drastic impact was seen even with high levels of preexpressed protein that cannot be controlled pre-translationally by ns1 suggests that bst-2 does not necessarily act as a cross-species barrier for transmission of low pathogenic avian influenza viruses to humans. furthermore, as these effects were surprisingly not observed to the same extent with tested human and human-derived viruses, we hypothesize that the avian pa segment may exhibit a different pa-pa-x balance that would translate into a disproportionately large impact on balancing host-shut off with viral replication compared to human viruses. human embryonic kidney (hek) 293t and madin-darby canine kidney (mdck) cells were maintained in opti-mem (life technologies) supplemented with 10% fetal bovine serum (fbs). cells were passaged twice weekly by detaching with 0.25% trypsin, and kept in a 37°c incubator at 5% co 2 . influenza viruses, and their proteins, studied in this work comprise a/puerto rico/8/34 (h1n1) (pr8) and a/duck/hong kong/365/78 (h4n6), gifts from dr. robert g. webster, st. jude children's research hospital, tennessee, usa (hoffmann et al., 2000) the human bst-2 coding sequence was amplified from phw-bst-2 (narkpuk et al., 2014) with primers carrying the bamhi and noti restriction sites for cloning into the lentiviral vector psin-csgw-ubem (a gift from dr. yasuhiro ikeda, mayo clinic, minnesota, usa). hek293t cells were transfected with the lentiviral vector along with vsv-g-expressing pmd-g (naldini et al., 1996) and the packaging plasmid pcmv-δr8.91 (zufferey et al., 1997) , and supernatants containing lentiviral particles were harvested 48 h post-transfection. the supernatants were clarified by centrifugation and used to infect mdck cells seeded in a 60-mm dish. polybrene (sigma-aldrich) was added to the infectious supernatant at a concentration of 8 μg/ml. single clones of transduced mdck cells were prepared and expression of bst-2 by these clones (mdck-bst-2) was verified by western blotting. mdck-bst-2 cells were plated at a density of 5 × 10 4 cells in a lab-tek ii chamber slide (thermo scientific) and incubated overnight. cells were washed with phosphate-buffered saline (pbs) and fixed with 4% formaldehyde for 5 min. after washing with pbs, cells were blocked with blocking buffer (pbs supplemented with 10% fbs and 1% bsa) for one hour at room temperature, probed with a 1:1000 dilution of a rabbit anti-bst-2 antibody (santa cruz biotechnology) in blocking buffer for one hour, washed in pbs, and incubated with a 1:1000 dilution of alexa-488-conjugated goat anti-rabbit igg (abcam) for another hour. after a final washing step with pbs, cells were stained with dapi and imaged by fluorescence microscopy. viral rna from the a/duck/hong kong/365/78 (h4n6) and a/ duck/suphanburi/ai157/2007 (h6n1) viruses were extracted using the viral nucleic acid extraction ii kit (geneaid). the rna was used as a template for rt-pcr using takara's one step rt-pcr kit (clontech) along with primers containing the bsmbi restriction sites and specific for the 5′ and 3′ untranslated regions of each genomic rna segment (hoffmann et al., 2001) . amplified cdna products were digested with bsmbi and inserted into the phw2000 vector (hoffmann et al., 2000) . selected clones were sequenced and compared to the official sequence reports on genbank. any nucleotide position that did not match sequence data on genbank was corrected by site-directed mutagenesis. pa mutants were generated following the mutagenesis approach reported by jagger et al. (2012) . the δc mutants were generated with primers covering the frameshift motif with a single c598 nucleotide deletion. the fs (frameshift) mutants were generated with primers covering the frameshift motif with mutations of residues 597-600 from ucgu to caga. six-well plates were seeded with co-cultures of 7.5 × 10 5 hek293t cells and 2.5 × 10 5 mdck cells overnight prior to transfection with eight phw2000-based plasmids encoding the eight influenza virus genomic segments (500 ng each). twenty-four hours after transfection, the cells were washed and replenished with serum-free opti-mem supplemented with 1 μg/ml tpck-treated trypsin. reverse genetics supernatants were harvested once extensive cell death was observed (typically 48 h post-transfection) and clarified by centrifugation at 230 ×g prior to injection into 10-day old embryonated chicken eggs. the eggs were incubated at 37°c for 48 h before the allantoic fluid was collected for virus. allantoic fluid was clarified by centrifugation at 230 ×g and sterilized by passage through a 0.20 µm syringe filter before storage at −80°c. to verify the sequence of these reverse genetics-derived viruses, rna from the viruses were extracted using the viral nucleic acid extraction ii kit. rt-pcr was performed using the superscript iii onestep rt-pcr system (invitrogen) and the products were then submitted for sequencing (1st base, malaysia). to study growth kinetics, mdck or mdck-bst-2 cells were plated at a density of 5 × 10 5 cells per well in 6-well plates overnight and infected with virus at an moi of 0.001. after an hour of virus adsorption at 37°c, the cells were washed with pbs and replenished with serumfree opti-mem supplemented with 1 μg/ml tpck-treated trypsin. supernatant samples were collected at various times points for plaque titration. to assess the impact of chimeric and mutant viruses on bst-2 expression, an moi of 5 was used for infection and cells were incubated in the absence of tcpk-treated trypsin for 18 h prior to harvest. mdck cells were plated at a density of 8 × 10 5 cells per well in 6well plates and incubated overnight. influenza viruses from cell culture supernatants were prepared as serial 10-fold dilutions and appropriate dilutions added to seeded cells. cells were incubated at 37°c for 1 h and then washed with pbs before being overlaid with agar in minimum essential medium (mem) supplemented with 1 μg/ml tpck-treated trypsin. after another 48-72 h at 37°c, the agar was removed and the cells fixed with 0.1% crystal violet in formaldehyde. viral titers were calculated by counting the number of plaques in the well containing 10-100 plaques and then multiplying that number by the dilution factor. log transformation was applied to viral titer readouts prior to assessment of statistical significance with student's two-tailed t-test. infected or transfected cells were lysed in radioimmunoprecipitation (ripa) buffer (50 mm tris-cl ph 7.4, 120 mm nacl, 1% triton x-100, 0.5% sodium deoxycholate, 1% sodium dodecyl sulfate) supplemented with 1% proteoblock protease inhibitor cocktail (thermo scientific)). lysates were loaded into 10 or 12% polyacrylamide gels for sds-page, and subsequently transferred onto nitrocellulose membranes. membranes were blocked with 5% skim milk in 0.1% tween 20-tris-buffered saline (tbs) for at least one hour before probing with antibody. human bst-2 was detected with a rabbit anti-human bst-2 antibody (santa cruz biotechnology), influenza virus pa with a rabbit anti-pa antibody (thermo scientific), and β-actin with a mouse anti-β-actin antibody (santa cruz biotechnology). secondary antibodies (horseradish peroxidase-conjugated goat anti-rabbit igg and goat anti-mouse igg antibodies) were purchased from santa cruz biotechnology. bands were visualized using the clarity western ecl substrate chemiluminescence kit (bio-rad) by film exposure. films were scanned using the molecular imager chemidoc xrs+ imaging system (bio-rad) and analyzed by densitometry using imagej. infected mdck and mdck-bst-2 cells were harvested in pbs at various time points. half the harvested cells were used for genomic dna extraction using the genejet genomic dna purification kit (thermo scientific), and the remaining half used for rna extraction using the genejet rna purification kit (thermo scientific). extractions were carried out according to manufacturer instructions. quantitative pcr (qpcr) was performed on the genomic dna to assess relative fibronectin copy number using the ssoadvanced universal sybr green supermix (bio-rad) and the two-step qpcr protocol according to manufacturer instructions. rt-qpcr was used to assess mrna levels of the human bst-2 transgene using the itaq universal sybr green onestep kit (bio-rad) and the associated two-step protocol according to manufacturer instructions. the bio-rad c1000 touch thermal cycler was used to run these qpcr experiments. statistical significance was assessed with student's two-tailed t-test. the novel influenza a virus protein pa-x and its naturally deleted variant show different enzymatic properties in comparison to the viral endonuclease pa release of filamentous and spherical influenza a virus is not restricted by tetherin structural basis for suppression of a host antiviral response by influenza a virus bst-2/tetherin: a new component of the innate immune response to enveloped viruses a single amino acid mutation in the pa subunit of the influenza virus rna polymerase inhibits endonucleolytic cleavage of capped rnas twenty amino acids at the c-terminus of pa-x are associated with increased influenza a virus replication and pathogenicity tetherin sensitivity of influenza a viruses is strain specific: role of hemagglutinin and neuraminidase pa-x protein decreases replication and pathogenicity of swine influenza virus in cultured cells and mouse models amino acid residues in the nterminal region of the pa subunit of influenza a virus rna polymerase play a critical role in protein stability, endonuclease activity, cap binding, and virion rna promoter binding influenza a virus protein pa-x contributes to viral growth and suppression of the host antiviral and immune responses early vertebrate evolution of the host restriction factor tetherin a dna transfection system for generation of influenza a virus from eight plasmids universal primer set for the full-length amplification of all influenza a viruses pa-x: a key regulator of influenza a virus pathogenicity and host immune responses pa-x decreases the pathogenicity of highly pathogenic h5n1 influenza a virus in avian species by inhibiting virus replication and host response bst-2 restricts iav release and is countered by the viral m2 protein an overlapping protein-coding region in influenza a virus segment 3 modulates the host response broad-spectrum inhibition of retroviral and filoviral particle release by tetherin tetherin-mediated restriction of filovirus budding is antagonized by the ebola glycoprotein selective degradation of host rna polymerase ii transcripts by influenza a virus pa-x host shutoff protein bst-2/ hm1.24 is a raft-associated apical membrane protein with an unusual topology modulation of an ectodomain motif in the influenza a virus neuraminidase alters tetherin sensitivity and results in virus attenuation in vivo influenza virus partially counteracts restriction imposed by tetherin/bst-2 influenza virus non-structural protein ns1: interferon antagonism and beyond in vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector an unconventional bst-2 function: down-regulation of transient protein expression tetherin inhibits retrovirus release and is antagonized by hiv-1 vpu tetherin inhibits hiv-1 release by directly tethering virions to cells infectious lassa virus, but not filoviruses, is restricted by bst-2/ tetherin shutoff of host gene expression in influenza a virus and herpesviruses: similar mechanisms and common themes inhibition of lassa and marburg virus production by tetherin adaptive mutation in influenza a virus non-structural gene is linked to host switching and induces a novel protein by alternative splicing sh3 binding motif 1 in influenza a virus ns1 protein is essential for pi3k/akt signaling pathway activation severe acute respiratory syndrome coronavirus orf7a inhibits bone marrow stromal antigen 2 virion tethering through a novel mechanism of glycosylation interference the interferon-induced protein bst-2 restricts hiv-1 release and is downregulated from the cell surface by the viral vpu protein strain-specific antagonism of the human h1n1 influenza a virus against equine tetherin bst2/cd317 counteracts human coronavirus 229e productive infection by tethering virions at the cell surface influenza a virus does not encode a tetherin antagonist with vpu-like activity and induces ifn-dependent tetherin expression in infected cells budding capability of the influenza virus neuraminidase can be modulated by tetherin multiply attenuated lentiviral vector achieves efficient gene delivery in vivo we thank the members of the virology and cell technology laboratory for their insightful comments and critique. this work was funded by the young fellow grant from the national center for genetic engineering and biotechnology (biotec), thailand. the authors declare no conflict of interest. supplementary data associated with this article can be found in the online version at doi:10.1016/j.virol.2018.09.016. key: cord-272045-v1wt5t7m authors: wilson, lauren; gage, peter; ewart, gary title: hexamethylene amiloride blocks e protein ion channels and inhibits coronavirus replication date: 2006-09-30 journal: virology doi: 10.1016/j.virol.2006.05.028 sha: doc_id: 272045 cord_uid: v1wt5t7m all coronaviruses encode a small hydrophobic envelope (e) protein, which mediates viral assembly and morphogenesis by an unknown mechanism. we have previously shown that the e protein from severe acute respiratory syndrome coronavirus (sars-cov) forms cation-selective ion channels in planar lipid bilayers (wilson, l., mckinlay, c., gage, p., ewart, g., 2004. sars coronavirus e protein forms cation-selective ion channels. virology 330(1), 322–331). we now report that three other e proteins also form cation-selective ion channels. these e proteins were from coronaviruses representative of taxonomic groups 1–3: human coronavirus 229e (hcov-229e), mouse hepatitis virus (mhv), and infectious bronchitis virus (ibv), respectively. it appears, therefore, that coronavirus e proteins in general, belong to the virus ion channels family. hexamethylene amiloride (hma) – an inhibitor of the hiv-1 vpu virus ion channel – inhibited the hcov-229e and mhv e protein ion channel conductance in bilayers and also inhibited replication of the parent coronaviruses in cultured cells, as determined by plaque assay. conversely, hma had no antiviral effect on a recombinant mhv with the entire coding region of e protein deleted (mhvδe). taken together, the data provide evidence of a link between inhibition of e protein ion channel activity and the antiviral activity of hma. coronaviruses (order nidovirales, family coronaviridae, genus coronavirus ), are enveloped, single-stranded, positive-sense rna viruses with a genome of about 30 kb. all coronaviruses encode the envelope (e) protein, which is a small hydrophobic membrane protein. while the exact functions and mechanisms of the coronavirus e protein are yet to be fully characterized, the e protein has been shown to be important for coronavirus replication, with roles in viral assembly and morphogenesis (fischer et al., 1998; kuo and masters, 2003; ortego et al., 2002) . in general, while the different coronavirus e proteins share little sequence homology, their basic structures are similar (shen et al., 2003) . all e proteins are small hydrophobic proteins, with a single putative transmembrane α-helix and a hydrophilic c terminal domain (shen et al., 2003; siddell, 1995) . previously, we demonstrated that the e protein from severe acute respiratory syndrome coronavirus (sars-cov) forms cation-selective ion channels in planar lipid bilayers that are about 10-fold more selective for na + than for k + ions. the first 40-amino-acid residues of sars-cov e protein, encompassing the putative transmembrane (tm) domain, were sufficient for formation of ion channels with similar properties to the fulllength peptide . this was the first coronavirus e protein shown to belong to the virus ion channel family. madan et al. (2005) have recently reported that the mhv e protein induces membrane permeability changes in e. coli and mammalian membranes, indicating that it has viroporin-like activity (madan et al., 2005) . therefore, it is possible that ion channel formation is a function common to all coronavirus e proteins. it is not yet known whether the sars-cov e protein ion channel activity has an important role in coronavirus replication. however, for the two best characterized virus ion channels -m2 protein from influenza a and vpu from hiv-1ion channel activity has been strongly linked to roles in the virus's life cycles by studies with mutants and channel blocking drugs (hout et al., 2005a (hout et al., , 2005b pinto et al., 1992; sakaguchi et al., 1996; schubert et al., 1996) . the m2 ion channel activity is inhibited by amantadine and some of its derivatives, and these are currently used in the clinical treatment of influenza a infections (fleming, 2001; pinto et al., 1992) . we have demonstrated that the vpu ion channel activity in planar lipid bilayers is blocked by the amiloride derivate hma, and that hma also inhibits replication of hiv-1 in cultured human macrophages . for some other viral ion channels, inhibitors have also been discovered (griffin et al., 2003; pavlovic et al., 2003; premkumar et al., 2005; premkumar et al., 2004) , although evidence of antiviral activity of the compounds has not yet been published. the goals of our research were, therefore, to identify other coronavirus e protein ion channels, find blockers of the channel activity and characterize the effect of channel-blocking compounds on viral replication. in this paper, we report that e proteins from three coronaviruseshuman coronavirus 229e (hcov-229e, a group 1 coronavirus); mouse hepatitis virus (mhv, group 2), and infectious bronchitis virus (ibv, group 3)have ion channel activity in planar lipid bilayers. furthermore, hcov-229e and mhv e protein ion channel conductance was inhibited by the amiloride derivative hma, but not by amiloride itself. channelblocking activity correlated with inhibition of replication of these viruses, as demonstrated by plaque assays. conversely, hma had no antiviral activity against the recombinant mhv with the entire e protein deleted, indicating that e protein is the possible antiviral target. synthetic peptides corresponding to the e proteins of hcov-229e, mhv, and ibv, were prepared using solid phase chemistry. the full-length peptides, with predicted molecular weights of 9092.9, 9660.0, and 10475.4 da, respectively, were excised from sds-polyacrylamide gels and purified as described in materials and methods. western blot analysis of the purified peptides ( fig. 1) showed that all three preparations contained discrete protein bands electrophoresing at approximately the expected size of the full-length products and recognized by the appropriate peptide-specific antibodies. hcov-229e e protein ran as a single discrete band at about its expected molecular weight, indicating that the sample contained mostly full-length protein and did not contain significantly truncated products. the mhvand ibv e samples both yielded discrete doublet bands on the blots, with in each case the smaller of the two bands running at about the expected molecular weight. it is possible that, due to their highly hydrophobic nature, the proteins may electroporese more slowly than expected, and it may be the larger of the two bands that corresponds to the full-length (or at least very near full-length) protein and the smaller band represents a truncated species. the mhv e protein was detected with an antibody that recognizes an epitope within the first 19 n-terminal amino acids (referred to as, anti-mhv en). since, peptide synthesis proceeds from the c-terminus to the n-terminus and because it would require about 6-amino-acid residues to form an epitope to react with the anti-mhv en antibody, the western blot indicates the smaller immunoreactive peptide would be truncated at the nterminus by maximally about 13 amino acids. as predicted from mhv e protein hydropathy plot, the e protein n-terminus consists of about a 14-amino-acid hydrophilic region so even the smaller of the two immunoreactive species would contain an intact tm putative ion channel domain. the ibv e protein also ran as a doublet, and again, it is likely that the larger band represents the full-length product and the smaller band is ibv e protein with several amino acids truncated from the n-terminus. for all three synthetic peptides western blots confirmed the presence of proteins of at least sufficient length to contain the tm putative ion channel-forming domain. the e protein from group 1 coronavirus hcov-229e forms potassium preferring ion channels in planar lipid bilayers addition of about 3 μg of purified hcov-229e e protein to the cis chamber in the planar lipid bilayer rig resulted in the formation of ion channels in the bilayer. fig. 2a shows typical current trace in a solution containing a 10-fold gradient of nacl (500 mm cis: 50 mm trans) at a range of holding potentials. fig. 2b shows the current-voltage (i-v) plot for this experiment, which reveals a reversal potential of about +26 mv. in nineteen similar experiments where the theoretical equilibrium potential for na + ions was +54 mv, the average measured reversal potential was +22 ± 7 mv (mean ± sem). this indicates that the channel is weakly selective for na + ions over cl − ions. using ion activities in the goldman, hodgkin, and katz (ghk) equation the p na +/p cl − ratio was calculated to be 3, indicating that the hcov-229e e protein ion channel is only 3 times more permeable to na + than cl − ions. for these 19 experiments, the average conductance was 19 ± 4 ps, and the maximum conductance measured was 91 ps. fig. 2c shows typical current traces for hcov-229e e protein in asymmetrical kcl solution, in this experiment the k + ion current was observed to reverse at +42 mv (fig. 2d ). in thirteen comparable experiments with a theoretical k + equilibrium potential of +54 mv, the average measured reversal potential was +38 ± 4 mv. by the ghk equation, hcov-229e e protein is 12 times more selective for k + than for cl − ions. for the 13 experiments, the average conductance was 23 ± 5 ps. the ion selectivity for hcov-229e e protein ion channel is thus, k + > na + > cl − and the channel is about 4 times more permeable to k + than to na + ions (table 1) . the group 2 coronavirus mhv e protein forms ion channels that are sodium selective mhv e protein was tested for its ability to form ion channels in planar lipid bilayers, and experiments were done to determine the selectivity of mhv e protein for na + ions over cl − ions and for k + ions over cl − . fig. 3a shows characteristic current traces for mhv e protein ion channel activity in asymmetrical nacl solution. in this experiment the ion current reversed at +42 mv ( fig. 3 b) . in 14 equivalent experiments, the average reversal potential was +49 ± 1 mv, which is close to the na + equilibrium potential of +54 mv. thus, the mhv e protein ion channel is highly selective for na + ions and the p na +/p cl − ratio is 138. for the 14 experiments the conductance varied between 235 and 7 ps, with an average conductance of 61 ± 16 ps. fig. 3c illustrates representative current traces of mhv e protein ion channel activity in a 10-fold concentration gradient of kcl. in this experiment, k + ion flow was observed to reverse at +25 mv (fig. 3d ), but after eleven similar experiments, the average reversal potential was calculated to be +13 ± 6 mv, indicating that mhv e protein ion channel is only 2 times more selective for k + over cl − ions. the conductance for the 11 experiments ranged from 60 to 6 ps with an average of 18 ± 5 ps. the ion selectivity order for mhv e protein ion channel was na + ≫ k + > cl − , with the channel being about 69 times more permeable to na + than k + ions. thus, the mhv e protein ion channel distinguishes between monovalent cations and is highly selective for na + ions (table 1) . the group 3 coronavirus ibv e protein forms ion channels that prefer sodium ibv e protein also forms ion channels in planar lipid bilayers and experiments conducted in nacl and kcl solutions demonstrated that ibv e protein channels have a slight preference for na + ions over k + ions, similar to e protein channels from the group 2 coronaviruses mhv and sars-cov . fig. 4a shows typical current traces of ibv e protein ion channel conductance in asymmetrical nacl solution. in this experiment, the na + ion flow was observed to reverse at +30 mv (fig. 4b ). in 13 independent experiments, the average reversal potential was +36 ± 5 mv and the ibv e protein p na+ /p cl − ratio was 10. for the 13 experiments, the conductance was variable, the highest conductance measured was 77 ps, with an average conductance of 24 ± 5 ps. the i-v relationship for the ibv e protein in nacl solution was nonlinear, indicating rectification of the ion channel. the channel conductance was smaller when the trans chamber was held at positive potentials relative to the cis chamber than when the polarity was reversed. typical current traces for ibv e protein in asymmetric kcl solution are shown in fig. 4c ; the i-v plot indicates that the k + ion flow reversed at +28 mv (fig. 4d ). in 20 equivalent experiments, the average reversal potential was +22 ± 3 mv, thus, the ibv e protein was only 3 times more permeable to k + than cl − ions. the average conductance for the 20 experiments was 22 ± 6 ps. thus, the ibv e protein ion selectivity series is na + > k + > cl − and the channel is about 3 times more selective for na + than k + ions ( table 1) . rectification of the ibv e protein channel was not seen in kcl solutions. we currently do not have an explanation as to why the ibv channel shows rectification in na + but not k + solutions. rectification indicates that ions flow more readily through the channel in one direction than the other and the mechanism by which this is achieved in ibv e protein channels is likely to be related to specific properties of the protein quaternary structure forming the gate and selectivity filters of the channel. the in vivo significance of a rectifying channel is that ion conductance will occur preferentially in one direction, i.e., extracellularintracellular, or vice versa, which could have important implications for the physiological functions of the channel. we have previously shown that ion channel conductance in planar lipid bilayers is dependent on addition of channelforming peptides and is not due to addition of solvent or buffers alone (ewart et al., 1996; premkumar et al., 2004; wilson et al., 2004) . to demonstrate that the ion channel activities we were observing were specifically due to the hcov-229e or mhv e proteins, rather than a contaminant in samples, purified epitopespecific antibodies recognizing these proteins were utilized. in some previously published cases, such antibodies have been shown to inhibit ion channel activity directly in bilayer experiments. this inhibition depends on binding of the antibody to an exposed epitope and either physical blocking of the ion channel by the antibody or induction of a conformational change in the ion channel structure that inactivates it. however, for many antibodies, simple binding of the antibody may not be sufficient to inactivate the target ion channel. so alternatively, the antibodies can be used to remove the ion channel-forming peptide from solution (ewart et al., 1996; melton et al., 2002) . addition of between 2 and 7 μg of affinity purified antibody, that recognizes the hcov-229e c-terminal end of the tm domain (anti-hcov-229e e antibody), to the cis (n = 8 experiments) or trans (n = 12) chambers did not noticeably reduce the hcov-229e e protein ion channel conductance (data not shown). however, when this antibody was used to remove hcov-229e e protein from solutions (using a seize x protein a immunoprecipitation kit) samples of the remaining solution did not form ion channels, even after prolonged periods of stirring for over 15 min (n = 5, results not shown). depletion of the hcov-229e e protein from the samples was confirmed by western blot analysis using the anti-hcov-229e e polyclonal antibody (data not shown). in contrast, samples containing the purified synthetic hcov-229e e protein always resulted in ion channel conductance within 15 min of the addition of the peptide to the cis chamber. hence, the immunoprecipitation experiments confirmed that the ion channel conductance observed after addition of the purified synthetic hcov-229e e protein to planar lipid bilayers was dependent on presence of the full-length hcov-229e e protein. in the case of the mhv e protein, an antibody directed to the e protein first 19 amino acids of the n-terminus (anti-mhv en antibody) significantly inhibited ion channel conductance when added to the cis chamber (p ≪ 0.01; n = 5, t test to test the difference between the means). these data confirm that the channel-forming species in the samples was the mhv e protein ( fig. 5a1-2) . conversely, addition of the anti-mhv en antibody to the trans chamber did not affect channel conductance ( fig. 5a3-4) . this result indicates that the mhv e protein inserts in the bilayer in an orientation-specific manner with the n-terminal domain facing the cis chamber. since, the anti-mhv en antibody significantly inhibited mhv e protein ion channel activity it confirms that the ion channel-forming species contains the mhv e protein n-terminus and is thus likely to be full-length peptide. another mhv e antibody, the anti-mhv e47-67, which does not bind directly adjacent to the putative tm domain, did not inhibit mhv e protein ion channel conductance when added to the cis (n = 8) or the trans chamber (n = 7) ( fig. 5b1-4) . however, similarly to the hcov-229e e protein, the anti-mhv e47-67 antibody was used to immunoprecipitate the full-length mhv e protein from solution. western blot analysis of the immunoprecipitate flowthrough demonstrated that the mhv e protein had been removed from the solution (data not shown). addition of the mhv e depleted solution to the cis chamber did not result in ion channel formation in planar lipid bilayers (n = 5 experiments), even after stirring for up to 1 h and 40 min. these data further demonstrate that the ion channel conductance detected was specifically due to the purified full-length mhv e protein. unfortunately, we were not able to obtain enough anti-ibv e protein antiserum to conduct ion channel inhibition and immunoprecipitation experiments with the ibv e protein. nevertheless, the anti-ibv e antibody directed to the cterminal was used to demonstrate that the synthetic purified ibv e protein contained full-length peptide (fig. 1) . we tested amiloride, plus its derivative, hma for their ability to inhibit hcov-229e, mhv, and ibv e protein ion channels in planar lipid bilayers. once ion channel conductance was detected, 100-200 μm of compound was added to the cis chamber while stirring to facilitate binding of the compound to the channel. fig. 6 demonstrates that hma significantly reduced hcov-229e e protein (p ≪ 0.01, n = 5) and mhv e protein (p ≪ 0.01, n = 5) current across the bilayer, while, hma had no significant affect on ibv e protein channel conductance (p = 0.14, n = 10). amiloride itself did not have a significant effect on any of the coronavirus e proteins ion channel activity (data not shown). furthermore, addition of the compound solvent, 50% dmso: 50% methanol, alone did not affect the e protein ion channel conductances (data not shown), indicating that channel inhibition was dependent on the presence of hma. to determine if hma inhibits mhv e protein in a dosedependent manner, hma was titrated on mhv e protein channel activity in planar lipid bilayers. fig. 7 demonstrates that, with increasing concentration of hma, there is a subsequent decrease in channel activity. the percent inhibition was plotted against the concentration of hma and from the equation of the line the effective concentration 50 (ec 50 ) of hma on mhv e protein channel activity in bilayers was calculated to be 10.2 μm. these data further confirm that the inhibition observed with hma is specifically due to addition of the compounds and not to spontaneous reductions in the channels macroscopic conductance. occasionally, channel activity in bilayers can go quiet in the absence of inhibitors, this could be due to the lipids encompassing the peptides, reduction in the macroscopic channel activity or open probability. this spontaneous reduction in channel activity would not occur in a linear dose-dependent fashion, as observed in fig. 7a . to determine if amiloride and hma inhibit mhv replication in cultured cells, they were tested for their ability to reduce the number of mhv plaques in l929 cells without plaque overlay. mhv infection in l929 cells without plaque overlay, results in the newly emerged virus diffusing from the original infected cell and starting a new plaque. therefore, in the presence of antiviral compound, there is a reduction in plaque number. l929 cells were infected with mhv at a multiplicity of infection (moi) of 0.01 and treated with various concentrations of hma or amiloride (see materials and methods). the number of plaques per well was counted, and the percent reduction in plaque number was calculated from the no compound control. the percent reduction was plotted against the compound concentra-tion, and the ec 50 was calculated from the equation of the line. the ec 50 of hma on mhv replication was calculated to be 3.91 μm (table 2) , in reasonable agreement with the ec 50 measured in bilayer experiments. amiloride did not inhibit mhv replication in cultured cells (table 2) , which correlates with the inability of amiloride to inhibit ion channel activity in bilayers. to investigate the plaque phenotype of mhv in the presence of hma, l2 cells with a plaque overlay was used. the plaque overlay on the mhv infected l2 cells slows diffusion of the newly emerged virus, which then infect adjacent cells, resulting in larger plaques than in the l929 cells. hma was tested on mhv infection in l2 cells at 20 μm, which is significantly above the ec 50 measured in l929 cells (table 2 ), but removed from toxicity (as determined by mtt cytotoxicity assay). to determine if the mhv e protein was the antiviral target, hma was also tested for its ability to inhibit the recombinant mhv with the entire e protein deleted (mhvδe). mhvδe replicates to low titer and has a small plaque phenotype in l2 cells (kuo and masters, 2003) . therefore, it is expected if compounds were targeting the mhv e protein, replication of mhvδe would not be affected. furthermore, if hma was targeting the mhv e protein the plaque phenotype of mhv wild-type in the presence of hma would be comparable to the mhvδe small plaque phenotype. fig. 8 shows that the mhv wild-type virus in mouse l2 cells has a large plaque phenotype of about 3-4 mm in diameter in the absence of antiviral compound (fig. 8a1) . in the presence of 20 μm hma the plaque size is reduced to about 1 mm (fig. 8a2) , about the same size as the mhvδe plaques (fig. 8b1) . conversely, 20 μm of amiloride did not reduce mhv plaque size (fig. 8a3) . moreover, none of the amiloride derivatives tested notably inhibited mhvδe plaque formation ( fig. 8b1-3) , indicating that mhv e protein is the probable antiviral target. hma, which blocked hcov-229e e protein ion channel conductance in planar lipid bilayers, also inhibited replication of hcov-229e in cultured cells (fig. 8c2) . the ec 50 of hma on hcov-229e was calculated to be 1.34 μm (table 2) . additionally, amiloride, which did not block hcov-229e e protein ion channel current amplitude, had no antiviral activity on hcov-229e replication in cultured cells (fig. 8c3) . previously, we reported that the sars-cov e protein is a member of the viral ion channel family , and in this paper, we extend the list of known coronavirus ion channels by three. our data demonstrate that the e proteins from hcov-229e, mhv, and ibvrepresentative of coronavirus taxonomic groups 1, 2, and 3, respectivelyform cationselective ion channels in planar lipid bilayers. in the cases of hcov-229e and mhv, epitope-specific antibodies were used to confirm that the channel-forming species in test samples was indeed the purified synthetic e protein. the anti-hcov-229e e antibody, which binds on the c-terminal side of the transmembrane helix, did not inhibit channel activity in bilayers. however, when this antibody was used to immunoprecipitate the e protein, the residual supernatant did not form ion channels. the anti-mhv en antibody inhibits the mhv e ion channel activity when added to one chamber (but not the other) of the bilayer rig, indicating that the n-terminus of the peptide is exposed on the cis side of the bilayer. further, this shows that the peptides are aligned in a parallel, rather than antiparallel, conformation in the presumed homo-oligomer that forms the ion channel. herein, we also show that hma inhibits the ion channels formed by hcov-229e and mhv e proteins (see fig. 6 ). hma, an amiloride analogue, also blocks other viral ion channels including; vpu from hiv-1 (ewart et al., 1996) ; p7 from hepatitis c virus (hcv) (premkumar et al., 2004) and m protein from dengue (premkumar et al., 2005) . such broad-spectrum inhibition by hma suggests that the drug-binding sites of these channels from diverse viruses share structural similarities. another example of one compound inhibiting different ion channels is amantadine, which blocks both influenza a m2 (pinto et al., 1992; sugrue and hay, 1991) , hcv p7 (griffin et al., 2003) and dengue m protein channels (premkumar et al., 2005) . hma does not block the ibv e protein ion channel suggestive of a more divergent structure of this group 3 coronavirus e protein. in this regard, models of the membrane topology of the ibv and mhv e proteins are quite different (corse and machamer, 2000; maeda et al., 2001) . the rectified i-v plot observed in nacl solutions (fig. 4b) is also indicative of unique properties of the ibv e protein channels that warrant further investigation. for some viral ion channels, like influenza a m2 and hiv-1 vpu, ion channel activity is known to be important for virus replication (reviewed in fischer and sansom, 2002; gonzalez and carrasco, 2003) . amantadine's clinical use against influenza a infections is the clearest evidence of this and the mechanism of action of the m2 channel in virus replication has been well characterized. we have shown that hmaa blocker of the vpu ion channelinhibits replication of hiv-1 in cultured human primary macrophages , but in this case, the underlying mechanistic links remain to be uncovered. in this manuscript, we report that in addition to blocking e protein ion channels, hma also inhibits replication of hcov-229e and mhv. conversely, hma at 20 μm does not inhibit the recombinant virus, mhvδe, with the entire e protein deleted (see . taken together, the data imply that the antiviral target of hma is the e protein, and further, that the e protein ion channel activity is important for coronavirus replication. other groups have previously provided evidence for the importance of e protein for replication of mhv and transmissible gastroenteritis coronavirus (tgev), a group 1 coronavirus (kuo and masters, 2003; ortego et al., 2002) . deletion of the open reading frame encoding the e protein from the genome of mhv results in a strongly attenuated virus with a small plaque phenotype (kuo and masters, 2003) . this indicates that, while the e protein has important roles in the mhv life cycle, it is not essential for in vitro replication of mhv. in contrast, the e gene is thought to be essential for replication of tgev (ortego et al., 2002) . hma inhibited mhv replication in cultured cells with an ec 50 of 3.94 μm and the ec 50 of hma on mhv e protein ion channel activity in planar lipid bilayers was calculated to be 10.2 μm. the two ec 50 s are in good agreement, particularly given the vast differences in the physico-chemical environments the channels are exposed to in the bilayer compared to infected cells and the fact that membrane insertion and channel assembly are not regulated by the same natural processes in the artificial bilayer. the e protein channels described here have different ion selectivities (see table 1 ), but all e proteins tested hitherto prefer monovalent cations over chloride ions. the channel formed by the hcov-229e e protein (group 1 coronavirus) is about four times more selective for k + than na + , while the channels from mhv (group 2) and ibv (group 3) prefer na + ions. the two e proteins from the group 2 coronaviruses tested thus far, mhv and sars-cov (subgroups 2a and 2b, respectively) are the most similar and have the highest selectivity for sodium. others have previously noted a relatively high degree of amino acid sequence similarity between mhv and sars-cov e proteins, especially in the tm domain (shen et al., 2003) . as more e protein channels are characterized, it will be interesting to see how the channel properties vary with sequence divergence and whether ion selectivity properties co-segregate with taxonomic groupings. complementation studies suggest that the e proteins from coronavirus groups 2 and 3 may be functionally more similar than for the group 1 e proteins. in a recombinant mhv (group 2) construct, the ibv e protein (group 3)as well as the other group 2 e proteins from bovine coronavirus and sars-covcould substitute for the mhv e protein and enhance replication of the recombinant viruses. on the other hand, the tgev (group 1) e protein could not functionally replace the mhv e protein (lili kuo and paul masters, personal communication) . in similar experiments, group 1 and group 2 e proteins were not able to substitute for each other in the formation of heterologous virus-like particles (vlps) (baudoux et al., 1998) . it is possible that the functional similarities and differences may be related to the ion channel selectivities of the e protein channels, which we found to be more similar between groups 2 and 3 e channels (prefer na + ions) than group 1 (prefers k + ions). of course, more ion channel selectivity and complementation studies are required to confirm these early observations. in summary, the data presented here demonstrate that coronavirus e proteins belong to the growing family of virus ion channels. hma blocks the hcov-229e and mhv e protein channels, which correlated with its ability to inhibit coronavirus replication in cultured cells. conversely, hma did not affect mhvδe recombinant virus replication in cultured cells. taken together, the data suggest that e protein ion channel activity is important for coronavirus replication and e protein blockers could have potential anticoronaviral therapeutic use. peptides were synthesized corresponding to the e protein sequences from three coronaviruses in the genbank database ( table 3 ). the peptides were synthesized at the biomolecular research facility, john curtin school of medical research, on a symphony/multiplex (protein technologies inc. woburn, ms) multiple peptide synthesizer using fmoc chemistry and solid phase peptide synthesis. the full-length peptides were purified from any truncated products using proteoplus kit (qbiogene, inc., ca), following manufactures instructions. briefly, e peptides were dissolved in loading buffer (60 mm tris-hcl ph 8.3, 6 m urea, 5% sds, 10% glycerol, 0.2% bromophenol blue, and 100 mm βmercaptoethanol) and approximately 3 μg of the e peptides were run with molecular weight markers (mbi fermentas, hanover, md) on a 4-20% gradient polyacrylamide gels (gradipore, nsw, australia). the band corresponding to the full-length peptides, of about 9.1, 9.7, or 10.5 kda for hcov-229e, mhv, or ibv e proteins, respectively, were excised from the gel, placed in a proteoplus tube, and subjected to electrophoresis. the purified peptides were dried in a speedy-vac and resuspended in 2,2,2-trifluoroethanol (tfe). the peptide concentration was determined by bradford assay (pierce, rockford, il), following manufacturer's instructions. attempts to characterize the purified e peptides by mass spectrometry were unsuccessful, possibly due to the hydrophobic nature of the peptides. three synthetic peptides were also made for production of antibodies recognizing the hcov-229e and mhv e proteins. the sequences of these shorter peptides are indicated as underlined bold-face segments of the full-length sequences in table 3 . the antigenic peptides were chemically synthesized and coupled to a poly-lysine core via their terminal cysteine to prepare multiple antigenic peptides (map) (lu et al., 1991) . individual, new zealand white rabbits were immunized with the map conjugated peptides with initial immunization of 200 μg peptide in freund's complete adjuvant (imject®, pierce, rockford, il). boosters of 200 μg of peptide were given every 2 weeks in freund's incomplete adjuvant (imject®, pierce, rockford, il), until sufficient antibody titers were attained. about 15 ml of blood was collected from the ear vein 2, 4, 6, 8, and 10 weeks post-immunization and antisera prepared. antiserum was assayed for antibody production by western blotting with the appropriate full-length e peptide. epitopespecific antibodies were purified from antisera by sulfolink™ columns coupled to the equivalent peptide (pierce, rockford, il), following manufactures instructions. for western blot analysis, samples were prepared in loading buffer (60 mm tris-hcl ph 8.3, 6 m urea, 5% sds, 10% glycerol, 0.2% bromophenol blue, and 100 mm βmercaptoethanol) and run with molecular weight markers (mbi fermentas, hanover, md) on 4-20% gradient polyacrylamide gels (gradipore, nsw, australia). peptides were transferred to polyvinylidene difluoride membranes (invitrogen, vic, australia), using a semi-dry transfer apparatus (amersham biosciences, vic, australia). nonspecific sites were blocked with skim milk proteins in tris-buffered saline containing 1% tween-20. peptides were detected with purified primary antibody to hcov-229e e protein tm domain cterminal (anti-hcov-229e e); mhv e protein n-terminal antibody (anti-mhv en); and ibv e protein anti-c-terminal antibody (anti-ibv ec) (corse and machamer, 2000) (kind gift from carolyn machamer, john hopkins university, baltimore). the primary antibodies were detected with goat antirabbit igg alkaline phosphatase-conjugated antibody (dako, nsw). color development was visualized with western bluestabilized substrate for alkaline phosphatase (promega, nsw, australia). the purified hcov-229e, mhv, and ibv e proteins were resuspended to 1 mg/ml in tfe and their ability to form ion channels was tested on a warner bilayer rig (warner instruments, inc. 1125 dixwell avenue, hamden, ct 06514), as described previously . briefly, a lipid mix of 3:1:1 1palmitoyl-2-oleolyl phosphatidyl ethanolamine:1-palmitoyl-2oleolyl phosphatidyl serine:1-palmitoyl-2-oleolyl phosphatidyl choline in chloroform were dried under n 2 gas and resuspended in n-decane. bilayers were painted across a circular hole of approximately 100 μm diameter in a delrin cup separating aqueous solutions in the cis and trans chambers. for testing e protein selectivity for na + and cl − ions, the solution in the cis chamber consisted of 500 mm nacl, and 5 mm hepes (ph 7.2), while the solution in the trans chamber contained 50 mm nacl and 5 mm hepes (ph 7.2). for testing e protein selectivity for k + and cl − ions, the solution in the cis chamber consisted of 500 mm kcl and 5 mm hepes (ph 7.2), while the solution in the trans chamber contained 50 mm kcl and 5 mm hepes (ph 7.2). currents were amplified using a warner model bd-525d amplifier with sampling rate of 5 khz and filtered at 1 khz before being digitally recorded directly using small aliquots (about 3 μg) of the e peptides dissolved in tfe were added to the cis chamber, while stirring to facilitate spontaneous insertion of the hydrophobic peptides into the lipid bilayer. typically, when present, ion channel activity was detected after about 5-15 min of stirring. the cis chamber was earthed and the trans chamber was held at a range of potentials between +100 mv to −100 mv. the voltage (v m ) vs. the current (i) were plotted (i-v plot) to determine the reversal potential (v r ), which indicates the ion selectivity of the channel. the theoretical v r for a na + or k + selective channel in our experimental set up was +54 mv, whereas the theoretical v r for a cl − channel was −54 mv (using activities in the nernst equation). the v r was used in the goldman-hodgkin-katz (ghk) equation to determine the relative permeability ratios (p na +/p cl − or p k +/p cl −) of the ion channel. to determine antibody blocking, after ion channel activity was detected, affinity-purified polyclonal antibody was added to the cis or trans chamber to a final concentration of about 50-100 μg/ml, while stirring for 30 s. bilayer currents were recorded before ion channel formation, after ion channel formation, and after the addition of the purified antibody. a t test (microsoft excel) was used to determine if there was significant difference between the mean current recorded before and after addition of the epitope-specific antibody. affinity purified anti-hcov-229e e and anti-mhv e47-67 antibodies were used to immunoprecipitate the hcov-229e and mhv e proteins, respectively, using the seize x protein a immunoprecipitation kit (pierce, rockford, il), following manufactures instructions. briefly, the antibodies were bound and cross-linked to protein a column and 1 mg/ml of corresponding e protein was immunoprecipitated, from the solution. the flow-through, which should not contain any e protein, and eluate, which contains the immunoprecipitated e protein were analyzed by western blot. the hcov-229e and mhv e protein flow-through were tested for their ability to form ion channels in planar lipid bilayers as described above. stock solutions of amiloride and hma (sigma, australia) at 50 mm were prepared in 50% dmso:50% methanol. to determine if the amiloride derivatives blocked the e proteins ion channel conductance in planar lipid bilayers, after ion channel currents were detected, 100-200 μm of compound was added to the cis chamber while stirring to facilitate binding of the compound to the channel. the current across the bilayer was recorded prior to addition of the e protein, after detection of ion channel conductance, and after addition of the compound. a t test (microsoft excel) was used to test the difference between the normalized mean currents before and after addition of the compounds. mouse l2, l929, and 17cl1 cells lines (atcc) were grown in dmem (invitrogen, vic, australia) supplemented with 10% fcs (invitrogen, vic, australia). mhv-a59 (atcc) and mhv with the entire e protein deleted (referred to as mhvδe) (kind gift from paul masters, wadsworth centre, albany), were amplified in 17cl1 cells. the mhv wild-type and mhvδe recombinant virus were plaque assayed in l2 cells with plaque overlay for observation of plaque phenotype or assayed in l929 cells without overlay for the easy counting of plaque numbers. human mrc-5 cell lines (atcc) were grown in dmem (invitrogen, vic, australia) supplemented with 10% fcs (invitrogen, vic, australia). the hcov-229e (atcc) was two times plaque purified in mrc-5 cells and plaque assayed in mrc-5 cells with plaque overlay. for determining if hma or amiloride had antiviral activity on mhv and for calculating the compounds ec 50 the plaque assay was done in l929 cells, without plaque overlay. mhv infection in l929 cells without plaque overlay results in the newly emerged plaques disseminating from the original infected cell and starting a new plaque. therefore, in the presence of antiviral compound, there is a reduction in plaque number, which can easily be counted. the l929 cells were infected with mhv wildtype at a multiplicity of infection (moi) 0.01 for 1 h at 37°c in 5% co 2 , then 10 μm, 7.5 μm. 5 μm, 2.5 μm, 1.25 μm, 0.625 μm, 0.3125 μm, or 0 μm of amiloride or hma were added. after 24-h incubation at 37°c in 5% co 2 , the culture media were removed, and the cells were stained with 0.1% crystal violet in 20% methanol. the plaque number per well was counted, and the percent reduction was calculated from the no compound control. the compound concentration was plotted against the percent reduction in plaques, and the ec 50 was calculated from the equation of the line. the plaque phenotype of mhv wild-type or mhvδe in the presence or absence of antiviral compound was studied in mouse l2 cells with plaque overlay (1% seaplaque in mem, 10% fcs). the l2 cells were plated in 6-well plates and grown to confluence, then infected with mhv wild-type at a moi of 0.01 or mhvδe (moi 0.1) for 1 h. higher moi of mhvδe was used so that the number of plaques per well were comparable between the mhv wild-type and mhvδe, which has a lower titration. after 1-h incubation, the virus was removed and replaced with 1% seaplaque overlay in mem supplemented with 10% fcs and 20 μm or 0 μm (no compound control) of testantiviral compound dissolved in 50% dmso: 50% methanol. after 48-h incubation at 37°c in 5% co 2 , the cells were stained with 0.1% crystal violet in 20% methanol and the average plaque size determined. hcov-229e was plaque assayed in human mrc-5 cells. the mrc-5 cells were grown to confluence in 6-well plates and then infected with hcov-229e at a moi of 0.01 for 1 h at 35°c in 5% co 2 . after 1-h incubation 5 μm, 2.5 μm, 1 μm or 0 μm (no compound control) of test-antiviral compound dissolved in 50% dmso: 50% methanol was added to each well. the assay was incubated for 6 days at 35°c in 5% co 2 , until plaques were visible and then stained with 0.1% crystal violet in 20% methanol. the concentrations of the compounds used in the antiviral assays were not significantly toxic as determined by the mtt cytotoxicity assay (sigma, australia), following manufactures instructions. note, that for the mrc-5 cells the compounds were found to be considerable more toxic, which is most likely due to the elongated nature of the mrc-5, resulting in greater absorbance of the compounds. therefore, the amiloride derivatives in the hcov-229e plaque assay were used at a lower concentration, which was not toxic for the mrc-5 cells. coronavirus pseudoparticles formed with recombinant m and e proteins induce alpha interferon synthesis by leukocytes infectious bronchitis virus e protein is targeted to the golgi complex and directs release of virus-like particles the vpu protein of human immunodeficiency virus type 1 forms cation-selective ion channels amiloride derivatives block ion channel activity and enhancement of virus-like particle budding caused by hiv-1 protein vpu potential new anti-human immunodeficiency virus type 1 compounds depress virus replication in cultured human macrophages viral ion channels: structure and function analysis of constructed e gene mutants of mouse hepatitis virus confirms a pivotal role for e protein in coronavirus assembly managing influenza: amantadine, rimantadine and beyond a comparative sequence analysis to revise the current taxonomy of the family coronaviridae the p7 protein of hepatitis c virus forms an ion channel that is blocked by the antiviral drug substitution of the transmembrane domain of vpu in simian-human immunodeficiency virus (shiv (ku1bmc33)) with that of m2 of influenza a results in a virus that is sensitive to inhibitors of the m2 ion channel and is pathogenic for pig-tailed macaques scrambling of the amino acids within the transmembrane domain of vpu results in a simian-human immunodeficiency virus (shivtm) that is less pathogenic for pig-tailed macaques the small envelope protein e is not essential for murine coronavirus replication chemically unambiguous peptide immunogen: preparation, orientation and antigenicity of purified peptide conjugated to the multiple antigen peptide system viroporin activity of murine hepatitis virus e protein membrane topology of coronavirus e protein alphavirus 6k proteins form ion channels generation of a replication-competent, propagation-deficient virus vector based on the transmissible gastroenteritis coronavirus genome the hepatitis c virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives influenza virus m2 protein has ion channel activity cation-selective ion channels formed by p7 of hepatitis c virus are blocked by hexamethylene amiloride dengue virus m protein cterminal peptide (dvm-c) forms ion channels the ion channel activity of the influenza virus m2 protein affects transport through the golgi apparatus the two biological activities of human immunodeficiency virus type 1 vpu protein involve two separable structural domains small envelope protein e of sars: cloning, expression, purification, cd determination, and bioinformatics analysis the small-membrane protein structural characteristics of the m2 protein of influenza a viruses: evidence that it forms a tetrameric channel sars coronavirus e protein forms cation-selective ion channels the authors would like to thank frank bowden for his critical reading of the manuscript and paul masters and carolyn machamer for their helpful discussions. this paper is dedicated to the memory of prof. peter gage. key: cord-278578-vq5fy8m5 authors: stodola, jenny k.; dubois, guillaume; le coupanec, alain; desforges, marc; talbot, pierre j. title: the oc43 human coronavirus envelope protein is critical for infectious virus production and propagation in neuronal cells and is a determinant of neurovirulence and cns pathology date: 2017-12-26 journal: virology doi: 10.1016/j.virol.2017.12.023 sha: doc_id: 278578 cord_uid: vq5fy8m5 the oc43 strain of human coronavirus (hcov-oc43) is an ubiquitous respiratory tract pathogen possessing neurotropic capacities. coronavirus structural envelope (e) protein possesses specific motifs involved in protein-protein interaction or in homo-oligomeric ion channel formation, which are known to play various roles including in virion morphology/assembly and in cell response to infection and/or virulence. making use of recombinant viruses either devoid of the e protein or harboring mutations either in putative transmembrane domain or pdz-binding motif, we demonstrated that a fully functional hcov-oc43 e protein is first needed for optimal production of recombinant infectious viruses. furthermore, hcov-oc43 infection of human epithelial and neuronal cell lines, of mixed murine primary cultures from the central nervous system and of mouse central nervous system showed that the e protein is critical for efficient and optimal virus replication and propagation, and thereby for neurovirulence. coronaviruses are widespread rna viruses of the nidovirales order, coronaviridae family, most often associated with human and veterinary respiratory infections (de groot et al., 2012) . of the six human-infecting coronavirus strains, four (hcov-229e, hcov-nl63, hcov-hku1 and hcov-oc43) are currently co-circulating and elicit respiratory illnesses (vabret et al., 2009) . coronaviruses also represent a significant public health concern due to the recent zoonotically emerged, highly pathogenic species, sars corovonavirus (sars-cov) (drosten et al., 2003; ksiazek et al., 2003 ksiazek et al., ) in 2002 ksiazek et al., -2003 ksiazek et al., and, since 2012 , middle-east respiratory syndrome coronavirus (mers-cov) (zaki et al., 2012) , localized to the arabian peninsula, but with sporadic travel-related outbreaks worldwide. in addition to their respiratory tropism, human coronaviruses have been detected concurrently with severe and acute neurological symptoms (arabi et al., 2015; morfopoulou et al., 2016; yeh et al., 2004) and shown to naturally infect the central nervous system (cns) (arbour et al., 2000; gu et al., 2005; xu et al., 2005) with neurons demonstrated as the main target of infection in hcov-oc43 (bonavia et al., 1997; favreau et al., 2012; jacomy et al., 2006; jacomy and talbot, 2003) and sars-cov (gu et al., 2005; xu et al., 2005) . coronaviruses represent the largest known enveloped rna (single-stranded positive sense) viruses with a genome of approximately 30 kb (de groot et al., 2012) . the viral envelope is composed of four or five proteins, the spike (s), membrane (m), envelope (e) and hemagglutinin-esterase protein (he), the latter in some β coronaviruses genus, such as hcov-oc43. coronavirus e proteins are 74-109 amino acids in length, 84 amino acids for hcov-oc43, and share only a small amount of sequence identity between coronavirus species. however, its secondary structure, composed of a short n-terminal domain followed by a single hydrophobic transmembrane domain (tmd) and hydrophilic cytoplasmic tail, remains overall conserved and is suggested to be more important than sequence for function (kuo et al., 2007; torres et al., 2005) . the importance of the presence of the e protein in the viral envelope is emphasized by the fact that there are only about twenty e molecules incorporated within the virion structure (godet et al., 1992; liu and inglis, 1991; yu et al., 1994) and deletion of the protein can either completely prevent the production of detectable infectious virions (almazán et al., 2013; curtis et al., 2002; ortego et al., 2007 ortego et al., , 2002 or significantly reduce infectious virus titers (dediego et al., 2008 (dediego et al., , 2007 kuo et al., 2007; kuo and masters, 2003) . the majority of the coronavirus e protein in the infected cell is localized within the secretory pathway between the membranes of the endoplasmic reticulum (er), golgi and intermediate compartment between them (ergic) (cohen et al., 2011; nieto-torres et al., 2011; venkatagopalan et al., 2015) . it is in this intracellular region that additional functions mediated by various domains of the coronavirus e proteins are proposed to occur. homo-pentameric oligomerization of the e protein tmd in membranes to form ion channels, called viroporins, has been predicted for several coronaviruses (torres et al., 2005) and extensively studied for species such as sars-cov (nietopervushin et al., 2009) or avian infectious bronchitis virus (ibv) (ruch and machamer, 2012; westerbeck and machamer, 2015) . another domain found at the extreme c-terminal end of the e protein, a pdz-domain binding motif (pbm), has also been predicted for several coronavirus species . this protein-protein interaction motif capable of interrupting normal cellular functions, has been demonstrated in other viruses to play important roles in replication, dissemination in the host and pathogenesis (javier and rice, 2011) . the multiple properties of coronavirus e proteins have not yet been fully investigated or explained, and can at times differ between coronavirus species. the multifunctionality of the e protein could be explained by the presence of two distinct pools (monomeric versus homo-oligomeric states) present in the infected cell (westerbeck and machamer, 2015) . furthermore, the different motifs found within the protein could mediate different specific functions. the coronavirus e protein was also recently recognized as an important virulence factor for the sars-cov , where deletion of the whole or part of the protein led to an attenuated pathology in mouse lungs (dediego et al., 2008 (dediego et al., , 2007 , attenuation which was later linked to the e protein tmd and pbm . hcov-oc43 represents a circulating strain of human coronavirus causing respiratory illness, which is naturally capable of invading the cns where neurons are preferentially targeted for infection. in this study, we demonstrate that the fully functional hcov-oc43 e protein (harboring specific tmd and pbm) is critical in infectious virus production and dissemination in epithelial and neuronal cell cultures and in the murine cns and that it is a determinant of neurovirulence, a first demonstration for this coronavirus species. in order to evaluate the importance of the hcov-oc43 e protein in infectious virion production, a stop codon was introduced at the beginning of the e gene of our cdna infectious clone, pbac-oc43 fl (st-jean et al., 2006) , preventing corresponding full-length e protein production in the resultant recombinant virus (fig. 1a) . transfection of bhk-21 cells with the pbac-oc43fl led to the detection of reference hcov-oc43 recombinant infectious virus (roc/atcc) whereas transfection with the pbac-oc43-e-stop mutant did not lead to any detectable infectious virus (roc/e-stop) (fig. 1b) . to confirm that the inability to detect infectious viral particles was due to the lack of e protein expression, we wished to verify whether viral production could be rescued with wild-type e protein. transfection of a plasmid containing the reference hcov-oc43 e gene, pcdna(oc-e), in bhk-21 cells clearly showed via western blot assay (wb) that the e protein was produced compared to an empty plasmid condition (fig. 1c ). subsequently, a transient co-transfection was conducted in the same cells with pbac-e-stop and pcdna(oc-e) and, by making use of a monoclonal antibody against the s protein of hcov-oc43, we confirmed that the co-transfection did not affect transfection efficiency, and that the viral s protein was produced at equivalent levels in cells transfected with pbac-oc43 fl alone or pbac-e-stop with pcdna(oc-e) or empty plasmid (fig. 1d ). following the co-transfection, infectious particles production was rescued to detectable levels in a dose-dependent manner (fig. 1e ). viral rna was harvested and cdna sequenced to confirm that the infectious particles detected after transfection corresponded to roc/atcc and roc/e-stop mutant (data not shown). as we were able to rescue infectious particles production through transient complementation, we wondered whether this resultant virus, still lacking the e gene, could be amplified further in subsequent passages. to this end, we amplified the viral stocks of all transfected plasmids three times without trans-complementation on hrt-18 epithelial cells, each time normalizing to the lowest detectable viral titer to infect cells at an identical moi for all recombinant viruses (fig. 1f) . throughout the amplification process we were consistently unable to detect infectious viral particles issuing from viral stocks of roc/e-stop. amplifications of initially complemented viral stocks of roc/e-stop led to detectable titers which decreased over the course of each subsequent amplification compared to roc/atcc. sequencing of viral rna confirmed that the e gene in the viral roc/e-stop stocks contained the introduced stop codon at each amplification step (data not shown). these results demonstrate that production of progeny infectious hcov-oc43 virions is still possible in the absence e protein, however the efficiency of the process is dramatically diminished. interestingly, when conducting independent experiments following the same experimental approach, the titers of initially complemented roc/e-stop sometimes increased substantially after two or three amplifications, approaching reference virus titer levels after three rounds of amplification on hrt-18 cells (fig. 1f ). sequence analysis of the e gene of the corresponding viral stocks revealed that a reversion of sequence appeared at the position where the stop codon had been initially introduced; representing reversion to wild-type or new amino acids (fig. 1g ). taken together, these data demonstrate that the hcov-oc43 e protein is critical for efficient infectious virion production in epithelial cells. hcov-oc43 is neuroinvasive (arbour et al., 2000) and neurotropic, with the neuron being the main target of infection in the cns jacomy and talbot, 2003) . therefore, we sought to investigate whether the absence of the e protein would modify these neurotropic capacities by infecting a susceptible differentiated human neuronal cell line or mixed primary cultures of murine cns cells. initially complemented roc/e-stop, previously recovered from transfection on bhk-21 cells (p0), was used for infection and infectious viral titers determined over a period of 72 h post-infection (hpi). this revealed an important decrease of infectious virus production for human cells ( fig. 2a) , which was exacerbated in primary murine cells, where virus titers were under the limit of detection (fig. 2b ). however, in these primary cultures, low levels of infected cells were visualized by immunofluorescence (ifa) where we detect the viral s protein, suggesting that infection was possible even for the complemented roc/e-stop virus but that production of new infectious progeny and eventual propagation were severely inhibited compared to wild type virus (fig. 2c ). the transmembrane domain (tmd) of some coronavirus e protein is known to homo-oligomerize in membranes and appears to modulate infectious virus production . in order to determine the effect of hcov-oc43 e protein tmd on virus production in cell culture, pbac-oc43fl was modified at a key amino acid position previously identified in other coronaviruses to be critical for the stability of this specific domain ruch and machamer, 2012 ) and compared against wild-type virus during infection of cells. the large, polar glutamine at position 17 of the hcov-oc43 e protein putative tmd was modified into a smaller, non-polar alanine (pbac-e-tm-q17a) in order to diminish any possible ion channel selectivity conveyed by this amino acid (pervushin et al., 2009) at the opening of the putative ion channel. transfection of transmembrane mutant in bhk-21 cells yielded detectable virus titers of roc/e-tm q17a , which was further amplified on hrt-18 cells (p1) to significantly lower titers compared to reference virus (fig. 3a) . infection of human la-n-5 cells and mixed primary cultures of mouse cns cells showed a similar virus production kinetic infectious viral titer differences observed between experiments, revealed, by sequencing (g), the appearance of reversions at the position in the e gene where a stop codon was introduced are indicated by bold and underline. lod, limit of detection. ┼ (cross) indicates appearance of reversion(s) in the hcov-oc43 e gene in viral stocks as detected by sequencing. j.k. stodola et al. virology 515 (2018) 134-149 with an initial delay over the first 24hpi in the cell-free fraction ( fig. 3b and c, left panels). however, in the cell-associated fractions, the amount of recovered infectious virus particles was almost identical to those of the reference virus ( fig. 3b and c, right panels), suggesting a possible defect in virus release. in order to ascertain this potential defect, human la-n-5 cells were infected with either roc/atcc or roc/ e-tm q17a , and both infectious titer and viral rna copies (associated to total viral particles) in the cell-free fraction were quantified at 16 h post-infection (fig. 3d ). only the infectious titer was quantified in the cell-associated fraction as (with our system of q-rt-pcr), we are not able to differentiate between viral genomic and subgenomic rna and between viral rna that would be inserted in virion or free in the infected cell. similar levels of viral infectious particles were measured in the cell-associated fractions for both viruses at 16hpi indicating that the cells have been infected with the same efficiency and that the viral replication early steps are not affected by the alteration of the tmd. however infectious viral titer in the supernatant (cell-free fraction) of cells infected with roc/e-tm q17a was significantly reduced compared to cells infected with roc/atcc, although both viruses produced similar amount of total viral particles. these findings are supported by immunofluorescence assay showing a delay in propagation of roc/e-tm q17a over 72 hpi compared to reference virus (fig. 3e , figure s1a and b). bioinformatics analysis and modeling suggest that several coronavirus species possess a pdz-domain binding motif (pbm) at the extreme c-terminus of their e protein that could interact with cellular and viral proteins . we sought to investigate whether the putative four-amino acid e protein pbm of hcov-oc43 modulates production of infectious particles and infection of susceptible cells. to this end, we modified our cdna infectious clone to change the two key amino acids of the putative pbm motif, at the −0 and −2 positions from the c-terminal end respectively, into inert alanines, and thereby abrogated putative motif recognition by potential interaction partner(s). single amino acid mutants (pbac-oc-e-pbm-d82a and pbac-oc-e-pbm-v84 a) or double mutant (pbac-oc-e-pbm-d82a-v84a) (fig. 1a) , were transfected in bhk-21 cells and amplified on hrt-18 cells at the same multiplicity of infection and compared to reference virus (fig. 4a ). amplification on hrt-18 cells demonstrated that viral titers of the double mutant were significantly decreased compared to other viruses. to investigate whether the ability to infect susceptible cells, replicate and disseminate is affected by the putative c-terminal pbm in the context of the cns, la-n-5 or mixed primary cultures of murine cns cells were infected with single or double mutant pbm viruses and viral titers and propagation were analyzed. in la-n-5 cells, after 18 hpi, the titers of roc/e-pbm d82a-v84a were significantly decreased in the cell-free and cell-associated virus fraction compared to single pbm mutants or reference viruses and total infectious virus titers of the double mutant was severely altered over 72 h (fig. 4b ). this trend was exacerbated in primary mixed murine cns cultures, which showed no detection of infectious roc/e-pbm d82a-v84a compared to single mutant pbm and reference viruses (fig. 4c ). as no differences were observed between both single pbm mutant (roc/e-pbm d82a and roc/e-pbm v84a ) and reference virus, we continued the characterization of the potential pbm only with the double mutant virus (roc/e-pbm d82a-v84a ). immunofluorescence analysis indicated that cells could be infected by all viruses but we detected a significant difference in propagation for both la-n-5 cells (fig. 4d , figure s2a ) and primary mixed murine cns cultures ( figure s2b) , showing a significantly reduced propagation for the double pbm mutant roc/e-pbm d82a-v84a . given that the coronavirus e protein is known to play an important role in infectious virion formation and maturation (de groot et al., 2012) , we then looked if its deletion or the alteration of its putative functional domains could also alter the total virion production. using a rt-qpcr approach, the quantity of total viral particles in stocks of roc/ atcc, roc/e stop , roc/e-tm q17a or roc/e-pbm d82a-v84a were evaluated and compared to the infectious titer of the corresponding viruses (fig. 5a ). whereas the reference virus possesses a ratio of infectious virion to total viral particles of approximately 1:100, the roc/ e stop mutant has a ratio of 1:56 000 correlating with the default in infectious virion production previously observed. surprisingly, the quantity of total viral particles (evaluated as total rna copy number by rt-qpcr) was close to the reference virus. the same defect was observed for roc/e-tm q17a but to a lesser extent with a ratio of 1:1065, but it was absent for the roc/e-pbm d82a-v84a mutant. indeed, even though this latter mutant produces less virions compared to the reference virus, a very high proportion of them are infectious, as the ratio of infectious over total particles is about 1:2 (fig. 5a ). these observations of modified ratio were confirmed by immunofluorescence comparing the percentage of infected cells when the quantity of virus used for the infection of la-n-5 cells was normalized either to the number of infectious virions or to the number of total viral particles (fig. 5b ). all these findings demonstrate that the hcov-oc43 e protein is critical for efficient replication in epithelial and neuronal cells, and that its functional domains play important and potentially distinct roles during the production of new infectious virions. as hcov-oc43 is naturally neuroinvasive and neurovirulent in mice (brison et al., 2011; desforges et al., 2014; le coupanec et al., 2015) and that the e protein is important for efficient propagation in neuronal cells (figs. 3 and 4), we wished to investigate the importance of the two functional domains of e in the process of neuroinvasion. seven-day-old c57bl/6 mice were infected and rt-qpcr performed at 5 days postinfection on complete brain revealed that mutant viruses were still neuroinvasive compared to the reference virus, however the copy number of viral rna was significantly lower for both mutants (fig. 6a ). this indicates that the tmd and pbm are not essential for hcov-oc43 neuroinvasion. moreover, it is interesting to note that the 4 mice (3 infected with the reference virus, and one with the tmd mutant) with a high number of viral rna copy (> 10 13 ) were the only ones to show signs of illness at 5 dpi. to investigate the role of the e protein in the induction of hcov-oc43-induced neurological pathology, 22-day-old c57bl/6 mice were intracerebrally infected with either roc/atcc, roc/e-tm q17a or roc/ e-pbm d82a-v84a , and the development of illness was monitored for 21 days after infection. during this period, only mice infected with the reference virus died, all other mice survived the infection (fig. 6b) . moreover, mice infected with roc/e-pbm d82a-v84a did not show any significant differences of weight gain compared to sham infected mice (fig. 6c ), nor did they show any sign of neurological disease compared to mice infected with the reference virus (fig. 6d) . however, mice infected with roc/e-tm q17a showed an intermediate weight gain profile between mice infected with the reference virus and the sham infected mice (fig. 6c) , suggesting that these mice were developing a disease, which was confirmed by the neurological symptoms developed by several mice, although to a lesser extent than mice infected with the reference virus (fig. 6d ). mice infected with roc/e stop did not show any signs of illness whether in terms of weight gain, or neurological symptoms ( figure s3a -c). 2.7. hcov-oc43 e protein and its tmd and pbm are essential for efficient replication in the murine cns as hcov-oc43 e protein and its functional domains modulate viral replication and propagation in human and murine neuronal cultures (figs. 3, and 4) , we examined if the differences in neurovirulence observed ( fig. 6 ) were also to be associated with defective infectious virus productions and propagation in the cns. infection of 22-day-old c57bl/ 6 mice with reference and e protein mutants revealed that the infectious titer in the brain (fig. 7a ) and the spinal cord ( fig. 7b ) was significantly reduced for the tmd mutant. this altered replication in the brain correlates with an extremely weak production in the spinal cord, where infectious virions were detected in only one mouse at 5 dpi. production of infectious particles was under the limit of detection in mice infected with the pbm mutant, correlating with the total absence of neurovirulence (fig. 6) . however, the pbm mutant rna was detected in the brain at 5 and 9 dpi, although at a lower level than the reference virus or the tmd mutant (fig. 7c) , indicating that this virus was capable of replicating at a low level in the brain. similar observations were obtained following mouse infection with roc/e stop (fig. s3d ). viral rna of each mutant was extracted at 5 and 9 dpi and sequenced for the e and m gene. no reversion in the targeted genes were observed (data not shown), indicating that the inserted mutations are stable during replication in the murine brain for at least 9 days. it is interesting to note than when infected with a higher viral dose (10 2.5 tcid 50 /10 µl vs 10 1.5 tcid 50 /10 µl), infectious virions of the pbm mutant could be detected in low amount in the brain of mice ( figure s4a ). our system only allows to detect infectious titer over 10 2.5 . thus, the apparent (but very low) increase of infectious virus production observed at 10-11 days post-infection may be explained by the fact that the pbm mutant replicates at very low level between 3 and 11 days pi and that, by doing this, this mutant could be able to avoid detection by the immune system and delay its clearance from the cns. viral rna was also detected in both the brain and spinal cord ( figure s4b and c). sequencing of the e and m genes again revealed the absence of reversion, again suggesting that the absence of detectable infectious virion production previously observed (fig. 7) was probably due to the low infectious dose. taken together, these data demonstrate that a fully competent e protein (with both functional tmd and pbm) is essential for hcov-oc43 neurovirulence in association with efficient replication in the cns, correlating with the early observations in neuronal cells (figs. 2-4) . 2.8. the putative tmd decreases hcov-oc43 propagation in the murine brain when infection was performed at a 10 1.5 tcid 50 /10 µl dose, only the e protein tmd mutant produced detectable virions in the brain (infectious virus is under the limit of detection for pbm mutant). however, this production was significantly lowered compared to reference virus. therefore, based on data obtained in neuronal cultures (figs. 2-4), we investigated if the tmd mutation also induced a defect in propagation within the brain. brains of mice infected intracerebrally with roc/atcc or roc/e-tm q17a were harvested at 3 and 7 dpi, and viral spreading was observed by immunofluorescence. whereas the reference virus had already infected the hippocampus at 3 days postinfection (with positive cells around the lateral ventricle and in the hypothalamus), and then continued to spread until the rest of the brain was infected at 7 dpi ( fig. 8 left panels) , the roc/e-tm q17a presented an important delay in spreading as only a few number of infected cells were visible at 3 dpi except for a small focus of infection around the hippocampus and the lateral ventricle. spreading then occurred to the same brain regions compared to reference virus but to a much lower extent (fig. 8 right panels) . this suggests that although the tmd mutant seems to follow the same spreading path in the murine brain, its spreading is greatly delayed and underlines the fact that hcov-oc43 e protein tmd is important for efficient propagation in the murine brain. in this study, by modifying a full-length cdna infectious clone of the human hcov-oc43 virus, we demonstrate that e protein is critical for the production of infectious virions, as transient complementation with wild type e protein rescued infectious viral production and a strong selection pressure to revert to a functional e protein was observed. moreover, mutations of specific domains revealed that a fully functional protein participate in the efficient viral spreading, associated with neuropathogenesis. deletion of the e protein leads to varying degrees of defects for coronaviruses. indeed, whereas murine hepatitis virus (mhv) and sars-cov are attenuated, showing a reduced ability to produce infectious virus without e protein, in a cell-type specific manner (dediego et al., 2008 (dediego et al., , 2007 jimenez-guardeño et al., 2015; kuo and masters, 2003) , transmissible gastroenteritis virus (tgev) (ortego et al., 2002) and mers-cov (almazán et al., 2013) are replication competent, but completely propagation defective, with no detectable infectious virus production when the e protein is deleted. similarly, we were able to rescue infectious roc/e stop production by providing wild-type e protein in trans, as the recovery of initially complemented roc/e stop through complementation, and amplification on epithelial cells yielded detectable infectious virus (fig. 1) . as suggested for mers-cov (almazán et al., 2013) , the apparent low titer detected after the first passage on hrt-18 cells (especially at p1), could be due to a transfer of detached cells transfected with the initially complemented pbac-oc43-e-stop. furthermore, the production of infectious particles (for initially complemented mutant lacking the e protein) was low and decreased with j.k. stodola et al. virology 515 (2018) 134-149 subsequent amplification attempts. on the other hand, these results also suggest that production of infectious virions without e protein is possible but with severely affected efficiency, underlining the requirement of a fully functional e protein. coronavirus e protein has been suggested to allow correct virion formation in part by inhibiting m protein aggregation (kuo et al., 2007) , or by inducing scission events at the ergic (fischer et al., 1998) . our data indicate that while reducing dramatically the quantity of infectious viral particles formed, the complete abrogation of hcov-oc43 e protein did not significantly alter the quantity of total viral particles produced compared to the reference virus (fig. 5) , suggesting that the protein is important for efficient virion maturation that lead to good infectivity. this concept is emphasized by the appearance of recombinant hcov-oc43 e protein revertants at a very low passage number (p2 or p3) on hrt-18 cells. indeed, the appearance of revertants with different e sequences after only a few rounds of amplification on hrt-18 cells indicates that low level of viral particles must have been produced earlier during the process as we already observed previously for hedeleted recombinant hcov-oc43 (desforges et al., 2013) . we observed strong selective pressure at the position where we introduced a stop codon where nucleotide changes led to reversion to reference e sequence or to another amino acid residue (tryptophan or leucine). it was previously described that sars-cov and mhv (kuo and masters, 2010 ) e protein deletion mutants underwent compensatory mutations after a few passages in culture to utilize a partially duplicated version of the adjacent m protein to recover partial virus production. a second type of reversion was observed for sars-cov e protein deletion mutant after an intranasal infection of susceptible mice as the small transmembrane ion channel forming 8a was modified to incorporate a potential pbm associated with increased infectious virus production compared to e protein deletion mutant (jimenez-guardeño et al., 2015) . our results support the hypothesis fig. 6 . a fully functional hcov-oc43 e protein is associated with optimal neuroinvasion and increases neurovirulence. (a) 7-day-old c57bl/6 mice received 10 3 tcid 50 /20 µl of roc/ atcc, roc/e-tm q17a or roc/e-pbm d82a-v84a by the in route. neuroinvasion was detected and quantified by rt-qpcr on brain rna at 5 days post-infection, each point represents a single mouse. (b) twenty-two-day-old c57bl/6 mice received 10 2.5 tcid 50 /10 µl of roc/atcc, roc/e-tm q17a or roc/e-pbm d82a-v84a by the ic route and were observed for survival over 21 days following the injection. (c) infected mice were weighted every 2 days for a period of 21 days following infection. the weight gain is shown as percentage of d0 set at 100%. (d) evaluation of the clinical scores (percentage of mice at each level of the scale) of mice infected by roc/atcc, roc/e-tm q17a or roc/e-pbm d82a-v84a based on neurological symptoms described in clinical score scale between level 0 and 4 over a period of 21 days (see materials and methods). representative of three different experiments. (* p < 0.05; ** p < 0.01; *** p < 0.001). j.k. stodola et al. virology 515 (2018) 134-149 that there is selective pressure to specifically restore the e protein functionality itself without partial duplication of the m gene (data not shown), however, it is important to note that we only introduced a stop codon at the beginning of the e gene instead of deleting part or all of the gene, as it was done for sars-cov and mhv (jimenez-guardeño et al., 2015; kuo and masters, 2010) . a recombinant hcov-oc43 in which the e gene would have been deleted could have used the same reversion process in other viral gene. production of infectious virus was reduced after infection of neuronal cell cultures with initially complemented roc/e stop compared to reference virus. immunofluorescence assay for viral proteins confirmed these latter results showing no defect in entry for the initially complemented roc/e stop virus compared to reference virus at 16 hpi, while observations at 48 h indicated an important defect of viral propagation (fig. 2) . a decrease in virus spread was also reported for mhv (kuo et al., 2007; kuo and masters, 2003) and sars-cov (dediego et al., 2007) δe mutants which formed smaller and less numerous plaques. the coronavirus e protein is now considered as a virulence factor (reviewed extensively in ) and there have been extensive efforts to characterize the different domains of this relatively small transmembrane protein and the possibility that it acts as a viroporin, with ion channel activity. the tmd of several coronavirus e proteins (including hcov-oc43) was predicted (torres et al., 2005) and fig. 7 . the e protein tm and pbm domain are essential for optimal replication in the murine brain and spinal cord. infectious viral particles were quantified in (a) the brains and (b) the spinal cord of 22-day-old c57bl/6 mice infected by the ic route with 10 1.5 tcid 50 /10 µl roc/atcc, roc/e-tm-q17a or roc/e-pbm-82-84 over a period of 15 days. (c) viral rna was detected and quantified by rt-qpcr in the brain of infected mice at 5 and 9 days post-infection. lod, limit of detection. representative of three different experiments. fig. 8 . hcov-oc43 e protein putative tm domain is required for efficient spreading in mouse cns. viral spreading in mice brain was examined by immunofluorescence at 3 and 7 days post-infection. the virus was detected using an antibody against the hcov-oc43 s protein (green) and nuclei were detected using dapi (blue). small white arrows point to isolated infected cells. brain regions: 1, olfactory bulb; 2, lateral ventricle; 3, hippocampus; 4, brainstem; 5, hypothalamus. j.k. stodola et al. virology 515 (2018) 134-149 shown to form ion channels permeable to small cations in artificial membranes for species such as ibv, mhv, hcov-229e (wilson et al., 2006) , mers-cov (surya et al., 2015) and sars-cov (wilson et al., 2004) . furthermore, it was demonstrated that the ibv e protein can exist in a penta-oligomeric state (westerbeck and machamer, 2015) , and that for mers-cov (surya et al., 2015) and sars-cov (pervushin et al., 2009; torres et al., 2006; verdiá-báguena et al., 2013 , it forms pentameric channels in lipid membranes. chemical inhibition of ion channel activity (wilson et al., 2006) , destruction of the integrity of (almazán et al., 2014; regla-nava et al., 2015; ye and hogue, 2007) or replacement of the tmd with those of other viral species (ruch and machamer, 2011) all led to reduced viral titers for other coronaviruses species. the e protein of coronaviruses is largely localized within the secretory pathway (cohen et al., 2011; nieto-torres et al., 2011; venkatagopalan et al., 2015) where it has recently been described for ibv to exist in two different pools; one of monomeric e proteins that disrupt the secretory pathway and a second pool in an oligomeric state, likely serving to facilitate the assembly of progeny virions (westerbeck and machamer, 2015) . the transient delay in infectious virus release observed for the tmd mutant compared to the reference virus (fig. 3) suggests a defect in virus release that could be the result of damaged infectious particles as it was observed for ibv, for which a tmd mutant of the e protein induces a similar defect associated with increased quantity of non-infectious viral particles released possessing a cleaved s protein near the virion surface (ruch and machamer, 2011) . considering the higher proportion of non-infectious virions released by cells infected with the tmd mutant compared to cells infected with the reference virus (figs. 3d and 5a), a similar process consisting in an alteration of infectious virions in the secretory pathway may also be involved here, suggesting an important role for tmd within the cellular secretory pathway. furthermore, mutation at homologous position t16 of ibv did not have an effect on vlp formation but rather was required for secretory pathway disruption (ruch and machamer, 2012; westerbeck and machamer, 2015) . these results suggest that mutation q17a (homologous to ibv t16a) in the hcov-oc43 e protein putative tmd plays a role in modulating infectious virus release from infected cells. a four-amino acid c-terminal pbm protein-protein interaction motif has been predicted for hcov-oc43 . through the replacement of the key amino acids of this motif by alanines, we demonstrated its importance in infectious virion production in the epithelial and neuronal cells tested (fig. 4) . defective propagation without an effect on the ability to infect cells was observed with the recombinant virus with an abrogated putative pbm. deletion of the pbm in sars-cov e protein led to slight decreases in viral titers in some cell types while in others viral titers remained unaffected . our data on hcov-oc43 reveal significant infectious virus production defects in an epithelial cell line which was further accentuated in neurons, but only after infection by the double mutant roc/e-pbm d82a-v84a , suggesting that hcov-oc43 replication in cultured cells can tolerate a slight flexibility in the putative pbm sequence. among coronavirus e proteins, only the sars-cov e protein has been shown to per se possess such a functional motif, which interacts with pals1 to disturb secretory pathway membranes to alter tight junction formation (teoh et al., 2010) and syntenin to play a role in the exacerbated inflammatory response typical of infection via p38 mapk activation . for sars-cov e protein, the pbm is suggested to be important in two independent functions: virus stability and virulence/pathogenesis rather than virus production regla-nava et al., 2015) . given that the functions of pbm is dependent on their sequence and surrounding sequence context (ye and zhang, 2013) , further study of the hcov-oc43 e protein neuronal interactome is warranted and could provide new insights on the precise function of the protein in infected cells. prevention of a functional interaction between hcov-oc43 e protein pbm and its pdz domain-containing ligand could conceivably function in the same vein as seen in case of neurotropic encephalitic rabies virus, for which differences in disease phenotype, rapid versus attenuated spread of virus infection, was attributed to differences in pbm sequences on the c-terminal of the rabies envelope glycoprotein g leading to different cellular interaction partners to mediate either neuronal cell survival or death (préhaud et al., 2010) . surprisingly, the pbm mutant forms less total viral particles but almost all of them are infectious. as protein-protein interaction motifs, viral pbm are involved in a variety of processes, including viral particle assembly and maturation (javier and rice, 2011) . abrogation of the e pbm, could prevent critical cellular and/or viral interaction necessary for the rapid and efficient formation and maturation of viral particles, making propagation highly inefficient. the sars-cov e protein is critical for neuroinvasiveness in susceptible mice (dediego et al., 2008) . hcov-oc43 e protein seems to differ from its sars-cov homolog as viral rna is present in the brain of all mice intranasally infected with either the reference virus, the tmd mutant or the pbm mutant (fig. 6-a) , indicating that the fully functional protein is not essential for neuroinvasion. on the other hand, the amount of viral rna was significantly lower in the brain of e mutantinfected mice. although we cannot rule out that these data represent a difference in replication and propagation once the virus is already in the brain, optimal hcov-oc43 neuroinvasion may necessitate a fully functional e protein. the sars-cov e protein was recently described as an important virulence factor during infection of the lungs, being in part responsible for the immune response exacerbation , the lung epithelium destruction (teoh et al., 2010) , and edema accumulation in the lungs . we demonstrate here that hcov-oc43 e protein deletion as well as abrogation of the putative pbm prevented neurological symptoms following cns infection, correlating with replication and propagation observations in epithelial and neuronal cells (fig. 6) . disappearance of respiratory tract disease was observed for sars-cov in which e pbm was abrogated, preventing interaction of the viral protein with the pdzcontaining protein syntenin regla-nava et al., 2015) . the absence of neurological symptoms following infection by either roc/e stop or roc/e-pbm d82a-v84a could potentially be linked to glutamate excitotoxicity that we have previously observed in mice (brison et al., 2011) , possibly by interfering with pdz-domain containing proteins found in neuronal cells (feng and zhang, 2009 ). we also demonstrate that hcov-oc43 putative tmd plays a role in the neuropathogenesis following the cns infection, albeit to a lesser extent than its pbm. indeed, infected mice showed some neurological symptoms, even if their severity and frequency were lower than for the reference virus. this attenuated phenotype was also associated with reduced viral particles production and propagation in the cns. homologous mutation at the position 15 of sars-cov e protein also led to an attenuated pathology, although infectious viral particle production was not significantly affected compared to wild-type virus . taken together, these results demonstrate that hcov-oc43 e protein is a virulence factor, with tmd and pbm being important determinants in that matter. in summary, the current study demonstrates the critical importance of a fully functional hcov-oc43 e protein in infectious virus production and efficient spread in both epithelial and neuronal cells. modifications to key amino acids in putative functionally important domains modulated infectious virion production and delayed virus spread in human and murine neuronal cells and within mouse cns. this points towards the presence of a true tmd, which has a role in the secretory pathway, as seen for other coronaviruses and that the hcov-oc43 e protein putative c-terminal pbm plays a significant role in infectious virion production as well as efficient virus spread. in addition, this study establishes a clear link between both putative functional domains of hcov-oc43 e protein and cns pathogenesis. functional viroporins and viral pbm are associated with viral pathogenesis for a growing number of viruses and their studies to better understand virus-host interaction represent an emerging field (javier and rice, 2011; nieva et al., 2012; scott and griffin, 2015) . considering that the hcov-oc43 e protein seems to possess both functions and that they seem to be important for the induction of disease, future studies regarding their functionality and underlying mechanisms resulting in hcov-oc43 neuropathogenesis previously described (brison et al., 2011; jacomy et al., 2006; le coupanec et al., 2015) are warranted and necessary as they will help to identify virus-host interfaces which could represent therapeutic target. all animal experiments were approved by the institutional animal care and use ethics committee (iacuc) of the institut national de la recherche scientifique (inrs) and conform to the canadian council on animal care (ccac). animal care and used protocols numbers 1304-02 and 1604-02 were issued by the iacuc of inrs for the animal experiments described herein. the bhk-21 cell line (atcc-ccl10) was cultured in minimal essential medium alpha (mem-α; life technologies) supplemented with 10% (vol/vol) fetal bovine serum (fbs; paa ge healthcare) and used for transfection. the hrt-18 cell line (a gift from the late david brian, university of tennessee) was cultured in the same medium and used for virus infections/amplifications. the la-n-5 cell line (a kind gift of stephan ladisch, george washington university school of medicine) was cultured in rpmi medium supplemented with 15% (vol/vol) fetal bovine serum (fbs), 10 mm hepes, 1 mm sodium pyruvate, and 100 μm non-essential amino acids (gibco -invitrogen). the la-n-5 cells were differentiated into human neurons as previously described (hill and robertson, 1998) . briefly, cells were seeded in 24-well plates precoated with 0.1% gelatin (1.25 × 10 3 cells/well) in rpmi medium supplemented with 10% (vol/vol) fbs, 10 mm hepes, 1 mm sodium pyruvate, and 100 μm non-essential amino acids. the next day and every 2 days for 6 days, the medium was replaced with the same medium supplemented with 10% (vol/vol) fbs and 10 μm all-trans retinoic acid (sigma-aldrich). mixed primary cultures of mouse cns cells were prepared as previously described (le coupanec et al., 2015) . briefly, embryos at 15 days of gestation were removed from pregnant anesthetized cd1 mice and their cortex and hippocampus were harvested and placed in hanks balanced salt solution (hbss) medium, without ca 2+ and mg 2+ , supplemented with 1 mm sodium pyruvate and 10 mm hepes buffer. tissues were gently pipetted up and down with a pasteur pipette to dissociate the cells. after a decantation step of 3 min at room temperature, supernatants were transferred into a 50-ml tube with 36 ml of neurobasal medium (invitrogen) supplemented with 0.5 mm glu-tamax-i (life technologies), 10 mm hepes buffer, b27 supplement (life technologies), gentamycin and 10% (vol/vol) of horse serum (life technologies). cells were then seeded at 1 × 10 5 cells/cm 2 and grown on 50 µm poly-d-lysine-treated 12-well plates containing glass coverslips (for immunofluorescence) or not (for evaluation of infectious virus production) in the same medium, which was replaced by fresh neurobasal medium without horse medium the next day. the medium was changed every 2 days after and the cultures were ready for infection after 7 days in culture. using our full-length, cdna infectious clone pbac-oc43 fl (st-jean et al., 2006) the recombinant hcov-oc43 virus (roc/atcc) was generated. in parallel, a series of recombinant mutant viruses were produced by site-directed mutagenesis using the quikchange multi site-directed mutagenesis kit (stratagene) and a variety of primers (table 1 was also produced using the pbac-oc43-e-pbm-d82a as a dna template for a second-round of mutagenesis reaction with oc-e-pbmtable 1 primers used to introduce nucleotide substitutions in the e gene of pbac-oc43 fl (st-jean et al., 2006) for recombinant hcov-oc43 virus production with amino acid modifications within the e protein (section a). primers used to verify sequences of the full hcov-oc43 e and m gene of recombinant viruses (section b). bold and underlined sections represent newly introduced nucleotide substitutions. underlined section in mutant e-pbm 82-84 represents previously introduced nucleotide substitutions. j.k. stodola et al. virology 515 (2018) 134-149 d82a-v84a primer to introduce a second mutation at nucleotide position 251; amino acid 84. prior to transfection of bhk-21 cells, all samples were sequenced to make sure that only the introduced mutations were present and that no other mutations appeared. in order to insert the hcov-oc43 e gene into the pcdna3.1(+) expression vector (pcdna; invitrogen) and allow for e protein expression upon transient co-transfection with pbac-oc43 infectious clones in bhk-21 cells, restriction enzymes nhei and bamhi were added to the 5' (primer: 5'-gctagc atg ttt atg gct gat gct ta-3') and 3' (primer: 5'-ggatcc cta aac gtc atc cac at-5') ends of the e gene respectively. the e gene with added restriction enzyme sites was pcramplified from cdna originating from a hcov-oc43 reference strain (atcc) infection on hrt-18 cells using accuprime pfx supermix (life technologies) with 1 cycle at 95°c for 5 min, followed by 35 cycles at 94°c for 15 s, 48°c for 30 s and 68°c for 1 min and 1 cycle at 68°c for 4 min and then introduced into the pcdna plasmid. the bhk-21 cells were cultured in mem-α supplemented with 10% (vol/vol) fbs and used for transfection of pbac-oc43 cdna infectious clones with lipofectamine 3000 reagent (life technologies) according to the manufacturer's instructions. briefly, for production of recombinant viruses, bhk-21 cells were seeded in 6-well cell culture plates at 6×10 5 cells/well. the next day, when cells were 70-90% confluent, the medium was replaced and cells were transfected with 7.5 µl lipofectamine 3000 transfection reagent, 10 µl p3000 reagent, 5 μg of pbac-oc43 fl or other modified pbac dna, and 2 μg of pcdna(oc-e) or empty pcdna plasmid per well. for semi-quantitative determination of transfection efficiency by immunofluorescence assay (ifa), bhk-21 cells were seeded at 5 × 10 4 cells/well onto glass coverslips in 24-well plates and transfected with 1.5 µl lipofectamine 3000 transfection reagent, 6 µl p3000 reagent, 1.5 μg of pbac-oc43 fl or other modified pbac dna, and 500 ng of pcdna(oc-e) or empty pcdna plasmid per well. the plates were incubated at 37°c for 8 h and then medium replaced with mem-α supplemented with 10% (vol/vol) fbs and 0.01% (vol/vol) gentamycin and incubated for 3 days. the cells from 6-well culture plates were harvested either to recover total rna or total protein while the supernatant (p0) was recovered by aspiration after centrifugation at 500 × g for 7 min and then clarified at 1000 × g for 10 min. the supernatant (p0) served to inoculate hrt-18 cells in order to amplify the viral stocks. the supernatant from this firstround amplification (p1) served for a second round of viral amplification on hrt-18 cells from which supernatant was recovered (p2) and in some cases, was repeated again for a third round of amplification (p3). the production of infectious viral particles corresponding to the different pbac-oc43 cdna clones was titrated by an immunoperoxidase assay (ipa) prior to each amplification step in order that titers could be normalized to the lowest detectable titer and replication rates be compared. the hrt-18 and la-n-5 cells as well as mixed primary cultures of mouse cns cells were infected at a moi equivalent to the lowest detectable titer of the series of recombinant virus stock used during each experiment or mock-infected and then incubated at 33°c (hrt-18) or 37°c (la-n-5 cell line and primary cns cultures), for 2 h (for virus adsorption), and incubated at 33°c with fresh mem-α supplemented with 1% (vol/vol) fbs (for hrt-18 cells), at 37°c with fresh rpmi medium supplemented with 2.5% (vol/vol) fbs (for la-n-5 cells) or at 37°c with fresh neurobasal medium with b27-glutamax-i (for primary murine cns cell cultures) for different periods of time before fixing cells for immunofluorescence detection or harvesting the cell-associated and/or cell-free medium fractions for infectious virus titer determination by ipa. for relative infectivity and release assay, la-n-5 cells were infected at a moi equivalent to the lowest titer of the compared recombinant viruses and incubated at 37°c for 16 h with fresh rpmi medium supplemented with 2.5% (vol/vol) fbs and 200 nm chloroquine (n 4 -(7-chloro-4-quinolinyl)-n 1 ,n 1 -dimethyl-1,4-pentanediamine diphosphate salt, sigma, cas number 50-63-5) in order to prevent re-infection. infection of 22-day-old female or 7-day-old male and female c57bl/ 6 mice (charles river) were performed as previously described (le coupanec et al., 2015) . briefly, mice were inoculated respectively by the ic route with 10 1.5 or the intranasal route with 10 3 of 50% tissue culture infective doses (tcid 50 ) recombinant virus. groups of 10 mice infected by each recombinant virus were observed on a daily basis over a period of 21 dpi, and survival and weight variations were evaluated. clinical scores were evaluated using a scale with 5 distinctive levels 0-4); where 0 was equivalent to the asymptomatic mouse; 1 for mice with early hunched backs; 2 for mice presenting slight social isolation, weight loss, and abnormal gait; 3 for mice presenting total social isolation, ruffled fur, hunched backs, weight loss and almost no movement; and number 4 was attributed to mice that were in moribund state or dead. the ipa was performed on hrt-18 cells, as previously described (lambert et al., 2008) . briefly, the primary antibody used was mab 4.3e4 (hybridoma supernatant; ½ dilution) directed against the s protein of hcov-oc43. the secondary antibody was horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (kpl; 1/500). immune complexes were detected by incubation with 0.025% (w/v) 3,3'-diaminobenzidine tetrahydrochloride (bio-rad) and 0.01% (vol/ vol) hydrogen peroxide in pbs and infectious virus titers were calculated by the karber method, as previously described (lambert et al., 2008) . bhk-21 and lan-5 cells as well as mixed primary cultures of mouse cns were fixed onto glass coverslips with 4% (wt/vol) paraformaldahyde for 30 min at room temperature and permeabilized for 5 min with 100% methanol at −20°c. for la-n-5 and bhk-21 cells, to detect hcov-oc43 spike (s) protein, one-hour incubations of primary 4.3.e.4 (hybridoma supernatant; ½ dilution;) followed by secondary antibody alexafluor 488 donkey anti-mouse igg (h + l) (1/1000; life technologies-molecular probes) were conducted with three pbs washes between steps. for primary mouse cns cultures, after blocking with a pbs-bsa 2% (wt/vol) solution for one hour at room temperature, primary antibody polyclonal rabbit anti-s protein (dilution 1/1000) and mouse monoclonal antibody against the neuron-specific map2 protein (1/1000; bd pharmagen, catalog no. 556320) were diluted in pbs + 0.1% triton x-100 and incubated on cells for one hour at room temperature followed by three pbs washes. cells were then incubated one hour at room temperature with anti-rabbit alexa fluor 568-and antimouse alexa fluor 488-conjugated secondary antibodies (1/1000; life technologies-molecular probes) in pbs. for all cell types, nucleus detection was accomplished by a 5-min incubation with 4',6-diamidino-2phenylindole (dapi; 1 μg/ml; life technologies). triplicate samples were mounted on glass slides with immuno-mount medium (fisher scientific). immunofluorescent staining was observed under a nikon eclipse e800 microscope with a qimaging retiga-exi fast 1394 digital camera using procapture system software. percentage of infected cells was quantified from immunofluorescence pictures with the cellprofiler software (carpenter et al., 2006) . for immunofluorescence on brain section, perfusion with 4% (wt/ vol) paraformaldehyde (pfa) was performed on infected c57bl/6 mice for each virus, at 3 and 7 dpi. murine brains were carefully harvested and conserved in 30% (wt/vol) sucrose at 4°c for 72 h. prior to section, harvested brains were embedded in tissue-tek oct compound (sakura finetek, wwr) at −20°c. sagittal brain sections were prepared at a thickness of 60 µm with a microtome cryostat hm 525; microm. serial sections were collected and prior to staining, sections were incubated with a solution of two droplets of h 2 o 2 in pbs for 10 min at rt and washed with pbs. sections were then permeabilized with a solution of 0,1% triton in pbs for 2 h at rt, and blocked with a solution of pbs containing 1 droplet of horse normal serum according to the manufacturer's protocol (abc kit vectastain, vector laboratories) for 1 h at rt. for detection of viral antigens, sections were incubated overnight at 4°c in a 1/500 dilution of polyclonal rabbit anti-s protein of bovine coronavirus (bcov). after three washes with pbs, sections were incubated in the dark for 1 h at room temperature with the secondary fluorescent antibodies alexa fluor 488 anti-mouse (1/500; life technologies). after three pbs washes, sections were incubated for 5 min at room temperature with 4',6-diamidino-2 phenylindole (dapi; 1 μg/ml; life technologies), washed once with pbs and water and then mounted with immuno-mount mounting medium (fisher scientific). immunofluorescent staining was observed under a zeiss lsm780 confocal microscope. after transfection of bhk-21 cells or infection of hrt-18 cells, cells were scraped from wells or plates, centrifuged at 500 × g for 7 min at 4°c, medium was removed and cell pellet resuspended with 1.5 ml icecold pbs and centrifuged at 1000 × g for 10 min at 4°c. pbs was aspirated and the dry pellet stored at −80°c until use. total rna was extracted using the rneasy mini kit (qiagen) with qiashredder spin columns (qiagen) to lyse cells according to manufacturer's instructions. rna quality was verified using the agilent 2100 bioanalyzer using the agilent rna 6000 nano assay protocol according to manufacturer's instructions and concentration measured using a nd1000 spectrophotometer (nanodrop). to produce cdna, 5 μg of total extracted rna was reverse transcribed using the superscript iii first-strand synthesis supermix kit using oligo(dt) primer (invitrogen) according to manufacturer's instructions. pcr was conducted using accuprime pfx supermix (life technologies) with one cycle at 95°c for 1 min, followed by 40 cycles at 95°c for 35 s, 50°c for 45 s and 68°c for 2 min, followed by one cycle at 68°c for 7 min using several sets of primers to amplify the e gene (forward primer, oc-ns5-116-e-for, oc-e-222-for, oc-m-172-for or oc-m-439-for; reverse primers, oc-m127-e-rev, oc-m-514-rev, oc-m241-e-rev or oc-n-60-rev) and gapdh gene as control (forward primer, gapdh-for; reverse primer, gadph-rev). a complete list and description of all primers is presented in table 1 , section b. real time rt-pcr for the absolute quantification of viral rna in viral stocks and during infection of murine cns, was modified from vijgen and collaborators (vijgen et al., 2005) using the taqman technology and the use of crna standards for the generation of a standard curve and to evaluate the copy number of viral rna in samples with the megashortscript kit (ambion/life technologies) (fronhoffs et al., 2002; vijgen et al., 2005) . briefly, total rna was extracted with the qiazol reagent (qiagen) for hrt-18 cell culture supernatant and mouse tissue to evaluate the amount of viral rna in virus stock and in mouse tissue respectively. crna standards were constructed exactly as described elsewhere made as previously described (vijgen et al., 2005) . rna concentrations were evaluated in all samples and quantified using a nd1000 spectrophotometer (nanodrop). real-time quantitative rt-pcr was performed with the taqman-rna-to-ct 1-step kit (applied biosystems/life technologies) in a 20 µl reaction mixture with 10 µl of 2× taqman rt-pcr mix (containing rox as a passive reference dye), 900 nm of forward and reverse primers, and 200 nm of fam bhq1-tp probe. four µl of rna for supernatant samples and crna standards (serial dilutions), were used for the reaction. amplification and detection were performed in a steponeplus realtime pcr system apparatus and analysis were performed with the stepone software version 2.3 (applied biosystems). to confirm e protein production after transfection of pcdna(oc-e) in bhk-21 cells, proteins from whole cell lysates were extracted. harvested cells were pipetted up and down into ripa buffer (150 mm nacl, 50 mm tris, ph 7.4, 1% (v/v) np-40, 0.25% (wt/vol) sodium deoxycholate, 1 mm edta) supplemented with protease cocktail inhibitor (sigma). lysates were incubated on ice for 20 min and centrifuged at 17,000 × g for 10 min at 4°c. supernatants were harvested, aliquoted and stored at −80°c until further analyzed. protein concentrations were determined using a bicinchoninic acid (bca) protein assay kit (novagen) according to the manufacturer's instructions. ten μg of protein was loaded on a tris-glycine 4-15% gradient gel, transferred to pvdf membrane with a semi-dry trans-blot apparatus (bio-rad). membranes were blocked overnight at 4°c with tbs buffer containing 1% (vol/vol) tween (tbs-t) and 5% (wt/vol) non-fat milk. the following day all steps were conducted at room temperature, with, or solutions diluted in, tbs-t and milk. a primary rabbit polyclonal antibody was used to detect either the hcov-oc43 e protein (1/5000) or gapdh (1/10 000) for 1 h, followed by three 10 min washes. anti-rabbit igg horseradish peroxidase linked whole antibody (from donkey) (ge healthcare) was the secondary antibody used, followed by three 10 min washes. detection was performed using 1/1 solution of clarity western ecl substrate (bio-rad) for one minute followed by membrane exposure on cl-x-posure film (thermo scientific). for cell experiments (percentage of infection), statistical analysis were conducted by one-way analysis of variance (anova), followed by tukey's post hoc test, or a t-test. for mice experiments, results were compared using two non-parametric statistical tests: kruskal-wallis and mann-whitney. statistical significance was defined as p < 0.05 and is indicated with * (student's t-test p value < 0.05), ** (student's t-test p value < 0.01) or *** (student's t-test p value < 0.001). gratefully acknowledges a masters studentship from the fondation universitaire armand-frappier de l'inrs. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. this study was supported by discovery grant 42619-2009 from the national sciences and engineering research council of canada and operating grant mt-9203 from the institute of infection and immunity of the canadian institutes for health research to p.j.t. who is the holder of the tier-1 (senior) canada research chair in neuroimmunovirology award. j.k.s. gratefully acknowledges a masters studentship from the fondation universitaire armand-frappier de l'inrs. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. a vaccine candidate east respiratory syndrome coronavirus as a vaccine candidate coronavirus reverse genetic systems: infectious clones and replicons severe neurologic syndrome 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mutations in the e protein are attenuated and promising vaccine candidates the hydrophobic domain of infectious bronchitis virus e protein alters the host secretory pathway and is important for release of infectious virus a single polar residue and distinct membrane topologies impact the function of the infectious bronchitis coronavirus e protein viroporins: structure, function and potential as antiviral targets recovery of a neurovirulent human coronavirus oc43 from an infectious cdna clone mers coronavirus envelope protein has a single transmembrane domain that forms pentameric ion channels the sars coronavirus e protein interacts with pals1 and alters tight junction formation and epithelial morphogenesis the transmembrane oligomers of coronavirus protein e model of a putative pore: the pentameric alpha-helical bundle of sars coronavirus e protein in lipid bilayers coronavirus humains (hcov) coronavirus envelope (e) protein remains at the site of assembly coronavirus e protein forms ion channels with functionally and structurally-involved membrane lipids analysis of sars-cov e protein ion channel activity by tuning the protein and lipid charge development of one-step, real-time, quantitative reverse transcriptase pcr assays for absolute quantitation a coronavirus e protein is present in two distinct pools with different effects on assembly and the secretory pathway (jvi.01237-15) sars coronavirus e protein forms cation-selective ion channels hexamethylene amiloride blocks e protein ion channels and inhibits coronavirus replication detection of severe acute respiratory syndrome coronavirus in the brain: potential role of the chemokine mig in pathogenesis structures and target recognition modes of pdz domains: recurring themes and emerging pictures role of the coronavirus e viroporin protein transmembrane domain in virus assembly detection of coronavirus in the central nervous system of a child with acute disseminated encephalomyelitis mouse hepatitis virus gene 5b protein is a new virion envelope protein isolation of a novel coronavirus from a man with pneumonia in saudi arabia we thank jessie tremblay for excellent technical assistance with confocal microscopy. j.k.s. and g.d. wishes to thank mathieu meessen-pinard, mathieu dubé, and especially marc desforges and pierre j. talbot for their advice and support throughout the project. additionally, j.k.s. and g.d. wishes to thank m.d. and p.j.t. for guidance in the preparation of this manuscript. this study was supported by discovery grant 42619-2009 from the national sciences and engineering research council of canada and operating grant mt-9203 from the institute of infection and immunity of the canadian institutes for health research to p.j.t. who is the holder of the tier-1 (senior) canada research chair in neuroimmunovirology award. j.k.s. j.k. stodola et al. virology 515 (2018) 134-149 the authors have declared that no competing interests exist. supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2017.12.023. key: cord-291611-cfe8yujp authors: zhang, xuming; kousoulas, konstantin g.; storz, johannes title: comparison of the nucleotide and deduced amino acid sequences of the s genes specified by virulent and avirulent strains of bovine coronaviruses date: 1991-07-31 journal: virology doi: 10.1016/0042-6822(91)90154-4 sha: doc_id: 291611 cord_uid: cfe8yujp abstract the entire nucleotide sequences of the spike glycoprotein (s) genes of the highly virulent bovine coronavirus (bcv) strain bcv-ly138, the avirulent bcv-l9 and related norden vaccine (bcv-vaccine) strains were determined using the polymerase chain reaction (pcr) to amplify cdnas obtained by reverse transcription of viral rna, and to produce single strand cdnas for dna sequencing. the s gene sequences of these viral strains were compared with those of recently published strains bcv-mebus, bcv-quebec, and bcv-f15. an open reading frame of 4092 nucleotides, encoding a protein of 1363 amino acid residues, was found in all six strains. frameshifts and insertions or deletions were not observed except for the bcv-fl 5. the s gene sequences were more than 98% conserved overall inspite of different origins of the six viruses. there were 45 to 56 nt differences between the virulent and avirulent groups while there were 6 to 14 nt differences among four avirulent strains. comparison of the deduced amino acid sequences indicated that the s proteins had typical properties of membrane glycoproteins. nineteen winked glycosylation sites were predicted in five strains, and 18 of them were conserved in the avirulent strain bcv-l9. the sequence krrsrr at the predicted proteolytic cleavage site was identified in five strains while the sequence krrsvr was found in bcv-fl 5. substitutions of few amino acids in the putative fusogenic domains and two prolines at 507 and 567 in the antigenic domains may cause altered immunogenic and other functional properties of the s proteins specified by the virulent and avirulent bcv strains. nine amino acid substitutions between the virulent and avirulent groups may correlate with bcv virulence. bovine coronavirus (bcv) is a member of coronaviridae, causing severe diarrhea in newborn calves and winter dysentery in adult cattle ( 1, 2) . it possesses a single-stranded, nonsegmented rna genome with positive polarity (3). the virion contains four major structural proteins: the nucleocapsid protein (n), the transmembrane protein (m), the hemagglutinin/ esterase protein (he) and the spike protein (s) (4). the s glycoprotein is a predominant peplomeric structure forming the typical coronavirus morphology. it is synthesized as a high molecular weight (mol. wt.) precursor (gpl90) which is cleaved to yield two comigrating subunit polypeptides: the n-terminal half (sl ) and the c-terminal half (s2) with an approximate mol. wt. of 100 kda (3, 5) . the s glycoprotein functions in virus attachment to permissive cells, virus-induced cell fusion, elicitation of neutralizing antibodies and cell-mediated immunity (6). two antigenic domains responsible for neutralization of bcv-quebec and bcv-l9 have been identified on the s protein (ref. (7); hussain et a/., submitted). however, the location of the neutralizing epitopes on the bcv s protein has not yet been determined. comparison of the s proteins with s-spe' to whom reprint requests should be addressed cific monoclonal antibodies (mabs) revealed that different neutralizing and non-neutralizing epitopes as well as conformational epitopes were present in the s glycoproteins specified by the virulent and avirulent strains (hussain et al., submitted) . in order to understand the molecular basis of the observed antigenic diversity of the s glycoproteins we successfully amplified, directly sequenced, and cloned the entire s genes of different bcv strains using the polymerase chain reaction (pcr). we report here the sequence characteristics of one virulent and two avirulent bcv strains and their comparison with the recently published sequences of three other bcv strains. the strain bcv-l9 was derived from bcv-mebus and passaged through different nonpolarized and highly polarized cells over 80 passages (8). the norden vaccine strain (bcv-vaccine) was also derived from bcv-mebus and used asvaccine by norden laboratories (omaha, ne) ( 1, 9) . the highly virulent wildtype strain bcv-ly 138 was isolated from diarrhea1 fluid of a diseased calf in 1965 in utah/usa ( io), and maintained in calves through oral inoculation since then (8, 11) . this strain replicates only in hrt-18 cells but not in numerous bovine cells (8). all strains were propagated in hrt-18 cells as described previously (8). after 24 h.p.i., cells were washed twice with phos-phate buffered saline (pbs), and rna was isolated using isothiocyanate/cesium chloride gradients according to the method described previously ( 12). as control, rna was isolated from uninfected cells. the first-strand cdna synthesis was carried out in a volume of 25 ~1 containing 50 mh/l tris-hci (ph 8.3), 50milikci,3mmmgci,, lommdlt, 1 mmeachof the four dntps, 25 u rnasin (bethesda research laboratories (brl)), 2'pm 3' primer, 1 pg total rna, 200 u moloney murine leukemia virus reverse transcriptase (m-mlv rt) (brl) for 1 hr at 42", and the reaction was stopped by heating samples to 95" for 10 min and then cooled on ice. the rna was denatured after adding 20 m 11/1 methylmercuric hydroxide ( mehgoh) and 70 m m 2-mercaptoethanol for 7 and 5 min at room temperature, respectively, incubated with oligonucleotide primer at 65" for 2 min, and then chilled on ice before adding other reagents. two s gene specific primers were designed both for cdna synthesis and pcr amplification according to the published sequence ( 13). the a3' primer is antisense representing the sequence at the positions 2 165-2 187 (5'-7ttggatccagg-ttgcagctgtcgtgaaaga-3'). the b3' primer corresponds to the sequence downstream of the s gene (5'-aacggatccaatatatcgtcaggagcc-aata-3'). both primers contain an extra barnhi linker in the 5' end for cloning purposes. the primers were synthesized in the gene assembler (pharmacia-lkb, piscataway, nj) in our laboratory according to the manufacturer's instructions, and purified using poly-pak (glen research, herndon, va) or oligo-pak column (millipore, burlington, ma). the reaction of second strand cdna synthesis and amplification was carried out in pcr (microcycler, epperidot-f inc., fremont, ca) with 5 ~1 of the reverse transcription reaction in a total volume of 100 ~1 containing 20mmtris-hcl(ph8.3),25mmkci, 1.5mmmgci,, 0.1% tween 20, 10 pg of bovine serum albumin (bsa), 2.5 u of taq dna polymerase (brl), 200 &l each of the four dntps, and 20 pmol each of the primers. the sense a5' primer corresponds to the sequence upstream of the s gene (5'-gctgaancgataatggt-actaggctgcatgat-3'), and the b5' primer represents the sequence at the positions 2 165-2 187 (5'-gctgaa ttctctttcacgacagctgcaacct-3 '). these primers contain an extra ecorl linker on the 5' end as indicated. each cycle consisted of a 1 -set denaturation at 96", followed by 30-set annealing (at 59") and 2-min extention (at 72") steps. after 30 to 35 cycles, the final products were extended for 7 min at 72". single-strand cdna fragments were generated in a second pcr for sequencing. briefly, one-tenth of the double stranded pcr product and a single primer with 40 pmol were used. other reagents were the same as in the previous pcr. the cycle profile of 20 to 25 cycles was: 1-set at 96", 30-set at 50", 1-set at 60", 2-min at 72" (fig. 1) . we sequenced the entire s genes of one virulent and two avirulent bcv strains which display distinct biological properties and epitopes as defined by mabs to the reference strain bcv-l9. with the exception of 40 nt on the 5'-end and 50 nt on the 3'-end, the s gene sequences were determined in both directions at least once, as illustrated in fig. 1 . these sequences were compared with the recently published sequences for the strains bcv-mebus ( 14), bcv-quebec ( /5), and bcv-f15 ( 13), and their alignment is presented in fig. 2 (origins of these strains are described in the legend). all of the s genes contained an orf of 4092 nucleotides. the only variations among these sequences consisted of nucleotide substitutions. frameshift, deletion, or insertion, and nonsense mutations were not observed except for bcv-f15. the few nucleotide substitutions (91 nt) represented approximately 2% of the sequence, and they seemed to be distributed randomly. by comparison with other strains, the following differences were detected in two regions in bcv-f15: the deletion of a base (t) at nucleotide 2316 resulted in a frameshift for 2 aa followed by the insertion of an a at nucleotide 2320 which returned the amino acid sequence to a homologous alignment (at aa position 772 within the cleavage site; also see second-strand, pcr-mediated dna synthesis (c); double-stranded dna amplification by pcr (d); single-stranded dna synthesis by pcr using an excess of 5' primer (e) and 3' primer (f); dna sequencing strategies(g). a5'and 85' indicate two 5'primers containing an extra ecorl linker; a3' and b3' indicate two 3' primers containing an extra barnhi linker. ( 13), respectively. the origins and properties of these strains are as follows: the strain bcv-mebus was isolated from a calf with enteritis, and adapted to bovine fetal kidney cells described previously (25) . the strain bcv-quebec was isolated from a calf with enteritis by cultivation in vero and mdbk cells in quebec in 1979. its biological properties were similar to bcv-mebus (26) . the wild-type strain bcvf15 was isolated in hrt cells from a calf with enteritis in france in 1979 (27) . the consensus sequences are underlined, and the start and stop codes are marked by asterisks. most similar sequences were those of bcv-l9 and bcv-vaccine (6 nt differences) in paired comparisons, and the most distant were those of bcv-quebec and bcv-f15 (74 nt differences). the virulent wild-type strain bcv-ly138 was evolutionally distant from the avirulent strains (total number of differences between 45 to 56 nt). the french strain bcv-f15 also differed from the avirulent strains (total number of differences between 55 to 74 nt). although there were 55 nt differences between bcv-ly138 and bcv-f15, the same nucleotide substitutions occurred in 29 nt. an alignment of the deduced amino acid sequences of the s proteins of the six bcv strains is shown in fig. 3 . all six s genes encoded a predicted protein of 1363 aa residues, having a mol. wt. of approximately 150 kda. nineteen potential n-linked glycosylation sites were predicted in 5 strains while 18 of them were conserved in bcv-l9. fifty-seven cysteine residues were found in bcv-l9 while 56 of them were conserved in the other 5 strains. the bcv-f15 had an additional cysteine at position 840. the glycosylated protein had an estimated mol. wt. of approximately 190 kda. all six s proteins possessed at least 98% sequence identity in paired comparisons. as with the nucleotide sescv-l9 ldhnlnmfli llislpmafa vigdlkcttv sindvdtgu tyyvld rvylnitlll ngwptscst yrnmalkgtl llsrlwtkpp bcv-vacc sistd$m nglg -bcv-meb en web -2 bcv-y v bcv-f15 : t t bcv-l9 ecv-vacc plsdfingif akvkntkvik kgvnyseppa itigstfvnt syswvqpht tnldnklqgl leisvcqytm ceypbticbp ni&yvew rwdxvvscl i= bcv-meb bcv quences, the most related proteins were those of bcvgent proteins were those of bcv-mebus and bcv-f15 mebus and bcv-quebec (4 aa differences) or bcv-l9 or bcv-quebec and bcv-f15 (26 aa differences, reand bcv-vaccine (6 aa differences); the most diverspectively). the alignment reveals that the strain bcv-quebec is closely related to the avirulent strains bcv-mebus, bcv-l9, and bcv-vaccine (6 to 14 nt and 4 to 11 aa differences, respectively), suggesting that this strain is a variant of the prototype bcv-mebus. in contrast to the nucleotide sequences, the amino acid sequences of bcv-ly138 and bcv-f15 were more homologous (13 aa differences). as shown in fig. 3, 9 amino acids in the avirulent strains were substituted in both bcv-ly138 and bcv-f15: a to v (38), r to t (94),kton(121),ltom(254),htod(476),vtoa (749),vtoe(980),atov (1106),htop(1247),andi to k (1347). this suggests that the wild-type strain bcv-f15 is possibly a virulent strain, and that the 9 amino acids, especially the proline (at 1247) and lysine (at 1347) on the carboxy-terminal, may relate to bcv virulence. amino acid differences between the virulent and avirulent groups varied from 16 to 26 aa. apparently, most of these substitutions occurred in the sl peptide (fig. 3) . comparisons of the amino acid sequences in the antigenic region ( 14, 16) suggested that two proline substitutions may cause the antigenic differences: at amino acid position 507, a proline in the avirulent strain bcv-l9 was substituted by a serine in the virulent strain bcv-ly138, and another proline (at 567) was substituted by a threonine in all other strains. proline is considered a helix-breaking residue, which may influence significantly the conformation and secondary structure of peptide molecules ( 17) . the proline substitutions resulted in decreased hydrophilicity, surface probability, and antigenicity, but increased flexibility of the peptide in this region (data not shown). our previous study indicated that one neutralizing epitope of bcv-l9 was present in bcv-vaccine but absent in the virulent strain bcv-ly 138, and one non-neutralizing epitope was present only in the avirulent strain bcv-l9 (hussain et a/., submitted). the number of proline substitutions in this region coincides with the number of conformational epitopes absent in bcv-ly 138 and bcv-vaccine lead us to conclude that substitutions of these prolines to other amino acids may alter the conformation of the s protein, and cause the loss of conformation-dependent epitopes in bcv-ly138 and bvc-vaccine. this hypothesis is currently under investigation. it is unclear whether substitutions of these prolines and other amino acids in this region also correlate with bcv virulence. our previous studies revealed that the biological properties of fusion, plaque formation and host cell range were evidently different between the virulent and avirulent groups of bcv strains (8). the fusion activity of bcv is believed to be associated with the s polypeptide and the cleavage of the s into sl and s2 subunits (5, 18) . the sequence krrsrr at the predicted proteo-iytic cleavage site was conserved in the virulent strain bcv-ly138 and in the avirulent strains while the sequence krrsvr occurred in the wild-type bcv-f15 (fig. 3) . the cleavage site of bcv s protein is located in a hydrophilic area, in contrast to paramyxoviruses and myxoviruses, in which the cleavage site is located in hydrophobic domains (20-30 aa) of the fusion proteins ( 19, 20) . these observations imply that there might be a different mechanism involved in bcv-induced cell fusion. parker et al. (14) indicated that a sequence vlgclgsac (905-913) on the s protein of bcv-quebec may constitute a portion of the fusogenic domain within the bcv s2 subunit. this stretch is similar to the sequence llgcigstc of mhv-a59, which contains a neutralizing epitope (25) . by comparing with paramyxovirus and retrovirus, chambers et a/. (26) proposed that the hydrophobic region adjacent to heptad repeat sequences may be the potentially fusion-related domain in the s proteins of mouse hepatitis virus, infectious bronchitis virus, and transmissible gastroenteritis virus. after multiple alignments it is found that the heptad repeat sequences are located in the s2 at positions 999-l 038 which are conserved in all strains, and the hydrophobic regions are located at 961-998 (see fig. 3 ). however, a direct involvement of these sequences in bcv fusion has yet to be demonstrated. similarly, the el protein of semliki forest virus (sfv) does not contain a hydrophobic n-terminal region. it has been proposed, however, that in el an internal uncharged stretch of 17 residues, located about 80 aa from the n-terminal, might act as the putative fusogenic domain (27) . interestingly, we found three patterns of amino acid substitution in the putative fusogenic domain: at position 971, v in the avirulent strains was substituted by e in the virulent strains; w (898) and a (993) were substituted by i and v in bcv-mebus and bcv-quebec, respectively. it is unclear, however, whether these amino acid changes in the predicted cleavage sites and the putative fusogenic domains reflect any functional differences, such as fusion activities among different bcv strains. proteins enterites virales/viral enteritis in human and animals this work was supported by grants 86-crsr-2-2871 and 89. 34116-4675 from the united states department of agriculture, and louisiana education quality support fund of the board of regents, state of louisiana, to j.s. and k.g.k.. and grant al87886 to k.g.k. we thank mamie burrell and li-ju t. huang for excellent technical assistance, susan newman for computer assistance, and dr. b. kaltenboeck for valuable discussion on pcr technique. key: cord-256769-flfycl7i authors: stoermer, kristina a.; morrison, thomas e. title: complement and viral pathogenesis date: 2011-03-01 journal: virology doi: 10.1016/j.virol.2010.12.045 sha: doc_id: 256769 cord_uid: flfycl7i the complement system functions as an immune surveillance system that rapidly responds to infection. activation of the complement system by specific recognition pathways triggers a protease cascade, generating cleavage products that function to eliminate pathogens, regulate inflammatory responses, and shape adaptive immune responses. however, when dysregulated, these powerful functions can become destructive and the complement system has been implicated as a pathogenic effector in numerous diseases, including infectious diseases. this review highlights recent discoveries that have identified critical roles for the complement system in the pathogenesis of viral infection. have been identified, such as interaction of c1q with the c-type lectin sign-r1 expressed on macrophages (kang et al., 2006) , c5 activation by thrombin (huber-lang et al., 2006) , and the properdin-activated pathway (kemper et al., 2010) . the classical pathway is primarily activated by igm and certain igg isotypes bound to antigen. these immune complexes interact with the complement component c1q. c1q binding leads to the activation of two serine proteases associated with c1q, c1r and c1s. c1s cleaves c4 into c4a and c4b resulting in the exposure of a reactive thioester that allows covalent attachment of c4b on surfaces. c2 binds c4b and is also cleaved by c1s to form the classical pathway c3 convertase (c4bc2a). c3 convertases cleave c3 to amplify complement activation and lead to the generation of ligands for a variety of complement receptors. the lectin pathway is initiated by pattern recognition receptors such as mannose-binding lectin (mbl) and the ficolins (fig. 1) . mbl and ficolins contain carbohydrate-recognition domains that recognize carbohydrate patterns on the surfaces of cells or invading microorganisms. mbl and the ficolins are in a complex with enzymes known as mbl-associated serine proteases (masps). similar to c1s, masp-2 activates the complement system by cleaving both c4 and c2 to form the c4bc2a c3 convertase. the alternative pathway is activated by spontaneous hydrolysis of c3 (c3-h 2 o) (fig. 1) . this pathway also functions as an amplification loop for the cleavage of c3 initially triggered by other mechanisms. c3-h 2 o or c3b bound to target surfaces are bound by the protease factor b (fb). factor d (fd) is a serine protease that cleaves c3-h 2 o or c3b-bound fb, resulting in the generation of bb and formation of the alternative pathway c3 convertase (c3bbb). both the classical and alternative convertases function to cleave c3 to c3a and c3b. similar to c4, cleavage of c3 exposes a reactive thioester bond in c3b that allows for the covalent attachment of c3b to target surfaces. in addition, c3b can bind to either the classical or alternative c3 convertases resulting in a change of the substrate specificity of the convertases from c3 to c5. these c5 convertases cleave c5 to c5a and c5b. release of c5b promotes assembly of the c5b-c9 membrane attack complex (mac) which can directly lyse pathogens or pathogen-infected cells. the anaphylatoxins c3a and c5a interact with specific receptors to promote chemotaxis and regulate effector functions of cells of both the innate and adaptive immune response. a variety of soluble and membrane-associated proteins regulate complement activation. factor h (fh), c4b-binding protein (c4bp), and c1 inhibitor (c1-inh) are soluble proteins that regulate activation of the complement system. fh promotes the dissociation of c3bbb convertases and acts as a cofactor for factor i (fi)-mediated cleavage of c3b. c4bp functions similar to fh in that it promotes dissociation of c4bc2a convertases and acts as a cofactor for fi-mediated cleavage of c4b. the c1-inh is a serine protease inhibitor that inactivates both the c1q-associated proteases of the classical pathway (c1r and c1s) as well as the masps of the lectin pathway. membrane-associated regulators of complement activation include decay accelerating factor (daf/cd55), membrane cofactor protein (mcp/cd46), complement receptor 1 (cr1/cd35), cd59, and crry (rodent-specific). these proteins function to destabilize both the classical and alternative complement convertases, act as cofactors for fi-mediated cleavage of c3b and c4b, and prevent assembly of the mac on cell surfaces. the complement system is increasingly recognized as a mediator of protection or pathology in a variety of viral infections. furthermore, the continued identification of novel mechanisms of viral antagonism of complement highlight the important role this system has in viral pathogenesis. here, we review recent studies of viral interactions with a variety of components of the complement system and emphasize fig. 1 . schematic of the complement system. complement is activated by three major pathways. the classical pathway is primarily activated when c1q interacts with igm and certain igg isotypes bound to antigen. c1q-associated c1s cleaves c4 and c2 to form the classical pathway (cp) c3 convertase (c4bc2a). the lectin pathway (lp) is initiated by carbohydrate pattern recognition receptors such as mannose-binding lectin (mbl) and the ficolins (f) which are in a complex with enzymes known as mbl-associated serine proteases (masps). masp-2 activates the complement system by cleaving both c4 and c2 to form the c4bc2a c3 convertase. the alternative pathway (ap) is activated by spontaneous hydrolysis of c3 (c3-h 2 o). this pathway also functions as an amplification loop for the cleavage of c3 initially triggered by other mechanisms. c3-h 2 o or c3b bound to target surfaces are bound by the protease factor b (fb). factor d (fd) is a serine protease that cleaves c3-h 2 o or c3b-bound fb, resulting in the generation of bb and formation of the alternative pathway c3 convertase (c3bbb). both the classical and alternative convertases function to cleave c3 to c3a and c3b. cleavage of c3 exposes a reactive thioester bond in c3b that allows for the covalent attachment of c3b to target surfaces. in addition, c3b can bind to either the classical or alternative c3 convertases resulting in a change of the substrate specificity of the convertases from c3 to c5. these c5 convertases cleave c5 to c5a and c5b. release of c5b promotes assembly of the c5b-c9 membrane attack complex (mac) which can directly lyse pathogens or pathogen-infected cells. c3b is further cleaved by factor i (fi), a function enhanced by factor h (fh), to generate degradation products such as ic3b and c3dg. c3b and its degradation products interact with cellular receptors to regulate effector functions such as phagocytosis and b cell activation. the anaphylatoxins c3a and c5a interact with specific receptors to promote chemotaxis and regulate effector functions of cells of both the innate and adaptive immune response. those findings that describe how these interactions impact the development of virus-induced disease. perhaps the best evidence that complement has an important role in the outcome of virus infection is the identification of specific mechanisms evolved by viruses to evade the complement system (fig. 2 ). one mechanism employed by viruses is to directly encode proteins that have structural and functional homology to host proteins that function as regulators of complement activation. gammaherpesviruses, including kaposi's sarcoma-associated herpesvirus, herpesvirus saimiri, and murine γ-herpesvirus 68 (γhv68) encode homologs of complement regulatory proteins (albrecht and fleckenstein, 1992; mullick et al., 2003a; spiller et al., 2003; virgin et al., 1997) . the γhv68 regulator of complement activation (rca) protein is expressed on the surfaces of infected cells and is detected in supernatants of γhv68infected cells, thus it has both membrane-associated and soluble forms (kapadia et al., 1999) . recombinant γhv68 rca protein and supernatants from γhv68-infected cells blocked c3 deposition on zymosan examples of viral evasion of the complement system. a.) virally encoded proteins allow viruses to evade complement-mediated destruction. human astrovirus (hastv) coat protein (cp) binds mbl and c1q, inhibiting the activation of both the lectin and classical pathways. influenza a matrix (m1) protein also binds c1q. flavivirus nonstructural protein 1 (ns1) binds c4 and c1s, leading to enhanced cleavage of c4 to c4b, as well as factor h (fh), leading to increased cofactor activity of fh for factor i (fi)-mediated cleavage of c3b into ic3b. additionally, membrane-bound flavivirus ns1 decreases deposition of c3b and the mac on cell surfaces. the murine gammaherpesvirus-68 (γhv68) regulator of complement protein (rca) blocks c3 deposition, whereas the hsv-1 glycoprotein (gc) binds c3b and blocks the binding of properdin and c5 to c3b. the variola virus inhibitor of complement enzymes (spice), the vaccinia virus complement control protein (vcp), the monkeypox virus inhibitor of complement enzymes (mopice), and the ectromelia virus inhibitor of complement enzymes (emice) function as cofactors for fi-mediated cleavage of c3b by binding to c3b and c4b. viruses, with their corresponding proteins that interfere with the complement cascade in parentheses, are indicated in red. b.) some viruses recruit host complement regulatory proteins into their virions. human immunodeficiency virus-1 (hiv-1), human t-lymphotropic virus-1 (htlv-1), and human cytomegalovirus (hcmv) incorporate the host complement control proteins cd55/daf and cd59 into their virions, while simian virus 5 (sv5) and mumps virus (muv) recruit cd46/mcp into their virions. physiological complement regulatory proteins are shown in green. viruses that incorporate these regulatory proteins into their virions are indicated in red. beads by both the classical and alternative activation pathways (kapadia et al., 1999) . to directly test the role of the γhv68 rca protein in pathogenesis, kapadia et al. generated a γhv68 deleted for the rca protein and evaluated the outcome of infection in both wildtype and c3-deficient mice (kapadia et al., 2002) . deletion of the γhv68 rca protein resulted in decreased virulence following intracranial inoculation of weanling mice or intraperitoneal inoculation of adult ifn-γ −/− mice, models of acute γhv68-induced meningoencephalitis and γhv68 persistent infection, respectively. genetic deletion of c3 in the host was able to restore virulence of the rca protein-deleted virus in the model of acute meningoencephalitis and enhanced its persistent replication, confirming that rca protein interaction with the host complement system is a critical regulator of γhv68 pathogenesis. interestingly, although wild-type mice had a reduced number of latently infected cells compared to c3 −/− mice, indicating that complement regulates the establishment of γhv68 latent infection, this effect was not counteracted by the γhv68 rca protein (kapadia et al., 2002) . poxviruses, such as variola virus, vaccinia virus, monkeypox virus, and ectromelia virus, also encode complement regulatory proteins that have structural and functional homology to host encoded regulators of the complement pathway (mullick et al., 2003b) ( fig. 2a ). the variola virus inhibitor of complement enzymes (spice), the vaccinia virus complement control protein (vcp), the monkeypox virus inhibitor of complement enzymes (mopice), and the ectromelia virus inhibitor of complement enzymes (emice) all bind to c3b, as well as c4b, and function as cofactors for fi-mediated cleavage of c3b moulton et al., 2010; rosengard et al., 2002; sahu et al., 1998) . a vcp-deleted vaccinia virus produced smaller skin lesions in rabbits compared to wild-type vaccinia virus (isaacs et al., 1992) and pathogenesis studies in cynomolgus monkeys with different strains of monkeypox virus revealed that mopice is deleted from the less virulent strains (chen et al., 2005) . furthermore, spice is a much more potent inhibitor of human complement compared to vcp and mopice, correlating with the increased virulence of variola virus in humans compared to vaccinia virus and monkeypox virus rosengard et al., 2002) . taken together, these studies and the findings that multiple components of the complement pathway are required for mice to survive ectromelia virus infection (discussed below), indicate that complement activation and viral evasion of the complement system are critical determinants of poxvirus pathogenesis. in contrast to herpesviruses and poxviruses, flaviviruses encode a protein that antagonizes the complement system despite the lack of any sequence homology to known regulators of the complement system. the nonstructural protein 1 (ns1) encoded by flaviviruses is a glycosylated protein detected within infected cells, on cell surfaces, and secreted from infected cells (alcon-lepoder et al., 2006) . ns1 accumulates in the serum of dengue virus-infected individuals and high circulating levels are associated with severe disease (avirutnan et al., 2006; libraty et al., 2002) . in an attempt to purify wnv ns1 from cell supernatants, chung et al. observed that fh copurified with ns1 (chung et al., 2006) . soluble wnv ns1 was found to enhance the cofactor activity of fh for fi-mediated cleavage of c3b to ic3b, while cell surface-associated ns1 decreased deposition of c3b and the c5b-c9 membrane attack complex on cell surfaces ( fig. 2a) . flavivirus ns1 also binds to c4 and c1s (avirutnan et al., 2010) ( fig. 2a) . these activities enhanced the cleavage of c4 to c4b and resulted in reduced activity of the classical c3 convertase (c4b2a) and reduced c4b and c3b deposition on cell surfaces (avirutnan et al., 2010) , providing an additional mechanism by which flaviviruses can evade complement-dependent neutralization. soluble ns1 has also been reported to bind the complement inhibitory factor clusterin, which normally inhibits the formation of the c5b-c9 membrane attack complex (kurosu et al., 2007) , however, a functional consequence of this interaction has not been reported. many other viruses employ a similar complement evasion strategy by encoding proteins that bind and inhibit or sequester complement components. for example, the coat protein of human astrovirus type 1 (hastv-1) suppresses complement activation by binding c1q, functionally displacing the protease tetramer, and thus inhibiting classical pathway activation (bonaparte et al., 2008; hair et al., 2010) ( fig. 2a) . this was further demonstrated for serotypes 2 and 4 (bonaparte et al., 2008) . the astrovirus coat protein also bound mbl and inhibited mannan-mediated activation of the lectin pathway (hair et al., 2010) . as discussed below, c1q enhances the neutralizing and hemagglutination inhibition activity of anti-influenza antibodies. experimental evidence suggests that the matrix (m1) protein of influenza a virus has evolved to counteract this host response, as m1 prevents complement-mediated neutralization of influenza virus in vitro by binding c1q and blocking the interaction between c1q and igg (zhang et al., 2009 ). herpes simplex virus 1 (hsv-1) encodes several immune modulators, including glycoprotein c (gc), which inhibits activation of the complement cascade by binding c3 and c3 fragments (friedman et al., 1984; fries et al., 1986; kostavasili et al., 1997; tal-singer et al., 1991) and by blocking binding of properdin and c5 to c3b (fries et al., 1986; hung et al., 1994; kostavasili et al., 1997) . these effects are mediated by two distinct domains in gc: one domain blocks properdin and c5 binding to c3b, and the other directly binds c3 and c3 fragments (hung et al., 1994) . in vitro, gc protects hsv-infected cells from complement-mediated lysis (harris et al., 1990 ) and cell-free virus from complement-mediated neutralization (friedman et al., 1996; hidaka et al., 1991) . it was further demonstrated that natural igm antibody binds and neutralizes hsv-1 and hsv-2 gc-null viruses via a c1q-, c3-, and c5-dependent mechanism (hook et al., 2006) . studies in animal models have confirmed the importance of gc-mediated complement inhibition in hsv-1 pathogenesis. when inoculated intravaginally into wild-type guinea pigs, but not c3-deficient guinea pigs, a gc-null hsv-1 replicated less efficiently and caused significantly less severe vaginitis compared to wild-type hsv-1 (lubinski et al., 1998) . the c3-dependent attenuated phenotype of gc-null hsv-1 was confirmed by inoculating wild-type and c3-deficient mice via skin scratch and evaluating hsv-1-induced zosteriform disease (lubinski et al., 2002 (lubinski et al., , 1998 . further studies with this murine model demonstrated that both domains of gc that modulate complement activation are critical virulence factors; however, hsv-1 specifically lacking the c3 binding domain was more attenuated compared to hsv-1 specifically lacking the c5/ properdin inhibitory domain (lubinski et al., 1999) . importantly, the gc mutant viruses were as virulent as wild-type virus in c3-deficient mice, indicating that the gc interactions with the complement system regulate hsv-1 pathogenesis (lubinski et al., 1999) . most recently, the knowledge gained from these studies was utilized to improve vaccine efficacy against hsv-1 infection. awasthi and colleagues demonstrated that combined immunization with the hsv-1 glycoprotein d (gd), a potent immunogen, and gc prevented hsv-1 evasion from complement, due to the development of an anti-gc antibody response, and enhanced the protection provided by gd immunization (awasthi et al., 2009) . a number of viruses evade the complement system by recruiting host complement regulatory proteins into their virions (fig. 2b ). for instance, human immunodeficiency virus-1 (hiv-1), human t-lymphotropic virus-1 (htlv-1), and human cytomegalovirus (hcmv) incorporate the complement control proteins cd55/daf and cd59 into their virions, thus protecting virions from complement-mediated destruction spear et al., 1995) . simian virus 5 (sv5) and mumps virus (muv), both paramyxoviruses, can be neutralized in a c3-dependent manner resulting in virion aggregation and virion lysis, respectively (johnson et al., 2008) . further studies revealed that both sv5 and muv virions contained cd46/mcp, a membrane-associated protein that has cofactor activity for fi-mediated cleavage of c3b and c4b. the incorporation of cd46 into sv5 and muv conferred virion-associated cofactor activity and increased resistance of sv5 and muv to complement-dependent neutralization ). thus, these viruses have evolved to usurp host complement regulatory proteins to avoid complement-mediated destruction. mannose binding lectin (mbl) is a c-type lectin that plays an important role in innate immunity by binding to carbohydrates on a wide range of pathogens (fujita, 2002) . recognition of carbohydrates is mediated via a c-terminal carbohydrate recognition domain. once bound, mbl can activate complement, due to its association with masps, or act directly as an opsonin. polymorphisms in the promoter and structural regions of the human mbl2 gene affect mbl oligomer formation and circulating levels of the protein (madsen et al., 1995) . due to these different mutations, humans exhibit a 1000-fold variation in circulating mbl levels that occur with varying frequencies in different populations. recently, a number of studies have demonstrated that direct interactions between mbl and virus particles can neutralize infection. mbl has been found to bind directly to virions from a number of different virus families, including human immunodeficiency virus (hiv), severe acute respiratory syndrome coronavirus (sars-cov), ebola virus, dengue virus (denv), and west nile virus (wnv) (fig. 3) . the finding that the hiv envelope glycoprotein, gp120, is modified with high mannose oligosaccharides led researchers to test the potential of hiv and gp120 to function as ligands for mbl. mbl was shown to bind directly to hiv-infected cells and recombinant gp120 (ezekowitz et al., 1989) . the importance of these interactions was demonstrated by experiments showing mbl could inhibit infection of specific target cells by cell culture-derived hiv. although primary isolates of hiv bound to mbl (saifuddin et al., 2000) , mbl binding inefficiently inhibited infection of peripheral blood mononuclear cells (ying et al., 2004) . however, mbl binding did lead to efficient opsonization and uptake of hiv by mononuclear phagocytes (ying et al., 2004) . despite these findings, the role of mbl in hiv pathogenesis is still unclear. different studies investigating the association between mbl levels and hiv infection have found no association, increased susceptibility among individuals with low mbl levels, and increased susceptibility among individuals with high mbl levels (ji et al., 2005a) . however, this area of research has led to an effort to identify lectins that interact with hiv and inhibit infection as a novel therapeutic approach to prevent hiv infection and disease (alexandre et al., 2010; hoorelbeke et al., 2010; huskens et al., 2010) . mbl has also been reported to bind to sars-cov or retroviral particles pseudotyped with the sars-cov spike glycoprotein (sars-s) (ip et al., 2005; zhou et al., 2010) (fig. 3) . similarly, mbl binds efficiently to retroviral particles pseudotyped with ebola virus or marburg virus glycoproteins (ji et al., 2005a) (fig. 3) . importantly, all of these studies showed direct mbl-mediated neutralization of virus infection (ip et al., 2005; ji et al., 2005b) . mbl binding to sars-s was dependent on a single n-linked glycosylation site in sars-s (n330) (zhou et al., 2010) . mbl binding blocked the interaction of sars-s with dc-sign, previously identified to interact with sars-s (yang et al., 2004) , but not with the major sars-cov receptor angiotensinconverting enzyme 2 (ace2) (li et al., 2003) . serum levels of mbl were reported to be significantly lower in patients with sars than in control patients; there was, however, no association between mbl genotypes and mortality related to sars (ip et al., 2005) . a second study also reported that mbl gene polymorphisms associated with reduced mbl levels were significantly associated with susceptibility to sars-cov infection (zhang et al., 2005) . however, a third study reported that mbl genotypes and allele frequencies do not influence the outcome of infection with sars-cov (yuan et al., 2005) . finally, studies showed that mbl binds n-linked glycans on the structural proteins of wnv and denv, resulting in neutralization through a c3-and c4-dependent mechanism that occurred, in part, by blocking viral fusion . these findings indicated that mbl opsonization was not sufficient for neutralization, but rather deposition of c3 or c4 onto virions was also required. for wnv, neutralization occurred only with virus produced in insect cells; for denv, neutralization occurred with insect and mammalian cellderived virus . experiments in mice demonstrated an accelerated intravascular clearance of denv or of a wnv mutant with two n-linked glycans on its e protein, but not wild-type wnv, that was mbl-dependent . the complement system enhances humoral immunity by a number of different mechanisms. complement regulates effector functions of both natural and immune antibodies, complement component c3 and its receptors participate in the capture and transport of antigen to the b cell compartments of secondary lymphoid tissue (gonzalez et al., 2010) , and complement receptor 2 (cr2, cd21) and complement receptor 1 (cr1, cd35) expression by follicular dendritic cells function to retain antigen in the lymphoid follicles, which is required for the generation of a normal humoral immune response (fang et al., 1998) . on b lymphocytes, cr2 forms a complex with other proteins, such as cd19, to activate signal transduction pathways that regulate b cell activation. coligation of the b cell receptor and cr2, which binds the cleavage products ic3b, c3dg, and c3d covalently attached to antigen, lowers the threshold of b cell activation (bradbury et al., 1992; hebell et al., 1991; heyman et al., 1990) . in fact, linking c3d to a model antigen generated a fusion protein that was 100-1000 fold more immunogenic (dempsey et al., 1996) . numerous studies have established a critical role for the complement system in regulating humoral immunity to virus infection. below, we highlight recent findings with a particular emphasis on studies that have investigated these pathways in the context of viral pathogenesis. natural antibody and complement neutralize virus. the humoral immunity of naïve individuals consists primarily of natural igm antibodies (ochsenbein and zinkernagel, 2000) . natural igm is polyreactive and it is thought that endogenous antigens drive the generation of natural igm. igm, which exists in circulation primarily as a pentamer, has a 1000-fold greater binding affinity for c1q compared with igg, making it a potent activator of the complement system (ehrenstein and notley, 2010) . initial studies revealed the presence of natural igm specific for various viral pathogens, including lymphocytic choriomeningitis virus (lcmv), vesicular stomatitis virus (vsv), and vaccinia virus (vv), in the sera of naïve mice. by reconstituting rag-1 −/− mice with sera from naïve mice, researchers demonstrated that natural antibodies protected mice from both vsv dissemination and disease (ochsenbein et al., 1999a) . earlier studies had shown that a natural igm antibody to a vsv antigen forms an immune complex that activates c1 and initiates complement activation via the classical pathway at the viral surface, thus neutralizing vsv (beebe and cooper, 1981) . this neutralization was associated with c3b deposition on the viral envelope that likely interferes with vsv attachment to susceptible cells. natural igm and components of the classical activation pathway have also been shown to neutralize influenza virus (jayasekera et al., 2007) . interestingly, rather than resulting in lysis of virions, these interactions resulted in coating and aggregation of virus particles. to test the significance of these interactions on influenza pathogenesis, rag-1 −/− mice were reconstituted with natural igm and then challenged with the mouse-adapted pr8 strain of influenza. reconstitution prolonged mouse survival following influenza challenge, suggesting that natural igm and complement also protect the host from influenza infection. furthermore, both complement and igm were required for the development of protective immunity against influenza virus after immunization (fernandez gonzalez et al., 2008) . similar to influenza virus, sera from naïve mice neutralizes ectromelia virus by a mechanism dependent on both complement and natural antibodies (moulton et al., 2008) . further analyses revealed that opsonization of ectromelia virus particles with c3b and c4b was the primary mediator of neutralization. consistent with these findings, ectromelia virus dissemination was more efficient and viral loads in tissues were greater in mice deficient in c3. in mortality studies, the genetic deficiency in the complement components c3, c4, or fb resulted in a higher mortality rate compared to wild-type mice. in addition, reconstitution of b cell-deficient μmt mice with sera containing natural antibodies significantly increased survival following inoculation of ectromelia virus. in sum, these studies have revealed the important role of natural antibodies and complement in protection from virus infection. complement-mediated enhancement of b lymphocyte responses protects from virus-induced disease. the study of humoral responses to herpesvirus infections has yielded important insights into complement-mediated regulation of b cell function. mice deficient in c3, c4, or cr2 (which in mice encodes both cr1 and cr2) had significantly diminished igg responses following herpes simplex virus-1 (hsv-1) infection, suggesting that activation of the classical pathway plays a key role in regulating the antibody response to hsv-1 infection (da costa et al., 1999) . interestingly, c3-deficient mice reconstituted with bone marrow from wild-type mice developed normal antibody responses following hsv-1 infection, while wild-type mice reconstituted with bone marrow from c3-deficient mice had significantly diminished igg responses (verschoor et al., 2003 (verschoor et al., , 2001 . these experiments indicated that c3 derived from bone marrow cells was essential to enhance b cell activation in response to hsv-1 infection and were some of the first studies to describe an important function for complement factors synthesized locally by a non-hepatic cellular source. in contrast to hsv-1 infection, igg responses to vsv infection, which is neurotropic in mice and can cause paralysis and death, were similar in c3 −/− mice compared to wild-type mice (ochsenbein et al., 1999b) . instead, researchers discovered that complement components c3 and c4, but not cr2, were critical for initial anti-vsv igm responses. further experiments suggested that complement was critical for targeting vsv antigens to marginal zone macrophages in lymphoid tissues to stimulate b cells and igm production. c3 −/− mice, but not cr2 −/− mice, were more susceptible to lethal vsv infection, suggesting that the depressed igm responses were critical to limit vsv spread and replication in the central nervous system. unlike vsv infection, the genetic absence of c3 or cr2 resulted in increased wnv-induced mortality, earlier wnv entry into the central nervous system, and greater viral loads of wnv in the brains of mice, suggesting complement-mediated regulation of b cell responses was critical to control wnv dissemination, replication, and disease (mehlhop et al., 2005) . in fact, c3 and cr1/cr2 were required for normal anti-wnv igm and igg responses in mice and passive transfer of immune serum protected c3 −/− mice from lethal wnv infection. in further studies, antibody responses to wnv were found to be normal in mice deficient in fb and fd, components of the alternative complement activation pathway (mehlhop and diamond, 2006) . these findings were consistent with previous findings in which igg responses to a t-dependent antigen were normal in fb −/− mice (matsumoto et al., 1997) and indicated that components of the alternative complement activation pathway are not required for the development of an anti-wnv antibody response. in contrast, components of the classical and lectin complement activation pathways, c4 and c1q, were required for normal humoral responses to wnv infection (mehlhop and diamond, 2006) . interestingly, c4, but not c1q, was required for normal anti-wnv igm responses, whereas both c4 and c1q were required for normal anti-wnv igg responses. these findings suggest that distinct complement pathways regulate igm vs. igg responses, at least in the context of a viral infection. importantly, similar to c3 and cr2 deficient mice, c4-and c1qdeficient mice were much more susceptible to lethal wnv-infection, providing further evidence that the host's complement system is critical to limit the severity of wnv disease (mehlhop and diamond, 2006) . c1q regulates anti-viral antibody effector mechanisms. a number of recent studies have identified a critical role for complement, and in particular the complement component c1q, in the regulation of antiviral antibody effector mechanisms. researchers investigating the serum-specific enhancement of influenza virus hemagglutinin (ha)specific monoclonal antibodies discovered that c1q could enhance both neutralization activity and hemagglutination inhibition (hi) activity (feng et al., 2002) . c1q more strongly influenced hi activity, and this enhancement did not require c3, suggesting that the effects were independent of c1q-mediated complement activation. c1qmediated enhancement of hi activity and neutralizing activity was dependent on the antibody isotype and epitope specificity (feng et al., 2002; mozdzanowska et al., 2006) . antibody-dependent enhancement (ade) of infection has been described for multiple viruses . in the case of dengue viruses, ade is associated with more severe disease (balsitis et al., 2010; kyle and harris, 2008; zellweger et al., 2010) . utilizing in vitro assays designed to measure ade of dengue virus infection, researchers discovered that the presence of fresh serum could completely abolish ade (mehlhop et al., 2007; yamanaka et al., 2008) . further analyses demonstrated that the inhibitory effect of fresh serum on ade was c1q-dependent and antibody isotypespecific (mehlhop et al., 2007; yamanaka et al., 2008) . however, in separate studies the suppression of ade by fresh serum was found to be c3-dependent or c3-independent, thus the extent to which other complement components participate in mediating these effects is unclear. in addition to suppressing ade, c1q was shown to enhance the neutralizing activity of anti-wnv antibodies (mehlhop et al., 2009) . mechanistic studies revealed that c1q reduced the number of antibodies required to bind a wnv virion and neutralize infectivity. interestingly, c1q reduced the number of antibodies required for neutralization to levels below the minimum number required for ade. importantly, the ability of an anti-wnv antibody to protect from lethal infection was decreased in c1q −/− mice compared to wild-type mice, indicating that c1q enhancement of neutralizing activity regulates wnv pathogenesis. in contrast to the effect of c1q on ade of flavivirus infection, ade of ebola zaire virus infection is c1qdependent . the glycoprotein of the reston strain of ebola virus, which is less pathogenic in humans, induces less enhancing activity compared to the zaire strain, suggesting that ade may also play a role in ebola virus pathogenesis. regulation of t cells by the complement system. increasing evidence from various experimental systems indicates that complement regulates cd4 + and cd8 + t cell activation and effector functions (dunkelberger and song, 2010; kemper and atkinson, 2007) . numerous complement receptors and membrane complement regulatory proteins can be expressed by antigen-presenting cells (apcs) and t cells. engagement of these receptors on apcs regulates apc effector functions, such as chemokine and cytokine gene expression, and modulation of apc function influences t cell activation during antigen presentation. direct action of complement on t cells is less well understood. ligation of cr1 on t cells has been shown to inhibit t cell proliferation (wagner et al., 2006) , while the c5a receptor (c5ar) has been shown to regulate t cell trafficking (tsuji et al., 2000) . in addition, several membrane complement regulatory proteins have been linked to t cell regulation. for example, cd46, which binds c3b and c4b and serves as a cofactor for their proteolytic inactivation, was found to regulate the proliferation and effector functions of cd4 + and cd8 + t cells (marie et al., 2002) . critically, complement regulates the effector function of activated t cells by regulating the development of th1, th2, and th17 helper cells. for example, c3ar-deficient mice were protected against th2-dependent airway hypereactivity and this has been linked to both decreased th2 cell responses and enhance-ment of il-17 producing helper t cells (drouin et al., 2001; lajoie et al., 2010) . in contrast c5 provides protection against airway hypereactivity by regulating th17 cytokine production (lajoie et al., 2010) . finally, evidence suggests that complement also regulates the termination of t cell responses by inducing the development of regulatory t cells (kemper et al., 2003) . utilizing mouse models, researchers discovered that c3 was required for normal t cell responses to influenza virus, lymphocytic choriomeningitis virus (lcmv) infection, and friend virus (fv) infection (banki et al., 2010; kopf et al., 2002; suresh et al., 2003) . in vitro, dendritic cells exposed to complement-opsonized fv or hiv were better inducers of cd8 + t cell activation (banki et al., 2010) . these effects were also observed with complement opsonized fv in mice. furthermore, c3-deficient mice had reduced numbers of fv-specific cd8 + t cells and higher numbers of infected spleen cells compared to wild-type controls, suggesting complement-mediated regulation of t cell responses functions to control fv replication (banki et al., 2010) . similarly, mice deficient in c3 had delayed clearance of influenza virus and increased titers in the lungs. these findings correlated with a dramatic decrease in the recruitment of virusspecific cd4 + and cd8 + t cells producing ifn-γ to the lung of influenza virus-infected c3 −/− mice. following either lcmv or influenza virus infection, c3 was required for normal priming of cd4 + and cd8 + t cells and mice deficient in cr1 and cr2 (cr2 −/− mice) did not show any of these defects, indicating that c3 regulates t cell responses in a cr1/cr2-independent manner (kopf et al., 2002; suresh et al., 2003) . in lcmv-infected mice, the effects of c3 on cd8 + t cell priming and expansion were epitopedependent and influenced by the mouse genetic background, suggesting that c3 may regulate epitope selection. subsequent experiments indicated that the c5ar may have an important role in the regulation of t cell responses to influenza virus infection as treatment of mice with a c5ar antagonist reduced the number of influenza virus-specific cd8 + t cells in both the lungs and lymphoid tissue (kim et al., 2004) . studies utilizing a mouse model of wnv infection and disease identified distinct complement pathways that regulate antiviral t cell responses (mehlhop and diamond, 2006) . mice deficient in fb were found to be highly susceptible to lethal wnv infection, despite normal anti-wnv antibody responses. instead, fb −/− mice, but not c1q −/− mice, had reduced cd8 + t cell responses in the spleen and impaired trafficking of cd4 + and cd8 + t cells to the cns. similar findings were observed in wnv-inoculated c4 −/− mice, suggesting that both the lectin and alternative complement activation pathways contribute to regulation of t cell functions. thus, similar to fv, influenza virus, and lcmv infections, complement activation was required for normal t cell priming and trafficking following wnv infection. the role of complement regulatory proteins in regulating t cell responses to viral infection has also been investigated. mice deficient in decay accelerating factor (daf; cd55), which regulates formation and inactivation of the c3 and c5 convertases, had an increased expansion of cd8 + t cells with greater killing capacity following lcmv infection and this correlated with lower viral titers. these enhanced cd8 + t cell responses were reversed in mice deficient for daf and c3 (daf-1 −/− ; c3 −/− ) or daf and the c5ar (daf-1 −/− ; c5ar −/− ). in contrast, following vaccinia virus infection, cd4 + , but not cd8 + , t cell responses were enhanced in mice deficient in cd59a, which blocks formation of the membrane attack complex (longhi et al., 2005) . taken together, these studies suggest that membrane complement regulators regulate both cd4 + and cd8 + t cell immunity. however, cd4 + t cell responses following vaccinia virus infection were enhanced in both cd59a −/− and cd59a −/− ; c3 −/− mice, indicating that the effects of cd59a on t cell responses were likely independent of complement activation, at least following vaccinia virus infection. hepatitis c virus (hcv) appears to exploit the complement system to establish persistence. hcv core protein is the first protein expressed during the early phase of hcv infection and free hcv core particles are found in the blood of hcv-infected patients (maillard et al., 2001) . by evaluating various vaccinia virus (vv)/ hcv recombinants in mice, large et al. discovered that a recombinant vv expressing the hcv core protein produced a lethal infection in mice and suppressed the vv-specific cd8 + t cell response (large et al., 1999) . using a yeast two-hybrid approach, kittlesen and colleagues identified that the hcv core protein bound the gc1q receptor (gc1qr) specific for the globular heads of the c1q protein (kittlesen et al., 2000) (fig. 3) . binding of gc1qr by the hcv core protein inhibited human peripheral blood t cell proliferation in standard one-way mixed lymphocyte reactions (mlr) or following mitogen stimulation (kittlesen et al., 2000) , a similar effect to the binding of c1q, the natural ligand for the gc1qr. this suppression was reversed by the addition of anti-gc1qr or anti-core antibodies in the t-cell proliferation assay (kittlesen et al., 2000) . more specifically, binding of hcv core protein to gc1qr on activated human peripheral blood t cells decreased il-2 and ifn-γ production and decreased il-2r and cd69 expression (yao et al., 2001) . a recent clinical study investigated the impact of hcv infection on t cell responses in acute as compared to resolved versus chronic infection (cummings et al., 2009) . during the acute phase of hcv infection, the frequency of gc1qr + cd4 + t cells increased compared to healthy controls. six months later, the frequency of gc1qr + cd4 + t cells remained elevated in chronic patients compared to that in resolved patients. chronic patients also had higher levels of circulating hcv core protein. tcr stimulation increased the frequency of gc1qr + cd4 + t cells, resulting in core-induced inhibition of t cell responses in both resolved and chronic patients. these results suggest that hcv infection expands gc1qr + cd4 + t cells, increasing the susceptibility to core-mediated immune dysregulation (cummings et al., 2009) . the gc1qr is also expressed by other immune cells, such as dendritic cells (dcs) and b cells. dcs isolated from patients chronically infected with hcv display a reduced capacity to induce t cell activation and to produce th1 cytokines (auffermann-gretzinger et al., 2001; bain et al., 2001; dolganiuc et al., 2003; kakumu et al., 2000; kanto et al., 1999 kanto et al., , 2004 . waggoner and colleagues demonstrated that binding of the hcv core protein to gc1qr on human monocytederived dcs (mddcs) inhibited tlr-induced il-12 production but not the production of other tlr-induced cytokines (waggoner et al., 2007) . in addition, hcv core protein engagement of gc1qr on mddcs promoted the production of th2 cytokines such as il-4 by cocultured cd4 + t cells. these results suggest that engagement of gc1qr on dcs by hcv core limits the induction of th1 responses (waggoner et al., 2007) . in contrast to the effects on t cells and dcs, hcv core protein binding to gc1qr on b cells leads to b cell activation and proliferation: increased costimulatory and chemokine receptor expression, cell proliferation, igm and igg production, and stat1 phosphorylation, and down-regulation of socs-1 expression (yao et al., 2008) . these data suggest that hcv exploits the complement system to dysregulate the host immune response via various mechanisms in order to establish persistence. to further investigate the role of the hcv core protein in hcv pathogenesis, transgenic mice were developed with tetracycline-regulated conditional hcv core protein expression (chang et al., 2009) . these mice develop liver inflammation, steatosis, and fibrosis. microarray analyses of inflamed liver showed induction of many components of both the complement and coagulation pathways, and administration of cd55 (daf) decreased hepatic inflammation, suggesting that the hcv core and the complement system contribute to hepatic inflammation associated with hcv infection. cryoglobulinemia is a systemic vasculitis that damages small and medium-sized arteries and veins of the skin, kidneys, and peripheral nerves (dammacco et al., 2001) , and evidence suggests that the deposition of immune complexes on the vessel wall activates complement and mediates this damage. mixed cryoglobulinemia (mc), which involves multiple immunoglobulin isotypes, is observed in 10-70% of hepatitis c virus (hcv)-infected patients and is associated with increased duration of hcv infection (charles and dustin, 2009 ). in hcv-positive patients with mc, circulating, nonenveloped hcv core protein was detected in cryoprecipitable immune complexes (sansonno et al., 2003) . as discussed above, the hcv core protein interacts with the gc1qr. the gc1qr can be proteolytically cleaved and released from the cell surface (kittlesen et al., 2000) . studies with hcv-positive mc patients revealed that mc following hcv infection correlated with significantly higher levels of circulating gc1qr compared to hcv-infected patients without mc (sansonno et al., 2009 ). in addition, higher serum gc1qr levels negatively correlated with circulating levels of the c4d fragment, which was instead sequestered in the vascular bed from skin biopsies of mc patients, indicative of complement activation in the vascular bed (sansonno et al., 2009) . these data suggest that following hcv infection, dysregulated shedding of gc1qr molecules contributes to vascular cryoglobulin-induced damage via a complement-mediated pathway (sansonno et al., 2009 ). hcv has also been linked to pulmonary diseases such as asthma and chronic obstructive pulmonary disease (copd) (moorman et al., 2005a) . in in vitro studies using normal human lung fibroblasts, moorman and colleagues found that the hcv core protein induced il-8 mrna and protein expression via gc1qr (moorman et al., 2005b) . taken together, these studies suggest that the hcv core protein interaction with the gc1q receptor of the complement system contributes to the pathogenesis of multiple diseases associated with hcv infection. further investigation into the proinflammatory role of the hcv core protein may provide a clearer understanding of the pathogenesis of hcv-associated diseases. chronic hcv infection is characterized by persistent complement activation, and can lead to liver fibrosis despite antiviral therapies. by interbreeding fibrosis-susceptible and fibrosis-resistant strains of mice, c5 was identified to be associated with hepatic fibrosis, and small molecule inhibitors of the c5a receptor had antifibrotic effects in vivo (hillebrandt et al., 2002) . polymorphisms of the human gene c5 were associated with advanced liver fibrosis, as compared with mild fibrosis, in chronic hcv infection (hillebrandt et al., 2005) (fig. 3) . moreover, the at-risk c5 haplotype in humans was associated with high serum c5 levels (hillebrandt et al., 2005) . these data suggest that c5 has a causal role in non-hcv and hcv-associated hepatic fibrosis across species and that c5-targeted therapeutics could be a beneficial treatment strategy. although denv ns1 can inhibit activation of the complement system (avirutnan et al., 2010) , evidence suggests that complement activation may contribute to increased disease severity following denv infection (fig. 3) . dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) are serious clinical conditions that typically occur after a second dengue infection by a different viral serotype or after a primary infection in infants born to dengue-immune mothers. lower levels of c3, c4, and fb, and higher levels of c3 cleavage products, were detected in severely ill dhf patients and correlated with signs of shock, suggesting that complement activation contributes to the more severe forms of dengue virus-induced disease (bokisch et al., 1973; churdboonchart et al., 1983; nascimento et al., 2009 ). in addition, dhf patients have been found to have higher levels of fd, which cleaves fb to yield the active (c3bbb) c3 convertase, and lower levels of fh, which inactivates the (c3bbb) c3 convertase, compared to patients with df (nascimento et al., 2009) . other studies have shown that plasma levels of sns1 and products of complement activation, including those with a known vascular effect such as c3a, c5a, and the terminal sc5b-9 complement complex, were present at higher levels in dhf patients before plasma leakage took place (avirutnan et al., 2006) , further supporting the hypothesis that complement activation is involved in the development of severe disease. comparison of global gene expression profiles in peripheral blood mononuclear cells (pbmcs) of patients with df or dhf discovered that the complement inhibitor cd59 was more strongly up-regulated in pbmcs from df patients than in pbmcs from dhf patients. furthermore, the wild-type mbl genotype, but not mbl genotypes associated with reduced mbl levels, was associated with the development of dengue-related thrombocytopenia and more severe disease (acioli-santos et al., 2008) . this is interesting given the recent findings that mbl binds denv resulting in complement activation and virus neutralization . these studies implicate the complement system in the pathogenesis of the more severe forms of dengue virus associated disease, dhf/dss; however, the mechanisms by which the complement system contributes to disease severity are not well understood. arthritogenic alphaviruses, including ross river virus (rrv) and chikungunya virus, are mosquito-borne viruses that cause debilitating musculoskeletal inflammation and pain in humans. evidence of complement activation was detected in synovial fluid from rrvinfected patients (morrison et al., 2007) . similarly, in a mouse model of rrv-induced disease, complement activation products were detected in inflamed joint and muscle tissues of wild-type mice (morrison et al., 2007) . rrv-infected c3-deficient mice exhibited less severe destruction of skeletal muscle tissue and less severe disease signs, indicating an important role for complement in rrv pathogenesis (fig. 3) . mice deficient in cr3 (cd11b −/− ) also developed less severe tissue damage and disease signs following rrv infection (morrison et al., 2008) (fig. 3) . interestingly, neither c3 nor cr3 deficiency prevented recruitment of inflammatory leukocytes to musculoskeletal tissues, suggesting that c3 and cr3 may act downstream of inflammatory cell invasion to promote tissue damage during rrv infection (morrison et al., 2007 (morrison et al., , 2008 . the genetic absence of c3 and cr3 significantly diminished rrv-induced expression of s100a9, s100a8, and il-6 within inflamed skeletal muscle tissue, indicating that the induction was regulated, at least in part, by cr3 interaction with its c3-derived ligand ic3b (morrison et al., 2008) . furthermore, mice deficient in mbl (mbl-dko) also developed less severe rrv-induced disease and these mice had reduced mbl and c3 deposition on tissues (unpublished results), providing evidence that rrv infection leads to complement activation via the lectin-dependent activation pathway. taken together, these studies suggest that interfering with the complement cascade and/or complement receptor signaling may represent a useful route for therapeutic intervention of rrv-induced disease. finally, recent evidence suggests that the complement system may contribute to seizures induced by virus infection. infection of wildtype mice with theiler's murine encephalomyelitis virus (tmev) results in acute seizures (libbey et al., 2008 (libbey et al., , 2010 . in contrast, c3 −/− mice developed far fewer seizures following tmev infection which correlated with reduced numbers of activated microglia and macrophages in the hippocampus and dentate gyrus as well as decreased neuronal cell loss (libbey et al., 2010) (fig. 3) . interestingly, depletion of systemic complement with cobra venom factor did not impact tmev-induced seizures, suggesting that locally produced complement within the central nervous system is sufficient to enhance tmev-induced seizures (libbey et al., 2010) . it is clear that the complement system is a critical determinant of the outcome of infection by a variety of different viruses. our understanding of the mechanisms by which complement protects from virus-induced disease has improved dramatically. research in this area will not only continue to contribute to our knowledge of viral pathogenesis, but will continue to provide insight into the regulation of immune responses, and lead to improved therapeutic and vaccine approaches for both viral and non-viral pathogens. perhaps less well understood are the mechanisms by which complement functions as a pathogenic effector in some virus-induced diseases. further progress towards identifying the signals and pathways that lead to complement activation, which are not understood for many viruses, particularly in vivo, and a deeper understanding of the impact of complement activation on host immune responses to viral infection may shed light. 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with complements from innate immunity activation of human cd4 + cells with cd3 and cd46 induces a t-regulatory cell 1 phenotype properdin: emerging roles of a patternrecognition molecule complement c5a receptor is essential for the optimal generation of antiviral cd8 + t cell responses interaction between complement receptor gc1qr and hepatitis c virus core protein inhibits t-lymphocyte proliferation complement component c3 promotes t-cell priming and lung migration to control acute influenza virus infection mechanism of complement inactivation by glycoprotein c of herpes simplex virus secreted complement regulatory protein clusterin interacts with dengue virus nonstructural protein 1 global spread and persistence of dengue complement-mediated regulation of the il-17a axis is a central genetic determinant of the severity of experimental allergic asthma suppression of host immune response by the core protein of hepatitis c virus: possible implications for hepatitis c virus persistence angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus seizures following picornavirus infection role for complement in the development of seizures following acute viral infection high circulating levels of the dengue virus nonstructural protein ns1 early in dengue illness correlate with the development of dengue hemorrhagic fever structure and regulatory profile of the monkeypox inhibitor of complement: comparison to homologs in vaccinia and variola and evidence for dimer formation cutting edge: murine cd59a modulates antiviral cd4 + t cell activity in a complementindependent manner herpes simplex virus type 1 glycoprotein gc mediates immune evasion in vivo in vivo role of complement-interacting domains of herpes simplex virus type 1 glycoprotein gc herpes simplex virus type 1 evades the effects of antibody and complement in vivo interplay between promoter and structural gene variants control basal serum level of mannan-binding protein nonenveloped nucleocapsids of hepatitis c virus in the serum of infected patients linking innate and acquired immunity: divergent role of cd46 cytoplasmic domains in t cell induced inflammation complement and coagulation: strangers or partners in crime? modulation of the antitumor immune response by complement abrogation of the alternative complement pathway by targeted deletion of murine factor b protective immune responses against west nile virus are primed by distinct complement activation pathways complement activation is required for induction of a protective antibody response against west nile virus infection complement protein c1q inhibits antibody-dependent enhancement of flavivirus infection in an igg subclass-specific manner complement protein c1q reduces the stoichiometric threshold for antibody-mediated neutralization of west nile virus hepatitis c virus and the lung: implications for therapy induction of p38-and gc1qr-dependent il-8 expression in pulmonary fibroblasts by soluble hepatitis c core protein complement contributes to inflammatory tissue destruction in a mouse model of ross river virus-induced disease complement receptor 3 promotes severe ross river virus-induced disease surviving mousepox infection requires the complement system ectromelia virus inhibitor of complement enzymes protects intracellular mature virus and infected cells from mouse complement enhancement of neutralizing activity of influenza virus-specific antibodies by serum components kaposi's sarcomaassociated herpesvirus (human herpesvirus 8) open reading frame 4 protein (kaposica) is a functional homolog of complement control proteins herpes and pox viral complement control proteins: 'the mask of self alternative complement pathway deregulation is correlated with dengue severity natural antibodies and complement link innate and acquired immunity control of early viral and bacterial distribution and disease by natural antibodies protective t cell-independent antiviral antibody responses are dependent on complement complement: a key system for immune surveillance and homeostasis variola virus immune evasion design: expression of a highly efficient inhibitor of human complement interaction of vaccinia virus complement control protein with human complement proteins: factor i-mediated degradation of c3b to ic3b1 inactivates the alternative complement pathway role of virion-associated glycosylphosphatidylinositol-linked proteins cd55 and cd59 in complement resistance of cell line-derived and primary isolates of hiv-1 interaction of mannose-binding lectin with primary isolates of human immunodeficiency virus type 1 non-enveloped hcv core protein as constitutive antigen of cold-precipitable immune complexes in type ii mixed cryoglobulinaemia role of the receptor for the globular domain of c1q protein in the pathogenesis of hepatitis c virus-related cryoglobulin vascular damage host cell-derived complement control proteins cd55 and cd59 are incorporated into the virions of two unrelated enveloped viruses. human t cell leukemia/lymphoma virus type i (htlv-i) and human cytomegalovirus (hcmv) complement regulation by kaposi's sarcoma-associated herpesvirus orf4 protein complement component 3 is required for optimal expansion of cd8 t cells during a systemic viral infection antibody-dependent enhancement of viral infection: molecular mechanisms and in vivo implications antibody-dependent enhancement of ebola virus infection herpes simplex virus glycoprotein c is a receptor for complement component ic3b early local generation of c5a initiates the elicitation of contact sensitivity by leading to early t cell recruitment cutting edge: myeloid complement c3 enhances the humoral response to peripheral viral infection myeloid c3 determines induction of humoral responses to peripheral herpes simplex virus infection complete sequence and genomic analysis of murine gammaherpesvirus 68 hcv core protein interaction with gc1q receptor inhibits th1 differentiation of cd4 + t cells via suppression of dendritic cell il-12 production the complement receptor 1, cr1 (cd35), mediates inhibitory signals in human t-lymphocytes infection-enhancing and -neutralizing activities of mouse monoclonal antibodies against dengue type 2 and 4 viruses are controlled by complement levels ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign hepatitis c virus core protein inhibits human t lymphocyte responses by a complement-dependent regulatory pathway differential regulation of socs-1 signalling in b and t lymphocytes by hepatitis c virus core protein interaction of mannose-binding lectin with hiv type 1 is sufficient for virus opsonization but not neutralization influence of fcgammariia and mbl polymorphisms on severe acute respiratory syndrome enhanced infection of liver sinusoidal endothelial cells in a mouse model of antibody-induced severe dengue disease association between mannose-binding lectin gene polymorphisms and susceptibility to severe acute respiratory syndrome coronavirus infection influenza a virus m1 blocks the classical complement pathway through interacting with c1qa a single asparagine-linked glycosylation site of the severe acute respiratory syndrome coronavirus spike glycoprotein facilitates inhibition by mannose-binding lectin through multiple mechanisms complement regulators and inhibitory proteins an enormous body of work has contributed to the knowledge of the complement system and viral interactions with the complement system. with that said, we acknowledge the research that was not specifically mentioned in this review. we thank michelle davis for her essential and outstanding contributions to the figures in this review. this work was supported by nih/niaid research grant k22 ai079163 awarded to t.e.m. k.a.s. was supported by the predoctoral nih institutional training grant t32 ai052066. key: cord-290993-bsnja161 authors: mcauliffe, josephine; vogel, leatrice; roberts, anjeanette; fahle, gary; fischer, steven; shieh, wun-ju; butler, emily; zaki, sherif; st. claire, marisa; murphy, brian; subbarao, kanta title: replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys date: 2004-12-05 journal: virology doi: 10.1016/j.virol.2004.09.030 sha: doc_id: 290993 cord_uid: bsnja161 sars coronavirus (sars-cov) administered intranasally and intratracheally to rhesus, cynomolgus and african green monkeys (agm) replicated in the respiratory tract but did not induce illness. the titer of serum neutralizing antibodies correlated with the level of virus replication in the respiratory tract (agm>cynomolgus>rhesus). moderate to high titers of sars-cov with associated interstitial pneumonitis were detected in the lungs of agms on day 2 and were resolving by day 4 post-infection. following challenge of agms 2 months later, virus replication was highly restricted and there was no evidence of enhanced disease. these species will be useful for the evaluation of the immunogenicity of candidate vaccines, but the lack of apparent clinical illness in all three species, variability from animal to animal in level of viral replication, and rapid clearance of virus and pneumonitis in agms must be taken into account by investigators considering the use of these species in efficacy and challenge studies. severe acute respiratory syndrome (sars) was recognized in late 2002, and by the end of the outbreak in july 2003 more than 8000 cases and 774 deaths were attributed to sars worldwide . a new coronavirus, termed the sars coronavirus (sars-cov), was isolated from patients with sars ksiazek et al., 2003; poutanen et al., 2003) . soon after the disease was recognized, the ability to experimentally infect and induce interstitial pneumonitis in cynomolgus macaques with sars-cov was demonstrated, thus fulfilling koch's postulates and confirming that sars-cov was the causative agent of sars kuiken et al., 2003) . efforts to develop an animal disease model for sars have continued. balb/c mice have been established as a good rodent model for the replication of sars-cov and viral replication has also been demonstrated in ferrets and cats (martina et al., 2003) . although pigs and chickens could be experimentally infected with sars-cov, these species did not support efficient virus replication (weingartl et al., 2004) . in addition to the importance of establishing the microbial etiology of a disease, animal models are critical to the study of disease pathogenesis and the evaluation of treatment and control strategies. non-human primates have been useful in evaluating vaccines and studying disease pathogenesis for several respiratory viruses including influenza, respiratory syncytial virus, and human parainfluenza viruses (crowe et al., 1993; durbin et al., 2000; murphy et al., 1982; rimmelzwaan et al., 2001; skiadopoulos et al., 2002) . these studies demonstrated that the susceptibility of different monkey species to specific respiratory viruses is variable. however, assessment of the level of virus replication in the respiratory tract of these models has been very informative. the primary purpose of this study was to identify the most permissive non-human primate species that could serve as a reproducible and informative model for the evaluation of sars vaccines and immunotherapy. we evaluated the level of virus replication and serologic response to infection with sars-cov in three species of old world monkeys. we first undertook a comparison of the level of replication of sars-cov administered via the respiratory route to three species of old world monkeys (4 african green, 4 rhesus and 4 cynomolgus monkeys). clinical signs of the febrile respiratory illness that defined sars in humans or afebrile respiratory tract illness were not observed in any of the monkeys following administration of 10 6.3 tissue culture infectious doses (tcid 50 ) of sars cov. rectal temperatures of all monkeys remained in the normal range (98-102 8f) for the duration of the study with the exception of one african green monkey (agm) that had a fever of 105 8f on day 3. the level of replication of sars-cov in the upper and lower respiratory tract of monkeys of all the three species detected from combined nose and throat (nt) swabs and tracheal lavage (tl) samples is presented in fig. 1 . the level of viral replication in agms was greater than that in cynomolgus monkeys, which was greater than that in rhesus monkeys. mean peak virus titers in agms were 10 3.1 and 10 3 tcid 50 /ml in the upper and lower respiratory tract, respectively (panels e and f). plasma, urine, and fecal specimens from rhesus monkeys and agms did not yield infectious virus but viral genome was detected by reverse-transcriptase polymerase chain reaction (rt-pcr) from fecal samples of the four sarsinfected agms between days 8 and 20 (data not shown). laboratory evaluations in agms revealed two findings that were not observed in rhesus or cynomolgus monkeys and were suggestive of systemic infection and may warrant further investigation. first, the hemoglobin concentrations and hematocrit of the four agms decreased by a mean of 25% by days 8-10 following infection and returned to baseline by day 27. the total white blood cell and platelet counts did not demonstrate a similar pattern. second, liver enzyme elevations (alanine amino transferase and isocitrate dehydrogenase) were noted in the serum of two agms with peak levels at day 4 post-infection; these values returned to baseline by day 27. serum igg antibody titers against recombinant baculovirus-expressed spike (s) protein of sars-cov were measured in an elisa assay (table 1) . four-fold rises in titer were detected in all monkeys following primary infection but titers in pre-infection (day 0) sera ranged from 1:640 to 1:2560, suggesting that although the elisa assay detected four-fold rises following infection, it also detected some pre-existing cross-reactive antibodies. all monkeys were seronegative (titer b 1:8) for sarsspecific neutralizing antibodies (nt ab) prior to virus administration ( table 1 ). the mean titers of nt ab achieved on day 28 in the three species of monkeys correlated with the levels of virus replication in the respiratory tract; the mean titer was 1:27 in rhesus monkeys, 1:31 in cynomolgus monkeys, and 1:57 in african green monkeys (table 1 ). in our assay, the mean neutralizing antibody titer in postinfection sera from mice was 1:25 to 1:49 and the titer in a convalescent human serum sample was 1:71 (unpublished data). serologic evidence of infection, defined as a four-fold rise in nt ab titer, was observed in 4 of 4 rhesus, 3 of 4 cynomolgus, and 4 of 4 agms. although the study described above indicated that all three species of monkeys were infected with sars-cov, there were significant discrepancies between our findings and published reports of cynomolgus macaques infected with sars-cov; kuiken et al. (2003) reported clinical illness and pathologic evidence of disease in cynomolgus macaques. we did not euthanize the monkeys in our first study, so we were unable to determine whether there were any histologic changes associated with virus replication. in order to resolve these differences, we performed an additional study in six agms, where four agms received the same dose (10 6.3 tcid 50 ) of sars-cov by intranasal and intratracheal administration and two were mock-infected. two infected and 1 mock-infected animal were euthanized and necropsied on day 2 and the remaining two infected and one mock-infected animals were euthanized and necropsied on day 4. combined nt swabs were collected daily and tl fluids were collected every other day till the animals were euthanized, so that we could determine how accurately nt swabs and tl samples reflected the level of virus replication in the lungs. at necropsy, trachea, nasal turbinates, and 12 to 14 samples from each animal's lungs were assayed for virus titration and histopathology and samples from the liver and spleen were examined for histopathologic changes. in three of four sars-cov-infected agms, virus titers in tl samples did not accurately reflect the titer of virus present in tracheal or lung tissue (table 2 ) and consistently higher titers of virus were seen in lung, tracheal, or nasal turbinate tissue homogenates than in nt swabs or tl samples (table 2) . moderate (agm id #443) to high (agm id #400) titers of virus were present in the tissue samples from the right lung on day 2, but the detection of virus was more patchy and titers were significantly lower on day 4 (table 2 ). virus titers in samples from the right lung were higher than in samples from the left lung, presumably because the right main stem bronchus is straighter and more of the intratracheally administered virus inoculum reached the right lung. there was no difference between the titer of virus in samples collected from the hilar region compared to those from the periphery of the lungs (data not shown). microscopic examination of lungs of agms sacrificed on day 2 post-infection revealed focal interstitial mononuclear inflammatory infiltrates (fig. 2) and edema in the lung. immunohistochemical assays (ihc) showed focal distribution of viral antigens in tracheal and bronchiolar epithelial cells, pneumocytes (figs. 3a-c), and macrophages on day 2 post-infection. ihc with double staining confirmed that the majority of sars-cov-antigen-positive cells were type i pneumocytes (co-labeled with cytokeratin and not colabeled with surfactant, fig. 4a ) and some scattered macrophages (co-labeled with antibodies to cd68, fig. 4b ). the degree of inflammation and the amount of viral antigen were significantly reduced in the lungs of animals fig. 1 . sars-cov replication in the respiratory tract of monkeys. mean titers of virus (expressed as log 10 tcid 50 /ml of sample; y axis) detected on indicated days (x axis) in the upper respiratory tract (left panels, a, c, and e, closed symbols) and lower respiratory tract (right panels, b, d, and f, open symbols) of rhesus (panels a and b, x, w), cynomolgus (panels c and d, e, 4), and african green (panels e and f, n, 5) monkeys following intranasal and intratracheal administration of 10 6 tcid 50 of sars-cov. error bars associated with each data point indicate standard error, and the dotted line indicates the lower limit of detection of virus (10 0.5 tcid 50 /ml). sacrificed 4 days post-infection compared to findings on day 2 post-infection (data not shown). prominent histopathologic changes and immunostaining were not observed in liver and spleen on day 2 or day 4 post-infection. the ability of primary infection to prevent re-infection was evaluated in the agms. the four agms from the first study were challenged with the same dose (10 6.3 tcid 50 ) of sars-cov administered via the intratracheal and intranasal route 2 months following primary infection. this study was performed before we established that virus titer in tissue homogenates exceeded titers seen in respiratory secretions, so only nt swabs and tl were collected from these animals following challenge. virus replication was highly restricted in the respiratory tract upon challenge; virus was not isolated from the upper respiratory tract of any monkey and was isolated at a single time point (day 2) at the lowest detectable titer in the lower respiratory tract of 2 agms (id #240 and #304). the possibility that this low titer represents residual virus inoculum cannot be ruled out. the agms had measurable nt ab present prior to challenge, but two of the agms (id #131 and #240) developed a four-fold rise in titer (data not shown) suggesting that, although virus replication was restricted in the respiratory tract upon challenge, at least two monkeys were re-infected. we compared the responses of three species of old world monkeys to experimental infection with sars-cov and established that although clinical illness was not present in any of the three species, the rhesus, agms and all but one cynomolgus monkey could be infected. sars-cov replicated to a higher titer and for a longer time in the respiratory tract of agms than in rhesus or cynomolgus monkeys, and the nt ab response correlated with the level of virus replication detected in the respiratory tract. consistently higher titers of virus were seen in tissue homogenates at necropsy than in nt swabs or tl fluid collected before the animals were sacrificed, indicating that more virus was present in the tissues than in secretions. this observation is consistent with the clinical finding that severe lung disease can occur early in sars cases, at a time when virus is not easily detected in respiratory secretions . histopathologic examination from agm lungs revealed interstitial pneumonitis in association with sars-cov on day 2 that was resolving by day 4. ihc staining for sars-cov antigens and double staining showed that tracheal and bronchial epithelial cells and type 1 pneumocytes were involved early in infection. these findings are consistent with our findings in mice and hamsters infected with sars-cov, where antigen is detected in epithelial cells early in infection (roberts et al., in press; subbarao et al., 2004) . 1:10240 a monkeys received 1 ml each of 10 6 tcid 50 of sars-cov by intranasal and intratracheal administration on day 0 and sera were collected before virus administration and 28 days later. b sera were tested starting with a dilution of 1:8. serum samples that did not neutralize virus infectivity at the starting dilution were assigned a titer of 1:4 in determining four-fold rises. c four-fold rises in titer are indicated in bold type. four agms received 10 6.3 tcid 50 of sars-cov by intranasal and intratracheal administration on day 0. b virus titers are expressed as log 10 tcid 50 /ml from secretions and as log 10 tcid 50 /g from 10% w/v tissue homogenates. c titers in lung are expressed as mean f standard error for 4-8 samples obtained from each lobe of the lung. d virus was not detected; the lower limit of detection in tissue homogenates was 10 1.5 tcid 50 /g and in nt swabs and tl was 10 0.5 tcid 50 /ml. these findings are consistent with those reported by kuiken et al. and haagmans et al., in appearance and patchiness, though we see more evidence of pneumonitis at an earlier time point post-infection than reported by or haagmans et al. (2004) (day 2 instead of day 6 or 4, respectively). kuiken et al. (2003) also found that one of four macaques did not have detectable virus in the lungs at day 6. the key difference that remains unresolved between our findings and those of kuiken et al. is that we did not observe lethargy, skin rash, or respiratory distress in cynomolgus macaques (or rhesus or agms) infected with the urbani strain of sars-cov. kuiken et al. (2003) infected cynomolgus monkeys by administration of a hong kong sars-cov strain into the nose, trachea, and conjunctiva, while our studies were done with the urbani strain administered into the nose and trachea. possible reasons that could account for the differences in our findings and those reported by kuiken et al. (2003) in cynomolgus monkeys are differences in the strains of sars-cov used and differences in the subspecies of monkeys in the two studies. both strains of sars-cov were isolated from fatal human cases and subsequently underwent a similar number of passages in vero cells. however, it is possible that the strain used in the studies of kuiken et al. is more virulent for cynomolgus monkeys; this question can be resolved by evaluating both strains in parallel. there are concerns about the risk of enhanced disease on re-exposure to sars, as has been described with feline infectious peritonitis virus infection (fipv) in cats, where accelerated and enhanced disease can occur on re-exposure to fipv in seropositive cats (pederson and boyle, 1980; scott, 1981a, 1981b) . infection of macrophages by fipv is believed to be important in the pathogenesis of accelerated disease. sars-cov primarily infects epithelial cells in the lungs of agms but there is evidence of infection in some macrophages as well that warrants further study. however, the lack of clinical illness and active viral replication on challenge of agms is reassuring. we followed the agms for 2 months after challenge and saw no evidence of enhanced disease and no evidence of virus replication in the lungs, liver, kidneys, spleen, or intestines at necropsy 2 months following challenge. these observations are consistent with our findings in mice and hamsters, where primary infection with sars-cov confers resistance to reinfection and we have not detected evidence of enhanced disease upon re-exposure to sars-cov (roberts et al., in press; subbarao et al., 2004) . we have carefully considered the optimal use of these three non-human primate species in the context of other available animal models for sars including mice, hamsters, and ferrets. serologic data from our study confirm that nonhuman primates will be useful for vaccine immunogenicity studies (gao et al., 2003) and our virologic data and histopathologic findings indicate that agms can be used for vaccine efficacy studies (bukreyev et al., 2004) . sars-cov titers in lung tissue homogenates were high in one agm and moderately high in the other at day 2 but were significantly lower in both animals sampled on day 4. an early peak of viral replication, seen in the respiratory tract of agms, has also been reported in mice , ferrets, and cats (martina et al., 2003) . it is not entirely clear how these kinetics compare with those in humans because the only report of viral load in respiratory secretions of sars patients was based on samples from subjects who were treated with steroids and ribavirin . agms support replication of sars-cov, with associated evidence of pneumonitis for at least 2 days but both viral infection and pneumonitis are patchy and not as consistent as the corresponding findings in experimentally infected mice and hamsters (roberts et al., in press; subbarao et al., 2004) . the animal-to-animal variability, rapid clearance of virus from the lungs, and rapid resolution of pneumonitis in agms are considerations that preclude this species from being the ideal model for the evaluation of the efficacy of vaccines against viral challenge. drs. l. j. anderson and t. g. ksiazek from the centers for disease control and prevention (cdc), atlanta, ga kindly provided the sars-cov (urbani strain) used in this study (ksiazek et al., 2003) . the virus was isolated and passaged twice in vero e6 cells at the cdc and was passaged twice in vero cells in our laboratory. the vero cells were maintained in optipro sfm (invitrogen, carlsbad, ca). all in vitro manipulations with infectious virus were performed inside a biosafety cabinet, in a biosafety containment level 3 facility, and personnel wore powered air purifying respirators (3m hepa airmate, saint paul, mn) as previously described . virus titers in secretions and tissues were determined in vero cell monolayers as previously described and are expressed as tcid 50 /ml or tcid 50 /g and the lower limits of detection were 10 0.5 tcid 50 /ml and 10 1.5 tcid 50 /g, respectively. the nih animal care and use committee approved the monkey studies that were carried out in an animal biosafety level 3 facility. all personnel entering the facility wore powered air-purifying respirators (3m hepa airmate, saint paul, mn). four juvenile african green monkeys (cercopithecus atheiops or chlorocebus sabeus), four rhesus monkeys (macaca mulata) and four cynomolgus monkeys (macaca fasicularis) were inoculated intranasally and intratracheally with 1 ml of 10 6 tcid 50 of sars-cov in each site. nt samples from the upper respiratory tract were collected daily from day 0 (prior to inoculation) to day 10 and on days 12, 14, 20, 23, and 27 post-inoculation. tl samples from the lower respiratory tract were collected on days 2, 4, 6, 8, 10, 14, 20, plasma, and urine samples were collected on days 2, 4, 6, 8, 10, 14, 20 , and 27 post-inoculation. duplicate samples were flash frozen and stored at ã�70 8c until all samples were available for virus titration. nt, tl, plasma, urine and fecal samples were assayed for presence of infectious sars-cov. piperacillin (sigma aldrich co., st. louis, mo), gentamicin (invitrogen, grand island, ny) and amphotericin b (quality biological, gaithersburg, md) were added to the tissue culture medium at final concentrations of 0.4, 0.1, 5 mg/l, respectively, when determining virus titers from monkey samples. monkeys were observed daily for signs of clinical illness from day 0 to day 10, and on days 12, 14, 20, 23, and 27 post-inoculation. rectal temperatures of monkeys were recorded, and blood was collected on days 0, 2, 4, 6, 8, 10, 14, 20 , and 28 for a complete blood count and alanine amino transferase (alt), isocitrate dehydrogenase (icd), and gamma glutamyl transferase (ggt) levels. to determine whether primary infection protected monkeys from subsequent challenge, agms were inoculated intranasally and intratracheally with 10 6 tcid 50 of sars-cov 2 months after the end of the primary infection study. nt and tl were collected and titered as described above for primary infection. the rhesus and cynomolgus monkeys were euthanized 2 months after primary infection and the agms were euthanized 2 months following challenge infection and tissue samples were obtained from liver, spleen, kidneys, and lungs for virus isolation. an additional study was undertaken in agms in order to determine whether the replication of sars-cov was associated with histopathologic changes in the lungs and to compare the level of virus replication in lung tissue with that seen in nt swabs and tl fluid. four agms were inoculated intranasally and intratracheally with 10 6 tcid 50 of sars-cov, and two were mock infected with leibovitz 15 medium (invitrogen) on day 0. two days later, two sars-cov infected and one mock-infected agm were euthanized and the remaining two sars-cov infected and one mock-infected agm were euthanized on day four. monkeys were observed daily for signs of clinical illness, rectal temperatures were recorded, and blood was collected on days 0, 2, and 4 for a complete blood count and a determination of the level of liver enzymes. nt swabs were collected daily and tl fluids were collected every other day till the animals were euthanized. at necropsy, trachea, nasal turbinates, and tissue blocks of 1 cm 3 from the hilar and peripheral regions of each lobe of the lungs were frozen and assayed for virus titration. similar lung tissue blocks were fixed in 2.5% glutaraldehyde for electron microscopy and the remaining tissues were placed in formalin for histopathologic examination. additional tissue samples that were collected for histopathologic examination include liver, spleen, kidney, heart, bone marrow, and intestines. fecal samples from agm and cynomolgus monkeys were thawed and 0.1 to 0.2 g of feces was suspended in sterile phosphate buffered saline for a final 10% w/v solution. rna was extracted as per manufacturer's protocol (qiagen viral rna kit, valencia, ca) from clarified supernatants and stored at ã�80 8c. fecal samples from rhesus monkeys were not tested. reverse transcription and real-time pcr analysis was performed using the stratagene brilliant plus two-step quantitative rt-pcr core reagent kit as per manufacturer's protocol on a mx4000 thermocycler (stratagene, lajolla, ca). primers and taqman probe (synthesized by qiagen) were used to amplify and detect a small 70-bp fragment located in the sars cov n-orf (accession number ay278741). the sequences of the primers used were as follows: forward: 5v-ccg aag agc tac ccg acg-3v ; reverse: 5v-gaa gta cca tct ggg gct gag-3vand probe 5v -hex-ctc ttt cat ttt gcc gtc acc acc ac-bhq1-3v . samples from which the 70-bp fragment was consistently amplified in two or more independent assays (each performed in duplicate or triplicate) were reported as positive for detection of sars-cov viral rna. serum samples were assayed for the presence of neutralizing antibody in a microneutralization assay starting with a dilution of 1:8. the serum dilution that completely neutralized infectivity of 100 tcid 50 of virus was calculated by the method of reed and muench as described previously . serum samples that did not neutralize virus infectivity at the starting dilution were assigned a titer of 1:4 to determine four-fold rises in titer. serial four-fold dilutions of heat-treated sera were assayed for the presence of igg antibodies against the spike (s) protein of sars-cov in an elisa assay. serum dilutions beginning at 1:40 were applied to wells of immulon 1b plates (dynex technologies, inc., chantilly, va) that were coated with 40 ng per well of purified recombinant baculovirus-expressed sars s protein (protein sciences, inc. meridian, ct). the second antibody used was rabbit anti-monkey igg at a dilution of 1:30,000 (icn, aurora, oh) and the elisa was developed using alkaline phosphatase-labeled goat anti-rabbit antibody at 1:8000 (icn) followed by substrate. the elisa antibody titer recorded was the serum dilution at which the optical density reading at 405 nm exceeded 0.2 and exceeded the reading from wells that were not coated with antigen by two-fold. a colorimetric immunoalkaline phosphatase ihc method was used as previously described . double-stain ihc was performed by using peroxidase polymer (dakocytomation inc., carpinteria, ca) labeled antibodies against cytokeratin, surfactant, or cd68, followed by the mouse anti-sars-cov antibody labeled with immunoalkaline phosphatase polymer. log transformed virus titers were compared in a twotailed t test and statistical significance was assigned to differences with p values b0.05. mucosal immunization of nonhuman primates with an attenuated parainfluenza virus expressing the sars coronavirus spike protein for the prevention of sars a comparison in chimpanzees of the immunogenicity and efficacy of live attenuated respiratory syncytial virus (rsv) temperature-sensitive mutant vaccines and vaccinia virus recombinants that express the surface glycoproteins of rsv identification of a novel coronavirus in patients with severe acute respiratory syndrome african green monkeys provide a useful nonhuman primate model for the study of human parainfluenza virus types-1,-2, and-3 infection aetiology: koch's postulates fulfilled for sars virus effects of a sarsassociated coronavirus vaccine in monkeys pegylated interferonalpha protects type 1 pneumocytes against sars coronavirus infection in macaques a novel coronavirus associated with severe acute respiratory syndrome sars virus infection of cats and ferrets virulence of avian influenza a viruses for squirrel monkeys immunologic phenomena in the effusive form of feline infectious peritonitis clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study national microbiology laboratory, canada and canadian severe acute respiratory syndrome study team pathogenesis of influenza a (h5n1) virus infection in a primate model sars coronavirus infection of golden syrian hamsters the recombinant chimeric human parainfluenza virus type 1 vaccine candidate, rhpiv3-1cp45, is attenuated, immunogenic, and protective in african green monkeys prior infection and passive transfer of neutralizing antibody prevent replication of sars coronavirus in the respiratory tract of mice susceptibility of pigs and chickens to sars coronavirus pathogenesis of feline infectious peritonitis: nature and development of viremia pathogenesis of feline infectious peritonitis: pathologic changes and immunofluorescence we thank dr. randy elkins for advice and assistance in procuring animals and thank tammy tobery, steve harbaugh, kenny stockman and josh moore from bioqual inc. for expert technical assistance in the primate studies. key: cord-289248-6mx4o0eb authors: wang, yilong; liu, rongxian; lu, mijia; yang, yingzhi; zhou, duo; hao, xiaoqiang; zhou, dongming; wang, bin; li, jianrong; huang, yao-wei; zhao, zhengyan title: enhancement of safety and immunogenicity of the chinese hu191 measles virus vaccine by alteration of the s-adenosylmethionine (sam) binding site in the large polymerase protein date: 2018-05-01 journal: virology doi: 10.1016/j.virol.2018.02.022 sha: doc_id: 289248 cord_uid: 6mx4o0eb the live-attenuated measles virus (mv) vaccine based on the hu191 strain has played a significant role in controlling measles in china. however, it has considerable adverse effects that may cause public health burden. we hypothesize that the safety and efficacy of mv vaccine can be improved by altering the s-adenosylmethionine (sam) binding site in the conserved region vi of the large polymerase protein. to test this hypothesis, we established an efficient reverse genetics system for the rmv-hu191 strain and generated two recombinant mv-hu191 carrying mutations in the sam binding site. these two mutants grew to high titer in vero cells, were genetically stable, and were significantly more attenuated in vitro and in vivo compared to the parental rmv-hu191 vaccine strain. importantly, both mv-hu191 mutants triggered a higher neutralizing antibody than rmv-hu191 vaccine and provided complete protection against mv challenge. these results demonstrate its potential for an improved mv vaccine candidate. measles virus (mv) is an enveloped virus with a non-segmented, negative-sense (nns) rna genome in the family paramyxoviridae, order mononegavirales . in developing countries, measles is still a leading cause of mortality in children (griffin and oldstone, 2009; tangy and naim, 2005) , though vaccination is an effective, economical, and safe way to prevent outbreaks (bester, 2016; de vries et al., 2008) . in early 1960, a live-attenuated vaccine based on the hu191 strain of mv was developed and is currently widely used for immunization in all provinces of china (zhang et al., 2009) . while this vaccine is efficacious, it has associated adverse effects. many vaccinated infants and children in china experienced side effects ranging from skin rashes, itching, swelling, and to high fever (bester, 2016; shu et al., 2011) . additionally, outbreaks of measles have been increasing significantly in the past a few years in china, particularly the increasing proportion of adult and infant cases (ma et al., 2016; zhang et al., 2016) . the infected adults had received measles vaccination during childhood; still remain susceptible to infection with the measles virus, as the population immunity against measles after vaccination gradually reduces with time (abad and safdar, 2015; gao et al., 2017; ma et al., 2016; zhang et al., 2016) . thus, there is an increasing urgency to develop a safer, more efficient mv vaccine for eradication of measles in china. reverse genetics system has been established for many nns rna viruses including the vesiculovirus, morbillivirus, respirovirus, and pneumovirus (neumann et al., 2002) . similar to other nns rna viruses, the minimal machinery for mv transcription and replication is the ribonucleoprotein (rnp) complex, which consists of the nucleocapsid (n)-rna template tightly associated with the rna-dependent rna polymerase, the large (l) protein and the phosphoprotein (p). assembly of replication-competent rnps is essential to the rescue of nns rna viruses (bukreyev et al., 1996; clarke et al., 2000; garcin et al., 1995; gassen et al., 2000; jin et al., 1998; lawson et al., 1995) . this can be achieved by co-transfection of a plasmid encoding a fulllength antigenomic cdna together with plasmids encoding n, p, and l genes. previously, several groups have already successfully rescued infectious mv from cdna clones (duprex et al., 1999; kovacs et al., 2003; nakatsu et al., 2006; parks et al., 1999; radecke et al., 1995; sidhu et al., 1995) . the reverse genetics system can facilitate the rational design of safer, more efficient measles vaccine candidates. the l protein of nns rna viruses possesses the majority of enzymatic activities for transcription and replication (ferron et al., 2002; poch et al., 1990; whelan et al., 2004) . during transcription, nns rna viruses synthesize mrnas that are capped and methylated at the 5'end and polyadenylated at the 3' end. recent studies have shown that the entire mrna capping and methylation machinery of nns rna viruses is distinct from their host (ferron et al., 2002; furuichi and shatkin, 2000; ogino and banerjee, 2007; zhang et al., 2014) . using vesicular stomatitis virus (vsv) as a model, it was found that vsv mrna capping is catalyzed by an rna:gdp polyribonucleotidyltransferase (prntase) in the l protein that transfers a monophosphate rna onto a gdp acceptor (li et al., 2008; ogino and banerjee, 2007) . the mrna cap methylation in nns rna viruses is also unusual in that a single region in the l protein catalyzes both guanine-n-7 (g-n-7) and ribose 2′-o (2′-o) methylation (li et al., 2006; rahmeh et al., 2009) . thus, mrna cap formation is an excellent target for development of antiviral drugs and live vaccine candidates for nns rna viruses. based on the sequence alignments, the l protein contains six conserved regions (cr) numbered i to vi. recent studies showed that cr v of the l protein possesses an mrna capping enzyme whereas cr vi is responsible for mrna cap methyltransferase (mtase) activity (li et al., 2008; ogino et al., 2005) . it was shown that mutations to the capping enzyme were lethal to the virus. however, mutations to mtase region yielded recombinant viruses that were attenuated in vitro and in vivo. this suggests mrna cap mtase is a novel target for rational design of live attenuated vaccines for nns rna viruses. this novel concept has recently been tested in several nns rna viruses including vsv, avian metapneumovirus (ampv), human metapneumovirus (hmpv), and rabies virus (rabv) (li et al., 2006; sun et al., 2014; tian et al., 2015; zhang et al., 2014) . it was shown that recombinant viruses lacking mtase activity are highly attenuated in vitro and in vivo, yet retain optimal immunogenicity. we hypothesized that engineering mutations to the mtase region of mv l protein would lead to further attenuation of the current live attenuated vaccine strain, enhancing the safety of mv vaccine. to test this hypothesis, we established a robust reverse genetics system based on a chinese mv vaccine strain mv-hu-191, allowing us to recover recombinant mv in bhk cells stably expressing t7 rna polymerase (xu et al., 2011; zhang et al., 2014) . subsequently, two recombinant mvs with amino acid (aa) substitutions in the s-adenosylmethionine (sam) binding site of l protein (rmv-hu191-g1788a and rmv-hu191-g1792a) were successfully recovered. these two mtase-defective mutants had delayed replication kinetics, grew to high titers, and were genetically stable through 15 passages in cell culture. both mv mutants were significantly more attenuated in vitro and in vivo compared to the parental vaccine strain. interestingly, both mutants induced significantly higher neutralizing antibody titers compared to the parental fig. 1 . construction of a full-length cdna clone for mv-hu191. the t7 promoter, 3′ and 5′ non-coding termini (nct), antigenomic hdv ribozyme and t7 terminator were assembled in several rounds of fusion pcr, and inserted into pyes-2 using a "seamless" cloning strategy, resulted in the construction of p107109-mv(+) (a). eight overlapping fragments containing the full-length mv genome were assembled into p107109-mv(+), creating pyes-mv(+) (b). a spontaneous mutation (c to u) in the h gene that distinguishes the lab-propagated parental virus and rescued recombinant virus was marked by "*" (b). virus. these results demonstrate that alteration of sam binding sites in mv l protein enhances both the safety profile and the immunogenicity of the mv vaccine. thus, mrna cap mtase can serve a novel approach for rational design of a safer and more efficacious mv vaccine. a full-length cdna clone of mv strain hu191, pyes-mv(+), was constructed by a novel methodology using the geneart™ high-order genetic assembly system. the full-length cdna clone of mv-hu191 was successfully assembled by a single step ligation, without the need for restriction endonucleases. the 15,896-nt antigenomic mv cdna was cloned under the control of a t7 rna polymerase promoter, a hepatitis delta virus (hdv) ribozyme sequence, and a t7 terminator ( fig. 1 ; table 1 ). to recover infectious mv, bhk-sr19-t7 cells stably expressing t7 rna polymerase were co-transfected with full-length cdna clone pyes-mv(+) and the support plasmids expressing ribonucleoprotein (pt7-hu191-n, pt7-hu191-p, and pt7-hu191-l). three days post-transfection, cell monolayers were trypsinized and co-cultured with fresh vero cells. mv-induced syncytia were observed 2-3 days later ( fig. 2a , b and c). the successful recovery of rmv-hu191 was further confirmed by detection of n protein expression in vero cells infected with the rescued rmv-hu191 by an immunofluorescence assay (fig. 2d ). when extensive syncytia were observed, cells were harvested and the supernatants were used for further passage in vero cells. after 3-4 passages, recombinant mv was plaque purified, and a large stock of virus was prepared. there is a spontaneous mutation (c to u) at nucleotide (nt) position 8763 within the h gene of the lab-propagated parental virus, which is different from the published hu191 sequence (genbank accession no. fj416067). this site in pyes-mv(+) was mutated back to c by site-directed mutagenesis with pcr, which distinguished the parental virus in the lab but is identical with the published sequence. to confirm the recovered recombinant virus (rmv-hu191) originated from pyes-mv(+) and not from cross-contamination of the parental mv-hu191 grown in our laboratory, a region of the rmv h gene was amplified by rt-pcr and sequenced. the result showed that rmv-hu191 contained the "c" mutation. having the establishment of robust reverse genetics for rmv-hu191, we next tested the hypothesis that the rmv-hu191 vaccine strain can be further attenuated by alteration of the sam binding site in mv l protein. previously, this strategy was used in the rational design of live attenuated vaccine candidates for vsv, ampv, and hmpv (li et al., 2006; sun et al., 2014; zhang et al., 2014) . the sam-dependent mtase superfamily typically contains a conserved g-rich motif for binding the sam molecule, the methyl donor for rna methylation (mcilhatton et al., 1997; schluckebier et al., 1995) . sequence alignment revealed that a gxgxgx motif was conserved in cr vi of the l proteins of all paramyxoviruses and most of the mononegavirales (fig. 3) (li et al., 2006; mcilhatton et al., 1997; poch et al., 1990; zhang et al., 2014) . sequence analysis found that aa residues corresponding to the gxgxgx motif of the mv l protein includes g1788, g1790, and g1792. therefore, these amino acids were individually mutated to alanine in an infectious cdna clone of mv, pyes-mv(+ ). three recombinant mv clones with a single point mutation (g1788a, g1790a or g1792a) in their sam binding sites were constructed. using the reverse genetics system, two recombinant mvs, rmv-hu191-g1788a and rmv-hu191-g1792a, were successfully rescued and viral titer gradually increased when they were passaged in vero cells. the rmv mutants were confirmed by detection of n protein expression in vero cells infected with the rescued rmv mutants by immunofluorescence (figs. 2e and 2f). the rmv-hu191-g1790a mutant was viable, but it grew poorly in vero cell and further passages of this mutant did not increase viral titer (data not shown). next, rmv-hu191-g1788a and rmv-hu191-g1792a were plaque purified. the sizes of virus-induced plaques differed between the rescued parental and mutant viruses. as demonstrated in fig. 4 , after 6 days of incubation, the parental rmv-hu191 formed plaques were 1.43 ± 0.22 mm in diameter, whereas the average plaque for rmv-hu191-g1788a and rmv-hu191-g1792a was significantly smaller (0.98 ± 0.16 mm and 0.79 ± 0.13 mm, respectively; p < 0.05). this suggests that the two mv mutants likely had impaired growth kinetics that caused the plaque sizes to be reduced. finally, the entire genome of each mv mutant was amplified by rt-pcr and sequenced. result showed that each mutant retained the desired mutation. in addition, no other mutations were found in the genome. we next compared the replication kinetics of the rmv-hu191 mutants and the parental virus in vero cells in the time course of 120 h after infection (fig. 5 ). parental rmv-hu191 reached a peak titer (6.86 ± 0.14 log 10 pfu/ml) at 72 h post-inoculation (hpi), while peak titers for the two mutants at 96 hpi. importantly, rmv-hu191-g1788a was delayed in replication but reached a peak titer of 6.9 ± 0.06 log 10 pfu/ml at 96 hpi, which was comparable to the parental virus (p > 0.05). the peak titer achieved by rmv-hu191-g1792a was 6.06 ± 0.20 log 10 pfu/ml at 96 hpi, which was significantly lower than that of parental rmv-hu191 at 72 hpi (p < 0.05). both mv mutants had delayed cytopathic effects (cpe) compared to the parental virus. the parental rmv-hu191 developed extensive cell-to-cell fusion and large syncytia at 48 hpi and reached maximum cpe at 72 hpi, whereas mutants rmv-hu191-g1788a and rmv-hu191-g1792a had a delay in formation of syncytia and reached maximum cpe at 96 hpi (fig. 6) . these data suggest that rmvs carrying mutations in the sam binding site were more attenuated in vero cells than the parental mv vaccine strain. to investigate whether rmv-hu191-g1788a and rmv-hu191-g1792a were genetically stable in vitro, each virus was passaged in vero cells for 15 times. the mutated region in the l gene was sequenced for each of the first 10 passages. virus in each passage retained the desired mutation. at passage 15, the entire genome of each mutant was sequenced, showing no additional mutations in the genome. table 1 sequences of the oligonucleotides used for pcr. sequence (5′−3′) virology 518 (2018) 210-220 2.5. rmv-hu191 carrying mutations in the sam binding site are more attenuated in vivo than the parental vaccine strain four-to-six-week-old specific-pathogen-free (spf) cotton rats were inoculated intranasally with parental and mutant rmv-hu191 in order to determine their replication in vivo. no clinical symptoms of respiratory tract infection were found in cotton rats inoculated with any of the rmvs. at day 4 post-inoculation, cotton rats were terminated, and viral titer in the lungs was determined ( table 2 ). the parental virus replicated efficiently in lungs with an average titer of 3.75 log 10 pfu/g lung tissue. recombinant rmv-hu191-g1788a had an average titer of 3.08 log 10 pfu/g lung tissue, which was significant lower than rmv-hu191 (p < 0.05). however, rmv-hu191-g1792a was the most attenuated mutant. only 1 out of 5 cotton rats had detectable viral titer in the lung with a titer of 2.56 log 10 pfu/g. these results show that the two mutant rmvs were more attenuated in viral replication in vivo compared to the parental vaccine strain. to ensure that each mutant was stable in vivo, total rna was extracted from each lung sample, and the regions harboring mutations were amplified by rt-pcr. the samples from each animal were sequenced, respectively. the result showed that the desired mutation was retained in rmv-hu191-g1788a or rmv-hu191-g1792a from each animal. no additional mutations were detected in the sequenced region. 2.6. rmv-hu191 mutants induce higher neutralizing antibodies than the parental vaccine strain and provide complete protection against mv challenge the immunogenicity of rmv-hu191 mutants was assessed in cotton rats. briefly, 4-6-week-old spf cotton rats were intranasally inoculated with 1.0 × 10 6 pfu of each mv, and were challenged with 1.0 × 10 7 pfu of rmv-hu191 at week 4 post-immunization. the two mutant rmvs induced high levels of neutralizing antibodies as early as 1 week after vaccination, and antibodies gradually increased from weeks 2-4. however, antibodies produced by the parental rmv-hu191 peaked at week 2, and declined during weeks 3 and 4. overall, the antibodies induced by rmv-hu191-g1788a and rmv-hu191-g1792a were comparable to those generated by wild-type rmv-hu191 at weeks 1-3 (p > 0.05) (fig. 7a) . however, at week 4, neutralizing antibodies induced by rmv-hu191 mutants were significantly higher than those from parental rmv-hu191 (p < 0.05; fig. 7b ). this suggests that rmv-hu191 mutants were more immunogenic compared to the parental vaccine strain. at week 4 post-vaccination, cotton rats were challenged with 1.0 × 10 7 pfu of rmv-hu191 and all cotton rats were terminated at day 4 post-challenge. no infectious virus was detected in the lung tissue of any of the vaccinated cotton rats (table 3 ). in contrast, an average titer of 4.02 ± 0.37 log 10 pfu/g was detected in lung tissue from unvaccinated but challenged controls. these results show that rmv-hu191-g1788a and rmv-hu191-g1790a provide complete protection from mv challenge. in this study, we successfully generated two recombinant measles viruses with amino acid substitutions in the sam binding site of l protein and examined the effects of these mutations on viral replication, safety, and immunogenicity. we found that both rmv-hu191-g1788a and rmv-hu191-g1792a were significantly more attenuated compared to parental rmv-hu191, the widely used vaccine in china. rmvs carrying mutations in the sam binding site were genetically stable, formed significantly smaller viral plaques, and had delays in cpe and replication kinetics. recombinant rmv-hu191-g1788a grew to high titer in vero cells that was comparable to rmv-hu191 but exhibited significantly more attenuation in cotton rats. recombinant rmv-hu191-g1792a grew to a relatively lower titer in vero cells but had a greater degree of attenuation in cotton rats compared to rmv-hu191-g1788a. both recombinant viruses triggered significantly higher neutralizing antibody compared to rmv-hu191, and provided complete protection against mv challenge. this indicates that alteration of sam binding site in mv l protein enhances the safety and immunogenicity of the rmv-hu191 vaccine strain. we established a more efficient method to assemble a full-length cdna clone of mv-hu191 without using restriction endonucleases. the mv genome was divided into eight overlapping fragments and assembled into a full-length plasmid using the geneart™ high-order genetic assembly system. the traditional method for assembly of an infectious cdna requires multiple cloning steps involved in restriction enzyme digestion and ligation, which are time consuming, labor extensive, and technically challenging. the traditional cloning strategy also often leads to some unexpected deletions, insertions, and mutations in the viral genome, which hamper the subsequent virus rescue. our assembly strategy was highly efficient, allowing us to obtain full-length cdna clones in a single step. previously, vaccinia virus vtf-7 or mva-t7 providing t7 rna polymerase had often been used to rescue mv in reverse genetics system. however, there was difficulty in separating the rescued mv and the helper viruses. bhk cells stably expressing t7 rna polymerase, instead, were used to rescue hmpv and bovine respiratory syncytial virus (buchholz et al., 1999; zhang et al., 2014) . in our study, bhk-sr19-t7 cells were co-transfected with a plasmid expressing antigenomic mv cdna and support plasmids expressing the mv n, p, and l proteins, allowing for efficient recovery of infectious mv in a vaccinia virus-free cell system. the primary advantage of this system is the elimination of the potential contamination by the vaccinia virus. this rescue system was highly efficient as we were able to recover many mutants in the cr vi of l protein including the two recombinant viruses with aa substitutions in the sam binding site reported in this study. a live attenuated vaccine is a very promising vaccine for most human paramyxoviruses, as it does not cause enhanced lung diseases upon re-infection by the same virus. a live-attenuated vaccine based on the hu191 strain of mv has been developed and is widely used for immunization in chinese infants and children (zhang et al., 2009 ). however, epidemiological study showed that this vaccine still causes some adverse effects that may cause public health burden. in addition to the safety issue, measles outbreaks have been increasing in recent years, likely due to gradual reducing of the population immunity against measles after vaccination with time (abad and safdar, 2015; gao et al., 2017; ma et al., 2016; zhang et al., 2016) . in this study, we sought to improve the safety and efficacy of current mv vaccine by mutating the sam binding site in the l protein. using a robust reverse genetics for rmv-hu191, rmv-hu191 carrying mutations in the sam binding site, rmv-hu191-g1788a and rmv-hu191-g1792a, were successfully rescued. these rmv-hu191 mutants produced smaller plaques, had delayed growth kinetics, and had delayed syncytia formation compared to parental rmv-hu191. clearly, both mutants were significantly more attenuated in vero cells than the parental rmv-hu191. in cotton rats, rmv-hu191-g1788a had a significantly lower viral titer in the lungs than rmv-hu191 (p < 0.05). recombinant rmv-hu191-g1792a was even more attenuated, as only 1 out of 5 inoculated cotton rats had detectable viral titer in the lung. despite the high attenuation phenotype, rmv-hu191-g1788a grew to high titer compared to parental vaccine virus in vero cells, the who approved cell line for vaccine production. although rmv-hu191-g1792a grew to a slightly lower titer (0.5 log less) in vero cells, it is still economically feasible for vaccine production. another advantage of using rmv-hu191-g1792a is that it had a greater degree of attenuation in vitro and in vivo compared to rmv-hu191-g1788a. interestingly, both mutants triggered a higher level of neutralizing antibodies than parental rmv-hu191, suggesting their greater immunogenicity. finally, cotton rats vaccinated with both mutants were completely protected from the mv challenge. thus, recombinant rmv-hu191 carrying mutations in the sam binding site are potentially improved vaccine candidates for mv. a novel finding is that recombinant rmv-hu191 carrying mutations in the sam binding site triggered a higher neutralizing antibody compared to the parental rmv-hu191 strain. although the detailed mechanism is not explored in this study, it is possible that these mv mutants may trigger a higher innate immunity, which in turn triggered a more robust adaptive immunity. in fact, it was shown that coronavirus lacking 2'-o methylation significantly enhanced type i interferon response, which is another advantage of using viral mrna cap mtase as a target in developing live vaccine candidates. previously, it was found that two pneumoviruses (ampv and hmpv) carrying mutations in the sam binding site in cr vi of l protein were specifically defective in ribose 2′-o methylation but not g-n-7 methylation, and were significantly attenuated but retained wild-type levels of immunogenicity. in addition, it was found that all 2′-o mtase-defective hmpvs were highly sensitive to ifn-α and ifn-β treatment (sun et al., 2014; zhang et al., 2014) . given the fact that the mtase domain is highly conserved in l proteins of all nns rna viruses, the general mechanism of attenuation of viruses that lack 2′-o methylation may be similarly conserved in all nns rna viruses. future experiments should investigate the mechanisms by which mv mutants enhance immunogenicity. we have established a novel and efficient strategy for assembly of a full-length cdna clone of mv-hu191 and established an efficient vaccinia virus-free reverse genetics system for mv-hu191. we generated two recombinant mv-hu191 carrying mutations in the sam binding site, which not only grew to high titer in vero cells and were genetically stable but also were significantly more attenuated and immunogenic compared to the currently used chinese mv vaccine strain. these two recombinant viruses may serve as improved vaccine candidates for mv. vero cells (african green monkey, atcc-ccl-81) and bhk-sr19-t7 cells (kindly offered by apath, llc, brooklyn, ny) were grown in dulbecco's modified eagle's medium (dmem; life technologies) supplemented with 10% fetal bovine serum (fbs). the chinese hu191 vaccine strain of mv (obtained from dr. yiyu lu, zhejiang cdc) was passaged in vero cells. viral rna was extracted from 200 µl of mv-hu191 using an rneasy mini-kit (qiagen), and reverse-transcribed using super script® iii reverse transcriptase (invitrogen) and random primer mix (neb). the genome was amplified in eight overlapping fragments by q5® high-fidelity 2 × master mix (neb), using eight pairs of mv-specific primers (table 1) , and cloned into the peasy-blunt vector (transgen) according to the manufacturer's instructions. the resultant eight plasmids containing the full-length mv-hu191 genome (peasy-n, peasy-p, peasy-m1, peasy-m2, peasy-f, peasy-h, peasy-l1, peasy-l2) were sequenced, and found to be identical with the published sequence of mv-hu191 (genbank accession no. fj416067), except for a single point change (c to u) at nt 8763 within the h gene. this mutation in peasy-h was corrected by site-directed mutagenesis with specific primers ( table 1 ). the resultant plasmid was named peasy-h-m. several rounds of amplification and "in-fusion" pcr were used to assemble five fragments [the t7 promoter, mv 3′ and 5′ non-coding termini (nct) (3′−107 nt and 5′−109 nt, respectively) were amplified from peasy-n and peasy-l2, respectively, hepatitis delta virus (hdv) ribozyme (84 nt of anti-genomic hdv sequence), and the t7 terminator], and subsequently inserted them into the pyes-2 plasmid using the geneart™ seamless cloning and assembly kit (invitrogen; fig. 1a ), creating plasmid p107109-mv(+). the primer sequences and approaches used in the pcr assays are available upon request. the ten fragments were successfully assembled into a full-length cdna clone using the geneart™ high-order genetic assembly system according to the manufacturer's manual (fig. 1b) , creating plasmid pyes-mv(+). eight mv-hu191 genomic fragments were amplified with specific primers from peasy-n, peasy-p, peasy-m1, peasy-m2, peasy-f, peasy-h-m, peasy-l1, and peasy-l2. the p107109-mv(+) insert (containing the t7 promoter, mv 3′ and 5′ ncts, hdv ribozyme, and t7 terminator) was divided into two fragments by pcr amplification with specific primers (f:5′-ttctgccgcctgcttcaaaccg-3', r:5′-ctcggatatccctaatcc-3'; f:5′-ttggttgaactccggaac-3', r: 5′-cagaatgggcagacattacgaatgc-3'). a backbone vector pt7, which contains the t7 rna polymerase promoter, encephalomyocarditis (emc) virus internal ribosome entry site (ires), and the t7 terminator sequences, was used to construct plasmids encoding mv-hu191 n, p, and l genes. the open reading frames (orfs) of the mv hu191 n and p genes were amplified from peasy-n and peasy-p using primer pairs mv-cds-n (+)/(-) and mv-cds-p (+)/(-), respectively, whereas the orf of the mv hu191 l gene was amplified from peasy-l1 and peasy-l2 using two primer pairs, mv-cds-l1 (+)/(-) and mv-cds-l2 (+)/(-). the mv n, p, and l genes were inserted into the pt7 vector between the ires and polya table 2 replication of rmv-hu191 mutants in cotton rats. % infected animals viral titer (log 10 pfu/g) b,c rmv-hu191 100 3.75 ± 0.12 a rmv-hu191-g1788a 100 3.08 ± 0.33 b rmv-hu191-g1792a 20 2.56 b dmem 0 nd nd: not detected. a each cotton rat was inoculated intranasally with 5.0 × 10 6 pfu of rmv-hu191 or rmv-hu191 mutants in a volume of 100 µl. at day 4 post-infection, the cotton rats were sacrificed, and lungs were collected for both virus titration and rt-pcr. b the viral titer was determined by plaque assay. c five cotton rats were tested in each group. values within a column followed by different capital letters (a and b) are significantly different. fig. 7 . neutralizing antibody titers produced by cotton rats after inoculation with recombinant mv. cotton rats were intranasally inoculated with 1.0 × 10 6 pfu of rmv-hu191 or rmv-hu191 mutants in 0.1 ml of opti-mem medium. (a) weekly blood samples were collected from each cotton rat by facial vein retro-orbital bleeding, and serum was tested for neutralizing antibodies by a plaque-reduction neutralization assay. (b) recombinant mvs carrying mutations in the sam binding site elicited significantly higher levels (p < 0.05) of neutralizing antibodies at 4 weeks post-inoculation. * =p < 0.05; * *=p < 0.01; *** =p < 0.001. sequences using a "seamless" cloning strategy, resulted in the construction of pt7-hu191-n, pt7-hu191-p, and pt7-hu191-l, respectively. the primer sequences used in the pcr assays are available upon request. to recover recombinant mv, bhk-sr19-t7 cells were grown overnight in six-well plates to approximately 90% confluence, and were transfected with 5 µg of pyes-mv(+), 1.5 µg of pt7-hu191-n, 1.5 µg of pt7-hu191-p, and 0.5 µg of pt7-hu191-l using previously described procedure (carsillo et al., 2009; kovacs et al., 2003; singh and billeter, 1999) . at 72 h post-transfection, cell monolayers were trypsinized and directly transferred onto vero cell monolayers (p0) at 75% confluence and co-cultured at 37°c for 3-5 days. cells were subjected to three freeze-thaw cycles when extensive cpe (mv-induced syncytia) was observed. after a brief centrifugation, supernatants (p1) were harvested and used for further passages on confluent vero cell monolayers. at p2 or p3, the recovered viruses were plaque purified and sequenced. vero cells grown in 24-well tissue culture plates were infected with rmv or rmv-mutant. after 1 h incubation, the cells were washed two times with pbs before cultivating them in dmem containing 2% fbs. at 24 or 48 h postinfection, the cells were fixed with 4% paraformaldehyde in pbs for 20 min at rt, permeabilized with 0.1% triton x-100 (merck millipore) in pbs for 10 min at rt, and blocked with 1% bsa in pbs containing 0.05% tween 20 for 1 h at rt. the cells were stained with mouse anti-measles virus n antibody (ab106292, abcam) for 1 h at rt. after washing with pbs, alexa fluor ® 594 donkey anti-mouse igg (h+l) (a21203, invitrogen) were added and incubated for 1 h at rt. then, the cells were stained with 4,6-diamidino-2-phenylindole (dapi) for 10 min at rt. images were obtained using a zeiss clsm780 confocal laser scanning microscope and zen 2012 software. amino acids (g1788, g1790, and g1792) in the sam binding site in the hu191 l protein were mutated to alanine individually (ggt to gct at aa posi tion 1788, gga to gca at aa position 1790, and ggt to gct at aa position 1792; the mutated nucleotides were underlined) in an infectious cdna clone of mv-hu191 [pyes-mv(+)] using a q5® sitedirected mutagenesis kit (neb) according to the manufacturer's instructions. the resultant plasmids were named pyes-mv(+)-g1788a, pyes-mv(+)-g1790a, and pyes-mv(+)-g1792a. mutations were confirmed by dna sequencing. the titers of rmv-hu191 viruses were determined by plaque assay in vero cells. briefly, vero cells were seeded in six-well plates at a density of 5 × 10 5 cells per well, incubated for 18 h, and the medium was removed prior to infection of cell monolayers with serial dilutions of rmv-hu191. after 1 h of adsorption with constant shaking, the medium was removed and cell monolayers were covered with 2.5 ml of eagle's minimal essential media (mem) containing 2% agarose, 0.75% sodium bicarbonate(nahco3), 5% fbs, 20 nm hepes, 2 mm l-glutamine, and 4 mg/ml of streptomycin. at 6 dpi, cells were fixed in 4% (vol/vol) paraformaldehyde for 2 h, and the plaques were visualized by staining with 0.05% (wt/vol) crystal violet. confluent vero cells in six-well plates were infected with rmv-hu191 viruses at a multiplicity of infection (moi) of 0.01. after 1 h incubation, the inoculum was removed and the cells were washed three times with pbs. fresh maintenance media (dmem supplemented with 2% fbs) was added, and the infected cells were incubated at 37°c. at different time points post-infection, the cells were harvested by three freeze-thaw cycles, and the supernatant collected by centrifugation at 3000×g in an allegra 6r centrifuge (beckman coulter) for 15 min. virus titers were determined by plaque assay in vero cells. confluent vero cells in t25 flasks were infected with each rmv-hu191 mutant at an moi of 0.01, and cell culture supernatants were collected after appearance of cpe and used to infect new confluent vero cells in fresh t25 flasks. each mutant was serially passaged 15 times in vero cells. viral rna was extracted from cell culture supernatant harvested from each passage. the cr vi of the l gene was amplified by rt-pcr and sequenced. additionally, the entire genome of each recombinant virus was amplified by rt-pcr and sequenced at passage 15. all the plasmids, viral stocks, and virus isolates from the lungs of cotton rats were sequenced. viral rna was isolated using an rneasy mini-kit (qiagen) according to the manufacturer's instructions. viral rna was treated with dnase i to eliminate possible contamination from original transfecting plasmid dna, and no-rt pcr controls were carried out to confirm complete digestion of plasmid dna. a 1.2 kb dna fragment of the h protein gene was amplified by a one-step rt-pcr kit (qiagen) using primers rmv-h-8583-forward (5'gttcagggatggacctatac-3') and rmv-l-9793-reverse (5'ggtgtgtgtctcctcctat-3'). pcr products were sequenced to ensure that the isolated virus was rescued from pyes-mv(+) and not from the contamination of the wild type mv-hu191 grown in our laboratory. a 1.1 kb dna fragment spanning cr vi of the mv l-protein was amplified by a one-step rt-pcr kit (qiagen) using primers rmv-l-14128-forward (5'-gaccggtagagaaatgtgcag-3') and rmv-l-15222-reverse (5'-gcttaatggataggatgtgac-3'). pcr products were sequenced to confirm that each recombinant virus contained the desired mutation. the rmv-hu191 stocks for use in animal experiments were grown in vero cells and purified by ultracentrifugation. twenty t150 flasks with confluent vero cells were infected with each rmv at moi of 0.01. after 1 h of adsorption with constant shaking, 15 ml of dmem (supplemented with 2% fbs) was added to each flask and incubated at 37°c until extensive cpe was observed. the cells were harvested using a cellscraper, and suspensions were clarified by centrifugation at 3000×g for table 3 immunogenicity of mtase-defective rmv-hu191 mutants in cotton rats a . inoculum a % infected animals viral titer (log 10 pfu/g) b rmv-hu191 0 nd rmv-hu191-g1788a 0 nd rmv-hu191-g1792a 0 nd dmem 100 4.02 ± 0.37 nd: not detected. a cotton rats were intranasally inoculated with 1.0 × 10 6 pfu of rmv-hu191 or mutants. at 28 days post-infection, rats were challenged with 1 × 10 7 pfu of rmv-hu191. at day 4 post-challenge, cotton rats were sacrificed and lungs were collected for both virus titration and rt-pcr. b the viral titer was determined by plaque assay. 20 min at 4°c in an allegra 6 r centrifuge (beckman coulter). the cell pellets were resuspended in 2 ml of dmem and subjected to three freeze-thaw cycles, clarified by low-speed centrifugation, and the supernatants were combined. the virus was pelleted by ultracentrifugation at 30,000×g in a beckman ty 50.2 rotor for 2 h, and resuspended in 0.3 ml of dmem, aliquoted, and stored. viral titer was determined by plaque assay. twenty 4-6 week-old female specific-pathogen-free (spf) cotton rats (envigo, indianapolis, in) were randomly divided into four groups (5 cotton rats per group), and housed within the ular facilities at the ohio state university according to iacuc policies and guidelines (animal protocol no. 2009a0221). each inoculated group was separately housed in rodent cages under biosafety level 2 conditions; rats were anesthetized with isoflurane before virus inoculation. cotton rats in groups 1-3 were inoculated with parental rmv-hu191, rmv-hu191-g1788a, and rmv-hu191-g1792a, respectively. cotton rats in group 4 were mock-infected with dmem, and served as uninfected controls. each cotton rat was inoculated intranasally with 5 × 10 6 pfu of virus in a volume of 100 µl. at 4 dpi, cotton rats were sacrificed and lungs were collected for virus titration and rt-pcr. for the immunogenicity study, twenty five 4-6 week-old cotton rats (envigo) were randomly divided into five groups (5 cotton rats per group). cotton rats in groups 1 were mock-infected with dmem and served as uninfected unchallenged control. cotton rats in groups 2, 3 and 4 were intranasally inoculated with 1.0 × 10 6 pfu of rmv-hu191, rmv-hu191-g1788a, and rmv-hu191-g1792a, respectively. cotton rats in groups 5 were mock-infected with dmem and served as uninfected challenged control. after immunization, the cotton rats were evaluated daily for mortality, and blood samples were collected from each cotton rat weekly by facial vein retro-orbital bleeding, and the serum was used for detection of neutralizing antibodies. at 4 weeks post-immunization, the cotton rats in groups 2-5 were challenged with 1.0 × 10 7 pfu of parental rmv-hu191 via intranasal route, and evaluated twice daily for the presence of any clinical symptoms. at 4 days post-challenge, all cotton rats were euthanized by co 2 asphyxiation, and their lungs were collected for virus titration. the immunogenicity of rmv-hu191 mutants was assessed based on their ability to trigger neutralizing antibodies and the ability to protect mv replication in lungs. serum neutralization of virus was performed using an endpoint dilution plaque reduction assay; and (ii) quantification of lung viral titers was done by plaque assay. mv-specific neutralizing antibody was determined using an endpoint dilution plaque reduction assay. briefly, cotton rat sera were collected weekly until challenge. the serum samples were heat inactivated at 56°c for 30 min. two-fold dilutions of the serum samples were mixed with an equal volume of dmem containing approximately 100 pfu/well rmv-hu191 in a 96-well plate, and the plate was incubated at room temperature for 1 h with constant rotation. the mixtures were then transferred to confluent vero cells in a 6-well plate in triplicate. after 1 h of incubation at 37°c, the virus-serum mixtures were removed and the cell monolayers were covered with 2.5 ml of eagle's minimal essential media (mem) containing 2% agarose, 0.75% sodium bicarbonate(nahco3), 5% fbs, 20 nm hepes, 2 mm l-glutamine, and 4 mg/ml of streptomycin. then, the cells were incubated for another 6 days before virus plaque titration as described above. the plaques were counted, and 50% plaque reduction titers were calculated as the mv-specific neutralizing antibody titers. statistical analysis was performed by one-way multiple comparisons; two-way multiple comparisons (anova) using prism statistical analysis software (version 8.0). p value of < 0.05 was considered statistically significant. measles and measles vaccination: a review generation of bovine respiratory syncytial virus (brsv) from cdna: brsv ns2 is not essential for virus replication in tissue culture, and the human rsv leader region acts as a functional brsv genome promoter recovery of infectious respiratory syncytial virus expressing an additional, foreign gene cytokine imbalance after measles virus infection has no correlation with immune suppression rescue of mumps virus from cdna measles vaccination: new strategies and formulations observation of measles virus cell-to-cell spread in astrocytoma cells by using a green fluorescent protein-expressing recombinant virus viral rna-polymerases -a predicted 2'-o-ribose methyltransferase domain shared by all mononegavirales viral and cellular mrna capping: past and prospects the measles epidemic trend over the past 30 years in a central district a highly recombinogenic system for the recovery of infectious sendai paramyxovirus from cdna: generation of a novel copy-back nondefective interfering virus establishment of a rescue system for canine distemper virus measles. history and basic biology recombinant human respiratory syncytial virus (rsv) from cdna and construction of subgroup a and b chimeric rsv enhanced genetic rescue of negative-strand rna viruses: use of an mva-t7 rna polyrnerase vector and dna replication inhibitors recombinant vesicular stomatitis viruses from dna a conserved motif in region v of the large polymerase proteins of nonsegmented negative-sense rna viruses that is essential for mrna capping a unique strategy for mrna cap methylation used y. wang et al by vesicular stomatitis virus measles transmission among adults with spread to children during an outbreak: implications for measles elimination in china mrna cap methylation influences pathogenesis of vesicular stomatitis virus in vivo nucleotide sequence analysis of the large (l) genes of phocine distemper virus and canine distemper virus (corrected sequence) rescue system for measles virus from cloned cdna driven by vaccinia virus lister vaccine strain a decade after the generation of a negative-sense rna virus from cloned cdna -what have we learned? unconventional mechanism of mrna capping by the rna-dependent rna polymerase of vesicular stomatitis virus sendai virus rna-dependent rna polymerase l protein catalyzes cap methylation of virus-specific mrna enhanced measles virus cdna rescue and gene expression after heat shock sequence comparison of five polymerases (l proteins) of unsegmented negative-strand rna viruses: theoretical assignment of functional domains rescue of measles viruses from cloned dna ribose 2'-o methylation of the vesicular stomatitis virus mrna cap precedes and facilitates subsequent guanine-n-7 methylation by the large polymerase protein universal catalytic domain structure of adomet-dependent methyltransferases measles vaccine adverse events reported in the mass vaccination campaign of sichuan province, china from rescue of synthetic measles-virus minireplicons -measles genomic termini direct efficient expression and propagation of a reporter gene a recombinant measles virus expressing biologically active human interleukin-12 methyltransferase-defective avian metapneumovirus vaccines provide complete protection against challenge with the homologous colorado strain and the heterologous minnesota strain live attenuated measles vaccine as a potential multivalent pediatric vaccination vector critical role of k1685 and k1829 in the large protein of rabies virus in viral pathogenicity and immune evasion transcription and replication of nonsegmented negative-strand rna viruses rescue of wild-type mumps virus from a strain associated with recent outbreaks helps to define the role of the sh orf in the pathogenesis of mumps virus rational design of human metapneumovirus live attenuated vaccine candidates by inhibiting viral mrna cap methyltransferase genetic characterization of chinese measles vaccines by analysis of complete genomic sequences measles outbreak among previously immunized adult healthcare workers, china, 2015. can the authors declare that they have no competing interests. key: cord-303497-s3zs1oxf authors: breuning, astrid; scholtissek, christoph title: characterization of a cold-sensitive (cs) recombinant between two influenza a strains date: 1983-10-15 journal: virology doi: 10.1016/0042-6822(83)90117-4 sha: doc_id: 303497 cord_uid: s3zs1oxf abstract recombinants between fowl plague virus (fpv, h7n1) and the hong kong (h3n2) or singapore (h2n2) influenza virus strains carrying the hemagglutinin of fpv and the neuraminidase of the human strains form only very tiny plaques at 33°, but normal plaques at 37°. one recombinant (113/ho) has been studied in more detail. it multiplies only very slowly at 33°, the nonpermissive temperature. adsorption and penetration are normal at 33°, but synthesis of protein is impeded. temperature-shift experiments suggest that the synthesis of viral mrna is slowed at 33°. 113/ho does not agglutinate chicken erythrocytes at 40°, as the parent viruses do. 113/ho can be adapted to grow normally at 33°. the frequency of adaptation is comparable to reversion of a single point mutation (ca. 10−5). recombinants which grow well at 37° but not at 33° are called cold-sensitive (cs) recombinants. temperature-sensitive (ts) mutants and recombinants of influenza a viruses have been isolated and characterized in several laboratories. these mutants and recombinants multiply at low temperatures as well as the wild-type strains, but are restricted in multiplication at temperatures around 40". with these isolates the functional significance of various genes has been studied (for a review see mahy, 1983) . in this communication we will describe and characterize recombinants which are restricted in multiplication at 33", while they multiply to normal titers at 3'7 or 40". by analogy to ts recombinants, we call these isolates cold-sensitive (cs) recombinants. cs mutants of poliovirus (wright and cooper, 1973) , of a murine sarcoma virus (somers and kit, 19'73) , and of a murine coronavirus (stohlman et a& 19'79) have been isolated and their properties have been described. materials and methods virus strains, recombimnts, and cells. the following virus strains were investi' to whom reprint requests should be addressed. gated: a/fpv/rostock/34, h7n1, plaque isolate 145 (f); a/pr/8/34, hlnl (p); a/ singapore/i/57, h2n2 (s); a/hong kong/ l/68, h3n2 (ho); a/chick/germany/ "n"/49, hlon7 (n); a/equine/miami/68, h3n8, eq2 (e); a/swine/1976/31, hlnl (sw); a/turkey/england/63, h7n3 (t). recombinants between fowl plague virus (fpv) and these strains were obtained by double infection of chick embryo cells either with specific ts mutants of fpv or fpv wild-type and the other prototype strains, and extended plaque purifications as described by scholtissek et al (1976) and rott et al (1979) . they are listed in table 1 . primary chick embryo cells were used for multiplication of viruses and for most of the plaque tests. in a few experiments mdck cells were also investigated. biological tests. hemagglutinating activity was determined with 1% chicken erythrocytes either at room temperature or at 40'. neuraminidase activity was determined according to seto and rott (1966) at 33,37, or 40". plaque tests were carried out in chick embryo cells at different temperatures (klenk et d, 1972) . the hemadsorption test was performed according to nakamura and homma (1974) . a the morphology of the plaques was determined after 3 days incubation at the corresponding temperature. between 50 and 100 plaques of each recombinant were examined. because of chance delay the plaque population is not completely homogeneous. therefore, the maximum sizes, which are the sizes of about 50% of the plaques examined, are listed. the error width of the measurement is between 20 and 40%, depending on the plaque size. *most of the recombinants were obtained by rescue of specific ts mutants of fpv (f) (represented by the first number) by corresponding prototype strains (scholtissek et al, 1976 and fpv, and picking plaques at random . 'the vrna segments 1, 2, and 3 code for the three polymerase proteins pb2, pbl, and pa, respectively. segment 4 = hemagglutinin gene; segment 5 = nucleoprotein gene, segment 6 = neuraminidase gene; segment 7 = membrane protein gene; segment 8 = nonstructural protein gene. labeling of viral proteins by [""s]methionine and polyacrylamide gel electrophoresis. chick embryo cells were infected with 10 to 50 pfu/cell under different temperature conditions. at 4 hr or later after infection 40 &i [35s]methionine (800 ci/mmol; amersham, england) per culture was added. three hours thereafter cells were processed and the proteins separated by polyacrylamide gel electrophoresis (bosch et al, 1979) . when plaque tests were performed on chick embryo cells at 37 or 40" with recombinants between fpv and other prototype influenza virus strains it was found that the plaque morphology of most of these recombinants was similar to that of fpv. however, at 33", 2 out of 21 recom-binants selected formed only microplaques. it is shown in fig. 1 and table 1 that these recombinants carry the neuraminidase (na) of the n2 serotype (s and ho) and the hemagglutinin (ha) of fpv. these recombinants also have, in addition to the na genes, rna segment 1 derived from the hong kong or singapore strains, respectively. unfortunately, there is no recombinant available in which only rna segment 6 of fpv is replaced by the corresponding gene of the hong kong or singapore strains. however, recombinants inwhich only rna segment 1 is replaced form plaques at 33" of 2 mm in diameter (table 1) . thus, replacement of segment 1 by itself is not responsible for microplaque formation, although the overall gene constellation has a certain influence on the plaque morphology (see table 1 ). furthermore, several independently isolated recombinants with gene constellations identical to 113/ho 1 and 113/s 1 all were able to produce only microplaques at 33" and normal plaques at 37" (not shown here). in the following, the isolate 113/ho has been studied in more detail. if an allantoic fluid obtained after infection with 113/ho at 37' is titrated at 33" and at 37", the 113/ho 1 ls/ho/ad. plaque titer at 37" is up to a factor of 100 higher than at 33". furthermore, at 33" the plaque number does not correlate with the dilution. for example, when at a given dilution the number of countable plaques was about 200, in the next dilution by a factor of 10 only between 2 and 5 plaques were visible, although 20 plaques had been expected. these observations suggest that most of the plaques at 33' are so tiny that they cannot be seen by the naked eye. only if several plaques are located close enough to fuse to a larger one do they become visible. indeed, if the plaque test at 33" was read not after 3 days but after 6 days, the number of plaques increased by a factor of at least 10. however, under these conditions the plaques became somewhat heterogeneous, which possibly is due to the appearance of "adapted" virus particles within a plaque. if after incubation at 37" plaques were picked, these viruses again formed microplaques at 33". the same observation concerning the plaque morphology of 113/ho and 113/s at the various temperatures was made when mdck cells were investigated. thus, the choice of the host cell seems not to play an important role in this phenomenon. after 36 hr at 33" the virus yield was extremely low when titrated at 33". the morphology of the residual plaques, however, was similar to that of fpv at 33" (fig. 1) . microplaques were not seen, presumably because the titer of the original virus was too low (see titer at 40" in table 2 ). in the second and third correspondingly diluted passages the titers of virus forming plaques of about 2 mm in diameter at 33" increased (table 2) . virus from the latter passage is called 113/ho ad. (adapted). it was further plaque purified at 33". the gene constellation is identical to that of the original 113/ho. when infectious allantoic fluids obtained by nine independent plaque isolates of the original 113/ho were titrated at 33 and 37", in two cases 1 out of lo4 infectious particles formed a plaque at 33" with a diameter of 2 mm. in the other seven fluids too many microplaques were seen at lo5 infectious particles per dish to recognize plaques of normal size at 33". thus, the frequency of adaptation is roughly the same (ca. 10p5) as the rate of reversion of a ts point mutation (scholtissek and spring, 1981) . in a single-cycle multiplication experiment (infection at a multiplicity of lo-50 pfu per cell) at 33", the yield of infectious 113/ho was very low when compared with the multiplication at 37" or with the adapted strain or fpv (fig. 2) . also, by the hemadsorption test after infection with 113/ho at 33" no significant hemagglutinin could be detected in the plasma membrane at 6 hr after infection. at 37 or 40" the hemagglutinin expressed at the cell surface was the same as after infection with the parent viruses or 113/ho ad. at any of the three temperatures (not shown). in a multiple-cycle experiment (infection at a multiplicity of <l pfu per cell), the results are very similar (fig. 3) . there is not only a retardation in the multiplication of 113/ho at 33", but also the final yield is very low. first it was examined whether the ratelimiting step at 33" during the multiplication of 113/ho is adsorption and/or penetration. therefore, chick embryo cells were infected with 113/ho either at 33" or at 37". after half an hour of incubation, they were washed with ice-cold 0.9% nacl at ph 2.5 to inactivate most of the virus 6. at the surface which had not yet penetrated (n. dimmock, personal communication; koennecke et al, 1981) . after two further washings with pbs, the cells were incubated at either 33 or 37" or were shifted after 1 hr incubation at 37" down to 33" according to the schedule outlined in table 3 . ten hours later, the ha titers and pfu were determined. as can be seen in table 3 , if cells were infected at 33" and after washing at ph 2.5 were further incubated at 37' the yield was the same as if the cells were incubated all the time at 37". if the cells were incubated for 1 hr at 37" immediately after infection at 33" and further incubated at 33", the yield was as low as if the cells were incubated all the time at 33". these results indicate that adsorption and penetration proceed normally at 33", but a step thereafter is impeded at 33". one observation deserves attention: if 113/ho is grown in chick embryo cells in culture at 37", ha can be detected by the hemagglutination test despite the fact that the infectivity titer is relatively low (table 3 , upper part, lines a and c). this implies either that more ha is synthesized compared with infectious particles, or that the notes. cells were infected with 10 to 50 pfu/cell. (a) infection at 37o, incubation at 37o; (b) infection at 33", incubation at 33"; (c) infection at 33". incubation at 37'; (d) infection at 33", incubation for 1 hr at 37"; thereafter shift to 33". all cells were treated at ph 2.5 after infection and processed at 10 hr after infection. quality of ha is different from that of the parent strain under identical conditions. next, protein synthesis has been studied by incorporation of [?s]methionine into viral proteins. as can be seen in fig. 4 , in 113/ho-infected cells at 33" very little protein is labeled when compared with cells infected with the adapted strain or the parent viruses. about 10 times more isotope has to be applied in order to get a comparable incorporation (not shown here). under these conditions, it was found that at 33" the ha of 113/ho is cleaved into ha1 and haz. labeling of 113/ho protein did not increase if the ?s pulse was applied later in the infectious cycle (not shown here). these results indicate that a step before virus maturation is impeded at 33' in 113/ho-infected cells, since labeling of viral proteins is already slowed down at this temperature. to further define the step which is slowed down at 33", corresponding temperature shift experiments have been performed. after infection, the cells were kept for 4 hr at either 33 or 37" prior to the pulse at 37 or 33" for 3 hr, respectively. as can be seen in fig. 5 , if cells infected by 113/ho were kept at 33" and the pulse was given at 37", the viral proteins were much less labeled when compared with conditions the other way around. for comparison, data on the adapted strain are included. the results suggest that at 33" the synthesis of viral mrna is slowed down rather than that of viral protein itself. otherwise, after preincubation at 33" and pulse at 37" labeling of viral proteins should not have been impeded. since all recombinants tested with the surface antigen composition h7n2 formed only micro, tiny, or turbid plaques at 33", the neuraminidase and hemagglutination activities of 113/ho have been compared with those of fpv and the adapted strain. when the neuraminidase activity tested at different temperatures was correlated to the ha titers or pfu of egg-grown virus, no significant differences between these and scholtissek strains were found. however, if the hemagglutination test was performed at 40" with 113/ho no agglutination of chicken erythrocytes was visible, in contrast to the adapted strain, or fpv, or ho, furthermore, erythrocytes once agglutinated at room temperature by 113/ho form a central pellet in the cups of the test plate after about 1 hr, depending on the virus dilution, while the agglutination patterns of the other strains stay stable for at least 6 hr or longer. since there is no difference in neuraminidase activity, it is suggested that, when compared with the other strains, 113/ho crosslinks red blood cells less firmly after release of some neuraminic the structure of the hemagglutinin, a determinant for the pathogenicity of influenza virus. vi?-ology 95 on the structure of influenza virus envelope isolation and properties of a temperaturesensitive mutant (ts 412) of an influenza virus recombinant with a ts lesion in the gene coding for the nonstructural protein mutants of influenza virus cell fusion by hela cells persistently infected with hemadsorption type 2 virus correlation of pathogenicity and gene constellation of influenza a viruses. iii. non-pathogenic recombinants derived from highly pathogenic parent strains correlation between rna fragments of fowl plaque virus and their corresponding gene functions suppressor recombinants and suppressor mutants functional significance of sialidase during influenza virus multiplication. vi?-ologg temperature-dependent expression of transformation by a cold-sensitive mutant of murine sarcoma virus characterization of the cold-sensitive murine hepatitis virus mutants rescued from latently infected cells by cell fusion isolation of cold-sensitive mutants of poliovirus this work was supported by the deutsche forschungsgemeinschaft, sonderforschungsbereich 47, and by the fonds der chemischen industrie. acid from the cell receptors. thus, the observed difference between 113/ho and the parent strains seems to be related, rather, to the hemagglutinin moiety. discussion recombinants and mutants of influenza viruses which multiply well at low but not at the elevated temperatures have been isolated in many laboratories. it might be assumed that certain gene products of these isolates either by themselves or in cooperation with other gene products are impeded in their function by some kind of a melting process or conformational changes at the elevated temperature. however, there are no reports on influenza virus isolates with the opposite property; this means normal multiplication at body temperature but restriction at lower temperatures when compared with the parent strains. such isolates, if they exist, are expected to have defects in the function of glycoproteins or the membrane (m) protein, which interact with lipid bilayers during their multiplication or assembly, since membranes become more rigid at low temperatures.in this communication, we describe recombinants between fpv and human influenza a strains carrying a n2 neuraminidase which multiply normally at 37" but only very slowly at 33" when compared with the parent strains. this property seems to be related to a specific combination of the surface glycoproteins, h'7n2. recombinants with this gene constellation produce only very tiny or turbid plaques at 33" (see table 1 ). recombinants between fpv and the hong kong strain (113/ho) have been studied in more detail. at 33", they adsorb and penetrate normally, although the hemagglutinin of these recombinants binds less firmly to red blood cells. since this latter property is more strongly expressed at elevated temperature, it is not clear whether it is rather circumstantial and not related to the slower multiplication of 113/ho at low temperatures.at 33" labeling of viral proteins of 113/ ho is significantly slowed down. however, since after preincubation at 33" and pulse at 37" viral proteins are much less labeled than under conditions the other way around (fig. 5) , it is suggested that viral mrna synthesis is impeded at low temperatures rather than that of viral proteins. the possible impairment of viral mrna synthesis at 33" is difficult to explain, interaction of viral glycoproteins during their synthesis on internal membranes might play a role in the regulation of the synthesis of viral mrna in a way which is not yet understood.it is possible that during reassortment a new "gene constellation" is generated, as in the case of 113/ho, in which an intrinsically cs protein that has been suppressed on the parental strain is now allowed to express a cs phenotype. the recombinant 113/ho can easily be adapted to grow normally at 33". the frequency of adaptation corresponds roughly to that of reversion of a single point mutation to wild type (ca. 10e5). thus, a single mutation might bring either one of the glycoproteins or another gene product into such a conformation that it cooperates normally again at 33". key: cord-292019-rfu0bkag authors: gómez, n.; carrillo, c.; salinas, j.; parra, f.; borca, m. v.; escribano, j. m. title: expression of immunogenic glycoprotein s polypeptides from transmissible gastroenteritis coronavirus in transgenic plants date: 1998-09-30 journal: virology doi: 10.1006/viro.1998.9315 sha: doc_id: 292019 cord_uid: rfu0bkag abstract the use of transgenic plants as vaccine production systems was described recently. we report on the immunological response elicited by two recombinant versions of the glycoprotein s from the swine-transmissible gastroenteritis coronavirus (tgev) expressed in transgenic plants. arabidoposis plants were genetically transformed with cdnas constructs encoding either the n-terminal domain (amino acid residues 1–750) or the full-length glycoprotein s of tgev, responsible for the neutralizing antibody induction against the virus, under the control of the cauliflower mosaic virus 35s (camv 35s) promoter. genomic dna and mrna analyses of leaf extracts from transformed plants demonstrated the incorporation of the foreign cdna into the arabidopsis genome, as well as their transcription. expression of recombinant polypeptides were observed in most transgenic plants by elisa using specific antibodies. mice immunized with leaf extracts from transgenic plants developed antibodies that reacted specifically with tgev in elisa, immunoprecipitated the virus-induced protein, and neutralized the virus infectivity. from these results, we conclude that transgenic plants expressing glycoprotein s polypeptides may possibly be used as a source of recombinant antigen for vaccine production. swine-transmissible gastroenteritis virus (tgev) is the causative agent of acute diarrhea of newborn piglets that provokes high mortality rates in affected farms. protective immunity against this disease must be developed in pregnant sows to confer passive protection to the piglets through colostrum and milk. neutralizing antibodies against the virus are directed mainly to glycoprotein s (garwes et al., 1978; jimenez et al., 1986) , and relevant epitopes in neutralization have been mapped into the n-terminal domain of this protein . four major antigenic sites have been described in glycoprotein s, of which site a is the immunodominant (de diego et al., 1992; delmas et al., 1990; sa â nchez et al., 1990) . glycoprotein s from tgev has been expressed using different vectors with tropism that favored antigenic presentation in the mucosal surfaces (smerdou et al., 1996; torres et al., 1995) . these vaccination approaches promoted systemic and mucosal antibody induction and, in the case of adenovirus vector, conferred protection to suckling piglets (torres et al., 1996) . the development of genetic transformation technology in plants has made possible the expression of foreign genes in different plant species, making reasonable the idea of using plants as bioreactors to produce recombi-nant proteins. the concept of vaccine production in transgenic plants was first introduced by mason et al. in 1992 . proteins involved in protective immune response can be produced at a low cost and easily purified from plant extracts for parental inoculation. in addition, oral immunization by edible vaccines produced in transgenic plants could stimulate immune responses at the portal entrance of many pathogens, facilitating the design of large-scale immunization programs. the presence of specific antigens into plants, even at low levels, can raise by the oral route immune reactions comparable to those raised by conventional vaccines (haq et al., 1995; mason et al., 1996) . hepatitis b surface antigen (thanavala et al., 1995) , escherichia coli heat-labile enterotoxin (lt-b) antigen (haq et al., 1995; tacket et al., 1998) , norwalk virus capsid protein (mason et al., 1996) , vp1 antigen from foot and mouth disease virus (carrillo et al., 1998) , and cholera toxin b subunit (arakawa et al., 1998) are the vaccine antigens expressed in transgenic plants and tested for the immune response elicited in immunized animals. additionally, rabies virus glycoprotein was expressed in transgenic tomatoes, but the immune response induced by administration of these plants to animals was not tested (mcgarvey et al., 1995) . in the present study, we investigated the feasibility of expressing the glycoprotein s from tgev in transgenic plants, as well as the antigenicity and immunogenicity of the plant-derived protein. the s protein is an excellent model for developing oral vaccines against enteric pathogens of mammals because of its immunogenicity and resistance to degradation in the gut. the binary prok i and prok ii recombinant plasmids ( fig. 1) , carrying a cdna coding for the n-terminal region or the full-length glycoprotein s respectively, were obtained by subcloning the corresponding sequences from previously obtained constructs. recombinant prok plasmids allow selection of transformants on media containing kanamycin and stable integration into nuclear chromosomal dna from the plant. prok uses the cauliflower mosaic virus 35s (camw 35s) promoter for nominally constitutive transcription of the cloned genes. plant transformation with prok i and ii was carried out as described in materials and methods by agrobacterium tumefaciens-mediated transformation. the transgenic plants resistant to the selective medium appeared similar in morphology to the nontransgenic arabidopsis plants. more than 20 different lines of transformants containing each construct were obtained and self-pollinated to obtain f2 lines. all lines were positive when screened for the presence of the recombinant genes by polymerase chain reaction (pcr) analysis ( fig. 2a ). most plants harboring recombinant genes showed specific transcription of foreign genes by reverse transcription (rt)-pcr analysis (fig. 2b ). to rule out the possibility of amplification of contaminant dna sequences present in the rna preparations, we treated the purified rna with ribonuclease before foreign gene amplification by using taq polymerase. no amplified dna fragments were detectable under those conditions, assessing the rna dependence of the reaction (fig. 2b ). the presence of the recombinant polypeptides in the plants harboring and expressing the foreign genes was investigated in four plants of each construct, selected to be analyzed by elisa and western blotting using an anti-tgev polyclonal serum. results demonstrated that leaf extracts from all selected plants were positive on elisa (fig. 3) . however, no specific reaction on western blotting was detected in any of the plant extracts analyzed (data not shown), probably due to the low levels of recombinant protein expression and to the conformational nature of most of the immunodominant epitopes present in this protein. from a titration elisa using different virus dilutions and a monospecific anti-glycoprotein s antibody, we found that ϳ30±60 g of soluble leaf protein contains a glycoprotein s antigenic mass equivalent to that contained in 0.02 g of purified tgev. the percentage of the total soluble protein corresponding to recombinant glycoprotein s polypeptides accumulated in the leaves of arabidopsis transformants could represent 0.06±0.03% of the total soluble leaf protein. leaf extracts from transgenic plants expressing the n-terminal (plants 1±4) or full-length glycoprotein s (plants 5±8) were used to immunize mice. a control mouse was immunized with a leaf extract from a plant transformed with prok2 plasmid. after three immunization doses, the specificity of mice sera was tested by an elisa using purified tgev as antigen. figure 4a shows that all sera reacted with the virus showing, as expected, different titers. a kinetic of antibody induction in an immunized mouse (number 3) was studied by immunoprecipitation of glycoprotein s induced by tgev in infected st cells. this mouse serum immunoprecipitated specifically the virus protein after two immunizations (fig. 4b) . finally, sera from all immunized mice were tested in a tgev neutralization assay. both glycoprotein s polypeptides produced in transgenic plants elicited virus-neutralizing antibodies (neutralization indexes of 2.2±3.5; fig. 4c ). serum from a nonimmunized mouse (not shown) or from the mouse immunized with the plant transformed with prok 2 plasmid did not show virus neutralization activity (fig. 4c ). in this report, we show that full-length or the globular part (n-terminal domain) of tgev spike protein (glycoprotein s) expressed in transgenic plants retained the antigenic properties and elicited neutralizing antibodies when used to immunize animals. expression in eukaryotic hosts is required for antigenic determinants that are dependent on glycosylation. of the three major antigenic sites defined on glycoprotein s involved in the induction of tgev-neutralizing antibodies, sites a and b are complex, conformational, and glycosylation dependent. site d can be represented by synthetic peptides, although glycosylation has a minor effect on its conformation (gebauer et al., 1991) . several genetically engineered vaccines using prokaryotic vectors have failed against tgev. glycoprotein s expressed at high levels in escherichia coli and used to inoculate animals did not induce neutralizing antibodies or confer protection in vivo (hu et al., 1987) . plant cells present differences in protein glycosylation with respect to animal cells that could determine the lose of antigenic determinants in antigens expressed in transgenic plants. glycosylation in plants may differ in the extent of glycosylation, processing, or both of n-linked oligosaccharide side chains (faye et al., 1993) . furthermore, the complex glycans of plants are often smaller than those of animals, in part due to the absence of sialic acid (faye et al., 1993) . the only precedent of a glycoprotein expressed in plants for vaccine development is the glycoprotein g of rabies virus (mcgarvey et al., 1995) . this protein expressed in tomato plants showed a molecular mass ϳ4 and ϳ6 kda less than that obtained from virus-infected cells but still larger than the protein size predicted for the unglycosylated polypeptide chain (mcgarvey et al., 1995) . the molecular mass of glycoprotein s expressed in arabidopsis thaliana could not be determined because we were not able to detect the recombinant protein on western blotting. however, antigenic determinants with strong dependence of glycosylation seem to be preserved because the plant-derived antigens induced neutralizing antibodies in immunized animals, indicating that critical antigenic sites are at least in part correctly glycosylated in plants. this work demonstrates the feasibility of expressing glycoprotein s polypeptides in plants. because the site of insertion of the transferred dna into the cellular chromosomal dna is random, different levels of protein expression in independent transformants are expected. we obtained expression levels similar to that described with equivalent constructs expressing hepatitis b surface antigen or rabies virus glycoprotein (mason et al., 1992; mcgarvey et al., 1995) . more recently, expression levels of norwalk virus capsid protein in tobacco have been shown to be higher than the above mentioned antigens (up to 0.23% of total soluble protein; mason et al., 1996) . we have not found significant differences in foreign antigen plant expression between the two forms of glycoprotein s studied. the use of different promoters, the use of plant-derived leader sequences and signal peptides, and mainly the modification of the codon usage of this protein could improve expression levels in plants. the demonstration that many proteins from pathogens, including some expressed in transgenic plants (haq et al., 1995; mason et al., 1996) , are immunogenic when administered orally, encourages the study of other antigens expressed in plants to develop edible vaccines. glycoprotein s from tgev is an interesting model because this protein is resistant, at least when incorporated into the viral particle, to gut degradation. in addition, the protective immune responses against tgev have to be stimulated at the mucosal surfaces to induce secretory and lactogenic immunity (de diego et al., 1992 saif and bohl, 1979; wesley et al., 1988) . once we have determined the feasibility of expressing immunological active polypeptides from tegv glycoprotein s in plants, studies on the immune response of plant-derived glycoprotein s polypeptides in pigs are necessary. seeds of arabidopsis thaliana (heynh, ecotype columbia) were sown in pots containing a mixture of universal substrate and vermiculite (3:1). to synchronize germination, pots were placed at 4°c for 48 h in darkness and then transferred to a growth chamber at 20°c with a 16-h photoperiod. irrigation was carried out with distilled water and, occasionally, with a mineral nutrient solution (haughn et al., 1986) . a 2250-pb cdna fragment (nucleotides 1±2250; fragment i) and a 4344-pb cdna fragment (nucleotides 1±4344; fragment ii) encoding for the n-terminal and full-length glycoprotein s from tgev purdue strain, respectively, were amplified by rt-pcr from viral rna and cloned into pbacpak9 plasmid (clontech). the rt primers used were 5ј-cccaactatggtaccatcaat aacagc-3ј (complementary primer to nucleotides 2225±2250) and 5ј-cgcgggatccttaatggacgtg-cactttttc-3ј (complementary primer to nucleotides 4313±4344). then, the cdna was synthesized by using the primer 5ј-gcgcggatccatgaaaaactatttgt-gg-3ј. subsequently, dna fragments i and ii were subcloned in the binary prok2 plasmid (baulcombe et al., 1986) under the control of the camw 35s promoter, yielding the recombinant plasmids prok i and prok ii, respectively (fig. 1) . plasmids prok i and prok ii were used for arabidopsis plant transformation as described elsewhere (bechtold et al., 1993) with slight modifications. a. tumefaciens (c58c1 strain) containing prok i or prok ii plasmids was grown in 600 ml of lb medium containing 50 g/ml kanamycin until an od 600 value of 2 was reached. after centrifugation, bacteria were resuspended in 200 ml of 2.35 g/l murashige and skoog medium containing 10 g/l 6-benzilaminopurine and 5% sucrose. the 6±7-week-old plants were immersed in the a. tumefaciens suspension by inversion of the pots, and vacuum infiltration was performed in a vacuum chamber at 50 mb for 15 min. infiltrated plants were rinsed with water and placed in the greenhouse until attaining maturity. transgenic t1 seeds were selected by germination in petri dishes containing gm [4.7 g/l murashige and skoog, 1% sucrose, 0.5 g/l 2-(n-morpholino)ethanesulfonic acid (mes), 8 g/l agar, ph 5.7] and 50 g/ml kanamycin. two-week-old transgenic plants were transplanted into soil and allowed to attain maturity. the plants were self-pollinated to obtain t2 plants and used for further analysis. the presence of the foreign cdna sequences in generated transgenic arabidopsis was detected by pcr. plant extracts were prepared by macerating leaves (ϳ10 mg) with pestle and mortar in 300 l of a buffer containing 200 mm tris±hcl, ph 8.5, 250 mm nacl, 25 mm edta, and 0.5% sds. the resulting extract was mixed with 150 l of 3 m ch 3 coona, ph 5.2, and incubated for 10 min at ϫ20°c. then, samples were centrifuged, and the dna contained in the supernatant was precipitated and resuspended in 30 l of te buffer. pcr was performed on 0.5 g of dna with a pair of primers that specifically amplify a 1389-bp fragment of the glycoprotein s gene (sense primer, 5ј-gcgcggatccatgaa-aaactatttgtgg-3ј; antisense primer, 5ј-gcgcgg-tacccgatgtgaagctattg-3ј). glycoprotein s mrna in transgenic plants was analyzed by rt-pcr. total rna from the leaves of transformed plants was isolated using the fast rna kit (bio 101) proteins from leaves were obtained by homogenization of leaves in a blender with liquid nitrogen, and the resulting powder was resuspended in buffer (0.3 g of fresh wt/ml) containing 10 mm mes, ph 6, 10 mm nacl, 5 mm edta, 0.6% triton x-100, 0.25 m sucrose, 0.15 mm spermine, 0.5 mm spermidine, 10 mm dtt, and 1 mm phenylmethylsulfonyl fluoride. the extract was filtered and centrifuged 10 min at 12,000 g, and the resulting supernatant was used for glycoprotein s polypeptides expression analyses. elisa plates were coated with 100 l (10 g/ml of pbs) of a mixture of two monoclonal antibodies, 6ac3 and 8dh8 (kindly provided by dr. l. enjuanes, centro nacional de biotechnologõ âa, csic, spain), recognizing the antigenic sites a and d of the glycoprotein s, respectively . antibodies were incubated for 12 h at 4°c, and then plates were washed and blocked 1 h at 37°c with 5% fetal bovine serum in pbs containing 0.05% tween 20. after washing the plates, leaf proteins from transgenic plants (15 g of total soluble protein per well, diluted in 200 l of pbs, ph 7), containing full-length or the n-terminal domain of glycoprotein s, were added to react with the previously adsorbed antibodies in the microtiter elisa plates during 12 h at 4°c. plates were then washed six times with 0.05% tween 20 in pbs, and 100 l of rabbit anti-s protein, obtained after three immunization doses with the baculovirus-expressed n-terminal fragment of glycoprotein s and diluted at 1:100 in pbs containing 0.05% tween 20, was added per well and left to react for 1 h at 37°c. plates were washed again six times with pbs±tween 20 buffer, and immunocomplexes were incubated with protein a±peroxidase (sigma) diluted 1:1000 in pbs±tween 20 for 1 h at 37°c. finally, plates were washed again, and 200 l of a freshly prepared solution of o-phenylenediamine dihydrochloride (sigma) and h 2 o 2 was added. reactions were stopped with 2 n h 2 so 4 , and the absorbance was measured at 492 nm. balb/c mice (one per arabidopsis plant) were immunized intramuscularly on days 0, 15, and 30 with leaf extract in pbs (40 g of total protein per animal per injection) in complete freund's adjuvant for the first inoculation and in incomplete adjuvant for the others. mice sera were evaluated for anti-glycoprotein s-specific antibodies by elisa using purified tgev as antigen. coated elisa plates with 100 l of pbs, ph 7.4, containing 0.2 g of virus were blocked as described above with 5% fetal bovine serum, and after washing of the plates six times, sera diluted 1:10 in pbs±tween 20 were added (100 l per well) and incubated for 1 h at 37°c. then, plates were washed again to remove unbound antibodies, and goat anti-mouse antibodies (1:500) were added to reveal immunocomplexes. after being washed and developed with o-phenylenediamine dihydrochloride substrate as described above, reaction was stopped with 2 n h 2 so 4 , and plates were read at 492 nm. immunoprecipitation of glycoprotein s by sera from a mouse after different immunization doses was carried out essentially as previously described for mouse antibodies (bullido et al., 1996) . briefly, st cells infected with tgev (m.o.i. 5) were incubated for 14 h, pulse labeled for 2 h with 200 ci/ml of 35 s-methionine (800 ci/mmol; amersham international, amersham, england)/ml, and lysed with lysis buffer (10 mm tris±hcl, 150 mm nacl, 1 mm edta, 1% nonidet p-40, ph 7.4, 1 mg/ml bovine serum albumin, and 1 mm phenylmethylsulfonyl fluoride). the lysate (10 6 cpm) was incubated with a control mouse serum (15 l) for 1 h and precleared with a 25% (v/v) suspension of protein g±sepharose (pharmacia, sweden) in lysis buffer. the precleared 35 s-labeled cell extract was incubated with mice sera (15 l) for 2 h at 4°c, and immunocomplexes were incubated with 25% suspension of protein g±sepharose for 1 h with gentle mixing. beads were washed three times with lysis buffer and boiled in sds±electrophoresis buffer. the antigen± antibody complexes were analyzed in 7.5% sds±page. a plaque reduction assay with sera from immunized mice was performed as described previously (jime â nez et al., 1986) . the neutralization index of each serum was expressed as the log 10 of the ratio of the pfu/ml of virus obtained using a normal serum and that observed in the presence of a given anti-glycoprotein s mouse serum. efficacy of a food plant-based oral cholera toxin b subunit vaccine expression of biologically active viral satellite rna from nuclear genome of transformed plants agrobacterium mediated gene transfer by infiltration of adult arabidopsis thaliana plants monoclonal antibody 2f4/11 recognizes the ␣ chain of the porcine ␤ 2 integrin involved in adhesion and complement mediated phagocytosis protective immune response to foot-and-mouth disease virus with vp1 expressed in transgenic plants antigenic structure of e2-glycoprotein of transmissible gastroenteritis coronavirus epitope specificity of protective lactogenic immunity against swine transmissible gastroenteritis virus characterization of the iga and subclass igg responses to neutralizing epitopes after infection of pregnant sows with the transmissible gastroenteritis virus or the antigenically related porcine respiratory coronavirus four major antigenic sites of the coronavirus transmissible gastroenteritis virus are located on the amino-terminal half of spike glycoprotein s detection, biosynthesis and some functions of glycans n-linked to plant secreted proteins antigenicity of structural components from porcine transmissible gastroenteritis virus residues involved in the antigenic sites of transmissible gastroenteritis coronavirus s glycoprotein oral immunization with a recombinant bacterial antigen produced in transgenic plants sulfonylurea-resistant mutants of arabidopsis thaliana studies of tgev spike protein gp195 expressed in e coli and by a tgevvaccinia virus recombinant critical epitopes in transmissible gastroenteritis virus neutralization expression of hepatitis b surface antigen in transgenic plants expression of norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice expression of the rabies virus glycoprotein in transgenic tomatoes passive immunity in transmissible gastroenteritis of swine: immunoglobulin classes of milk antibodies after oral-intranasal inoculation of sows with a live low cell culturepassaged virus antigenic homology among coronaviruses related to transmissible gastroenteritis virus characterization of transmissible gastroenteritis coronavirus s protein expression products in avirulent s. typhimurium ⌬cya ⌬crp: persistence, stability and immune response in swine immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato immunogenicity of transgenic plant-derived hepatitis b surface antigen tropism of human adenovirus type 5-based vectors in swine and their ability to protect against transmissible gastroenteritis coronavirus induction of antibodies protecting against transmissible gastroenteritis coronavirus (tgev) by recombinant adenovirus expressing tgev spike protein lack of protection in vivo with neutralizing monoclonal antibodies to transmissible gastroenteritis virus we thank j. c. oliveros, p. go â mez puertas, and a. brun for helpful discussions and suggestions and covadonga alonso for critical reading of the manuscript. this work was supported by grant bio96±1172 from comision interministerial de ciencia y tecnologõ âa of spain and by grant bid 802/oc-ar pid 168 from secyt-conicet, rep. argentina. key: cord-269094-6aka052v authors: ortego, javier; ceriani, juan e.; patiño, cristina; plana, juan; enjuanes, luis title: absence of e protein arrests transmissible gastroenteritis coronavirus maturation in the secretory pathway date: 2007-11-25 journal: virology doi: 10.1016/j.virol.2007.05.032 sha: doc_id: 269094 cord_uid: 6aka052v a recombinant transmissible gastroenteritis coronavirus (rtgev) in which e gene was deleted (rtgev-δe) has been engineered. this deletion mutant only grows in cells expressing e protein (e(+) cells) indicating that e was an essential gene for tgev replication. electron microscopy studies of rtgev-δe infected bhk-papn-e(−) cells showed that only immature intracellular virions were assembled. these virions were non-infectious and not secreted to the extracellular medium in bhk-papn-e(−) cells. rna and protein composition analysis by rnase-gold and immunoelectron microscopy showed that rtgev-δe virions contained rna and also all the structural tgev proteins, except the deleted e protein. nevertheless, full virion maturation was blocked. studies of the rtgev-δe subcellular localization by confocal and immunoelectron microscopy in infected e(−) cells showed that in the absence of e protein virus trafficking was arrested in the intermediate compartment. therefore, the absence of e protein in tgev resulted in two actions, a blockade of virus trafficking in the membranes of the secretory pathway, and prevention of full virus maturation. since the identification of a novel coronavirus associated with severe acute respiratory syndrome (sars), and the discovery of new human coronaviruses such as hcov-nl63 and hcov-nh, associated with respiratory illnesses, the interest on coronaviruses has clearly increased (esper et al., 2005a (esper et al., , 2005b kuiken et al., 2003; van der hoek et al., 2004) . the design of antiviral drugs interfering with coronavirus replication (hertzig et al., 2004) and the development of replication-competent propagation-deficient virus vectors (ortego et al., 2002) are potential powerful tools to prevent and control coronavirus infections. transmissible gastroenteritis coronavirus (tgev) is a member of the coronaviridae family within the nidovirales order (enjuanes et al., 2000b) . tgev is an enveloped virus with a single-stranded, positive-sense rna genome of 28.5 kb. about two-thirds of the genome encode the replicase gene, which comprises open reading frames 1a and 1b, the last one being expressed by ribosomal frameshifting (brierley et al., 1989; penzes et al., 2001) . the 3′ one-third of the genome includes structural and nonstructural genes, in the order 5′-s-3a-3b-e-m-n-7-3′ (enjuanes et al., 2000a) . in the coronaviridae family, the viral envelope contains at least three structural proteins. the most abundant is the membrane (m) protein, spanning the membrane three or four times and interacting with the nucleocapsid (n) and spike (s) proteins during assembly (escors et al., 2001; rottier, 1995) . the second most abundant is the s protein, a large type itransmembrane glycoprotein that forms peplomers and is responsible for cell receptor binding and membrane fusion (lewicki and gallagher, 2002; sui et al., 2004; suñé et al., 1990; suñé et al., 1991) . the third is the small envelope (e) protein, a transmembrane protein detected as a minor structural component (godet et al., 1992; liu and inglis, 1991; shen et al., 2003; yu et al., 1994) . another essential constituent of the virion is the n protein, an internal phosphoprotein that interacts with the genomic rna to form the viral nucleocapsid (escors et al., 2001; kapke and brian, 1986; narayanan and makino, 2001) . coronavirus maturation takes place at the cis-golgi network also known as endoplasmic reticulum-golgi intermediate compartment (ergic) (salanueva et al., 1999; tooze et al., 1984 tooze et al., , 1987 . the e protein plays an important role during virus budding and transiently resides in a pre-golgi compartment before progressing to the golgi apparatus (corse and machamer, 2000; maeda et al., 1999; raamsman et al., 2000) . it has been proposed that the e protein induces virus envelope curvature in pre-golgi membranes during mhv and sars-cov infections (arbely et al., 2004; kuo et al., 2002) . studies on the assembly of coronavirus structural proteins by heterologous mammalian expression systems have shown that coexpression of e and m proteins from bovine coronavirus (bcov), mhv, tgev, ibv, and sars-cov results in the formation of virus like-particles (vlps) that are morphologically identical to spikeless virions (baudoux et al., 1998; corse and machamer, 2000; hsieh et al., 2005; kuo et al., 2007; mortola and roy, 2004; vennema et al., 1996) . in addition, it has been described that both the mhv and ibv e proteins are sufficient for the generation of vlps (corse and machamer, 2000; maeda et al., 1999) . these observations suggested that neither the n nor the s proteins are needed for viral budding. in contrast, recent studies (huang et al., 2004) described that m and n proteins are necessary and sufficient for the formation of sars-cov pseudoparticles. therefore, the role of e protein in coronavirus morphogenesis requires additional studies. the construction of coronavirus full length cdna clones (almazán et al., 2000; casais et al., 2001; thiel et al., 2001; yount et al., 2000 yount et al., , 2003 or strategies of targeted recombination (koetzner et al., 1992; kuo et al., 2000; masters, 1999) have allowed the manipulation of viral genomes to study coronavirus morphogenesis. recently, a recombinant mhv virus with the entire gene e deleted was constructed (kuo and masters, 2003) . this virus replicates with a low infectious titer, indicating that e protein is critical, but not essential for mhv replication in vitro. at variance, our laboratory has described an essential role for the e protein during tgev replication. rtgev-δe virus was rescued by complementation within e + packaging cell lines (ortego et al., 2002) . in this article, we confirm that e protein is essential for tgev replication in different cell lines, and report the first evidence that tgev virions containing rna are generated in absence of e protein, although virus maturation was arrested in the budding compartment, and immature virions were accumulated between the rough endoplasmic reticulum and cis-golgi. these results provide evidence of an essential role of the e protein during tgev morphogenesis and in the intracellular virus trafficking through the secretory pathway. a replication-competent propagation-deficient rtgev-δe virus has been developed. this virus was rescued by complementation within e + packaging cell lines (ortego et al., 2002) . to determine whether tgev propagation deficiency was dependent on the infected cell line, a collection of e − cell lines permissive for tgev replication (st, crfk, bhkpapn, and llc-pk1) were infected with rtgev-δe. virus production was undetectable in infected bhk-papn, llc-pk1, st, and crfk e − cell lines either after 48 h postinfection or during four consecutive cell passages. in contrast, infection with rtgev-δe of e + cell lines produced infective virus, with titers up to 10 7 pfu/ml in bhk-papn-e + and 10 6 pfu/ml in llc-pk1-e + . these data indicated that the lack of infectious rtgev-δe virus production was independent of the cell line. to confirm the propagation deficiency of rtgev-δe, st-e − cells were infected with this virus and the propagation analyzed by plaque assay and immunofluorescence microscopy. lysis plaques were observed in monolayers of st-e − cells infected with rtgev-wt at 24 h post-infection, whereas no plaques were generated by rtgev-δe in these cells up to 96 h post-infection ( (risco et al., 1998) but with a higher diameter (larger than 80-nm). mature intracellular virions were not observed in rtgev-δe infected bhk-papn-e − cells. to determine whether mature or immature virions were secreted from rtgev-δe infected bhk-papn-e − cells, the presence of virions in the supernatants of infected bhk-papn-e + and e − cells was analyzed after virion purification by silverstaining sds-page and negative-staining electron microscopy ( fig. 2) . no structural tgev proteins were detected in the supernatants of bhk-papn-e − cells infected with rtgev-δe at 48 h post-infection, whereas s, m, and n proteins were detected in the supernatants of infected cells expressing e protein (fig. 2b ). the absence of secreted virions in bhk-papn-e − cells infected with rtgev-δe was confirmed by negative-staining electron microscopy on 200-fold concentrated supernatants, whereas virions were observed in supernatants of bhk-papn-e + infected cells (fig. 2c ). these observations demonstrated that rtgev-δe infected bhk-papn-e − cells did not secrete mature or immature virions. the composition of the large immature viral particles assembled in bhk-papn-e − cells was determined by immunogold detection in sections of freeze-substituted samples. mabs specific for s, m, and n proteins provided a positive signal on the rtgev-wt and rtgev-δe virions assembled on bhk-papn-e − cells, whereas only the rtgevwt virions bound the mab specific for e protein, as expected ( fig. 3a) . to study whether rtgev-δe virions incorporated rna, bhk-papn-e − cells infected with rtgev-δe or rtgev-wt were analyzed by rnase-gold electron microscopic cytochemistry. rnase-gold complex showed similar binding levels to virions assembled on bhk-papn-e − cells infected with rtgev-δe or rtgev-wt ( fig. 3b (+)) indicating that the rtgev-δe virions incorporated rna. binding of rnasegold to ribosomes of the rough endoplasmic reticulum (rer) was also clearly observed ( fig. 3b (+)), whereas the rnasetreated negative controls lacked any labeling either on ribosomes or on viral particles ( fig. 3b (−)), confirming binding specificity. the intracellular localization of tgev structural proteins was studied in bhk-papn-e − and e + cells infected with the rtgev-δe by indirect immunofluorescence using mabs specific for tgev s, m, n, and e proteins. the immunofluorescent signal associated with the tgev structural proteins m, n, and s was similar in bhk-papn-e − and e + cells ( fig. 4) with some significative differences. thus, proteins s and m were accumulated in the perinuclear region in infected bhk-papn-e − cells, whereas specific signals for these two proteins were extended to the cytoplasm in infected bhk-papn-e + cells. immunofluorescence specific for n protein was observed in the cytoplasm of bhk-papn-e − and e + cells but, in addition, a punctuate immunofluorescence pattern that corresponded to the accumulation of secretory vesicles in late infection stages was also observed in bhk-papn-e + cells (salanueva et al., 1999) . the changes observed in the subcellular localization of the tgev structural proteins, in the absence of e protein, suggested an arrest in tgev intracellular transport, whereas in bhk-papn-e + cells rtgev-δe virions progressed through the golgi to release from the cell by exocytosis. previous studies (salanueva et al., 1999) suggested that tgev morphological transformation from large immature particles into more condensed mature virus particles takes place during its transport along the exocytic pathway. to determine the effect of e gene deletion on tgev infection of bhk-papn-e − cells, the subcellular localization of virus particles and endoplasmic reticulum (protein disulfide-isomerase, pdi), intermediate compartment and cis-golgi (kdel receptor), and trans-golgi (giantin) specific markers were compared by immunofluorescence laser scanning confocal microscopy in rtgev-wt and rtgev-δe infected bhk-papn-e − and e + cells at different times post-infection (0, 8, 14, and 24 h post-infection). the immunofluorescence pattern observed with endoplasmic reticulum markers was similar in bhk-papn-e + and e − cells infected with rtgev-wt or rtgev-δe, and in non-infected cells, at all the time points analyzed, indicating that the overall distribution of this organelle in the cell cytoplasm was not significantly affected by any of these two viruses (fig. 5 ). only the time points 0 and 24 h are shown for simplicity. kdel receptor immunofluorescence specific signal in the intermediate compartment and cis-golgi was almost undetectable in rtgev-wt infected bhk-papn-e − and e + cells (fig. 6 ). in contrast, the kdel receptor specific signal in bhk-papn-e − cells infected with rtgev-δe showed a progressive and strong accumulation at 24 h post-infection (fig. 6) . immunoblots were performed and an increase in the amount of kdel-r was observed in rtgev-δe infected e − cells, consistent with the result obtained by confocal microscopy (data not shown). similar effect has been described (salanueva et al., 1999) when tgev infection is blocked with monensin, a drug that selectively affects the golgi complex interrupting the secretory pathway (data not shown). furthermore, the infection with rtgev-δe in presence of e protein provided in trans showed no accumulation of kdel receptor (fig. 6) . these data indicated that the accumulation of ergic vesicles could be a consequence of a membrane trafficking blockade in the secretory pathway leading to the inhibition of tgev morphogenesis in the absence of e protein. staining for the golgi complex in approximately 90% of rtgev-wt infected bhk-papn-e + and e − cells showed fragmentation and dispersion of golgi membranes at 24 h post-infection (fig. 7) , whereas this disruption of golgi was observed in only 20% of rtgev-δe infected bhk-papn-e + cells at 24 h post-infection probably due to the delay in the infection progress. in fact, later in the infection, the disruption golgi was up to 100% in rtgev-δe infected bhk-papn-e + (data not shown). in contrast, disorganization of golgi was not observed in rtgev-δe infected bhk-papn-e − cells at the analyzed times, indicating that the virus maturation did not progress from ergic to trans-golgi in the absence of e protein. taken together, these data indicate an arrest in the virus maturation in ergic, which is the tgev budding compartment (salanueva et al., 1999) . to confirm that the virus particles were blocked in the ergic in the absence of e protein, immunoelectron microscopy was developed to analyze the markers of the membranebound compartments containing virus particles. a different it has been shown that e protein is essential for tgev morphogenesis. in tgev-δe infected bhk-papn-e − cells virions containing rna were assembled, virus transportation was arrested in the ergic and full virus maturation was blocked and mature infectious virus was not secreted to the extracellular medium. previous studies have shown that, in the group 2 coronavirus mhv and sars-cov (dediego et al., 2007; kuo et al., 2002; kuo and masters, 2003) , e protein is not essential for virus replication, although δe defective mutants have a reduced growth in cell culture and in animal models. in contrast, for group 1 coronavirus tgev, we and others (curtis et al., 2002; ortego et al., 2002) have shown that cells transfected with an e gene-deleted tgev infectious cdna do not produce infectious virus. similarly, a gene e knockout mutant of the arterivirus equine arteritis virus (eav), is unable to produce infectious progeny (snijder et al., 1999) . kuo and masters have suggested that the apparent lethality of e gene knockouts in eav and tgev may reflect a slow infection kinetics of these viruses that would allow uninfected cells to overgrow infected cells and obscure the detection of δe mutants of some nidovirus (kuo and masters, 2003) . our results clearly showed, in this and in a fig. 6 . detection of kdel-r and rtgev in bhk-papn-e + and e − cells by immunofluorescence. tgev infection and the ergic marker kdel-r were monitored in bhk-papn-e + and e − cells infected with rtgev-wt and rtgev-δe at 0 and 24 h p.i. by immunofluorescence microscopy using a rabbit polyclonal antibody specific for tgev and a kdel-r specific mab. previous publication (ortego et al., 2002) , that e protein is essential for tgev replication and that no infectious virus was produced at all. a simple explanation is that these viruses belong to different cov groups and have different assembly requirements. the generation of infectious rtgev-δe virus was dependent on e protein expression and independent of the infected cell line. thus, rtgev-δe infected cells did not secrete virus to the extracellular medium, and the virus did not spread to neighboring uninfected cells, and therefore did not generate plaques in infected st-e − cells up to 96 h postinfection, confirming that rtgev-δe is a propagation-deficient virus in the absence of e protein provided in trans. furthermore, no virus amplification was observed using supernatants or cell extracts from bhk-papn-e − cells infected with rtgev-δe virus when used to infect fresh bhk-papn-e + cells, although these cells could have amplified minute amounts of rtgev-δe virus. these data confirmed that e protein was essential for tgev virus replication. therefore, a different behavior is shown by coronavirus from groups 1 and 2 in the requirement for e protein during virus assembly. the requirement of e and m proteins for the formation of virus-like particles has been previously described (vennema et al., 1996) . co-infection with recombinant baculoviruses showed that the membrane and envelope proteins are sufficient for the efficient formation of coronavirus vlps (mortola and roy, 2004) . in contrast, huang and coworkers reported that two viral genes, encoding the sars-cov m and n proteins, are necessary and sufficient for formation of vlps by cotransfection of expression plasmids (huang et al., 2004) . this discrepancy could be explained by the different cell types used in these studies (insect sƒ9 and human 293t cells, respectively). in our study, electron microscopy analysis of bhk-papn-e − cells fig. 7 . detection of giantin and rtgev in bhk-papn-e + and e − cells by immunofluorescence. tgev infection and the golgi apparatus marker giantin (gig) were monitored in bhk-papn-e + and e − cells infected with rtgev-wt and rtgev-δe at 0 and 24 h p.i. by immunofluorescence microscopy using a rabbit polyclonal antibody tgev specific and a giantin specific mab. infected with rtgev-δe demonstrated that only immature tgev virions were assembled in the absence of e protein. these virions contained rna, and also all the structural tgev proteins (s, m, and n) except the deleted e protein. these results indicate that the e protein is essential for the formation of mature tgev virions. in the absence of e protein, the assembled virions displayed a morphology different from the full-length virus, were non-infectious, accumulated in the cytoplasm of the infected cells, and were not released to the extracellular medium or transported to neighboring cells. tgev replication depends on the exocytic pathway to complete its morphogenesis (salanueva et al., 1999) . virions were assembled as large particles with annular morphology at perinuclear compartment of the cells, and changed their morphology into small dense virions during the transport along the exocytic route (risco et al., 1998; tooze et al., 1987) . in cells infected with rtgev-δe, an accumulation of the kdel receptor and the lack of fragmentation and dispersion of golgi membranes were observed in absence of e protein. these effects and rtgev-δe virus assembly into mature particles were recovered by providing e protein in trans. in addition, immunoelectron microscopy studies with an intermediate compartment specific marker confirmed that, in the absence of the e protein, the maturation of the virus is arrested in the ergic. a similar effect has been described when tgev infection is blocked with monensin, a drug that selectively affects the golgi complex interrupting the secretory pathway. monensin blocks the viral transport from ergic, leading to the accumulation of numerous large viral particles in dilated pre-golgi ergic elements of vacuolar morphology (salanueva et al., 1999) . the comparable deficiencies observed in viral morphogenesis in the absence of e protein and as a result of treatment with monensin suggest an aberrant assembly process due to the absence of e protein. in principle, e protein could act directly on the ergic and golgi compartments, on the virus itself, or in both. it has been postulated that e protein prepares the membrane or other viral proteins occupying strategic positions within the budding pathways (salanueva et al., 1999) , as proposed for small acylated glycoproteins of alphaviruses and orthomyxoviruses that also display ion channel activity (gaedigk-nitschko et al., 1990; ivanova et al., 1995; liao et al., 2006; wilson et al., 2004; zebedee and lamb, 1988) . the absence of e protein would inhibit the progression through the ergic-golgi compartments, resulting in the accumulation of virions in the ergic and of the ergic itself. e proteins from mhv and sars-cov have viroporin activity (liao et al., 2004; madan et al., 2005) . viroporins act at late stages of the viral cycle promoting the exit of new virus particles from the cell (gonzalez and carrasco, 2003) . the exact mechanism by which viroporins alter the permeability of the plasma membrane is unknown, and it is possible that this activity is displayed both at the plasma membrane and on cellular organelles (aldabe et al., 1997; van kuppeveld et al., 1997) . coronavirus e protein localizes in membranes of ergic, where it could modify their permeability, leading to the disruption of ion gradients at intracellular organelles (madan et al., 2005) , facilitating the transport of the virions along the exocytic pathway and their maturation. the absence of this viroporin activity mediated by the e protein possibly leads to the arrest of the transport of virions through the secretory pathway, the accumulation of non-mature virions in ergig, and the absence of release of new infectious virus from the cell. the construction of e protein mutants affecting e protein ion channel activity could assess whether this postulate is correct. e proteins from the different coronavirus groups show different channel selectivity. the e protein from the group 1 coronavirus hcov-229e is k + selective, whereas from the group 2 mhv and sars-cov, and group 3 coronavirus ibv, e protein is na + selective (wilson et al., 2006 (wilson et al., , 2004 . the diverse channel selectivity among the coronavirus groups could be translated in differences in the effect of e protein in the secretory pathway and may explain the discrepancies among the gene e-defective mutants from the three coronavirus groups, and why e protein is essential for replication of group 1 viruses and non-essential for replication of group 2 viruses. additional analysis of the gene e-defective mutants during assembly is required to shed light on the functions of the e protein in coronavirus morphogenesis in the three virus groups of the coronaviridae family. in conclusion, the arrest of tgev maturation and the accumulation of virions in the secretory pathway due to the absence of the e protein confirm that this protein is essential for tgev transportation and morphogenesis. the ability to generate tgev-δe mutants that are replication-competent but propagation-deficient by complementation in packaging cell lines, supports the potential use of these mutants as vaccine vectors. recombinant rtgev-mlui-fsei (rtgev-wt) and rtgev-δ3abδe (rtgev-δe) (ortego et al., 2002 (ortego et al., , 2003 were grown as described (jiménez et al., 1986) . baby hamster cells (bhk-21) stably transformed with the gene coding for the porcine aminopeptidase n (bhk-papn) (delmas et al., 1992) were grown in dulbecco's modified eagle's medium (dmem) supplemented with 2% fetal calf serum (fcs) containing geneticin g418 (1.5 mg/ml) as a selector agent. porcine kidney cells, llc-pk1 (european collection of cell cultures no. 86121112), were grown in medium 199 supplemented with 2 mm glutamine and 10% fcs. cat kidney cells, crfk (atcc ccl 94), were grown in dmem supplemented with 2% fcs. bhk-papn-e + and llc-pk1-e + cells were grown as described previously (ortego et al., 2002) . standard virus titrations were performed in porcine swine testis (st) cells. titrations of virus with the e gene deleted were performed in llc-pk1 cells expressing the e protein. the murine mabs 1d.g3, 3b.b3, and 3b.d10, specific for tgev s, m, and n proteins, respectively, have been previously described (gebauer et al., 1991; risco et al., 1995; sánchez et al., 1990) . the murine mab q3, specific for tgev e protein, was a kind gift of h. laude (inra, jouy-en josas, france). the rabbit serum specific for pdi and the murine mab specific for giantin was generously provided by a. nieto (centro nacional de biotecnología, csic, madrid, spain) and m. renz (institute of immunology and molecular genetics, karlsruhe, germany), respectively. the murine mab specific for kdel receptor and the rabbit polyclonal antibody specific for ergic-53/p58 were purchased from stressgen biotechnologies corp. (victoria, bc, canada) and sigma-aldrich (st. louis. mo, usa), respectively. tgev virions were sedimented through a 20% sucrose cushion in ten (tris-hcl 10 mm [ph 7.4], edta 1 mm, nacl 1m) 0.2% tween 20 by centrifugation in an sw28 beckman rotor at 25,000 rpm for 2 h at 4°c. sucrose was removed and the pellet was washed with ten. pellet was recovered by suspending it in ten-0.2% tween 20 and sedimented by centrifugation in a sw 41 beckman rotor for 1 h at 25,000 rpm. virions were recovered by suspending the pellet in tne (tris-hcl 10 mm [ph 7.4], edta 1 mm, nacl 100 mm) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and silver staining or negative staining and electron microscopy. monolayers of bhk-papn cells expressing tgev e protein and control cells were infected with rtgev-wt or rtgev-δe virus. the cells were fixed in situ at 12, 16, and 24 h post-infection with a mixture of 2% glutaraldehyde and 1% tannic acid in 0.4 m hepes buffer (ph 7.2) for 2 h at room temperature. the fixed monolayers were removed from the dishes in the fixative and transferred to eppendorf tubes. after centrifugation in a microcentrifuge, the cell pellets were washed with hepes buffer and processed for embedding in eml-812 (taab laboratories, berkshire, united kingdom) as described (risco et al., 1998) . the cells were post-fixed with a mixture of 1% osmium tetroxide and 0.8% potassium ferricyanide in distilled water for 1 h at 4°c. after four washes with hepes buffer, samples were incubated with 2% uranyl acetate, washed again, and dehydrated in increasing concentrations of acetone (50, 70, 90, and 100%) for 15 min each at 4°c. infiltration in the resin eml-812 was done at room temperature for 1 day. polymerization of infiltrated samples was done at 60°c for 2 days. ultrathin (50-to 60-nm-thick) sections of the samples were stained with saturated uranyl acetate and lead citrate by standard procedures. cultures of cells were subjected to a mild fixation with a solution of 4% paraformaldehyde containing 0.1% glutaraldehyde in pbs at 4°c for 30 min. small pellets of chemically fixed cells were cryoprotected with glycerol, applied to small pieces of filter paper, blotted, and quick frozen in liquid ethane. vitrified specimens were transferred to a reichert-jung afs freeze-substitution unit (leica, vienna, austria) and maintained for 48 h at − 90°c in a mixture of methanol and 0.5% (w/v) uranyl acetate. after freeze-substitution, samples were infiltrated in lowicryl k4m (eml laboratories) at − 30°c and polymerized with uv light. ultrathin sections of the samples were either stained or processed for immunogold labeling. immunogold detection of tgev proteins on ultrathinsections of infected bhk-papn cells was performed at room temperature with mabs specific for m, n, s, e, and ergic-53 proteins by established procedures (risco et al., 1995) . sections collected on formvar coated gold electron microscopy grids were incubated for 30 min with tris buffer-gelatin and then floated on drops of diluted primary antibodies for 75 min. after jet washing with pbs, samples were incubated for 45 min with secondary antibodies conjugated with 10-nm-diameter gold particles and washed again with pbs and distilled water. samples were then allowed to dry on filter paper before being stained with saturated uranyl acetate for 25 min, followed by 1 min with lead citrate. all samples were analyzed with a jeol 1200 ex ii electron microscope. for ultrastructural detection of rna, a complex of rnase and 10-nm-diameter colloidal gold (ey laboratories, san diego, ca) was used as described previously (risco et al., 1998) . ultrathin sections from epon-included samples were collected on gold electron microscopy (em) grids covered with collodion and incubated for 40 min at room temperature with the gold conjugated rnase diluted 1:20 in pbs. after being washed with pbs and distilled water, samples were stained with uranyl acetate and lead citrate. to unmask viral rna molecules, sections were subjected to a treatment before incubation with rnase-gold consisting on 15 min incubation at 37°c with proteinase k (10 μg/ml in tris-edta), washing with te, 10 min fixation with 4% paraformaldehyde in pbs, washing again with pbs, and incubation for 10 min with 0.2 m nh 4 cl. as a cytochemical control, some sections were pre-incubated for 30 min at 37°c with a solution of non-conjugated rnase (20 μg/ml) before being treated with the rnase-gold conjugated. virus from supernatants of bhk-papn-e + and e − cells infected with rtgev-δe virus, concentrated 200-fold, were analyzed by negative staining as described (bremer et al., 1998) . briefly, samples were adsorbed to uv light-activated copper grids for 2 min at room temperature. grids were washed two times in h 2 o (escors et al., 2001) and stained with 2% uranyl acetate for 1 min. samples were visualized in a jeol 1200 exii transmission electron microscope. cells were plated on glass coverslips. infections were performed at an m.o.i.=1 pfu/cell at 37°c in dmem containing 2% fcs. the inoculum was removed at 90 min and the cells were maintained in dmem 2% fcs. at the times indicated, cells were washed with pbs and fixed by addition of 4% paraformaldehyde for 30 min at room temperature. for dual-labeling experiments in which, one primary antibody was derived from mouse and the other one from rabbit, both antibodies were combined in a pbs-fcs 20% diluent containing 0.2% saponin (superfos-biosector, vedback, denmark). antibodies were allowed to adsorb for 90 min at room temperature, and washed three times with pbs-fcs 2%. cells were then incubated for 30 min at room temperature with a mixture of anti-rabbit and anti-mouse secondary antibodies conjugated to alexa 488 or alexa 594. the coverslips were washed five times with pbs-fcs 2%, mounted on glass slides, and analyzed with a laser scanning confocal system radiance 2100 (bio-rad) on a zeiss axiovert 200 microscope. 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channels hexamethylene amiloride blocks e protein ion channels and inhibits coronavirus replication strategy for systematic assembly of large rna and dna genomes: the transmissible gastroenteritis virus model reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus mouse hepatitis virus gene 5b protein is a new virion envelope protein influenza a virus m2 protein: monoclonal antibody restriction of virus growth and detection of m2 in virions we thank s. zuñiga, i. sola, j.l. moreno, m.l dediego, and e. alvarez for critically reading the manuscript and helpful discussions. we are also grateful to d. dorado, and r. arranz for their excellent technical assistance. this work was supported by grants from the comisión interministerial de ciencia y tecnología (cicyt), the consejería de educación y cultura de la comunidad de madrid, fort dodge veterinaria, and the european communities (projects fmdv vaccine. qrlt-2001-00825 and dissect, sp22-ct-2004-511060). key: cord-279346-7del8d2p authors: callendret, benoît; lorin, valérie; charneau, pierre; marianneau, philippe; contamin, hugues; betton, jean-michel; van der werf, sylvie; escriou, nicolas title: heterologous viral rna export elements improve expression of severe acute respiratory syndrome (sars) coronavirus spike protein and protective efficacy of dna vaccines against sars date: 2007-07-05 journal: virology doi: 10.1016/j.virol.2007.01.012 sha: doc_id: 279346 cord_uid: 7del8d2p the sars-cov spike glycoprotein (s) is the main target of the protective immune response in humans and animal models of sars. here, we demonstrated that efficient expression of s from the wild-type spike gene in cultured cells required the use of improved plasmid vectors containing donor and acceptor splice sites, as well as heterologous viral rna export elements, such as the cte of mazon-pfizer monkey virus or the pre of woodchuck hepatitis virus (wpre). the presence of both splice sites and wpre markedly improved the immunogenicity of s-based dna vaccines against sars. upon immunization of mice with low doses (2 μg) of naked dna, only intron and wpre-containing vectors could induce neutralizing anti-s antibodies and provide protection against challenge with sars-cov. our observations are likely to be useful for the construction of plasmid and viral vectors designed for optimal expression of intronless genes derived from cytoplasmic rna viruses. the mechanisms of sars-cov introduction into humans are largely unknown. sars-cov-like viruses have been isolated from caged himalayan palm civets and raccoon dogs in live-animal markets in china (guan et al., 2003; kan et al., 2005) . evidences for direct transmission of sars-cov-like viruses from these animals to humans have been reported the chinese sars molecular epidemiology consortium, 2004) . however, recent studies suggested that civets may have served only as an amplification host for sars-cov and the existence of an upstream wild animal reservoir has been proposed (tu et al., 2004; wu et al., 2005) . consistent with this hypothesis, sars-cov like viruses sharing more than 88% nucleotide identities with sars-cov have been isolated from horseshoe bats in mainland china and hong virology 363 (2007) 288 -302 www.elsevier.com/locate/yviro kong sar (lau et al., 2005) . thus, re-emergence of sars-cov is a matter of concern and an efficient vaccine would be the most effective way to control a new epidemic. similar to other coronaviruses, sars-cov is an enveloped positive-strand rna virus. its large single-stranded genome, 29.7 kb in length, encodes the replicase polyproteins, small accessory proteins and four major structural proteins: the nucleoprotein (n), the small envelope protein (e), the membrane protein (m), and a large, club-shaped spike protein (s). the sars-cov spike protein is a type-i transmembrane glycoprotein consisting of 1255 amino acids (a.a.), with a molecular weight of ∼ 180 kda. as predicted by sequence analysis, it consists of four domains: a signal sequence (a.a. 1 to 14), a large ectodomain comprised of amino-acids 15 to 1190 with 23 potential n-glycosylation sites, a transmembrane domain (a.a. 1191 to 1227), and a short cytoplasmic tail of 28 a.a. rota et al., 2003) . the s protein mediates many of the biological properties of the virus, including viral entry: binding to cellular receptors, ace2 and l-sign (jeffers et al., 2004) , penetration and fusion between the viral and cellular membranes (matsuyama et al., 2005; petit et al., 2005; simmons et al., 2005) . it is also associated with host range and tissue tropism (giroglou et al., 2004; qu et al., 2005) , as for other coronaviruses (casais et al., 2003; haijema et al., 2003; kuo et al., 2000) . the s protein of sars-cov is a candidate antigen for vaccine development, as it is the main target for neutralizing antibodies in human patients nie et al., 2004; temperton, 2005; traggiai et al., 2004) . moreover, passive immunization studies demonstrated that spike-specific neutralizing antibodies are protective in the mouse and ferret (ter meulen et al., 2004) animal models. accordingly, several candidate vaccines relying on the induction of spike-specific neutralizing antibodies have been reported to induce a protective immune response in various animal models (bisht et al., 2004 (bisht et al., , 2005 buchholz et al., 2004; bukreyev et al., 2004; chen et al., 2005; yang et al., 2004) . efficient expression of intronless genes from plasmid or viral vectors may require the presence of additional cis-acting regulatory modules within the expression cassettes, such as splice sites (ss), retroviral constitutive transport elements (cte) and hepadnaviral posttranscriptional regulatory elements (pre). the most studied of these latter viral elements are the cte of mason-pfizer monkey virus (mpmv-cte) (rizvi et al., 1996) and the pre of woodchuck hepatitis virus (wpre) (donello et al., 1998) and both have been successfully used for that purpose (loeb et al., 1999; tan et al., 1995; wodrich et al., 2000; zufferey et al., 1999) . however, the optimal choice of posttranscriptional enhancer modules depends on the type of cdna to be expressed . whether this strategy may apply to the expression of genes from cytoplasmreplicating rna viruses, which mrnas are not naturally processed by splicing and nuclear-export machineries, remains to be determined. in the present study, we report the construction of a series of dna expression vectors containing the wild-type s coding sequence. the influence of ss, mpmv-cte or wpre on expression of s protein in transfected cells and on induction of a protective sars-cov-specific immunity in mice after naked dna immunization are compared. we found that wpre together with ss allowed most efficient expression of s in 293t and veroe6 cells whereas cte had a slight to moderate effect depending on the cell line. furthermore, dose range experiments demonstrated that, when administered to mice, dna plasmid vectors with wpre induced the highest levels of anti-s antibodies and protection towards a challenge with live sars-cov. the coding region of sars-cov s protein was amplified by rt-pcr from the broncho-alveolar lavage of a patient hospitalized with probable sars at the hanoï french hospital (vietnam) in 2003 and cloned into pcdna3.1 and pci expression vectors under the control of the cytomegalovirus (cmv) immediate/early promoter, yielding pcdna-s and pci-s plasmids, respectively (see fig. 2a ). we initially tested protein expression driven by these vectors in transiently transfected vero e6 cells, which are permissive for sars-cov replication, thus allowing mature expression of essential sars-cov genes. pcdna-s did not induce detectable levels of s protein either by immunoblotting using an sspecific rabbit antiserum (fig. 1 , lane 2) or by indirect immunofluorescence (data not shown). this was not due to inadequate sensitivity of the detection methods, since s as fig. 1 . transient expression of the sars-cov spike protein following plasmid dna transfection. subconfluent monolayers of vero e6 cells were transfected with empty (−) or s (+) expression plasmids. at 48 h post-transfection, whole cell extracts were prepared in laemmli sample buffer, separated by sds-page as indicated (lanes 1 to 4) and analyzed by western blot as described in materials and methods using rabbit polyclonal antibodies directed against the s protein. alternatively, veroe6 cells were infected with vvtf7-3 at a moi of 5, transfected 1 h later with the indicated construct and analyzed for s expression 18 h thereafter (lanes 5 and 6). as controls, whole cell extracts prepared from veroe6 cells either mock infected (lane 7) or infected with sars-cov (lane 8) were analyzed. the position of the molecular weight markers is shown on the right of the gel (kda). expressed in sars-cov infected cells was readily detected at the expected size of ∼ 180 kda (fig. 1, lane 8 ). in addition, potential defect in the pcdna-s construct, low transfection efficiency or other flaws could be excluded, since pcdna-s induced high levels of s expression from the t7 rna polymerase promoter, when t7 rna polymerase was expressed in vero e6 cells after superinfection with vv-tf7.3 recombinant vaccinia virus (fig. 1, lane 6) . in contrast, s protein expression could be detected in vero e6 cells after transfection of the pci-s construct (fig. 1, lane 4) . similar data were obtained in 293t cells (see fig. 2b ). the pci-s construct differs markedly from the pcdna-s construct by the presence of a chimeric intron between the cmv promoter and the s coding sequence. the inserted intron is composed of the donor site from the first intron of the human β-globin gene and the branch and acceptor site from the intron of an immunoglobulin gene (promega, january 2004, revision date) . these results suggested that the presence of an intron is required for expression of the sars-cov s gene, when the corresponding mrna is synthesized in the nucleus of transfected cells. however, expression levels driven by pci-s remained low as compared to the levels observed in the t7 rna polymerase based expression system (fig. 1, compare lanes 4 and 6) . in the latter (fuerst et al., 1986) , transcription takes place primarily in the cytoplasm, suggesting that s mrna might be abnormally processed after synthesis in the nucleus or inefficiently exported to the cytoplasm. cis-acting viral export elements such as the cte of mason-pfizer monkey virus (mpmv-cte), and the pre of woodchuck hepatitis virus (wpre) have been shown to increase the nuclear export of mrnas, resulting in higher protein expression levels (bray et al., 1994; donello et al., 1998; gruter et al., 1998; popa et al., 2002) . therefore, mpmv-cte and wpre sequences were inserted in both pcdna-s and pci-s plasmids downstream of the s coding sequence and upstream of the polyadenylation sequence ( fig. 2a) . immunoblot analysis of cell lysates prepared from transfected veroe6 and 293t cells are shown in figs. 2b and c, respectively. s expression remained below detectable levels whatever viral export element inserted in the pcdna-s plasmid. in marked contrast, both cte and wpre increased s expression when inserted in the pci-s plasmid. the most dramatic effect was observed upon pci-s-wpre transfection into either veroe6 or 293t cells. similar data were obtained in simian frhk-4 and hamster bhk cells (data not shown). to evaluate more accurately the impact of cte and wpre on s production, protein expression levels were quantified from the luminescence signals recorded with a cooled ccd camera ( table 1) . as compared to pci-s, a 12-to 42-fold increase for the wpre-containing pci plasmid was observed, regardless of the cell lines used, while cte had more impact on s expression in veroe6 cells (9-to 26-fold increase) than in 293t cells (4-fold increase). expression of s at the cell surface was examined by facs analysis performed on non-permeabilized 293t cells at 48 h post-transfection. less than 5% of cells transfected with either plasmid from the pcdna-s series were stained by polyclonal mouse anti-s antibodies (fig. 3 , upper left panel and data not shown), while 50% of cells transfected with the pci-s-wpre construct were found positive (fig. 3 , lower right panel). in agreement with immunoblotting data, wpre was much more efficient than cte in enhancing s expression from pci-s dnas at the surface of 293t cells. levels of s produced upon transfection of pci-s-cte and pci-s-wpre were 2-and 11fold higher, respectively, than those produced upon transfection of pci-s. to analyze whether enhanced s expression correlated with increased s mrna levels, we performed northern blot analysis of cytoplasmic fractions prepared 24 or 48 h post-transfection of veroe6 and 293t cells. s mrnas were detected with a mixture of three s-specific probes and their relative abundance was calculated using β-actin mrna levels as an internal control. a representative experiment is shown in fig. 4 for mrnas prepared 48 h after transfection of 293t cells. no s-specific mrna was detected in the cytoplasm of cells transfected with any pcdna-s-derived plasmid or empty plasmid, in agreement with the lack of s expression. s mrnas of the expected size were observed with plasmids of the pci-s series. thus, cytoplasmic accumulation of s mrna and hence s protein expression appeared to be dependent on correct splicing. surprisingly, cte had no impact on the amount of cytoplasmic s mrnas in veroe6 and 293t cells, neither at 24 h nor at 48 h post-transfection ( fig. 4 and data not shown). in contrast, wpre increased mrna levels in 293t cells by 3fold at both 24 h (data not shown) and 48 h (fig. 4) posttransfection, and transiently increased mrna levels in veroe6 cells at 24 h post-transfection (2.7 fold increase; data not shown). altogether, our results showed that both cte and wpre have a greater effect at the protein level than at the mrna level (compare fig. 3 and table 1 to fig. 4 ) and thus promote sars-cov spike protein expression by increasing mrna translation. in all cell lines examined, the impact of wpre on s protein expression was consistently better than that of cte, reflecting synergistic effects of this element on cytoplasmic mrna levels and mrna translation. at 48 h post-transfection, cytoplasmic rna was prepared and analysed by northern blot for the presence of s mrnas with a mixture of negative sense riboprobes specific for s sequences, as described in materials and methods. the position of molecular weight markers is indicated on the side. for normalization, northern blot analysis was performed in parallel using a negative sense β-actin specific riboprobe. the amounts of s specific rnas of the expected size (stars) and β-actin rnas were quantified and the calculated s/β-actin specific mrna ratios are indicated below the images (1 = ratio found in pci-s transfected cells). having shown that the sars-cov s protein can be efficiently expressed at the cell surface by using the wpre element, we sought to further test whether the s protein expressed in this system was functional and antigenically relevant. we first evaluated the capacity of the wpre-expressed s protein to induce receptor-dependent cell-to-cell fusion. we took advantage of the fact that, under our experimental conditions, co-transfection of 293t cells with two different expression plasmids allowed expression of both encoded proteins in more than 90% of the transfected cell population (data not shown). thus, target cells were prepared by cotransfection of 293t cells with pcdna-dsred and pcdna3-ace2, which allow expression of the fluorescent dsred protein and the ace2 sars-cov receptor, respectively . concurrently, effector cells were prepared by transfection of 293t-gfp cells with the pci-s-wpre plasmid. the fusion of co-cultured green effector cells and red target cells was evaluated by facs analysis (fig. 5) . we found that no cell-tocell fusion occurred when s-expressing effector cells were mixed with mock-transfected target cells (panel b) or when mock-transfected effector cells were mixed with ace2expressing target cells (panel c), as the percentage of greenand red-stained cells did not increase above background (panel a). in contrast, more than 45% of target cells expressing ace2 did fuse with effector cells expressing s (panel d). next, we evaluated the antigenicity of the wpre-expressed s protein using convalescent human sera. veroe6 cells were transfected with pci-s-wpre or pci as a control, and immunofluorescence assays (ifa) were carried out using three sars convalescent human sera and two normal human sera (fig. 6 ). s-expressing veroe6 cells were readily stained with convalescent sera from the sars cases (panels e, f and data not shown) but not with sera from healthy blood donors (panel d as an example), while control vero cells showed no staining (panels a, b). in addition, this s-protein based ifa allowed to monitor seroconversion of patients for sars, using serum samples taken during the acute and convalescent phases of the disease (panels c and f, respectively as an example). altogether, our results indicate that wpre-mediated expression of s is qualitatively and quantitatively relevant, since the resulting polypeptide is capable of inducing cell-to-cell fusion and is efficiently recognized by convalescent human sera. in order to compare the capacity of cte and wpre to enhance the efficacy of naked dna vaccine, we performed a dose response experiment. female balb/c mice were immunized by intramuscular injection of 2, 10 or 50 μg of pci-s, pci-s-cte or pci-s-wpre naked plasmid dna. this dose range extended from suboptimal to optimal doses of typical plasmid dna vaccine in mice and was selected according to our preliminary experiments (data not shown) and published data (fu et al., 1999; ulmer et al., 1994; wang et al., 2000) . as control, a group of mice was injected with 50 μg of dna from the parental pci plasmid. mice were boosted at week 4 with the same dose of dna and the resulting immune responses were analyzed 3 weeks later. anti-s serum igg antibody titers were determined for each individual mice by indirect elisa using sars-cov-infected veroe6 cell lysates as native antigens (fig. 7) . at the optimal 50-μg dose, each of the three s expression plasmids induced a high anti-s igg response. the log10 serum igg titers were 3.7 ± 0.2 for both pci-s and pci-s-cte-immunized mice and 3.9 ± 0.1 for pci-s-wpre-immunized mice, indicating that pci-s-wpre induced significantly higher (∼ 2-fold, p < 0.05) anti-s igg levels than pci-s and pci-s-cte. at the 10-μg suboptimal dose of plasmid dna, pci-s-wpre induced a much higher and more consistent response across mice (3.6 ± 0.2 log10 titer) than pci-s (2.8 ± 0.4 log10 titer, p < 0.01) and pci-s-cte (2.4 ± 1.0 log10 titer, p < 0.03). at the lowest dose (2 μg plasmid dna per mouse), both pci-s and pci-s-cte failed to induce any detectable anti-s antibodies (log10 titer < 0.9) with the exception of a single mouse (1.4 log10 titer) out of eight pci-s-cte-immunized mice, whereas anti-s igg titers remained high (3.1 ± 0.5 log10 titer) for all pci-s-wpre-immunized mice. thus, the anti-s antibody levels induced by pci-s-cte were comparable to those induced by the pci-s construct, with no statistically significant differences at any dose tested (p > 0.3). this dose response experiment clearly demonstrates that wpre strongly enhances the immunogenicity of an s-based anti-sars naked dna vaccine in mice, whereas cte has no effect. the most remarkable effect was observed at the very low dose of 2 μg naked dna per mouse, for which the wpreenhanced dna vaccine only was able to induce high levels of anti-sars antibodies. to assess whether a low dose of the wpre-enhanced dna vaccine could induce protective immunity against sars, balb/c mice were injected three times with 2 μg of plasmid dna at 4-week intervals. the mice received the various dna constructs as described above. first, sera were sampled at 3 weeks post-immunization, and we examined whether the antibody response was capable of sars-cov neutralization. neutralizing antibody titers remained below the detection level fig. 7 . wpre enhances anti-s antibody responses in mice immunized with low doses of plasmid dna. groups of 6 balb/c mice were injected twice intramuscularly at 4-week interval with 2, 10 or 50 μg of s expression plasmid dna as indicated, or with 50 μg of pci plasmid dna as control. immune sera were collected 3 weeks after the second injection. sars-cov specific igg antibody titers were determined by indirect elisa using sars-cov infected veroe6 cell lysates as the capture antigen, as described in materials and methods. values for each individual mouse are represented with black circles, and means with horizontal bars. the detection limit of the assay is indicated by a dotted line. after injection of either pci-s or pci-s-cte plasmid dna (fig. 8a ). in contrast, significant levels of sars-cov neutralizing antibodies were measured in 7 out of 8 mice immunized with pci-s-wpre plasmid dna. next, at 8 weeks post-immunization, mice were transferred to a bsl-4 animal facility and challenged by intranasal inoculation of 10 5 pfu of sars-cov. viral loads in the lungs were evaluated 2 days later (fig. 8b ). no significant difference was found in the viral titers observed in mice injected with pci-s (5.8 ± 0.5 log10 pfu/lungs), pci-s-cte (5.9 ± 0.3 log10 pfu/lungs) or with control pci (5.3 ± 0.6 log10 pfu/lungs). in sharp contrast, immunization with pci-s-wpre plasmid dna conferred almost complete protection with no infectious virus detectable in 7 out of 8 mice (log10 pfu/ lungs <1.8) and a very low residual titer in a single mouse (p < 10 − 8 ). these studies clearly indicate that the wpre-enhanced sars dna vaccine is most effective in inducing complete protective immunity, providing protection against sars-cov challenge after low-dose injection of naked plasmid dna. to our knowledge, the vast majority of published studies relying on the transient expression of the spike protein of all coronaviruses before the sars-cov epidemic was recognized, involved the use of either t7 rna polymerase-based expression systems or viral expression vectors such as recombinant sindbis or vaccinia viruses (horsburgh and brown, 1995; krueger et al., 2001; narayanan et al., 2003; pulford and britton, 1991; vennema et al., 1996; yoo and deregt, 2001) . this may be indicative of the difficulty to express efficiently the long coronavirus s gene following transient transfection of mammalian cells with conventional dna expression vectors, that lead to mrna synthesis in the cell nucleus. consistent with this hypothesis, we also found that the sars-cov s gene is not expressed at detectable levels when cloned into the classical pcdna mammalian expression vector under the control of a nuclear polymerase ii promoter (fig. 1) , whereas it is expressed efficiently from a recombinant vaccinia virus (v. lorin, b. callendret, s. van der werf and n. escriou, unpublished data). likewise, it has been reported that the s protein from sars-cov is poorly expressed following dna transfection (chang et al., 2006; hofmann et al., 2004; qin et al., 2004; simmons et al., 2004) , hence most published studies used t7 rna polymerase-based expression systems (schwegmann-wessels et al., 2004; xiao et al., 2003) or synthetic genes optimized for human codon usage qin et al., 2004; zhi et al., 2005) . here, we describe the design and use of a modified dna expression vector to enhance sars-cov s expression in mammalian cells. this dna vector contains the cmv immediate/early promoter, a chimeric intron and a cis-acting cte or wpre element. we demonstrate that this vector permits the expression of high levels of s at the cell surface of transiently transfected cells, that are compatible with functional and antigenic characterization of the s protein. in addition, this low-cost and easy-to-use expression vector allowed us to develop a cell-to-cell fusion assay and an immunofluorescence assay, which may prove useful for the study of cellular and viral determinants of sinduced membrane fusion and for the sensitive and specific routine diagnosis of sars seroconversion. detectable expression of both spike mrna and protein in transfected cells required the use of plasmid vectors containing fig. 8 . wpre is required for protection of mice from sars-cov challenge upon immunization with low-dose plasmid dna. groups of 8 balb/c mice were injected three times intramuscularly at 4-week interval with 2 μg of s expression plasmid dna as indicated, or with 2 μg of pci plasmid dna as control. immune sera were collected 3 weeks after the third injection and assayed for neutralizing antibodies against sars-cov. neutralization titers (a) were calculated as the reciprocal of the highest dilution of each individual serum, which completely prevented sars-cov cpe in 50% of the wells, as described in materials and methods. eight weeks after the third dna injection, mice were challenged intranasally with sars-cov (10 5 pfu/mouse). two days after inoculation, mice were euthanized. lung homogenates were prepared and titrated for infectious sars-cov by plaque assay on veroe6 cells. viral titers were expressed as log 10 (pfu/lungs) for individual mice (b). values for each individual mouse are represented with black circles, and means with horizontal bars. the detection limits of the assays are indicated by a dotted line. data shown are from one experiment representative of two. an intron upstream of the s orf ( figs. 1 and 2 ). our findings are consistent with data reported by hofmann et al. (2004) , simmons et al. (2004) and broer et al. (2006) , who employed the pcagg or phcmv vectors to achieve detectable s expression. both pcagg and phcmv vectors also harbor donor and acceptor splice sites downstream of a polymerase ii promoter. altogether, these data indicate that cytoplasmic accumulation of s mrna, hence s protein expression, are absolutely dependent on splicing. most genes in higher eucaryotes contain at least one intron and many studies have clearly shown that constitutively spliced introns or the presence of a heterologous intron of either viral or cellular origin may be required for optimal gene expression in a variety of systems, including transient expression in mammalian tissue culture cells. the importance of splicing for expression of a given gene appears to depend on several factors such as intron and exon identity and cell type (buchman and berg, 1988; petitclerc et al., 1995) . albeit much less documented, the expression of intronless genes of bacterial and viral origin can also be enhanced by including introns within mammalian vectors (huang and gorman, 1990; huang and yen, 1995) . although it has been suggested that introns alter the nucleocytoplasmic distribution of mrnas, more recent data indicate that splicing rather enhances both mrna production and translational efficiency (lu and cullen, 2003; nott et al., 2003) . our results extend these observations and indicate that the nuclear expression of naturally intronless genes derived from rna viruses that replicate in the cell cytoplasm may follow the same requirements (presence of an intron) for efficient mrna accumulation in the cytoplasm. although cis-acting viral export elements could compensate for the lack of an intron in the expression of the human β-globin cdna from a retroviral vector , cte and wpre did not permit efficient expression of the sars-cov s gene from the pcdna vector and both s mrna and protein remained undetectable. however, a remarkable boost in s expression was observed when cte or wpre was combined with an intron within the pci vector (figs. 2 and 3) . interestingly, we did not observe any effect of cte at the level of s mrna production in 293t and veroe6 cells. this was unexpected because cte-enhanced expression has been linked to cte-mediated export of mrnas, conferring rev-independence to hiv-1 gag expression (bray et al., 1994; and contributing to overcome inhibitory elements of hpv-16 l1 mrna (tan et al., 1995) . our results rather support the hypothesis raised by schambach et al. (2000) , according to which cte is much more efficient in promoting export of unspliced intron-containing retroviral rna transcripts (ernst et al., 1997a; gruter et al., 1998; kang and cullen, 1999) , than in facilitating the transport of spliced mrna. in contrast, wpre enhanced cytoplasmic s mrna levels by 3-fold in both 293t and veroe6 cells. both cte and wpre have an export function, but they differ by the mechanisms involved in that cte binds to the tap nuclear mrna export factor (gruter et al., 1998) , while wpre recruits the cellular crm1 rna export factor (popa et al., 2002) . the observation that tap is also implicated in the export of cellular spliced mrnas from the nucleus suggests that cte rna and spliced mrnas share a common nuclear export pathway (braun et al., 1999) . this may explain the absence of synergy between intron and cte in enhancing the cytoplasmic levels of spliced s mrna in both 293t and veroe6 cells (fig. 4) . in contrast, wpre rna and spliced mrnas use different factors for nuclear export and our data indicate that these may act synergistically to enhance the cytoplasmic expression of an intron-containing wpre rna (fig. 4) . a similar observation was made by schambach et al., who reported that elevated expression of the human multidrug resistance gene (mdr1) mrna in sw480 cells depends on the presence of both an intron and wpre . the effect of cte and wpre was more pronounced on s protein levels than on s cytoplasmic mrnas levels (compare fig. 3 and table 1 to fig. 4) , indicating that, in addition to their initially recognized role in mrna export, cte and wpre also act by promoting efficient mrna translation. such a role for cte in mrna translation has been recently suggested by the groups of boris-lawrie and hammarskjöld (coyle et al., 2003; hull and boris-lawrie, 2003; jin et al., 2003) . in contrast, it is not in full agreement with the common view that wpre exerts its effect on protein expression mainly by enhancing 3′ mrna processing and nucleocytoplasmic transport (donello et al., 1998; loeb et al., 1999; zufferey et al., 1999) . to our knowledge, a single report suggested that wpre may contribute to enhanced cytoplasmic utilization of an intronless egfp mrna in addition to its effect on nuclear rna processing and rna export . interestingly, the closely related hepatitis b virus pre has also been reported to enhance translation of the rna harboring this element (lu and cullen, 2003) . in conclusion to our in vitro studies, we demonstrated that the marked enhancing effect of wpre on s protein expression from spliced s transcripts is due to the synergistic effect of increased levels of cytoplasmic messenger rna and increased cytoplasmic utilization of these mrnas. our data provide a likely explanation to the observations of wang et al. (2003) who reported that wpre improved the production of the e2 protein of the type 2 bovine diarrhea virus (a cytoplasmic rna virus) when expressed in transiently transfected cells or from a recombinant herpesvirus. our results have important implications for the construction of plasmid and viral vectors optimally designed for the expression of intronless genes from cytoplasmic rna viruses. successful expression of full-length s protein in mammalian cells was alternatively achieved by others by using codonoptimized synthetic genes qin et al., 2004; zhi et al., 2005) . codon-usage adaptation most often involves the substitution of nearly every wobble position for a g or c nucleotide (nakamura, october 2006, posting date; nakamura et al., 2000) , resulting in genes with a much diverse nucleotide composition and decreased a/t content. wild-type genes from viruses with a nuclear life cycle such as hiv-1 or hpv-16 contain regulatory elements evolved to inhibit mrna cytoplasmic accumulation. for hiv-1 gag, vpu, vif (graf et al., 2000; kotsopoulou et al., 2000; nguyen et al., 2004) or hpv-16 l1 and l2 proteins (collier et al., 2002; oberg et al., 2003) , codon optimization of corresponding genes has been shown to result in the inactivation of such elements, thus leading to increased viral protein expression from nuclear dna expression vectors. cytoplasmic viruses, such as sars-cov, may contain cryptic splice sites, mrna instability motifs such as a/ u-rich elements (ares), or putative cis-active inhibitory sequences, which may explain why dna vector-based expression of genes from these viruses is dependent on the presence of heterologous intron and transport elements. it can be hypothesized that codon-usage adaptation is a successful strategy to express high levels of cytoplasmic virus proteins as the result of removal of these yet uncharacterized inhibitory elements. in s-based dna vaccination experiments, we found that the presence of an intron was mandatory to induce high titers of protective neutralizing antibodies in immunized mice (data not shown). this is in agreement with the absence of s polypeptide expression in cultured cells from the pcdna-s plasmid (figs. 1-3) and with the weak antibody responses observed by others in mice immunized with pcdna-based s constructs (wang et al., 2005b) . in addition, among published reports describing full-length-s-based dna vaccines, only those based on a codon-optimized gene proved capable of inducing high titers of neutralizing antibodies in mice yi et al., 2005) or rabbits (wang et al., 2005a) . it is noteworthy that similar observations were reported recently for replication-defective human adenoviral vectors containing the wild-type s gene under the control of the cmv promoter; such recombinant vectors failed to induce cellular immune responses against spike epitopes, whereas vectors encoding codon-optimized s protein proved to be potent immunogens (zhi et al., 2005) . since expression from adenoviral vectors relies on nuclear transcription of the foreign gene, this corroborates our hypothesis that wild-type s mrna does not accumulate in the cytoplasm in the absence of a heterologous intron. in the present study, we demonstrated that a combination of an intron and wpre, but not of an intron and cte, acted synergistically in enhancing the immunogenicity of an s-based anti-sars dna vaccine in balb/c mice. we showed that 10 μg of pci-s-wpre induced a sars-cov neutralizing antibody response (log 10 titer = 2.2 ± 0.2) similar to that induced by 25 μg of a codon-optimized expression vector, as described by yang et al. (2004) . moreover, our in vivo data clearly indicate that immunization with the wpre-enhanced vaccine induced complete protective immunity against challenge infection with sars-cov at doses as low as 2 μg at which the more conventional dna vaccine (including intron sequence but lacking wpre) did not (fig. 8) . to our knowledge, this is the first report of a successful dna vaccination protocol using the full-length, wild-type s gene of sars-cov, without codon optimization. whether wpre could further enhance the protective efficacy of a dna vaccine based on a codonoptimized gene of the sars-cov s protein, such as that reported by yang et al., is an interesting question which would deserve additional studies. it has been reported recently that the presence of wpre also markedly enhanced the efficacy of two other dna vaccines, a viral vaccine based on ha expression in a mouse model of influenza (garg et al., 2004) , as well as a tumor vaccine in a mouse model of neuroblastoma metastases (pertl et al., 2003) . the three studies with the sars (our study), influenza (garg et al., 2004) and neuroblastoma (pertl et al., 2003) models show remarkable complementarity in demonstrating that wpre enhanced the protective efficacy of dna vaccines encoding antigens from the fully-spliced cdna of a tumor gene and two naturally intronless genes, one derived from a nucleusreplicating negative strand rna virus (influenza virus) and the other from a cytoplasm-replicating positive strand rna virus (sars-cov). the mechanisms involved in the enhanced induction of protective immunity remain stricto sensu to be investigated but wpre very likely improves the level of antigen expression in vivo, as it does in vitro in transient transfection experiments. in summary, our data provide valuable information with respect to the improvement of s protein expression from the wild-type sars-cov gene by using both an intron and the wpre post-transcriptional enhancer, and resulting improvement of the immunogenicity of an s-based dna vaccine. from a practical point of view, this may help decrease the amount of plasmid dna in immunization protocols. as noted by others (garg et al., 2004) , by lowering the amount of dna used in immunization protocols, concerns raised by the use of dna vaccines, such as risks posed by integration events into the host genome or the induction of autoimmune responses (cui, 2005) , are minimized. moreover, one of the principal limitations to dna vaccine efficacy in large animals, like nonhuman primates, may be related to the limited distribution of dna within the injected tissue and limited uptake to the nucleus following injection of dna vaccine in a relatively small volume (dupuis et al., 2000; otten et al., 2004) . these limitations may be circumvented by the use of efficient dna vectors based on a combination of intron and wpre enhancer, such as those we describe here. thus, these improved plasmid vectors might possibly contribute to the development of a safe and efficient dna vaccine against sars in humans. further studies in larger animals, such as cynomolgus macaques or african green monkeys, which were recently described as suitable animal models for sars lawler et al., 2006; mcauliffe et al., 2004; rowe et al., 2004) could help validate the intron and wpre based vectors. frhk-4 (fetal rhesus monkey kidney), cv-1 and vero e6 (african green monkey kidney) cells were grown at 37°c under 5% co 2 in complete dmem [dulbecco's modified eagle medium with 4.5 mg/ml l-glucose, 100 u/ml penicillin and 100 μg/ml streptomycin], supplemented with 5% heatinactivated fetal calf serum (fcs) (dmem-5). 293t (human kidney) cells were grown in complete dmem supplemented with 10% fcs (dmem-10). sars-cov ffm-1 strain , was kindly provided by h.w. doerr (institute of medical virology, frankfurt university medical school, germany). viral stocks were produced at 35°c by passage on vero e6 cell cultures at a multiplicity of infection (m.o.i.) of 0.01 in complete dmem with 2% fcs. all viral stocks were stored at − 80°c and titrated in a standard limiting dilution assay on vero e6 cell monolayers in 96-well microtiter plates. infectious titers were determined as 50% tissue culture infective doses (tcid50) according to reed and muench (reed and muench, 1938) . all work involving infectious sars-cov was performed in an enhanced biosafety level 3 containment laboratory with rigorous safety procedures according to who guidelines. vv-tf7.3 is a recombinant vaccinia virus encoding the t7 rna polymerase (fuerst et al., 1986) and was provided by b. moss (national institute of health, bethesda, u.s.a.). vv-tf7.3 was propagated at 35°c by passage on cv-1 monolayers at an moi of 0.1 and titrated by a standard plaque assay. the s gene of sars-cov was obtained directly from the viral rna extracted with the qiaamp viral rna mini kit (qiagen) from the broncho-alveolar lavage (#031589 specimen) of a patient hospitalized with a diagnosis of probable sars at the hanoï french hospital, vietnam. briefly, after reverse transcription of the rna, overlapping s cdna fragments were produced by nested pcr and the cdna fragment representing the complete s gene sequence (nt 21406-25348) was assembled from clones harboring the consensus protein sequence as deduced by direct sequencing of the amplicons from specimen #031589 (nal et al., 2005) . the resulting plasmid psars-s was used as the source of s cdna for subsequent cloning into mammalian expression vectors. comparison of its sequence with the s sequences of the tor2 (genbank accession no. ay274119.3) and urbani (genbank accession no. ay278741.1) isolates revealed one amino-acid difference at position 577: alanine for tor2 and serine for urbani and 031589. the dna sequence coding for the s protein was amplified by pcr with the pwo polymerase (roche) using psars-s plasmid as a template and oligonucleotides 5′-ataggatcca ccatgtttat tttcttatta tttcttactc tcact-3′ and 5′-atactcgagt tatgtg-taat gtaatttgac acccttg-3′ containing bamhi and xhoi restrictions sites. after digestion with bamhi and xhoi, the resulting dna fragment was inserted at the corresponding sites of the pcdna3.1(+) vector (invitrogen), yielding plasmid pcdna-s. next, the s insert was subcloned between the nhei and xhoi sites of the pci plasmid (promega) , yielding plasmid pci-s. plasmids containing the constitutive transport element of mazon-pfizer monkey virus (cte) (ernst et al., 1997b) or the post-regulatory element of the woodchuck hepatitis virus (wpre) (donello et al., 1998) were kindly provided by y. jacob and p. charneau respectively (institut pasteur, paris, france). the cte fragment was obtained by digestion of the relevant plasmid with xhoi and bamhi. the wpre fragment was obtained by digestion of the relevant plasmid with xhoi and kpni. these fragments were then inserted downstream the s orf sequence between the xhoi and xbai sites either in pcdna-s or in pci-s plasmids, yielding plasmids pcdna-s-cte, pcdna-s-wpre, pci-s-cte and pci-s-wpre respectively. all constructs' inserts were verified by the sequencing of positive clones using a big dye terminator sequencing kit and an automated sequencer (applied biosystems). the cdna encoding amino acids 475 to 1193 of the s protein was amplified by pcr using the s plasmid as a template and oligonucleotides 5′-cccatatgag tgaccttgac cggtgcacca c-3′ and 5′-cccccgggtt taatatattg ctcatatttt ccc-3′ containing ndei and xmai restriction sites. after digestion with ndei and xmai, the resulting dna fragment was inserted at the corresponding sites of the bacterial expression vector pivex2.4d (roche), yielding plasmid piv2.4s c . the final recombinant fragment of s (s c ) contained an extraneous n-terminal polyhistidine tag. the construct's insert was confirmed by dna sequencing using a big dye terminator sequencing kit and an automated sequencer (applied biosystems). exponentially growing cultures of escherichia coli bl21(de3)pdia17 cells harboring the piv2.4s c expression construct were induced to synthesize the s c polypeptide by addition of 1 mm isopropyl-β-d-thiogalactoside. cells were allowed to grow for 2 h at 30°c. under these conditions, the s c recombinant polypeptide was recovered as inclusion bodies: cells were harvested and lysed in lysis buffer (0.1 m tris-hcl ph 7.5, 1 mm edta) using a french press at 1200 psi. inclusion bodies in the cell lysate were pelleted by centrifugation at 20,000×g for 15 min and washed in lysis buffer supplemented with 2% triton x100 and 10 mm β-mercaptoethanol and then in 10 mm tris-hcl, 7 m urea, ph 7.5 buffer for 30 min under gentle shaking, before final resuspension in 10 mm tris-hcl, ph 7.5. the s c protein was obtained at a purity of more than 90% as determined by sds-page and coomassie blue staining. new zealand white rabbits were immunized by intradermal inoculation with 0.5 mg of recombinant s c polypeptide emulsified in incomplete freund's adjuvant (sigma) and boosted twice at 3 to 4-week intervals. this was followed 3 weeks later by another injection of recombinant s c polypeptide, which had been further purified on a sucrose gradient and washed with 2% triton x100. immune sera were collected 5 weeks after the last boost. the antibodies reacted specifically with the s protein expressed in sars-cov infected veroe6 cells. anti-s antibodies were also raised in mice against the ectodomain of the s protein, expressed in mammalian cells as a soluble polypeptide (b. callendret, v. lorin, p. charneau, s. van der werf and n. escriou, unpublished data). the first serum sample (sample #033168) was collected 38 days after the onset of symptoms from patient a with probable sars, according to the who case definition (who, october 2004, posting date) . the second set of sera consisted of one pair of samples that was collected from patient b with probable sars, 8 days (sample #032703) and 29 days (sample #033153) after the onset of symptoms. for both patients a and b, respiratory tract specimens were tested positive for the sars-l gene by nested rt-pcr . as a control, serum samples collected before the 2002 sars epidemic were obtained from healthy blood donors (#tv262 and #tv263). all serum samples were inactivated by two cycles of heating for 30 min at 56°c and clarified by centrifugation for 5 min at 15,000×g. igg antibodies against the sars-cov were present in both the #033168 and the #033153 serum samples as assayed by indirect elisa using crude lysates from sars-cov infected cells as the coating antigen (see below), and there was no non specific binding of the #tv262 and #tv263 sera to sars-cov antigens. analysis of s protein expression in transiently transfected cells 10 6 vero e6 cells or 1.4 × 10 6 293t cells in 35 mm petri dishes under 2 ml of dmem-5 or dmem-10 culture medium respectively were transfected with 2 μg of plasmid dna and 6 μl of fugene6 reagent (roche), according to manufacturer's instructions and incubated at 37°c under 5% co 2 . medium was replaced 24 h later and s expression analyzed at the indicated time by western blot, cytofluorometry or immunofluorescence assays. alternatively, vero e6 cells (10 6 in 35 mm petri dishes) were infected at an moi of 5 with vv-tf7.3 and transfected 1 h later with 2 μg of either pcdna-s or pcdna plasmid dna as described above. vero e6 cells grown in 12 cm 2 flasks were infected with sars-cov ffm-1 strain at an moi of 2 and incubated in complete dmem supplemented with 2% fcs at 35°c under 5% co 2 . total cell lysates were prepared 18 h after vv-tf7.3 infection or 24 h after sars-cov infection, and s expression was analyzed by western blot. for western blot assays, total cell extracts were harvested 48 h post-transfection in 300 μl of laemmli sample buffer. samples were denatured by heating at 95°c for 10 min and sonicated. proteins were separated by electrophoresis on 8% sds-polyacrylamide (prosieve 50, cambrex) gels, and transferred onto a pvdf membrane (amersham). the membrane was incubated with pbs-0.1% tween-5% unfatted milk for 1 h at room temperature prior to immunoblotting with rabbit anti-s polyclonal antibodies. following incubation with peroxydase conjugated anti-rabbit secondary antibody (na934v, amersham) the immunoblot was revealed with enhanced chemiluminescence (ecl+, amersham), according to the manufacturer's instructions and by autoradiography on hyperfilm (amersham). alternatively, the luminescence signals were captured with a cooled ccd camera (fluor-s multimager, bio-rad). protein expression levels were quantified from gel images using the quantity one software (v 4.2.3, bio-rad). veroe6 and 293t cells grown in 35-mm dishes were transfected as described above. at 24 or 48 h post-transfection, cell monolayers were washed twice in d-pbs (invitrogen) and cellular rnas were prepared. total cellular rnas were isolated by trizol-ls extraction (invitrogen) according to the manufacturer's recommendations. for cytoplasmic rna extraction, cells were lysed on ice in 10 mm tris-hcl ph 8.0, 0.14 m nacl, 1.5 mm mgcl 2 , 1 mm dtt, 0.5% np-40. after 10 min of incubation at 4°c, samples were centrifugated at 10,000×g for 2 min, and the rnas were purified from the supernatant by phenol/chloroform extraction and isopropanol precipitation. the rnas were treated with dnase i using the dna-free kit (ambion) according to the manufacturer's recommendations and stored at − 80°c until use. after denaturation at 70°c in 50% formamide, 2.2 m formaldehyde, 1× running buffer [40 mm mops, 10 mm sodium acetate, 2 mm edta, ph 7.0], samples (2 μg) were run on a 1.2% agarose-0.6 m formaldehyde gel, blotted onto a nylon membrane (hybond n, amersham), and fixed by uv irradiation. the membranes were hybridized with a mixture of 3 negative sense s-specific 32 p-labeled riboprobes corresponding to nt 21492-22830, 22830-24374 and 24374-25256 of the sars-cov genome. for normalization, the membranes were hybridized with a β-actin-specific riboprobe. hybridizations were performed at 65°c in a 50% formamide, 5× ssc, 5× denhardt, 0.5% sds solution. the membranes were washed 3 times in 2× ssc, 0.1% sds at room temperature and another 3 times in 0.1× ssc, 0.1% sds at 70°c. finally, the membranes were exposed on a storm 820 phosphorimager (molecular dynamics) and analyzed using the image quant program (v 1.2, molecular dynamics). for cytofluorometry assays, subconfluent monolayers of veroe6 cells and 293t cells were transfected as described above. 48 h post-transfection, cells were washed once in d-pbs and detached with 1 ml of cell dissociation solution (sigma). cells were stained with polyclonal mouse anti-s antibody for 45 min at 4°c and, after washing, incubated with secondary antibody (anti-mouse fitc-conjugated, bd bioscience pharmingen) for 45 min at 4°c. cells were then washed and fixed with pbs-1% paraformaldehyde. events were acquired on a facscalibur fluorocytometer (beckton dickinson) and analyzed with the bd cellquest pro software (v 5.2, beckton dickinson). for indirect immunofluorescence assays, subconfluent monolayers of veroe6 cells were transfected as described above. 24 h post transfection, cells were dissociated with 1 ml of cell dissociation solution, plated on 20 mm glass coverslips in a 12 well-plate and incubated in dmem-5 at 37°c under 5% co 2 . 24 h later, cells were washed in d-pbs and fixed with pbs-4% paraformaldehyde for 15 min. coverslips were then incubated for 45 min with patient serum diluted 1/300 in pbs-2% bsa. after subsequent incubation with a fitc-conjugated anti-human igg secondary antibody (jackson immunoresearch), the samples were mounted on slides with dapicontaining vectashield (vector laboratory) and analyzed under an axioplan 2 epifluorescence microscope (zeiss). pictures were acquired with an axiocam mrm camera and processed with the axiovision software (v 4.2, zeiss). plasmid pcdna3-ace2 encoding the ace2 molecule, which is a receptor for sars-cov, was obtained from michael r. farzan . 293t cells, constitutively expressing green fluorescent protein (293t-gfp) were obtained by transduction of 293t cells with the trip-gfp lentiviral vector (zennou et al., 2000) . 293t-gfp effector cells were transfected either with plasmids encoding the s glycoprotein or the pci plasmid as a control, as described above. in parallel, 293t target cells were co-transfected with 3 μg of dsred plasmid dna and 3 μg of either pcdna or pcdna-ace2 plasmid dna using 10 μl fugene6 transfection reagent. medium was replaced 24 h after transfection. effector and target cells were detached 12 h later with cell dissociation solution, resuspended in fresh dmem-10 and counted. 6 × 10 5 per well of each target and effector cells were plated together in a 12 well-plate with 1 ml of dmem-10 in each well and co-cultured for 12 h at 37°c under 5% co 2 . the cells were then trypsinized, fixed with pbs-1% paraformaldehyde and analyzed on a facscalibur fluorocytometer. fusion was measured as the ratio between double-stained and red target cells using the bd cellquest pro software. female balb/c mice (cerj) 7 to 8 weeks of age were housed and handled according to the pasteur institute guidelines in compliance with european animal welfare regulations. mice were injected intramuscularly with 100 μl pbs (50 μl in each tibialis anterior muscle) containing plasmid dna. mice were injected with either 50 μg of pci plasmid dna as a control, or variable amounts (2, 10 or 50 μg) of pci-s, pci-s-cte or pci-s-wpre plasmid dna. booster injections were administered at 3 or 4-week intervals. dna used for injection was prepared using the endofree plasmid mega kit (qiagen) and tested for the absence of endotoxin (< 5 eu/mg), as measured with the qcl-1000 endotoxin kit (biowhittaker). blood from mice was collected 1 week before immunization and 3 weeks after each injection. the induction of sars-cov specific antibodies in immunized mice was measured by indirect elisa and neutralization assay. for elisa, microtiter plates were coated with sars-cov infected or mock-infected vero e6 crude cell lysates, which had been prepared in a 50 mm boric acid, 120 mm nacl, 1% triton x-100 solution at ph 9.0, sonicated in a cup-horn tip, clarified by centrifugation at 15,000×g for 10 min and inactivated frozen by gamma irradiation with 50 kgy from a 60 co source. after washing in pbs-0.1% tween, plates were incubated with three-fold serial dilutions of mouse sera in pbs-tween-3% dry skimmed milk (w/v) starting at 1:16.7 or 1:50. bound antibodies were revealed with horseradish peroxidaseconjugated anti-mouse igg secondary antibody (amersham) and tmb (3,3′-5,5′-tetramethylbenzidine, kpl). the absorbance was measured at 450 nm (reference wavelength: 620 nm). readings from wells coated with mock lysates were substracted from wells with sars-cov lysates and sars-cov specific igg titers were calculated as the reciprocal of the highest dilution of individual serum, giving an absorbance difference of 0.5. for neutralization assays, two-fold serial dilutions of heatinactivated (56°c, 30 min) serum samples were mixed with 100 tcid50 of sars-cov in 100 μl complete dmem, incubated at 37°c for 60 min and added to a subconfluent monolayer of frhk-4 cells in a 96-well microtiter plate. each dilution of serum as well as positive control (100 tcid50 of sars-cov) and negative cell controls were tested in quadruplicate and cytopathic effect (cpe) endpoints were read up to 7 days after inoculation. neutralizing antibody titers were determined according to the reed and muench method (reed and muench, 1938) as the reciprocal of the highest dilution of each serum, which suppressed cpe in at least 2 out of 4 wells. eight weeks after the third plasmid dna injection, mice were lightly anaesthetized with isoflurane (mundipharma) and inoculated intranasally with 10 5 pfu of sars-cov in 40 μl pbs. mice were euthanized 48 h after challenge infection and whole lungs crushed in 500 μl dmem supplemented with 2% fcs using glass beads and a mm200 mixer mill (retsch). after clarification by low-speed centrifugation for 5 min, lung homogenates were titrated for infectious virus on veroe6 cells monolayers in a standard plaque assay. challenge infection of mice and subsequent analysis were done in the "jean merieux" biosafety level 4 containment laboratory. three mice died before transfer to the bsl-4 facility, thus could not be challenged. statistical analysis was performed on the log10 of the viral titers measured for individual mice using the student's independent t-test, with the assumptions used for small samples (normal distribution of the variables and same variance for the populations to be compared). loth and sandra lacôte for expert technical assistance in performing the sars-cov challenge experiment. the assistance of the staff of the who collaborative center for reference and research on influenza and other respiratory viruses (institut pasteur, paris, france) in initial analysis of the sars specimens and sera is gratefully acknowledged. this work was supported in part by grants from glaxo-smithkline biologicals and bio-rad laboratories. b.c. was supported by a fellowship from glaxosmithkline biologicals. severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice neutralizing antibody and protective immunity to sars coronavirus infection of mice induced by a soluble recombinant polypeptide containing an n-terminal segment of the spike glycoprotein tap binds to the constitutive transport element (cte) through a novel rna-binding motif that is sufficient to promote cte-dependent rna export from the nucleus a small element from the mason-pfizer monkey virus genome makes human immunodeficiency virus type 1 expression and replication rev-independent important role for the transmembrane domain of severe acute respiratory syndrome coronavirus spike 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papillomavirus type 16 l1 open reading frame results in production of high levels of l1 protein in human epithelial cells sam68 enhances the cytoplasmic utilization of intron-containing rna and is functionally regulated by the nuclear kinase sik/brk dna vaccine woodchuck hepatitis virus contains a tripartite posttranscriptional regulatory element identification of a novel coronavirus in patients with severe acute respiratory syndrome distribution of dna vaccines determines their immunogenicity after intramuscular injection in mice a structured retroviral rna element that mediates nucleocytoplasmic export of introncontaining rna secondary structure and mutational analysis of the mason-pfizer monkey virus rna constitutive transport element aetiology: koch's postulates fulfilled for sars virus dose dependence of ctl precursor frequency induced by a dna vaccine and correlation with protective immunity against influenza virus challenge eukaryotic transientexpression system based on recombinant vaccinia virus that synthesizes bacteriophage t7 rna polymerase the hybrid cytomegalovirus enhancer/chicken beta-actin promoter along with woodchuck hepatitis virus posttranscriptional regulatory element enhances the protective efficacy of dna vaccines retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus s protein concerted action of multiple cis-acting sequences is required for rev dependence of late human immunodeficiency virus type 1 gene expression tap, the human homolog of mex67p, mediates cte-dependent rna export from the nucleus isolation and characterization of viruses related to the sars coronavirus from animals in southern china switching species tropism: an effective way to manipulate the feline coronavirus genome identification of a critical neutralization determinant of severe acute respiratory syndrome (sars)-associated coronavirus: importance for designing sars vaccines s protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients cloning, sequencing and expression of the s protein gene from two geographically distinct strains of canine coronavirus intervening sequences increase efficiency of rna 3′ processing and accumulation of cytoplasmic rna role of the hepatitis b virus posttranscriptional regulatory element in export of intronless transcripts generation of synthetic severe acute respiratory syndrome coronavirus pseudoparticles: implications for assembly and vaccine production analysis of synergy between divergent simple retrovirus posttranscriptional control elements cd209l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus tap and nxt promote translation of unspliced mrna molecular evolution analysis and geographic investigation of severe acute respiratory syndrome coronavirus-like virus in palm civets at an animal market and on farms the human tap protein is a nuclear mrna export factor that contains novel rna-binding and nucleocytoplasmic transport sequences a rev-independent human immunodeficiency virus type 1 (hiv-1)-based vector that exploits a codon-optimized hiv-1 gag-pol gene variations in disparate regions of the murine coronavirus spike protein impact the initiation of membrane fusion a novel coronavirus associated with severe acute respiratory syndrome retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats cynomolgus macaque as an animal model for severe acute respiratory syndrome angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus bats are natural reservoirs of sars-like coronaviruses enhanced expression of transgenes from adeno-associated virus vectors with the woodchuck hepatitis virus posttranscriptional regulatory element: implications for gene therapy analysis of the stimulatory effect of splicing on mrna production and utilization in mammalian cells protease-mediated enhancement of severe acute respiratory syndrome coronavirus infection replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys homo sapiens codon usage table codon usage tabulated from international dna sequence databases: status for the year 2000 differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins nucleocapsidindependent specific viral rna packaging via viral envelope protein and viral rna signal codon optimization of the hiv-1 vpu and vif genes stabilizes their mrna and allows for highly efficient rev-independent expression neutralizing antibodies in patients with severe acute respiratory syndromeassociated coronavirus infection a quantitative analysis of intron effects on mammalian gene expression mutational inactivation of two distinct negative rna elements in the human papillomavirus type 16 l2 coding region induces production of high levels of l2 in human cells enhancement of dna vaccine potency in rhesus macaques by electroporation immunotherapy with a posttranscriptionally modified dna vaccine induces complete protection against metastatic neuroblastoma genetic analysis of the sarscoronavirus spike glycoprotein functional domains involved in cell-surface expression and cell-to-cell fusion the effect of various introns and transcription terminators on the efficiency of expression vectors in various cultured cell lines and in the mammary gland of transgenic mice crm1-dependent function of a cis-acting rna export element revision date. pci and psi mammalian expression vectors. technical bulletin intracellular processing of the porcine coronavirus transmissible gastroenteritis virus spike protein expressed by recombinant vaccinia virus silencing of sars-cov spike gene by small interfering rna in hek 293t cells identification of two critical amino acid residues of the severe acute respiratory syndrome coronavirus spike protein for its variation in zoonotic tropism transition via a double substitution strategy a simple method of estimating fifty percent endpoints role of mason-pfizer monkey virus (mpmv) constitutive transport element (cte) in the propagation of mpmv vectors by genetic complementation using homologous/heterologous env genes macaque model for severe acute respiratory syndrome context dependence of different modules for posttranscriptional enhancement of gene expression from retroviral vectors a novel sorting signal for intracellular localization is present in the s protein of a porcine coronavirus but absent from severe acute respiratory syndrome-associated coronavirus characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry 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assembly of coronavirus-like particles by co-expression of viral envelope protein genes enhanced type i immune response to a hepatitis b dna vaccine by formulation with calcium-or aluminum phosphate a hepadnavirus regulatory element enhances expression of a type 2 bovine viral diarrhea virus e2 protein from a bovine herpesvirus 1 vector identification of two neutralizing regions on the severe acute respiratory syndrome coronavirus spike glycoprotein produced from the mammalian expression system immune responses with dna vaccines encoded different gene fragments of severe acute respiratory syndrome coronavirus in balb/c mice summary of probable sars cases with onset of illness from 1 posting date. who guidelines for the global surveillance of severe acute respiratory syndrome (sars) multiple copies of the mason-pfizer monkey virus constitutive rna transport element lead to enhanced hiv-1 gag expression in a context-dependent manner civets are equally susceptible to experimental infection by two different severe acute respiratory syndrome coronavirus isolates the sars-cov s glycoprotein: expression and functional characterization a dna vaccine induces sars coronavirus neutralization and protective immunity in mice evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses single amino acid substitutions in the severe acute respiratory syndrome coronavirus spike glycoprotein determine viral entry and immunogenicity of a major neutralizing domain a single amino acid change within antigenic domain ii of the spike protein of bovine coronavirus confers resistance to virus neutralization hiv-1 genome nuclear import is mediated by a central dna flap identification of murine cd8 t cell epitopes in codon-optimized sars-associated coronavirus spike protein woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors we thank pr. dr. h.w. doerr (institute of medical virology, frankfurt university medical school, germany) for providing us with the sars-cov ffm-1 strain and dr. m.r. farzan (department of medicine, harvard medical school, boston, ma) for providing us with plasmid pcdna3-ace2. we also thank drs. annette martin and nadia naffakh for critical reading of the manuscript and helpful suggestions, philippe key: cord-296416-q0rsfzgw authors: lavi, ehud; wang, qian; weiss, susan r.; gonatas, nicholas k. title: syncytia formation induced by coronavirus infection is associated with fragmentation and rearrangement of the golgi apparatus date: 1996-07-15 journal: virology doi: 10.1006/viro.1996.0382 sha: doc_id: 296416 cord_uid: q0rsfzgw abstract coronavirus mouse hepatitis virus (mhv) possesses a membrane glycoprotein (m) which is targeted to the golgi apparatus (ga). we used immunocytochemistry with an organelle-specific antiserum to investigate the morphologic changes of the ga during infection of l2 murine fibroblasts with mhv-a59. twenty-four hours after infection the ga was fragmented and translocated in the center of syncytia, while the microtubular network was also rearranged displaying radiating elements toward the center of syncytia. two fusion-defective mutants, which contain an identical amino acid substitution in the cleavage signal sequence of the spike glycoprotein (s), induced fragmentation of the ga. however, the ga migrated only partially to the centers of syncytia during infection with these mutants. revertant viruses, in which the above mutation was corrected, had fusion properties and ga staining similar to wtmhv-a59. experiments with brefeldin a (bfa), which induces redistribution of the ga into the rough endoplasmic reticulum (rer), revealed that an intact ga for a period of 4–16 hr postinfection, is required for coronavirus replication and syncytia formation. thus, during mhv infection, syncytia formation is associated with fragmentation of the ga, followed by a previously undescribed phenomenon of migration of the organelle into the centers of syncytia. the fragmentation of the ga, however, may occur without the formation of syncytia. therefore, two distinct mechanisms may be responsible for the fragmentation of the ga and its subsequent migration to the center of syncytia. undergo endocytosis into the trans-golgi-network (tgn), while shiga toxin is internalized into the ga and the rer polypeptides synthesized in the rough endoplasmic (sandvig et al., 1991; . pretreatment of cells reticulum (rer) and destined for plasma membranes, with bfa protects them against the lethal effects of cerlysosomes, and secretion are transported through the tain toxins, implying that the toxin's entry into tgn and golgi apparatus for posttranslational modifications and the ga is necessary for translocation into their cytosolic targeting (farquhar and palade, 1981; mellman and sitargets (yoshida et al., 1991) . in the human disease mons, 1992; rothman and orci, 1992) . the ga is a dyamyotrophic lateral sclerosis (als), the ga of spinal cord namic organelle which undergoes morphologic and funcmotor neurons is fragmented into numerous small eletional modifications under physiologic and pathologic ments which resemble the dispersion of the organelle conditions. in mitotic hela cells the ga disperses in early induced by agents depolymerizing microtubules (moureprophase and reaggregates in telophase (robbins and latos et al., 1990, 1993; gonatas et al., 1992) . a similar gonatas, 1964) . during mitosis, the golgi apparatus fragfragmentation of the ga has been observed in motor ments into numerous small groups of vesicles which neurons of transgenic mice expressing a mutant cu,zn have been referred to as the mitotic form of the organelle superoxide dismutase (mourelatos et al., 1996) . (lucocq et al., 1987) . in interphase cells treated with migration and rearrangements of the ga have been drugs which depolymerize microtubules, the ga fragreported during syncytia formation and fusion of vero ments into small randomly distributed elements (robbins cells infected with sindbis virus (ho et al., 1990) . within and gonatas, 1964; turner and tartakoff, 1989) . in many 3-5 hr after infection, individual elements of the ga, cells the secretion blocker brefeldin a (bfa) reversibly which are associated initially with separate microtubuleredistributes membranes and enzymes of the ga back organizing centers in perinuclear areas of fused cells, into the rer, but does not inhibit endocytosis (doms et congregate in the center of syncytia and form an exal., 1989; lippincott-schwartz et al., 1989; johnston et al., tended network of nondisrupted intact golgi complexes 1994). ricin, cholera toxin, and wheat germ agglutinin (ho et al., 1990) . in contrast to sindbis infections, the golgi apparatus is fragmented in cells infected with herpes simplex virus 1 (campadelli et al., 1993) . is characterized by retention of the viral envelope glyco-1991). the primary structure of mg-160 revealed significant homology with a chicken fibroblast growth factor protein m (previously known as e1) within the ga (tooze et al., 1988; klumperman et al., 1994; krijnse-locker et receptor and a ligand to e-selectin (gonatas et al., 1995; steegmaier et al., 1995) . in chicken, mg-160 appears al., 1994) and budding of virions from internal membranes (holmes and behnke, 1981; tooze et al., 1988) . the close early in development and the gene coding the protein, named glg1, has been assigned to chromosome 16 interaction between coronavirus particles with the ga provides an unusual opportunity to study morphologic (stieber et al., 1995; mourelatos et al., 1995) . and functional properties of the ga and various aspects of virus-cell interactions. in this study, we used organ-immunohistochemistry elle-specific antibodies, immunohistochemistry, and transmission electron microscopy to examine the fate of cells, grown on poly-d-lysine-treated coverslips, were the ga during cell fusion and syncytia formation in mouse fixed with 2% paraformaldehyde for 20 min at room tem-l-2 cells infected with mhv-a59 and fusion defective perature, washed three times in pbs, then incubated for mutants. fragmentation of the ga into small immuno-30 min in 0.05% saponin/10% goat serum (gs) in pbs stained elements occurred prior to their migration into and washed three times in 10% gs in pbs. cells were the centers of syncytia. experiments with bfa revealed then incubated with primary antibody (1:1000 dilution in that the period of 4-16 hr postinfection is critical for pbs of immunoaffinity-purified rabbit anti-mg-160 anticoronavirus replication and syncytia formation. the rebodies or with a supernatant from the anti mg-160 hysults of studies with fusion defective mutants suggest bridoma (10a8) overnight at room temperature. cultures that different mechanisms are responsible for the initial were then washed, incubated with a biotinylated goat fragmentation of the ga and its subsequent migration to anti-rabbit igg antibody, incubated with the avidin-biotin the centers of syncytia. complex (abc), and stained with diaminobenzidine tetrahydrochloride (dab) (5 mg dab/10 ml tris-saline con-materials and methods taining 10 mm imidazole and 0.03% h 2 o 2 ), according to standard methods (graham and karnovsky, 1966; gues-cell cultures don et al., 1979) . l-2 cells (murine fibroblasts), originally obtained from american type culture collection (atcc; rockville, md), electron microscopy were used to maintain viral growth cultures, preparation of viral stocks, and viral plaque assays. in some experi-cells grown on thermanox plastic (em sciences, fort ments 17cl-1 murine fibroblasts were used. the cultures washington, pa), were fixed overnight at 4њ with 2.5% were maintained in dmem/heat-inactivated 10% fetal boglutaraldehyde / 1% paraformaldehyde in 0.1% cacodylvine serum/1% pen/strep/4500 mg/liter d-glucose/l-gluate buffer, ph 7.4, / 0.002% cacl 2 . subsequently cultures tamine. were postfixed in 1% osmium tetroxide / 1.5% potassium ferrocyanide, dehydrated in ethanol, and embedded in viruses and infections araldite. sections (8-10 nm thick) were stained with lead and uranyl salts and viewed in a transmission electron three times plaque purified mhv-a59 stock virus was microscope (jeol 100 cx) at 80 kv (karnovsky, 1971; used as previously described (lavi and weiss, 1989) . stieber et al., 1987) . fusion defective mutants and revertants were prepared as previously described (gombold et al., 1993; hingley et al., 1994) . infections of cultures were done by incuba-viral infectivity assay tion of virus with cells for 1 hr at a multiplicity of infection (m.o.i.) of 1, followed by washing of cells with fresh me-viral titers were determined by duplicate plaque dium three times. assays of several 10-fold dilution of samples in l-2 cell grown in six-well plates (lavi et al., 1984) . mg-160 is a conserved sialoglycoprotein of the medial brefeldin a (bfa) treatment cisternae of the ga. the preparations of the anti-mg-160 monoclonal antibody (10a8), and the immunoaffinity bfa (sigma) stock solution (5 mg/ml in ethanol) was diluted in pbs and applied to cultures at a concentration purified anti-mg-160 polyclonal antibodies, were described in previous publications (gonatas et al., 1989; of 5 mg/ml for periods of 1 hr or longer, as specifically indicated in the text (fig. 3) . in control experiments culcroul et al., 1988 croul et al., , 1990 . the preparation of the monoclonal antibody 2h1, a rer marker of a 60-to 65-kda tures were incubated for the same time periods with the same dilution of ethanol used to dissolve bfa. protein, has been previously described (chen et al., and cyncytia formation when infected with mhv-a59. following infection, cells were fixed and examined by immu-changes of the ga during mhv infection and nohistochemistry with identical methods used for l2 formation of syncytia cells. the changes in the ga observed in these cells were identical to those seen in l2-infected cells. morphological changes of the ga during coronavirus infection of l2 cells (4, 8, 16, 24 , and 48 hr) were examined by immunocytochemistry using organelle-specific changes of the ga following infection of l2 cells with antibodies (croul et al., 1988 (croul et al., , 1990 gonatas et al., 1989) . fusion-defective mutants and a fusion-negative strain typical infection of l2 cells with mhv-a59 caused syncyof mhv tia formation of the entire monolayer within 24 hr and complete cytolysis within 48 hr. at 16 hr postinfection, to investigate whether the fragmentation of the ga is dependent on cell fusion, we examined the organelle in cell borders were indistinct and the nuclei were aggregated; by 24 hr postinfection, the syncytia acquired their l2 cells infected with two fusion defective mutants of mhv-a59. the c12 and b11 mutants, used in these ex-typical morphology consisting of a ring of nuclei surrounding a cytoplasmic center devoid of nuclei. between periments, were isolated from primary cultures of glial cells infected with mhv-a59. these mutants are fusion-24 and 48 hr postinfection, cells underwent pyknosis, karyorhexis, and then died and detached from the culture delayed and defective but not fusion-negative and produce reduced number of syncytia formations. thus at dish. the immunostained ga of uninfected cells formed con-any given time point postinfection with the mutant viruses, only a small percentage of l2 cells form syncytia, tiguous coarsely granular focal or ring-like perinuclear profiles (figs. 1a and 1d). following infection of l2 cells as compared to cells infected with mhv-a59 (gombold et al., 1993) . these fusion-defective mutants have a histi-with mhv-a59, the coarsely granular stain of the ga became fine and smaller individual elements of the ga dine to aspartic acid mutation (h716d) within the cleavage signal of the spike (s) glycoprotein. cleavage of s were discernible. at 16 hr postinfection, the majority of the syncytia consisted of clustered nuclei within a cyto-is necessary for efficient cell fusion during mhv-a59 infection (gombold et al., 1993) . infection with these mutant plasmic mass, lacking distinguishable cell borders; in those syncytia, the immunostained ga appeared as viruses does not affect the efficiency of viral replication or titers of infectious virus (gombold et al., 1993) . immu-strands of finely granular elements forming a honeycomb-like network with interspersed nuclei (figs. 1b and nostaining of the ga in l2 cultures infected with the fusion defective mutants b11 and c12 showed fragmen-1e). however, before full syncytia formation, in cells surrounding the forming syncytia the distribution of the ga tation of the ga similar to that found in cells infected with mhv-a59. however, the translocation and rear-was perinuclear, similar to that of controls but with finer granular ga elements (compare figs. 1e and 1d) . this rangement of the ga during infection with these two mutant viruses was distinctly different from the changes phenomenon probably represented an early stage of fragmentation and rearrangement of the ga. at 24 hr of the organelle seen during infections with the wild-type mhv-a59. specifically, cells infected with either b11 or postinfection, the process of fragmentation of the ga and its relocation in the centers of syncytia, as well as the c12 mutants displayed both central and peripheral aggregates of finely granular ga (figs. 1h and 1i). the rearrangement of the nuclei in a ring formation within the syncytia, was complete (figs. 1c and 1f) . the honey-granular stain of the ga at the periphery of syncytia, and specifically around the peripherally located nuclei, was comb morphology due to the interspersed nuclei was replaced by a typical central aggregate of finely granular observed only in cultures infected with these two mutant viruses. this pattern of ga distribution was not seen in or fragmented ga, surrounded by a rim of nuclei (compare figs. 1f with 1e). furthermore, the nuclei in the any of the stages of mhv-a59 viral replication (compare figs. 1i and 1f ). in order to rule out the possibility that syncytium were not surrounded by any residual ga. thus, in the fully developed syncytium, all elements of the frag-the fusion-defective variants showed only delay in translocation of the ga into the center of cyncytia, immunocy-mented ga had moved into the center, while the more peripherally arranged nuclei formed a ring devoid of adja-tochemical analysis was performed at 36 and 48 hr after viral infection. these experiments showed that the typical cent elements of the ga (fig. 1f) . staining of syncytia with rabbit anti-mhv polyclonal antibodies revealed an complete central translocation of the ga, similar to that seen in a59-infected cultures, never occurred in cultures abundance of viral proteins in the center of syncytia (not shown). infected with the two fusion defective mutants, even prior to or at the stage of complete cell death. in order to determine whether the fragmentation and rearrangement of the ga was cell-type dependent, an-to further explore the link between fragmentation of the ga and fusion we then infected l2 cells with mhv-other cell line, 17cl-1 murine fibroblasts, was infected with mhv-a59. this cell line supports both cell fusion 2, a fusion-negative strain of mhv. l2 cells are susceptithe network of microtubules is rearranged during mhv-a59 infection pletely destroyed after 48 hr with titers similar to mhv-a59. however, there was no cell fusion or syncytia forma-since the ga is associated with microtubules of tion in these cultures. infection with mhv-2 produced interphase cells (robbins and gonatas, 1964; turner dispersion and fragmentation of the ga in individual and tartakoff, 1989) , we investigated whether microtucells, some of which appeared to be balooned after 24 bules are affected during coronavirus infection. spehr (fig. 1g) . these observations are consistent with the cifically, we investigated whether the fragmentation of conclusion that the fragmentation of the ga during infecthe ga within the centers of the syncytia is associated tion with mhv is independent of cell fusion. however, with a similar change of the microtubules. the immuthe translocation of the ga in the center of the syncytia nocytochemical staining of the ga at 4, 8, 16, 24, and is probably linked to cell fusion since fusion-defective 48 hr after mhv-a59 infection was compared with the mutants were also defective in their ability to induce immunostaining of microtubules with antibodies translocation of the ga to the center of the syncytia. against alpha and beta tubulin. while the ga appeared fragmented early during infection and syncytia forma-ultrastructural changes of the ga in mhv-infected tion, fragmentation and disintegration of microtubules cells occurred late (48 hr), when cells die. the kinetics of to further investigate the morphologic aspects of the the microtubule changes after infection with mhv-a59 fragmented ga in syncytia of l2 cells infected with mhvwas depicted by immunofluorescence on l-2 cells a59, an electron microscopic examination was perafter infection with mhv-a59 (m.o.i. å 1 pfu/cell) and formed in cells 24 hr after infection and at a multiplicity after staining with anti-tubulin antibodies and fitcof infection (m.o.i.) of 1 plaque forming unit per cell. areas conjugated secondary antibodies (fig. 3) . in uninof syncytia with a typical peripheral rim of nuclei were fected cells and at 4 hr postinfection the microtubules selected from semithin (0.5-1 mm) sections. in uninwere distributed throughout the entire cytoplasm of fected cells the ga was seen in a perinuclear location the individual cells. at 16 hr postinfection the syncytia and consisted of several groups of stacked cisternae were beginning to form and the microtubules were surrounded by numerous coated and uncoated vesicles still distributed within the entire cytoplasm. at 24 hr (fig. 2b) . in contrast to this typical morphology of the ga, postinfection, when the ga was fragmented and transin infected cells the stacks of the cisternae were marklocated to the centers of syncytia, the microtubules edly diminished in size and were replaced by numerous were rearranged in a characteristic pattern (fig. 3) . tubulovesicular structures, some of which containing vi-specifically, at the periphery of the syncytia, an inrus particles ( fig. 2a) . furthermore, in infected cells the tense stain for tubulins suggested that the nuclei were region containing remnants of ga cisternae and the surrounded by a rich network of microtubules. at the abundant tubulovesicular structures was rich in intermediate zone, between the periphery and the centransversing microtubules ( fig. 2a) . ter of syncytia, the microtubules formed a radiating network. at the center of the syncytia, the immunostain the distribution of the rer is not affected by mhvfor alpha and beta tubulin was less intense and amor-a59 infection phous (fig. 3) . these changes suggest that during coronavirus infection the microtubules undergo rear-the distribution of the rer in mhv-a59-infected and rangement and perhaps provide guidance for the control l2 cells was investigated by immunocytochemtranslocation of the fragmented elements of the ga istry with the organelle-specific monoclonal antibody into the center of the syncytia. 2h1 . there was no detectable difference between the infected and uninfected cells in the the effect of brefeldin a on coronavirus infection immunostaining of the rer. in both cases the fine granular staining of the rer was evenly distributed since coronavirus infection is associated with the processing of viral proteins through the ga, including within the entire cytoplasm including the centers of syncytia (not shown). within the organelle, we investigated the effect of bfa 24 hr later. uninfected cells, however, had completely recovered from the bfa effect 16 -24 hr later. these on the morphology and kinetics of coronavirus infection. as summarized in fig. 4 , bfa was introduced at findings suggest that virus infection accentuates and prolongs the bfa effect (fig. 4) . various time points during infection of l2 cell with mhv-a59. in uninfected l2 cells incubated for 1 hr when cells were treated continuously with bfa during infection there was dispersion of the ga, lack of syncytia with 5 mg/ml of bfa, immunostaining with anti-mg-160 showed a diffuse cytoplasmic pattern consistent formation, and no detectable viral titers (fig. 4) . if bfa treatment began at 4 or 6 hr after viral inoculation, the with the known redistribution of mg-160 and other golgi markers within the rer (doms et al., 1989; lipeffect of this treatment on infection was similar to the effect observed after continuous bfa treatment (i.e., re-pincottschwartz et al., 1989; johnston et al., 1994) . following 1 hr of bfa treatment at the beginning of duced syncytia formation and no viral titers). however, when the bfa treatment began 12 hr or more after viral infection cells had diffuse staining of the ga when observed 8 hr postinfection as did uninfected cells inoculation, there were relatively minimal effects on syncytia formation, and viral titers were 1-2 logs lower as following a similar treatment with bfa. however, at 16 hr postinfection (and 1 hr of treatment with bfa at the compared to viral titers without bfa treatment (which usually fluctuate about 1-1.5 logs in various experi-beginning of infection) there was incomplete recovery from the bfa effect. in uninfected cells treated in a ments). when bfa was introduced 20 min or an hour before similar fashion with bfa, the recovery was complete. therefore, when cells were infected with mhv-a59 inoculation and continued through the entire infection or 1, 3, or 5 hr after inoculation, there was no significant prior to or at the same time of treatment with bfa, the effect of the drug on the ga was not abolished 16 or effect on the ability of the virus to form syncytia 24 hr by cisternae derived from the intermediate compartment between the er and the golgi stacks, thus acquiring two membranes in one step (sodeik et al., 1993 (sodeik et al., , 1995 . the second wrapping cisternae in vaccinia virus assembly is derived from the trans golgi network (schmelz et al., 1994) . in cmv, ultrastructural as well as biochemical studies suggested that short-term exposure of infected cultures to bfa during the late infectious cycle primarily prevented golgi-dependent processes, e.g., envelopment of naked cytoplasmic nucleocapsids in the trans-golgi network (tgn) and normal processing of glycoprotein b (eggers et al., 1992) . in uukuniemi virus, a member of the bunyaviridae, immunofluorescent staining indicated that g1 glycoprotein expressed alone localized to the ga. g2 expressed alone was associated with the rer (melin et al., 1995) . coronavirus mhv m glycoprotein (previously known as e1) is targeted to the ga and contains a retention signal for the ga (machamer et al., 1990; swift and machamer, 1991; armstrong and patel, 1991) . previous reports have shown that the ga undergoes rearrangement (ho et al., 1990) and fragmentation (campadelli et al., 1993) in viral infections. the data presented here detail the changes in the morphology of the ga in cells infected by a virus which induces the formation of syncytia. in coronavirus infection, the virus displays cultures were incubated with virus (") for 1 hr and exposed to bfa (ٖ) for various periods. at the end of 24-hr incubation the cultured cells complex and close interactions with the ga. in coronaviwere stained for mg-160 by immuohistochemistry and the supernatants rus-induced syncytia formation there is a unique translowere titered for virus by plaque assay. titers labeled 0 indicate levels cation and aggregation of the ga into the center of the below 50 pfu/ml, which is the lowest level of detection in this assay. syncytia which is not accompanied by similar changes of the rer and cytoskeleton (fig. 1f) . the observed fragmentation of the ga is not related to the formation of postinfection and viral titers at that time were only 1-1.5 syncytia as demonstrated by the experiments using a logs lower than without treatment. thus bfa did not fusion-negative strain of mhv and fusion-defective mublock the endocytosis and the initial processing of the tants in which fragmentation of the ga also occurs. fragvirus into cells. bfa reduced viral replication only by mentation and translocation of the ga may be important 1-2 logs when introduced at 12-24 or 16-24 hr after in the life cycle of coronavirus replication. further studies infection. the results of the exposure to bfa at the beginare necessary to determine whether there are viruses ning of infection for 4 hr and for various periods at the that replicate within cells without causing alteration of end of infection (fig. 4) indicated that the bfa effect on ga morphology. the ga during 4-16 hr postinoculation was associated central translocation and aggregation of the ga may with reduced viral replication. if bfa was introduced after be unique to coronaviruses as it was not associated with or before this stage (4-16 hr), there was no effect on other fusion and syncytia forming viruses such as sindbis viral replication, assembly, and maturation. these results virus infection (ho et al., 1990) or herpes simplex virus suggest that the interval of 4-16 hr postinfection is the infection (campadelli et al., 1993) . since the aggregation most significant period requiring an intact ga for viral of the fragmented ga in the centers of syncytia has not replication and syncytia formation. this conclusion is been previously reported, we investigated the ga in cells based on infection at an m.o.i. of 1 pfu/cell which may infected with lacrosse bunyavirus, which is known to be an asynchronous infection. in a more synchronous induce the formation of syncytia (gonzalez-scarano et infection the period of bfa effect may terminate earlier al., 1984) . immunostaining of the ga in bhk cells infected than 16 hr postinfection. with lacrosse virus for 24 hr showed network formation and fragmentation of the ga, without the aggregation of discussion the ga into centralized zones and without the formation of amorphous centers of syncytia which is characteristic the ga plays an important role in the life cycle of many viruses such as vaccinia, cmv, bunyaviruses, and of mhv infection (unpublished observations). thus the translocation of the ga to the center of the syncytia may coronaviruses. vaccinia virus dna becomes enwrapped evolution of a coronavirus during persistent infection in vitro use of ferrocyanide-reduced osmium tetroxide cessed through the golgi apparatus after infection with herpes simin electron microscopy coronavirus m proteins accumulate in the 60-65 kd membrane polypeptide of the rough endoplasmic reticulum golgi complex beyond the site of virion budding an anti-organelle antibody in pathology. the chromatolytic reaction studied with a characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the rer to the golgi complex rothman experimental demyelination produced by the a59 strain of ture and toxic effect assembly of vaccinia virus: the second lar aspects of neurotropic viral infections assembly of vaccinia virus: incorporation of p14 treated with brefeldin a: evidence for membrane cycling from golgi and p32 into the membrane of the intracellular mature virus form of the golgi apparatus in hela cells assembly of vaccinia virus: role of the intermediate compartment between the endoplasmic reticulum and the golgi stacks proteolytic cleavage golgi complex ronnholm, r., and fusion activity virus contains a signal for localization to the golgi complex the golgi complex: in vitro veritas? fragmentation of the golgi apparatus of motor anti-organelle antibody neurons in amyotrophic lateral sclerosis revealed by organelle-spe-stieber mg-160, a membrane protein of the golgi apparatus assignment of the glg1 gene for mg-160, a fibroblast ligand for e-selectin, is found only in the golgi apparatus and apgrowth factor and e-selectin binding membrane sialoglycoprotein of pears early in chicken embryo development 354-355. membrane-spanning domain of coronavirus e1 protein fusion formation by the uncleaved spike protein of in transgenic mice expressing mutant cu,zn superoxide dismutase murine coronavirus jhmv variant cl-2 acetyl-galactosamine to the e1 glycoprotein of mouse hepatitis virus-mourelatos histochemical and ultrastrucand membrane traffic in golgi complex organization the ultrastructure of a mammaof the golgi apparatus by brefeldin a inhibits the cytotoxicity of ricin, modeccin, and pseudomonas toxin monoclonal antibody against the golgi apparatus. am. j. pathol. 133, be related to a unique interaction between coronavirus 355-362. proteins with the membranes of the ga. croul, s. e., mezitis, s. g. e., stieber, a., chen, y., gonatas, j. o., goud, since mutant viruses that are defective in their ability b., and gonatas, n. k. (1990) . immunohistochemical visualization of to cause efficient fusion are also inefficient in translocatthe golgi apparatus in several species, including human, and tissue ing the ga to the center of the syncytia, the translocation with antiserum against mg-160, a sialoglycoprotein of rat golgi apparatus. j. histochem. cytochem. 38, [957] [958] [959] [960] [961] [962] [963] of the ga appears to be linked to the ability of the virus doms, r. w., russ, g., and yewdell, j. w. (1989) . brefeldin a redistribto cause fusion. although the fusion property has been utes resident and itinerant golgi proteins to the endoplasmic reticuassociated with the s gene and the site encoding cleavlum. j. cell biol. 109, [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] [72] age of the s protein (gombold et al., 1993) , other reports eggers, m., bogner, e., agricola, b., kern, h. f., and radsak, k. (1992). showed that cleavage of the s gene in coronaviruses is inhibition of human cytomegalovirus maturation by brefeldin a. j. gen. virol. 73, 2679 -2692 not an absolute requirement for fusion (stauber et al., farquhar, m. g., and palade, g. e. (1981) . the golgi apparatus (com-1993; taguchi et al., 1993) . however, fragmentation of plex)useful in future studies of aspects of both viral replication gonzalez-scarano, f., pobjecky, n., and nathanson, n. (1984) . lacross and the biology of the ga.bunyavirus can mediate ph-dependent fusion from without. virology 132, 222-225. key: cord-296364-7rp60d2m authors: youn, soonjeon; leibowitz, julian l.; collisson, ellen w. title: in vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication date: 2005-02-05 journal: virology doi: 10.1016/j.virol.2004.10.045 sha: doc_id: 296364 cord_uid: 7rp60d2m molecular clones of infectious bronchitis virus (ibv), derived from the vero cell adapted beaudette strain, were constructed, using an in vitro assembly method. in vitro transcribed rna from a cdna template that had been constructed from seven cdna fragments, encompassing the entire genome of ibv, was electroporated into bhk-21 cells. the cells were overlaid onto the susceptible vero cells and viable virus was recovered from the molecular clone. the molecularly cloned ibv (mibv) demonstrated growth kinetics, and plaque size and morphology that resembled the parental beaudette strain ibv. the recombinant virus was further manipulated to express enhanced green fluorescent protein (egfp) by replacing an open reading frame (orf) of the group-specific gene, orf 5a, with the egfp orf. the rescued recombinant virus, expressing egfp (gibv), replicated to lower viral titers and formed smaller plaques compared to the parental virus and the mibv. after six passages of gibv, a minority of plaques were observed that had reverted to the larger plaque size and virus from these plaques no longer expressed egfp. direct sequencing of rt-pcr products derived from cells infected with the plaque-purified virus, which had lost expression of egfp, confirmed loss of the egfp orf. the loss of egfp expression (δ5a ibv) was also accompanied by reversion to growth kinetics resembling the standard virus and intact recombinant virus. this study demonstrates that the 5a orf is not essential for viral multiplication in vero cells. infectious bronchitis virus (ibv), a coronavirus, continues to be one of the most economically important pathogens in the poultry industry. coronaviruses are enveloped viruses with positive sense, 5vcapped and 3vpolyadenylated rna genomes, that range from 27.6 to 32 kb (lai and cavanagh, 1997) . two thirds of the coronavirus genome encodes the replicase activity, including a viral rna-dependent rna polymerase (rdrp), helicase, and viral proteinases. the remaining one third of the genome encodes the structural proteins and small group-specific orfs (lai and cavanagh, 1997) . ibv has four essential structural proteins, the three membrane proteins, the spike (s), integral membrane (m), and small envelope (e) proteins, and a phosphorylated, nucleocapsid (n) protein. the s protein interacts with cellular receptors and induces cell and viral membrane fusion (bosch et al., 2003) . n proteins, which interact with viral genomic rna, forming ribonucleocapsid (rnp) complexes, have been associated with replication and transcription (baric et al., 1988; compton et al., 1987; robbins et al., 1986) . the e and m proteins are localized in er-golgi intermediate compartment and play critical roles in viral budding (de haan et al., 1998; fischer et al., 1998) . coronaviruses are classified into three distinct groups by antigenic cross-reactivity and nucleotide sequence analysis (fields, 1996) . the group i coronaviruses include human coronavirus 229e, transmissible gastroenteritis virus (tgev), feline coronavirus, and feline infectious peritonitis virus. the group ii coronaviruses include human coronavirus oc43, murine hepatitis virus (mhv), and bovine coronavirus. ibv belongs to the group iii coronaviruses, all of which are avian specific. severe acute respiratory syndrome coronavirus (sars cov) respiratory disease in humans that resembles ibv in poultry may be distantly related to group ii coronaviruses (snijder et al., 2003; stavrinides and guttman, 2004) . coronavirus genomes also encode group-specific orf that have been reported to express in infected cells (lai and cavanagh, 1997; liu and inglis, 1992; liu et al., 1991) . functions of these genes are not well understood, however, deletion of combinations of several group-specific genes in mhv resulted in in vivo attenuation (de haan et al., 2002) . although the functions of the four ibv group-specific orf, 3a, 3b, 5a, and 5b are not known, a vero cell passaged ibv mutant with a truncated 3b was viable in vitro and in vivo (shen et al., 2003) . reverse genetic systems for human coronavirus 229e, tgev, mhv, sars cov, and ibv have been developed using three distinct methodologies. bacterial artificial chromosomes (bac) and vaccinia virus vectors, both of which can accommodate the large coronavirus genomes, have been utilized to carry a full-length cdna clone of the genome (almazan et al., 2000; casais et al., 2001; gonzalez et al., 2002; thiel et al., 2001) . a third system relied on the in vitro assembly of a modest number of cloned cdnas. instead of cloning the entire genome of tgev into vectors, yount et al. (2000) amplified the viral genome into several fragments by rt-pcr and the cloned amplicons were ligated through unique restriction sites for in vitro assembly of the entire genome. this strategy was further modified to construct an infectious cdna clone of mhv with their bno see'mq technology, in which restriction endonuclease sequences were incorporated into amplicons, such that upon enzyme treatment, the endonuclease sites were eliminated prior to in vitro ligation. the assembled cdna product was used as template for in vitro transcription to generate genomic rna (yount et al., 2002) . more recently, this strategy was used to construct a reverse genetic system for the sars cov (yount et al., 2003) . in the current study, molecular clones of ibv were generated using the in vitro assembly of cdna fragments as a template for transcription of full-length genomic rna. we further have demonstrated that the 5a orf can be eliminated or replaced by the egfp orf while maintaining virus viability. therefore, this gene is not essential for viral replication and its orf may potentially be used as a site for heterologous gene delivery. cdna was constructed that encompassed the entire ibv genome the cloning strategy for a full-length ibv-beaudette construct is illustrated in fig. 1a . five rt-pcr fragments were produced that cumulatively amplified the entire genome of the vero cell adapted beaudette ibv strain. amplicons, overlapping by about 10 nucleotides, were designated a through e and the fragment lengths were as follows; a, 2269 nts; b, 6356 nts; c, 6895 nts; d, 5030 nts; and e, 7058 nts (fig. 1b) . however, consistent with the report that these regions were toxic or unstable in bacteria, difficulties arose in cloning the b and c fragments into bacterial plasmid based cloning vectors (casais et al., 2001) . therefore, the b and c fragments were each cloned as two smaller fragments b1 and b2, and c1 and c2. fragments a through c2 were cloned into the psmart vector and fragments d through e2 were cloned into pcr-xl-topo cloning vectors. in order to determine the consensus sequence of the genome, two to five independent clones of each amplicon were sequenced using a panel of internal primers. clones with consensus sequences were used for in vitro assembly of full-length cdna template. there were several sequence differences, distributed throughout the entire genome of ibv, between our standard cell adapted ibv strain and the published beaudette sequence (table 1 ). the standard parental strain of ibv beaudette used for these studies had a five nucleotide insertion at position 25,414 between the m and 5a orfs, resulting in a genome size of 27,613, instead of 27,608 nt. the sequence differences were confirmed by sequencing three independent clones and direct sequencing of rt-pcr products. a nucleotide insertion was found in an intergenic sequence of our strain compared to the original published sequence. in addition, an e amplicon with a single unique nucleotide change of a to g at position 25,793 nt, resulting in amino acid change from q to r, was used as a marker for the molecular clone. full-length cdna was in vitro assembled with amplicons conforming with our established consensus sequence. to increase full-length in vitro ligation efficiency, ordered ligation reactions were used. the appropriately sized ligation reaction products were purified from 1% agarose gels before further ligating with neighboring inserts (fig. 1c) . a full-length genomic transcript of ibv was produced in vitro using t7 rna polymerase with the t7 rna promoter, incorporated at the 5vend of fragment a. the final transcript product was verified by northern blot analysis using a probe against 3v utr (data not shown). any transcript of a size comparable to viral genomic rna with a detectable 3vutr presumably contained the entire genome of ibv because only the a fragment had a t7 rna promoter and thus could initiate transcription. the transcripts from the full-length cdna (along with the n transcript alone), total cellular rna from ibv infected vero cells (positive control), and pbs (negative control) were electroporated into non-permissive bhk-21 cells, which were then cultured with vero cells. three days after transfection, typical cytopathic effects (cpe), including syncytia formation, were seen in cells transfected with the positive control. supernatant from the cells, which were overgrown by 3 days after transfection, were harvested and passaged into fresh vero cells. one day after passage, the positive control showed cpe. two days after passage, cells transfected with the transcript of the full-length ligated cdnas showed cpe. cpe was never observed in the negative control cells. we were unable to rescue virus from full-length ibv rna transcript without co-transfection of n transcript, confirming the possible essential role of n transcript as described by previous reports (casais et al., 2001; yount et al., 2000 yount et al., , 2002 . the virus generated by the molecular cdna clone was referred to as mibv. the derivation of mibv from the ligated cdna was confirmed by rt-pcr sequence identification of the marker mutation of a g at nt 25,793. mibv was further characterized after three rounds of plaque purification. mibv formed plaques which were indistinguishable in size and morphology from standard beaudette cell adapted viral plaques (figs. 2a and b) . the growth kinetics of mibv and the standard virus were also compared and found to be similar (fig. 3) . the reverse genetic strategy we adapted for ibv was used to evaluate whether a small group-specific gene was necessary for the survival of ibv in cell culture. furthermore, the replacement with a foreign orf would be a first step in developing constructs of ibv as expression vectors. because it has been shown that the expression level of coronavirus genes can be modified by their transcription regulatory sequences (trs), rather than add an additional trs, a reporter gene was used to replace most of the 5a orf, thus maintaining the 5a trs sequence. based on results with mhv, we hypothesized that ibv group-specific genes would not be necessary for viral replication in cell culture. the 5a orf was used rather than the 3a, 3b region which contains an internal ribosomal entry site (ires) needed for expression of the essential structural, e protein (le et al., 1994) . the egfp orf was used to replace the 5a orf, beginning at the start codon, while the 3v end of 5a orf, that included the stop codon, was kept intact because this region overlaps with the 5b orf. pcr mutagenesis and the bno see'mq methodology were used to replace orf5a with egfp. the cloning strategy used to replace most of the 5a orf with egfp (d5a/egfp) is illustrated in fig. 4a . three regions, including the egfp orf (fig. 4b, lane 1) , and sequences between a region upstream of 5a, including orf m (fig. 4b, lane 2) , a region downstream of 5a through the 3v utr (fig. 4b , lane 3), were pcr amplified using primers that incorporated the bsmbi restriction enzyme recognition sequence (table 2) . these three pcr products were cloned into the pstblue cloning vector and the inserts were excised from the vectors with the bsmbi restriction enzyme. the e amplicon was replaced with the d5a/egfp amplicon and the full-length infectious cdna template was assembled and transcribed as described previously. three days after electroporation into bhk cells and coculturing with vero cells, cpe was observed in the positive control and in the cells transfected with the gibv rna transcript (fig. 5a ). the cells that showed cpe by gibv infection also expressed egfp which was easily detected under a uv microscope (fig. 5b) . interestingly, some cells expressed egfp before even showing virus infection induced morphological changes. following nucleotide sequencing analysis, it was confirmed that no unintentional mutations were incorporated into the amplicons used to construct the recombinant gibv. therefore, not only is 5a expression not essential for viral replication but also it is possible to replace the 5a with a functional foreign orf. a-g -22,441 c-g l-v a according to our vero-adapted ibv derived sequence. the entire sequence has been submitted to genbank. b left nucleotide according to the previously published sequences and the right nucleotide from our vero cell-adapted derived strain. c left amino acid from the previously published sequences and the right amino acid from our vero cell-adapted derived strain. -indicates that there was no amino acid difference. to confirm the stability of the recombinant gibv, the virus was subsequently passaged in vero cells. egfp expression was detected in each of seven sequential passages. gibv was further characterized by comparing plaque morphology and one step growth curves. compared to our standard virus and the virus from the cloned mibv, gibv-generated plaques were much smaller in size (fig. 6a) . the growth of gibv was similar to the standard ibv with maximum growth at 14 h post-infection. however, gibv grew to a 10-fold lower titer compared with the standard beaudette ibv or mibv (fig. 3) . beyond passage five in vero cells, the plaque assays of the gibv stocks began to demonstrate plaques similar in size to those formed by our standard virus. viruses derived from these plaques had lost the ability to express egfp. to explain the phenomena, the region of egfp insertion/5a replacement from several plaque purified viruses was rt-pcr amplified and compared with rt-pcr products of mibv and the gibv expressing egfp (forming smaller plaques). the rt-pcr product of the latter was smaller than the gibv and mibv (fig. 7a ). in addition, the region across the insertion site of egfp orf for ten gibv derived strains that had lost egfp expression was amplified by rt-pcr. sequencing of the rt-pcr products confirmed that these stains had either partially or completely lost the egfp orf. the 10 strains examined could be categorized into four distinct groups, depending on the deletion pattern of the egfp orf (fig. 7b) . the 5b orf of all was maintained and completely intact while the start codon at the insertion site of the egfp was deleted in all of these phenotypic revertants. interestingly, the size of plaques with the 5a deletions, including a revertant that had completely lost the egfp orf, was comparable to plaques generated by our standard beaudette cell-adapted beaudette virus (fig. 6b ). when compared with the standard virus, growth kinetics and maximum titer of the revertants with the 5a deletion was similar to the standard cell adapted beaudette virus (fig. 3) . molecular clones of several coronaviruses have been constructed using three different methodologies, vaccinia virus vectors and bac as cloning systems, and the in vitro assembly strategy. in this study, we constructed an infectious cdna clone of ibv and recombinant ibv expressing egfp instead of 5a protein, using in vitro assembly which is less labor intensive and allows for easy manipulation, such as the introduction of targeted mutations. we chose to use highly attenuated strain of ibv, the vero cell-adapted beaudette us strain as the cdna backbone because we hope to eventually use ibv as a gene transfer vector, as well as to elucidate mechanisms of pathogenesis and generate new appropriate vaccines. contrary to the difficulties stated in a previous report (casais et al., 2001) , our study has demonstrated that the in vitro assembly method used to generate infectious rna from in vitro assembled cloned cdnas of tgev, mhv, and sars coronavirus also can be applied to ibv. this is also the first report that the group iii coronavirus group-specific 5a gene is not essential for viral replication. based on studies with mhv and tgev, in which deletion of multiple group-specific genes resulted in attenuation of virulence, group-specific genes of ibv are reasonable candidates for viral attenuation and foreign gene replacement (de haan et al., 2002; sola et al., 2003) . however, it has been shown that these small orf, including 5a, are conserved among the various strains of ibv (brooks et al., 2004) . therefore, it is likely that 5a is important, if not critical, for in vivo replication. further studies will determine the in vivo role of 5a in pathogenesis and whether its deletion results in attenuation. gibv produced smaller sized plaques compared to our standard parental virus. although many recombinant viruses have been constructed to express egfp or gfp without known reports that egfp is toxic to virus replication, egfp was shown in at least one study to induce cellular apoptosis (liu et al., 1999) . in the current study, it is possible that infected cells died by apoptosis because of high expression of egfp prior to producing maximum amounts of virus viral replication. this is consistent with the finding that we could detect expression of egfp before cells showed cpe. alternatively, manipulation of orf 5a could affect orf 5b expression. it has been assumed that gene 5b is expressed by leaky scanning mechanism (liu and inglis, 1992) . the egfp orf contained an appropriate kozak sequence and may have reduced the expression of 5b. therefore, reduction in 5b expression could potentially have been associated with the observed change in viral replication. gibv phenotypic revertants, which lost the egfp orf, maintained an intact 5b orf, and regained standard beaudette cell adapted virus plaque size, growth kinetics and titer. based on sequence analysis, four distinct deletion events were indicated. it has been shown that the gfp gene has homology with the mhv trs and hinders expression of gfp because of abortive transcription (fischer et al., 1997) . however, we do not favor this explanation because, contrary to fisher et al., we had sufficient expression of egfp, with recombinant virus during the first several passages, to be easily detectable and no sequence similarity could be identified in or around the deleted region between the ibv trs and the egfp gene. non-homologous recombination events during ibv fig. 6 . egfp deletion revertant of gibv showed comparable plaques to standard ibv or mibv. after six serial passages of gibv, heterogeneoussized plaques were observed. the arrow indicates typical gibv plaques with the smaller size compared to mibv and the arrow head indicates a larger plaque from the gibv stock that was comparable to the standard cell adapted ibv, and plaques purified from the larger plaques in a are shown in panel b. replication likely accounts for the deletion of a gene, such as egfp, that is not necessary for the viral replication. it is not clear whether the foreign gene itself or its specific location in the viral genome causes this spontaneous deletion event. furthermore, we cannot conclude whether deletion of the egfp gene, under the control of the 5a trs, is a general phenomenon of recombinant ibv or a result of egfp toxicity. coronaviruses have several attractive traits as gene delivery systems, including their unusually high packaging capacity, exclusive replication in cytoplasm, excluding any chance of viral genome incorporation into host chromosomes, and the ability to express multiple genes which has been shown by thiel et al. (2003) . the stability of a foreign orf replacing the 5a orf should be further examined with genes that encode non-toxic proteins, in order to determine the replication status and pathogenesis of these recombinant viruses in vivo. furthermore, this methodology provides with a convenient reverse genetic tool for correlating the pathogenesis with the group-specific genes of ibv. a vero cell-adapted strain of ibv beaudette us, from american type culture collection (manassas, va) was plaque purified three times before use. the virus was propagated in an african green monkey kidney vero cell line, obtained from viromed laboratory (minnetonka, mn) and maintained in dulbecco's modified eagle medium (dmem) containing 5% fetal bovine serum (fbs) supplemented with penicillin g (100 units/ml) and streptomycin (100 ag/ml). the baby hamster kidney cell line, bhk-21, was fig. 7 . gibv phenotypic revertant had lost the egfp orf. the absence of the egfp orf was confirmed by rt-pcr and nucleotide sequencing of the region corresponding to nucleotides 25,162 to 25,613 of the standard cell adapted ibv. compared to gibv, the gibv revertant produced a smaller rt-pcr product, smaller than the e amplicon, suggesting that the revertant had lost the egfp and 5a orfs. (a) rt-pcr and pcr products of the region corresponding to nucleotides 25,162 to 25,613 of gibv, the phenotypic 5agfp deletion revertant and the control, e amplicon. the m lanes represent the dna molecular size marker; lane 1, rt-pcr product of gibv expressing egfp; lane 2, the rt-pcr product of the phenotypic revertant that had lost expression of egfp, and 3, as a control, the pcr product from the e fragment. (b) alignment of the nucleotide sequences of the region spanning the 5a/egfp orf of d5agfp viruses identified four distinct types of egfp deletions. italicized nucleotides and the positions starred below the sequence alignment represent conserved sequences. every mutant lost the egfp start codon but maintained the 5b start codon (underlined). also obtained from viromed and maintained in dmem containing 10% fbs supplemented with antibiotics as described for vero cells. total cellular rna was extracted from ibv-beaudette infected vero cells with trizol reagent (invitrogen, carlsbad, ca), according to the manufacturer's directions. reverse transcription was performed with superscript ii (invitrogen) and reverse direction primers p1r, p2r, p3r, beaussr, and gnrt (table 2) , containing bsmbi and sapi restriction endonuclease recognition sequences. the ibv sequence, as reported in genbank (accession number m95169), was used for primer design and nucleotide sequencing. each dna fragment was amplified from cdna templates by pcr using expand long polymerase (boehringer mannheim biochemical). in order to transcribe rna using t7 rna polymerase, the t7 rna promoter sequence was incorporated into the t7p1f primer. pcr primer pairs used to amplify genomic regions are listed in table 2 . pcr amplification of cdna fragments was performed using the following conditions; denaturation at 94 8c for 2 min, 10 cycles at 94 8c for 30 s, 55 8c for 30 s, and 68 8c for 2-6 min depending on the size of the fragment and 18 cycles of 94 8c for 30 s, 55 8c for 30 s, and 68 8c for 2-6 min (with an additional 20 s for each subsequent cycle). the pcr products were isolated from agarose gels and cloned into pcr-xl-topo (invitrogen) or psmart (lucigen, middleton, wi) vectors, according to the manufacturer's directions. two to four independent clones of each amplicon were isolated and sequenced by using specific primers and the abi dye termination sequencing method. a consensus sequence was determined by comparison of direct pcr product sequencing from each independent clone and amplicons, encoding the consensus sequence of each region of ibv, were cloned using standard recombinant dna techniques (sambrook et al., 1989) . the entire ibv n orf, including the 3v utr, was amplified by rt-pcr from total cellular rna extracted from ibv infected vero cells, and the rt-pcr product was cloned into a transcription vector, pgem-3zf(+) (promega, madison, wi). the egfp orf (f fragment) was pcr amplified from plegfp-n1 (clontech laboratories, inc., palo alto, ca), using the gfpsf and gfper primer pair, and cloned into pstblue ( table 2 ). the region upstream of the 5a orf (g fragment) was pcr amplified using the e amplicon as a template with the 5asr and bmf primer pair and the product cloned into pstblue (novagen, darmstadt, germany), referred to as 5as. the region downstream of the 5a orf (fragment h which included the 3vutr and polyadenylated tail) was pcr amplified using the e amplicon and the 5aef and gnrt primer pair before cloning into the pcr-xl-topo vector (referred to as 5ae). the g fragment was prepared by digesting 5as with xhoi and bsmbi restriction enzymes. the f fragment was digested from the vector with bsmbi. the g and f fragments were ligated into the 5ae amplicon which was digested with xhoi and bsmbi 5v -cgtctctaagcgtatacgcccaccca-3v a as described in fig. 1 . b as derived from the sequence of our vero cell-adapted, laboratory beaudette strain. c the t7 rna polymerase recognition sequences shown in bold. underlined are bsmbi restriction enzyme recognition sequences. restriction enzymes. replacement of 5a sequences with egfp orf was confirmed by sequencing of the d5a/egfp amplicon. each amplicon, having the consensus sequence, was prepared from an overnight bacterial culture. plasmid was isolated and digested with the indicated restriction endonucleases according to the manufacturer's directions. briefly, the ibv a amplicon was digested with xhoi and treated with calf intestine alkaline phosphatase (cip), before digesting with the sapi enzyme. the ibv e amplicon was digested with ecori, cip treated and digested with bsmbi. the b1, b2 and c1 amplicons were digested with bsmbi at 55 8c and then digested with sapi at 37 8c. the c2 and d amplicons were digested with bsmbi. inserts of each amplicon were resolved by electrophoresis on 1% agarose gels containing crystal violet and isolated with qiaquick gel extraction kit (qiagen inc., valencia, ca) according to the manufacturer's directions. one picomole of each cdna insert was ligated in an ordered reaction (a + b1, b2 + c1, c2 + d, e1 + e2 or ed5a/egfp + e2). the appropriately sized ligation reaction products were purified from 1% agarose gels and further ligated with neighboring inserts for 4 h, pooled and ligated overnight (fig. 1c) . the final ligation product was purified by extracting with a phenol/chloroform/isoamyl alcohol (25:24:1) mixture and precipitated with ethanol before using as a template for in vitro transcription reactions. full-length transcripts of the ibv cdna constructs were generated in vitro using the mmessage mmachine t7 ultra kit (ambion, austin, tx) according to the manufacturer's direction with certain modifications. the in vitro transcription reaction was performed at 37 8c for 2 h in 20 al reaction mixtures, supplemented with 3 al of 30 mm gtp, resulting in a 1:1 ratio of gtp to capping analog. a similar reaction was performed for the n transcript using a 1:3 ratio of gtp and capping analog. before electroporation, the transcripts were treated with dnase i and analyzed by 1% denaturing agarose gel (containing 2.2m formaldehyde) electrophoresis. the identity of the full-length in vitro transcript was confirmed by northern blot analysis using a probe against 3vutr of ibv. after bhk-21 cells were grown to subconfluence (80%), treated with trypsin, and washed with cold depc treated pbs twice, they were resuspended in depc-treated pbs at a concentration of 10 7 cells/ml. rna transcripts were added to 400 al of the bhk-21 cell suspension in microfuge tubes on ice, gently pipetted and transferred to electroporation cuvettes. three consecutive electrical pulses of 850 v at 25 af were given, using the electro-cell manipulator 600, btx (genetronics, inc., san diego, ca). the transfected bhk-21 cells were diluted 1 to 20 with complete dmem in 100 mm cell culture petri dishes and co-cultured with 2 â 10 6 vero cells/dish. triplicate wells of vero cells in six-well plates were infected with virus at a multiplicity of infection (m.o.i.) of 2 to 3. the cells were harvested every 4 h for 24 h and stored at à80 8c until they were used for quantification by titration. the cells were three times frozen and thawed, and 10-fold serially diluted in dmem without serum. two hundred microliters of each dilution were inoculated into six-well plates for an hour, washed with pbs and overlaid with dmem, containing 2% fbs, 0.8% agarose, before incubating at 37 8c for 3 days. the plaques were counted after removing the agarose overlay from the cells and staining the monolayer with crystal violet. engineering the largest rna virus genome as an infectious bacterial artificial chromosome interactions between coronavirus nucleocapsid protein and viral rnas: implications for viral transcription the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex comparisons of envelope through 5b sequences of infectious bronchitis coronaviruses indicates recombination occurs in the envelope and membrane genes reverse genetics system for the avian coronavirus infectious bronchitis virus in vitro replication of mouse hepatitis virus strain a59 coronavirus particle assembly: primary structure requirements of the membrane protein the group-specific murine coronavirus genes are not essential, but their deletion, by reverse genetics, is attenuating in the natural host coronaviridae: the viruses and their replication analysis of a recombinant mouse hepatitis virus expressing a foreign gene reveals a novel aspect of coronavirus transcription analysis of constructed e gene mutants of mouse hepatitis virus confirms a pivotal role for e protein in coronavirus assembly stabilization of a full-length infectious cdna clone of transmissible gastroenteritis coronavirus by insertion of an intron the molecular biology of coronaviruses distinct structural elements and internal entry of ribosomes in mrna3 encoded by infectious bronchitis virus identification of two new polypeptides encoded by mrna5 of the coronavirus infectious bronchitis virus a polycistronic mrna specified by the coronavirus infectious bronchitis virus is green fluorescent protein toxic to the living cells? rna-binding proteins of coronavirus mhv: detection of monomeric and multimeric n protein with an rna overlay-protein blot assay molecular cloning: a laboratory manual emergence of a coronavirus infectious bronchitis virus mutant with a truncated 3b gene: functional characterization of the 3b protein in pathogenesis and replication unique and conserved features of genome and proteome of sarscoronavirus, an early split-off from the coronavirus group 2 lineage engineering the transmissible gastroenteritis virus genome as an expression vector inducing lactogenic immunity mosaic evolution of the severe acute respiratory syndrome coronavirus infectious rna transcribed in vitro from a cdna copy of the human coronavirus genome cloned in vaccinia virus multigene rna vector based on coronavirus transcription strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a59 reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus we thank dr. ralph baric for providing valuable advice in developing the in vitro assembly procedure for ibv. this work was supported in part by grants from the u.s. poultry and egg association (297) key: cord-287777-ogs4mq0v authors: lindner, holger a. title: deubiquitination in virus infection date: 2007-06-05 journal: virology doi: 10.1016/j.virol.2006.12.035 sha: doc_id: 287777 cord_uid: ogs4mq0v post-translational modification of proteins and peptides by ubiquitin, a highly evolutionarily conserved 76 residue protein, and ubiquitin-like modifiers has emerged as a major regulatory mechanism in various cellular activities. eukaryotic viruses are known to modulate protein ubiquitination to their advantage in various ways. at the same time, the evidence for the importance of deubiquitination as a viral target also is growing. this review centers on known viral interactions with protein deubiquitination, on viral enzymes for which deubiquitinating activities were recently demonstrated, and on the roles of viral ubiquitin-like sequences. post-translational modification of proteins and peptides by ubiquitin (ub), a highly evolutionarily conserved 76 residue protein, and ubiquitin-like modifiers (ubls) (fig. 1 ) has emerged as a major regulatory mechanism in various cellular activities including signal transduction, transcription, membrane protein trafficking, nuclear transport, autophagy, and immune responses (d' azzo et al., 2005; haglund and dikic, 2005; welchman et al., 2005) . protein modifications by ub and ubls, such as nedd8, isg15, and sumo, modulate protein-protein interactions (kerscher et al., 2006) , while ubiquitin-like atg8 homologs become lipidated and attached to cellular membranes (tanida et al., 2004) . ub and most ubls are produced as precursor proteins, and only carboxy-terminal processing after recognition sequence motifs by deubiquitinating enzymes (dubs) generates the active modifiers (amerik and hochstrasser, 2004) . conjugations of ub and ubls to their targets relies on analogous enzymatic cascades comprising the sequential action of three enzymes (passmore and barford, 2004) : a modifier activating enzyme (e1), one of several modifier carrier enzymes (e2s), and a member of the large and diverse group of modifier-target ligases (e3s), which chiefly determine target specificity. the enzymatic trio transfers the carboxy terminal glycine of ub to the epsilon-nh2 group of an internal lysine residue of the target protein, or less often to its terminal amino group. in contrast to the known ubl modifications, ub can further be assembled into polymeric chains (polyubiquitination). one out of five internal lysine residues at position 6, 11, 29, 48, and 63 of ub, but mostly lysine-48 or lysine-63, is used for the attachment of additional ub units. while attachment of at least four lysine-48 linked ub molecules usually promotes the degradation of a protein by the ub-proteasome system (ups), the most important machinery for the degradation of cytoplasmic and nuclear proteins, chain formation via other lysine residues, or conjugation of individual ub molecules mediates largely non-proteolytic functions of ub (ciechanover, 2006) . ub and ubl modifications are reversed through the isopeptidase activities of dubs (fig. 1) , with most studied dubs deconjugating only a small number of targets (nijman et al., 2005) . in fact, deubiquitination, a term used here inclusive of ub and ubl deconjugation, is an emerging regulatory process in signaling pathways, chromatin structure, endocytosis, and apoptosis (nijman et al., 2005) and is important for physiological activities including neuronal function, development, and immunity (evans, 2005) . viruses of eukaryotes are known to take advantage of protein ubiquitination in various ways. entry or release of different viruses, for example, was shown to depend on the proteasome or on certain cellular e3s (banks et al., 2003; bieniasz, 2006; ros and kempf, 2004; yu and lai, 2005) . what is more, many viruses manipulate protein ubiquitination in order to overcome host cell defense mechanisms, including apoptosis, the type 1 interferon (ifn) response, and major histocompatibility complex (mhc) class 1 antigen presentation. to this end, numerous viruses encode proteins that redirect cellular e3s of the ups to proteins with antiviral activity (fig. 1, step 4) , including for example the tumor suppressor protein p53 (induction of apoptosis) and the signal transducers and activators of transcription (ifn response). alternatively, some viruses express their own e3s (fig. 1, step5) , which commit cellular defense proteins, such as p53 or mhc class 1 molecules, to degradation. such viral strategies were reviewed recently by shackelford and pagano (2005) , gao and luo (2006) , and barry and früh (2006) and are not considered here in detail. different from ubiquitination, only few examples for the targeting of protein deubiquitination (fig. 1, step 6 ) by viruses have been described to date. they include the potential recruitment of dubs for the stabilization of β-catenin in epstein-barr virus (ebv)-infected b cells (ovaa et al., 2004; shackelford et al., 2003) , and the specific targeting of the cellular dub ubiquitin-specific protease 7 (usp7) by the epstein-barr nuclear antigen 1 (ebna1) and the herpes simplex virus type 1 (hsv-1) regulatory protein icp0 (everett et al., 1997; holowaty and frappier, 2004) . despite the compelling biochemical evidence for the specificity of these two interactions, the importance of viral targeting of usp7 remains vague. the possibility that modulation of deubiquitination is, nevertheless, a more common viral strategy has gained support by the recent in vitro demonstration of deubiquitinating activities for three viral enzymes: the adenovirus protease adenain (balakirev et al., 2002) , the papain-like protease (plpro) of severe acute respiratory syndrome coronavirus (sars-cov) (barretto et al., 2005; lindner et al., 2005) , and a protease domain contained in the n-terminal fragment of the large tegument protein ul36 (ul36 usp ) from several herpesviruses, namely, hsv-1, ebv, and mouse and human cytomegalovirus (mcmv and hcmv) schlieker et al., 2005; wang et al., 2006) . however, the roles of these deubiquitinating activities during virus infection remain elusive. here, the known viral interactions with protein deubiquitination are reviewed, and potential roles of viral dubs are considered. the majority of the dubs from all kingdoms of life (excluding archaea), including the known and predicted viral enzymes, represent cysteine protease homologs, with the remainder forming a separate family of metalloproteases (rawlings et al., 2004) . at least six structural classes (families) of cysteine protease dubs have been identified: the ubiquitinspecific protease (usp), autophagin (atg), ubiquitin cterminal hydrolase (uch), ovarian tumor-related protease (otu), josephin-domain protease (jd), and ubiquitin-like general schematic for ubl conjugation and deconjugation and points of viral interference. proteolytic maturation of ubl (including ub) precursor proteins by dubs (step 1) exposes a new c-terminus in the modifier, which is then activated by e1 in an atp-dependent manner (step 2) and next transferred to the e2 (step 3). an e3 generally recognizes the target protein (step 4) and facilitates the ligation of the ubl to it (step 5), altering its protein interaction repertory and thereby its function. polyubiquitination as a particular example of ubl modification of a protein can lead to its proteasomal degradation. ubl deconjugation by dubs reversely regulates target protein function and, additionally, replenishes the pool of free ubl (step 6). points of viral interference are marked (*). viruses are known to intervene with the ub conjugation pathway (at steps 4 and 5, see text), but also target ubl deconjugation (step 6), inclusively referred to as deubiquitination, which is the principal theme of this review. viruses may also interfere with ubl precursor processing (step 1), as suggested by the ability of some viral dubs to process the isg15 precursor protein (see text). protein-specific protease (ulp) families (amerik and hochstrasser, 2004; nijman et al., 2005; sulea et al., 2006) . they all feature structural variations of the papain fold and display canonical papain-like spatial arrangement of catalytic centers. many are further characterized by additional variable aminoand/or carboxy-terminal sequences, for which a growing number of structures and functions, including localization, substrate recognition, and/or activation of catalytic activity, are being reported (nijman et al., 2005; reyes-turcu et al., 2006; sulea et al., 2006) . despite recent advances in the understanding of dub structure-function relationships (amerik and hochstrasser, 2004; nijman et al., 2005; sulea et al., 2006) , inference of biological importance for uncharacterized dubs from sequence information alone remains challenging. the human genome encodes around 90 cysteine protease dubs and 12 metalloprotease dubs (rawlings et al., 2004) . the number of catalytically active human cysteine protease dubs, inclusive of putative alternative splice isoforms, amounts to over 100. in order to further the functional characterization of dubs on a large scale, ploegh and co-workers synthesized specific probes for the proteomic profiling of cysteine protease dub activities (borodovsky et al., 2002) . they used an intein-based chemical ligation method to modify the free c-terminus of a hemagglutinin (ha)-tagged ub with a series of thiol-reactive groups, such as vinylmethylsulfone (vme) or bromoethylamine. after incubation of cellular lysates with some of these active sitedirected suicide substrates and subsequent anti-ha immunoprecipitation, tandem mass spectrometry facilitated the simultaneous identification of multiple dubs and some associated proteins (borodovsky et al., 2002) . haubvme exhibited the broadest reactivity, and, in the following, was used to detect active cysteine protease dubs and associated proteins in ebvinfected b cells (ovaa et al., 2004; shackelford et al., 2003) , and human papillomavirus (hpv)-infected cervical carcinoma cells as well as hpv e6/e7 immortalized keratinocytes (rolén et al., 2006) . in a study by ovaa et al. (2004) , lymphoblastoid transformation of freshly isolated b cells by in vitro ebv infection led to an increase in the activities of six cellular dubs, identified as uch37, uch-l3, uch-l1, usp7, usp9x, and usp15, at different times postinfection. interestingly, usp9x was previously shown to interact with the bifunctional armadillo repeat protein β-catenin in vitro as well as in cultured epithelial cells and was able to stabilize β-catenin in these cells (taya et al., 1999) . β-catenin is further known to be stabilized in latency type iii ebv-transformed b cells (see below). β-catenin is a component of cell-cell adherens junctions. at the same time, a low-level cytoplasmic pool of this protein functions in canonical wnt signaling (reviewed by brembeck et al., 2006) . in the absence of wnt signaling, cytoplasmic βcatenin is continuously phosphorylated by casein kinase followed by glycogen synthase kinase 3-β, which together with the two scaffold proteins axin and the adenomatosis polyposis coli tumor-suppressor protein constitute the so-called degradation complex. these consecutive phosphorylations events initiate the polyubiquitination of β-catenin by a ub ligase complex, known as skp1/cullin/f box protein β-trcp (scf β-trcp ) (liu et al., 2001 , and references therein), and its rapid proteasomal degradation. binding of the wnt ligand to its cell surface receptors is thought to trigger a series of phosphorylation events that result amongst others in the degradation of axin, causing disassembly of the abovementioned degradation complex and consequently the stabilization of β-catenin which then accumulates in the nucleus. here, β-catenin acts as a coactivator of wnt target genes that regulate cellular proliferation and differentiation during animal development and tissue homeostasis (städeli et al., 2006) . β-catenin stabilization in latency type iii ebv-transformed b cells occurs by mechanisms that involve ebv latent membrane proteins 1 (lmp1) and 2a (lmp2a) (hayward et al., 2006) . this does, however, not necessarily entail its nuclear accumulation, indicating that effects of ebv induced β-catenin stabilization are dependent on the cellular context and in lymphoid cells may be distinct from wnt activation (morrison et al., 2004) . lately, pagano and coworkers identified lmp1 as a transcriptional down-regulator of another ub ligase complex that targets β-catenin for proteasomal degradation and is called seven in absentia homolog-1 (jang et al., 2005) . in any event, the observed up-regulation of dubs in ebv transformed b cells by ovaa et al. (2004) , together with the almost simultaneous demonstration that β-catenin in the type iii latently infected b cell line sav iii exists in a complex with active, yet unidentified dubs by shackelford et al. (2003) , strengthens the possibility that dubs contribute to ebv induced β-catenin stabilization. usp7 is an evolutionary conserved mammalian dub of the usp family, which was originally identified by its ability to bind to two different herpesviral proteins, namely, ebna1 of ebv, and the hsv-1 regulatory protein icp0 for which it has also been coined herpes virus-associated ubiquitin-specific protease (hausp) (everett et al., 1997; holowaty and frappier, 2004) . usp7 displays debranching activity against lysine-48 linked polyubiquitin chains and was shown to play an important part in the dynamic regulation of nuclear p53 turnover (brooks and gu, 2006; cheon and baek, 2006) . in unstressed cells, p53 is constitutively polyubiquitinated, which leads to its proteasomal degradation, keeping p53 levels low (gomez-lazaro et al., 2004) . p53 levels dramatically increase upon various types of stress, including viral infection, triggering either growth arrest or apoptosis. virus mediated gain of p53 ubiquitinating activity as a means of apoptosis avoidance was already mentioned above. expression of usp7, the target protein of ebna1 and icp0, however, effectively promotes increased levels of p53 by antagonizing proteasomal degradation of p53 through deubiquitination (li et al., 2004) . as may be expected, partial reduction of usp7 levels by rna interference (rnai) was shown to destabilize p53 (li et al., 2004) . with the collaboration of the adapter protein daxx (death domain associated protein) (tang et al., 2006) , usp7 also deubiquitinates the mouse double minute 2 (mdm2) oncogene, one of several e3s that mediate p53 proteasomal degradation. this counteracts autoubiquitination of mdm2 and its proteasomal degradation (li et al., 2004) . in fact, a genetic knockout of usp7 caused depletion of mdm2 and, despite the aforementioned rescue effect of usp7 on p53, effectively stabilized p53 (cummins et al., 2004; li et al., 2004) . brooks and gu (2006) have recently proposed that the predominant role of mdm2 during cellular stress is to ubiquitinate p53, and to keep its levels in check in order to maintain growth arrest while avoiding default execution of the apoptotic program and thereby to afford cell survival in the event of successful cellular repair and overcoming of the original challenge. in view of these activities, usp7 appears to be well-positioned to relay signals that regulate the p53-mdm2 pathway (fig. 2 ). but it also becomes clear that the possible reduction of p53 levels through viral inhibition of usp7 would likely need to be wellbalanced in order not to achieve the opposite. recent structurefunctional studies on the ebv protein ebna1 have brought to light how this protein may accomplish this and are outlined in the following. the role of the icp0-usp7 interaction in p53 metabolism during hsv-1 infection has as yet proven complex to delineated (see below). for a long time, ebna1 has been known as a regulator of both transcription and replication of the ebv genome, as well as being required for the segregation of ebv genomes with chromosomes during mitosis (frappier, 2004; wang and sugden, 2005) . ebna1 is essential for viral persistence, promotes cellular immortalization and is found consistently expressed in ebv-associated human malignancies including burkitt's lymphoma, hodgkin's lymphoma, and nasopharyngeal carcinoma. functional studies by saridakis et al. (2005) have indicated that an interaction of transfected ebna1 with usp7 fosters cellular degradation of p53, presumably by preventing p53 deubiquitination through usp7, and confers apoptosis resistance to uv-irradiated cells. structural analyses from the laboratories of shi and frappier have recently provided insight into the molecular mechanism by which ebna1 modulates p53 turnover and likely contributes to the antiapoptotic and survival factor function of ebna1 in vivo. crystal structures for short ebna1, p53, and mdm2 peptides, respectively, in complex with the n-terminal tumor necrosis factor-receptor associated factor (traf)-like domain of usp7 show that these three proteins use a consensus tetrapeptide recognition sequence to engage in structurally conserved contacts with the same surface groove of the traf-like domain of usp7 (hu et al., 2006; saridakis et al., 2005; sheng et al., 2006) . the buried contact surface areas and extent of directed interactions, as well as the measured binding affinities of corresponding peptides to the usp7 traf-like domain increase in the order of p53 < mdm2 < ebna1 (hu et al., 2006; sheng et al., 2006) . it appears that the competitive binding to usp7 of ebna1 versus p53 and mdm2 prevents deubiquitination of the lowest affinity binder (p53) sufficiently to reduce cellular p53 levels, while it permits deubiquitination of the intermediate binder (mdm2) to continue, avoiding mdm2 depletion and p53 stabilization, as observed in the usp7 knockout (fig. 2) . otherwise, i.e., in case mdm2 levels also fig. 2 . model for regulation of the p53-mdm2 pathway by usp7 and interference by ebna1. the e3 enzyme mdm2 catalyses both autoubiquitination and ubiquitination of p53 leading to proteasomal degradation in both cases. deubiquitination by usp7 stabilizes p53 and mdm2, with the adaptor protein daxx directing usp7 to mdm2. partial reduction of usp7 activity by rnai destabilizes p53 through reduced deubiquitination. contrarily, genetic knockout of usp7 increasingly commits mdm2 to proteasomal degradation thus leading to an important reduction in p53 ubiquitination and, indirectly, effective p53 stabilization. the ebv protein ebna1, mdm2, and p53 compete for same binding site on usp7, with affinities decreasing in this order. hence, inhibition of usp7 by ebna1 may exhibit selectivity and contribute twofold to p53 destabilization. by blocking the deubiquitination of p53 more efficiently than the deubiquitination of mdm2, it may allow for sufficient levels of mdm2 that maintain p53 ubiquitination. dramatically drop in the presence of ebna1, one of the remaining e3s for p53 (brooks and gu, 2006) may play an important role in keeping p53 levels low in ebv-infected cells. the immediate-early hsv-1 gene product icp0 is required for efficient initiation of lytic infection by stimulating the reactivation of quiescent viral genomes (hagglund and roizman, 2004) . icp0 functions as an e3 whose putative in vivo targets include p53. early in hsv-1 infection, icp0 associates with nuclear domain 10 (nd10), nuclear substructures that are found juxtaposed to the genomes of many dna viruses including herpesviruses and adenoviruses (everett, 2006) . nd10 have been implicated in dna repair, the ifn response, and the regulation of p53 activity (everett, 2006; takahashi et al., 2004) and might be preferred sites of transcription and replication of dna viral genomes (ching et al., 2005) . icp0 induces the proteasomal degradation of the nd10 organizing promyelocytic leukemia (pml) protein and promotes rapid dispersal of nd10 (everett, 2006) . this has been proposed to alleviate pml protein mediated anti-hsv-1 effects of ifn (chee et al., 2003) . recent results by everett et al. (2006) , obtained by the use of rnai to reduce pml levels, indeed argue for the contribution of pml to a cellular antiviral repression mechanism that is countered by icp0. everett and coworkers (1997) had previously demonstrated that icp0 increases the proportion of usp7 localized to nd10. contrary to ebna1, p53, and mdm2, the binding of icp0 to usp7 was mapped to a domain of unknown structure, located c-terminally to the catalytic core domain of usp7 (holowaty et al., 2003) . possible effects of icp0 on p53 metabolism were shown not to depend on the interaction of icp0 with usp7 but on the particular cell type under investigation . instead, the significance of icp0 binding to usp7 possibly lies in the ability of usp7 to counteract autoubiquitination of icp0 and to protect it from proteasomal degradation (canning et al., 2004) . although usp7 is conversely ubiquitinated and marked for proteasomal degradation by icp0, the biological net effect of the reciprocal activities between icp0 and usp7 is thought to be the stabilization of icp0 early during hsv-1 lytic infection or reactivation from latency, when icp0 levels are low (boutell et al., 2005) . interestingly, the aforementioned usp7-mdm2 adaptor protein daxx (tang et al., 2006 ) is a major nd10 component (everett et al., 2006) , raising the question whether it also has an adaptor function for usp7 in this nuclear compartment. deubiquitinating activities for viral enzymes have directly been demonstrated for the adenovirus protease adenain, sars-cov plpro, and herpesviral ul36 usp . structural aspects of these specificities have been reviewed recently by sulea et al. (2006) . in the following, potential roles for these confirmed viral dubs are considered against the background of their established functions and properties. during adenovirus infection, adenain is first made in an essentially inactive form of 23 kda which localizes to both the cytoplasm and the nucleus (reviewed by mangel et al., 2003) . in the nucleus, binding to viral dna partially activates the enzyme inside nascent virions, allowing it to cleave an eleven amino acid peptide, named pvic, from the precursor of the viral dna binding capsid protein vi (pvi). pvic binding fully activates adenain, and the peptidic cofactor becomes eventually disulphide-linked to the enzyme. activated adenain is thought to subsequently complete the proteolytic maturation of altogether six virus capsid precursor proteins inside the virion. the stepwise activation of adenain by viral dna and pvic prevents precursor protein cleavage before virion assembly and the generation of immature capsids. maturation of the capsid proteins is important for their ability to promote low-ph activated endosomal lysis and cytoplasmic entry of viral capsids during the next infection cycle (cotten and weber, 1995) . cytoplasmic adenain is believed to contribute to cell lysis and release of virions by the cleavage of cytoskeletal proteins, including cytokeratin 18 and actin. in contrast to nuclear adenain, the cytoplasmic form has no access to either of the two capsid bound viral cofactors, viral dna and pvic. instead, the c-terminal sequence of actin, which is highly homologous to pvic, efficiently replaces pvic acting as a cellular cofactor (brown and mangel, 2004) . within the extracellular virus, adenain resides in an oxidized and dormant state, but becomes activated again up on infection and re-entry into a reducing cellular environment. adenain eventually participates in the final steps of the viral uncoating program, i.e., dissociation of the viral dna from the capsid at the nuclear pore complex, by the digestion of pvi (greber et al., 1996) . using a biotinylated form of the specific dub inhibitor ubaldehyde as a probe, balakirev et al. (2002) retrieved adenain from a lysate of adenovirus-infected hela cells. they showed that the enzyme accounted for a time-dependent increase in global and, especially, nuclear deubiquitinating activity in late phase adenovirus-infected cells compared to mock-infected cells, as judged by the decline of the ub conjugate pool analyzed by western blotting with anti-ub antibodies (balakirev et al., 2002) . additionally, the authors overexpressed in the same cell line hexahistidine-tagged ub together with either adenain or an autocleavable fusion of the enzyme to its activating peptide cofactor pvic, followed by enrichment of ub conjugates from cell lysates over a metal affinity resin. anti-ub western blotting of the resulting fractions demonstrated, again, an overall reduction in ub conjugates with both versions of the transfected enzyme. enzymatic in vitro assays with recombinant purified adenain and substrate proteins, and chemically synthesized pvic peptide showed that adenain, indeed, exhibits debranching activity against lysine-48 linked polyubiquitin and could also process the isg15 precursor protein, but not a fusion of the yeast sumo homolog smt3 to the green fluorescent protein. this specificity, seemingly, conflicts with the structural classification of adenain as a member of the ulp family of desumoylating enzymes (balakirev et al., 2002) but agrees well with the molecular binding site features of the enzyme (reviewed by sulea et al., 2006) . the described prevalence of adenain-dependent deubiquitination of nuclear proteins in infected cells (balakirev et al., 2002) is in accordance with the presence of adenain in this subcellular compartment, where it is involved in virion maturation. at the same time, however, activation of nuclear adenain is thought to occur only inside the nascent virion particle, with about 70 adenain-pvic complexes remaining incorporated per particle (mangel et al., 2003) . while this may suggest that virion resident adenain deubiquitinates capsid proteins, no change in the overall pattern of the anti-ub staining in comparison to nuclei of mock-infected cells was observed (balakirev et al., 2002) , indicating that the overall decline in nuclear ub conjugates, for the most part, reflected deconjugation of cellular proteins. this could mean that activated adenain partially escapes from nascent virions and acts on proteins in the surrounding nucleoplasm (the possibility that nuclear adenain is activated in a virion independent manner is considered below). as already mentioned, besides hsv-1, adenoviruses belong to the dna viruses whose genomes associate with nd10. interestingly, nd10 in turn are associated with nuclear aggresomes, sites that recruit chaperones, ub, and proteasomes and that may be specialized in protein degradation (reviewed by wileman, 2006) . moreover, nd10 have been proposed to present passageways for proteins, including viral proteins that are destined for proteasomal degradation in the proximity of nd10 (bailey and o'hare, 2005; hay, 2005) . in this light, it is tempting to speculate that deubiquitination by adenain safeguards the delivery of viral proteins to nascent capsids by protecting them from proteasomal degradation at close-by aggresomes, much as cellular usp7 is thought to stabilize icp0 of hsv-1 (boutell et al., 2005) . it is also noteworthy that the plasmid mediated overexpression of adenain in hela cells, both with and without its peptidic cofactor, resulted in an overall loss of cellular ub conjugates (balakirev et al., 2002) . the strong dependence of adenain catalytic activity on cofactor complex formation and the ability of the c-terminal sequence of the traditionally cytoplasmic protein actin to act as a cofactor (brown and mangel, 2004) may suggest that predominantly cytoplasmic adenain activity was detected in this experiment. alternatively, a notable fraction of adenain may have entered the nucleus together with actin derived peptide cofactor. actin itself has also been identified in the nucleus as a component of protein complexes active in various aspects of gene transcription (grummt, 2006; miralles and visa, 2006) . actin may therefore also be involved in a virion independent stimulation of adenain activity in the nucleus. after entry of the coronaviral single-stranded positive-sense rna genome into the cytoplasm, the viral replicase gene is translated directly from it. two coronaviral proteases, 3c-like protease (3clpro) and plpro, are part of the replicase polyprotein, the precursor of the altogether sixteen non-structural proteins (nsps) that form the viral rna replication complex thiel et al., 2003) . 3clpro, on the one hand, is contained in nsp5, and after autocleavage, releases all downstream replicase subunits. plpro, on the other hand, originally referred to a domain of around 24 kda within nsp3, whose boundaries are defined by homology to the papain-fold (herold et al., 1999) . plpro processes the amino-proximal nsps (harcourt et al., 2004) . in accordance with previous structural bioinformatics prediction , the crystal structure of sars-cov plpro recently established the enzyme's membership in the usp family of dubs (ratia et al., 2006) . additionally, it revealed an unexpected ub-like domain at the n-terminus of the plpro catalytic core domain. ub-like domains are defined by a common β-grasp threedimensional structure (kiel and serrano, 2006) . within the multidomain nsp3, plpro is further preceded by an acidic sequence forming the n-terminus of nsp3, a macro-domain with adp-ribose-1″-phosphatase activity, which is potentially involved in viral rna modification (saikatendu et al., 2005) , and a so-called sars unique domain . plpro is followed by a hydrophobic domain with putative transmembrane regions (harcourt et al., 2004) . plpro cotranslationally liberates nsp1 to 3 in this order (harcourt et al., 2004) . all three cleavage products become part of the replication complex, which is found bound to double-membrane vesicles that are characterized by autophagosome markers (prentice et al., 2004) . a more recent ultrastructural study, however, points to the endoplasmic reticulum as the direct origin of the membranes associated with sars-cov replication complex, including nsp3 (snijder et al., 2006) . sars-cov plpro, which now refers to the enzyme's catalytic core domain plus the n-terminal ub-like domain, was recombinantly expressed and purified (barretto et al., 2005; lindner et al., 2005) . as predicted based on the similarity of its catalytic core domain to the corresponding domain of usp7 , sars-cov plpro displays dub activity. specifically, it debranches lysine-48 polyubiquitin chains, very efficiently hydrolyzes the general dub substrate ub-7-amino-4methylcoumarin (ub-amc) (barretto et al., 2005; lindner et al., 2005) , and exhibits isg15 precursor processing activity . although this proves the enzyme's proficiency as a dub in vitro, it is not clear whether sars-cov plpro can gain access to potential deubiquitination targets, other than replicase polyprotein sequences themselves, during its synthesis as part of the replicase polyprotein, cotranslational autoprocessing, and incorporation into the membrane bound replication complex. it is noteworthy that the synthesis of both negative-and plus-strand coronavirus rna requires ongoing viral protein production (kim et al., 1995; perlman et al., 1986; sawicki and sawicki, 1986) , and it is conceivable that concomitant deubiquitination by sars-cov plpro protects replicase subunits against proteasomal degradation. prompted by the known interaction of icp0 with cellular usp7, ploegh and coworkers made use of the active-site directed probe haubvme, mentioned above, to monitor dub activity in lysates of primary fibroblasts infected with hsv-1 . a major haub-adduct corresponding to an ∼ 47-kda protein occurred late in infection and persisted. the protein was identified as n-terminal fragment of the essential large (3164 amino acid residues) tegument protein ul36 (also called vp1/2 or icp1/2). further labeling attempts with similar ubl probes, and in vitro enzymatic assays using a 533 residues recombinant n-terminal fragment of ul36, established that the enzyme, baptized ul36 usp , is specific for ub, but exhibits relatively low catalytic efficiency. recombinant hsv-1 ul36 usp disassembled lysine-48 but not lysine-63 polyubiquitin chains which may implicate the enzyme in protein stabilization . sequence alignment of ul36 homologs from α-, β-, and γherpesvirus genomes identified a putative cysteine box around the haubvme reactive cysteine residue, and a histidine box located around 130 amino acids further downstream . although none of the ul36 usp sequences shows similarity to any known dub, the conservation of cysteine box and histidine box motifs is characteristic of cysteine protease dub families (amerik and hochstrasser, 2004) . in addition to hsv-1 (αsubfamily), analogous labeling experiments confirmed the presence of dub activity and specificity for ub in recombinant ul36 usp from ebv (γ-subfamily), and mcmv and hcmv (βsubfamily) (schlieker et al., 2005; wang et al., 2006) . the sequence of the recombinant ul36 usp variant examined for ebv extended only little beyond the histidine box motif, demonstrating that an n-terminal ul36 fragment of just under 22 kda carries the ul36 usp specificity (schlieker et al., 2005) . wang et al. (2006) detected haubvme labeling of fulllength ul36 in hcmv, simian cmv (scmv) and hsv-1infected fibroblasts. they also reported the detection of the 47-kda fragment of ul36 for hsv-1 previously described by kattenhorn et al. (2005) but ascribed its occurrence to uncontrolled proteolysis . the authors went ahead to confirm catalytic activity in isolated wild-type and mutant hcmv extracellular particles. mutation of either the putative catalytic cysteine or histidine residue abolished catalytic activity, identifying ul36 usp as the sole source of dub activity in the virion. for additional mutants of several conserved cysteine box and histidine box residues, the spread and development of cytopathic effects as well as virus yields in virusinfected cells were wild-type like. only the respective cysteine and histidine mutants gave notably lower virus yields and delayed the development of cytopathic effects. however, the cysteine mutation caused no apparent changes during cell infection as judged by electron microscopy. overall, this indicates that ul36 usp is important for optimal hcmv replication but is not essential in cell culture . so far, we can only speculate on possible roles for ul36 usp considering the known implications of the large tegument protein ul36, which according to earlier studies shows equal cytoplasmic and nuclear distribution (mcnabb and courtney, 1992) . ul36 was demonstrated to interact with a region of the viral α sequence, which is required for cleavage and packaging of the viral genome in the nucleus (chou and roizman, 1989) into icosahedral capsids (see pomeranz et al., 2005 , for a review of the α-herpesvirus life cycle). studies of α-herpesvirusinfected cells using microscopic techniques coupled with protein labeling methods have led to two different theories that aim at explaining how nucleocapsids exit the nucleus, obtain the viral tegument, and acquire an envelop. they imply different pathways for the capsid attachment of ul36, which is thought to form the innermost, capsid-proximal layer of the tegument. according to the more prevalent of the two theories (reviewed by mettenleiter, 2004; , viral capsids reach the cytoplasm by successive envelopment and deenvelopment at the inner and outer membrane of the nuclear envelope, respectively. primary attachment of ul36 in the cytoplasm then affords further assembly of inner tegument proteins. the inner tegument subsequently coalesces with the outer tegument, which assembles independently at future sites of budding into the exocytic pathway, likely into the trans-golgi network (mettenleiter, 2006) . the alternative theory (leuzinger et al., 2005; wild et al., 2005) assumes a dual pathway for nuclear egress of capsids. capsids may either undergo nuclear envelopment followed by intraluminal transport to the golgi, or they may leave the nucleus directly through dilated nuclear pores and afterwards bud from the cytoplasm into compartments of the exocytic pathway. here, the deposition of tegument is thought to occur during the budding at the inner nuclear membrane or at cytoplasmic membranes (leuzinger et al., 2005; wild et al., 2005) , hence presumably involving nuclear or cytoplasmic ul36, respectively. it has however to be noted that ul36 has not yet been detected as a component of virions inside the lumen of the nuclear envelope (mettenleiter and minson, 2006) . wherever herpesvirus tegumentation occurs, tegument proteins are known to engage in many complex protein-protein interactions (reviewed by mettenleiter, 2006) . yeast two-hybrid analyses suggest that ul36 uses a domain downstream of ul36 usp to directly bind to ul37, another inner tegument protein (klupp et al., 2002; vittone et al., 2005) . fusion of golgi derived vesicles with the plasma membrane eventually releases infectious virions from the cell. after infection by fusion of the viral envelope with the cell membrane, ul36 initially stays associated with the capsid. ul36 is in fact emerging as a main candidate among herpesviral proteins that may dock incoming capsids to the dynein motor during transport along microtubules (mts) to the nuclear pores (granzow et al., 2005) and during retrograd axonal transport (antinone et al., 2006; luxton et al., 2005) . in this regard, it is interesting to note that the minus end-directed transport of misfolded proteins to the microtubule-organizing center during aggresome formation was shown to require the interaction of the dynein motor with the cellular dub ataxin-3 (burnett and pittman, 2005) . as ul36 usp , ataxin-3 debranches lysine-48 polyubiquitin chains, but possible targets of this activity during retrograd transport are unknown. last of all, a temperature sensitive mutation in hsv-1 ul36 impeded the release of viral dna from the capsid into the nucleus at the nuclear pore (batterson et al., 1983) . taken together, during herpesvirus infection ul36 reportedly localizes to the nucleus, the cytoplasm, and the lumen of the nuclear envelope as well as the exocytic pathway where it is eventually part of the infectious virion, whose yield is reduced by mutational inactivation of ul36 usp . it remains speculative at this point whether deubiquitination of viral or cellular proteins by its ul36 usp domain plays part in any of the processes that have been associated with the presence of ul36 in these locations, i.e., nucleocapsid formation, tegumentation, budding, viral egress from and entry into cells, mt-dependent capsid transport, and nuclear entry of the viral genome. the genomes of some viruses encode ub or ub-like sequences. the baculovirus autographa californica nucleopolyhedrovirus, for example, expresses a viral ub precursor protein during the late phase of infection (guarino, 1990) . although non-essential for replication in cell culture, the gene is required for optimal virus production (reilly and guarino, 1996) . the baculoviral ub is functional in protein conjugation in vitro but inhibits the formation of more extended lysine-48 linked polyubiquitin chains necessary for proteasomal targeting (haas et al., 1996) . the authors speculated that this protects otherwise short-lived viral proteins from proteasomal degradation. viruses also express ub or ub-like sequences as part of multidomain polyproteins. intriguingly, ub and ubls including nedd8, sumo, and atg8 homologs represent the most frequent inserts in the polyprotein of several strains of bovine viral diarrhea virus (bvdv), where their presence is associated with a viral cytopathogenic phenotype (baroth et al., 2000; meyers et al., 1998; qi et al., 1998; tautz et al., 1993) . in fact, these inserts function as polyprotein processing signals that allow cellular proteases, presumably dubs, to process the polyprotein at positions corresponding to the precursors processing sites of the respective cellular ub or ubl sequences. in the case of an atg8 sequence insert in the bvdv isolate jacp, the processing enzyme was indeed identified as a specific cellular dub, namely, autophagin-1 (atg4b) (fricke et al., 2004) . compared to ub and ubls, the already mentioned n-terminal domain of sars-cov plpro (ratia et al., 2006) for example represents a different but not less common type of ub-like domain (kiel and serrano, 2006) . here, the lack of a c-terminal recognition sequence motif precludes proteolytic processing by dubs and subsequent conjugation to other molecules. nevertheless, such intrinsic ub-like domains also serve in mediating protein-protein interactions in a variety of multidomain proteins (see kiel and serrano, 2006, and references therein) . these include players of the ups, such as specialized ub receptors , and last but not least dubs (nijman et al., 2005; zhu and sulea, unpublished data) . the domain arrangement in sars-cov plpro is in fact reminiscent of usp14 and its homologs, where the ub-like domain likewise precedes the catalytic core domain. usp14 and its yeast homolog ubp6 both bind to the regulatory subunit of the proteasome via their ub-like domain which greatly stimulates their catalytic activities (hu et al., 2005; schmidt et al., 2005) . comparison of the crystal structures for the free and ub bound catalytic core domain of usp14 suggests that the enzyme is activated by conformational translocation of two enzyme surface loops, which block access of the ub c-terminus to the active site in the free enzyme (hu et al., 2005) . proteasome binding may promote this activation step. usp14 was further shown to debranch lysine-48 linked polyubiquitin chains from the distal end (hu et al., 2005) . usp14/ubp6 is thought to prevent the translocation of ub from incoming substrates into the inner core particle of the proteasome and to contribute to the homeostasis of the cellular pool of free ub (schmidt et al., 2005) . it is tempting to speculate that the ub-like domain of sars-cov plpro may similarly anchor the enzyme to a larger protein complex. there is, however, no indication from the available crystal structure of the free enzyme, like for usp14, that its catalytic activity may require activation (ratia et al., 2006) . the binding of icp0 and ebna1 to different domains of the cellular dub usp7 contributes to the maintenance of a productive life cycle for hsv-1 and the establishment of latency for ebv, respectively, representing the only well established examples of viral interference with deubiquitination so far. interestingly, however, infection by ebv, hcmv, and hpv modulates the activities of several cellular dubs (ovaa et al., 2004; rolén et al., 2006; wang et al., 2006) the significance of which remains to be established. dubs, such as usp9x, for instance, might be involved in β-catenin stabilization in latency type iii ebv-transformed b cells. it will further be important to establish the roles of viral deubiquitinating activities. like usp7, the three recently confirmed viral dubs, adenain, sars-cov plpro, and herpesvirus ul36 usp , all exhibit lysine-48 linked polyubiquitin debranching activities. it is conceivable that viruses avail themselves of this activity in order to stabilize viral gene products or host cell proteins whose proteasomal degradation promotes anti-viral responses such as iκb (evans, 2005) . the ability of adenain and sars-cov plpro to additionally cleave the isg15 precursor protein has led to the speculation that these enzymes mimic cellular usp18 (sulea et al., 2006) , a dub with delsgylating activity and a negative regulator of the ifn response (dao and zhang, 2005) . recent data by zhang and coworkers (malakhova et al., 2006) revealed, however, that usp18 attenuates jak-stat signaling, and thereby the type 1 ifn response, in a non-enzymatic manner, i.e., by directly competing with jak1 for binding to the ifnar2 subunit of the type 1 ifn receptor. as for sars-cov plpro, the importance of the deubiquitinating activity of usp18 remains to be determined. table 1 summarizes the demonstrated and potential roles in viral infection of both cellular and viral dubs discussed thus far. the review of the possible subcellular sites of action for the three confirmed viral dubs presented here suggests that their deubiquitinating activities could, similar to the polyprotein processing activities of adenain and sars-cov plpro, also contribute to more basic viral needs such as viral genome replication and packaging, or viral egress and entry. similar to adenain (greber et al., 1996) , ul36 usp may for example exhibit digestive activity during the release of viral dna from the capsid, explaining the prevention of this step in a temperature sensitive mutant of hsv-1 ul36 (batterson et al., 1983) . the example of ul36 usp of hcmv has shown that infection of cell cultures with mutant viruses does not necessarily cause a tangible phenotype. for adenain and sars-cov plpro it may prove difficult to dissect their essential functions in polyprotein cleavage from the significance of their dub activities. for sars-cov, the actual importance of the nsp1/ 2, nsp2/3, and nsp3/4 cleavages carried out by the plpro is still not clear. nevertheless, in cell culture infection nsp1 appears to suppress host gene expression by promoting host mrna degradation (kamitani et al., 2006) , and nsp2, although dispensable, was shown to be required for optimal virus replication (graham et al., 2005) . in order to, however, more comprehensively study potential roles of cellular and viral dubs in virus pathogenicity, e.g., through the modulation of the host antiviral immune responses, animal models of viral infection are needed (cf. arrode and davrinche, 2003; cantin et al., 1999; cinatl et al., 2005; jogler et al., 2006) . the acquisition of ub and ubl sequences by strains of bvdv as polyprotein processing signals is intriguing and suggests a possible scenario for the evolutionary origin of viral dubs. in bovine cells, the essential processing of the atg8 insert in the bvdv isolate jacp was demonstrated to be carried out by the dub atg4b. this processing step also occurred when an atg8 containing bvdv polyprotein sequence was recombinantly expressed in avian, fish, and insect cells, as well as in a rabbit reticulocyte lysate (fricke et al., 2004) . the utilization of a ub or ubl sequence as polyprotein processing signal may present an advantageous viral strategy because a newly infected host cell is very likely to already express a dub that can perform the processing reaction. it is tempting to speculate that ub or ubl sequence containing viruses originally have happened to insert sequences of cellular dubs into their genomes as functional protein processing enzymes, thereby becoming independent of the cellular enzymes. as a fitness advantage, this could have enhanced the viral host cell spectrum. table 1 demonstrated and potential roles of dubs in virus infection demonstrated roles a adenovirus protease adenain release of its own activating peptidic cofactor, maturation of capsid precursor proteins, promotion of cell lysis by cleavage of cytokeratin 18 and actin, support of viral uncoating, and dna release by capsid protein digestion at the nuclear pore mangel et al. (2003) sars-cov plpro processing of nsp1 to 3 from the viral replicase polyprotein harcourt et al. (2004) potential roles b usp9x stabilization of β-catenin in ebv-infected b cells stabilization of viral proteins kattenhorn et al. (2005) a among the viral enzymes with deubiquitinating activity, proteolytic roles during virus infection have only been demonstrated for the adenovirus protease adenain and sars-cov plpro. in both cases, however, they involve hydrolysis of polypeptides at regular peptide bonds by these enzymes and not isopeptide bond cleavage. b dubs have been proposed to benefit viral infection by stabilizing viral gene products or, selectively, cellular proteins by protecting them from proteasomal degradation. c the delsgylating activities of adenain and sars-cov plpro may mimic usp18, a demonstrated negative regulator of the interferon response (dao and zhang, 2005 ). yet, an isopeptidase independent mechanism for this function of usp18 was discovered recently (malakhova et al., 2006) , and the significance of its de-isgylating activity remains unclear. mechanism and function of deubiquitinating enzymes the herpesvirus capsid surface protein, vp26, and the majority of the tegument proteins are dispensable for capsid transport toward the nucleus dendritic cells and hcmv cross-presentation comparison of the sumo1 and ubiquitin conjugation pathways during the inhibition of proteasome activity with evidence of sumo1 recycling deubiquitinating function of adenovirus proteinase viruses and the 26s proteasome: hacking into destruction insertion of cellular nedd8 coding sequences in a pestivirus the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity viral modulators of cullin ring ubiquitin ligases: culling the host defense molecular genetics of herpes simplex virus: viii. further characterization of a temperature-sensitive mutant defective in release of viral dna and in other stages of the viral reproductive cycle late budding domains and host proteins in enveloped virus release chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family herpes simplex virus type 1 infection induces the stabilization of p53 in a usp7-and atm-independent manner reciprocal activities between herpes simplex virus type 1 regulatory protein icp0, a ubiquitin e3 ligase, and ubiquitin-specific protease usp7 balancing cell adhesion and wnt signaling, the key role of β-catenin p53 ubiquitination: mdm2 and beyond interaction of actin and its 11-amino acid cterminal peptide as cofactors with the adenovirus proteinase the polyglutamine neurodegenerative protein ataxin 3 regulates aggresome formation a ring finger ubiquitin ligase is protected from autocatalyzed ubiquitination and degradation by binding to ubiquitin-specific protease usp7 role for gamma interferon in control of herpes simplex virus type 1 reactivation promyelocytic leukemia protein mediates interferon-based anti-herpes simplex virus 1 effects hausp as a therapeutic target for hematopoietic tumors pml bodies: a meeting place for genomic loci? characterization of dna sequence-common and sequence-specific proteins binding to cis-acting sites for cleavage of the terminal a sequence of the herpes simplex virus 1 genome the ubiquitin proteolytic system: from a vague idea, through basic mechanisms, and onto human diseases and drug targeting development of antiviral therapy for severe acute respiratory syndrome the adenovirus protease is required for virus entry into host cells tumour suppression: disruption of hausp gene stabilizes p53 isg15: a ubiquitin-like enigma e3 ubiquitin ligases as regulators of membrane protein trafficking and degradation delivery of ubiquitinated substrates to proteinunfolding machines regulation of pro-inflammatory signalling networks by ubiquitin: identification of novel targets for anti-inflammatory drugs interactions between dna viruses, nd10 and the dna damage response a novel ubiquitin-specific protease is dynamically associated with the pml nuclear domain and binds to a herpesvirus regulatory protein pml contributes to a cellular mechanism of repression of herpes simplex virus type 1 infection that is inactivated by icp0 viral plasmids in mammalian cells processing of a pestivirus protein by a cellular protease specific for light chain 3 of microtubuleassociated proteins the ubiquitin-proteasome pathway in viral infections p53: twenty five years understanding the mechanism of genome protection the nsp2 replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for viral replication entry of pseudorabies virus: an immunogold-labeling study the role of the adenovirus protease on virus entry into cells actin and myosin as transcription factors identification of a viral gene encoding a ubiquitin-like protein functional characterization of the ubiquitin variant encoded by the baculovirus autographa californica role of icp0 in the strategy of conquest of the host cell by herpes simplex virus 1 ubiquitylation and cell signaling identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity sumo: a history of modification notch and wnt signaling: mimicry and manipulation by gamma herpesviruses a human rna viral cysteine proteinase that depends upon a unique zn2+-binding finger connecting the two domains of a papain-like fold hausp/usp7 as an epstein-barr virus target protein interaction domains of the ubiquitin-specific protease, usp7/ hausp structure and mechanisms of the proteasome-associated deubiquitinating enzyme usp14 structural basis of competitive recognition of p53 and mdm2 by hausp/usp7: implications for the regulation of the p53-mdm2 pathway up-regulation of β-catenin by a viral oncogene correlates with inhibition of the seven in absentia homolog 1 in b lymphoma cells replication properties of human adenovirus in vivo and in cultures of primary cells from different animal species severe acute respiratory syndrome coronavirus nsp1 protein suppresses host gene expression by promoting host mrna degradation a deubiquitinating enzyme encoded by hsv-1 belongs to a family of cysteine proteases that is conserved across the family herpesviridae modification of proteins by ubiquitin and ubiquitin-like proteins the ubiquitin domain superfold: structure-based sequence alignments and characterization of binding epitopes coronavirus protein processing and rna synthesis is inhibited by the cysteine proteinase inhibitor e64d pseudorabies virus ul36 tegument protein physically interacts with the ul37 protein herpes simplex virus 1 envelopment follows two diverse pathways a dynamic role of hausp in the p53-mdm2 pathway the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme siah-1 mediates a novel β-catenin degradation pathway linking p53 to the adenomatous polyposis coli protein from the cover: targeting of herpesvirus capsid transport in axons is coupled to association with specific sets of tegument proteins ubp43 is a novel regulator of interferon signaling independent of its isg15 isopeptidase activity specific interactions of the adenovirus proteinase with the viral dna, an 11-amino-acid viral peptide, and the cellular protein actin analysis of the ul36 open reading frame encoding the large tegument protein (icp1/2) of herpes simplex virus type 1 budding events in herpesvirus morphogenesis intriguing interplay between viral proteins during herpesvirus assembly or: the herpesvirus assembly puzzle egress of alphaherpesviruses herpesvirus assembly: a tale of two membranes insertion of a sequence encoding light chain 3 of microtubule-associated proteins 1a and 1b in a pestivirus genome: connection with virus cytopathogenicity and induction of lethal disease in cattle actin in transcription and transcription regulation differential signaling pathways are activated in the epstein-barr virusassociated malignancies nasopharyngeal carcinoma and hodgkin lymphoma a genomic and functional inventory of deubiquitinating enzymes activity-based ubiquitin-specific protease (usp) profiling of virusinfected and malignant human cells getting into position: the catalytic mechanisms of protein ubiquitylation mhv nucleocapsid synthesis in the presence of cycloheximide and accumulation of negative strand mhv rna molecular biology of pseudorabies virus: impact on neurovirology and veterinary medicine. microbiol identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins insertion of a bovine smt3b gene in ns4b and duplication of ns3 in a bovine viral diarrhea virus genome correlate with the cytopathogenicity of the virus severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme merops: the peptidase database the viral ubiquitin gene of autographa californica nuclear polyhedrosis virus is not essential for viral replication the ubiquitin binding domain znf ubp recognizes the c-terminal diglycine motif of unanchored ubiquitin activity profiling of deubiquitinating enzymes in cervical carcinoma biopsies and cell lines the ubiquitin-proteasome machinery is essential for nuclear translocation of incoming minute virus of mice structural basis of severe acute respiratory syndrome coronavirus adp-ribose-1″-phosphate dephosphorylation by a conserved domain of nsp3 structure of the p53 binding domain of hausp/usp7 bound to epstein-barr nuclear antigen 1: implications for ebv-mediated immortalization coronavirus minus-strand rna synthesis and effect of cycloheximide on coronavirus rna synthesis a deubiquitinating activity is conserved in the large tegument protein of the herpesviridae proteasome-associated proteins: regulation of a proteolytic machine targeting of host-cell ubiquitin pathways by viruses epstein-barr virus activates {beta}-catenin in type iii latently infected b lymphocyte lines: association with deubiquitinating enzymes molecular recognition of p53 and mdm2 by usp7/ hausp unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex transcription under the control of nuclear arm/[beta]-catenin deubiquitination, a new function of the severe acute respiratory syndrome coronavirus papainlike protease? structural aspects of recently discovered viral deubiquitinating activities pml nuclear bodies and apoptosis critical role for daxx in regulating mdm2 lc3 conjugation system in mammalian autophagy processing of poly-ubiquitin in the polyprotein of an rna virus the deubiquitinating enzyme fam interacts with and stabilizes beta-catenin mechanisms and enzymes involved in sars coronavirus genome expression determination of interactions between tegument proteins of herpes simplex virus type 1 origins of bidirectional replication of epstein-barr virus: models for understanding mammalian origins of dna synthesis high-molecular-weight protein (pul48) of human cytomegalovirus is a competent deubiquitinating protease: mutant viruses altered in its active-site cysteine or histidine are viable ubiquitin and ubiquitin-like proteins as multifunctional signals impairment of nuclear pores in bovine herpesvirus 1-infected mdbk cells aggresomes and autophagy generate sites for virus replication the ubiquitin-proteasome system facilitates the transfer of murine coronavirus from endosome to cytoplasm during virus entry the comments on the text by traian sulea and robert ménard are gratefully acknowledged. this is nrcc publication no. 47552. key: cord-298934-vtrfqozl authors: makino, shinji; shieh, chien-kou; soe, lisa h.; baker, susan c.; lai, michael m.c. title: primary structure and translation of a defective interfering rna of murine coronavirus date: 1988-10-31 journal: virology doi: 10.1016/0042-6822(88)90526-0 sha: doc_id: 298934 cord_uid: vtrfqozl abstract an intracellular defective-interfering (di) rna, disse, of mouse hepatitis virus (mhv) obtained after serial high multiplicity passage of the virus was cloned and sequenced. disse rna is composed of three noncontiguous genomic regions, representing the first 864 nucleotides of the fend, an internal 748 nucleotides of the polymerase gene, and 601 nucleotides from the 3′ end of the parental mhv genome. the disse sequence contains one large continuous open reading frame. two protein products from this open reading frame were identified both by in vitro translation and in di-infected cells. sequence comparison of disse and the corresponding parts of the parental virus genome revealed that disse had three base substitutions within the leader sequence and also a deletion of nine nucleotides located at the junction of the leader and the remaining genomic sequence. the 5′ end of disse rna was heterogeneous with respect to the number of ucuaa repeats within the leader sequence. the parental mhv genomic rna appears to have extensive and stable secondary structures at the regions where di rna rearrangements occurred. these data suggest that mhv di rna may have been generated as a result of the discontinuous and nonprocessive manner of mhv rna synthesis. mouse hepatitis virus (mhv), a member of the coronaviridae, contains a single-stranded, positive-sense rna of approximately 6 x lo6 da (lai and stohlman, 1978; wege eta/., 1978) . in infected cells, the genomic rna of mhv is first translated into an rna-dependent rna polymerase (brayton et al., 1982 mahy et a/., 1983) which is responsible for the synthesis of a genomic-sized negative-stranded rna (lai et a/., 1982b) . the negative-stranded rna then serves as the template for the synthesis of six subgenomic and a genomic-sized mrna lai et a/., 198213) . these mrnas are arranged in the form of a 3' coterminal "nested" set, i.e., the sequence of each mrna is contained entirely within the next larger mrna (lai et a/., 198 1; leibowitz et al., 1981) . in addition, each mrna has a common leader sequence, which is derived from the 5' end of the genome (lai et a/., 1982a (lai et a/., , 1983 spaan et al., 1983) . several pieces of evidence demonstrated that mhv utilizes a novel mechanism of leader rna-primed transcription, in which a free leader rna species derived from the 5' end of genomic rna is utilized as a primer for the transcription of subgenomic mrnas (baric eta/., 1983 (baric eta/., , 1985 makino et a/., 1986b) . another unusual feature of coronavirus rna synthesis is that the virus undergoes rna-rna recombination at a very high frequency (makino eta/., 1986a) . the ' to whom requests for reprints should be addressed. unusually high frequency, approaching 10% under some circumstances (makino et al., 1986a) , of coronavirus rna recombination suggests that discontinuous rna transcripts might be generated during coronavirus rna synthesis. these incomplete rna intermediates may rejoin the original or different rna template to continue rna synthesis, resulting in rna recombination in the latter case. the detection of such rna intermediates in mhv-infected cells (baric et a/., 1985 (baric et a/., , 1987 suggests that coronavirus genomic rna synthesis involves a discontinuous and nonprocessive mechanism, which may account for the high frequency of recombination via a copy choice mechanism. defective-interfering (di) particles are naturally occuring deletion mutants that have been described for many virus groups. characteristically, di particles (a) lack part of the viral genome, (b) contain normal viral structural proteins, (c) replicate only with the aid of a helper standard virus, and (d) interfere with replication of homologous standard virus. deletion of genomic sequence can occur in various regions of the genome; however, all of the di rnas apparently retain signals for rna replication since they can be replicated in the presence of helper virus. the generation of di rna can be viewed as the result of abnormal rna replication or illigitimate rna recombination. therefore, the structure of di rna is of particular interest in elucidation of the mechanism of viral rna replication and recombination. we have previously reported the generation of di particles during high multiplicity passages of the jhm 0042-6822188 $3.00 copyright q 1999 by academic press. inc. all rights of reproduction in any form reserved. in a 1% agarose gel without denaturation. numbers 1, 2, 3, 6, and 7 represent the major mhv-jhm-specific mrna species. strain of mhv (mhv-jhm) (makino eta/., 1984a) . in diinfected cells, the synthesis of most of the standard viral mrnas is inhibited. instead, three distinct virusspecific rna species could be detected (makino eta/., 1985) (fig. 1 ). the first species, dlssa, is equivalent to dl virion rna in length and is eventually incorporated into virus particles. this rna differs from the standard virus genome in that it contains multiple deletions distributed throughout the genome, except for the 5' and 3' ends of the genomic rna (makino et a/., 1985) which encode rna polymerase (gene a) and nucleocapsid (n) protein, respectively. surprisingly, dlssa rna can replicate by itself in the absence of helper virus infection, suggesting that dlssa codes for functional rna polymerases (makino et a/., 1988) . thus, dlssa is not a defective rna in a strict sense. the second major rna species found in di-infected cells is indistinguishable from the mrna 7 made by the standard virus. the synthesis of this mrna and its product n protein is not inhibited in di-infected cells. the third rna species is a novel single-stranded polyadenylated di rna species of varying size. oligonucleotide fingerprinting studies suggest that it represents se-quences derived from various noncontiguous parts of the genome. the size of this rna varies with the di passage level (makino et a/., 1985) . one of these rnas, dlsse, which is the smallest di rna detected, has been analyzed in greater detail (makino et al., 1988) . in contrast to dlssa, dlsse rna synthesis requires helper virus coinfection (makino et al,, 1988) . only a trace amount of it is incorporated into virus partcles to serve as a template for rna replication (makino et a/., 1988) . thus, it may lack packaging signals. on the other hand, since it is efficiently replicated in diinfected cells, dlsse rna must contain the sequences essential for viral rna replication. in the present study, we analyzed the primary structure of dlsse rna. the results revealed that dlsse consists of three noncontiguous regions of mhv-jhm genomic rna, including 5'end leader rna and the 3'end of genomic rna. one large open reading frame (orf) was demonstrated and the product of this orf was identified both in infected cells and by in vitro translation. possible mechanisms of di rna generation are discussed. viruses and cell culture mhv-jhm was used as a nondefective standard virus. serially passaged mhv-jhm stock at passage level 17 was used as the source of di particles (makino et al., 1985) . all viruses were propagated in dbt cells as described previously (makino et al., 1984a ). mhv-specific intracellular rna was extracted by procedures described previously (makino et a/., 1984b) . poly(a)-containing rna was obtained by oligo(dt)-cellulose column chromatography (makino et a/., 198413) . agarose gel electrophoresis 32p-labeled virus-specific rna was analyzed by electrophoresis on 1% agarose gels without denaturing as described previously (makino et al., 1988) . poly(a)-containing rna was purified by preparative gel electrophoresis in 1 o/o urea-agarose gels as previously described (makino et a/., 1984a) . the rna was eluted from gel slices by the methods of langridge et al., (1980) . cdna cloning of dlsse cdna cloning followed the general method of gubler and hoffman (1983) . five hundred nanograms of oli-go(dt),2-,8 was mixed with 2 pg of gel-purified dlsse rna in 10 ~1 of distilled water. the rna and oligo(dt) mixture was heated at 70"for 3 min and chilled quickly. the rna-dna hybrid was then incubated in 50 ~1 of first-strand cdna synthesis buffer containing 60 units of rnasin (promega biotec), 50 mll/l tris-hci (ph 8.3 at 42') 100 mll/l kci, 10 ml\/l mgc12, 10 mm dlt, 1.25 mmeach of datp, dctp, dgtp, and ltp, and 20 units of avian myeloblastosisvirus reverse transcriptase (life science) at 42" for 1 hr. the cdna synthesis was stopped by adding 4.4 ~1 of 250 mm edta. nucleic acids were extracted with phenol-chloroform-isoamyl alcohol and precipitated with ethanol. second-strand synthesis was carried out in a reaction volume of 100 ~1 containing 20 ml\/l tris-hci (ph 7.4), 5 mfl/l mgc12, 100 m/l/l kci, 50 pg/ml of bsa, 10 mm (nh&s04, 0.15 mlll /3-nad, 100 plm dntps, 25 units of dna polymerase i, 2 units of escherichia co/i dna ligase, 0.8 units of rnase h, and the product from the first strand reaction. the mixture was incubated at 12" for 1 hr, and then at 22" for 1 hr. the reaction was stopped by adding 8.7 ~1 of 250 mm edta, and products were extracted with phenol-chloroform-isoamyl alcohol, and precipitated with ethanol. doublestranded dna was dc-tailed in a 1 ~-pi reaction mixture containing 10 units of terminal transferase, 200 mm potassium cacodylate, 0.5 rnn/l coci,, 25 mm tris-hci (ph 6.9) 2 mm dlt, 250 pglml bsa, and 50 pi\/i dctp at 37" for 4 min. the dc-tailed double-stranded dna was annealed to 200 ng of dg-tailed pstl-cut pbr322 plasmid in 20 ~1 of a buffer containing 10 mm tris-hci (ph 7.4), 100 mlli naci, and 0.25 mm edta. the dna mixture was heated at 68" for 5 min and then cooled slowly overnight for annealing. the annealed molecules were used to transform e. co/i mc 106 1 as described (dagert and ehrlich, 1979) . identification of large cdna clones containing dlsse sequence 32p-labeled mhv-jhm gene a cdna clones c96 and f82 (shieh eta/., 1987) and 5'end 32p-labeled leader-specific 72-mer derived from leader sequence of mhv were used for colony hybridization to isolate dlsse-specific cdna clones. colonies yielding a strong signal were further analyzed by southern hybridization (maniatis et a/., 1982) . the gel-purified rnas were incubated in 8 ~1 of distilled water containing 10 mn/r methyl mercury. after 10 min incubation at room temperature, rna was incu-bated in 50 ~1 of first-strand cdna synthesis buffer with 28 mm /3-mercaptoethanol and 5' end-labeled oligodeoxyribonucleotides at 42" for 1 hr. reaction products were extracted with phenol-chloroform-isoamyl alcohol, precipitated with ethanol, and analyzed by electrophoresis on 6% polyacrylamide gels containing 8.3 m urea and were eluted from the gels according to the published procedures (maxam and gilbert, 1980) . sequencing was carried out by sanger's dideoxyribonucleotide chain termination method (sanger et a/., 1977) and maxam-gilbert chemical modification procedure (maxam and gilbert, 1980) as described previously . sequence analysis and predicted rna secondary structures were obtained with the lntelligenetics sequencing program. an mrna-dependent rabbit reticulocyte lysate (new england nuclear) was used as previously described . antisera a monoclonal antibody, j.3.3, directed against the mhv-jhm n protein has been described (fleming et a/., 1983) . the anti-p28 antibody was generated in rabbits against a synthetic peptide representing a portion of the mhv-jhm p28 protein (soe et a/., 1987) and will be described in detail elsewhere (s. c. baker et a/., manuscript in preparation). labeling of intracellular proteins, immunoprecipitation, and sds-polyacrylamide gel electrophoresis dbt cells were infected with either wild type mhv-jhm or mhv-jhm containing di particles at 2 pfu per cell. at 7.5 hr postinfection, cells were labeled in methionine-free medium containing 30 pci of l-[35s]methionine/ml (icn translabel) for 30 min. cell extracts were prepared by treatment with lysolecithin (l-a-lysophosphatidylcholine, palmitoyl; sigma) at 125 pg/ml for 1 min at 4". the treated cells were scraped in 300 ~1 hnd buffer (0.1 m hepes, ph 8.0, 0.2 m nh&i, 0.005 m dtt), disrupted by pipetting with a pastuer pipet, and then centrifuged at 800 g for 5 min to remove nuclei and cell debris. the resulting supernatant was used for immunoprecipitation. lmmunoprecipitation was performed by the methods of kessler (198 1) . the cell-free extracts were incubated with 3 ~1 of antisera for 4 hr at 4". the antigen-antibody complexes were collected by binding to pansorbin (calbiochem, la jolla, ca) and washed three times with washing buffer (50 mll/ltris-hci, ph 7.4, 150 mlll naci, 5 mm edta, and 0.5% np-40) and eluted by boiling for 2 min in electrophoresis sample buffer (0.1 n/rpmercaptoethanol, 1 o/o sds, 0.08 11/1 tris-hci, ph 6.8, and 10% glycerol). the bacteria were removed by centrifugation and proteins were analyzed by electrophoresis on 5 to 15% sds-polyacrylamide gels (laemmli, 1970) . cdna cloning and sequencing of dlsse rna to understand the primary structure of dlsse rna, dlsse-specific cdna clones were generated according to the general method of gubler and hoffman (1983) using oligo(dt) as a primer and gel-purified dlsse rna. since previous oligonucleotide fingerprinting analysis suggested that dlsse rna contains the leader sequence and the 5' end region of genomic sequence (makino et al., 1985) cdna clones were screened by colony hybridization using 5' end-labeled, leader-specific 72-mer, and two cdna clones f82 and c96, which correspond to the 5' end of genomic rna of mhv-jhm . several large cdna clones were isolated and their structure was further analyzed. a diagram representing the structure of the dlsse genome and that of mhv-jhm genomic rna and the strategy used for sequencing the cdna clones are shown in fig. 2 . the dlsse sequence obtained is shown in fig. 3 . sequence analysis of dlsse cdna clones revealed that dlsse rna consists of three different regions of mhv-jhm genomic rna. the first region represents 864 nucleotides from the 5' end of the genomic rna. the second region, 748 nucleotides in length, is a region within the polymerase gene that corresponds to the region at 3.3 to 4 kb from the 5' end of genomic rna (shieh, unpublished observation) , and the third region contains a sequence of 601 nucleotides derived from the extreme 3'end of the genomic rna. the entire sequence of dlsse rna is identical to that of the corresponding regions of mhv genomic rna shieh et a/., unpublished data) , with some exceptions in the leader sequence region (see below). the cdna clones obtained does not appear to have a complete sequence at its extreme 5' end. to understand the complete 5' end sequence of dlsse, we performed primer-extension studies on dlsse rna using a specific primer (5'~aatgtcagcactatgaca-3') complementary to nucleotides 123-140 from the 5' end of the genome of mhv-jhm . the ei'end-labeled primer was hybridized to gel-purified dlsse rna and extended with reverse transcriptase. primer extension products were then analyzed by electrophoresis on 6% polyacrylamide gels containing 8 m urea. as shown in fig. 4a , two cdna products of 136 and 131 nucleotides were obtained, indicating heterogeneity at the 5'end sequence of dlsse. these primerextended products were sequenced by the maxam-gilbert method. the sequences of both cdna products were identical except that the faster migrating cdna products contained three ucuaa repeats at the 3'end of the leader sequence, while the slower migrating species contained four ucuaa repeats (fig. 4b ). in addition, the 5'end sequences of dlsse and mhv-jhm genomic rna showed several differences. within the leader sequence, 3 bases were substituted in dlsse rna (fig. 4b , asterisks) and nine nucleotides (uuuau-aaac) were deleted in dlsse at the junction between the leader rna and the remaining genomic sequences. the significance of the heterogeneity in the number of ucuaa repeats and of the nine-nucleotide deletion will be discussed below. another significant feature of dlsse rna is the presence of a single large orf (fig. 3) . this orf is expected to share amino acid sequence identity with three different regions of the standard mhv-jhm. the first 218 amino acids correspond to the n terminus of the mhv polymerase. this region represents the part of the n-terminus of the polymerase protein which is cleaved into a p28 protein (denison and perlman, 1986; soe et al., 1987) . the following 250 amino acids were derived from the region of the polymerase at 3.3 to 4 kb from the 5'end of the genome. the 3'end region of the orf of dlsse rna is the same as the orf utilized for the n protein . thus, the predicted product of this orf should contain the n-terminus of p28 and the c-terminus of the n protein. the predicted molecularweight mass of this orf product is 62,538. to examine whether the orf of dlsse rna is utilized for translation, we first performed in vitro translation in a rabbit reticulocyte lysate of dlsse rna purified from the di-infected cells. two proteins with an apparent molecular mass of approximately 88,000 (88k) and 79,000 (79k) were detected (fig. 5a ). both proteins were immunoprecipitated with anti-n protein monoclonal antibody and anti-p28 antibody (fig. 5a, lanes 2 and 3) . therefore, these two proteins were likely the translation products of dlsse rna. a minor band of approximately 60 kda had the same electrophoretic mobility as the n protein of mhv-jhm, and was precipitated with anti-n monoclonal antibody, but not with anti-p28 antibody (fig 5a, lanes 2 and 3) . thus, this protein is most likely the n protein translated from the contaminated mrna 7 in the dlsse rna preparation. the synthesis of dlsse-specific protein in di-infected cells was then examined. dbt cells were mock-infected (fig. 5b, lanes 1 and 4) , infected with mhv-jhm (fig. 5b, lanes 2 and 5) or infected with mhv-jhm containing di particles (fig. 5b, lanes 3 and 6) . both 88k and 79k proteins were specifically immunoprecipitated with anti-n monoclonal antibody and anti-p28 antibody from di-infected cells. the amount of these two proteins was low as compared to the n protein. nevertheless, they were reproducibly detected in di-infected cells. thus, the dlsse rna is a functional mrna. the relationship between the two protein species detected is not clear. the discrepancy between the predicted and observed molecular weights of the translation products of dlsse could be due to post-translational modification of the protein or aberrant migration of the protein. a small amount of p28 was immunoprecipitated with anti-p28 antibody in mhv-jhm-infected cells (fig. 5b, lane 5) . however, this protein was hardly detectable in di-infected cells (fig. 58, lane 6) . the absence of detectable amount of p28 in di-infected cells may be due to the inhibition of mhv-jhm genomic rna synthesis by di particles (makino et al,, 1985) . possible secondary structure at the di rna rearrangment sites sequence analysis revealed that dlsse rna consisted of three noncontiguous regions of mhv-jhm genomic rna. we have previously proposed that coronavirus rna synthesis proceeds by a discontinuous, nonprocessive mechanism, being interrupted at sites with hairpin loops (baric et a/., 1987) . this transcriptional interruption could account for the generation of soe et al., 1987) was 3zp-labeled at the 5' end, hybridized to the gel-purified dlsse rna, and extended with reverse transcriptase. the products were electrophoresed on 6% polyacrylamide gels containing 8 m urea. 0, origin of the gel. two primer-extended products are shown as a and b. (b) the dna sequences of these primerextended products were determined by the maxam-gilbert method. the 5'.end sequence of mhv-jhm genomic sequence was obtained from previous studies soe ef a/., 1987) . the letters a and b represent the canonical seven-nucleotide sequence ucuaaac and imperfectly repeated sequence of uauaaac, respectively. a bold solid line represents the nine-nucleotide sequence which is deleted in dlsse but present in mhv-jhm. dlsse (a) and dlsse (b) correspond to the sequences of primer-extended products, a and b, in fig. 4a , respectively. three base substitutions are indicated by asterisks. di rnas. we therefore examined whether any significant secondary structure existed at rearrangement sites on mhv-jhm genomic rna. the nucleotide sequences surrounding deleted regions of mhv-jhm genomic rna were analyzed by an rna secondary structure program of zuker and stiegler (1981) . the predicted secondary structures of these rearrangement regions are shown in fig. 6 . all four genomic deletion sites have extensive and stable secondary structures. the free energies of these structures range from -73.0 to -114.2 kcal/mol. furthermore, as previously described for the standard mhv-jhm, the sequence surrounding the junction of leader rna and the remaining 5'-end genomic sequence also contains a stable secondary structure (soe eta/., 1987) . this junction region includes the nine-nucleotide deletion detected in dlsse rna (fig. 4b) . thus, an extensive and stable secondary structure exists at each parental mhv-jhm genomic region where deletion occurred. the present study demonstrated that the smallest di-specific rna, dlsse, is composed of three discontiguous parts of the viral genome, including the 5' end and 3'end of genomic rna. this structure is similar to many di rnas of other viruses, which typically retain both ends of the standard nondefective viral rnas. our previous study has demonstrated that dlsse is replicated from its negative template in the presence of helper virus (makino et al., 1988) . therefore, the dlsse sequence likely contains essential recognition signals for mhv rna replication. the structure of dlsse rna supports the likelihood that the recognition signals for the synthesis of negative-strand rna and positivestrand rna are localized at the 3' end and 5' end of genomic rna, respectively. one of the unique features of coronavirus di rna is that subgenomic di rna was poorly incorporated into virus particles (makino et a/., 1988) . one of the possible explanations is that the di subgenomic rnas lack a packaging signal. since all mhv-specific subgenomic mrnas contain the leader sequence, yet only genomic-sized rna is efficiently packaged into virus par-ticles, the packaging signal is probably located in gene a but not in leader sequence. the present study indicates that dlsse rna has a nine-nucleotide (uuuau-aaac) deletion at the junction between the leader rna and the remaining genomic rna sequence. however, this deletion is not likely to account for the failure of efficient di rna packaging into virus particles since dlssa and the genomic rna of a mutant mhv-jhm, both of which are packaged into virus particles, also have similar nine-nucleotide deletions (s. makino, unpublished data). thus, the packaging signals may be localized downstream of the 5' end 864 nucleotides. recently we found that another intracellular di-specific rna, dlssf, could be packaged more efficiently than dlsse (s. makino, unpublished data). the dlssf rna is approximately i .7 kb larger than dlsse and appears to contain more gene a sequences than dlsse, as determined from tl -oligonucleotide fingerprinting (makino et al., 1985) . sequence analysis of dlssf may reveal the possible reason for the poor incorporation of dlsse rna into virus particles. the data presented in this paper demonstrate extensive and stable secondary structures in the standard viral rna at sites where di rna underwent deletions. this observation is consistent with a model of di rna generation, in which rna transcription is interrupted at sites of hairpin loops on the template, and the rna intermediates then fall off and rebind at new sites on the template to generate an rna with extensive deletions. we have previously suggested that coronavirus rna synthesis may utilize a discontinuous, nonprocessive mechanism, in which rna transcription pauses at sites of secondary structures (baric et a/., 1987) . the incomplete rna intermediates dissociate from templates and then rejoin the temple for subsequent rna transcription. this mechanism is supported by the findings that mhv can undergo rna recombination at an extremely high frequency (makino et a/., 1986a) , and that free incomplete rna transcription products of various sizes were detectable in the cytoplasm of mhv-infected cells (baric et al., 1985 (baric et al., , 1987 . furthermore, the sizes of these rna products correspond to the lengths between the 5' end and the sites of hairpin loops (baric et al,, 1987) , in agreement with the notion that transcription pauses at these hairpin loops. thus, the potential hairpin loops present in the genomic rna at the di rna rearrangement sites could have interrupted rna transcription. the incomplete rna tran-script may join the rna template at the downstream rearrangement sites and create deleted rna as a result. however, there is no consensus sequence at the sites of rna deletion and reinitiation. it is not known how the reinitiation of rna synthesis occurred. the deletion of the nine nucleotides (uuuauaaac) at the 5' end where the leader rna joins the genomic rna may have been caused by the same discontinuous and nonprocessive transcription mechanism. it is interesting to note that the ucuaaac, which is the consensus sequence for the leader rna binding is imperfectly repeated (uauaaac) at nine nucleotides downstream . it is these nine nucleotides which were deleted in dlsse rna. similar nine-nucleotide deletions have also been noted in the genomic rna of dlssa, and that of an mhv-jhm mutant virus (s. makino, unpublished data) . this rna structure suggests that rna synthesis may pause at the first repeat, and then reinitiate at the second repeat because of the binding of the incomplete rna transcript to the second repeat. finally, the heterogeneity in the number of ucuaa repeats in di rnas also supports the discontinuous nature of coronavirus rna replication. similar heterogeneity has been noted in the genomic rna of several different mhv strains (s. makino and m. m. c. lai, manuscript in preparation) . thus, di rna may be a product of discontinuous, nonprocessive rna replication of coronaviruses. there was a significant difference between the apparent molecular mass of the dlsse-specific protein products, 88k and 79k, and the predicted molecular mass of the potential product of the large orf of dlsse rna. this difference could be due to unusual configurations affecting electrophoretic migration, or due to the presence of phosphorylation, since the n protein is phosphorylated (stohlman and lai, 1979) and protein translated in vitro could be phosphorylated (chattopadhyay and banerjee, 1987) . a similar difference between the predicted and actual molecular mass of the n protein has previously been noted . the relationship between the two protein species is not clear. the n protein has also been shown to consist of multiple species (robbins et al., 1986) . it is not clear whether these proteins play any functional roles in di-infected cells. typically, di rnas do not synthesize any protein; however, in the sindbis virus system, translation products have been detected from a di rna (migliaccio et al., 1985) . although mhv genomic rna and dlsse rna are the major rna species among mhv-specific mrna species in virus-infected cells (makino et a/., 1985 (makino et a/., , 1988 (fig. l) , the gene products of these two mrnas, rna polymerase and both the 79k and 88k proteins, were present in small quantities in virus-infected ceils (fig. 5b) . we have previously demonstrated that the presence of stable secondary structure at the 5' end noncoding regions of the polymerase gene reduced the amount of polymerse protein synthesized in vitro . also, as discussed previously, the presence of the small orf encoding eight amino acids (fig. 3 ) may reduce the number of ribosomes reaching the downstream optimal translation site . since dlsse rna has a 5' end structure similar to that of genomic rna, the dlsse rna may provide a tool to better our understanding of the mechanism of translational control of mhv rnas. furthermore, the fusion protein synthesized by dlsse rna may be useful for understanding the functional and structural domains of the mhv polymerase and n protein. analysis of intracellular small rnas of mouse hepatitis virus: evidence for discontinuous transcription characterization of replicative intermediate rna of mouse hepatitis virus: presence of leader rna sequences on nascent chains. 1. viral characterization of leader-related small rnas in coronavirus-infected cells: further evidence for leader-primed mechanism of transcription characterization of two rna polymerase activities induced by mouse hepatitis virus. 1. i/irol further characterization of mouse hepatitis virus rna-dependent rna polymerases phosphorylation within a specific domain of the phosphoprotein of vesicular stomatitis virus regulates transcription in vitro prolonged incubation in calcium chloride improves the competence of escherichia co/i cells translation and processing of mouse hepatitis virus virion rna in a cell-free system antigenic relationships of murine coronaviruses: analysis using monoclonal antibodies to jhm (mhv-4) virus a simple and very efficient method for generating cdna libraries use of protein a-bearing staphylococci for the immunoprecipitation and isolation of antigens from cells cleavage of structural proteins during the assembly of the head of bacteriophage t4 characterization of leader rna sequences on the virion and mrnas of mouse hepatitis virus, a cytoplasmic rna virus mouse hepatitis virus a59: mrna structure and genetic localization of the sequence divergence from hepatotropic strain mhv-3 presence of leader sequences in the mrna of mouse hepatitis virus further characterization of mrnas of mouse hepatitis virus: presence of common 5'-end nucleotides. f. viral replication of mouse hepatitis virus: negative-stranded rna and replicative form rna are of genome length rna of mouse hepatitis virus extraction of nucleic acids from agarose gels the virus-specific intracellular rna species of two murine coronaviruses: mhv-a59 and mhv-jhm rna-dependent rna polymerase activity in murine coronavirusinfected cells structure of the intracellular defective viral rnas of defective interfering particles of mouse hepatitis virus high-frequency rna recombination of murine coronaviruses defective-interfering particles of murine coronavirus: mechanism of synthesis of defective viral rnas leader sequences of murine coronavirus mrnas can be freely reassorted: evidence for the role of free leader rna in transcription defective interfering particles of mouse hepatitis virus analysis of genomic and intracellular viral rnas of small plaque mutants of mouse hepatitis virus molecular cloning: a laboratory manual sequencing end-labelled dnawith base-specific chemical cleavages mrna activity of a sindbis virus defective-interfering rna rna-binding proteins of coronavirus mhv: detection of monomeric and multimeric n protein with an rna overlay-protein blot assay dna sequencing with chain-terminating inhibitors the 5'-end sequence of the murine coronavirus genome: implications for multiple fusion sites in leaderprimed transcription coronovirus jhm: nucleotide sequence of the mrna that encodes nucleocapsid protein sequence and translation of the murine coronavirus 5'-end genomic rna reveals the n-terminal structure of the putative rna polymerase coronavirus mrna synthesis involves fusion of non-contiguous sequence phosphoproteins of murine hepatitis viruses. f. viral genomic rna of the murine coronavirus jhm optimal computer folding of large rna sequences using thermodynamics and auxiliary information we thank ming-fu chang for his valuable suggestions for dna sequencing and david vannierfor excellent technical assistance. we also thank carol flores for typing the manuscript. this work was sup key: cord-275403-g4rohhtt authors: bautista, elida m.; faaberg, kay s.; mickelson, dan; mcgruder, edward d. title: functional properties of the predicted helicase of porcine reproductive and respiratory syndrome virus date: 2002-07-05 journal: virology doi: 10.1006/viro.2002.1495 sha: doc_id: 275403 cord_uid: g4rohhtt abstract porcine reproductive and respiratory syndrome virus (prrsv) is a member of the positive-strand rna virus family arteriviridae. although considerable research has focused on this important pathogen, little is known about the function of most prrsv proteins. to examine characteristics of putative nonstructural proteins (nsp) encoded in orf1b, which have been identified by nucleotide similarity to domains of equine arteritis virus, defined genomic regions were cloned and expressed in the prset expression system. one region, nsp10, encoded a protein with a putative helicase domain and was further examined for functional helicase-like activities. prrsv nsp10 was found to possess a thermolabile and ph-sensitive ntpase activity that was modulated by polynucleotides and to unwind dsrna in a 5′ to 3′ polarity. these results provide the first evidence of the functional properties of prrsv helicase and further support the finding that nidovirus helicases possess properties that distinguish them from other viral helicases. porcine reproductive and respiratory syndrome virus (prrsv) emerged in the late 1980s and became a pathogen of scientific interest due to its economic importance. analysis of prrsv structure, genomic organization, and expression and its biological, physicochemical, and pathogenic properties has shown that prrsv is related to equine arteritis virus (eav), lactate dehydrogenaseelevating virus of mice (ldv), and simian hemorrhagic fever virus (shfv) collins et al., 1992; meulenberg et al., 1993; plagemann and moennig, 1992) . these viruses are grouped in a new family designated arteriviridae and placed with the coronaviridae family within the order nidovirales (cavanagh, 1997) . prrsv has been revealed to consist of two distantly related genotypes, north american (na, 15.4 kb) and european (eu, 15.1 kb), based on sequence analysis (allende et al., 1999; meulenberg et al., 1993; murtaugh et al., 1995 murtaugh et al., , 1998 nelsen et al., 1999) . the genome of prrsv encodes at least eight open reading frames (orfs) that are expressed in the infected cell as a nested set of subgenomic mrnas. orf1, translated from the full-length rna template, codes for replicase-related nonstructural proteins and is the focus of this study. orfs 2 to 7 encode mostly structural polypeptides. orf1 contains two long open reading frames, designated orf1a and orf1b, that are autocatalytically processed by virally encoded proteases (meulenberg et al., 1993) . orf1b is only expressed by ribosomal ϫ1 frame shifting due to a slippery sequence and pseudoknot structure in the overlapping orf1a/orf1b junction (allende et al., 1999; den boon et al., 1991; brierly et al., 1989; meulenberg et al., 1993; nelsen et al., 1999; wootton et al., 2000) . the catalytic domains and cleavage sites for eav orf1 have been identified. the eav proteases, located within nsp1, nsp2, and nsp4, have been shown to cleave eav orf1ab into 12 nonstructural proteins (nsps), 8 in orf1a and 4 in orf1b (den boon et al., 1995; snijder et al., 1992 snijder et al., , 1993a snijder and meulenberg, 1998; van dinten et al., 1996 . eav nsp1 autocatalytically cleaves the orf1 protein immediately downstream between g260 and g261; nsp2 is proposed to cleave far downstream between two glycine residues located at eav amino acids 1367 and 1368, and nsp4, the major protease, cleaves orf1 between amino acids glutamine and glycine (eg) or glutamine and serine (es) (ziebuhr et al., 2000) . based on significant homology in the catalytic domains and in predicted cleavage sites, it was surmised that prrsv polymerase may undergo similar proteolytic processing as that of eav (snijder and meulenberg, 1998; wassenaar et al., 1997; ziebuhr et al., 2000) . the four predicted orf1b nonstructural polypeptides comprise a putative rna-dependent rna polymerase (pol or nsp9), a protein (nsp10) containing a putative metal-binding domain (mbd) and nucleoside triphos-phate binding or helicase motif (hel), and two proteins of unknown function (nsp 11 and 12). nsp11 contains a domain (corona) that is conserved in all nidoviruses (allende et al., 1999 (allende et al., , 2000 den boon et al., 1991; godeny et al., 1993; gorbalenya et al., 1989; herold et al., 1996; meulenberg et al., 1993; nelsen et al., 1999; shen et al., 2000; snijder and meulenberg, 1998; van dinten et al., 1996 wootton et al., 2000) . despite extensive research on the molecular and biological properties of prrsv, there are important aspects of the disease and biology that remain unclear. the role of the nonstructural genes encoded in orf1ab in viral replication, pathogenesis, and immunity have not been demonstrated. this information is necessary to devise effective strategies to prevent and control prrsv infection of swine. the knowledge available for the genes encoded in orf1ab is limited to sequence information for a few virus isolates (allende et al., 1999 (allende et al., , 2000 jiang et al., 2000; meulenberg et al., 1993; nelsen et al., 1999; shen et al., 2000; wootton et al., 2000) and predictions on their function based on sequence homology and studies on eav and coronaviruses (gorbalenya et al., 1989; herold et al., 1996; snijder and meulenberg, 1998; . to elucidate the role of the nonstructural genes of arteriviruses in virus replication, previous studies have been done with eav (12.7 kb) to develop and characterize infectious clones and their derivative mutants (de vries et al., 2000; van dinten et al., 1997 van dinten et al., , 2000 van marle, 1999) . these studies have provided important insights as to the critical role of the genes encoded in orf1ab in arterivirus replication. however, prrsv is genetically divergent from eav, possessing only approximately 41% (orf1a) to 48% (orf1b) nucleotide identities to the sequenced eav cell-adapted avirulent strain . in addition, infectious clones of prrsv have been more difficult to prepare and analyze, apparently due to the increased genomic size of prrsv (15.1-15.4 kb), the restricted cell tropism of this virus, and the difficulty in transforming these permissive cells with nucleic acids . we have chosen a virulent field isolate of prrsv to explore phenotypic properties of this virus as it exists in nature. to demonstrate and characterize prrsv orf1b polypeptides, we have generated recombinant individual nsp proteins that were then analyzed under controlled conditions. this article describes the first functional studies of the putative prrsv helicase. two overlapping orf1b gene segments were amplified by rt-pcr and designated cf and ih. these gene segments were cloned and confirmed to be prrsv-14specific nucleotides by sequence analysis. the nucleotide sequence of prrsv-14 orf1b has been deposited in genbank (accession no. af298771). nucleotide sequence comparison of prrsv-14 orf1b revealed that this isolate is closely related to prrsv-na strains (data not shown). sequence analysis also determined that the predicted cleavage sites for prrsv orf1b were maintained in isolate prrsv-14 (allende et al., 1999; . these predicted cleavage sites appear to be well conserved among prrsv strains (table 1) . genome clones cf and ih both contained an overlapping unique saci site that was used to generate a 5262-nt ch fragment, which comprised all orf1b flanked by minimal orf1a sequence at the 5ј end and by a small amount of orf2 sequence at the 3ј end. the ch fragment was cloned and used as a template to generate pcr-amplified orf1b nsp gene fragments (fig. 1a) . these gene fragments were cloned into prset-b plasmids for protein expression and designated ptprrsv14 nsp9-12. western blot analysis with t7.10 ab demonstrated that the produced recombinant proteins corresponded to polypeptides of the predicted molecular size (fig. 1b) . to confirm the specificity of the individual nsps, antipeptide rabbit sera were generated as described under materials and methods. western blot analyses with nsp10 antipeptide rabbit sera (␣-hel, fig. 1b ) or other gene-specific anti-peptide sera (data not shown) demonstrated the specificity of prrsv-14-specific individual nsps. experiments were conducted to obtain soluble recombinant prrsv-14 orf1b proteins. optimal conditions for nsp10, which is predicted to have rna helicase and atpase functions (meulenberg et al., 1993) , were obtained with the escherichia coli strain bl21(de3)plyse, which has been designed to have more stringent expresnote. orf1b amino acid alignment of north american and european isolates (genbank accession nos. are indicated in the text) was performed using the pileup program of seqweb version 1.2 of the wisconsin package version 10.1. the predicted cleavage sites are underlined and based on sequence homology to the predicted sites described for eav (meulenberg et al., 1993; nelsen et al., 1999; van dinten et al., 1996 . lowercase indicates amino acid difference at that position compared to the reference na strain. sion of the t7 polymerase. in addition, culturing of transformed bacteria at 22°c and the use of reduced-nutrient media further improved expression. under these conditions and after 3 h of iptg induction, approximately 60-80% of expressed recombinant nsp10 protein was soluble. efforts were directed to obtain purified nsp10 and to characterize its predicted ntpase function and demonstrate its helicase activity. to obtain purified recombinant prrsv-14 helicase, proteins in soluble extracts from bacteria transformed with ptprrsv14-nsp10 were adsorbed by affinity to cobalt or nickel resins and eluted as described. samples obtained in the last elution fractions from nickel columns provided a highly purified helicasesoluble protein preparation (ͼ95% pure based on coomassie blue staining, fig. 2 ). protein preparations obtained with cobalt resins also yielded adequate yields but had reduced atpase activity compared to nickel affinity-purified protein (data not shown). therefore, nickel affinity-purified nsp10 helicase was used for the characterization of its inherent atpase and helicase activities. with similar conditions, soluble nsp11 and nsp12 were obtained (data not shown) and used as controls in the subsequent experiments as indicated. as initial proof of helicase-like activity, experiments were performed to test the ability of purified prrsv-14 nsp10 protein to hydrolyze radiolabeled ribonucleotides in the presence or absence of polyribonucleotides. from the autoradiographic films of the chromatographs (fig. 3) , it was apparent that prrsv nsp10 helicase hydrolyzed all four nucleotides in the absence of polynucleotides. polynucleotides did not appear to significantly affect the level of nucleotide hydrolysis, except for a slight reduction in the level of hydrolysis of ctp in the presence of poly(u). to further characterize the ntpase function of prrsv nsp10 helicase, the subsequent studies were performed with atp as substrate. primers used for amplification and the numbers in italics represent the predicted molecular size of the amplified products. the ih-and cf-amplified gene segments were selected for cloning and sequencing. the cloned ih and cf gene fragments were consequently ligated at the saci restriction site to generate the ch fragment (represented by the cross-hatched bar) that contains the complete orf1b gene of prrsv-14. the recombinant polypeptides from prrsv-14 orf1b that were expressed in the prokaryotic system are presented by lines. the number in italics on the lines represent the predicted molecular size in kilodaltons (kda) of prrsv-14 orf1b polypeptide fragments. the histidine (his)-t7.10 tag derived from the expression vector increased the molecular size in approximately 3.6 kda. (b) western blot analysis of the expressed recombinant proteins with the indicated antibodies described in the text. molecular markers (mm) are indicated by the numbers at the left in kda. a positive control protein for the monoclonal antibody t7.10 was included (t7ϩc). as negative control, bacterial extracts that were transformed with empty plasmids (prset) were included to test each antibody. the protein bands that were specifically recognized by the antibodies and had the expected molecular size of the predicted polypeptide are indicated by arrows. the level of atpase activity in recombinant prrsv helicase was compared to the atpase activity in control samples. the control samples were obtained following the same procedure used for purification of recombinant helicase and consisted of protein preparations (equalized by protein concentration) obtained from bacteria transformed with one of the following plasmids: (1) empty prset, (2) ptprrsvnsp11, or (3) ptprrsvnsp12. the results demonstrated that the hydrolysis of atp appeared specific for recombinant nsp10 (prrsv helicase) and not for possible contaminating bacterial proteins. these results also established that both predicted nsp11 and predicted nsp12 products lacked a specific atpase activity (fig. 4) . the atpase activity of prrsv helicase was shown to be dependent on recombinant protein concentration (fig. 4 ). prrsv helicase atpase activity was also determined to be dependent on ph (optimal ph range was between 7.5 and 8.5) and influenced by the assay buffer used (fig. 5a ). with tris buffer, the atpase activity of prrsv helicase was stable over a broader ph range than with the other buffers tested (fig. 5a ). there was no significant differences seen in the level of atpase activity using tris buffer concentrations in the range of 10 to 100 mm (data not shown). further characterization of the atpase activity was performed using 25 mm tris buffer at ph 8.0. prrsv helicase activity was dependent on the presence of divalent ions, as the presence of edta inhibited the recombinant proteins' atpase activity in a dose-dependent manner (fig. 5b ). in addition, there was no atpase activity observed in the absence of divalent ions (fig. 5c ). optimal atpase activity was detected at 1-2 mm for all the four divalent ions tested. whereas the level of atpase activity of prrsv helicase consistently remained stable over a broader range of mgcl 2 concentrations, increasing concentrations of mnso 4 inhibited the activity in a dose-dependent manner and to a greater extent than the other divalent ions tested (fig. 5c) . lastly, the atpase activity of prrsv helicase was very sensitive to temperature. optimal activity was detected between 25 and 30°c. the atpase activity was significantly reduced at 37.5°c and was only detected at background levels at temperatures at and above 40°c and completely abolished at 60°c (fig. 5d ). kinetic analysis of recombinant helicase atpase activity revealed dependence on time (fig. 6a ) and substrate concentration (fig. 6b) . the michaelis-menten constant (k m ) for the atpase activity of prrsv helicase was determined to lie in the range of 6-13 mm atp, with a maximum velocity of 1.3-2 mm atp hydrolyzed per min (fig. 6c ). to assess whether the level of atpase activity would be affected by the presence of polynucleotides, kinetic analysis was performed in the presence or absence of poly(u). poly(u) significantly enhanced the atpase activity of prrsv helicase at a given atp level (fig. 7a ). although maximum velocity of enzymatic activity was not reached, comparison of the initial velocities of the relative atpase activities showed that the presence of poly(u) increased the k m and v max at least 19-and 24fold, respectively. however, at low atp concentrations, poly(u) did not have an effect or tended to reduce the atpase activity ( table 2 ). the fold increase in the level of atp hydrolyzed induced by poly(u) was in a close linear proportion (r 2 ϭ 0.9817) to the concentration of atp substrate (fig. 7b) . these data indicate that the effect of polynucleotides on the atpase activity of prrsv is dependent on atp substrate concentration. this result also explains our initial findings (fig. 4) , in which no apparent effect of the polynucleotides was detected when the atp concentration was limited and the level of atpase activity present in the assay was nearly 100% of the substrate. last, the effect of the presence of other mononucleotides on the level of atpase activity was ascertained using the bioluminescence detection method (mcelroy and deluca, 1983 ). this assay has been shown to spefig. 4 . specificity of the atpase activity of prrsv-14 helicase. the specificity of the atpase activity was determined by testing the indicated protein concentrations of purified recombinant proteins prepared as described in the text. the atpase assay was performed using 0.1 m of atp substrate and the level of atp remaining in the samples was measured after 30 min incubation. the level of atpase activity is expressed as the percentage of atp hydrolyzed in the atpase assay. data represent the mean ϯ standard deviation of triplicate samples. cifically detect atp (moyer and henderson, 1983) . in addition, we confirmed that the measurement of atp by the bioluminescence method was not affected by the presence of ctp, gtp, or utp (data not shown). as shown in fig. 8 , ctp, gtp, and utp inhibited the level of atp hydrolysis by prrsv helicase only when they were present at concentrations at least 10-fold higher than the atp substrate. this result confirms that prrsv nsp10 helicase protein can interact with all four nucleotides and, furthermore, this helicase protein may have a higher affinity for atp than for the other nucleotides. to demonstrate whether recombinant prrsv helicase possessed functional rna unwinding activity, various substrates were designed and tested in a standard helicase assay. these substrates consisted of two partially complementary rna strands containing single-stranded (ss) regions at the 3ј or 5ј ends of one or both strands. one set of substrates designed to contain single-strand regions at both 3ј and 5ј ends did not initially form the expected partial duplex structures under initial hybridiza-tion conditions. one explanation is that these long oligonucleotides are predicted to form secondary structures (energies of ϫ25 to ϫ30 kcal/mol; data not shown) and therefore might not have initially annealed to the other strand. however, duplexes or other higher ordered structures appear to form in the presence of recombinant helicase (data not shown). one suggestion for this result is that prrsv helicase first promoted unwinding of initial interoligonucleotide structures, then winding to form the expected duplexes, and/or introducing secondary structures in the labeled rna strand. to further analyze the predicted helicase activity of the prrsv nsp10 protein, experiments were performed using duplex substrates prepared with synthetic oligonucleotides containing single-strand regions at the 3ј or 5ј ends and short duplex regions (21 and 24 bp). results obtained with rna 3јduplex and 3ј3јduplex indicated that prrsv helicase lacked 3ј-to-5ј unwinding activity (fig. 9a ). in contrast, results with the rna 5јduplex revealed that prrsv helicase has unwinding activity in the 5ј-to-3ј orientation (fig. 9b) . the 5ј-to-3ј orientation of the unwinding activity of the prrsv helicase was further confirmed in experiments using substrates prepared with in vitro transcribed rna that contained singlestrand regions at the 5ј end of both strands and duplex regions of 67 bp (5ј5јduplex #1) and 85 bp (5ј5јduplex #2), as shown in fig. 9b . these results, together with the atpase data described above, demonstrate that the recombinant prrsv nsp10 helicase is functionally active and shares the properties reported for helicases of other members of the nidovirales (seybert et al., 2000a,b) . the nonstructural polypeptides encoded in nidovirales orf1b regions have been predicted to have essential roles in virus replication and gene expression (allende et al., 1999; gorbalenya et al., 1989; meulenberg et al., 1993; nelsen et al., 1999; van dinten et al., 1996) . evidence for the significant role of these proteins in arteriviruses has been derived using an infectious clone of eav (van dinten et al., 1997 (van dinten et al., , 2000 van marle et al., 1999) . however, as stated previously, eav is genetically distinct from other arteriviruses, possessing only approximately 48% nucleotide identity and 53.8% amino acid identity in the orf1b region when compared to prrsv-na . thus, it is crucial to gather basic information on distantly related arteriviruses to examine variability in orf1b nucleotide composition and function and to assess outcomes of potential orf1b region mutations. this study was completed to provide molecular tools to facilitate understanding of the biochemical and functional properties of prrsv-na-predicted orf1b-processed polypeptides. accordingly, we have cloned and expressed predicted orf1b protein products of prrsv-14 in a prokaryotic system and determined optimal conditions for functional expression of soluble nsp10, nsp11, and nsp12. of these three polypeptides, nsp10 was chosen for further biochemical characterization, as this predicted protein has been shown by sequence comparison to contain conserved ntpase and helicase domains (allende et al., 1999; nelsen et al., 1999; meulenberg et al., 1993) of the superfamily (sf) 1 of helicases (kadare and haenni, 1997; seybert et al., 2000a,b) . prrsv-na nsp10 bears only approximately 45% amino acid similarity to nsp10 of eav , illustrating remarkable interfamily divergence and rendering possible differences in precise dynamics for this critical protein. the described studies demonstrated that the predicted nsp10 product containing the metal binding and ntpase-helicase domains of prrsv orf1b has ntpase activity. the initial ntpase analysis, determined by thin-layer chromatography, revealed that prrsv helicase hydrolyzes the four ribonucleotides in the absence of polynucleotides. the interaction of the recombinant prrsv helicase with the nucleotides was confirmed by demonstrating that the presence of excess gtp, ctp, and utp inhibited the level of atp hydrolysis. the ability of prrsv helicase to catalyze hydrolysis of different nucleotides is a characteristic shared by various reported viral helicases (bisaillon et al., 1997; kadare et al., 1996; li et al., 1999; preugschat et al., 1996; rikkonen et al., 1994) . we demonstrated that the activity observed for the recombinant his-tagged nsp 10 is specific for the helicase because the recombinant prrsv orf1b nsp11 and nsp12 polypeptides that were expressed and purified under the same conditions as prrsv helicase did not have specific atpase activity. although atpase activity has been previously reported for the human coronavirus 229e helicase (heussip et al., 1997; seybert et al., 2000a) , a member of the order nidovirales, and more recently for eav (seybert et al., 2000b) , this is the first study reporting the characterization of the ntpase activity of the putative helicase of prrsv. the characterization of the atpase activity revealed some features of prrsv helicase that are common among known viral helicases such as the requirement for divalent cations and sensitivity to extreme ph and temperatures. in contrast to other viral helicases (bisaillon et al., 1997) , prrsv helicase appears to be extremely sensitive to temperatures above physiological levels. this finding suggests that viral or cellular factors may be required to stabilize prrsv helicase function. the atpase activity of prrsv helicase was also dependent 9 . prrsv helicase activities. rna substrates were generated by annealing two partially complementary rna strands for use in a standard helicase (hel) assay. rna substrate predicted duplex structures are indicated by diagrams above each assay result. a sample lacking prrsv helicase (ϫh) was included to indicate migration of intact radiolabeled duplex, and denatured substrates (ϫh, 95°c) were included to indicate migration of unduplexed labeled short strand. migration of substrates after the helicase assay is indicated on each electropherogram as single-stranded (ss) or double-stranded (ds) oligomers. (a) results of the helicase assay with substrates containing 3ј single regions at one (3јduplex) or both (3ј3јduplex) strands and duplex regions of 21 and 24 bp, respectively. (b) prrsv helicase activity displaced the labeled short strand of 5ј tailed rna substrates with duplex lengths of 21 (5јduplex), 67 (5ј,5јduplex-#1), and 85 bp (5ј5јduplex-#2). note. the atpase assay was performed as described under materials and methods at the indicated atp concentrations in the presence (ϩ) or absence (ϫ) of poly(u) in triplicate. * statistical significance was determined by paired two-sample t-test with alpha at 0.05. on protein concentration. although the presence of polynucleotides was not required for detecting the ntpase activity of prrsv helicase using the standard tlc method, poly(u) had a significant effect on this activity when the activity was measured at various atp substrate concentrations. interestingly, the effect of poly(u) in the atpase activity of prrsv helicase was dependent on the substrate concentration. at atp concentrations lower than the estimated k m , poly(u) had an inhibitory effect, whereas at higher atp concentrations, poly(u) had a stimulatory effect on the atpase activity of prrsv helicase. it was also interesting to note that the level of effect of poly(u) was in a close linear relationship with the atp substrate concentration. the substrate concentration-dependent modulatory effect of polynucleotides on the atpase activity of prrsv helicase found in this study has not been previously described. data reported for other viral helicases are consistent with a stimulatory effect that varies depending on the type of helicase and polynucleotide used (rikkonen et al., 1994; tamura et al., 1993) . the level of nucleic acid stimulation reported for the known members of the viral sf1 and sf3 helicases has only been in the range of a twofold increase, whereas higher levels of stimulation have been reported for sf2 helicases (kadare and haenni, 1997) . the atpase activity of the human coronavirus 229e, which belongs to the same nidovirales order as prrsv, was found to be highly stimulated with polynucleotides and the major stimulatory effect was found with poly(u), which increased the ntpase activity of the helicase up to 50-fold (heusipp et al., 1997; seybert et al., 2000a) . a 20-fold increase in the atpase activity by polynucleotides was found for the eav helicase (seybert et al., 2000b) . for prrsv helicase we found that whereas the overall effect of poly(u) on the k m and v max reached levels of 19-and 24-fold increase, respectively, the effect on the amount of atp hydrolyzed varied from ϳ0.6-fold (at 0.3 m atp substrate) to 6.5fold (at 100 m atp substrate). experimental data analyzing the effect of polynucleotides under the range of atp substrate concentrations used in this study have not been reported for other viral helicases. therefore, it is not clear whether our findings represent a unique feature of prrsv helicase. we suggest that the modulatory effect of poly(u) on the atpase activity of prrsv helicase discovered in this study may have biological relevance regarding virus replication and gene expression. it is tempting to speculate that if prrsv helicase is involved in the processes of its genome replication and transcription, as suggested by the studies with the mutant eav infectious clones (van dinten et al., 1997 (van dinten et al., , 2000 van marle et al., 1999) , prrsv helicase needs to be regulated to achieve a balance between nucleotide hydrolysis and viral rna synthesis. analysis of the crystal structure of prrsv helicase will be important to determine whether the regulatory effect of polynucleotides in the atpase activity of prrsv helicase is dependent on conformational changes differentially induced by various ratios of atp substrate to polynucleotide bound to the protein. to further characterize prrsv helicase function in terms of its major predicted role in rna winding, studies analyzing the ability of the protein to interact with various substrates were performed. the recombinant helicase did not exhibit 3ј to 5ј unwinding activity on synthesized oligomer duplexes of 22 or 24 bp and single-strand regions of 15-16 nucleotides. the prrsv helicase did demonstrate 5ј-to-3ј unwinding activity on duplexed substrates with single-stranded segments on one or both 5ј ends. these results are consistent with the 5ј-to-3ј unwinding properties found for eav (seybert et al., 2000b) and hcov (seybert et al., 2000a) , supporting the finding that nidovirales sf1 helicases are closely related with functional properties different to those of other viral helicase families. this study provides the first evidence on both atpase and helicase-like activities of prrsv helicase and suggests a regulatory role of polynucleotides on its atpase activity and unwinding properties. the molecular tools developed in this study will be useful for further studies directed to understand the role of the predicted intermediate orf1b gene protein products in prrsv replication. a prrsv isolate, designated prrsv-14, was obtained from the national veterinary services laboratories (nvsl, ames, ia). selection of isolate prrsv-14 was based on its clinical history and its ability to replicate well in the marc-145 cell line (kim et al., 1993) . the virus was isolated from the serum of an 8-day-old piglet exhibiting interstitial pneumonia. the piglet had come from an alabama farm experiencing a reproductive disease outbreak in march of 1996. prrsv-14 was propagated in marc-145 cells to passage level 9-10. culture fluid containing virions was concentrated by ultracentrifugation through a 0.5-m sucrose cushion and further purified by sucrose gradient centrifugation as described (bautista et al., 1996) . viral rna was extracted from the purified virus with trizol reagent (gibco-brl), concentrated by alcohol precipitation, suspended in rnase-free double distilled water, and stored at ϫ70°c until analyzed. rt-pcr, cloning, and sequencing analysis of prrsv-14 orf1b genomic segments prrsv-14 gene fragments containing orf1b sequences were amplified by rt-pcr. reverse transcription was performed in a 100-l reaction containing 1 g of purified viral rna, 0.25 g random hexamers, and 1000 u superscript ii reverse transcriptase (gibco-brl), following the manufacturer's recommendations. polymer-ase chain reactions (pcr) were performed in 50 l reactions using 2 l viral cdna, 200 m dntps, 1 u high-fidelity vent (new england biolabs), and various set of primers (table 3) according to the strategy depicted in fig. 1a . the reactions were performed by an initial denaturation for 5 min at 94°c, followed by 30 cycles and 10 min final extension at 72°c. the cycles consisted of denaturation at 95°c for 45 s, annealing at 58°c for 1 min, and extension at 73°c for 2.10 min ϩ 10 s/cycle. the extension time for gene products longer than 3000 nt was performed for 3.2 min ϩ 10 s/cycle. two overlapping fragments, designated cf and ih (fig. 1a) , were cloned into the pcrblunt vector system (invitrogen). plasmid dna from selected cf and ih clones were purified using a plasmid purification kit (qiagen) according to the manufacturer's instructions and confirmed by enzymatic restriction and sequence analysis. sequence analysis was performed with plasmid-specific primers at the automated sequence facility at eli lilly and co., indianapolis, in. prrsv sequences used for nucleotide and amino acid comparison included published genbank sequences u87392 (vr-2332; nelsen et al., 1999), af066183 (respprrs; yuan et al., 2001) , af066384 (primepac; yuan et al., 1999) , af184212 (sp, shen et al., 2000) , af176348 (pa8, wootton et al., 2000) , and m96262.1 (lelystad; meulenberg et al., 1993) . cloned cf and ih fragments were joined at a unique saci restriction site to generate a 5262-nt ch fragment (fig. 1a) , which comprised the last 182 nt of orf1a, all of orf1b, and the first 711 nt of orf2. four ch clones (ch2, ch11, ch18, and ch37) were confirmed for proper frame and orientation by restriction digest and sequence analysis. the ch2 clone was used as a template for further orf1b nsp gene subcloning. gene fragments encoding predicted orf1b nsps (nsp 9 to 12) were amplified by pcr and cloned into the pcrblunt vector using prrsv-14-specific primers (table 3 ) designed to contain restriction sites for cloning into the prokaryotic prset-b expression vector (invitrogen). prset-b bacterial expression vector cloning was performed following standard techniques (sambrook, 1989) . briefly, the gene fragments were inserted into the bamhi and kpni sites of the prset. the recombinant prset plasmids containing orf1b nsp genes were confirmed for proper orientation and frame by enzymatic restriction analysis and by sequencing the region at the insertion sites. the recombinant plasmids were designated pt-prrsvnspn, where n corresponded to the number of the cloned orf1b nsp. expression analysis of predicted orf1b nsp, optimization of protein expression, and protein purification the prset vector system allows the expression of recombinant proteins via transcription by iptg-induced t7 polymerase. orf1b nsps were expressed as recombinant proteins attached at their amino-terminus to a tag containing a polyhistidine peptide, the antigenic peptide t7.10, and an enterokinase cleavage site. ten to fifty nanograms of recombinant prset-b plasmid dna containing the corresponding orf1b fragments were placed in e. coli bl21(de3)plyss-or bl21(de3)plyse-competent cells by bacterial transformation following the manufacturer's recommendations (invitrogen). expression of soluble recombinant proteins was optimized by culturing transformed bacterial clones at various temperatures, in different culture media, and by varying iptg-induction times. recombinant protein expression was confirmed by western blot analysis of crude protein extracts with a chemiluminescence detection kit (boehringer mannheim) using selected antibodies, including a monoclonal antibody to the polyhistidine peptide (clontech), a t7.10 tag antibody (novagen), and peptide-specific polyclonal antisera generated to prrsv-14 nsp 9, 10, and 11 (described below). after optimal conditions were determined, soluble recombinant proteins were purified from bacterial extracts by affinity to cobalt (novagen) or ni-nta (qiagen) resins according the manufacturer's recommendations. recombinant proteins were eluted with 300 mm imidazole. eluted fractions were subjected to electrophoresis, followed by coomassie blue staining, and western blot analysis. fractions containing the purified recombinant protein of interest were pooled and dialyzed against 50 mm tris ph 7.5 containing 1% glycerol and 1 mm dtt. purified proteins were stored at ϫ20°c until further analysis. purified recombinant protein concentrations were determined by colorimetric assay (smith et al., 1985) using the bca protein assay reagent kit (pierce) with bovine serum albumin (bsa) as standard. antigenic regions for development of antipeptide sera were selected by computer analysis of peptide hydrophobicity/antigenicity indices of the predicted amino acid sequences of prrsv-14 nsp 9, 10, and 11 genes. the amino acid sequences of the peptides used to generate the antibodies were (c)hrpstypaknsmagingrfptkd for nsp9, (c)eqgltpldpgryqtrrg for nsp10, and (c)reylddrere for nsp11. peptide synthesis, khl conjugation, polyclonal rabbit antibody production, and affinity purification of antibodies were contracted with zymed laboratories, inc. affinity-purified antibodies were preadsorbed with lysates of porcine alveolar macrophages, marc-145, and e. coli cells at 4°c overnight, clarified by centrifugation, filtered through a 0.22-nm filter, and aliquots stored at ϫ20°c. optimal antibody concentration was determined by testing serial dilutions of antibodies by western blot of crude protein extracts. analysis and characterization of ntpase activity of prrsv-14 nsp10 (helicase) the functionality of the recombinant nsp10 helicase was initially determined by assessing ntpase activity on standard thin layer-chromatography (gwack et al., 1999) . purified protein samples were incubated with 0.1 ci of ␣-32 p-labeled ribonucleotide in 10 l of a solution containing 2 mm dtt, 1 mm nacl, and 100 g/ml bsa at different conditions. the samples were analyzed under various divalent ion concentrations, temperatures, time periods, protein concentrations, ph conditions, and buffer systems. the divalent ions tested included mgcl 2 , mgso 4 , mncl 2 , and mnso 4 . different ph conditions were tested using buffers containing 25 mm mes, 25 mm pipes, 25 mm hepes, or 25 mm tris. reaction products (2 l) were spotted onto polyethylenimine (pei)-cellulose f plates (em science). chromatographs were developed in 0.4 m kh 2 po 4 (ph 3.5), dried, autoradiographed, and analyzed by densitometry or phosphoimaging. the atp bioluminescence assay kit cls ii (boehringer mannheim) was used for quantitative analysis of nsp 10 atpase activity. in this assay, the bioluminescent signal emitted by the oxidation of luciferin is directly proportional to the atp concentration present in the test sample (mcelroy and deluca, 1983) . this assay was performed in triplicate at conditions described above (10-l reactions) with 0.1 to 1 m atp (promega) as substrate. for kinetic analysis, the level of atp hydrolysis produced by the recombinant nsp 10 helicase was measured at different concentrations of atp and at various incubation times. after incubation, the helicase was inactivated at 90°c for 5 min and samples were diluted with 19 vol of 50 mm tris-hcl ph 8.0 buffer containing 2 mm edta. diluted samples (50 l) were transferred in triplicate to microfluor b 96-well flat-bottom plates (dynex technologies inc. this assay was performed using 100 nm protein in 10 l reaction buffer containing 25 mm tris ph 8.0, 3 mm mgcl 2 , 100 g/ml bsa, 1 mm nacl, 2 mm dtt with various concentrations of atp at 35°c for 5 min. the 5-min incubation was selected because it was within the range at which the rate of atp hydrolysis was linear (fig. 7b) . the k m of prrsv-14 helicase atpase activity was determined by nonlinear regression analysis of the michaelis-menten equation using the prism program version 3.0 (graphpad software) according to the method described by motulsky (1999). duplexed rna substrates were prepared using both synthetic rna oligos and in vitro transcribed rna. the synthetic oligonucleotides 5ј-gggagtagctccaattcgccc-3ј (ss1), 5ј-tttttttttttttttgggcgaattggagctactccc-3ј (ls5ј), 5ј-gggcgaattggagctactccctttttttttt-ttttt-3ј (ls3ј), 5ј-cgagccattctaggtcaaaccaatt-gccgccgtcgactt-3ј (ss2-3ј), and 5ј-actagtggtt-tcacctagaatggctgcgtcccttcttttcctc-3ј (ls2-3ј) were purchased from annovis (aston, pa). the ss1 and ss2-3ј were labeled at the 5ј end with [␥-32 p]-atp (amersham, 3000 ci/mmol) using t4 polynucleotide kinase and purified using the qiaquick nucleotide removal kit (qia-gen). duplexes were generated by annealing the labeled short strands with 10-fold excess unlabeled long strands as described (seybert et al., 2000b) . the following substrates were obtained by annealing rna oligos for 10 min at 95°c: 5ј duplex (ss1 ϩ ls-5ј), 3јduplex (ss1 ϩ ls-3ј), 3ј3јduplex (ss2-3ј ϩ ls2-3ј). in vitro transcribed rna was generated using plasmids pgem-l and pgem-s as templates. dna fragments were generated by rt-pcr amplification of strain vr-2332 3ј-untranslated region from a previously prepared clone (ptvrsgorf7; unpublished data) using adapter primers (5ј-30-apai: 5ј-actag-ggcccttaacaaaaaaaaaaaaaaaaaaa; 3ј-utr-ndei: 5ј-actagcatatggcactagtgattccggaat; 5ј-65-apai: 5ј-actagggcccttaattggcgagaac-catgcggc) to generate 5ј-ttaattggcgagaaccat-gcggccgaaattaacaaaaaaaaaaaaaaaaaaagcg-gccgcgaattccggaatcactagtgcca-3ј (l fragment) and 5ј-ttaacaaaaaaaaaaaaaaaaaaagcggccgcgaatt-ccggaatcactagtgcca-3ј (s fragment). after apai and ndei digestion, these fragments were cloned into the respective sites of pgem-t to produce two plasmids (pgem-s and pgem-l). these plasmids were subsequently digested with either apai (sp6 orientation), saci (t7 orientation), or nsii (t7 orientation), purified using the qiaquick pcr purification kit (qiagen), and blunt-ended prior to in vitro transcription. in vitro transcription was performed with sp6 and t7 polymerases using ribomax large scale rna production system (promega). pgem-s was transcribed in the presence of [␣-32 p]-utp (amersham, 3000 ci/mmol). transcripts were treated with dnase i and purified by two rounds of phenolchloroform-isoamyl alcohol extraction, followed by two rounds of chloroform-isoamyl alcohol extraction. the substrates 5ј3јduplex#1 (l-sp6-apai ϩ s-t7-saci), 5ј5јduplex#1 (l-t7-saci ϩ s-sp6-apa1), 5ј5јduplex#2 (l-t7-nsii ϩ s-sp6-apai), and 5ј3јduplex#2 (l-sp6-apai ϩ s-t7-nsii) were generated by annealing the short radiolabeled transcript with 10-fold excess unlabeled long strand as described (seybert et al., 2000b) . the unwinding assay was performed in 20 l reactions containing 400 m protein, 12 m substrate in 25 mm tris buffer (ph 8.0), or hepes buffer (ph 7.4) containing 5 mm mgcl 2 , 2 mm dtt, 100 g/ml bsa, 10% glycerol, 5 mm atp, and 25 mm nacl. reactions were incubated at 30°c for 1 h and stopped by adding 5 l of 5ϫ loading buffer (5% sds/15% ficoll/100 mm edta/ 0.25% bromphenol blue). controls included in each assay were native (annealed) and denatured (heated at 95°c) substrate in the absence of helicase. the reaction products were analyzed by native tbe-polyacrylamide gel (4-20% gradient gel, bio-rad) electrophoresis and autoradiography. north american and european porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions mutations in the genome of porcine reproductive and respiratory syndrome virus responsible for the attenuation phenotype identification of the catalytic sites of a papain-like cysteine proteinase of murine coronavirus structural polypeptides of the american (vr-2332) strain of porcine reproductive and respiratory syndrome virus characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr-2332) characterization of the nucleoside triphosphate phosphohydrolase and helicase activities of the reovirus lambdal protein characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot nidovirales: a new order comprising coronaviridae and arteriviridae isolation of swine infertility and respiratory syndrome virus (isolate atcc vr-2332) in north america and experimental reproduction of the disease in gnotobiotic pigs molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group genetic manipulation of equine arteritis virus using full-length cdna clones: separation of overlapping genes and expression of a foreign epitope processing and evolution of the n-terminal region of the arterivirus replicase orf1a protein: identification of two papainlike cysteine proteases equine arteritis virus subgenomic mrna synthesis: analysis of leader-body junctions and replicative-form rnas complete genomic sequence and phylogenetic analysis of the lactate dehydrogenase-elevating virus (ldv) coronavirus genome: prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis rnastimulated atpase and rna helicase activities and rna binding domain of hepatitis g virus nonstructural protein 3 characterization of coronavirus rna polymerase gene products identification of an atpase activity associated with a 71-kilodalton polypeptide encoded in gene 1 of the human coronavirus 229e isolation and genome characterization of porcine reproductive and respiratory syndrome virus in p.r atpase, gtpase, and rna binding activities associated with the 206-kilodalton protein of turnip yellow mosaic virus virus-encoded rna helicases enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma-104 cell line the serine protease and rna-stimulated nucleoside triphosphatase and rna helicase functional domains of dengue virus type 2 ns3 converge within a region of 20 amino acids firefly and bacterial luminescence: basic science and applications lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav infectious transcripts from cloned genome-length cdna of porcine reproductive and respiratory syndrome virus analyzing data with graphpad prism nucleoside triphosphate specificity of firefly luciferase comparison of the structural protein coding sequences of the vr-2332 and lelystad virus strains of the prrs virus genetic variation in the prrs virus porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents analysis of orf 1 in european porcine reproductive and respiratory syndrome virus by long rt-pcr and restriction fragment length polymorphism (rflp) analysis lactate dehydrogenaseelevating virus, equine arteritis virus, and simian hemorrhagic fever virus: a new group of positive-strand rna viruses a steady-state and pre-steady-state kinetic analysis of the ntpase activity associated with the hepatitis c virus ns3 helicase domain atpase and gtpase activities associated with semliki forest virus nonstructural protein nsp2 molecular cloning. a laboratory manual biochemical characterization of the equine arteritis virus helicase suggests a close functional relationship between arterivirus and coronavirus helicases the human coronavirus 229e superfamily 1 helicase has rna and dna duplexunwinding activities with 5ј-to-3ј polarity determination of the complete nucleotide sequence of a vaccine strain of porcine reproductive and respiratory syndrome virus and identification of the nsp2 gene with a unique insertion measurement of protein using bicinchoninic acid the 5ј end of the equine arteritis virus replicase gene encodes a papainlike cysteine protease proteolytic processing of the n-terminal region of the equine arteritis virus replicase the coronaviruslike superfamily proteolytic processing of the replicase orf1a protein of equine arteritis virus the arterivirus nsp2 protease. an unusual cysteine protease with primary structure similarities to both papain-like and chymotrypsin-like proteases the arterivirus nsp4 protease is the prototype of a novel group of chymotrypsin-like enzymes, the 3c-like serine proteases the arterivirus replicase. the road from rna to protein(s), and back again the molecular biology of arteriviruses rna-stimulated ntpase associated with the p80 protein of the pestivirus bovine viral diarrhea virus processing of the equine arteritis virus replicase orf1b protein: identification of cleavage products containing the putative viral polymerase and helicase domains an infectious arterivirus cdna clone: identification of a replicase point mutation that abolishes discontinuous mrna transcription proteolytic processing of the open reading frame 1b-encoded part of arterivirus replicase is mediated by nsp4 serine protease and is essential for virus replication the predicted metal-binding region of the arterivirus helicase protein is involved in subgenomic mrna synthesis, genome replication, and virion biogenesis characterization of an equine arteritis virus replicase mutant defective in subgenomic mrna synthesis alternative proteolytic processing of the arterivirus replicase orf1a polyprotein: evidence that nsp2 acts as a cofactor for the nsp4 serine protease full-length sequence of a canadian porcine reproductive and respiratory syndrome virus (prrsv) isolate recombination between north american strains of porcine reproductive and respiratory syndrome virus complete genome comparison of porcine reproductive and respiratory syndrome virus parental and attenuated strains virus-encoded proteinases and proteolytic processing in the nidovirales we thank drs. stephen wessman and randall levings from the national veterinary services laboratories (nvsl, ames ia) for providing prrsv isolates and the marc-145 cell line. we appreciate dr. thomas w. molitor from the university of minnesota (st. paul, mn) for kindly providing prrsv isolates and anti-prrsv sera useful in the characterization of prrsv isolates. the technical advice of drs. elcira villareal and jesus gutierrez is deeply appreciated. we thank the personnel from the sequencing facility at eli lilly and co. for help in the automated nucleotide sequencing. the technical assistance of anita jackson and the preparation of material transfer agreements by the lilly's legal department are greatly appreciated. financial support for these studies was provided by elanco animal health, a subsidiary of eli lilly and co., greenfield, in. key: cord-281309-c9y7m5do authors: guo, baoqing; lager, kelly m.; henningson, jamie n.; miller, laura c.; schlink, sarah n.; kappes, matthew a.; kehrli, marcus e.; brockmeier, susan l.; nicholson, tracy l.; yang, han-chun; faaberg, kay s. title: experimental infection of united states swine with a chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus date: 2013-01-20 journal: virology doi: 10.1016/j.virol.2012.09.013 sha: doc_id: 281309 cord_uid: c9y7m5do the pathogenesis of type 2 highly pathogenic porcine reproductive and respiratory syndrome virus (hp-prrsv) in 10-week old swine in the united states was investigated. rjxwn06, rescued from an infectious clone of chinese hp-prrsv, replicated in swine with at least 100-fold increased kinetics over u.s. strain vr-2332. rjxwn06 caused significant weight loss, exacerbated disease due to bacterial sepsis and more severe histopathological lung lesions in pigs exposed to hp-prrsv than to those infected with vr-2332. novel findings include identification of bacterial species present, the degree of thymic atrophy seen, and the inclusion of contact animals that highlighted the ability of hp-prrsv to rapidly transmit between animals. furthermore, comprehensive detailed cytokine analysis of serum, bronchoalveolar lavage fluid, and tracheobronchial lymph node tissue homogenate revealed a striking elevation in levels of cytokines associated with both innate and adaptive immunity in hp-prrsv infected swine, and showed that contact swine differed in the degree of cytokine response. in 2006, investigators from several chinese provinces reported a unique syndrome in growing swine that was highlighted by the predominant clinical signs of high fever, anorexia, listlessness, red discoloration of skin, respiratory distress and very high morbidity and mortality rates (li et al., 2007; tian et al., 2007; tong et al., 2007; wu et al., 2009; zhou et al., 2008) . originally known as porcine high fever disease (phfd), this syndrome spread to vietnam in 2006 and to cambodia, laos, the philippines, bhutan, myanmar, thailand, south korea and russia in later years feng et al., 2008) . although there was concern that this syndrome may have been caused by a new disease agent, extensive diagnostic testing revealed only known pathogens. one consistent finding was the detection of porcine reproductive and respiratory syndrome virus (prrsv) with two discontinuous deletions in the replicase polyprotein known as nonstructural protein 2 (nsp2), and two single nucleotide deletions in the 5 0 and 3 0 untranslated regions (utrs) (zhou and yang, 2010) . experimental infection of chinese swine with the initial novel prrsv field isolates reproduced the clinical disease (li et al., 2007; tian et al., 2007; tong et al., 2007; wu et al., 2009; zhou et al., 2008) , providing strong evidence for its role as the causal agent of phfd. since the severity of the clinical disease was greater than expected for a typical prrsv infection, there remained the chance that an unknown agent in the prrsv isolates exacerbated the disease symptoms. this question was resolved when phfd was reproduced in chinese swine with virus derived from an infectious clone of the jx143 prrsv isolate (lv et al., 2008) . the prior studies demonstrated that prrsv isolates with a common genetic pattern had a causal role in phfd, leading to the new designation of this viral lineage as highly pathogenic prrsv (hp-prrsv). the presence of the unique nsp2 deletion motif was initially thought to contribute to the severity of disease (tian et al., 2007) . however, studies showed that the nsp2 region of hp-prrsv strain rjxwn06 containing the novel deletion could be replaced with 458 amino acids from low virulence strain hb-1/3.9 to result in a chimeric virus with only a minor delay in mortality (zhou et al., 2009) . there was also concern that there may be some unique aspect that may predispose asian pigs to a severe outcome, e.g., husbandry practices, endemic infections with other pathogens, climate, or host genetics. although a number of experiments have associated genetic changes in prrsv with an attenuation phenotype using point mutations (grebennikova et al., 2004; nielsen et al., 2001; storgaard et al., 1999) or by the construction of chimeric viruses (ellingson et al., 2010; kwon et al., 2008; wang et al., 2008; zhou et al., 2009 ), there appears to be no single locus for which mutations confer a predictable change in virulence. collectively, this area of study suggests the factors that contribute to prrsv pathogenicity are complex and viral strain-specific . as part of the single-strand positive-sense rna virus order nidovirales, family arteriviridae, prrsv genomes can vary between 15 kb to 15.5 kb, consist of at least 10 open reading frames (orfs) and replicate through a nested set of subgenomic rnas (van hemert and snijder, 2008) . members of this highly variable virus have been grouped into two genotypes, type 1 (european-like, prototype strain lelystad) or type 2 (north american, prototype strain vr-2332), which differ in nucleotide similarity by approximately 45% (nelsen et al., 1999) . both genotypes have representatives of varying pathogenicity, and intragenic nucleotide sequence variation in each can be as much as 20% (shi et al., 2010) . for this study, we imported a plasmid containing the cdna copy of the hp-prrsv strain rjxwn06 genome, a type 2 prrsv genome that is 15,321 bases in length and has 89% pairwise nucleotide identity to the prototype genome of vr-2332 (zhou et al., 2009) . to investigate the pathogenesis of hp-prrsv infection and the potential contribution of climate, host genetics, commensal bacteria, other environmental conditions and husbandry practices to the pathogenicity of hp-prrsv in u.s. swine, we compared and contrasted the pathogenicity of rescued jxwn06 (rjxwn06) virus to that of vr-2332 in 10-week old swine. we found that this hp-prrsv strain caused extreme morbidity, as was seen in asia, but novel to this study, resulted in up to 100x higher abundance of circulating virus when compared to vr-2332, caused extremely exacerbated thymic atrophy such that the thymus was often difficult to discern, and the host response was assessed in comparison to animals infected with strain vr-2332 for the first time by a swine protein array including 5 innate and 5 adaptive cytokines in serum, bronchoalveolar lavage fluid and lymph nodes. moreover, we completed bacterial speciation and loads after necropsy and specified the degree of weight loss seen. lastly, we showed that hp-prrsv readily transmitted to contact swine causing a different pattern of cytokine responses. as a result of our study, the remaining plausible contributing factors to the high virulence seen in asia were discredited, and thus hp-prrsv strains pose a serious threat to the u.s. swine industry. pigs challenged with chinese hp-prrsv strain rjxwn06 (group 2) began exhibiting clinical signs of disease within 2-3 days post exposure (dpe). as a group, the pigs developed fevers, became listless, anorexic, and began shivering and huddling together in a pile. clinical signs became more severe over the next few days, with pigs rapidly losing body weight (fig. 1) , becoming dehydrated and weak. respiratory distress, characterized by dyspnea, tachypnea and coughing was common in all pigs. erythema of the skin was present in most of the pigs, and several developed cutaneous hemorrhages and cyanotic extremities (blue ears) (fig. 2 ). on 7 dpe, one pig from group 2 was found dead. two others were euthanized due to severe weakness and moribund condition on dpe 8 and 9, and one was found dead on dpe 13. on 10 and 11 dpe, clinical signs of disease in some of the hp-prrsv challenged group began to decrease in severity. while pigs remained huddled in a pile with respiratory signs, approximately half of them were subjectively less listless, would move away when approached and began showing an interest in feed again. during the study, contact pigs displayed similar clinical signs as those challenged with hp-prrsv . pigs challenged with vr-2332 (group 4) were clinically normal until dpe 7, when they began to exhibit slightly increased respiratory rates and became a little less active than the control group. one pig from the vr-2332 group was found dead on dpe 9; however, the cause of death was attributed to gastric dilatation and volvulus, rather than to clinical disease typical of prrsv strain vr-2332 . control pigs (group 1) remained clinically normal for the duration of the study. postmortem examination revealed severe lesions in the hp-prrsv challenged pigs including: marked interstitial pneumonia, lymphadenopathy and thymic atrophy. necropsy findings in this group include: pulmonary edema, pleuritis, peritoneal and pericardial effusions, renal petechia, and fibrinous peritonitis that are delineated in table 1 . lungs and representative lesions are shown in fig. 2 . no pathologic lesions were identified in control pigs (group 1). pigs in the vr-2332 challenge group (group 4) had diffuse interstitial pneumonia, characteristic of an uncomplicated prrsv infection. although lung lesions were subjectively more severe in the hp-prrsv and contact groups, a statistically significant difference in lung scores was only found between the rjxwn06 contact group and the group inoculated with vr-2332 (groups 4 and 3, po0.003) (fig. 3a) . lymphadenopathy was present in all infected pigs (groups 2-4 ), with no statistically significant difference between hp-prrsv and vr-2332 infected groups (fig. 4a ). thymic atrophy was also a common finding in infected pigs and was significantly more severe in hp-prrsv infected and contact pigs compared to vr-2332 infected pigs (p ¼0.0003 and po0.0001; fig. 4b ). table 2 summarizes aspects of the clinical disease seen in this study. shown are adwg and associated standard errors for each of the four groups for days à 1-7 and days 7-13. sham inoculated control swine (group 1) and those challenged with vr-2332 (group 4) gained weight at an expected rate for their age group. group 2 swine challenged with hp-prrsv strain rjxwn06 and their contacts (group 3) lost body weight during the last week of the experiment. the adwg for group 2 on days à 1-7 includes only 11 pigs, and on days 7-13 includes only 8 pigs. the body weights for the contact animals were taken on the same day as pigs in the other groups. significance values are shown. microscopic lung lesions consisted of histiocytic interstitial pneumonia with increased numbers of macrophages in alveolar septa and lumina (fig. 5a , c). type ii pneumocyte hyperplasia was also present. these lesions were seen in the hp-prrsv challenge group (group 2), the hp-prrsv contact pigs (group 3) and the vr-2332 challenge group (group 4). mean interstitial pneumonia lesions were 3.33, 3.75, and 2.00 for chinese prrsv challenge group (group 2), contact chinese prrsv pigs (group 3) and vr-2332 challenge (group 4), respectively (fig. 3b ). lesions were significantly (p¼0.0002) more severe in the chinese prrsv challenge group (group 2) and contact chinese prrsv pigs (group 3) as compared to the vr-2332 challenge (group 4). two sham inoculated control pigs had mild lesions with a score of 1, while the remaining pigs had no lesions. immunohistochemistry labeling for prrsv nucleocapsid was positive in 100% of chinese prrsv challenge and contact pigs (groups 2 and 3) and 88% of vr-2332 challenge pigs (group 4), with mean ihc labeling scores of 2.08, 1.75, and 1.63, respectively (fig. 5) . positive labeling was present in alveolar macrophages and interstitial macrophages for all infected groups ( fig. 5b and c). no significant difference was present in the amount of prrsv labeling in the lung among the three treatment groups. immunohistochemistry labeling for prrsv in lung tissue was negative in all sham inoculated control pigs. sera collected at à 1, 7, and 13 dpe were tested for prrsv antibody. most swine infected with rjxwn06 (group 2) had seroconverted by 7 dpe (mean s/p ¼0.51, sd¼0.30), and all had seroconverted by 13 dpe (mean s/p ¼1.14, sd ¼0.26); however, the rjxwn06 contact animals (group 3) had not yet seroconverted by 5 dpe (mean s/p ¼0.00, sd ¼0.00) but did by 11 dpe (mean s/p¼0.99, sd¼0.12). vr-2332 infected pigs (group 4) were seronegative at 7 dpe (mean s/p ¼0.09, sd ¼0.12); but positive by 13 dpe (mean s/p¼ 1.05, sd ¼0.37). the sham inoculated control animals (group 1) remained negative for prrsv nucleocapsid antibody throughout the study. all serum and balf samples were screened on marc-145 cells for infectious virus, and vi positive samples were then titered. a significant increase in virus load (100-1000 fold) for the rjxwn06 challenge and rjxwn06 contact pigs was found in the serum when compared to the vr-2332 challenge group (fig. 6a ). no significant differences were found in serum virus titer between the rjxwn06 challenge and rjxwn06 contact groups at any time points even though the contact pigs were exposed to rjxwn06 infected pigs at 2 dpe. at necropsy, no significant differences in balf titers were found among the rjxwn06 challenge, rjxwn06 contact, or vr-2332 challenge groups (fig. 6b ). control serum and balf samples were vi negative throughout the experiment ( fig. 6a and b) . to examine the levels of prrsv rna and to strengthen the case for the apparent difference in viral loads between the hp-prrsv and vr-2332 inoculated animals seen above, qrt-pcr was completed on all serum samples as well as balf and lymph node tissue (fig. 7) . the level of viral rna detected in serum samples mirrors the results obtained by virus isolation on marc-145 cells (fig. 7a, pr0 .005 at all dpe). the qrt-pcr results from balf showed little or no significant difference between the infected lymph node weight (lnw) to body weight (bw) ratios showed pronounced lymphadenopathy in all infected animals (groups 2-4) as compared to control animals (group 1)(p¼ 0.02 or higher significance), but no statistical significance between prrsv infected swine. (b) thymus weight (tw) to bw ratios showed thymic atrophy in all infected animals (groups 2-4) as compared to control animals (p o 0.0001), and statistically significant differences between hp-prrsv inoculated (po 0.0001) and naturally infected (p ¼ 0.0003) animals compared to vr-2332 inoculated swine. groups (fig. 7b ). tbln samples suggested that swine inoculated with vr-2332 had significantly less viral rna present than those inoculated (p¼0.0205) or infected (p¼ 0.0017) with hp-prrsv (fig. 7c ). sham inoculated control serum, balf, and tbln tissue samples were viral rna negative throughout the experiment ( fig. 7a -c ). bacteria were isolated from the balf of 8 of the 12 rjxwn06 inoculated pigs (group 2) and 2 of the 4 rjxwn06 contact pigs (group 3) but from only 1 of 8 pigs in each of the sham inoculated control (group 1) and vr-2332 inoculated (group 4) groups. in the rjxwn06 challenge group pasteurella multocida was isolated from four pigs (ranging from 50 to 4 300 cfu/100 ml), p. multocida ( 4300 cfu/100 ml) and actinobacillus suis ( 4300 cfu/ 100 ml) were isolated from two pigs, p. multocida, a. suis and streptococcus suis (approximately 50, 100, and 25 cfu/100 ml, respectively) from one pig, and staphylococcus aureus and klebsiella pneumoniae (approximately 150 and 20 cfu/100 ml, respectively) were isolated from one pig. in the contact group, p. multocida and a. suis were isolated from one pig (4300 cfu/100 ml each) and p. multocida, a. suis, and arcanobacterium pyogenes (approximately 50, 4300, and 4300 cfu/100 ml, respectively) were isolated from a second pig. s. aureus and escherichia coli (4300 cfu/100 ml each) were isolated from 1 vr-2332 inoculated pig, and bordetella bronchiseptica (65 cfu/100 ml) was isolated from 1 sham inoculated control pig. results on 10 cytokines measured in serum, balf and tbln homogenates are presented in figs. 8 and 9 and the changes in cytokine levels relative to sham-inoculated controls are listed in table 3 . pigs directly inoculated with rjxwn06 (group 2) had significantly elevated average serum levels of ifna, il-1 b, il-2, il-10 and ifng, ranging from 6 to 11 times the levels detected in sera of sham inoculated controls (group 1). in pigs inoculated with rjxwn06, serum ifna levels were significantly elevated at 4 dpe in comparison to both controls and vr-2332 exposed pigs (po0.05) and remained significantly elevated at 7 dpe versus both groups. serum levels of il-10 were elevated by 7 dpe, with il-2, il-10, il-12 and ifng all signficantly elevated at 11 dpe compared to both controls and vr-2332 exposed pigs (po 0.05). compared with sham inoculated controls, contact pigs exposed to rjxwn06 (group3) developed significant serum level elevations (3 to 89 times greater) in 3 out of 5 innate immunity cytokines measured (ifna, tnfa and il-1 b) and elevated levels (3 to 21 times greater) for all 5 cytokines measured associated with adaptive immunity (il-2, il-4, il-10, il-12 and ifng). in pigs exposed to rjxwn06 by contact transmission, serum ifna levels were signifcantly elevated at 2, 5 and 9 dpe in comparison to controls (po0.05). in addition, contact pigs had significant elevations in il-1 b at 9 and 11 dpe (po0.05). contact pigs also had significantly elevated levels of all adaptive immunity cytokines at 9 and 11 dpe (p o0.05) with il-10 also significantly elevated at 5 dpe. in contrast, none of the 10 cytokines measured had significant elevations in serum levels detected in pigs inoculated with the north american prototype strain vr-2332 (group 4) when compared with sham inoculated controls. similarly, when compared to sham inoculated controls, balf of swine inoculated with rjxwn06 (group 2) had significantly elevated (7 to 22 times greater) levels of 3 out of 5 innate immunity cytokines measured (tnfa, il-1 b and il-8) and elevated levels (2 to 54 times greater) for all 5 cytokines measured associated with adaptive immunity (il-2, il-4, il-10, il-12 and ifng). compared with sham inoculated controls, contact swine exposed to rjxwn06 (group 3) had significantly elevated balf cytokine levels (4 to 21 times greater) in 4 out of 5 innate immunity cytokines measured (ifna, tnfa and il-8) and elevated levels (3 to 30 times greater) for all 5 cytokines measured associated with adaptive immunity (il-2, il-4, il-10, il-12 and ifng). in contrast, swine inoculated with vr-2332 (group 4) had significantly elevated balf cytokine levels (4 times greater) for only 2 out of 5 innate immunity cytokines measured (il-1b and il-8) and elevated levels (10 to 12 times greater) for 2 of 5 cytokines measured associated with adaptive immunity (il-10 and ifng). in this study, we compared the pathogenicity of chinese hp-prrsv strain rjxwn06 to north american prototype vr-2332 in u.s. high health swine under controlled conditions. the clinical disease induced in this study by the hp-prrsv virus was similar to what has been reported in asia for phfd and for experimental infections with the wild-type or rescued virus ( zhou et al., 2009; zhou et al., 2008) . likewise, the clinical disease induced by the vr-2332 virus was similar to that observed in previous reports (faaberg et al., 2010; rossow et al., 1994) . clinical disease and pathology were much more severe in the rjxwn06 group and their contacts than in the vr-2332 group. overall, gross pathology lesions in the rjxwn06 challenged and contact groups were much more extensive and not restricted solely to the respiratory tract and lymph nodes as was the case with the vr-2332 challenged pigs. the high occurrence of bacterial co-infections in the rjxwn06 challenge and contact swine likely played a prominent role in the difference in pathology and clinical disease between groups. bacterial co-infections in pigs naturally infected with prrsv have been documented, with susceptibility attributed to factors including prrsv strain differences, host genetics, management practices and environmental factors (rossow, 1998) . in this study, we used swine of high health status in a controlled research environment that were from 2-and 3-way crosses of commercial genetic lines. we believe the incidence and magnitude of bacteria isolated from the rjxwn06 challenge and contact groups when compared to the vr-2332 and control groups suggest the differences in secondary bacterial infection susceptibility are specific to viral strain. in this study, we observed a 30% mortality rate in rjxwn06 infected pigs by 13 dpe, which is less mortality than the original report (zhou et al., 2009) , but similar to other hp-prrsv strains used (li et al., 2007; lv et al., 2008; zhou et al., 2008) . possible explanations for differences may be the route and dose of inoculation, the age of pigs, the hp-prrsv strain utilized, and the time course of study. in addition, since the hp-prrsv lineage may exacerbate subclinical bacterial infections, it is possible different endemic bacterial infections played a role in apparent different mortality rates. in natural infections with chinese hp-prrsv, pulmonary interstitial hyperplasia with hemorrhage and edema is described, which suggests an acute septicemic process due to a secondary bacterial pathogen (tian et al., 2007; zhou and yang, 2010) . upon histopathologic examination of tissues from this study, north american pigs directly inoculated with rjxwn06, as well as contact pigs, had an interstitial pneumonia that was significantly more severe than the vr-2332 inoculated group, which is consistent with the severity of disease reported in china. although pulmonary lesions were more severe in the rjxwn06 challenge and contact pigs, the amount of antigen labeling was not significantly different from the vr-2332 inoculated swine. since levels of proinflammatory cytokines, including tnfa, il-1 b and il-8, were significantly increased in the balf of rjxwn06-inoculated and contact pigs, the host response to hp-prrsv may play a role in the augmented lung pathology seen. in addition, the increased incidence of secondary bacterial infections in rjxwn06 challenge and contact pigs may have contributed to increased cytokine production and resultant immunopathology. there are numerous reports about the interplay of prrsv with the swine immune system that describe variable responses to infection at the cellular and cytokine level (miguel et al., 2010; thanawongnuwech et al., 2004; thanawongnuwech and thacker, 2003; wang et al., 2011) . although a large part of this variability may result from differing methods, challenge viruses, outbred pigs, and experimental designs, there are consistent findings emerging among the studies of increases in levels of selected cytokines associated with both innate and adaptive immunity. here we report a comprehensive assessment of the effects of prrsv infection on levels of cytokines critical to innate (ifna/b, tnfa, il-1 b, il6 and il-8) and adaptive (il-2, il-4, il-10, il-12 and ifng) immune systems in serum, balf and tbln homogenates. it was demonstrated that infection with a highly pathogenic strain of prrsv elicited a significant elevation of all adaptive immunity cytokines measured in balf, as well as a majority of these cytokines in serum and tbln homogenates of the same groups of pigs. this observation is consistent with previous reports of table 3 changes in cytokine levels in sampled tissues relative to non-challenged control pigs (p-values in comparison to non-challenged controls). serum values are an area under the curve overall average level measured in samples collected after exposure to virus. geometric means back-calculated from log10 means were used to determine relative changes in tbln and balf cytokine levels compared to sham-inoculated controls. (sun et al., 2012) ]. in contrast, in pigs infected in this study with rjxwn06, we observed significant elevations of serum ifna as the first cytokine to peak following exposure to rjxwn06 in either the directly challenged or contact pigs, with the zenith occurring 4 and 2 dpe, respectively. however, consistent with a previous study (gomez-laguna et al., 2009 ), elevated serum ifna had little to no apparent effect on virus clearance as the viremia peaked in the rjxwn06 challenge and contact pigs on dpe 7 and 4, respectively, and remained above the serum virus levels of vr-2332 pigs until the end of the study (11 and 13 dpe). it is well documented that prrsv infection will increase the susceptibility of swine to co-infection with various bacteria (brockmeier et al., 2001; thanawongnuwech et al., 2004; thanawongnuwech and thacker, 2003; thanawongnuwech et al., 1997; thanawongnuwech et al., 2001; xu et al., 2010) , and based on previous findings with other hp-prrsv strains (xu et al., 2010) and our current rjxwn06 findings, it is clear that exposure to hp-prrsv greatly increases the likelihood of secondary bacterial infection due to commensal or pathogenic organisms typically found in the swine upper respiratory tract. whether the significant elevations of multiple cytokines that were measured in serum, balf and tbln following exposure to rjxwn06 were a direct result of the virus, an indirect effect mediated by the secondary bacterial infections, from extensive host tissue damage, or a combination of all of these events cannot be determined from our experiments. however, the pattern of multiple cytokines being elevated nearly simultaneously in serum (8 of 10 cytokines in rjxwn06 contact pigs and 5 of 10 cytokines in direct inoculated pigs) has not previously been reported and was not detected in vr-2332 infected swine. cytokines are a diverse collection of peptides that elicit a wide range of biological responses and are characteristically understood in the context of an immune response wherein inflammation and immunity are carefully orchestrated by sequential secretion of cytokines that coordinate innate and adaptive immune responses. macrophages and stressed or damaged cells typically initiate a cytokine cascade through secretion of chemokines and proinflammatory cytokines in order to initiate the innate immune response at sites of acute infection or damage. generally, they act locally at nano-to picogram levels with short half-lives and transient activity. however, inflammatory cytokine cascades classically comprise a sequential appearance and disappearance of proinflammatory cytokines (e.g., tnfa, il-1 and il-6) intended to activate immune cells and their recruitment to generate additional cytokines and chemokines. this initial wave of cytokine production is usually followed by anti-inflammatory cytokine production (mainly the il-10 family) to moderate or down regulate the pro-inflammatory cytokines. cytokine cascades are therefore usually sequential with transiently detectable levels in peripheral blood; any dysregulation of these cascades can lead to adverse immunopathological responses. while significant elevations of several cytokine levels in tissues such as balf and tbln were expected, near simultaneous elevation of several cytokines in serum was not entirely expected as part of a normal host immune response. moreover, the levels of cytokines detected were in several instances significantly elevated (usually several times more) over levels detected with the low virulence north american prototype prrsv strain, vr-2332 . prolonged elevations of serum ifng levels have been reported in swine infected with prrsv, a finding in contrast to serum ifng in pigs infected with influenza or respiratory coronavirus, where minimal transient detectable levels are observed (wesley et al., 2006) . in swine infected with rjxwn06, it appears the normal sequence of cytokine production (e.g., ifng, tnfa, and il-2 often being the first cytokines produced in response to a viral infection, followed by il-6 and il-10) leading to effective virus clearance and a normal immune response was dysregulated given that significant elevations of several serum cytokines levels were still evident at 7 to 11 dpe. two recent studies have reported hp-prrsv infection in swine results in down regulation of a key toll-like receptor adapter gene, sarm1 (sterile aand armadillomotif-containing protein) (zhou et al., 2011; zhou et al., 2012) . sarm1 normally dampens the proinflammatory immune response by attenuating nf-kb activation and decreasing expression of il1, il-8 and tnfa (carty et al., 2006) . previous studies in pigs infected with strains of hp-prrsv were reported to have swollen livers and petechial hemorrhages on the kidneys as well as immunohistochemical staining evidence of viral antigen in the liver and kidneys (among other tissues) (li et al., 2007; tian et al., 2007) . impaired hepatic and renal function as a consequence of viral or secondary bacterial disease could have a significant effect on clearance of the cytokines detected in serum, and contribute to an apparent severe cytokine release syndrome. whether our findings represent a parallel condition in swine to severe cytokine release syndromes or cytokine storms reported in humans that have been attributed to various causes cannot be proven with our data (descotes and gouraud, 2008; tarrant, 2010) . however, given prrsv causes polyclonal b cell activation, autoimmunity, lymphoid hyperplasia and hypergammaglobulinemia in pigs (lemke et al., 2004) , and it modulates multiple intracellular signaling pathways [reviewed in (sun et al., 2012) ], it is not surprising to find an exaggerated immune stimulation in swine infected with a particularly virulent strain of prrsv. early studies with cytokine administration to livestock species identified potential toxicities associated with systemic cytokine administration. interferon-g was found to have beneficial activity on immune function but was too toxic for practical usage due to febrile responses observed within 24 h of a single dose of 1.25 mg/ kg of body weight (roth and frank, 1989) . similarly, administration of 166 ng of bovine il-1 b/kg of body weight every 8 h for 4 days caused transient fever, inappetence, increased pulse and respiratory rate, and diuresis (goff et al., 1992) . blood cytokines have been proposed as biomarkers of in vivo toxicity associated with new drug development (tarrant, 2010) . under most disease scenarios where there is rapid resolution of the infection by the host immune response, much of the biological activity of cytokines will occur in the locally infected tissues and elevated levels of the cytokines may not be detected in serum. we cannot definitively state whether the elevated cytokine syndrome reported here contributes to the pathophysiology of the disease caused by this highly pathogenic prrsv isolate. however, the association between the elevated levels of multiple cytokines and the severe morbidity and high mortality reported is consistent with a multiple cytokine toxicity syndrome in humans associated with various therapeutics (tarrant, 2010) . adverse reactions ranging from mild-to-moderate flu-like reactions to severe cytokine release syndromes have been observed with many therapeutic proteins in current use in human medicine, and some result in severe and even potentially life-threatening syndromes (reviewed in (tarrant, 2010) ). macrophage activation syndrome and cytokine storm are different names for two syndromes that share many features including a massive inflammatory response, elevated serum cytokine levels, multiorgan system disease and often death (behrens et al., 2011) . although these syndromes may be clinically indistinguishable, the cytokines that predominate in each may differ with tnfa being dominant in bacterial sepsis and ifng predominate in the macrophage activation syndrome (behrens et al., 2011) . the exact pathophysiology of the systemic toxicity in these syndromes is not fully defined. in pigs infected by natural contact with rjxwn06 inoculated pigs, we detected significant serum elevations in ifna, tnfa, il-1 b, il-2, il-4, il-10, il-12 and ifng. however, given that prrsv infects the macrophage cell line in pigs, the elevated serum cytokine levels in pigs infected with hp-prrsv may represent both conditions (macrophage activation syndrome and cytokine storm) and contribute to the multiorgan damage and high mortality reported here. marc-145 cells were cultured in minimum essential medium (mem, safc 56416c) with 10% fetal bovine serum at 37 1c, 5% co 2 . wild-type (wt) type 2 prrsv strain vr-2332 (genbank u87392), passage 6 on marc-145 cells, was titrated, and used for the swine study. virus (rescued rjxwn06; rjxwn06) was rescued from a cloned cdna of chinese highly pathogenic type 2 prrsv strain jxwn06 in marc-145 cells [pwsk-jxwn; genbank ef641008, (zhou et al., 2009) ] and passaged 3 times on marc-145 cells for use in the swine study. the in vivo swine study described here was performed at the national animal disease center under approval from its animal care and use committee. thirty-two 10-week-old cross-bred pigs were obtained from a u.s. high-health status herd and were found to be free of prrsv antibodies by herdchek elisa, influenza virus antibodies by np elisa, and negative for porcine circovirus type 2 by quantitative real-time pcr (data not shown). one day prior to inoculation, pigs were bled, weighed and randomly assigned to one of four groups. group 1 (n ¼8) consisted of sham inoculated control pigs, which received an intranasal 2 ml sham inoculum of mem on day 0. group 2 pigs (n ¼12) were challenged intranasally with 2 ml of 1 â 10 6 50% tissue culture infective dose (tcid 50 )/ml of chinese prrsv strain rjxwn06 in animal biosafety level 3-agriculture (bsl3-ag) housing, where they remained for the duration of the experiment. group 3 consisted of naïve pigs (n ¼4) that were placed in contact with group 2 two days after group 2 was inoculated (dpe). group 4 pigs (n ¼8) were challenged intranasally on day 0 dpe with 2 ml of 1 â 10 6 tcid 50 /ml of type 2 prototype strain vr-2332 . groups 1 and 4 were housed in separate isolation rooms in an absl2 facility. clinical monitoring of pigs was performed daily throughout the study. specifically, observations were made regarding the pig's mental alertness, body condition, appetite, activity level, and clinical signs of respiratory or systemic disease. serum was collected on à 1, 4, 7, 11, and 13 dpe (sera from group 3 pigs was collected on à 3, 2, 5, 9 and 11 dpe), and pigs were weighed on à 1, 7 and 13 dpe (group 3 were weighed on à 3, 5 and 11 dpe). necropsy was scheduled on dpe 13 (dpe 11 for group 3), or sooner if pigs died or were euthanized due to severe disease. at necropsy, a thorough post-mortem examination was performed, and a complete set of samples was collected for evaluating disease severity. tracheobronchial lymph nodes (tbln) and thymic tissue were weighed (lnw and tw, respectively) and compared to respective body weights (bw) to derive a tissue mass index (lnw/bw, tw/bw) measuring the effect of prrsv infection on organ weight (mengeling et al., 2003) . upon removal, lungs were examined and extent of macroscopic lung lesions was estimated, as previously described, and reported as a percentage of lungs affected (halbur et al., 1996) . sections of tissues (lung, tracheobronchial lymph node, trachea, thymus, heart, tonsil, spleen, iliac lymph node, mesenteric lymph nodes, ileum, bone marrow, kidneys, liver, inguinal lymph node, cerebrum, brainstem, cerebellum and ventral midbrain) were collected into 10% neutral-buffered formalin for histopathology and immunohistochemistry. tbln were collected for rna extraction and cytokine protein assays. bronchoalveolar lavage fluid (balf) was collected after removing whole lungs from pigs and aseptically lavaging with 50 ml antibiotic-free mem. tissues were processed by routine histopathologic procedures and slides were stained with hematoxylin and eosin. a boardcertified veterinary pathologist blinded to treatment groups evaluated microscopic lesions. lung sections were scored on a 5-point scale that accounted for distribution and severity of interstitial pneumonia: 0-no lesions, 1-mild, focal to multifocal interstitial pneumonia (o50% of lung section affected), 2-moderate, multifocal to coalescing (50-75% of lung section affected), 3-severe, patchy to coalescing and extensive (75-90% of lung section affected), and 4-severe and diffuse ( 490% of lung section affected). immunohistochemistry prrsv-specific antigen was detected in lung tissues using a previously described immunohistochemical (ihc) method with minor modifications (halbur et al., 1995) . briefly, tissue sections were deparaffinized and hydrated in distilled water. slides were quenched in 3% hydrogen peroxide for 10 min, rinsed three times in distilled water and treated in 0.05% protease for 2 min. slides were then rinsed three times in distilled water. a primary monoclonal antibody (mab) cocktail of one part 1:800 prrsv sdow17 (rti, brookings, sd) and one part 1:500 prrsv sr30 (rti, brookings, sd) was applied to the slides and were incubated at room temperature for 1 h. bound mabs were stained with peroxidase-labeled anti-mouse igg followed by chromogen using the dako lsab2-hrp detection system (dako, carpinteria, ca) according to the manufacturer's instructions. 3,3 0 -diaminobenzidine (dab; vector laboratories, burlingame, ca) was applied to the slides for 5 min. the slides were rinsed in deionized water and counterstained with gill's hematoxylin. prrsv labeling was graded on a 4 point scale of 0: none, 1: mild scattered signals (1 to 20 cells in entire section), 2: moderate scattered signals (less than or equal to 50% of high power fields (hpf) containing immunolabeling) and 3: abundant scattered signals (greater than 50% of hpf contain labeling and/or there are at least 2-3 groups of 30 cells or more with staining). virus isolation was attempted on all serum and balf samples as described previously (faaberg et al., 2010) . those samples that were positive by virus isolation were then titered by serial dilution on marc-145 cells to determine the quantity of virus present to produce a cytopathic effect in 50% of inoculated tissue culture cells (tcid 50 ). quantitative rt-pcr (qrt-pcr), as previously described (faaberg et al., 2010) , was used to determine the amount of viral rna per ml of serum and balf, and per gram of tbln homogenized tissue prepared as described below. serum samples were tested on study days à1, 7 and 13 for evidence of seroconversion with the prrs 2xr enzyme-linked immunosorbent assay (herdchek elisa; idexx laboratories). a sample was considered positive for antibodies to prrsv nucleocapsid protein if the sample-to-positive (s/p) ratio was equal to or greater than 0.4. levels of tnfa, il-1 b, il-2, il-4, il-6, il-8, il-10, il-12p70 and ifng cytokine levels (pg/ml) were measured in serum, balf and tbln samples diluted 1:2 with dilution buffer supplied by the manufacturer using a searchlight (aushon biosystems, woburn, ma, usa) customized multiplex immunoassay (m-elisa) following the manufacturer's protocol. m-elisa samples were assayed in duplicate. the elisa limits of detection for tnfa, il-1 b, il-2, il-4, il-6, il-8, il-10, il-12p70 and ifng were 0.2, 2.4, 0.78, 0.39, 1.6, 0.098, 0.001, 0.098, and 0.78 pg/ml respectively. approximately 1 g of tbln was homogenized in 750 ml of lysis buffer containing 0.5% triton x-100, 150 mm nacl, 15 mm tris, 1 mm cacl 2 , and 1 mm mgcl 2 , ph 7.4, using a tissue homogenizer (biospec products, bartlesville, ok) (greenberger et al., 1995) . homogenates were incubated on ice for 30 min, then centrifuged at 1258 x g for 10 min. supernatants were collected, passed through a 0.45micron filter (gelman sciences, ann arbor, mi), then stored at à20 1c prior to assessment of cytokine levels. ifna protein was measured with a porcine ifna specific elisa by using f17 monoclonal antibody (mab) and k9 mab (r&d systems inc.) as previously described (miller et al., 2009) . mab k9 was conjugated with horseradish peroxidase (hrp) using a peroxidase labeling kit (roche molecular biochemical, indianapolis, in). immulux hb flat-bottomed 96-well plates (dynex technology, chantilly, va) were coated overnight at 4 1c with f17 at a concentration of 3 mg/plate in coating buffer (100 mm carbonate buffer, ph 9.6, sigma inc., st. louis, mo). after blocking with 1% non-fat dried milk, 0.05% tween 20 in phosphate buffered saline (pbs) for 1 h at 37 1c, the plates were washed three times with 0.05% tween 20 in pbs. samples (50 ml) were added into each well containing 50 ml of 1% non-fat dried milk, 0.05% tween 20 in pbs and incubated for 2 h at 37 1c. following three washes, 100 ml of peroxidase conjugated k9 was added to each well. after 1 h incubation, at 37 1c, and three washes, 100 ml of substrate solution, tetramethylbenzidine (kpl inc., gaithersburg, md), was added to each well. after 30 min, the reaction was stopped with tetramethylbenzidine stop solution (kpl inc., gaithersburg, md) and the optical density was measured at 450 nm by an elisa plate reader. quantified recombinant porcine ifna (rifna, r&d systems inc., minneapolis, mn) was used as a standard, and ifna concentrations were calculated based upon a standard curve. one unit/ml of rifna is equivalent to 26 pg/ml. bacterial culture was performed by plating 100 ml of balf both on a casman's agar plate supplemented with 0.01% nicotinamide adenine dinucleotide (nad) and 5% horse serum, and on a 5% sheep's blood agar plate. both agar plates were incubated for 48 h at 37 1c. bacterial identification was performed by 16s rrnaspecific pcr and dna sequencing. 16s rrna-specific pcr and dna sequencing whole-cell bacterial lysates, used as templates, were prepared by suspending a colony, $ 2 mm in diameter or the equivalent, in 50 ml of sterile water. the mixture was boiled for 10 min, placed on ice until chilled, and centrifuged at 16,000 â g for 1 min to pellet cell debris and stored at à 20 1c. supernatant (5 ml) was used as the template in each pcr. the forward primer (5 0 -agagtttgatcctggctcag-3 0 ), designated univ16s-3, is homologous to a highly conserved sequence from the 5 0 end of the 16s rrna gene and the reverse primer (5 0 -gcggctgctggcacg-3 0 ), designated univ16s-4, is homologous to a highly conserved sequence between the third and fourth variable regions of the 16s rrna gene. this previously described primer set generates an amplicon of approximately 520 bp (register and yersin, 2005) . reactions were carried out in a volume of 50 ml and contained 2 u amplitaq polymerase (applied biosystems, foster city, ca), 5 ml 10x buffer ii (100 mm tris-hcl , ph 8.3, 500 mm kcl), 5 ml dimethyl sulfoxide, 1.5 mm mgcl 2 , 0.5 mm primers, and 200 mm deoxynucleoside triphosphates. an initial denaturation step of 5 min at 95 1c was followed by 35 cycles of 20 s at 94 1c, 30 s at 55 1c, and 3 min at 72 1c, with a final extension step of 10 min at 72 1c. 10 ml of each pcr was analyzed by agarose gel electrophoresis and pcr products were purified with spin columns (promega, madison, wi) and sequenced directly by fluorescence-based cycle sequencing with amplitaq and bigdye terminators on an abi 377 sequencer at the national animal disease center genomics unit. sequences were analyzed using geneious 5.0 software (biomatters ltd, auckland, new zealand). quantitative virus copy numbers and serum cytokine levels were analyzed using a mixed linear model for repeated measures (proc mixed, sas 9.2 for windows, sas institute, cary, nc, usa). linear combinations of the least squares means estimates for each variable were used in a priori contrasts after testing for either a significant (po0.05) effect of prrsv challenge strain (vr-2332, rjxwn06, rjxwn06 contacts or sham inoculated controls). comparisons were made between groups at each time-point using a 5% level of significance (po0.05) to assess statistical differences. log10 transformed virus copy numbers and cytokine levels in balf and tbln homogenates were analyzed by analysis of variance using a general linear model for unbalanced data (proc glm, sas 9.2 for windows, sas institute, cary, nc, usa). a 5% level of significance (po0.05) was used to assess statistical differences. geometric mean back transformations were made for final data presentation in figures and tables. mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the u.s. department of agriculture. usda is an equal opportunity provider and employer. highly pathogenic porcine reproductive and respiratory syndrome virus repeated tlr9 stimulation results in 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china the authors would like to recognize ann vorwald, sarah anderson, deb adolphson and amanda burow for their excellent technical assistance, and jason huegel, brian pottebaum, and jason crabtree for their exceptional animal care. we also appreciate the suggestions on manuscript layout and data organization made by crystal loving. key: cord-283035-tpqf458q authors: thanthrige-don, niroshan; abdul-careem, mohamed f.; shack, l. allen; burgess, shane c.; sharif, shayan title: analyses of the spleen proteome of chickens infected with marek's disease virus date: 2009-08-01 journal: virology doi: 10.1016/j.virol.2009.05.020 sha: doc_id: 283035 cord_uid: tpqf458q marek's disease virus (mdv), which causes a lymphoproliferative disease in chickens, is known to induce host responses leading to protection against disease in a manner dependent on genetic background of chickens and virulence of the virus. in the present study, changes in the spleen proteome at 7, 14 and 21 days post-infection in response to mdv infection were studied using two-dimensional polyacrylamide gel electrophoresis. differentially expressed proteins were identified using one-dimensional liquid chromatography electrospray ionization tandem mass spectrometry (1d lc esi ms/ms). comparative analysis of multiple gels revealed that the majority of changes had occurred at early stages of the disease. in total, 61 protein spots representing 48 host proteins were detected as either quantitatively (false discovery rate (fdr) ≤ 0.05 and fold change ≥ 2) or qualitatively differentially expressed at least once during different sampling points. overall, the proteins identified in the present study are involved in a variety of cellular processes such as the antigen processing and presentation, ubiquitin–proteasome protein degradation (upp), formation of the cytoskeleton, cellular metabolism, signal transduction and regulation of translation. notably, early stages of the disease were characterized by changes in the upp, and antigen presentation. furthermore, changes indicative of active cell proliferation as well as apoptosis together with significant changes in cytoskeletal components that were observed throughout the experimental period suggested the complexity of the pathogenesis. the present findings provide a basis for further studies aimed at elucidation of the role of these proteins in mdv interactions with its host. marek's disease (md) in chickens is caused by gallid herpesvirus 2 (gahv-2) or marek's disease virus (mdv). hereafter, this virus is referred to as mdv. mdv is a highly prevalent alpha-herpesvirus and the disease it causes is characterized by transient neurological signs and immunosuppression at early stages that could subsequently be followed by lymphoma formation in various visceral organs in susceptible birds. upon infection via inhalation, in all infected birds, mdv is taken to various lymphoid organs, such as spleen, thymus and bursa of fabricius where early cytolytic infection in b cells that proceeds to latent infection of t cells occurs. while a lifelong latent phase could occur in genetically md-resistant birds and in those protected by vaccination, late reactivation of latent virus in susceptible birds could cause transformation of mainly cd4+ t cells, leading to lymphoma formation. meanwhile, active replication of mdv occurs in feather follicles of infected birds regardless of their genetic susceptibility rendering them a continuous source of infectious viruses (baigent and davison, 2004) . although md is currently controlled by vaccination, there have been periodical md outbreaks caused by new strains of mdv with increased virulence (witter, 1997) . the potential of mdv to evolve and overcome vaccinal immunity is considered as a major threat for a sustainable md control strategy. however, many aspects of mdv-host interactions are still being elucidated (baaten et al., 2004) . to date, several studies have been conducted to examine host gene expression in response to mdv infection on a relatively large scale using various genomic techniques, such as microarrays. changes in gene expression of chicken embryo fibroblasts (cef) infected with rb1b, a very virulent strain of mdv, were studied by morgan et al. (2001) using a microarray containing 1126 expressed sequence tags. these authors reported the differential expression of a number of host genes including those associated with inflammation, antigen presentation and cell growth. an in vivo study by our group using the same virus strain and a small-scale microarray has revealed significant changes in the expression of genes encoding cell surface molecules, transcription and signal transduction molecules as well as cytokines (sarson et al., 2006) . while studies of this nature, in the context of md in particular and various other viruses in general (reviewed in piersanti et al., 2004) would certainly enhance our understanding of hostpathogen interactions, further expansion of this knowledge with proteomic studies is still important (reviewed in burgess, 2004; zhang et al., 2005) . this is partly because of the possible inconsistency between the expression of genes at the transcript and protein levels (gygi et al., 1999) . furthermore, viruses can induce post-translational modifications in host proteins without affecting the mrna expression (liu et al., 2001) . fig. 1 . representative 2d gel images of mdv-infected and uninfected control spleen proteomes with their respective sampling points. arrows with accompanying spot numbers show successfully identified protein spots that were uniquely expressed (qualitative differences) in each group at corresponding time point. please refer to table 1 for identities of corresponding spot numbers and to fig. 2 for the map of quantitatively differentially expressed spots. in a recent in vitro proteomic study, ramaroson et al. (2008) inventoried 1460 and 1676 proteins expressed in mdv-infected and mock-infected cef, respectively. several other proteomic studies in the context of md have been conducted to model the proteome of mdvtransformed cd4+ t lymphocytes in vitro. notably, proteomic modeling of mdv-transformed cd30 hi cd4+ t lymphocytes has suggested that these cells exhibit a regulatory t cell phenotype (buza and burgess, 2007; shack et al., 2008) . further, such studies have been able to describe the fundamental differences between mdv-transformed t cells and their non-transformed healthy counterparts with regard to activated signalling pathways . with respect to viral protein expression, liu et al. (2006) identified a number of unique proteins expressed during the lytic phase of mdvinfected cef using a mass spectrometry-based proteomic approach. these studies highlight the potential of various proteomic tools in understanding the dynamics of host-pathogen interactions during various phases of md. the current study was intended to investigate the dynamics of host protein expression across the various phases of mdv life cycle in mdvinfected chickens. while we were able to detect more than 500 separate protein spots for each sample, here we report more than 60 significantly differentially expressed proteins identified using two-dimensional gel electrophoresis (2de) and mass spectrometry. putative importance of some of these proteins in the context of md is discussed. proteins from spleens of mdv-infected and uninfected control chickens at 7, 14 and 21 days post-infection (dpi) were extracted and analyzed by 2de in order to compare the protein expression profiles between each group. on average, 517 ± 84 distinct protein spots could be resolved by 2de using ph 3-10nl ipg strips loaded with fig. 2 . a representative gel image showing 2d gel electrophoresis map of the relative locations of spots that displayed significant quantitative differential expression (fdr ≤ 0.05 and fold change ≥ 2) at least once during different sampling times. this image represents the proteome of 7 dpi mdv-infected spleen. because of the absence of certain spots in this gel, arrows for spot 111, 968, 1576 and 1609 represent the relative positions only. please refer to table 1 for identities of corresponding spot numbers and to fig. 1 for maps of qualitatively differentially expressed spots. fig. 3 . comparison of total numbers of significantly differentially expressed protein spots in mdv-infected spleens at various sampling time points. in calculation of total number of spots in each category, newly induced protein spots were considered as upregulation and the absence of spots compared to uninfected controls was considered as down-regulation. 200 μg of total proteins. the molecular weights of spots ranged from 8 to 120 kda. differences in spot intensity were identified as either qualitative or quantitative changes (figs. 1 and 2). on average there were over 500 spots on each gel, however only 309, 375 and 341 spots from each group at consecutive sampling points were considered for statistical comparison. because, according to our selection criteria, only those spots which were present in at least 3 of 4 gels in both infected and control groups at a given sampling point were considered for quantitative comparisons. the highest number of qualitative differences in spots was detected at 7 dpi (n = 26). this number decreased in the subsequent sampling time point at 14 dpi (n = 19) and the lowest number was at 21 dpi (n = 7). although a similar pattern was seen in the total number of spot differences, the highest number of quantitatively significant differences (false discovery rate or fdr ≤ 0.05 and fold change ≥ 2) in spot expression was detected at 7 dpi (n = 33), which was followed by 21 dpi (n = 12) and 14 dpi (n = 2). among these spots, there were 16, 1 and 5 spots identified as significantly up-regulated at 7, 14 and 21 dpi, respectively. the rest were significantly down-regulated (fig. 3) . taken together, 61 protein spots were detected as either quantitatively or qualitatively differentially expressed at least once during different sampling points. in order to obtain the identities of the differentially expressed spots, the spots were manually excised from preparative gels prepared by loading 1 mg of total proteins and staining with coomassie blue. subsequently, trypsin-digested spots were identified by 1d lc esi ms/ ms. peptide identities with fdr b 0.01 were considered significant and, in total, the identity of proteins in 61 different spots representing 48 different proteins was determined (table 1) . moreover, several spots contained peptides generated from multiple proteins. while we have used the protein with the highest relative abundance under corresponding spot number throughout our discussion, the complete list of spots with multiple identities has been provided as supplementary table 1 . several proteins were differentially expressed at more than one time point, e.g. cathepsin d (cathd), natural killer cell enhancing factor isoform 4 (nkef), eukaryotic elongation factor 2 (eef2), aldolase b (aldob), membrane associated guanylate kinase (magi3), a predicted hypothetical protein (hp5) and beta actin (actb) at all three times. in total, 30 proteins were differentially expressed exclusively at 7 dpi. notably, several proteins involved in antigen presentation pathways, the ubiquitin-proteasome protein degradation system and a number of spots representing several cellular structural proteins were among those that were differentially fig. 4 . venn diagram summarizing the spots that were significantly differentially expressed in the spleen tissues of mdv-infected chickens according to their corresponding time of sampling. these identities include both quantitatively and qualitatively differentially expressed spots. the identities of spots which were commonly expressed were placed in overlapping areas accordingly. corresponding spot numbers are in parentheses. refer to table 1 for the respective protein names. expressed exclusively at 7 dpi. there was only one spot specific to each of 14 and 21 dpi. these spots were identified as glutathione-stransferase theta 1 (14 dpi) and proliferating cell nuclear antigen (21 dpi). the significant changes detected with the rest of the spots overlapped between each time point in varying numbers. interestingly, some of the spots representing a particular protein showed opposite directions of regulation even within the same group. for example, three spots with different molecular weights and pi were identified as cathd (table 1) . of these three spots, two spots with molecular weights of 13.3 and 28.4 kda were found to be up-regulated in infected birds at more than one time point. the summarized distribution of the identities of spots identified at each time point is presented in fig. 4 . forty-percent of the identified proteins were associated with the cytoplasm and the plasma membrane (terms go:0005737 and go:0005886 respectively). furthermore, there were 24% nuclear and nuclear envelope proteins (go:0005634 and go:0005635), 22% cytoskeleton-associated proteins (go:0005856) and 5% extracellular proteins (go:0005576). nine-percent of proteins did not have any go annotation with respect to their cellular compartment, hence, were categorized very broadly as being associated as a "cellular component" (go:0005575). for biological processes, the highest associations (18%) were with metabolic processes (go:0008152). another 16% associations were with nucleic acid metabolism (go:0006139), while 14% and 13% were associations with transport (go:0006810) and cell communication (go:0007154), respectively. among the remaining associations, 4% were associated with cell death (go:0008219) (fig. 5) . virus genome copy numbers in infected spleens were determined using quantitative real-time pcr (qpcr). using conventional pcr screening, mdv-meq gene could be amplified from dna of all infected spleens but not from any of the uninfected controls (data not shown). results of the subsequent qpcr analysis of infected samples are presented in fig. 6 . the average mdv-meq copy numbers were 3.82 × 10 6 ± 1.48 × 10 6 (n = 3), 2.66 × 10 7 ± 7.78 × 10 6 (n = 3) and 2.98 × 10 7 ± 1.22 × 10 7 (n = 4) per 100 ng of spleen dna at 7, 14 and 21 dpi, respectively (fig. 6 ). there was a statistically significant difference between genome copy numbers at 7 dpi compared to other time points. in the present study, we have profiled the global protein expression changes in the chicken spleen in response to mdv infection at various time points representing the different phases of mdv life cycle. furthermore, we have determined the identity of the spots that were differentially expressed between infected and uninfected birds using mass spectrometry. among the 48 proteins that were differentially expressed, there was considerable number of proteins that had multiple spots in 2d gels. in addition, there was some degree of disagreement between the expected and experimental molecular weights in case of some of the proteins. this could be due to several reasons. first, some proteins exist as different isoforms (therefore different pi) as well as different intermediate stages between translation and functionally mature form (i.e. pre-pro-and pro-forms of protein). this could change the molecular weight and/or the isoelectric point (for example, please see the discussion regarding different spots of cathd below). another reason is possible protein degradation between sample collection and processing. interestingly, no protein with multiple matches showed any specific migration pattern in our 2d gels (such as trains of spots). while current data are not enough to determine the exact reason for such discrepancies, it should be noted that several previous studies using 2d gels have also reported the presence of similar patterns of proteins with multiple spots (dupont et al., 2008; liu et al., 2008; saldanha et al., 2008; zheng et al., 2008) . analysis of viral load in spleen tissue revealed a significant increase over time. although viral genome load may not be a direct indicator for the degree of infection, there is a correlation between mdv genome load and the number of infected cells (bumstead et al., 1997) as well as with subsequent md incidence (islam et al., 2006) or protection conferred by vaccines against md (abdul-careem et al., 2007) . based on our previous observations, virus genome load of around 1 × 10 5 copies/100 ng of spleen dna is correlated with development of tumors in infected birds (abdul-careem et al., 2007) . therefore, it is conceivable that the virus copy numbers observed in the present study would be an indicative of a high level of infection. the proteins identified in the present study are involved in a variety of cellular processes, notably the antigen processing and presentation, ubiquitin-proteasome protein degradation, formation of the cytoskeleton, cellular metabolism, signal transduction and regulation of translation (table 1 ). the significance of these processes in chicken-mdv interaction is discussed below. although this was not determined in the present study, temporal changes in the cellular composition of the spleen may have, at least in part, influenced the whole spleen organ proteome. however, the advantage of our method is that the proteins that are differentially expressed could be used to point to cell subsets and/or mechanisms that have a potential involvement in mdv-host interactions and could be targeted for future studies. among the differentially expressed spots, several proteins were identified that are either directly or indirectly involved in the ubiquitin-proteasome (upp) and antigen presentation pathways (table 1 ). in our study, we identified three differentially regulated proteins, namely ubiquitin c-terminal hydrolase l3 (uchl3), proteasome (prosome, macropain) subunit beta type 7 (psmb7) and predicted protein similar to mouse proteasome 26s subunit atpase 5 (psmc5) (also known as msug1), which are involved in upp (table 1) . among them, chicken uchl3 (alias uch-6) has 86% amino acid similarity with its human counterpart (baek et al., 1999) , which is a deubiqutinating enzyme (dub) that functions as a negative regulator of ubiquitination of proteins as well as facilitates recycling of ubiquitin. increasing evidence suggests that dubs are important regulators of many cellular processes such as endocytosis, apoptosis and various signalling pathways (wing, 2003) . several virus-encoded proteins such as epstein-barr virus (ebv)-encoded epstein-barr nuclear antigen 1 and herpes simplex virus (hsv-1) regulatory protein icp0 have been shown to interact with dubs for viral survival in infected cells (reviewed in lindner, 2007) . although there is a high amino acid identity between human uch-l3 and chicken uch-6, they differ with respect to their substrate specificity and tissue distribution (baek et al., 1999) . therefore, the chicken ortholog of uch-l3 may not have a similar role in viral infections. more functional studies are needed to understand the significance of the down-regulation of uchl3 in mdv infection. down-regulation of psmb7 at 7 dpi probably indicates the modification of proteasome into immunoproteasome under the influence of interferon (ifn)-γ to enhance the generation of peptides for binding to major histocompatibility complex (mhc) class i molecules. in this process, three constitutive beta subunits of the 20s proteasome, namely delta, x and z (psmb7), are replaced by ifn-γ inducible low molecular mass peptide (lmp)-7, lmp-2 and multicatalytic endopeptidase complex-like (mecl) catalytic subunits, respectively (griffin et al., 1998) . although we could not detect upregulation of ifn-γ inducible subunits, given the reciprocal regulation between constitutive and ifn-γ inducible subunits (hisamatsu et al., 1996) , down-regulation of psmb7 may indicate the effect of induced ifn-γ. in line with that, mdv is known to induce ifn-γ in chickens as early as 3 dpi and remains up-regulated until at least 15 dpi (xing and schat, 2000) . moreover, although mdv is capable of down-regulating mhc-i expression in vitro, elevated ifn-γ has been shown to reverse the effect of mdv on mhc-i (levy et al., 2003) . our present observations do not provide evidence for differential regulation of mhc-i expression. however, given the possible enhancement of immunoproteasome activity, it is conceivable that there is an enhanced mhc-i-mediated antigen presentation. the 19s proteasome subunit sug1 up-regulates mhc-ii expression by interaction with class ii transactivator (ciita) gene and mhc-ii proximal promoter in human. mhc-ii expression is reduced in the absence of the expression of sug1 (bhat et al., 2008) . in agreement with this, the observed absence of predicted protein, msug1 in infected birds is associated with a significant decrease in the expression of the mhc class ii alpha chain (b-la) in infected spleens at 7 dpi. reduction of the mhc-ii expression is known to be a predominant way of evading the host immune response by a number of viruses including herpesviruses (hegde et al., 2003) . in agreement with our present observation of down-regulation of mhc-ii expression, previous work from our laboratory showed that mdv infection causes significant downregulation of invariant (ii) chain gene expression in the chicken spleen tissue (sarson et al., 2006) . in contrast to this observation, niikura et al. (2007) have shown an up-regulation of mhc-ii in bursa cells of chickens infected with md11, another very virulent strain of mdv. however, in contrast to our present infection model which used outbred spf chickens, they have used chickens from a cross between two inbred lines, 15i 5 and 7 1 , both of which are susceptible to md (bacon et al., 2000) . therefore, it is possible that the difference in genetic background of infected birds may have, at least in part, contributed to these contradicting observations. taken together, our observations suggest that there is enhanced antigen processing, presumably mediated by elevated ifn-γ for mhc class i pathway, while there is a down-regulation of mhc class iimediated antigen presentation at least in our experimental model at early stages of the disease. in the current study, altered spot profiles were observed for a number of cytoskeleton-associated proteins representing all three main categories of cytoskeleton proteins, namely microfilaments, intermediate filaments and microtubules. among the microfilament proteins, all detected actin spots, except for alpha-2 actin (acta2) and a predicted protein similar to coactosin-like 1 (cotl1) at 21 dpi, were either newly induced or significantly up-regulated across the experimental period. intermediate filament, lamin b2 (lmnb2) spots also were either up-regulated or newly induced in infected birds at all times. however, microtubule protein, tubulin β 2b (tubb2b) was significantly down-regulated in infected birds at 7 dpi (table 1) . changes in cytoskeleton proteins have been previously described in several other viral infections, such as ibdv (zheng et al., 2008) , severe acute respiratory syndrome (sars)-associated coronavirus (jiang et al., 2005) and human papillomavirus type 8 (akgül et al., 2009) . herpesviruses are known to interact with the actin filament system and its regulatory protein, rho gtpase, at various stages of infection (favoreel et al., 2007) . in addition to actins, here we have identified a rho gtpase regulatory protein, d4-gdi, as a protein that was differentially expressed in spleen of infected chickens (discussed in detail elsewhere in the discussion). this may indicate a putative role of the same system in the context of mdv infection. schumacher et al. (2005) showed that mdv-encoded us3 ortholog protein causes depolymerization of the actin stress fibers. however, its role in mdv replication and/or spreading is still unclear. stathmin 1 (stmn1), which also plays a role in the regulation of the microtubule system, has been shown to be up-regulated in ebv-infected b lymphocytes in human as early as 7 dpi (baik et al., 2007) . in the present study, we could not detect changes in stmn1 in spleen at 7 dpi. however, the expression of this protein was induced by 21 dpi. as a possible result of regulation by stmn1, tubb2b, which is a component of the microtubule filament system, also showed a similar pattern of expression in mdv-infected spleen tissues. however, if that change has any direct relationship with the stmn1 is not known. apart from its interaction with the microtubule system, stmn1 has been shown to interact with heat shock protein 70 (hsp70) proteins, particularly with heat shock cognate 70 (hsc70) (manceau et al., 1999) . in line with that, we have also identified significant changes in the expression of hsp70 proteins, including hsc70. another cellular structural protein, lmnb2, which is associated with the nuclear membrane, was up-regulated in infected birds throughout the experimental period as a probable result of the egress process of virus nucleocapsids from the infected nuclei. the nucleocapsids of herpesviruses acquire a temporary envelope from the inner nuclear membrane before egress by budding off from the infected cell nuclei (granzow et al., 2001) . in line with that, camozzi et al. (2008) have reported various biochemical and structural modulations, including mislocalization of lamin proteins in the nuclear envelope of cells infected with human cytomegalovirus. although camozzi et al. (2008) did not observe any quantitative changes in the expression of lamin proteins, qualitative changes in the expression of the same protein have been described in infections with several other viruses such as ibdv (zheng et al., 2008) , enterovirus 71 (leong and chow, 2006) and sars-associated coronavirus (jiang et al., 2005) . similar to the other members of the family, mdv particles egress from the nucleus of infected cells through budding (baigent and davison, 2004) . therefore, it is conceivable that the up-regulation of lmnb2 protein observed in the present experiment is, at least partly, the result of such interaction. proliferating cell nuclear antigen (pcna), which is one of the critical proteins in cell survival, was significantly up-regulated in infected spleens at 21 dpi. pcna is an essential component in dna synthesis process in the cell. therefore, it is likely that induction of pcna represents highly replicating cell populations in the spleen at 21 dpi. while it is possible that elevated pcna expression represents a transformed cell population, possible interactions between pcna and viral proteins might be also occurring. for example, hsv-1 encoded protein, icp34.5, interacts with pcna of infected cells, presumably preventing virus-induced translational arrest (brown et al., 1997; harland et al., 2003) . the possibility of a yet unidentified mdv protein interacting with pcna remains to be investigated. icp34.5 has also been shown to interact with phosphatase 1, preventing the inactivation of eukaryotic elongation factors (eef), a group of proteins which play an important role in protein biosyntheses, hence being important in various cell processes including proliferation (thompson and sarnow, 2000) . eef2 was among the proteins that were up-regulated at both 7 and 14 dpi. increased levels of eef2 have been shown in response to human immunodeficiency virus (hiv) protein, vpr, and have been shown to have the potential of preventing vpr-mediated apoptosis in cd4+ t cells (zelivianski et al., 2006) . d4-gdp-dissociation inhibitor (d4-gdi) (also known as ly-gdi) was differentially regulated in infected birds compared to uninfected controls. as mentioned above, ly-gdi represents a group of proteins i.e. gdi, which are involved in the regulation of another group of proteins, rho family gtpases. apart from its involvement in the organization of the cytoskeleton, rho-gtpases are also involved in cell signalling and proliferation. gdis are involved in the regulation of shifting of rho gtpase between the active gtp-bound form and the inactive gdp-bound form. our present observations showed a more than 5-fold increase of an 18.4 kda d4-gdi spot while there was a significant decrease of the presumably intact protein of 27 kda. while activation as well as apoptosis of different types of cells in response to viral infections has been well documented, our present observations may highlight some of the molecules involved in these processes in the context of mdv infection. cathepsin d (cathd), a lysosomal aspartic proteinase, was among the proteins that were differentially expressed in infected spleens at all three time points. there were two spots with molecular weight of approximately 13 and 28 kda which were up-regulated, while another one (39.2 kda) was down-regulated. cathd is synthesized as a single chain pre-pro-enzyme and after being cleaved into several successive intermediates, it forms the mature 34 + 14 kda form consisting of a heavy and a light chain in the lysosome (laurent-matha et al., 2006) . therefore, processing of the 39.2 kda intermediate into the mature enzyme appears to be induced by mdv infection. cathd has been shown to be an important mediator of apoptosis induced through the lysosomal pathway (guicciardi et al., 2004) . while cathd appears to be involved in the intrinsic pathway of the induction of apoptosis in activated t lymphocytes in human (bidere et al., 2003) , it may also play a significant role in the resolution of inflammation by inducing apoptosis in neutrophils (conus et al., 2008) . apart from its role in the induction of apoptosis, increasing evidence suggests that cathd plays a significant role in cancer progression and metastasis. in the context of md, several studies have shown the occurrence of cell death in mdvinfected organs such as the bursa of fabricius (st hill and sharma, 1999) , thymus (morimura et al., 1996) and in peripheral blood mononuclear cells (morimura et al., 1995) . while it is conceivable that up-regulated cathd may play a role in apoptosis during the early stages of md, it may also have a role in t cell transformation in the later stages of the disease; however, this needs to be further studied. transglutaminase 4 (tgm4) is among the proteins with the highest fold increase in spleens of infected chickens. generally, transglutaminases are involved in post-translational modification of proteins (beninati and piacentini, 2004) . tgm4 is highly expressed in the prostate gland of humans but little information is available about the function of this protein in chickens. tgm2, which is a ubiquitously expressed protein in human (also known as tissue tgm, ttgm), has been shown to play a significant role in stress response (ientile et al., 2007) notably in apoptotic cell death. while tgm2 is up-regulated in apoptotic cells, it acts as a molecular glue and appears to stabilize the dying cells to prevent release of intracellular molecules prior to clearance by phagocytosis (fesus and szondy, 2005) thereby preventing adverse effects, such as excessive inflammatory reactions. assuming chicken tgm4 has a similar role as that of human tgm2, above observations may explain the putative role of the former in the context of host-mdv interactions in the spleen to prevent collateral damage to the neighbouring cells. according to go classification based on the biological process, the highest association of identified proteins in the current study was with metabolic processes (19%) (fig. 5) . notably, several metabolic enzymes associated with glycolysis have been found differentially regulated. among them, a 38.76 kda spot representing aldolase b fructose-bisphosphate (aldob) was constantly up-regulated in infected spleens at all time points. interestingly, another spot with similar identity and molecular weight but slightly different pi was significantly down-regulated only at 7 dpi. while different pis for the same protein is possible with structural changes such as phosphorylation, functional relevance in this context needs to be elucidated. another two newly induced glycolytic enzymes, each at 7 and 21 dpi, were identified as triosephosphate isomerase 1 (tpi1) and phosphoglycerate mutase 1 (pgam1), respectively. viruses utilize host cell metabolic process for their replication process. in agreement with the present observations, significantly elevated levels of several serum enzymes, including aldolase b, in response to mdv infection in vivo has been previously described (ivanov et al., 1974) . further, mdvencoded protein pp38 has been shown to up-regulate cellular metabolic activities in vitro as determined by the enhanced activity of mitochondrial dehydrogenases (li et al., 2006) . similarly, human cytomegalovirus infection in fibroblasts has also shown overall upregulation of a number of glycolytic enzymes (munger et al., 2006) . in conclusion, findings of the present study highlight some of the mechanisms involved in the host response in the spleen to mdv infection during various time points representing different stages of mdv pathogenesis. although the functions of the proteins, which were identified here, were not studied, it is likely that all or some of them are involved in host-virus interactions. one of the limitations of the tools used in this study is the inefficiency of detecting low abundance proteins or those with low molecular weights, such as cytokines and chemokines. therefore, a more comprehensive study is needed to elaborate on our present observations and to further explore other proteins that may play a role in pathogenesis of the virus as well as host responses to this virus. all the chickens used in this experiment were one-day old specific pathogen free (spf) chickens obtained from the animal disease research institute, canadian food inspection agency (ottawa, ontario, canada). birds were kept in an isolation facility at the ontario veterinary college throughout this experiment. chickens were infected with the rb1b strain of very virulent marek's disease virus (passage 9) (schat et al., 1982) which was obtained from dr. k.a. schat (cornell university, ny, usa) . twenty-four, one-day old chicks were randomly divided into two groups and were housed in the isolation facility. one group of birds (n = 12) was given 750 plaque-forming units (pfu) of the rb1b strain of very virulent mdv intraperitoneally on day 5 of age. the rest (n = 12) were kept as uninfected controls. infected and uninfected control birds were kept in separate units with similar environmental conditions. on 7, 14 and 21 dpi, representing different stages of mdv pathogenesis, four chickens that were randomly selected from each group were euthanized using co 2 inhalation. at necropsy, a portion of spleen was collected from each bird and was snap-frozen in liquid nitrogen. subsequently, frozen tissues were kept at −80°c until further processing. another portion was preserved in rnalater (qiagen inc., missisauga, on, canada) . animal experiments were conducted in accordance with the guidelines provided by the canadian council on animal care. all experiments complied with institutional animal care guidelines and were approved by university of guelph animal care committee (protocol number 06r015). each frozen spleen tissue was briefly homogenized in a lysis buffer (ph 8.5) containing 30 mm tris-cl, 2 m thiourea, 7 m urea and 4% (w/v) chaps. the volume of lysis buffer used for each tissue sample was equal to 15 times of tissue mass. samples were further solubilized by sonication for 5 min on ice and insoluble tissue debris was removed by centrifugation under 18,000 ×g at 4°c. subsequently, supernatant was collected separately from each sample and protein concentrations were determined using the bio-rad protein assay as prescribed by the manufacturer. the spleen protein sample from each chicken was analyzed separately. there were a total of 24 chickens in two groups (infected and uninfected) i.e. 24 protein samples for analysis by 2d-page. each analytical 2d-page gel was prepared with 200 μg of proteins mixed with rehydration buffer (8 m urea, 2% chaps, 90 mm dtt, 5 μl/ml appropriate ipg buffer, 12 μl/ml destreak reagent (ge healthcare) and 0.005% bromophenol blue) to a total volume of 250 μl. the first dimension separation was performed in 13 cm, ph 3-10 non-linear immobiline drystrips (ge healthcare) using ettan ipgphor isoelectric focusing unit (ge healthcare). after rehydration at 30 v for 12 h, isoelectric focusing was performed at 500 v for 1 h, 1000 v for 1 h and 8000 v until a total of 57,000 volt hours was reached. each focused strip was incubated at room temperature, initially in 10 ml of equilibration buffer (50 mm tris-cl (ph8.8), 6 m urea, 30% (v/v) glycerol, 2% (w/v) sds and 0.005% bromophenol blue) containing 1% (w/v) dtt for 15 min and subsequently in a similar volume of equilibration buffer containing 2.5% (w/v) iodoacetamide for a similar time. for the second dimension separation, each ipg strip was placed on a 12.5% sds-polyacrylamide gel and four such gels were simultaneously run each time subjecting them to 25 ma/gel of current at 25°c in a ruby apparatus until the bromophenol blue dye front reach the opposite edge of the gel. each gel was subsequently fixed for 1 h in a solution containing 10% (v/v) methanol and 7% (v/v) acetic acid, stained with sypro ruby stain (bio-rad) overnight and destained in the fixing solution for 2 h. gel images were digitized using typhoon 9400 variable mode imager (ge healthcare) at 532 nm using a 610 nm filter. preparative gels were prepared in a similar manner, using 1 mg of protein from each sample and stained them with coomassie brilliant blue instead of sypro ruby. digitized gel images were used to estimate the expression of different proteins in each analytical gel using 2004 version of phoretix 2d software (nonlinear dynamics). the pixel volume of each detected spot was referred to as the spot volume and was used in the subsequent comparisons. background correction for pixel volumes of each spot was done using the mode of non-spot and normalized spot volumes were calculated as a fraction against the total volume of spots in each gel. the spots which were not present in the expected position or showed decreased intensity were considered "down-regulated", while the spots that appeared in only one group or showed enhanced intensity were considered "up-regulated". although this is an indicator of protein abundance, the possibility of post-translation modifications, hence changes in location of spots on the 2d-page gels, could not be ruled out in our study. there were eight 2d-page gels per time point: 4 derived from infected spleens and 4 from those of uninfected birds. only the spots that were present in all gels and those that were absent from a maximum of one analytical gel per group at a given time point were considered for the statistical comparison. statistical analysis was done using sas (version 9.1). normalized volumes of each corresponding spot from each group at similar time point were compared with student t-test. the resulting p values were used to calculate the fdr. spots that were having both p ≤ 0.01 and fold difference n2 in mean normalized volumes were considered as significantly differentially expressed. fdr for any selected spot was less than 5%. the spots that showed a significant difference, and those expressed only in a particular group at a given sampling point, were selected for identification by 1d lc esi ms/ms using a lcq deca xp plus mass spectrometer coupled with two thermo surveyor ms pump quaternary gradient pumps at the life science and biotechnology institute, mississippi state university as described below. each selected spot was excised manually from coomassie brilliant blue stained preparative gels and "in-gel" digested exactly as previously described (shevchenko et al., 1996) . proteins were reduced with 5 mm dtt at 65°c for 5 min and alkylated with 10 mm iodoacetamide at 30°c for 30 min. trypsin digestion was done using molecular biology grade porcine trypsin (2 μg; 37°c; 16 h; 50:1 ratio of protein:trypsin; promega corporation, madison, wi). liquid chromatography was done with a reverse phase (c18) lc column coupled directly in line with the mass spectrometer. peptides were loaded into a liquid chromatography gradient ion exchange system containing a thermo separations p4000 quaternary gradient pump (thermoelectron corporation; san jose, ca) coupled with a 0.18 × 100 mm biobasic c18 reverse phase liquid chromatography column of a proteome x workstation (thermoelectron). the reverse phase gradient used 0.1% formic acid in acetonitrile and increased the acetonitrile concentration in a linear gradient from 5% to 30% in 15 min and then 30% to 65% in 5 min followed by 95% for 5 min and 5% for 10 min. the mass spectrometer was configured to optimize the duty cycle length with the quality of data acquired by alternating between a single full ms scan followed by three tandem ms scans on the three most intense precursor masses (as determined by xcalibur software in real time) from the full scan. the collision energy was normalized to 35%. dynamic mass exclusion windows were set at 2 min and all of the spectra were measured with an overall mass/ charge (m/z) ratio range of 300-1700. identification of all spots was done as a single run in a randomized order. to prevent "carry-over" after the peptides generated from each spot were analyzed, and before those from the next spot were analyzed, the lc column was washed with 95% acn, a negative control sample was run to confirm no carryover and the lc column was washed again. resulting mass spectra were analyzed using bioworks 3.2 (thermoelectron) using a non-redundant proteome database containing protein from both chicken and mdv-rb1b (build 3.2) downloaded from the ncbi. we used the bioworks reverse database function to create the decoy database from this proteome database. the probability of the tandem mass spectrometry match occurred by chance was calculated using; (a) the decoy database searching exactly as described by elias and gygi (2007) and (b) the orthogonal p(pep) function in bioworks 3.2 (which calculates probability based on a theoretical y and b ion spectrum calculated based on theoretical amino acid dissociation). these two probabilities were the used to calculate the fdr as described by benjamini and hochberg (1995) and only peptides with fdr b 0.01 were considered as a significant and retained for protein identification. the probability of protein identity was then calculated from the peptide probabilities exactly as described (maccoss et al., 2002; nesvizhskii et al., 2003) . to calculate the percent protein coverage by identified peptides, the protein sequence was digested in silico using "peptidecutter" (http://us. expasy.org/tools/peptidecutter/ (gasteiger et al., 2005) ) and the total number of amino acids in peptides between 6 and 30 amino acids (the size range of 99% of the peptides detected by the mass spectrometer) was used as the denominator; the peptides identified were used as the nominator. for spots with multiple protein identities, the protein with the highest peptide coverage and highest protein score (i.e. σxcorr; a surrogate for the amount of precursor ion) (nanduri et al., 2005) was considered as the dominant protein and the most likely protein to contribute to the differential expression in the gel. subsequently, their biological relevance was discussed. spot identities were submitted to goretriever (http://www. agbase.msstate.edu/) to obtain the go annotations. if no annotation was returned, goanna was used to retrieve go annotations assigned depending on the sequence similarities. the resulting annotations were summarized based on the goa and whole proteome goslim set using goslimviewer (mccarthy et al., 2006) . dna extraction, conventional pcr confirmation of the presence of mdv-meq gene and subsequent determination of the absolute mdv genome copy number from infected spleen samples were essentially performed as previously described . quantitative real-time pcr reactions to determine viral genome load in samples were performed in duplicate. development of a real-time pcr assay using sybr green chemistry for monitoring marek's disease virus genome load in feather tips cytokine gene expression patterns associated with immunization against marek's disease in chickens proteomic analysis reveals the actin cytoskeleton as cellular target for the human papillomavirus type 8 study of host-pathogen interactions to identify sustainable vaccine strategies to marek's disease a review of the development of chicken lines to resolve genes determining resistance to diseases molecular cloning of chick uch-6 which shares high similarity with human uch-l3: its unusual substrate specificity and tissue distribution marek's disease virus. inbiology and life cycle identification of stathmin 1 expression induced by epstein-barr virus in human b lymphocytes the transglutaminase family: an overview: minireview article controlling the false discovery rate -a practical and powerful approach to multiple testing the 19s proteasorne atpase sug1 plays a critical role in regulating mhc class ii transcription cathepsin d triggers bax activation, resulting in selective apoptosis-inducing factor (aif) relocation in t lymphocytes entering the early commitment phase to apoptosis the herpes simplex virus virulence factor icp34.5 and the cellular protein myd116 complex with proliferating cell nuclear antigen through the 63-amino-acid domain conserved in icp34.5, myd116, and gadd34 quantification of marek's disease virus in chicken lymphocytes using the polymerase chain reaction with fluorescence detection proteomics in the chicken: tools for understanding immune responses to avian diseases modeling the proteome of a marek's disease transformed cell line: a natural animal model for cd30 overexpressing lymphomas different signaling pathways expressed by chicken naive cd4 + t cells, cd4 + lymphocytes activated with staphylococcal enterotoxin b, and those malignantly transformed by marek's disease virus remodelling of the nuclear lamina during human cytomegalovirus infection: role of the viral proteins pul50 and pul53 caspase-8 is activated by cathepsin d initiating neutrophil 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interferon gamma tissue transglutaminase and the stress response relationship between marek's disease virus load in peripheral blood lymphocytes at various stages of infection and clinical marek's disease in broiler chickens changes in the activity of several serum enzymes in chicks with marek's disease quantitative analysis of severe acute respiratory syndrome (sars)-associated coronavirus-infected cells using proteomic approaches -implications for cellular responses to virus infection processing of human cathepsin d is independent of its catalytic function and autoactivation: involvement of cathepsins l and b transcriptomic and proteomic analyses of rhabdomyosarcoma cells reveal differential cellular gene expression in response to enterovirus 71 infection major histocompatibility complex class i is downregulated in marek's disease virus infected chicken embryo fibroblasts and corrected by chicken interferon expression of marek's disease virus phosphorylated polypeptide pp38 produces 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report dynamics of the cellular metabolome during human cytomegalovirus infection proteomic analysis using an unfinished bacterial genome: the effects of subminimum inhibitory concentrations of antibodies on mannheimia haemolytica virulence factor expression a statistical model for identifying proteins by tandem mass spectrometry marek's disease virus up-regulates major histocompatibility complex class ii cell surface expression in infected cells use of dna microarrays to monitor host response to virus and virus-derived gene therapy vectors changes in the gallus gallus proteome induced by marek's disease virus differential proteome expression associated with urokinase plasminogen activator receptor (upar) suppression in malignant epithelial cancer transcriptional analysis of host responses to marek's disease viral infection characterization of 2 highly oncogenic strains of marek's disease virus the protein encoded by the u s 3 orthologue of marek's disease virus is required for efficient de-envelopment of perinuclear virions and involved in actin stress fiber breakdown the neoplastically transformed (cd30 hi ) marek's disease lymphoma cell phenotype most closely resembles t-regulatory cells mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels response of embryonic chicken lymphocytes to in ovo exposure to lymphotropic viruses regulation of host cell translation by viruses and effects on cell function deubiquitinating enzymes-the importance of driving in reverse along the ubiquitin-proteasome pathway increased virulence of marek's disease virus field isolates expression of cytokine genes in marek's disease virusinfected chickens and chicken embryo fibroblast cultures suppressive effect of elongation factor 2 on apoptosis induced by hiv-1 viral protein r host-pathogen interactions: a proteomic view proteomics analysis of host cells infected with infectious bursal disease virus key: cord-310218-fky0cm5e authors: yoo, dongwan; parker, michael d.; babiuk, lorne a. title: the s2 subunit of the spike glycoprotein of bovine coronavirus mediates membrane fusion in insect cells date: 1991-01-31 journal: virology doi: 10.1016/0042-6822(91)90045-d sha: doc_id: 310218 cord_uid: fky0cm5e abstract the hemagglutinin/esterase (he), spike precursor (s) and the s1 and s2 subunits of the spike precursor protein of bovine coronavirus were expressed in spodoptera frugiperda (sf9) cells, and the cell-fusing activity of each recombinant glycoprotein was examined. extensive syncytia formation was observed in cells infected with the s2 recombinant but not with the he or s1 recombinant baculoviruses. fusion of sf9 cells expressing the intact s protein precursor was evident after trypsin treatment. these results demonstrate that proteolytic cleavage of the s spike precursor is required for fusion induction and that the fusion is mediated by the s2 subunit. these observations may reflect the biological role of the s2 subunit in fusion-penetration during bovine coronavirus infection. the hemagglutinin/esterase (he), spike precursor (s) and the sl and s2 subunits of the spike precursor protein of bovine coronavirus were expressed in spodopfera frugperda (sf9) cells, and the cell-fusing activity of each recombinant glycoprotein was examined. extensive syncytia formation was observed in cells infected with the s2 recombinant but not with the he or si recombinant baculoviruses. fusion of sf9 cells expressing the intact s protein precursor was evident after trypsin treatment. these results demonstrate that proteolytic cleavage of the s spike precursor is required for fusion induction and that the fusion is mediated by the s2 subunit. these observations may reflect the biological role of the s2 subunit in fusion-penetration during bovine coronavirus infection. o 1991 academic press, i~c. the envelopes which surround many viruses are derived from host cell membranes and contain multiple copies of viral glycoproteins which form the surface projections of the virions required for host cell binding and penetration. two mechanisms for virus penetration of the target cell have been proposed (i). in the case of paramyxoviruses, for example, after initial binding of the cell surface, the viral envelope fuses with the plasma membrane directly, releasing the nucleocapsids into the interior of the cell (2) . in contrast, alphaviruses, rhabdoviruses, and ot-thomyxoviruses are internalized by endocytosis and fusion of the envelope with endosomal membranes is activated in an acidic, intracellular compartment (3) . bovine coronavirus (bcv) contains two major membrane glycoproteins; the hemagglutinin/esterase (he) and the spike(s) protein (4, 5) . the he glycoprotein is a disulfide-linked 140k homodimer of a 62k monomer, and exhibits hemagglutinating and sialic acid-containing receptor-destroying activities (6) (7) (8) . the 180k s glycoprotein is cleaved into sl and s2 subunits at amino acids 768-769 and forms the large club-shaped projections characteristic of coronaviruses (9, 10) . the s2 subunit is associated with the viral envelope and forms the stalk of the surface projection while the sl subunit is believed to form the bulbous head of the projection. studies using a series of monoclonal antibodies have demonstrated that bcv-neutralizing epitopes are located on the he glycoprotein (1 i) and the sl subunit of the s glycoprotein (i 1, 12). the biological significance of the s2 subunit of bcv has not been determined yet. although cell fusion has been observed in cultures ' to whom requests for reprints should be addressed. infected with many coronaviruses, there is no direct evidence as to which viral components are involved (13, 14) . one approach to elucidate the function of individual viral components is to express the genes for the individual protein and examine their biological activiti.es in the absence of other viral components. recently, the autograph californica nuclear polyhedrosis baculovirus has been developed as a helper-independent expression vector. the recombinant proteins expressed by baculovirus appear to retain the biological activities, such as immunogenicity, enzymatic activity, and other physicochemical properties (15, 16) . several viral fusion proteins expressed in insect cells have been also demonstrated to be biologically active (17, 18) . thus, in order to identify the viral membrane glycoprotein which induces cell fusion by bcv, we expressed the he, the s, and the sl and s2 subunits of the s glycoprotein using recombinant baculoviruses, and examined the cell-fusing activity of each recombinant polypeptide. the construction of recombinant plasmids of the he (8) s (9) and sl (12) genes of bcv have been described in detail elsewhere. briefly, in order to express the sl subunit, the s gene was digested with tthllll, which cleaved the s gene within the sequences encoding the proteolytic cleavage site of the s precursor glycoprotein (nucleotides 2294-2295). the 5' 2294nucleotide fragment was fused to a universal translational terminator and inserted into the barnhi site of baculovirus transfer vector pacym 1 (19). because the s2 subunit is derived by proteolytic cleavage of the s precursor polypeptide, the cdna sequences encoding the s2 subunit lack a translation initiation codon and membrane translocation sequence. therefore, the cdna sequence encoding the s2 subunit was fused to the amino-terminal signal sequence of the bcv he glycoprotein. plasmid pcve3 (20) , which contains the cloned he gene of bcv in the bamhl site of ptz18r (pharmacia), was digested with styl, blunt-ended by si nuclease treatment and digested with s&l. a 2.0-kb seal-sal1 fragment containing the cdna sequences encoding the s2 subunit was obtained from the plasmid pcve2, which contains the bcv s gene cloned in the bamhl site of ptzl8r and was ligated into the linearized pcve3. this recombinant plasmid was subsequently modified by site-directed mutagenesis, yielding plasmid pacds2, which contained the complete bcv he signal sequence (21) fused to the s2 subunit through the alanine residue located at the amino terminus of the mature s2 subunit (10) (fig. 1) . the cloned genes were introduced into the genome of a. californica nuclear polyhedrosis virus (acmnpv) by homologous recombination, and recombinant viruses were isolated by plaque hybridization and subsequent purification (22) . recombinant baculoviruses containing the he, s, sl, and s2 genes were designated bvlhe (8) vacs (9), vacs1 (12) , and vacs2, respectively. synthesis of bcv-specific polypeptides in spodoptera frugiperda (sf9) cells infected with the recombinant viruses was examined at 24 hr postinfection. rabbit antisera to bcv precipitated a 57k polypeptide when analyzed under reducing conditions in the cells infected with bvlhe (fig. 2, lane 2) . the 57k polypeptide represents the monomer of the he glycoprotein. cells infected with vacs produced a 170k polypeptide. this indicates that the s polypeptide was produced but proteolytic cleavage was not occurring to a significant extent in sf9 cells (fig. 2, lane 3) . a 95k and an 80k polypeptide of the sl and s2 subunits were immunoprecipitated from cells infected with vacs1 and vacs2, respectively (fig. 2, lanes 4, 5) . in order to determine if any of the recombinant polypeptides were capable of inducing cell fusion, sf9 cells were infected with the recombinant baculoviruses at an m.o.i. of 5-l 0 pfu per cell and incubated at 28" in tnm-fh media containing 10% fetal bovine serum (22) . at 36 hr postinfection, the medium was replaced with tnm-fh in which the ph varied between 5.0-6.5. in order to examine syncytia formation at alkaline ph and because tnm-fh becomes turbid at alkaline ph, replicates of the infected cells were overlayed with 1.5% agarose in pbs which had been adjusted to a ph range of 5.0-8.0. syncytia formation was monitored by phase-contrast microscopy. fusion was not detected in cultures infected with wild-type acmnpv, bvlhe, or vacs1 recombinant baculoviruses over the ph range examined (figs. 3a, 3b, 3d ). in contrast, fusion of vacs-infected cells was apparent within 2 hr after a ph shift to 5.3 (fig. 3c) . extensive syncytia formation was observed in vacs2-infected cells (fig. 3e ) and continued to increase over 8 hr of observation until giant syncytia composed of approximately 100-200 cells were observed (fig. 3f) . when polyclonal bcv antibodies were included in the media, the fusion by the s and s2 polypeptides was partially inhibited (table 1 ). these observations demonstrate that the s2 subunit of the spike glycoprotein of bcv can induce cell fusion in the absence of other viral components. 150 mm naci. 50 mm tris-hci, ph 7.4, 10 mm edta, 0.1% sds) and immunoprecipitated with bcv-specific rabbit antisera using a suspension of 10 mg protein a-sepharose (pharmacia). the immune complexes were dissociated with 10% sds, 25% glycerol, 10 mmp-mercaptoethanol, 10 mmtris-hci, ph 6.8, 0.02% bromphenol blue) and electrophoresed on a 7.5% discontinuous sds-polyacrylamide gel followed by autoradiography. the low level of fusion observed in vacs-infected vacs-infected cells to a level similar to that seen in cells (fig. 3c ) was thought to be due to a low level of vacs24nfected cells. this result demonstrates that cleavage of the s polypeptide. partial cleavage of the cleavage of the s precursor is required for induction of recombinant s polypeptide of bcv in sf9 cells has cell fusion as previously reported in coronavirus-inbeen demonstrated by a pulse-chase experiment (72). fected cells (23, 24) . in order to further determine the effect of proteolytic we have demonstrated that a polypeptide comcleavage on the cell fusing activity of the recombinant posed of signal sequence of the..bcv he glycoprotein s polypeptide precursor, vacs-infected cells were followed by the s2 subunit of the bcv s glycoprotein treated with 20 pg/ml of trypsin for 10 min in pbs, ph induces cell-cell fusion in sf9 cells. although it is not 8.0, prior to a shift to ph 5.3. as shown in table 1 , clear whether the he signal sequence is cleaved in trypsin treatment increased the extent of cell fusion in insect cells, the alanine residue at position 19 derived from the n-terminus of the s2 subunit provides a favorable context for cleavage of the he signal sequence (25) . the he signal peptide of 18 amino acids does not seem to contribute to the fusogenicity of the recombinant s2 polypeptide since the he polypeptide expressed with bvlhe did not induce fusion (fig.3b) . we have concluded that the domain responsible for the cell fusion is located on the s2 subunit. the location of fusion domain on the s2 subunit of bcv is consistent with previous suggestions that the crucial fusogenic domain in mhv resides in the c-terminal half (s2 portion) of the peplomer protein (26, 27) . fusion of insect cells infected with vacs was dramatically increased by trypsin treatment (table 1) . therefore, it is clear that proteolytic cleavage is required to induce the fusion activity of both the recombinant s polypeptide in insect cells and the authentic s polypeptide produced in bcv-infected cells (14) . it has been observed that fusogenic polypeptides which require proteolytic activation often contain a hydrophobic domain located at the n-terminus which is exposed as a result of proteolytic cleavage of the precursor (2) . in other instances, internal fusogenic domains that are masked at physiological ph are uncovered and activated by a conformational change which occurs in acidic compartments within the infected cell (28) . a hydropathy plot of the bcv s2 subunit indicates that there are seven hydrophobic domains in addition to the putative membrane anchor domain. the n-terminus of the s2 subunit of bcv appears to be largely hydrophilic. the first hydrophobic domain (vtidcsafv-cgdyaac) is located 54 amino acids downstream from the cleavage site of the s precursor. a similar situation has been observed for semliki forest virus where the fusogenic domain of the el glycoprotein is located at amino acids 80-96 from the n-terminus (29) . the significance of the internal hydrophobic domains of the bcv s2 subunit with respect to fusogenicity remains to be determined. recently, sturman et al. (30, 31) have demonstrated that the both sl and s2 subunit glycoproteins of mouse hepatitis coronavirus (mhva-59) undergo conformational changes at ph 8.0 and 37', resulting in the release of the sl from virions. furthermore, based upon the observation of mhv-induced cell fusion under mildly alkaline conditions, both of the subunits have been suggested to correlate with the coronavirus infectivity and cell fusion. however, we have not detected fusion with the sl subunit (fig. 3d) . in semliki forest virus, although both el and e2 glycoproteins undergo conformational changes at fusion ph, only el is necessary and sufficient to mediate membrane fusion (32) . similarly, a conformational change of the bcv sl may be important for exposing the fusogenic domain of the s2 subunit by releasing the sl rather than being directly involved in fusogenicity. acidic conditions required for fusion in insect cells by the recombinant s2 subunit seem to indicate the involvement of an acidic compartment in initiation of bcv infection. earlier evidence also suggested that mhv infection involved acidification of endosomal vesicles (33) . however, fusion of mammalian cells infected by bcv or other coronavirus has been observed at mildly alkaline conditions ranging from ph 7.5 to 8.0 (14, 30) , suggesting that coronavirus may enter the target cells by direct fusion of the viral envelope with the plasma membrane. in addition, we have previously observed dramatic differences in glycosylation of the bcv spike glycoproteins between insect cells and mammalian cells (12) . the glycosylation differences may result in the conformational changes and the altered optimum ph for fusogenicity of the recombinant s2 subunit. thus, expression of the s2 subunit polypeptide in mammalian cells should help to determine if posttranslational processing of the s2 subunit has a direct bearing on the ability to mediate cell fusion at alkaline ph. previous reports have indicated that the s2 subunit of the s polypeptides of other coronaviruses contain neutralizing epitopes as well as determinants of virulence and tissue tropism (26, (34) (35) (36) . deregt et al. (5) have produced a panel of monoclonal antibodies specific for the s polypeptide of bovine coronavirus, all of which bind the sl subunit (12) . as fusion of the viral envelope with cellular membranes is a critical requirement for virus infection, we are preparing antisera directed against specific regions of the s2 subunit in order to identify the amino acids mediating membrane fusion and to determine if the fusogenic domain of the s2 polypeptide constitutes an important immunological determinant. proc. nat/. acad. sci. usa proc. natl. acad. sci. usa key: cord-300810-a1skdp67 authors: lafay, f.; coulon, p.; astic, l.; saucier, d.; riche, d.; holley, a.; flamand, a. title: spread of the cvs strain of rabies virus and of the avirulent mutant avo1 along the olfactory pathways of the mouse after intranasal inoculation date: 1991-07-31 journal: virology doi: 10.1016/0042-6822(91)90145-2 sha: doc_id: 300810 cord_uid: a1skdp67 abstract after intranasal instillation in the mouse, rabies virus (cvs strain) selectively infected olfactory receptor cells. in the main olfactory bulb (mob), infection was observed in periglomerular, tufted, and mitral cells and in interneurons located in the internal plexiform layer. beyond the mob, cvs spread into the brain along the olfactory pathways. this infection is specific to chains of functionally related neurons but at the death of the animal some nuclei remain uninfected. cvs also penetrated the trigeminal system. the avirulent mutant avol, carrying a mutation in position 333 of the glycoprotein, infected the olfactory epithelium and the trigeminal nerve as efficiently as cvs. during the second cycle of infection, the mutant was able to infect efficiently periglomerular cells in the mob and neurons of the horizontal limb of the diagonal band, which indicates that maturation of infective particles is not affected in primarily infected neuronal cells. on the other hand, other neuronal cells permissive for cvs, such as mitral cells or the anterior olfactory nucleus, are completely free of infection with the mutant, indicating that restriction is related to the ability of avo1 to penetrate several categories of neurons. from these observations, we concluded that cvs should be able to bind several different receptors to penetrate neurons, while the mutant would be unable to recognize some of them. rabies is an enveloped rna virus which causes encephalitis in all mammals and is considered a strictly neurotropic virus. peripheral inoculation of the cvs strain of the virus results in a specific infection of the nervous system, especially of the neurons. viral material has also been demonstrated in striated muscle at the site of injection of street rabies virus (murphy and bauer, 1974) and in various tissues of experimentally infected animals (murphy et al., 1973; fekadu and shaddock, 1986) , including the salivary glands, the secretions of which are the major source of virus in animal vectors of rabies. the only external protein of the virion is the glycoprotein (g), a transmembrane protein of 505 amino acids. the g protein plays a pivotal role in the pathogenicity of the virus because of its interaction with the host cells ' abbreviations used: aon, anterior olfactory nucleus; cns, central nervous system; gaba, gamma aminobutyric acid; hdb, horizontal limb of the diagonal band; hrp, horseradish peroxidase; hsvl, herpes simplex type 1; ipl, internal plexiform layer: lc, locus coeruleus; ld50, lethal dose 50%; lpa. lateral preoptic area; mcpo, magnocellular preoptic nucleus; mhv, murine hepatitis virus; mob, main olfactory bulb; pfu, plaque-forming unit; p.i., postinfection; scg, superior cervical ganglion; vsv, vesicular stomatitis virus. during the adsorption-penetration process and because of its function in the stimulation of the immune system. few mutations which affect antigenic site ii, the immunodominant antigenic site of the glycoprotein, slightly reduce the pathogenic power of the virus (prehaud et a/., 1988) . in antigenic site iii, the other major antigenic site, substitution of the arginine in position 333 by an amino acid other than lysine completely abolishes the virulence of the virus for adult mice, whatever the dose and the route of inoculation (coulon et al., 1982a; dietzschold et al., 1983; seif et a/., 1985; tuffereau et al., 1989) . these avirulent mutants are useful tools for studying the specificity of viral tropism for the central nervous system (cns)'. avol , an antigenic mutant of the cvs strain, is currently the most studied mutant. its mutation consists of a substitution of the arginine 333 by a glutamine (seif et a/., 1985) . experiments in rats indicate that this avirulent mutant is restricted in infection of the cns. inoculation of cvs virus in the anterior chamber of the eye results in infection of both the trigeminal and the parasympathetic nerve ganglia and the retinopetal route, whereas avol is only able to penetrate into the trigeminal nerve (kucera et a/., 1985) . a similar antigenic mutant called rv-194-2 has been isolated in another laboratory and was also used for experiments in animals (dietzschold et a/., 1983) . the virulent and avirulent viruses have been compared in their ability to penetrate and propagate in the nervous system of the mouse along different pathways dietzschold et a/., 1985; jackson, 1991) . after injection in the forelimb both kinds of viruses infect the motor and sensory neurons with the same efficiency . after intramasseter inoculation, avirulent mutant was also found in both motor and sensory nuclei, while cvs was found only in the motor route (jackson, 1991) . in both cases, the block in the propagation of the avirulent mutant was observed as early as the second cycle of multiplication. this block would be the consequence of either abnormal maturation of the mutant in the neurons or nonrecognition of a specific cellular receptor by the glycoprotein of avirulent mutant. on the contrary, dietzschold et a/. (1985) who injected the virus intracerebrally, published that the two viruses spread similarly in the brain, although the infection by the avirulent mutant would progress slower than the cvs one. in order to investigate more precisely the origin of the restricted multiplication of avol in the cns, we decided to follow the propagation of the virus in the olfactory pathways. lntranasal inoculation was used for several reasons. first, rabies virus is able to infect the cns via the nasal route, since a few examples of airborne transmission of rabies have been described (winkler, 1975) ; despite increasing interest for this route of infection, the mechanism of viral spread to the cns has never been carefully examined. in addition, the olfactory system is anatomically well studied with several categories of connected neurons relatively easy to identify. finally, the receptor cells of the olfactory epithelium are bipolar neurons whose dendrites are in contact with the external environment. thus, these cells can be directly infected by a virus after it has been dropped in the nasal cavity without preliminary multiplication in nonneural tissues. neuroreceptor axons terminate in the glomerular layer of the main olfactory bulb (mob) where they synapse with dendrites of the periglomerular cells, the tufted cells, and the mitral cells (halasz and shepherd, 1983) . the axons of neurons of the anterior olfactory nucleus (aon) (switzer et al., 1985) the horizontal limb of the diagonal band (hdb) (zaborsky et al., 1986) and the dorsal raphe nuclei (dr) (mclean and shipley, 1987) also terminate in the glomerular layer (fig. 1) . since the progression of the infection seems to occur only through synapses it should be possible to follow at least two successive cycles of multiplication after instillation of the virus in the nose. mice were infected by this route with cvs or avol, and the spread of the viruses in the cns was followed with immunohistochemical techniques. the cvs strain of rabies and its avirulent derivative avol were grown in bhk-21 cells. concentration of the viruses from cell culture supernatants was performed as described by coulon et a/. (1989) . six-week-old female of1 random bred mice (iffa-credo, st germain sur i'arbresle, france) were anesthetized with 100 ~1 of equithesin (4% chloral hydrate, 16% pentobarbitol). three microliters of concentrated cvs or avol viruses in td (tris-hci, ph 7.4; naci, 150 mh/l; kci, 5 mhll; na,hpo,, 0.7 mm + 1 m/l/l edta were instilled in the right nostril using a hamilton syringe connected to a stretched catheter. the mice were maintained on their backs for 15 min after instillation. at various times after infection the animals were successively perfused intracardially with pbs (phosphatebuffered saline) (naci, 150 m/l/l; na,hpo,, 7.4 mm; kh,po,, 2.4 mhll) (20 ml), 24/o paraformaldehyde in pbs (150 ml), and 10% sucrose in pbs (50 ml) using a peristaltic pump. after dissection, the brain was kept in pbs + 10% sucrose for 24 hr for cryoprotection; the nose was decalcified in pbs + 0.1 ili edta for 5 days and then kept for 24 hr in pbs + 10% sucrose at 4". the tissues were frozen at -70". serial 30-pm frontal sections of the entire brains were made in a cryostat microtome (bright, huntingdon, england). an average of 500 sections per brain was obtained. all the sections were treated for immunofluorescence and the total number of positive neurons was determined for each infected animal. in a few instances, sections were collected on three alternate series of gelatin-coated slides. a first series was treated for immunofluorescence, a second for immunoperoxidase, and a third was kept in reserve. several noses were also cut in totality and all sections were treated for immunofluorescence. the sections were permeabilized in pbs + 1% triton x-100 for 30 min at room temperature, washed three times in pbs, and then treated with fluorescein isothiocyanate-conjugated anti-nucleocapsid antibodies (pasteur production). after washing with tap water, the slides were mounted in elvanol (tris-po,, 50 mm; polyvinyl alcohol, 20%; glycerol, 20%; ph 8.2) and then individually examined using a uv microscope. lmmunoperoxidase the sections were permeabilized in pbs + 1% triton x-100 for 30 min and then washed three times with elisa iii buffer (naci, 150 mm; tris-hci, 50 mlli; edta, 1 mm; tween-20, 0.05%; bovine serum albumin, 0.1%; ph 7.4). after a 3-min incubation in 0.3% h,02 and three washes in elisa iii, the tissues were incubated for 24 hr at 4" with a rabbit anti-nucleocapsid antibody (l/300 in elisa ill). the sections were washed three times in elisa iii and incubated for 1 hr at 37" with an anti-rabbit biotinylated antibody (abc kit "elite," vector, burlingame, ca) diluted l/200 in elisa iii. the signal was amplified by incubation (20 min at 37') in a mixture of avidin and biotinylated peroxidase. after washing, the sections were incubated in a substrate of diaminobenzidine (sigma, st louis, mo) at 1 mg/ml in tris-hci, 0.1 m, ph 7.4, plus an equal volume of 0.02% h,o, in water. the reaction was stopped after discarding the mixture and washing with tap water. tissues were counterstained with giemsa. after washing and drying, the sections were mounted with dpx (bdh, poole, england). identification of the cerebral structures was performed according to the stereotaxic atlas of the mouse brain by lehmann (1974) and that of the rat brain by paxinos and watson (1986) . under our experimental conditions the olfactory route is an efficient route of infection for the cvs strain. the median lethal dose (ld50) was determined and corresponded to 50 pfu. by comparison, the intracerebral ld50 is 1 pfu, the intramasseter ld50 at least lo3 pfu (coulon et al,, 1982b) , and the ld50 after injection in the forelimb about 5 x 1 o4 pfu . as expected, the avol mutant was completely avirulent by the olfactory route even when 7.5 x 10' pfu of virus was instilled. undiluted cell culture supernatant containing cvs (5 x 1 o4 pfu or about 1000 intranasal ld50) was used in preliminary experiments. the first round of multiplication in the nasal cavity occurred only in the olfactory receptor cells. twenty-four hours after instillation few neurons (around 25 in the whole organ) contained enough material to be detectable by immunofluorescence. at 2 days postinfection, this number increased to reach a mean value of 150 and remained relatively stable during the following 2 days. the infected neurons were usually not adjacent (fig. 2b) . in order to increase the number of infected receptor cells, we instilled concentrated virus. following instillation of 4 x 10' pfu of cvs (8 x lo5 in ld50), the number of positive neurons reached 2 to 5 x 1 o3 at 2 days postinfection (p.i.), which represents less than 0.1% of the total number of olfactory receptor cells. the infected cells were consistently found mainly in the bottom of the turbinates ( fig. 2a ) and in the septal organ. complementary experiments using horseradish peroxidase (hrp) instead of virus showed that the epithelial areas where all cells were hrp-positive were also the infected ones (data not shown). since only a minority of the olfactory receptor cells were infected, it is not known if all the receptor cells are permissive for rabies. infected neurons were generally found in the right olfactory epithelium since the virus was instilled into the right nostril. in a few mice (about 1 of lo), some infected cells were also detected on the left nasal fossa, which was not unexpected since the fossae communicate. the avirulent mutant avol showed a comparable capability to infect receptor cells. the ratio between infected cells and pfu is the same for both viruses and the infected cells were found in the same epithelial areas (data not shown). at day 6 or 7 when cvs-infected mice die, olfactory receptor cells were still infected, but their number decreased. in the avol -infected mice which survived the infection, the olfactory epithelium was clear of virus after day 7. the vomeronasal organ and the respiratory epithelium never appeared to be infected, even at the latest stage of the infection. the first neurons detected as infected by immunofluorescence in the mob were observed 48 hr after instil-lation. this agrees with the occurrence of a first cycle of infection in the olfactory epithelium before reaching the bulb. at 2 and 2.5 days p.i. only a few cells located in the glomerular layer appeared infected in the ipsilateral mob (table 1) . at day 3, the number of infected periglomerular and/or external tufted cells significatively increased. as these cellular types were difficult to distinguish (fig. 3a ) they were counted together (table 1) . a spotted fluorescence was observed in some glomeruli even in the absence of surrounding infected cells, suggesting the presence of viral nucleocapsids in the axonal endings of receptor cells. mitral cells and large size neurons located in the internal plexiform layer (ipl) also appeared infected (fig. 3b) . these cellular elements were also counted together ( table 1 ). the use of the immunoperoxidase method allowed us to estimate that the mitral cells and ipl neurons were equally represented. few positive medium size neurons were found in the external part of the granular layer. no labeling was noted in the contralateral bulb, except when both fossae were primarily infected. from day 4 the contralateral mob became infected. in some animals the number of infected mitral cells could be higher in the contralateral bulb than in the ipsilateral one (table 1) . at day 5 nearly all the mitral cells in the contralateral bulb appeared infected (fig. 3c ). from this time to death it was not possible to count the infected cells because they were too numerous. nevertheless, very few cells in the granular layer contained viral nucleocapsids (fig. 3c) . at days 3 and 4 neurons appeared infected in the periglomerular region of the ipsilateral bulb, but their number was apparently reduced compared to that noted in the cvs infection at the same time. as for cvs infection, all the infected cells in the glomerular layer were counted together ( table 2) . accumulation of nucleocapsids in the glomeruli was also detectable (fig. 3d ). almost no labeling was observed in the mitral cells and the neurons located in the ipl. among 11 animals observed at day 3 or 4 p.i., only a single infected mitral cell was detected in each of two animals. beyond day 5 p.i., few mitral cells were detectable in almost all observed animals (table 2 ). these infected cells were found in the ipsi-or the contralateral bulb and their number remained low and did not account for more than 0.1% of the mitral cells [assuming a total number of mitral cells around 50,000 as estimated in the rat (meisami and safari, 1981) ]. other structures of the brain early infected with cvs at day 2 or 2.5 p.i., very few infected neurons were detected in two nuclei known to project into the glonote. mice were instillated with 4 x 10' pfu of cvs virus and sacrificed at various times after infection. the brains were processed as described under materials and methods. each line represents one infected animal in which the total number of infected neurons was determined. * only one of three sections was treated for immunofluorescence; the number of labeled cells obtained was tripled for the comparison with the other series. aon, anterior olfactory nucleus. hdb, horizontal limb of the diagonal band. mob, main olfactory bulb. merular layer: the aon and the hdb. in one animal sacrificed 2.5 days after instillation, 3 of the 4 infected cells found in the aon were located in the ipsilateral nucleus and the other in the contralateral one. at day 3 p.i., the aon was found to be consistently infected ipsilaterally and to a lesser extent contralaterally (fig. 4) . it is not surprising to find infected neurons in the contralateral aon, because the aon is an important contralateral source of mob afferents (carson, 1984) . there were between 18 and 664 infected cells in the right aon (table 1) ; between 0 and 168 infected neurons were detected in the left aon. like the aon, the ipsilateral hdb was consistently infected at day 3 p.i. (fig. 5a) . in some animals (three of seven), labeled cells were observed in the contralateral hdb (table 1 ). in the first animal the contralateral labeling of hdb was the consequence of a bilateral infection of the olfactory epithelium, since the contralateral mob was infected. this was not the case for the other two animals; given that, unlike the aon, hdb does not project contralaterally in the mob (carson, 1984) , infection in these animals was due to a third cycle of multiplication. in these animals, some third order labeled cells were also found in the piriform cortex (fig. 5a ). other regions of the brain were consistently found to be infected ipsilaterally at day 3 in addition to hdb and aon. they were the lateral preoptic area (lpa) and the magnocellular preoptic nucleus (mcpo) (fig. 5a ). a very different situation was found in avol -infected animals. at day 3 or 4 p.i., hdb appeared as efficiently infected with the mutant as with the wild type, while there was no infection in the aon ( table 2 ). the mcpo which was labeled with cvs was also permissive for the avol mutant (fig. 5b) , while the lpa showed no labeled ceils. in the aon few cells were labeled from the fourth day of infection, and in any case the number of infected cells remained very low compared to the wild type (less than 0.1%). the invasion of the brain by cvs after the third day of infection involved many nuclei which were not necessarily related to the olfactory pathways. a precise description will be published elsewhere; in this paper only some relevant points allowing a better understanding of mechanisms which could explain the difference in pathogenicity between cvs and the avol mutant will be described. several animals were examined at each day of infection, in order to distinguish the consistently infected areas from those rarely infected. two different situations were observed. (i) the same structures were infected by both strains, even if fewer neurons were often involved for the mutant strain. nuclei with at least 20% infection compared to the wild type were considered to be in this category. infection with the mutant could be observed at nearly the same time as for the wild type, like in the orbital cortex and in the amygdala. for most nuclei infected later, the appearance of antigen with the mutant was slightly delayed (up to 1 day). this was the case for the purkinje cells in the cerebel-lum and the ventromedial thalamic nucleus. (ii) some structures were infected with the wild type and not, or scarcely, infected with the mutant. this was the case for two nuclei related to the olfactory pathways, the septal area (one of the most heavily infected areas for the wild type) and the raphe nucleus. another example is the hippocampus which was infected by the wild type as early as day 4 p.i. [mainly pyramidal cells (fig. 6 )] and completely invaded by day 5. with the mutant strain only two of five animals showed a small focus of infected cells in this structure at day 5. of three animals sacrificed at day 6 or 7, two were free of infection in the hippocampus, while the latter showed 10 infected cells. moreover, in these small infected areas very few pyramidal cells were concerned, most of the infected cells being in the stratum lacunosum moleculare. among the other nuclei known to project directly to the mob, the locus coeruleus was never infected either by the wild type or by the avol mutant. in the course of our study, we observed that the trigeminal nerve was consistently infected even at the early stage of the infection with cvs. systematic observations of the gasser ganglions in immunofluorescence showed that this structure was infected at day 2 (data not shown). this indicates that the virus was able to penetrate the trigeminal endings without injury to the nasal cavity. the avol mutant directly infected the trigeminal nerve in the same manner as cvs (data not shown). at day 4, infected neurons were detected in the mesencephalic nucleus of the trigeminal nerve in both infections (fig. 7) but the number of labeled cells was lower in the avol than in the cvs infection. several neurovirulent viruses invade the brain along olfactory pathways: vesicular stomatitis virus (vsv) (lundh et a/., 1987 (lundh et a/., , 1988 murine hepatitis virus (mhv)-jhm (barthold, 1988; perlman et a/., 1989) and herpes simplex type 1 (hsvl) (mclean et al., 1989) . the rabies virus, under appropriate conditions, is also able to infect the cns of various mammals after instillation in the nose, but the mechanism of centripetal spread of the virus to the cns has never been clarified. here we provide a careful description of the first cycles of viral infection, including timing and identification of permissive neurones. we also precisely compared the cvs (pathogenic) and avol (avirulent) viruses for their ability to invade the cns of the mouse after nasal instillation. both viruses infect the olfactory epithelium but not the respiratory epithelium. the same specificity was previously reported for vsv, another rhabdovirus (lundh et al., 1987) . this is not the case for mhv-jhm infection in which the respiratory and olfactory epithelia are both infected (barthold, 1988) . a second route of infection is used by cvs and the avirulent mutant after nasal instillation. it is the trigeminal route which is followed by other neurotropic viruses like mhv-jhm (perlman et a/., 1989) and hsvl (mclean eta/., 1989) . as early as 2 days postinfection viral material was revealed by immunofluorescence in the gasser ganglion. this result confirms previous experiments in which cvs and avol viruses had been shown to infect the trigeminal ganglion after intraocular inoculation (kucera et al., 1985) . it also suggests that trigeminal endings are accessible to the virus without injury of the nasal fossae. very few infected neurons are detectable 24 hr p.i. in the olfactory epithelium, although the distance between the dendrite endings and the cell body is very short. the increase in the number of infected neuroreceptors observed between days 1 and 2 cannot be due to a second cycle of infection since infected cells are usually not adjacent. the timing of the first cycle of multiplication in the olfactory epithelium can thus be estimated at 48 hr. this is longer than in cell cultures or in other neurons. for example, after injection in the forelimb of the mouse, viral material can be detected in the spinal cord and the dorsal root ganglia as early as 18 hr p.i. . the longer cycle observed after intranasal exposure could be due to a lower temperature in the nasal cavity compared to that of the body or to a trapping of the virus during the passage across the mucus layer covering the epithelium. as already mentioned, we did not observe patches of infected neurons in the olfactory epithelium. this indicates that neurons adjacent to the primary infected cells could not be infected, either because there is no budding of virions at the cell body level or because there is no receptor for these virions at this level. a comparable observation was reported in dorsal root ganglia infected with cvs . these observations argue for strictly transsynaptic transmission of rabies virus. rabies virus is different in this respect from vsv, which has been shown to bud at the level of cell bodies of the olfactory receptor cells (lundh et a/., 1987) and from hsvl, which gives patchy label at least in the olfactory epithelium and the bulb, thus suggesting a transmission from cell body to cell body in addition to the transsynaptic transport (mclean et a/., 1989) . at the third day of infection with cvs, the three categories of bulbar neurons known to have dendritic terminals in the glomerular layer are infected. these are the periglomerular, tufted, and mitral cells (halasz and shepherd, 1983) . another category of cells which was found to be consistently infected in the ipl anatomically resembles the horizontal cells described by schneider and macrides (1978) . but, this latter category is not currently thought to make connections with olfactory neuroreceptors. these neuroanatomical connections identified with rabies virus used as a tracer will be described in more detail elsewhere. among the nuclei known to project into the glomerular layer, the aon and the hdb are infected by the wild type. all these neurons are secondarily infected with virions produced by the olfactory receptor cells. by comparison, in the mob, avol is only able to infect cells located around the glomeruli, the mitral and the horizontal cells not being infected with the mutant. in the brain, the avol mutant is able to infect secondarily the hdb. in contrast the aon is not permissive for the mutant. these results allow us to conclude that avol is able to replicate (because avo 1 -infected neurons are detect-able by immunohistochemistry) and to mature (because of the infection of synaptically connected neurons) in the olfactory epithelium and the trigeminal ganglion. unlike dietzschold et al. (1985) we concluded that the spread of the avirulent mutant does not proceed at a slower rate than the spread of cvs but rather implied less categories of neurons. why are certain neurons not permissive for avol? either the mutant is unable to replicate normally in these cells or it does not bind to a specific receptor(s) at the synaptic junctions of these cells. the first hypothesis seems unlikely because the mutation of avol affects the glycoprotein, and this protein is not known to be involved in the replication of rhabdoviruses (see wertz et a/., 1987 , for a review). the replication of avol could probably take place in any category of neurons normally permissive for cvs provided that the nucleocapsid arrives at the cell body. more probably, the restricted tropism of avol is related to the lack of recognition of receptors present at synapses of different groups of neurons. the strong tropism for neuronal cells suggests that viral receptors should be specific to neurons and could be a protein. the suggestion that the acetylcholine receptor could be the receptor for rabies virus has been made on the basis of competitive fixation experiments between rabies virus and a-bungarotoxin on cultured myotubes (lentz et a/., 1982) and sequence homology between the 189-2 14 region of the rabies glycoprotein and the 30-56 region of the long neurotoxins (lentz et a/., 1984) . since avol is able to penetrate several categories of neurons, and is restricted for others, the wild type should be able to bind to several different receptors, and thus the acetylcholine receptor cannot be the unique receptor for rabies. some of our results argue in favor of the acetylcholine receptor hypothesis: indeed, neurons of the hdb ending in the glomerular layer of mob are cholinergic (zgborszky et al., 1986) , and it is likely that the horizontal neurons of the ipl are the cholinoceptive neurons described by nickel1 and shipley (1988) . on the contrary, other permissive cells respond to various neurotransmitters: the periglomerular cells to gaba, dopamine, and enkephaline; the tufted cells to glutamate/aspartate, dopamine, and substance p; and the mitral cells to glutamate/aspartate (halasz and shepherd, 1983) . of course it is also possible that other molecules which are not implicated in the binding of neurotransmitters are used by rabies virus as receptors. interestingly, even during cvs infection, some categories of neurons are not infected by the virus. this is the case for the granular cells in the mob and the locus coeruleus (lc), a nucleus directly connected to the mob (shipley eta/., 1985) . these cells are secondarily (or later) infected in rats instilled with hsvl (mclean et al., 1989) or in mice instilled with vsv (lundh eta/., 1988) . if, as suggested by shipley(l985) , the lc is connected to the granular cells in the mob, it is not surprising that this nucleus was not infected early since granular cells are not permissive for the cvs strain of rabies. nevertheless the absence of infection of the lc even when the mouse was moribund suggests that these neurons are nonpermissive to the virus. the lc has a noradrenergic projection in the mob . another group of neurons responding to noradrenaline, the superior cervical ganglion (scg), does not seem permissive for the rabies virus. while injection of pseudorabies virus (herpesvirus) in the anterior chamber of the eye of rats results in infection of the scg (dolivo et a/., 1981), the injection of rabies virus at the same site does not result in the infection of this ganglion (tsiang et a/., 1983; kucera et al., 1985) . it has long been stated that the rabies virus is transported from the synapses to the cell bodies by retrograde axonal flow. our experiments confirm our previous results that the virus can travel via anterograde as well as retrograde flow. indeed, during the first cycle of infection the virus is transported in an anterograde fashion in the olfactory epithelium and in the trigeminal nerve. during the second cycle, the virus uses the two types of transport: (i) anterograde in the mitral and the periglomerular cells in the mob and (ii) retrograde in the horizontal cells of the mob and the neurons of the aon and the hdb. after intranasal inoculation, animals died with some regions of the brain free of virus. mice infected with avol survived, although several nuclei such as hdb or ventromedial thalamus were heavily infected. the death of cvs-infected animals is then probably due to the infection of nuclei which are permissible for cvs but not for avirulent mutants. such observations reduce the number of potential targets for the lethal effects of the virus. olfactory neural pathway in mouse hepatitis virus nasoencephalitis quantitative localization of neurons projecting to the mouse main olfactory bulb invasion of the peripheral nervous systems of adult mice by the cvs strain of rabies virus and its avirulent derivative avol molecular basis of rabies virus 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of virus to peripheral tissues two anatomically specific classes of candidate cholinoceptive neurons in the rat olfactory bulb the rat brain in stereotaxic coordinates spread of a neurotropit murine coronavirus into the cns via the trigeminal and olfactory nerves antigenic site ii of the rabies glycoproteln: structure and role in viral virulence laminar distributions of interneurons in the main olfactory bulb of adult hamster rabies virus virulence: effect on pathogenicity and sequence characterization of mutations affecting antigenic site ill of the glycoprotein transport of molecules from nose to brain: transneuronal anterograde and retrograde labeling in the rat olfactory system by wheat germ agglutinin-horseradish peroxidase applied to the nasal epithelium surprisingly rich projection from locus coeruleus to the olfactory bulb in the rat olfactory system an in viva and in vitro study of rabies virus infection of the rat superior cervical ganglia arginine or lysine in position 333 of era and cvs glycoprotein is necessary for rabies virulence in adult mice the role of protetns in vesicular stomatitis virus replication. ln "the rhabdoviruses airborne rabies. ln "the natural history of rabies cholinergic and gabaergic afferents to the olfactory bulb in the rat with special emphasis on the projection neurons in the nucleus of the horizontal limb of the diagonal band we thank g. payen for her technical assistance. this work was supported by the centre national de la recherche scientifique (lp a2431), and by the ministere de la recherche et de la technologie (contract 88-1896). key: cord-299976-36r794ow authors: o’brien, amornrat; mettelman, robert c.; volk, aaron; andré, nicole m.; whittaker, gary r.; baker, susan c. title: characterizing replication kinetics and plaque production of type i feline infectious peritonitis virus in three feline cell lines date: 2018-12-01 journal: virology doi: 10.1016/j.virol.2018.08.022 sha: doc_id: 299976 cord_uid: 36r794ow investigating type i feline coronaviruses (fcovs) in tissue culture is critical for understanding the basic virology, pathogenesis, and virus-host interactome of these important veterinary pathogens. this has been a perennial challenge as type i fcov strains do not easily adapt to cell culture. here we characterize replication kinetics and plaque formation of a model type i strain fipv black in fcwf-4 cells established at cornell university (fcwf-4 cu). we determined that maximum virus titers (>10(7) pfu/ml) were recoverable from infected fcwf-4 cu cell-free supernatant at 20 hours post-infection. type i fipv black and both biotypes of type ii fcov formed uniform and enumerable plaques on fcwf-4 cu cells. therefore, these cells were employable in a standardized plaque assay. finally, we determined that the fcwf-4 cu cells were morphologically distinct from feline bone marrow-derived macrophages and were less sensitive to exogenous type i interferon than were fcwf-4 cells purchased from atcc. feline coronaviruses (fcovs) are members of the alphacoronavirus genus that infect cats and can cause the highly lethal disease known as feline infectious peritonitis (fip) (pedersen, 2009 ). fcov infection is widespread among domestic feline populations, especially within multicat households and catteries, which can exhibit upwards of 96% seropositivity (addie et al., 2003; addie and jarrett, 1992; hohdatsu et al., 1992; pedersen, 2009 pedersen, , 1976 vennema et al., 1998) . despite the global burden of fcov infection, there are no currently approved therapeutics to treat fip; however, reports of direct inhibition of virus growth and treatment of individual cats using small molecule viral inhibitors have been promising (kim et al., 2016 (kim et al., , 2015 (kim et al., , 2013 (kim et al., , 2012 murphy et al., 2018; pedersen et al., 2017; st john et al., 2015) . fcovs are typically grouped into two biotypes (or pathotypes), which have been classified as feline enteric coronavirus (fecv) and feline infectious peritonitis virus (fipv), based on tissue tropism, disease progression, and genetic markers (reviewed in kipar and meli, 2014; pedersen, 2014 pedersen, , 2009 , although the range of disease signs and clinical outcomes are likely to extend beyond these two basic definitions. endemic fecv causes mild enteritis associated with loose stool and diarrhea and commonly leads to an asymptomatic, persistent infection (addie, 2011; addie et al., 2003; pedersen et al., 2008) . a subset of these infections (3-10%) result in lethal fip (addie and jarrett, 1992; pedersen, 1976) arising from a shift in virus tropism and systemic infection of monocytes and macrophages. perturbations of the host feline immune state leading to immune deficiency can allow virus replication to surge (pedersen, 2009; tekes and thiel, 2016) , resulting in the formation of a quasispecies and the genetic sampling required for progression of fecv to the second biotype, fipv. the internal mutation theory proposes that, within an individual animal, fipv arises directly from fecv due to accumulation of non-synonymous mutations in spike (s) (licitra et al., 2014 (licitra et al., , 2013 rottier et al., 2005) and group-specific proteins (chang et al., 2010; herrewegh et al., 1995; lin et al., 2009; pedersen et al., 2012; phillips et al., 2013; poland et al., 1996; vennema et al., 1998) . the resulting infection of monocytes and macrophages by fipv leads to systemic spread and development of immune-mediated fip (pedersen, 2009) . fcov biotypes are further defined by their viral s protein. classically, antigenicity of the s protein alone has been used to categorize fcovs into two serotypes (type i and type ii) (hohdatsu et al., specific grouping of fcovs into two clades via functionality-based sprotein sequencing (whittaker et al., 2018) . type i viruses account for the bulk (80-90%) of natural infections in domestic cats, while type ii fcov, a naturally-occurring recombinant between the type i and canine coronavirus (ccov) spike proteins (herrewegh et al., 1998) , is far less prevalent (< 10%) (addie et al., 2003; benetka et al., 2004; hohdatsu et al., 1992; kennedy et al., 2002) . a commendable number of studies focus on type ii fcov as these viruses are much more easily propagated in cell culture. however, it is difficult to know how accurately type ii laboratory strains reflect natural infections with type i viruses given that the bulk of what is known about type i is extrapolated from studies using a type ii virus. it is critical that we investigate type i fipv in laboratory cell culture in order to understand the basic virology of natural infection, characterize type i clinical isolates, test novel therapeutics, and develop effective feline vaccines with broader coverage. however, this has been challenging because type i fcovs cannot be easily adapted to laboratory cell culture; furthermore, the receptor for type i is not known (cham et al., 2017; dye et al., 2007; hohdatsu et al., 1998) , making the identification of highly permissive cell types difficult. select type i isolates, such as the fipv black strain used in this study, have been adapted to growth in tissue culture at the cost of reduced in vivo virulence (black, 1980; pedersen, 2009; tekes et al., 2007; thiel et al., 2014) . feline airway epithelial (ak-d) cells propagate fipv black (regan et al., 2012) ; however, these cells do not represent natural tropism for fipv. felis catus whole fetus 4 (fcwf-4) cells are a more physiologically-relevant feline macrophage-like cell line (jacobse-geels and horzinek, 1983), but these cells come with several technical drawbacks for studying type i fipv. first, fcwf-4 cell doubling time is slow (> 31 h) (american type culture collection, 2013) and cells do not grow to high density. second, previous studies report that type i fipv grows to low titers (< 10 5 pfu/ml) in these cells relative to type ii (> 10 6 pfu/ml) (tekes et al., 2012) and can be measured by determining the 50% tissue culture infectious dose (tcid 50 ) (ramakrishnan, 2016; reed and muench, 1938) or by plaque assay (tekes et al., 2012 (tekes et al., , 2010 . third, type i virus kinetics are variable in fcwf-4 cells, requiring between 15 and 72 h to achieve maximum titer (jacobse-geels and horzinek, 1983; tekes et al., 2012 tekes et al., , 2007 this study) . finally, some reports suggest that type i is highly cell-associated (jacobse-geels and horzinek, 1983; pedersen et al., 1984) and multiple freeze-thaw cycles may be required to recover virus. together, these factors have made investigation of type i fipv challenging. as part of this study, we characterized three feline cell lines-two from the american type culture collection (atcc) and one from cornell university-and evaluated the replication kinetics, efficiency of plaque formation, and responsiveness of these cells to interferon (ifn) in order to identify the optimal cell culture conditions for type i fipv black. we found that an fcwf-4 cell line established at cornell university college of veterinary medicine, designated fcwf-4 cu, propagates type i fipv to significantly higher titers in cell-free supernatant and with more rapid kinetics compared to commercially available fcwf-4 cells. we show that fcwf-4 cu cells are less responsive to exogenous type i interferon than fcwf-4 cells from the atcc and are permissive to infection by both biotypes of type ii fcov. to facilitate cell-free titer was determined from cell-clarified supernatants; cell-associated titer was determined from suspended cell monolayers following three freeze-thaw cycles alternating between −80°c and 37 o c. samples were taken at hours post-infection (hpi) just prior to, at, and following the maximum (max) virus titer for each cell type. titers determined by plaque assay on ak-d cells in triplicate; error bars ± sd. quantitation of fipv black, we established a standardized plaque assay method using fcwf-4 cu cells and commercially available ak-d cells and show that both cell types permit rapid and consistent quantitation of infectious titers of type i fipv as well as type ii fipv and fecv from cell-free supernatants. to determine the optimal cell type and conditions required to grow the type i fipv black strain, we evaluated virus growth kinetics using a standard infection time course. cells were infected at a multiplicity of infection (moi) of 0.1 and virus titer was determined by plaque assay from cell-free supernatants over 96 h. fipv black, a distinct type i lab strain that replicates in feline epithelial cells, replicated as expected in ak-d cells reaching a maximum titer > 10 6 pfu/ml at 36 h post-infection (hpi) (fig. 1a) . in our hands, using fcwf-4 cells purchased from the atcc, the replication of fipv black reached a maximum titer > 10 4 pfu/ml over 72-96 hpi (fig. 1a) . strikingly, fipv black replication kinetics and maximum titer were drastically different in an fcwf-4 cell line established at cornell university college of veterinary medicine (fcwf-4 cu). using these cells, the virus reached a significantly higher maximum titer of > 10 6 pfu/ml at 20 hpi. in other words, nearly 100 times more virus was produced from the fcwf-4 cu cells a full 2-3 days faster than in fcwf-4 atcc cells. to address whether the differences in titer observed between ak-d, fcwf-4 cu, and fcwf-4 atcc cells were due to differences in cell-free and cell-associated virus, we compared the cell-associated and cell-free virus titers from each cell type at the time points around the respective maximum titers. surprisingly, the cell-free virus titers were higher than the cell-associated titers at all time points and in all cell types assayed (fig. 1b) . this indicates that fipv black virions are released into cell supernatant during infection of cell culture and freeze-thaw cycles are not necessary to obtain high virus titers. although the maximum titers of fipv black were comparable between ak-d and fcwf-4 cu cells, the progression of cell cytopathic effects (cpe) induced by the virus differed. fipv black formed large, uniform syncytia in fcwf-4 cu cells, while individual cell-death-induced clearings were observed in infected ak-d cells ( fig. 2a ). of note, maximum titers from both cell types were obtained just prior to the appearance of major cpe, allowing a visual guide to virus collection. to further demonstrate this point, fcwf-4 cu and ak-d cells infected with fipv black were labeled with an anti-nucleocapsid antibody (ccv2-2) (poncelet et al., 2008) and visualized by immunofluorescence prior to the induction of major observable cpe. as expected, the majority of cells were positive for virus antigen (fig. 2b ) and the differences in cpe are clearly shown: note the syncytial membrane fusion in infected fcwf-4 cu cells and the maintenance of distinct cell membranes in infected ak-d cells (fig. 2b) . together, these results demonstrate the ability of the fcwf-4 cu cells to rapidly produce high levels of type i fipv black in cell-free supernatants. after observing the rapid and uniform development of cpe and release of virus into cell supernatants during infection of ak-d and fcwf-4 cu cells, we reasoned that these cells would be employable in a standardized plaque assay to consistently determine fipv black titer. to this end, we calculated the endpoint titer and compared the size, uniformity, and timing of virus plaque development over time in ak-d, fcwf-4 atcc, and fcwf-4 cu cells following infection with 10-fold dilutions of the same fipv black virus stock initially grown on fcwf-4 cu cells. a detailed description of the plaque assay is provided in the materials and methods; we note here that oxoid agar is critical for visualizing clear plaques. at 2 days post-infection (dpi), fipv black formed enumerable plaques on both ak-d and fcwf-4 cu cells with the latter cells producing more numerous and larger plaques at higher dilutions (fig. 3 top) . plaques were not observed in fcwf-4 atcc cells at 2 dpi (fig. 3 top) . plaques were detected at 3 dpi (fig. 3 middle) ; and were more clear at 4 dpi in fcwf-4 atcc cells (fig. 3 bottom) . calculated titers overall were higher in fcwf-4 cu (> 10 7 pfu/ml) than in ak-d cells (≥10 6 pfu/ml); however, we report that both cell types are useful for determining virus titer, whereas the fcwf-4 atcc cells are not ideal for use in this assay. the apparent differences in production and kinetics of fipv black virus in two fcwf-4 cell lines led us to ask if there are morphologic or functional differences between the two cell types. to answer this question, we first compared the single-cell morphologies of fcwf-4 atcc, fcwf-4 cu cells, and primary feline bone marrow-derived macrophages (fbmdms) by wright-giemsa staining. the typical morphologic characteristics of feline macrophages (large cytoplasmic inclusions, a non-dominant nucleus, a non-ruffled cell membrane) (bienzle et al., 2003) were observed for the fbmdms (fig. 4a) . comparison of the two fcwf-4 cell lines revealed stark differences in morphology. fcwf-4 atcc cells were large with a smooth cell membrane and had large and abundant cytoplasmic inclusions comparable to the fbmdms (fig. 4a ). fcwf-4 cu cells were more similar in size to fbmdms; however, the fcwf-4 cu cell line exhibited fewer cytoplasmic inclusions and more cell membrane ruffling (fig. 4a ) than either the fbmdms or fcwf-4 atcc cells. neither fcwf-4 cell line had a "true" macrophage morphology further corroborating their original "macrophage-like" description (jacobse-geels and horzinek, 1983). as macrophages are innate immune cells that restrict virus replication through production of interferon-stimulated genes (isgs) in response to type i ifn, we reasoned that differences in virus replication may be due to variation in cell ifn-responsiveness. therefore, we asked if fcwf-4 atcc and cu cell lines differed in responsiveness to exogenous type i ifn by measuring the resulting isg54 transcript production following treatment with ifn. remarkably, fcwf-4 atcc cells produced significantly higher isg54 transcripts in response to ifn stimulation compared to fcwf-4 cu cells (fig. 4b) , suggesting that the fcwf-4 cu cells are much less responsive to ifn. this is not to say, however, that fcwf-4 cu cells are insensitive to ifn since they also exhibit significant, dose-dependent isg54 transcript production. together, these data highlight the distinct morphology of the fcwf-4 cu cells and suggest that enhanced virus replication in these cells may be due, at least in part, to reduced ifnresponsiveness. due to the high titer and rapid kinetics of type i fipv black replication in the fcwf-4 cu cells, we next addressed whether these cells are permissive to type ii fcov infection. fcwf-4 cu cells were infected with type i fipv black or one of two type ii viruses, fipv 79-1146 or fecv 79-1683. cell cytopathic effects were observed over time in these cells, with the viruses all forming similar, large syncytia (fig. 5a) . cellfree virus titers were determined at 12 and 24 hpi. all three viruses accumulated to titers > 10 6 pfu/ml in supernatants by 24 hpi (fig. 5b) , with fipv 79-1146 reaching the highest titer. the kinetics of the type ii viruses were faster than the type i fipv black strain, producing higher titers by 12 hpi (fig. 5b) and inducing more substantial syncytial cpe by 16 hpi (fig. 5a) . next, we asked if the fcwf-4 cu cells could be utilized in a plaque assay for determining the titer of type ii fcov. indeed, both fipv 79-1146 and fecv 79-1683 formed clear, uniform, enumerable plaques at 24 hpi (fig. 5c) . thus, we have demonstrated that the fcwf-4 cu cells replicate both fcov types and biotypes to high titers and are employable in a plaque assay to consistently determine the titers of all viruses assayed. since the isolation of the fipv black strain in 1980 (black, 1980) it has remained a predominant model of type i fipv because it is cultivatable in commercially available fcwf-4 cells. however, different groups have reported major variations in the growth kinetics, maximum obtainable titer, and techniques for recovery of this virus from tissue culture (jacobse-geels and horzinek, 1983; pedersen et al., 1984; tekes et al., 2012 tekes et al., , 2007 thiel et al., 2014) . for example, maximal titers measured by determining the tcid 50 can range between > 10 3 (jacobse-geels and horzinek, 1983) and 10 4 tcid 50 /ml (takano et al., 2015) at 24-48 hpi, or measured by plaque assay between > 10 4 (36 hpi) (tekes et al., 2012 (tekes et al., , 2007 and > 10 7 pfu/ml (20 hpi) (this report). this suggests either high variability in the fcwf-4 cell lines used or co-adaptation between a particular virus and fcwf-4 cell line used during laboratory cultivation. indeed, we report significant differences in fipv black replication properties between an fcwf-4 cell line that was newly purchased from the atcc, and the fcwf-4 cells that were established at cornell university. the enhanced rate of fipv black virus growth and increased maximum titer (> 10 7 pfu/ml by 20 hpi) obtained from infected fcwf-4 cu cells, however, may not be due to coadaptation with our particular strain of fipv black, as these cells also replicated both biotypes of type ii viruses. instead, the fcwf-4 cu cells may be highly susceptible to fcov infection in general and therefore may be particularly useful in generating highly-permissive cell types to isolate and grow clinical type i fcovs. the increased virus infection of fcwf-4 cu cells could be due to any number of cellular factors; however, it is tempting to speculate that the reduced ifn-responsiveness of the fcwf-4 cu cells relative to the fcwf-4 atcc cells may significantly enhance infection in the former. further, these fcwf-4 cu cells may express a higher density of the yet unknown type i virus receptor (cham et al., 2017; dye et al., 2007) and therefore may be critical in identifying the receptor or other cellular characteristics that allow for enhanced type i virus replication. fipv black infection of fcwf-4 cells has also been reported to be highly cell-associated (jacobse-geels and horzinek, 1983; pedersen et al., 1984) , requiring suspension and freeze-thaw cycling to release infectious virus. in contrast, we found significantly higher titers of virus in cell-free supernatants and speculate that multiple freeze-thaw cycles may actually decrease virus titer by damaging the virus envelope. the release of virus into cell supernatants and the uniform cpe observed in ak-d and fcwf-4 cu cells were critical in establishing a standardized plaque assay using either cell type. further, we report that the stage of cpe development can be used as an indicator of when maximal virus titers can be recovered. one possible explanation for the varied reports on titer of fipv black is that laboratory lines of fcwf-4 cells have deviated from the original atcc stock. this is likely what occurred at cornell university to produce the fcwf-4 cu cell line, given that fcwf-4 cells were obtained from the atcc and then passaged for many years. to our knowledge, this is the first report describing the phenotypic differences between the original fcwf-4 cells available from the atcc and a distinct lineage that was derived from the original cells. many challenges are still associated with the growth of type i fcov in tissue culture, including the lack of a known cell-entry receptor and no highly permissive cell type that rapidly grows clinical samples of these viruses. however, our studies with the fcwf-4 cu cell line demonstrate that rapid, high titers of type i fipv black can be recovered from cell-free supernatants and enumerated using a standardized plaque assay. it is our hope that the fcwf-4 cu cells will alleviate some of the technical hardships associated with the growth of type i fcov and expedite investigation of a wider range of type i fcov strains. the fcwf-4 cu cells, due to their distinct growth kinetics and enhanced replication of fipv black virus, will be deposited at the atcc to facilitate their distribution to the research community. feline coronavirus strains including type i feline infectious peritonitis virus (fipv) , type ii fipv wsu 79-1146, and type ii feline enteric coronavirus (fecv) wsu 79-1683 were kindly provided by dr. fred scott, cornell university college of veterinary medicine, ithaca, ny. feline airway epithelial (ak-d) cells were purchased from the american type culture collection (atcc) (atcc ® ccl-150™) and maintained in dulbecco's modified eagle medium (dmem; gibco, #12100-046) containing 10% fetal bovine serum (fbs) (atlanta biologicals, #s11150), supplemented with 2.2 g/l of sodium bicarbonate (sigma, #s5761), 1% non-essential amino acids (hyclone, #sh30238.01), 1% hepes (hyclone, #sh30237.01), 1% sodium pyruvate (corning, #25-000-ci), 1% l-glutamine (hyclone, #sh30034.01), and 1% penicillin/ streptomycin (corning, #30-002-ci). when cells grew to a confluent monolayer, the medium was removed and the monolayer was rinsed with pbs. the cells were removed by addition of 2 ml of 0.25% trypsin (gibco, #15090-046) in versene solution (0.48 mm edta in pbs) for 1-2 min at room temperature. for routine passaging, approximately 5.0 × 10 5 -1.0 × 10 6 cells were transferred (1:5 split) to a new t-75 flask every 3 days. felis catus whole fetus (fcwf-4) cells were purchased from the atcc (atcc ® crl-2787™), designated fcwf-4 atcc cells. fcwf-4 atcc cells were maintained in minimal essential medium eagle (emem) (sigma, #m0268) containing 10% fbs, supplemented with 1.5 g/l sodium bicarbonate, 1% non-essential amino acids, 1% hepes, 1% sodium pyruvate, 1% lglutamine and 1% penicillin/streptomycin. as described by the atcc, the doubling time for these cells is > 31 h. when cells were confluent in t-25 flask, the monolayer was washed with pbs, then cells were removed by addition of 1.5 ml 0.25% trypsin-versene solution for 2-3 min at room temperature. for routine passaging, approximately 1.0 × 10 5 -5.0 × 10 5 cells were transferred (1:3 split) to a new t-25 flask every 3 days. a second source of felis catus whole fetus cells were provided by dr. edward j. dubovi, cornell university college of veterinary medicine, ithaca, ny, designated fcwf-4 cu, and maintained in the same medium as the fcwf-4 atcc cells. when cells were confluent, the monolayer was washed with pbs, then cells were removed by addition of 2 ml 0.25% trypsin-versene solution for 1-2 min at room temperature. for routine passaging, approximately 5.0 × 10 5 -1.0 × 10 6 cells were transferred (1:10 split) to a new t-75 flask every 3 days. all cells used in this study were monitored for mycoplasma contamination using a pcr-based assay. cell culture supernatants were routinely collected after 3 days of culture and then heat-inactivated at 95°c for 10 min. pcr amplification for mycoplasma detection was performed using a forward primer: 5'-ggc gaa tgg gtg agt aac acg -3' and a reverse primer: 5'-cgg ata acg ctt gcg acc tat g -3'. thermocycler settings were as follows: initial denaturation at 95°c for 10 min; 35 cycles consisting of denaturation at 95°c for 45 s, annealing at 60°c for 45 s, and extension at 72°c for 60 s; and a final extension at 72°c for 10 min. the pcr products were analyzed on 1% (w/v) agarose gel. dna fragments were visualized with a uv transilluminator after being stained with ethidium bromide. if mycoplasma was detected, the cells were treated for 7 days with 0.5 μg/ml of mycoplasma removal agent (bio-rad, #buf035). all results shown here are from mycoplasma negative cells. iacuc approved protocols. total bone marrow content was collected. red blood cells and fatty tissue were removed by lysis in ack lysis buffer and straining through a 0.70 µm filter (falcon). the remaining cells, predominantly hematopoietic stem cells, were cryopreserved at 5.0 × 10 7 cells/ml in 90% fbs and 10% dmso. feline bone marrow derived macrophages (fbmdms) were differentiated as previously reported (gow et al., 2013) with slight modification. briefly, 5.0 × 10 7 bone marrow cells were plated in 100 × 26 mm petri dishes (vwr, #25387-030) in dmem (corning, #10-017-cv) supplemented with 20% fbs, 10,000 iu/ml recombinant human (rh) m-csf (peprotech, #300-25) and 50 μm β-mercaptoethanol then incubated at 37°c and 5% co 2 . at day 3, supernatant was removed, clarified of cells, diluted 1:1 with fresh dmem supplemented with 20% fcs and 10,000 iu/ml rhm-csf, and returned to cells. fbmdms were recovered on day 6 by gentle aspiration in pbs following 30 min, 4°c incubation in pbs. the plaque assay technique was established using both ak-d and fcwf-4 cu cells. 6.5 × 10 5 cells per well were plated in 6-well plates or 3.0 × 10 5 cells per well were plated in 12-well plates. cells were infected with 10-fold serial dilutions of viral samples for 1 h at 37°c, followed by overlaying with a 0.5% oxoid agar (oxoid ltd, #lp0028)-dmem containing 1% fbs mixture. plates were incubated at 37°c for 48 h (or the indicated time) and fixed using 3.7% formaldehyde-pbs solution for 30 min. viral plaques were visualized by staining with 0.1% crystal violet for 30 min and photographed. we note plaques were clearly evident when we used oxoid agar, but not if we used noble agar. we analyzed the growth kinetics of type i fipv black in three cell lines: ak-d, fcwf-4 cu, and fcwf-4 atcc. type ii fcov titers were evaluated in fcwf-4 cu cells. for all cell types, 1.5 × 10 5 cells were plated in 24-well plates or 3.0 × 10 5 cells in 12-well plates. after incubating for 24 h, cells were infected with fipv in serum-free media at a multiplicity of infection (moi) of 0.1 at 37°c. after a 1 h incubation, the infectious media were replaced with fresh media containing 2% fbs. at the indicated time points, cell-free supernatants and/or infected cells were harvested and used for titration by plaque assay on ak-d (fipv black) or fcwf-4 cu (type ii fcovs) cells. the supernatant was collected from the cultures at the time indicated and subjected to centrifugation at 2200 ×g for 10 min at 4°c to remove any dead cells. this cell free supernatant was aliquoted and frozen at −80°c until use. to prepare the cell-associated sample, 0.5 ml of medium was added to the infected cells in the 24-well plate, and the entire plate was frozen at −80°c. the infected cells were then subjected to two additional rounds of freezing and thawing (37°c for 1-2 min). after the third thaw, the cells and medium were transferred to a centrifuge tube and centrifuged at 2200 ×g for 10 min at 4°c to remove cell debris. the supernatant containing the viruses released from the cells during the freeze-thaw process was designated the cellassociated virus sample. monolayers of 1.0 × 10 5 ak-d and fcwf-4 cu cells were cultured in 8-well chamber slides (nalge nunc international, #177445) at 37°c for 24 h. cells were infected with fipv black at a moi of 0.1 for 1 h at 37°c. at times indicated, the infected cells were fixed with 3.7% formaldehyde-pbs solution for 30 min, permeabilized with 0.1% triton x-100 in pbs for 10 min, and then incubated with blocking solution containing 5% normal goat serum and 0.1% triton x-100 in pbs at 4°c overnight. for immunofluorescence staining of fipv black-infected cells, the cells were incubated with mouse monoclonal anti-fipv nucleocapsid (ccv2-2) (poncelet et al., 2008 ) (bio-rad, #mca2594b) as a primary antibody at a dilution of 1:500 at room temperature for 1 h. cells were then incubated with a secondary antibody, alexa fluor 568conjugated goat anti-mouse igg (thermo fisher scientific, #a11004) at a dilution of 1:1000 in the presence of hoechst33342 (thermo fisher scientific, #h1399) at a dilution of 1:1000 for nucleus stain. after 30 min incubation with the secondary antibody at room temperature, the cells were then washed with pbs, mounted, and examined under a fluorescence microscope. 4.8. quantification of isg54 transcript production by rt-qpcr following stimulation with feline ifn cells (1.0 ×10 5 in a 24-well plate) were treated with 10, 100, or 1000 u/ml of purified feline ifn-alpha (pbl assay science, #15100-1) for 6 h. to determine isg54 and feline β-actin mrna production, total rna was extracted and an equal amount of rna (1000 ng) was used for cdna synthesis using rt 2 ht first-strand kit (qiagen, #330401). qpcr was performed with specific primers for feline β-actin transcript (fwd 5'-caa ccg tga gaa gat gac tca ga -3'; rev 5'-ccc aga gtc cat gac aat acc a -3') or isg54 transcript (fwd 5'-cct gag ctg cag cct ttc aga aca g -3'; rev 5'-cac gtg aaa tgg cat tta agt tgc cgc ag -3') using rt 2 sybr green qpcr mastermix (qiagen, #330502). a bio-rad cfx96 thermocycler was set as follows: one step at 95°c (10 min); 40 cycles of 95°c (15 s), 60°c (1 min), and plate read; one step at 95°c (10 s); and a melt curve from 65°c to 95°c at increments of 0.5°c/0.05 s. samples were evaluated in triplicate and data are representative of three independent experiments. the levels of mrna are reported relative to β-actin mrna and expressed as 2 −δct [δc t = c t(gene of interest) −c t(β-actin) ]. feline bone marrow-derived macrophages, fcwf-4 cu, or fcwf-4 atcc cells, grown for 24 h in 100 × 26 mm petri dishes (vwr, #25387-030), were washed with pbs, incubated in pbs for 30 min at 4°c, then collected with gentle pipetting. using a cytospin (shandon), 200 μl of cells (10,000 cells/ml) suspended in pbs supplemented with 2% bsa were spun onto glass coverslips (pre-treated with 2% bsa in pbs) at 115 xg for 6 min. cells were dried, rinsed in pbs, then fixed in absolute methanol for 1 min. wright-giemsa (thermo fisher scientific, #9990710) staining was performed per the manufacturer's instructions using a 3 min primary stain and a 2 min counter stain, and washed with 1) . b) fcov growth kinetics in fcwf-4 cu cells (moi=0.1) determined by c) plaque assay on fcwf-4 cu indicator cells. tenfold serial dilutions of cellfree virus inoculum were applied to cells. following oxoid agar/media overlay, cells were fixed in 3.7% formaldehyde and stained using 0.1% crystal violet after 24 h. fcov used were the type i strain fipv black and the type ii strains fipv wsu 79-1146 and fecv wsu 79-1683. plaque assay performed in triplicate; error bars ± sd. a. o'brien et al. virology 525 (2018) 1 ml rinse solution. slides were dried and imaged under oil immersion at 100x magnification. infectious diseases of the dog and cat. saunders a study of naturally occurring feline coronavirus infections in kittens persistence and transmission of natural type i feline coronavirus infection american type culture collection prevalence of feline coronavirus types i and ii in cats with histopathologically verified feline infectious peritonitis immunophenotype and functional properties of feline 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feline infectious peritonitis feline infectious peritonitis: role of the feline coronavirus 3c gene in intestinal tropism and pathogenicity based upon isolates from resident and adopted shelter cats comparative sequence analysis of fulllength genome of fipv at different tissue passage levels two related strains of feline infectious peritonitis virus isolated from immunocompromised cats infected with a feline enteric coronavirus detection of antigenic heterogeneity in feline coronavirus nucleocapsid in feline pyogranulomatous meningoencephalitis determination of 50% endpoint titer using a simple formula a simple method of estimating fifty per cent endpoints characterization of a recombinant canine coronavirus with a distinct receptor-binding (s1) domain acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein x-ray structure and inhibition of the feline infectious peritonitis virus 3c-like protease: structural implications for drug design differential effects of viroporin inhibitors against feline infectious peritonitis virus serotypes i and ii chimeric feline coronaviruses that encode type ii spike protein on type i genetic background display accelerated viral growth and altered receptor usage genome organization and reverse genetic analysis of a type i feline coronavirus a reverse genetics approach to study feline infectious peritonitis feline coronaviruses: pathogenesis of feline infectious peritonitis tackling feline infectious peritonitis via reverse genetics feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses improving virus taxonomy by recontextualizing sequence-based classification with biologically relevant data: the case of the alphacoronavirus 1 species we thank matthew hackbart, dr. xufang deng, dr. jean k. millet and javier jaimes for helpful discussions. we also thank dr. fred scott and dr. edward j. dubovi for the provision of reagents. tissue samples and associated phenotypic data were provided by the cornell veterinary biobank, a resource built with the support of national institutes of health (nih) grant r24 gm082910 and the cornell university college of veterinary medicine. this work was supported by a pilot project grant issued to s.c.b. by loyola university of chicago and a research grant from the winn feline foundation bria fund (#mtw17-022 to s.c.b. and g.r.w.). r.c.m. is supported by the (nih) t32 training grant for experimental immunology (#ai007508) and the arthur j. schmitt dissertation fellowship in leadership and service (arthur j. schmitt foundation). g.r.w. is supported by research grants from the cornell feline health center and the winn feline foundation. key: cord-302972-imtttzvr authors: feldmann, h.; will, c.; schikore, m.; slenczka, w.; klenk, h.-d. title: glycosylation and oligomerization of the spike protein of marburg virus date: 1991-05-31 journal: virology doi: 10.1016/0042-6822(91)90680-a sha: doc_id: 302972 cord_uid: imtttzvr abstract the oligosaccharide side chains of the glycoprotein of marburg virus (mw 170,000) have been analyzed by determining their sensitivity to enzymatic degradation and their reactivity with lectins. it was found that they consist of nand o-glycans. studies employing chemical cross-linking showed that the glycoprotein is present as a homotrimer in the viral envelope. marburg virus and the morphologically related ebola virus are negative-stranded rna viruses of the filoviridae family (1). both viruses cause a severe hemorrhagic fever with a mortality as high as 90% for ebola virus (2) and about 35% for marburg virus (3). marburg virus was first isolated in 1967 from laboratory workers, when it caused three outbreaks in europe at marburg, frankfurt, and belgrade. the workers were infected after contact with tissues of monkeys imported from uganda (3). since that time, four more episodes of marburg disease in africa have been reported (4). marburg virus is an enveloped, filamentous virus containing a negative-stranded rna genome with a molecular weight of approximately 4.2 x lo6 (5). the virion rna does not bind to oligo-dt-cellulose and is not infectious. sds-page profiles demonstrate the presence of seven virion proteins. these are the l protein (180k), the glycoprotein gp, the nucleoprotein np (96k), and proteins vp40 (38k), vp 35 (32k), vp30 (28k), and vp24 (24k). in analogy to ebola virus, vp35 may be a transcriptase component and vp30 a second nucleoprotein. the functions of vp40 and vp24 are still unknown (4, 6). the virion proteins are synthesized from monocistronic mrna species analogous to those of ebola virus (7). by in vitro translation, five different mrna species could be assigned to proteins np, vp40, vp35, vp30, and vp24. mrnas coding for l and gp could not be detected yet (4). gp is the only membrane glycoprotein of marburg virus and forms the surface projections on the viral envelope. the molecular weight of gp has previously been estimated to be 140,000 but reevaluation with appropriate protein markers indicates that 170,000 may be more accurate (fig. 1a) . to examine the type of glycan-protein linkages, we treated virion proteins ' to whom requests for reprints should be addressed. with different glycohydrolases. these treatments were performed overnight at 37" after denaturing of the proteins. as shown in fig. 1 b, the electrophoretic mobility of gp was slightly enhanced after incubation with endoglycosidase h (lane 4), and a distinct further increase was obtained by treatment with endoglycosidase f (lane 3) indicating that gp contains ai-glycans of the oligomannosidic, but mainly of the complex type. however, carbohydrate not linked by n-glycosidic bonds was also present. sensitivity of gp to endo-a-/vacetylgalactosaminidase ( fig. 1 b, lane 2) indicated that 0-glycans containing galactose-p(l-3)-n-acetylgalactosamine disaccharide units were present. the existence of 0-glycans was also suggested by n-acetylgalactosamine which was detected, in addition to n-acetylglucosamine, when purified gp was subjected to amino acid analysis (data not shown). to further assess the nature of the carbohydrate moiety, the affinity of various lectins to gp was analyzed. the lectins used were conjugated to the steroid hapten digoxigenin (boehringer, mannheim, germany), and their binding was assayed by immunoblotting. the results obtained with peanut agglutinin (pna) and datura stramonium agglutinin (dsa) are shown in fig. 2 . as indicated by control experiments with fetuin that contains n-and o-linked oligosaccharides and with asialofetuin containing the same glycans devoid of sialic acid, pna reacts specifically with the galactose-/3( l -3)-n-acetylgalactosamine cores of 0-glycans that must not be substituted with terminal n-acetylneuraminic acid. the observation that marburg virus gp is recognized by pna after incubation with endoglycosidase f or n-glycopeptidase f provides further support for the presence of 0-glycans. it was unexpected, however, that pna bound also to gp after incubation with endo-a-n-acetylgalactosaminidase. this suggests that galactose-p(l-3)-n-acetylgalactosamine cores substituted with nonsialic acid residues of 20 &i/ml, and labeled virus was harvested 24 hr thereafter. culture fluid was clarified by centrifugation at 5,000 rpm for 15 min at 4", and virus was pelleted at 25,000 rpm through a 20% sucrose cushion in tne (0.01 m tris-hci, ph 7.4; 0.15 m naci; 2 mm edta) for 2 hr at 4'. the pellet was resuspended in tne and further purified by gradient centrifugation in a sw28 rotor through 0 to 40% (w/v) potassium tartrate. 30 to 0% glycerol in tne at 27,000 rpm for 16 hr, followed by 20 to 70% sucrose in tne (containing 1 m naci) at 27,000 rpm for 4 hr at 4". the virus band was isolated, diluted in tne, and pelleted by centrifugation at 35,000 rpm for 30 min at 4". glycohydrolase treatments were performed at 37" overnight after denaturing of the proteins by boiling in buffer containing 0.1% sds, 0.5% octylglucoside, 0.5% p-mercaptoethanol, 50 mm sodium acetate, ph 7.0, and 5 mm edta. the proteins were separated by electrophoresis on polyacrylamide slab gels and detected by fluorography. virus was grown in e6 cells, labeled, and purified as described in the legend of fig. 1 . virus resuspended in triton-lysis buffer was incubated with dsp in dmso at a final concentration of 0.8 mm for 20 min at 15". the excess of dsp was adsorped by adding sodium hydrogen carbonate at a final concentration of 20 mm. virus was inactivated by boiling in buffer containing sds at a final concentration of 3%. but without @mercaptoethanol. the cross-linked samples were analyzed on a 3.5% polyacrylamide slab gel and detected by fluorography. b. two-dimensional sds-page analysis of [35s]methionine-labeled, cross-linked gp. growth of virus, labeling, and cross-linking were performed as described in the legend of fig. 1 and as in a above. proteins were first electrophoresed in a slab gel containing 5% acrylamide and 0.13% bisacrylamide (horizontal direction). the gel lane was cut out, soaked in a reducing solution of 5% fl-mercaptoethanol. 10 mm dtt, and 0.5 m tris-hci. ph 6.8, for 30 min. it was placed on top of a slab gel of 10% acrylamide and 0.26% bisacrylamide, using 1% agarose made in reducing solution to embed the gel lane, and then electrophoresed in the vertical direction (18). are present, that are not susceptible to the enzyme, yet still recognized by the lectin. dsa binds specifically to /v-glycans as demonstrated by the control experiments employing asialofetuin and fetuin. after incubation with endo-&v-acetylgalactosaminidase, marburg virus gp still binds this lectin, but is no longer able to do so after exposure to endoglycosidase f or n-glycopeptidase f. gp bound also weakly to galantus nivalis agglutinin (gna) which recognizes terminal mannose linked to mannose in a/-glycans. treatment with endoglycosidase h abolished reactivity with gna (data not shown) confirming the presence of high mannose type n-glycans on gp. the subunit organization of gp in the viral membrane was analyzed in cross-linking studies using the homobifunctional reagent dithiobis(succinimidylpropionate) (dsp) which is susceptible to cleavage by reducing agents such as /3-mercaptoethanol. purified virus was incubated in triton lysis buffer (1 x mnt (10 m/vi [nmorpholinolethanesulfonic acid), 1 mm phenylmethylsulfonyl fluoride, 10 mlll idodoacetamide, 1% triton x-l 00, 1:20 trasylole, 10 mm edta) with dsp at a final concentration of 0.8 mm for 20 min at 15" and analyzed by electrophoresis on 3.59/o polyacrylamide slot gels. figure 3a shows an autoradiogram of [3h]glucosamine-labeled virus analyzed under nonreducing conditions. whereas in the absence of dsp, only a single gp band was present, two additional bands of lower electrophoretic mobilities were detected, when cross-linking took place. by comparison with cross-linked phosphorylase b (sigma) and a,-macroglobulin from horse plasma (boehringer, mannheim) as markers, the three bands have been estimated to be 170, 330, and 550 kda in size, suggesting that they represent gp monomers, dimers, and trimers, respectively. in addition, the dsp-cross-linked, [35s]methionine-labeled complexes of high molecular weight were analyzed by two-dimensional sds-page. separation in the first dimension was performed under nonreduced conditions and in the second one under reduced conditions as described in the legend to fig. 3b. fig. 3b demonstrates that monomeric, dimeric, and trimeric forms of gp were free of other proteins. thus gp complexed only with itself, but not with other cellular or viral gene products. to further analyze the composition of the gp complexes, purified [35s]methionine-labeled virus was subjected to solubilization by nonionic detergent and sedimentation on sucrose density gradients, and the polypeptides present in the fractions obtained from the gradients were assayed by polyacrylamide gel electrophoresis under denaturing and reducing conditions. figure 4a shows the sedimentation profile of gp monomers (fractions 13-l 7) that were obtained by treating the virus with p-mercaptoethanol (5%, 10 min, 96"). most of the other viral proteins were insoluble after this treatment and were removed by lowspeed sedimentation, prior to gradient centrifugation. fig. 1 . cross-linking was performed as described in the legend of fig. 3 . the samples were applied onto a preformed 5 to 30% (w/v) sucrose gradient in mnt and 1% triton x-l 00 and centrifuged in a sw 41 rotor at 40,000 rpm for 16 hr at 4". fractions were collected from the bottom of the tube, tca precipitated, and analyzed by electrophoresis on a 10% polyacrylamide slab gel after boiling in buffer containing sds and p-mercaptoethanol at final concentrations of 3 and 5%, respectively. a. virus non-cross-linked and treated with @-mercaptoethanol (5%, 10 min, 96") prior to gradient centrifugation. b. virus cross-linked and not treated with p-mercaptoethanol. were obtained under nonreducing conditions, and their sedimentation profile is shown in fig. 4b . the sedimentation coefficients of monomeric and trimeric gp were approximately 5 and 11 s, respectively, relative to trimerit influenza virus (fpv) hemagglutinin (8.8 s). gp trimers have thus a higher sedimentation coefficient than hemagglutinin trimers, and this is in agreement with the higher molecular weight of gp. figure 4b shows also that the other viral proteins cosedimented under nonreducing conditions with gp monomers, and that fractions 5-l 1 containing gp complexes were free of other viral proteins. the data presented here indicate that gp of marburg virus is present as a homotrimer in the viral envelope. gp resembles therefore in its quaternary structure the hemagglutinin of influenza virus and the g protein of vsv which form also trimers. our data show also that gp contains not only n-glycans of the complex and the oligomannosidic type, but also carbohydrate side chains in 0-glycosidic linkages. the latter ones contain the core unit galactose-/-i(l-3)-n-acetylgalactosamine and therefore belong to the 0-glycans of the mucin type. whereas a/-glycans are very common with enveloped viruses, o-linked carbohydrate side chains have been detected so far only in a few viral glycoproteins. these are, on the one hand, the el glycoprotein of bovine and murine coronaviruses that contain only oglycans (8-10) and, on the other hand, herpes virus glycoproteins (1 l-l 3), vaccinia virus hemagglutinin (14), and the g-protein of respiratorysyncytial virus (15, 16) that contain both n-and o-linked side chains, like marburg virus gp. marburg virus disease the replication of negative strand viruses acknowledgment this work was supported by the deutsche forschungsgemeinschaft (ki 238/1-l). key: cord-287620-vuvgi8xx authors: butler, noah; pewe, lecia; trandem, kathryn; perlman, stanley title: murine encephalitis caused by hcov-oc43, a human coronavirus with broad species specificity, is partly immune-mediated date: 2006-04-10 journal: virology doi: 10.1016/j.virol.2005.11.044 sha: doc_id: 287620 cord_uid: vuvgi8xx the human coronavirus hcov-oc43 causes a significant fraction of upper respiratory tract infections. most coronaviruses show a strong species specificity, although the sars-coronavirus crossed species from palm civet cats to infect humans. similarly, hcov-oc43, likely a member of the same coronavirus group as sars-cov, readily crossed the species barrier as evidenced by its rapid adaptation to the murine brain [mcintosh, k., becker, w.b., chanock, r.m., 1967. growth in suckling-mouse brain of “ibv-like” viruses from patients with upper respiratory tract disease. proc natl acad sci u.s.a. 58, 2268–73]. herein, we investigated two consequences of this plasticity in species tropism. first, we showed that hcov-oc43 was able to infect cells from a large number of mammalian species. second, we showed that virus that was passed exclusively in suckling mouse brains was highly virulent and caused a uniformly fatal encephalitis in adult mice. the surface glycoprotein is a major virulence factor in most coronavirus infections. we identified three changes in the hcov-oc43 surface glycoprotein that correlated with enhanced neurovirulence in mice; these were located in the domain of the protein responsible for binding to host cells. these data suggest that some coronaviruses, including hcov-oc43 and sars-cov, readily adapt to growth in cells from heterologous species. this adaptability has facilitated the isolation of hcov-oc43 viral variants with markedly differing abilities to infect animals and tissue culture cells. until recently, human coronaviruses were mostly associated with mild upper respiratory tract infections (''the common cold''), and occasionally with outbreaks of gastroenteritis (vabret et al., 2003) . however, with the recognition that the severe acute respiratory syndrome (sars) was caused by a coronavirus, it became apparent that coronaviruses could also cause more significant disease in the human population (drosten et al., 2003; fouchier et al., 2003) . since then, two additional coronaviruses, hcov-nl63 and hcov-hku1, have been identified; these agents cause upper and lower respiratory tract diseases that are much less severe than sars (van der hoek et al., 2004; woo et al., 2005) . hcov-oc43 and hcov-229e are the etiological agents for many coronavirus-induced upper respiratory tract infections. hcov-oc43, harvested from a patient with an upper respiratory tract infection, was originally isolated after passage in human embryonic tracheal organ cultures; this virus caused neurological disease after only one passage in suckling mice and encephalitis within 2-4 passages (mcintosh et al., 1967) (termed hcov-oc43 nv ). hcov-oc43 nv was then propagated in tissue culture cells generating a tissue culture-adapted variant (termed hcov-oc43 tc ). hcov-oc43 showed increasing neurovirulence with passage through the murine brain (mcintosh et al., 1967) ; however, most recent studies have used cns-adapted viruses that were further propagated, at least for a few passages, in 0042-6822/$ -see front matter d 2005 elsevier inc. all rights reserved. doi:10.1016/j.virol.2005.11.044 tissue culture cells. for example, talbot and co-workers showed, using the mouse-adapted virus after passage in tissue culture cells (termed hcov-oc43 que herein;) that mice infected intranasally with 10 4 -10 5 tcid 50 developed encephalitis if inoculated 8 days but not 21 days postnatally (jacomy and talbot, 2003) . hcov-oc43 que was passaged 5-6 times in tissue culture prior to use in mice (personal communication, dr. pierre talbot, inrs-institut armand-frappier, laval, quebec) and consequently may be less virulent than virus isolated directly from infected suckling mouse brains. consistent with this possibility, we observed, in preliminary experiments, that virus directly harvested from suckling mouse brains caused a lethal infection after intranasal inoculation of 5-to 8-week-old mice. mice died 9 -11 days after inoculation, a time when the adaptive immune response to another coronavirus, mouse hepatitis virus (mhv), is maximal (bergmann et al., 1999) . to begin to understand these differences in virulence, we initiated a more complete study of the disease caused by the neurovirulent strain of hcov-oc43. this ability of hcov-oc43 to cross species barriers to infect mice and to gain virulence in the new host contrasts with the strict species specificity exhibited by most coronaviruses. for example, the group i coronavirus hcov-229e does not readily infect mice, even transgenic mice expressing human aminopeptidase n, the virus receptor for hcov-229e (wentworth et al., 2005) . however, the ability of the group ii coronavirus hcov-oc43 to adapt easily to replication within the murine brain suggests that it may be more lax in its species specificity than other coronaviruses. in that sense, it resembles another group ii coronavirus, sars-cov, which likely crossed the species barrier from animals such as palm civet cats to infect humans (csmec, 2004; guan et al., 2003; song et al., 2005) . unlike other coronaviruses, hcov-oc43 and the closely related bovine coronavirus (bcov) appear to bind to cells via n-acetyl-neuraminic acid (schultze and herrler, 1992; vlasak et al., 1988) , although there are data to suggest that hcov-oc43 can also employ mhc class i antigen as a host cell receptor (collins, 1993) . this use of a sugar moiety for entry would also be consistent with the ability to infect a broader range of species than most coronaviruses. this possibility was investigated by infecting tissue culture cells from several different animal species with hcov-oc43, using both the mouse-adapted and the tissue culture-adapted strains. the ability to rapidly gain virulence after passage in the murine brain is likely to occur via selection of mutations in the s protein that optimize binding and entry to target cells. passage of neurovirulent variants of mhv, in tissue culture, selects for viruses that are attenuated in vivo but enhanced for replication in vitro. these changes map to the surface (s) glycoprotein (gallagher and buchmeier, 2001; tsai et al., 2003) . in addition, infection of rats with uncloned stocks of mhv resulted in the selection of virulent strains of virus; again, virulence correlated with changes in the s protein (taguchi et al., 1985) . also, replacement of the s gene in the moderately virulent a59 strain of mhv with the gene encoding the s protein of the virulent jhm strain resulted in a gain in virulence in mice (phillips et al., 1999) . in other studies, several groups showed that the adaptation of the sars-cov to humans during the 2003 epidemic included several mutations in the s protein. these mutations were shown to enhance binding to human angiotensin converting enzyme 2 (ace 2), the host cell receptor for sars-cov (kan et al., 2005; li et al., 2005) . therefore, to investigate the role of the s protein in hcov-oc43 pathogenesis, we sequenced the s genes of hcov-oc43 tc and hcov-oc43 nv and compared the results to published sequences of the s genes of several other hcov-oc43 isolates. intranasal inoculation of hcov-oc43 nv , but not hcov-oc43 tc , is uniformly fatal to 5-to 8-week-old c57bl/6 (b6) mice in confirmation of our preliminary results, intranasal inoculation of hcov-oc43 nv resulted in 100% mortality in mice ranging from 5 to 8 weeks old (fig. 1a) . mice developed signs of acute encephalitis, including hunched posture, lethargy and wasting by days 6-7. mortality was associated with a 30 -35% loss of body mass (fig. 1b) . severe clinical encephalitis was associated with widespread mononuclear cell infiltration including perivascular cuffing and with loss of cns architecture (data not shown). in contrast, intranasal inoculation of 5week-old c57bl/6 mice with hcov-oc43 tc did not cause any clinical disease, including any weight loss (fig. 1) . consistent with the uniformly lethal outcome observed in fig. 1 . hcov-oc43 nv is uniformly lethal to wild type c57bl/6 mice. 5-weekold mice were inoculated intranasally with 30 ld 50 hcov-oc43 nv or 10 6 tcid 50 hcov-oc43 tc as described in materials and methods. mice were monitored daily for survival (a) and weight loss (b). (*indicates p < 0.0002). mice infected with hcov-oc43 nv , we detected high titers of virus in the cns of infected mice. hcov-oc43 nv grew poorly in tissue culture cells and we could only reliably titer infectious virus using suckling mice, as described in materials and methods. virus titers increased from days 5 to 7 (fig. 2) . to confirm these results, we also measured viral loads using a realtime rt-pcr assay. as shown in figs. 2a and b , there was a strong positive correlation between recovery of infectious virus and the detection of oc43 nucleocapsid rna in the brains of mice. also, and in agreement with the results of jacomy et al. (jacomy and talbot, 2003) , hcov-oc43 infected other organs to a small extent, such as the lungs and intestines, when viral rna was assayed by real-time rt-pcr (fig. 2c) . notably, the results also suggested that virus was in the process of clearance at the time of death since virus titers/rna levels declined between days 7 and 9 p.i. (fig. 2c) . in mice infected intranasally with neurovirulent strains of mhv, virus enters the cns via the olfactory nerves with subsequent transneuronal retrograde dissemination to distant connections of the olfactory bulb (perlman et al., 1989 (perlman et al., , 1990 . using in situ hybridization and immunohistochemistry to track hcov-oc43 rna and antigen, respectively, we detected overlap between the pathways of entry and spread used by mhv and those used by hcov-oc43 after intranasal inoculation. hcov-oc43 was detected in the olfactory bulb and the olfactory nucleus at 3 days p.i. but was cleared from these structures by day 5 p.i. at later time points (days 7 and 9 p.i.), hcov-oc43 rna and antigen were detected primarily in the brainstem and the spinal cord (fig. 3a , data not shown). unlike mhv, which is known to infect oligodendrocytes and is present throughout the white matter in the infected cns, hcov-oc43 was not detected in the white matter at any time point examined (shown at day 9 p.i. in figs. 3a, b). the ability of hcov-oc43 nv to cause lethal encephalitis in adult animals contrasted with that reported for hcov-oc43 que . jacomy et al. reported that hcov-oc43 que infection was primarily restricted to neurons in vivo (jacomy and talbot, 2003) . thus, one possible explanation for the enhanced neurovirulence of hcov-oc43 nv , relative to hcov-oc43 que , is an expanded cell tropism. to more directly assess this possibility, we used combined in situ hybridization and immunohistochemistry to identify cell types harboring viral rna and antigen. we found that viral product was restricted to cells with morphologies consistent with neurons (figs. 3c, d). moreover, in some animals evaluated at late times postinfection (day 9), we could clearly identify purkinje cells that harbored virus antigen (fig. 3e ). when we evaluated whether astrocytes were infected by hcov-oc43 nv in vivo, we found no evidence of colocalization of hcov-oc43 rna and the astrocyte-specific marker, gfap antigen (figs. 3f-h). we also performed a series of colocalization experiments for viral rna and f4/80, a macrophage/microglia-specific cell marker, but could not identify f4/80 + cells that were positive for viral product. to confirm these results, we sorted f4/80 + cd45 hi (macrophages) and f4/80 + cd45 int (microglia) cells from the cns of infected mice at 7 days postinfection, collected them onto glass slides and stained them for virus antigen. neither macrophages nor microglia stained positive for viral antigen, while infected tissue culture cells processed in parallel stained positively (data not shown). these data suggest that unlike mhv, hcov-oc43 does not infect astrocytes or macrophages/ microglia. based on the morphology of the infected cells as well as the lack of virus staining in the white matter or in gfap + or f4/80 + cells, we conclude that the predominant, if not sole, targets for hcov-oc43 nv are neurons. host adaptive immune response contributes to hcov-oc43-induced morbidity and mortality viral rna burden in the cns was diminishing at the time of death (fig. 2c ), consistent with a role for the host immune response in both virus clearance and disease. when we immunophenotyped mononuclear cell infiltrates from hcov-oc43 nv -infected mice 7 days postinfection, we found that a large fraction consisted of cd4 and cd8 t cells (fig. 4a ). one fig. 3 . neurons are the primary target of hcov-oc43 nv in vivo. mice were inoculated intranasally with 30 ld 50 of hcov-oc43 nv and brains and spinal cords were harvested 7 or 9 days p.i. tissue samples were prepared for in situ hybridization, antigen staining or luxol fast blue (lfb) staining and slides were examined with standard light, fluorescence or confocal microscopy as described in materials and methods. (a -b) hcov-oc43 antigen is localized in the gray matter of spinal cords. panel a depicts immunohistochemical detection of hcov-oc43 using the anti-oc43 s hybridoma o.4.3. panel b depicts a serial section stained with lfb to demarcate the white matter. (c -e) morphology of hcov-oc43 nv -infected cells is consistent with that of neurons. images are representative coronal sections of brain stems from mice harvested 7 -9 days p.i. sections were stained with o.4.3 followed by cy3-labeled goat anti-mouse. (f -h) combination in situ hybridization for hcov-oc43 nucleocapsid rna (cy3-labeled antisense probe, red) and immunohistochemistry for the astrocyte marker gfap (fitc-labeled anti-gfap, green). panel h is a merged image of panels f and g. original images are 20â magnification for panels a and b, or 40â magnification for panels c -h. hcov-oc43-specific cd4 t cell epitope recognized in c57bl/6 mice is known, spanning residues 133 -147 of the transmembrane (m) protein (epitope m 133 ). identification of this epitope was based on sequence homology with mhv. we found that 1 -3% of the infiltrating cd4 t cells at day 9 recognized this epitope (fig. 4b ). by contrast, 20 -25% of cd4 t cells in the mhv-infected cns responded to epitope m133 at 7 days p.i. (haring et al., 2001) . no hcov-oc43 cd8 t cell epitopes have been identified yet so the magnitude of the virus-specific cd8 t cell response could not be determined. to probe the role of the host adaptive immune response in pathogenesis, we infected immunodeficient mice lacking normal t and b cell responses (mice with genetic disruption of the recombination activating gene 1 (rag1 à/à )) and monitored these mice for weight loss and survival. hcov-oc43 nv -infected rag1 à/à mice developed signs of enceph-alitis (lethargy, hunching and weight loss) similar to those observed in infected wild type mice, but with delayed kinetics (fig. 4c, left panel) . infected rag1 à/à mice also survived longer than did their b6 counterparts (fig. 4c, right panel) . at the time of death, virus loads were 5 -7-fold higher than detected in moribund b6 mice, when measured by real-time rt-pcr (fig. 4d , left panel) or infectious virus titers (fig. 4d , right panel). at this time, viral antigen or rna, detected by immunohistochemistry or in situ hybridization, was detected primarily in the brainstem although more cells were infected than in b6 mice. as in wild type c57bl/6 mice, neurons were the primary target for infection, suggesting that the antiviral immune response was not responsible for the lack of infection of glial cells (data not shown). to confirm the pathological role of t cells, we adoptively transferred hcov-oc43-immune cells to rag1 à/à mice that had been infected with 30 ld 50 hcov-oc43 nv intranasally 4 days earlier. hcov-oc43 nv -infected rag1 à/à animals that received no cells died by days 16 -18, in line with experiments described above. in contrast, the adoptive transfer of hcov-oc43-immune splenocytes to rag1 à/à mice resulted in death of recipient animals by days 11 -13 (data not shown). divergent spike glycoprotein gene sequences between mouse cns-adapted, tissue culture-adapted and primary clinical isolates of hcov-oc43 while hcov-oc43 nv causes lethal encephalitis that is enhanced by the antiviral t cell response, the results described above show that hcov-oc43 tc is severely attenuated in mice (fig. 1) . hcov-oc43 que was reported to cause less severe disease than we observed in mice infected with hcov-oc43 nv . these differences in neurovirulence prompted us to determine the sequence of the s glycoproteins of hcov-oc43 nv and hcov-oc43 tc and compare them to the published hcov-oc43 que sequence, since the coronavirus s protein is often associated with virulence (thorp and gallagher, 2004) . included in this comparison are all available sequences submitted for a number of hcov-oc43 isolates. these additional isolates represent strains that were reported as having been minimally or extensively passaged in tissue culture cells, as well as several primary clinical isolates that were never passaged in tissue culture cells (supplementary fig. 1 ). the sequence alignment revealed substantial divergence in the primary amino acid sequence of these hcov-oc43 isolates. of note, there were three amino acid substitutions wholly unique to hcov-oc43 nv (table 1) . multiple other differences between the hcov-oc43 tc and hcov-oc43 que or hcov-oc43 nv strains are also present in both the s1 and s2 domains ( supplementary fig. 1) . the large number of differences will make it difficult to determine which substitutions are critical for tissue culture cell adaptation. of note, sequencing of the 3vterminal end of hcov-oc43 nv , including genes encoding non-structural 12.9 kda, the small envelope (e), the nucleocapsid (n) and the transmembrane (m) proteins and the 3v untranslated region, revealed no nucleotide differences between hcov-oc43 nv and hcov-oc43 que . broad species and cell-type tropism of hcov-oc43 tc and hcov-oc43 nv as described above, hcov-oc43 differs from most strains of coronaviruses in that it readily crossed species barriers to infect mice (mcintosh et al., 1967) . this observation raised that possibility that hcov-oc43, unlike most other coronaviruses, is able to infect a wide variety of species without substantial adaptation. to gain insight into this possibility, we infected a variety of tissue culture cells from a wide range of species (hamster, pig, human, mouse, rat, monkey and cat) with hcov-oc43 nv or hcov-oc43 tc . as shown in fig. 5 , immunocytochemical staining for viral antigen revealed that the tissue culture-adapted virus infected hamster, pig, human, mouse, monkey and cat cells, but not frt rat epithelium cells. the neurovirulent strain also exhibited a wide host range specificity, infecting hamster, pig, human, monkey, cat and mouse cells. for some cells lines, such as murine 17cl-1, and human kb and ht1080 cells, we detected infection only by the tissue culture-adapted variant. thus, both hcov-oc43 nv and hcov-oc43 tc showed broad species specificity, and not surprisingly, hcov-oc43 tc showed enhanced ability to infect all types of tissue culture cells. whether animals from these various species are also infectable by hcov-oc43 remains to be determined, although data suggesting a close relationship between hcov-oc43 and bcov (vijgen et al., 2005b) make it likely that the virus would be able to infect other species after minimal adaptation. hcov-oc43, as demonstrated herein, infects many cell types and readily adapts to cause a virulent infection in mice talbot, 2001, 2003) (fig. 1) . bcov, which is closely related to hcov-oc43, also infects mice (akashi et al., 1981; barthold et al., 1990) ; phylogenetic analyses suggest that the two viruses diverged as recently as 1891 (vijgen et al., 2005b) . these results suggest that hcov-oc43 or bcov crossed species from either humans or bovine to infect the other species, although the direction of spread is not known. this ability to cross species is also shared by sars-cov. sars-cov spread from exotic animals, most likely palm civet cats, to humans during contacts in ''wet markets'' in china (peiris et al., 2004) . also, as part of efforts to study the pathogenesis of this virus and to develop vaccines, several animal species, including mice, cats, ferrets and monkeys were shown to be susceptible to infection with the virus (peiris et al., 2004) . sars-cov did not cause reproducible disease in any of these species, suggesting that further adaptation was required for optimal virus replication in these heterologous species. hcov-oc43 and sars-cov differ from many coronaviruses, exemplified by hcov-229e, in their ability to cross species. fig. 1. fig. 5 . broad tissue and cell type tropism of hcov-oc43. a wide variety of tissue culture cells from hamster, rat, pig, human, mouse, monkey and cat were infected with 10 tcid 50 (10 5 smld 50 ) of hcov-oc43 nv , 30 tcid 50 of hcov-oc43 tc or mock-infected as described in materials and methods. after 3 days, virus-infected cells were detected with immunocytochemistry as described in materials and methods. nearly all of the cell lines were infected with both viruses, demonstrating that hcov-oc43 exhibits a broad species specificity in vitro. original images are 20â magnification. hcov-229e, a human respiratory virus, does not infect mice or even mice transgenic for human aminopeptidase n (hapn), the hcov-229e host cell receptor (wentworth et al., 2005) . however, infection occurs, albeit with mild clinical disease if virus is adapted for growth in murine cells expressing hapn and if mice are deficient in type i interferon responses (lassnig et al., 2005) . sars-cov and hcov-oc43, while both able to cross species, differ in regard to host cell receptor usage. the primary receptor for sars-cov is angiotensin converting enzyme 2 (ace2) and other molecules such as dc-sign serve to enhance binding to target cells (jeffers et al., 2004; li et al., 2003; marzi et al., 2004) . adaptation to growth in foreign species involves mutations that enhance binding to heterologous ace2 . no proteinaceous receptor for hcov-oc43 has been conclusively identified; rather, oacetylated sialic acid or a closely related compound appears to serve as the host cell receptor (schultze and herrler, 1992; vlasak et al., 1988) . while little is known about the changes required for efficient replication of hcov-oc43 in the murine cns, it is likely that this process includes mutations that affect binding to host cell sugar moieties. infection with the prototypic alphavirus, sindbis virus (sinv), or with a picornavirus, theiler's murine encephalomyelitis virus (tmev), is initiated by binding to a polyanionic polysaccharide, heparin sulfate (byrnes and griffin, 1998; klimstra et al., 1998; reddi and lipton, 2002) . sinv with enhanced binding to heparin sulfate is selected after passage in tissue culture cells. hcov-oc43 may undergo a similar set of changes during the course of adaptation to growth in tissue culture cells. the s protein of hcov-oc43 tc differs from that of hcov-oc43 nv at 20 positions ( supplementary fig. 1 ), making it difficult to determine which ones might contribute to enhanced ability to replicate in tissue culture cells. by contrast, hcov-oc43 nv includes a limited number of amino acids that are not present in any s proteins described in the literature (table 1 and supplementary fig. 1 ). in all laboratory isolates of hcov-oc43, viruses encoding these proteins were passaged at least a few times in tissue culture cells (kunkel and herrler, 1993b; kunkel and herrler, 1996; mounir and talbot, 1993; st-jean et al., 2004; vijgen et al., 2005b) . while no two sequences are identical, three substitutions, t33s, n491h and r759h, are unique to the hcov-oc43 nv strain (table 1 and supplementary fig. 1) . the receptor binding domain of hcov-oc43 has not been defined, but both t33s and n491h are in regions important for receptor binding by other coronaviruses (e.g., saeki et al., 1997; xiao et al., 2003) . thus, these two changes could potentially alter the association or stability between hcov-oc43 nv and host cell receptors in the murine cns. in other coronaviruses, cleavage of s into s1 and s2 domains is required for optimal virus replication. for example, disruption of the s1 -s2 cleavage site in mhv resulted in a viable virus that was attenuated for growth in tissue culture cells and mice (stauber et al., 1993; taguchi, 1993) . on the other hand, studies in bcov revealed no correlation between the presence of the furin cleavage motif and virulence (zhang et al., 1991) . of the several laboratory isolates of hcov-oc43 that have been sequenced, most contain the sequence rrsrg at the putative furin cleavage site. kunkel and herrler showed that s proteins containing this motif are not cleaved in infected cells, whereas two strains (designated oc43-cu and oc43-va, supplementary fig. 1 ) in which the s gene encoded a prototypic cleavage site (rrsrr) were cleaved (kunkel and herrler, 1993a) . the sequences of seven recently described primary clinical isolates also share the rrsrr motif (supplemental fig. 1) (vijgen et al., 2005a) . whether or not these primary isolates are cleaved and whether this motif contributes to clinical disease in humans are not known. as reported herein, the s protein of hcov-oc43 nv contains the sequence rrshg and presumably is not cleaved to a significant extent because it contains a glycine at position 5; it is even less likely that hcov-oc43 tc , in which the sequence at this site is irsrg, is cleaved. although the r759h substitution is unlikely to affect cleavability of hcov-oc43 nv s, as this is a conserved (charged-basic to charged-basic) change, it is formally possible that this unique substitution could alter cleavage and contribute to the enhanced virulence of hcov-oc43 nv . the position of these 3 mutations (t33s, n491h, and r759h) is consistent with a role in virulence, but to prove definitively their role in disease enhancement, it will be necessary to introduce these changes into the hcov-oc43 genome. this will require a reverse genetics system. an infectious clone for hcov-oc43 has not yet been reported, but it is likely that one or more infectious cdna clones will be available in the near future. while a comparison of our results with those of jacomy et al. suggest that hcov-oc43 nv is more virulent than hcov-oc43 que , both strains show a tropism for neurons (jacomy and talbot, 2003) . direct virus destruction of infected neurons is clearly important for much of the disease observed in mice infected with hcov-oc43 nv or hcov-oc43 que ; even though death is delayed in infected rag1 à/à mice compared to wild type mice, all mice still die by 16 days p.i. (fig. 4) . however, the delayed death of infected rag1 à/à mice suggests that hcov-oc43-induced encephalitis is in part mediated by the antiviral t cell response. of note, jacomy et al. previously concluded that the adaptive immune response did not contribute to pathology associated with hcov-oc43 infection, based on the observation that treatment of mice with cyclosporin (csa) prior to infection resulted in more rapid onset of disease and an increase in the percentage of mice that succumbed to the virus (jacomy and talbot, 2003) . these contrasting results may result from the different mechanisms of immunosuppression observed in rag1 à/à and csa-treated mice. neurons do not normally express mhc class i or ii antigen and express only low levels of the machinery required for loading peptide onto mhc class i antigen (joly and oldstone, 1992) . however, electrically silent or damaged neurons do express mhc class i antigen (medana et al., 2000; neumann et al., 1995 neumann et al., , 1997 and it is possible that infection with hcov-oc43 makes neurons into suitable targets for cd8 t cells. of note, even damaged neurons have never been reported to express mhc class ii antigen, making a direct effect of cd4 t cells on infected neurons unlikely. however, cd4 t cells are required for optimal function of cd8 t cells in the mhvinfected cns (stohlman et al., 1998) . sindbis virus (sinv), a prototypic alphavirus, also primarily affects neurons. while virus clearance is largely mediated by antiviral antibodies, t cells, by secreting ifn-g, are critical for clearance from spinal cord neurons (binder and griffin, 2001) . similar mechanisms may be involved in virus clearance from hcov-oc43 neurons, with subsequent immunopathology. future studies, directed at determining whether antiviral cd4 and cd8 t cell responses contribute to both virus clearance and to severe disease in hcov-oc43-infected mice, may also be relevant to understanding disease outcome in patients with sars, since neurons are infected in some patients (gu et al., 2005) . hcov-oc43 nv and hcov-oc43 tc (vr-759 and vr-1558, respectively), as well as the cell lines 17cl-1, l929, hela and ak-d, were obtained from the atcc (manassas, va). frt, cho-k1 and llck-pk cells were a generous gift from dr. michael welsh, university of iowa. ht-1080 and kb cells were a generous gift from dr. paul mccray, university of iowa. hcov-oc43 vr-759 was propagated in suckling mouse brain. hcov-oc43 vr-1558 was propagated in hrt-18 cells. pathogen-free male c57bl/6 mice were purchased from the national cancer institute (bethesda, md). rag1 à/à and suckling swiss mice were obtained from breeding colonies maintained by our laboratory. for intranasal infection, 5-to 8week-old mice were lightly anesthetized with halothane and droplets containing 30 ld 50 (10 7 smld 50 ) of hcov-oc43 nv or 10 6 tcid 50 of hcov-oc43 tc were administered to the nares. all procedures used in this study were approved by the university of iowa institutional animal care and use committee. monoclonal antibodies recognizing murine glial fibrillary acidic protein (gfap) or murine f4/80 were purchased from dako (carpinteria, ca) and caltag (burlingame, ca), respectively. monoclonal antibody (mab) directed against the surface glycoprotein of oc43 was prepared from the hybridoma o.4.3, a generous gift from dr. john o. fleming, university of wisconsin, madison, wi. cells were maintained in the appropriate growth medium at 37 -c in 5% co 2 . for oc43 infection, cells were washed twice with pbs, and virus was adsorbed for 2 h at 33 -c in dulbucco's modified eagle's medium (serum-free dmem) + 15 mm hepes, ph 7.5. cells were infected with 30 tcid 50 of hcov-oc43 tc (based on infection of hrt-18 cells) or 10 5 smld 50 (approximately 10 tcid 50 based on infection of hrt-18 cells). virus suspensions were aspirated and medium containing 2% fetal bovine serum was added to cells. cells were then cultured for an additional 2-5 days at 33 -c. whole brains were aseptically removed from mice at various times postinfection, homogenized in sterile pbs and clarified by centrifugation. 20 al of serial log 10 dilutions of brain homogenates was inoculated intracranially into 2-or 3day-old swiss pups. survival was monitored and viral titers were calculated using the formula of karber: negative log of the lowest dilution à [(sum of percentage positive / 100) à 0.5] â log interval. virus titers are expressed as the reciprocal of the highest dilution where the virus suspension killed 50% of inoculated suckling mice (smld 50 ). for immunohistochemistry, brain and spinal cord sections were processed as previously described (pewe et al., 2002) . primary antibody was mouse anti-s mab (o.4.3) and secondary antibody was biotinylated goat anti-mouse. sections were developed by sequential incubation with streptavidin -horseradish peroxidase (hrp) conjugate and dab (sigma, st louis, mo) or streptavidin -cy3 reagent (jackson immunoresearch, west grove, pa). for immunocytohemistry of oc43-infected adherent cells, samples were fixed for 30 min in 10% formalin, rinsed with 100 mm glycine and permeabilized in 0.5% triton x-100. wells were blocked with 10% ngs and incubated with o.4.3 overnight at 4 -c. wells were developed as described above. in situ hybridization 8 am brain and spinal cord sections were prepared as described above and in situ hybridization for viral rna was performed using a protocol adapted from gene detect (auckland, new zealand). briefly, formalin fixed, paraffin embedded tissue sections were permeabilized for 30 min at 37 -c in 250 ag/ml pepsin in 200 mm hcl and then blocked with prehybridization buffer for 2 h at 37 -c. 3v -biotinylated, hcov-oc43-specific oligonucleotide probes (antisense 5v-3v , gtattgacatcagcctggttgctag; sense 5v-3v , ctag-caaccaggctgatgtcaatac) were added to a final concentration of 200 ng/ml and sections were incubated for 18 h at 37 -c. after several washes of increasing stringency, streptavidin -hrp conjugate was added for 1 h at 25 -c followed by tyramide signal amplification (tsa) cyanine 3 reagent (perkin elmer, boston, ma). for some experiments, slides were also stained with anti-gfap followed by fitc-conjugated goat anti-rabbit secondary antibody prior to developing with tsa-cy3 reagent. sections were examined with a bio-rad (hercules, ca) lasersharp2000 confocal microscope. total rna was isolated using tri reagent (molecular research center, cincinnati, oh) following the manufacturer's instructions. 2 ag of total rna was reverse transcribed to cdna using retroscript rt-pcr kit (ambion, austin, tx) according to the manufacturer's instructions. the resulting cdna was subjected to pcr as follows. 2 al of cdna was added to a 23 al pcr cocktail containing 2â sybr green master mix (applied biosystems, foster city, ca) and 0.2 am of each sense and antisense primers (intergrated dna technologies, coralville, ia). amplification was then performed in an applied biosystems prism 7700 thermocycler. specificity of the amplification was confirmed using melting curve analysis. data were collected and recorded by the prism 7700 software and expressed as a function of threshold cycle (c t ). specific primer sets used for hcov-oc43 and murine housekeeping gene are as follows (5v to 3v ); hcov-oc43 nucleocapsid forward, ggtctcaacccccagctagt; oc43 nucleocapsid reverse, tgatgctctttaggctttcca; hprt forward, cctcatggactgattatggac; hprt reverse, cagattcaacttgcgctcatc. hcov-oc43 nucleocapsid rna abundance was calculated using methods described previously (pewe et al., 2005) . total rna from infected murine brain or a549 cells was reverse transcribed to cdna. primers were designed (based on genbank accession number ay585228) to generate overlapping amplimers, ensuring complete coverage of the s genes of hcov-oc43 nv and hcov-oc43 tc , as well as 3v terminal end of the hcov-oc43 nv genome. pcr products were sequenced directly by the university of iowa dna core. single cell suspensions of mononuclear cells from whole brain homogenates were prepared as previously described (pewe et al., 2005) . fc receptors were blocked with normal rat serum and anti-cd16/cd32 (clone 2.4g2, bd biosciences, san jose, ca). antibodies used to phenotype cells were fluorescein isothiocyanate-labeled anti-mouse cd4 and phycoerythrin-labeled anti-mouse cd8 (clones gk1.5 and 53 -6.7, bd biosciences, mountain view, ca). cd4 t cells recognizing an hcov-oc43-specific cd4 t cell epitope m 133 (spanning residues 133-147 of the m protein) were identified using intracellular cytokine staining as previously described (wu et al., 2000) . samples were analyzed on a facscan flow cytometer (bd biosciences, mountain view, ca). statistical analysis was done with unpaired (two-tailed) t tests. values in figures are expressed as mean t sem. values of p < 0.05 were considered significant and are indicated by an asterisk (*) in figures. propagation of the kakegawa strain of bovine coronavirus in suckling mice, rats and hamsters susceptibility of laboratory mice to intranasal and contact infection with coronaviruses of other species inverted immunodominance and impaired cytolytic function of cd8+t cells during viral persistence in the central nervous system interferon-gamma-mediated site-specific clearance of alphavirus from cns neurons binding of sindbis virus to cell surface heparan sulfate molecular evolution of the sars coronavirus during the course of the sars epidemic in china hla class i antigen serves as a receptor for human coronavirus oc43 identification of a novel coronavirus in patients with severe acute respiratory syndrome aetiology: koch's postulates fulfilled for sars virus coronavirus spike proteins in viral entry and pathogenesis multiple organ infection and the pathogenesis of sars isolation and characterization of viruses related to the sars coronavirus from animals in southern china high-magnitude, virus-specific cd4 t-cell response in the 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coronavirus receptor and viral determinants of sars-coronavirus adaptation to human ace2 dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus growth in suckling-mouse brain of ''ibv-like'' viruses from patients with upper respiratory tract disease mhc class i-restricted killing of neurons by virusspecific cd8+t lymphocytes is effected through the fas/fasl, but not the perforin pathway molecular characterization of the s protein gene of human coronavirus oc43 induction of mhc class i genes in neurons major histocompatibility complex (mhc) class i gene expression in single neurons of the central nervous system: differential regulation by interferon (ifn)-gamma and tumor necrosis factor (tnf)-alpha severe acute respiratory syndrome spread of a neurotropic murine coronavirus into the cns via the trigeminal and olfactory nerves effect of olfactory bulb ablation on spread of a neurotropic coronavirus into the mouse brain cd4 t-cell-mediated demyelination is increased in the absence of gamma interferon in mice infected with mouse hepatitis virus a severe acute respiratory syndromeassociated coronavirus-specific protein enhances virulence of an attenuated murine coronavirus pathogenesis of chimeric mhv4/mhv-a59 recombinant viruses: the murine coronavirus spike protein is a major determinant of neurovirulence heparan sulfate mediates infection of highneurovirulence theiler's viruses identification of spike protein residues of murine coronavirus responsible for receptor-binding activity by use of soluble receptor-resistant mutants bovine coronavirus uses n-acetyl-9-oacetylneuraminic acid as a receptor determinant to initiate the infection of cultured cells cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human proteolytic cleavage of the murine coronavirus surface glycoprotein is not required for fusion activity human respiratory coronavirus oc43: genetic stability and neuroinvasion ctl effector function within the central nervous system requires cd4+t cells fusion formation by the uncleaved spike protein of murine coronavirus jhmv variant cl-2 characterization of a variant virus selected in rat brains after infection by coronavirus mhv jhm diversity of coronavirus spikes: relationship to pathogen entry and dissemination amino acid substitutions within the heptad repeat domain 1 of murine coronavirus spike protein restrict viral antigen spread in the central nervous system an outbreak of coronavirus oc43 respiratory infection in normandy, france identification of a new human coronavirus circulation of genetically distinct contemporary human coronavirus oc43 strains complete genomic sequence of human coronavirus oc43: molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza c viruses cells of human aminopeptidase n (cd13) transgenic mice are infected by human coronavirus-229e in vitro, but not in vivo characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia cd4 and cd8 t cells have redundant but not identical roles in virus-induced demyelination the sars-cov s glycoprotein: expression and functional characterization comparison of the nucleotide and deduced amino acid sequences of the s genes specified by virulent and avirulent strains of bovine coronaviruses supplementary data associated with this article can be found in the online version at doi:10.1016/j.virol.2005.11.044. key: cord-300884-rqfxe0x1 authors: zhang, jianqiang; guy, james s.; snijder, eric j.; denniston, doug a.; timoney, peter j.; balasuriya, udeni b.r. title: genomic characterization of equine coronavirus date: 2007-12-05 journal: virology doi: 10.1016/j.virol.2007.06.035 sha: doc_id: 300884 cord_uid: rqfxe0x1 the complete genome sequence of the first equine coronavirus (ecov) isolate, nc99 strain was accomplished by directly sequencing 11 overlapping fragments which were rt–pcr amplified from viral rna. the ecov genome is 30,992 nucleotides in length, excluding the polya tail. analysis of the sequence identified 11 open reading frames which encode two replicase polyproteins, five structural proteins (hemagglutinin esterase, spike, envelope, membrane, and nucleocapsid) and four accessory proteins (ns2, p4.7, p12.7, and i). the two replicase polyproteins are predicted to be proteolytically processed by three virus-encoded proteases into 16 non-structural proteins (nsp1–16). the ecov nsp3 protein had considerable amino acid deletions and insertions compared to the nsp3 proteins of bovine coronavirus, human coronavirus oc43, and porcine hemagglutinating encephalomyelitis virus, three group 2 coronaviruses phylogenetically most closely related to ecov. the structure of subgenomic mrnas was analyzed by northern blot analysis and sequencing of the leader–body junction in each sg mrna. coronaviruses are mainly associated with respiratory and gastrointestinal disease in humans (drosten et al., 2003; holmes, 2001; ksiazek et al., 2003; peiris et al., 2003; van der hoek et al., 2004; woo et al., 2005) and respiratory, enteric, neurological, or hepatic disease in animals (holmes, 2001) . coronaviruses have also been isolated from bats, poultry and other birds (cavanagh, 2005; chu et al., 2006; poon et al., 2005; ren et al., 2006) . on the basis of antigenic and genetic analyses, coronaviruses are divided into three groups (gonzalez et al., 2003; gorbalenya et al., 2004; snijder et al., 2003) . group 1 viruses include human coronaviruses 229e (hcov-229e) and nl63 (hcov-nl63), canine coronavirus (ccov), feline coronavirus (fcov), porcine transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv), and bat coronavirus. group 2 viruses are subdivided into group 2a which includes murine hepatitis virus (mhv), human coronaviruses oc43 (hcov-oc43) and hku1 (hcov-hku1), bovine coronavirus (bcov), porcine hemagglutinating encephalomyelitis virus (phev), and rat coronavirus (rcov), and group 2b which includes sars-coronavirus (sars-cov). group 3 viruses include avian viruses, such as avian infectious bronchitis virus (ibv), and turkey coronavirus (tcov). members of the family coronaviridae are enveloped, positive-stranded rna viruses with exceptionally large, polycistronic genomes (27-32 kb). the 5′-proximal two-thirds of the genome comprises two open reading frames (orfs), orf1a and orf1b, which encode the replicase polyproteins (pp) 1a and pp1ab (ziebuhr, 2005) . expression of the pp1ab requires a − 1 ribosomal frameshift during translation of the genomic rna (brierley et al., 1987) . the two replicase polyproteins are processed extensively by two or three viral proteases encoded by orf1a to generate up to 16 end-products termed nonstructural proteins (nsp) 1 to 16 and multiple processing intermediates (ziebuhr, 2005; ziebuhr et al., 2000) . the n-proximal region of the polyproteins is processed by one or two papain-like proteases (pl pro ), whereas the central and c-proximal region is processed available online at www.sciencedirect.com virology 369 (2007) 92 -104 www.elsevier.com/locate/yviro by the viral main protease, 3c-like protease (3cl pro ) (ziebuhr, 2005; ziebuhr et al., 2000) . the 3′-proximal one-third of the genome encodes structural proteins and various accessory proteins. genes encoding the four structural proteins present in all coronaviruses occur in the 5′ to 3′ order as spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins (brian and baric, 2005; lai et al., 2006) . some coronaviruses contain an additional structural protein, the hemagglutinin-esterase (he) protein which is located upstream of the s protein gene (lai et al., 2006) . in contrast to the replicase proteins which are directly translated from the genomic rna, coronavirus structural and accessory proteins are expressed from a nested set of 3′ coterminal subgenomic (sg) mrnas that also possess a common 5′ leader sequence derived from the 5′ end of the genome (pasternak et al., 2006; sawicki et al., 2007) . the common 5′ leader is fused to the 3′ body segments through a mechanism that is presumed to involve discontinuous minus strand rna synthesis to produce subgenome-length templates for subgenomic mrna synthesis, with the transcription regulatory sequence (trs) elements determining the fusion sites of leader and body segments (see recent review of pasternak et al., 2006; sawicki et al., 2007 for details) . equine coronavirus (ecov) was first isolated from feces of a diarrheic foal in 1999 (ecov-nc99) in north carolina, usa (guy et al., 2000) . little is known about ecov and its clinical significance. molecular characterization of ecov and development of diagnostic and prophylactic reagents necessitate sequencing of ecov. in this study, we determined the fulllength nucleotide sequence of the ecov-nc99 strain of equine coronavirus. the viral genome and proteome were analyzed and the predicted features of ecov nonstructural, structural, and accessory proteins were compared to those of other coronaviruses. synthesis of sg mrnas in ecov-infected cells was analyzed by northern blotting. the leader-body junction sequence in each sg mrna was determined and the exact position of trs used for synthesis of each sg mrna was mapped on the genome. the evolutionary relationship between ecov and other phylogenetically closely related group 2a coronaviruses was explored. we report here the full-length genomic sequence of the first ecov isolate, the nc99 strain, and this is also the first reported complete genome sequence of ecov. the nucleotide sequence was determined by directly sequencing 11 overlapping cdna fragments which were rt-pcr amplified from viral rna. the ecov-nc99 genome comprises 30,992 nucleotides (nt), excluding the 3′ poly (a) tail, and has a gc content of 37.2%. the nucleotide sequence data have been deposited in genbank under accession number ef446615. both 5′ and 3′ ends of the ecov genome contain short untranslated regions (utr). the 5′ utr comprises 209 nt (1-209) and includes a potential short internal orf of 8 codons (nt 99-125). four stem-loop structures (i, ii, iii, and iv) were identified in the 5′ utr and a short stretch of nucleotides that are part of the orf1a (see supplementary fig. s1 ). the bulged stem-loop iii (96-115) and iv (189-208) closely resemble the stem-loop iii and iv that have been identified as replication signaling elements in bovine coronavirus and other group 2 coronaviruses (raman and brian, 2005; raman et al., 2003; wu et al., 2003) . the 3′ utr of the ecov genome comprises 289 nt (30, 992) and contains a putative bulged stem-loop structure (nt 30,703-30,770 ) and a putative pseudoknot structure (30,766-30,819) (see supplementary fig. s2 ). similar putative bulged stem-loop structure and pseudoknot structure have been identified in murine hepatitis virus and other group 2 coronaviruses; these have been shown to be essential for viral replication (goebel et al., 2004a,b; hsue and masters, 1997; hsue et al., 2000; williams et al., 1999) . analysis of the ecov-nc99 genome reveals 11 potential orfs (1a, 1b, 2-8, 9a and 9b) as shown in fig. 1 and table 1 . the orfs 1a and 1b encode the replicase polyproteins pp1a and pp1ab. the orfs 2-8, 9a and 9b encode structural and accessory proteins ns2, he, s, p4.7, p12.7, e, m, n, and i, respectively. the replicase orf1a (nt 210-13,499) and replicase orf1b (13,478-21,595) occupy 21.4 kb (69%) of the ecov-nc99 genome. the translation of orf1a generates a precursor pp1a of 4,429 amino acids. similar to other coronaviruses, translation of orf1b involves a − 1 ribosomal frameshift, generating a 7128amino acid pp1ab. the ribosomal frameshift is assumed to be directed by two signals in the orf1a/1b overlapping region: a slippery sequence 5′uuuaaac3′ (nt 13,472-13,478) and a predicted downstream rna pseudoknot structure (nt 13,484-13,559) (see supplementary fig. s3 ). the pp1a and pp1ab proteins are predicted to be proteolytically processed by viralencoded proteases into 16 non-structural proteins (nsp1-16, table 2 ) required for viral replication and transcription. by comparison to other coronaviruses, a number of putative functional domains are predicted in the ecov pp1a and pp1ab and these are summarized in fig. 1 and table 2 (gorbalenya et al., 1991 snijder et al., 2003; ziebuhr, 2005; ziebuhr et al., 2001) . enzymatic activities of nsp3, nsp5, nsp12, nsp13, nsp14 and nsp15 have been experimentally confirmed for some coronaviruses (barretto et al., 2005; cheng et al., 2005; guarino et al., 2005; heusipp et al., 1997; ivanov et al., 2004a,b; ivanov and ziebuhr, 2004; lindner et al., 2005; minskaia et al., 2006; putics et al., 2005 putics et al., , 2006 seybert et al., 2000 seybert et al., , 2005 tanner et al., 2003; ziebuhr, 2005; ziebuhr et al., 2001) . the 3cl pro (catalytic residues his-3333 and cys-3437) is predicted to cleave the c-terminal half of the ecov pp1a and the orf1b-encoded part of pp1ab. the putative pl1 pro (catalytic residues cys-1078 and his-1229) and pl2 pro (catalytic residues cys-1675 and his-1832) are predicted to process the n-proximal regions of the ecov pp1a ( fig. 1 and table 2 ). the most striking differences between the ecov replicase and other group 2 coronaviruses replicases were identified in nsp3. the ecov nsp3 protein has 3 aa deletions and 55 aa insertions compared to the nsp3 proteins of bcov, hcov-oc43, and phev, three viruses phylogenetically most closely related to ecov. these insertions and deletions are clustered at two regions: the ac domain and the region between the pl2 pro and the y domain. the functional significance of these insertions and deletions is unknown as yet; however, the functions of pl1 pro , pl2 pro , and adrp are not anticipated to be affected since insertions and deletions are not located in the functional domains of these enzymes (fig. 1) . orf2 (nt 21,610-22,446) of ecov-nc99 encodes the predicted ns2 protein with 278 amino acids. the ns2 of fig. 1 . schematic diagrams of ecov genome organization. the ecov entire genome organization is depicted (middle). the 5′ leader, orfs 1a and 1b encoding replicase polyproteins are shown, with the ribosomal frameshift site indicated. structural and accessory proteins are also indicated: ns2 protein (encoded by orf2), hemagglutinin esterase (he, orf3), spike protein (s, orf4), p4.7 protein (orf5), p12.7 protein (orf6), envelope protein (e, orf7), membrane protein (m, orf8), nucleocapsid protein (n, orf9a), and i protein (orf9b). predicted cleavage products (nsp1-nsp16) of the replicase polyproteins are depicted (bottom). arrows represent sites in the corresponding replicase polyproteins that are cleaved by papain-like proteases (white arrows) or the 3c-like cysteine protease (black arrows). a number of putative functional domains predicted in the ecov pp1a and pp1ab are indicated. pl1, papain-like proteinase 1 (aa 1059-1275); pl2, papain-like proteinase 2 (aa 1570-1867); x, x-domain which contains adenosine diphosphate-ribose 1ʺ-phosphatase (adrp) (aa 1276-1435); tm, transmembrane domain; 3cl, 3c-like proteinase; rdrp, rna-dependent rna polymerase; z, zinc-binding domain; hel, helicase domain; exon, exonuclease; n, nidoviral uridylatespecific endoribonuclease (nendou); mt, 2′-o-ribose methyltransferase (2′-o-mt). domains ac (aa 846-1058) and y (aa 2310-2796) are described by ziebuhr et al. (2001) . the spike protein (1363 amino acids) of ecov is represented by a black line (top). the n-terminal signal peptide (amino acid residues 1-14 or 17), the heptad repeat 1 (hr1, amino acid residues 991-1902), the heptad repeat 2 (hr2, amino acid residues 1259-1304), the transmembrane domain (amino acid residues 1308-1330), and the cytoplasmic domain (amino acid residues 1331-1363) are depicted. a potential cleavage recognition sequence (rrqrr) at residues 764-768 and the predicted cleavage site between residues 768 and 769 are indicated. the generated cleavage products s1 and s2 subunits are depicted. the positions of the receptor-binding domain on the s1 subunit and the fusion peptide on the s2 subunit are currently unknown. ecov has 67%, 67%, and 45% amino acid identity with the respective ns2 proteins of bcov, hcov-oc43, and phev. the lower amino acid identity with phev may be attributable to the fact that phev has a truncated ns2 protein (vijgen et al., 2006) . sequence analysis revealed that the ecov ns2 protein contains a domain (aa 46-135) with similarity to the putative cyclic phosphodiesterase (cpd, martzen et al., 1999) . the cpd domain has also been identified in the ns2 proteins of other group 2a coronaviruses as well as in the 3′end of the pp1a protein of toroviruses snijder et al., 1991 snijder et al., , 2003 . the ns2 of ecov was predicted to contain 9 potential phosphorylation sites. the ns2 of ecov does not contain a signal peptide and is a non-secretory protein. the function of the ns2 protein in coronaviruses has not been studied in detail. it is known that the ns2 gene is non-essential for mhv replication in transformed cells (schwarz et al., 1990) . however, a recent study showed that a point mutation in the ns2 of mhv led to its attenuation in mice in spite of its wild-type replication in tissue culture (sperry et al., 2005) . orf3 (nt 22,458-23,729) of ecov-nc99 encodes the predicted he protein containing 423 amino acids. nine potential n-glycosylation sites were predicted. signalp analysis revealed a signal peptide probability of 0.802 with a potential cleavage site between residues 17 and 18. it was predicted that the n-terminal 390 amino acids are located outside the cell surface or viral envelope with a transmembrane helix at amino acids 391-413 and an internal domain at amino acids 414-423. the putative active site for esterase activity, fgds (kienzle et al., 1990) , is present at amino acids 36-39 of the he protein in ecov. orf4 (nt 23,744-27,835) of ecov-nc99 encodes the predicted spike (s) protein containing 1363 amino acids. eighteen potential n-glycosylation sites were predicted. an n-terminal signal peptide was identified with a potential cleavage site between amino acids 14 and 15 predicted by signalp-nn or between amino acids 17 and 18 predicted by signalp-hmm. the ecov s protein was predicted to be a typical type i membrane protein with the n-terminal 1307 residues exposed on the outside of the cell surface or virus particle, a transmembrane domain near the c terminus (residues 1308-1330), followed by a cytoplasmic tail (residues 1331-1363). following multiple alignments with the s proteins of other group 2a coronaviruses, a potential cleavage recognition sequence (rrqrr) was identified at residues 764-768 which would predict a cleavage between amino acids 768 and 769, separating the ecov s protein into s1 and s2 subunits (fig. 1) . the ecov s1 subunit is expected to contain a receptor-binding domain whose position has not yet been determined. the s2 subunit is predicted to mediate membrane fusion. two heptad repeat (hr) regions, which are conserved in position and sequence among the three groups of coronaviruses and play important roles in membrane fusion (see reviews of eckert and kim, 2001; hernandez et al., 1996) , were identified in the ecov s2 subunit (hr1: aa 991-1092; hr2: aa 1259-1304) (fig. 1) . the ecov s2 subunit is anticipated to possess a fusion peptide whose position is yet unknown. some coronavirus s proteins have been shown to contain important neutralization epitopes (godet et al., 1994; kubo et al., 1994; yoo et al., 1991) and mutations in the s protein have been associated with altered viral antigenicity and pathogenicity (ballesteros et al., 1997; bernard domains ac and y are described by ziebuhr et al. (2001) . a nucleotide position means the location of the nucleotides encoding corresponding proteins in the entire genome of equine coronavirus-nc99 strain. b pl1 pro , papain-like proteinase 1; pl2 pro , papain-like proteinase 2; adrp, adenosine diphosphate-ribose 1ʺ-phosphatase (formerly known as 'x-domain'); 3cl pro , 3c-like proteinase; tm, transmembrane domain; gfl, growth factor-like domain; rdrp, rna-dependent rna polymerase; zbd, zinc-binding domain; hel, helicase domain; nendou, nidoviral uridylate-specific endoribonuclease; 2′-o-mt, 2′-o-ribose methyltransferase. and laude, 1995; dalziel et al., 1986; gallagher and buchmeier, 2001; leparc-goffart et al., 1997) . whether the s protein of ecov has such properties remains to be determined. orf5 (nt 27,825-27,947) of ecov-nc99 is predicted to encode a hypothetical protein of 40 amino acids with an estimated molecular weight of 4.7 kda (termed p4.7 protein). it was predicted to be a non-secretory protein and did not contain any transmembrane helix. this protein is not closely matched to any known protein based on a search using blastp, psi-blast, or fasta programs. orf6 (nt 28,076-28,405) of ecov-nc99 is predicted to encode a protein of 109 amino acids corresponding to the bcov 12.7 kda non-structural protein (p12.7). this orf overlaps by 15 nucleotides with the orf7 that encodes the e protein. no signal peptide or any transmembrane helix was present. no n-glycosylation site was found. orf7 (nt 28,392-28,646) of ecov-nc99 encodes the predicted e protein containing 84 amino acids. no n-glycosylation site was identified. it was predicted to contain a signal anchor (probability 0.999). one transmembrane domain was predicted at residues 18-36 by tmpred analysis or at residues 15-37 by tmhmm analysis. both programs predicted the n-terminus of the protein to be external to the cell surface or viral envelope. in the case of other coronaviruses, there is increasing evidence that the e protein together with the m protein is instrumental in viral assembly and budding; the cytoplasmic tails of both proteins have an important interactive role in this process (corse and machamer, 2000 , 2003 vennema et al., 1996) . orf8 (nt 28,661-29,353) of ecov-nc99 encodes the predicted m protein containing 230 amino acids. it was predicted to contain a signal anchor (probability 0.947). three transmembrane domains were predicted to be present at positions 25-46, 57-78, and 81-102 by tmpred analysis or at positions 25-44, 49-71, and 81-103 by tmhmm analysis. the n-terminal 24 amino acid residues were predicted to be outside and the c-terminal 127 or 128-amino acid hydrophilic domain was predicted to be inside the virus. one potential nglycosylation site was predicted at position 26 (nfs). the presence of potential o-glycosylation sites was predicted at the extreme n-terminus of the m protein (msstptpapgyt). whether these sites are glycosylated or not needs to be experimentally verified. previous studies have shown that the m protein of group 1 and 3 coronaviruses (e.g. tgev and ibv) are n-glycosylated, whereas the m protein of group 2 coronavirus mhv is only o-glycosylated (de haan et al., 2002; lai et al., 2006) . the m protein is the most abundant envelope component and plays a key role in coronavirus assembly by interacting with the e, s, n and he proteins (bosch et al., 2005; de haan and rottier, 2005 , and references therein). orf9a (nt 29,363-30,703) of ecov-nc99 encodes the predicted n protein containing 446 amino acids. it was predicted to contain 36 potential phosphorylation sites. no signal peptide or any transmembrane helix was present. the n protein of coronaviruses has been shown to be multifunctional, e.g. interaction with the viral rna genome to form a viral nucleocapsid, interaction with the m protein, and the ability for selfassociation (masters, 1992; narayanan et al., 2000 narayanan et al., , 2003 . recently it has also been reported that the n protein may play a role in coronavirus replication (almazan et al., 2004; schelle et al., 2005) . orf9b (nt 29,424-30,044) of ecov-nc99 encodes a hypothetical protein (i) containing 206 amino acids within orf9a which encodes the n protein. it was predicted to contain 10 potential phosphorylation sites. no signal peptide or any transmembrane helix was present. in the case of mhv, expression of the protein i has been detected in virus-infected cells but this protein is nonessential for viral replication and viral production (fischer et al., 1997) . it is generally accepted that the replicase proteins are directly synthesized from the coronavirus genome, whereas the structural and accessory proteins are expressed from a nested set of subgenomic mrnas. however, the number of sg mrnas and the characteristics and expression pattern of the proteins they encode (e.g. a sg mrna may sometimes express multiple proteins) varies for each virus. in order to investigate ecov sg mrna synthesis, northern blot analysis was performed to evaluate the synthesis of genomic and subgenomic rnas in ecov-infected cells. a digoxigenin-labeled rna probe complementary to the 3′ end (nt 30,660-30,946) of the ecov genome was used for a northern blot hybridization analysis. as shown in fig. 2 , nine mrnas were detected in ecov-infected hrt-18g cells at 72 h p.i. absence of such mrnas in mock-infected cells confirms that these mrnas are ecov-specific. according to the estimated sizes of the mrnas, it is reasonable to assume that sg mrnas 2-8 express the ns2, he, s, p4.7, p12.7, e, and m proteins, respectively and that mrna 9 expresses the n protein and probably the i protein as well. there is a general agreement that the trs elements determine the fusion sites of the 5′ leader and the 3′ body segments in coronavirus sg mrnas. in order to determine the precise location of the leader and body trss used for ecov sg mrna synthesis, the leader-body junction and flanking sequences of each ecov sg mrna were determined using sg mrna-specific rt-pcrs (see table 3 and materials and methods for details). the sg mrna sequences were aligned to the leader and corresponding 'body' genomes as shown in fig. 3 . analysis of the leader-body junction sequences revealed that the core sequence of the trs motifs is 5′ucuaaac3′. the leader trs (5′ucuaaac3′) and the body trs (5′ ucuaaac3′) used for synthesizing he mrna, s mrna, and n mrna exactly match each other. there is one mismatch between the leader trs and the body trs (5′ucuaaaa3′) used for generating the mrna of the ns2 protein. there is also one mismatch between the leader trs and the body trs (5′uccaaac3′) used for generating e mrna and m mrna. there are two mismatches between the leader trs and the body trs (5′uuaaaac3′) used for generating the mrna of the p4.7 protein. interestingly, in the case of the mrna of the p12.7 protein, the leader and the body segment is joined at the unusual consensus variant 5′uaaa-cuuuauaa3′. previously it has been shown that the mrna of the p12.7 protein of bcov also utilizes an unusual consensus variant for joining the leader and body segment . from the sequence data, we conclude that the ecov common leader on sg mrnas is the first 64 nucleotides of the ecov genome. phylogenetic analyses of ecovand other coronaviruses were performed based on the amino acid sequences of replicase polyprotein pp1a, the orf1b-encoded part of the pp1ab, s, e, m, and n. phylogenetic analysis clustered coronaviruses into three major groups (g1, g2a, and g3) irrespective of the gene used for analysis (fig. 4) . the sars-cov forms a separate branch and is classified as subgroup 2b (g2b) as suggested previously (gorbalenya et al., 2004; snijder et al., 2003) . phylogenetic analysis clearly demonstrated that ecov falls into the cluster of group 2a coronaviruses and is most closely related to bcov, hcov-oc43, and phev. to further explore the possible evolutionary relationships among ecov, bcov, hcov-oc43, and phev, the genetic distances of ecov, bcov, and phev to hcov-oc43 were determined over the entire genome using the simplot analysis (lole et al., 1999) . as shown in fig. 5 , the bcov strains and hcov-oc43 had lowest genetic distances over the complete genome; the genetic distance between phev and hcov-oc43 was similar to the distance between bcov and hcov-oc43 in most regions of the genome with exception of the spike gene where the genetic distance of phev to hcov-oc43 was significantly greater than the distance of bcov to hcov-oc43; the genetic distance of ecov to hcov-oc43 was significantly greater than the distance of either bcov or phev to hcov-oc43 in the regions of the first half of orf1a, the central part of orf1b, ns2 and he genes; the genetic distance with respect to the spike gene between ecov and hcov-oc43 was similar to the distance between phev and hcov-oc43 but greatly higher than the distance between bcov and hcov-oc43. the genetic distances of bcov and phev to hcov-oc43 observed in this study are consistent with previously reported findings (vijgen et al., 2005 (vijgen et al., , 2006 . vijgen et al. (2006 vijgen et al. ( , 2005 concluded that phev diverged from the common ancestor before bcov and hcov-oc43. our analysis suggested that ecov had diverged earlier than phev from a common ancestor. in summary, ecov had emerged earlier than phev, bcov, and hcov-oc43, notwithstanding the fact that ecov was not isolated until 1999 from a diarrheic foal in usa. in this study, we have determined the first complete genome sequence of ecovand provided the first comprehensive analysis of the ecov genome. completion of the genome sequence of ecov will contribute to our understanding of this virus at the molecular level and also enrich the database of coronaviruses. the sequence data are expected to aid in the development of diagnostic and prophylactic reagents. the sequence data of ecov-nc99 will also help identify and characterize other ecov isolates and enhance our understanding of the molecular epidemiology of coronavirus. neonatal enterocolitis is an economically significant disease for horse breeders. further studies are needed to determine the prevalence of ecov infection in equine populations and the relative role of ecov as a cause of enteric disease in horses. the human rectal tumor cell line hrt-18g (american type culture collection [atcc, crl-11663] ) was grown in dulbecco's modified eagle's medium (dmem) supplemented with 4 mm l-glutamine, 5% fetal bovine serum, and penicillin/streptomycin at 37°c in the presence of 5% co 2 . the equine coronavirus-nc99 (guy et al., 2000) was propagated once in hrt-18g cells to produce the working virus stocks. the complete genome of ecov was determined by sequencing 11 overlapping rt-pcr products encompassing the entire genome (nt 1-3615; nt 3446-5458; nt 4953-6600; nt 5497-9678; nt 9347-13,021; nt 12,451-15,736; nt 15,425-19,307; nt 19,039-22,812; nt 22,566-26,390; nt 26,065-29,662; and nt 29,363-30,992) . viral rna was isolated from ecov stocks using the qiaamp viral rna mini kit (qiagen). viral rna was first reverse transcribed with accuscript reverse transcriptase (stratagene) following the manufacturer's instructions. then, pcr amplification was performed with proof-reading pfuultra highfidelity dna polymerase (stratagene) in a volume of 50 μl: 5 μl pfuultra pcr buffer (10×), 1.0 μl dntp mix (10 mm each), 1 μl of each primer (20 μm), 2 μl cdna template, 1 μl pfuultra dna polymerase, and 39.0 μl nuclease-free water. the reaction mixtures were incubated at 95°c for 2 min, followed by 35 cycles of amplification at 95°c for 45 s, 50-53°c for 45 s, and 72°c for 4.5 min, with a final incubation at 72°c for 10 min. the pcr products were gel-purified using qiaquick gel extraction kit (qiagen). both sense and anti-sense strands were sequenced using the applied biosystems big dye terminator v3.0 sequencing chemistry on abi 3730 dna sequencers (davis sequencing center). partial genomic sequence (9487 nucleotides) of ecov had fig. 5 . genetic distance between ecov, bcov, phev and hcov-oc43. the average genetic distances were calculated over the entire genome using the simplot program with a sliding window size of 400 bp and a step size of 200 bp. each curve represents a comparison of the sequence data of ecov-nc99, the bcov strains, and phev-vw572 to the reference sequence data of the hcov-oc43 atcc strain vr759 (nc_005147). the sequence data of the bcov strains used for comparison are the 50% consensus sequence of six bcov strains: bcov-ent (nc_003045), bcov-alpaca (dq915164), bcov-db2 (dq811784), bcov-mebus (u00735), bcov-quebec (af220295), and bcov-lun (af391542). the linear representation of the ecov-nc99 genome was shown at the top of the diagram. been previously determined by two groups (guy et al., 2000, genbank accession number af251144; wu et al., 2003, af523846 and af523850. h.y. wu, j.s. guy, and d.a. brian, unpublished data, ay316300) . these regions were re-sequenced in this study. to determine the remaining genomic sequence of ecov-nc99, initial rt-pcr and sequencing primers were designed based on multiple alignments of the genomes of bcov (genbank accession number nc_003045), hcov-oc43 (nc_005147), phev (dq011855), and mhv (nc_001846); additional primers were designed based on the results of the first and subsequent rounds of sequencing. all of the primer sequences are attached in the supplementary table s1 . the nucleotide sequences were assembled and manually edited using codoncode aligner version 1.5.2 to produce the complete sequence of the viral genome. orf analysis was performed using vector nti advance 10 (invitrogen). rna secondary structures of 5′ and 3′ utrs and the ribosomal frameshift signals were predicted using the mfold program with the default parameter settings (mathews et al., 1999; zuker, 2003) . potential 3c-like protease cleavage sites were predicted using the netcorona 1.0 server (kiemer et al., 2004) . prediction of signal peptides and their cleavage sites was conducted using signalp 3.0 server (nielsen et al., 1997) . potential n-glycosylation sites, o-glycosylation sites, and phosphorylation sites were predicted using netnglyc, netoglyc, and netphos, respectively (blom et al., 1999; julenius et al., 2005) . prediction of transmembrane domains was performed using tmpred (hofmann and stoffel, 1993) and tmhmm server 2.0 (sonnhammer et al., 1998) . protein similarity searches were performed using blastp version 2.2.16, psi-blast against the protein data bank (pdb) (altschul et al., 1997; schaffer et al., 2001) and fasta version 34.26 against the uniprot protein database with the default parameter settings (pearson and lipman, 1988) . pairwise amino acid comparison was performed using emboss pairwise alignment algorithms with the default parameter settings (http://www.ebi.ac.uk/emboss/align). multiple sequence alignments were performed using clustalx version 1.83 (thompson et al., 1997) . phylogenetic analysis and unrooted neighbor-joining trees were carried out using paup version 4.0b10 with the default parameter settings. bootstrap analysis was carried out on 1000 replicate data sets. the genetic distance between genomes was determined using the simplot version 3.5.1 (lole et al., 1999) . one anti-sense rna probe base pairing to the 3′ end of the ecov genome (nt 30,660-30,946) was developed to evaluate the synthesis of genomic and subgenomic rnas in ecovinfected cells by northern blotting. the ecov rna was amplified using two primer pairs (forward primer 30660p: 5′ agcagatggatgatcccctc3′; reverse primer 30946n: 5′ actgggtggtaacttaacatgctg3′) and the qiagen one-step rt-pcr kit (qiagen). the gel-purified rt-pcr products were cloned into a linearized plasmid vector with overhanging 3′ t residues (pdrive cloning vector, qiagen). the authenticity and orientation of the insert was determined by sequencing both strands of dna with m13 reverse and forward primers. plasmid dna was linearized with bamhi (roche), phenol/chloroform extracted, ethanol precipitated, and resuspended in nuclease-free water. a digoxigenin (dig)-labeled rna probe was prepared using the dig rna labeling kit (roche) according to the manufacturer's instructions. intracellular rna was extracted at 72 h p.i. from ecovinfected hrt-18g cells using the rnaqueous-4pcr kit (ambion). northern hybridization with the dig-labeled rna probe was carried out following the protocols that had been previously described for equine arteritis virus (balasuriya et al., 2004) . the leader-body junction sites of all ecov sg mrnas were rt-pcr amplified and sequenced. briefly, intracellular rna was extracted from ecov-infected hrt-18g cells using the rnaqueous-4pcr kit (ambion). reverse transcription was carried out with an rt primer located downstream to the body trs region in a sg mrna (table 3) using superscriptiii reverse transcriptase (invitrogen) following the manufacturer's instructions. due to the nested nature of sg mrnas, such an rt primer also binds to the corresponding positions in all larger viral mrnas, including the genomic rna. subsequently, cdna was pcr amplified with a forward primer (1p) located in the leader sequence and a reverse primer located just upstream of the rt primer in the body of the mrna (table 3) . amplification was performed in a volume of 50 μl: 5 μl pfuturbo pcr buffer (10×), 0.4 μl dntp mix (25 mm each), 1 μl of each primer (20 μm), 2 μl cdna template, 1 μl pfuturbo® dna polymerase, and 39.6 μl nuclease-free water. the reaction mixtures were incubated at 95°c for 2 min, followed by 35 cycles at 95°c for 45 s, 50-56°c for 45 s, and 72°c for 3 min, with a final incubation at 72°c for 10 min. rt-pcr products corresponding to each mrna species could be distinguished by size differences on agarose gel. pcr products were gel-purified and sequenced to obtain the leader-body junction sequences for each sg mrna. the nucleotide sequence of ecov was deposited in genbank under the accession number ef446615. the nucleoprotein is required for efficient coronavirus genome replication gapped 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two paralogous papain-like proteases that cleave the same peptide bond mfold web server for nucleic acid folding and hybridization prediction this work was partly supported by funds from fort dodge animal health and kentucky agricultural experiment station, college of agriculture, university of kentucky. key: cord-296075-8axbkyyz authors: castro, raymond f.; perlman, stanley title: differential antigen recognition by t cells from the spleen and central nervous system of coronavirus-infected mice date: 1996-08-01 journal: virology doi: 10.1006/viro.1996.0415 sha: doc_id: 296075 cord_uid: 8axbkyyz abstract cd8+cytotoxic t lymphocytes (ctls) isolated from the central nervous system (cns) of c57bl/6 mice acutely infected with mouse hepatitis virus, strain jhm (mhv-jhm), and analyzed in a directex vivocytotoxicity assay recognize two epitopes (h-2dband h-2kb-restricted encompassing amino acids 510–518 and 598–605, respectively) within the surface (s) glycoprotein. in contrast, cd8+t cells isolated from the spleens of mice inoculated intraperitoneally with mhv-jhm and restimulatedin vitroonly respond to the h-2db-restricted epitope. in this report, the preferential recognition of the h-2db-restricted epitope is confirmed using splenocytes stimulatedin vitrowith either mhv-jhm-infected mc57 cells or with a cell line expressing the s protein and analyzed in secondary ctl assays. to determine whether these results represent a difference in epitope recognition between the spleen and cns, secondary ctl assays were performed using spleen cells coated with peptides encompassing the ctl epitopes as stimulators. under these conditions, both epitopes sensitized cells for lysis by spleen-derived ctls, suggesting that both epitopes were recognized by splenic cd8+t cells after infectionin vivo.furthermore, limiting dilution analysis indicated that the precursor frequency of splenic cd8+t cells specific for both the h-2kband h-2db-restricted epitopes were not significantly different. thus, the results suggest thatin vitrostimulation of splenocytes specific for the h-2kb-restricted epitope is inefficient after endogenous processing but that this inefficiency can be corrected if peptide is provided exogenously at sufficiently high concentrations. as a consequence, the results also show that cells responsive to both of the previously identified cns-derived cd8+t cell epitopes are present in the infected spleen at nearly the same frequency. (26) or s-specific immunogenic peptides s510-518 or s598-605 (unpublished observations) in direct ex vivo chronic demyelinating encephalomyelitis in susceptible ctl assays using cells isolated from the spleens of mice mice and rats (3-8). the chronic disease results either with acute encephalitis. bergmann et al., however, were if mice are infected with an attenuated variant of mhv able to isolate s protein-specific cd8 / t cell clones from or if they are protected from the acute encephalitis by a the spleens of b6 mice infected intraperitoneally with a passive infusion of anti-viral antibodies, cd4 / t cells or sublethal dose of virus. these clones only recognized cd8 / t cells (6, (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) . this chronic neurological disease one of the two epitopes which we identified in the direct has been studied as an animal model of human demyex vivo ctl assays. the same results were obtained elinating disease, including multiple sclerosis. when spleen cells were analyzed in ctl assays after both cd4 / and cd8 / t cells are required for virus secondary stimulation in vitro with mhv-infected spleen clearance (23-25). target proteins for cd8 / t cell cytocells (2). toxicity have been identified in mhv-jhm-infected balb/ in many cases, antigen-specific cytotoxicity by cells c and c57bl/6 (b6) mice. in balb/c mice, the majority of primed in vivo can only be demonstrated after secondary the ctl activity is directed against the nucleocapsid (n) in vitro stimulation with antigen. conversely, there are protein, whereas in b6 mice, the s glycoprotein is the no reports identifying epitopes which are observed in predominant target (26, 27). using cells isolated from the primary but not secondary cytotoxic t cell assays. the cns of b6 mice with acute encephalitis and analyzed results described above, therefore, would be most condirectly ex vivo in cytotoxicity assays, we identified two sistent with different epitope recognition between efimmunogenic cd8 / t cell epitopes within the s protein fectors harvested from peripheral lymphoid tissue and (1). in contrast to these results, we could not identify a those harvested from the site of inflammation, the brain. significant amount of ctl activity against the s protein alternatively, insufficient presentation of the h-2k b -restricted epitope in vitro and consequent inefficient expansion of lymphocytes primed in vivo to respond to this to determine if inadequate presentation of the h-2k brestricted epitope could be overcome by using a different source of antigen for stimulation, two alternative approaches were tried. first, irradiated (9000 rad), mhvinfected mc57 cells (h-2 b ) were used as stimulators. mc57 cells in general are not readily infected by mhv-jhm (28), but a line which was partially susceptible to the virus (approximately 10-20% of the cells were positive for viral antigen in an immunofluorescence assay) was developed. when these cells were used to stimulate control (el-4). the average spontaneous release for el-4 targets corresponded to õ13% and for mc57 cells õ25%. percentage of specific for this purpose, splenocytes were harvested 8 days release was calculated as described previously (26). after intraperitoneal inoculation and restimulated in vitro with el-4-s35 cells. when analyzed in ctl assays, these cells recognized the h-2d b , but not the h-2k b -restricted stricted epitopes. irradiated splenocytes were coated with both peptides at a concentration of 5 mm each, epitope (fig. 1b) . the results from these experiments using two different types of cells expressing s protein washed, and added to bulk cultures of spleen cells from mhv-infected mice. as shown in fig. 2 , splenocytes stim-as stimulators are the same as those obtained by bergmann et al. using mhv-jhm-infected syngeneic spleen ulated in vitro with both peptides for 5 days were able to efficiently lyse targets coated with either peptide in cells. they showed that when s protein was processed endogenously, the h-2k b -restricted epitope was not rec-secondary cytotoxicity assays. these results suggested that both epitopes were rec-ognized by spleen-derived lymphocytes in secondary ctl assays. ognized efficiently in the mouse and that stimulation in vitro was inefficient in the case of the h-2k b -restricted the results presented thus far are consistent with inefficient presentation of the h-2k b -restricted epitope either epitope. it was not possible from these secondary ctl assays using bulk cultures to determine if there were in vivo during the primary response or later in vitro. stimulation with exogenous peptide should circumvent diffi-quantitative differences in the number of cd8 / t cells responding in vivo to the two epitopes. such quantitative culties associated with presentation of a suboptimal concentration of the h-2k b -restricted epitope in vitro after differences could contribute to the results shown in figs. 1 and 2. if in vivo priming after intraperitoneal inoculation endogenous processing. to determine if this in fact occurred, splenocytes from intraperitoneally infected mice with mhv-jhm occurred more efficiently with the h-2d brestricted epitope, cells responding to the two epitopes were stimulated in vitro with splenocytes coated with peptides corresponding to the h-2d b -and h-2k b -re-should be present in the spleen at different concentra-of cultures that contained stimulators alone without responders. these assays using peptide-coated splenocytes as stimulators revealed comparable ctl precursor/effector frequencies for the h-2k b -(average 1/1291, range 1/927-1/1542 spleen cells) and h-2d b -restricted (average 1/987, range 1/380-1/1323 spleen cells) epitopes in these mice. as expected, the ctl precursor/effector frequencies obtained from infected b6 mice were significantly higher than those obtained from naive b6 mice (average 1/171, 872 spleen cells and 1/660,803 spleen cells for the h-2k b -and h-2d b -restricted epitopes, respectively). therefore, in vivo priming was equally effecshown in fig. 4 , similar amounts of the two h-2 molecules were present on el-4-s35 cells, suggesting that preferential down-regulation of the h-2k b molecule was not the tions. on the other hand, if inefficient expansion occurring in vitro is the sole explanation for the different explanation for the differences in recognition between the h-2d b -and h-2k b -restricted epitopes. results presented above, precursors to the two epitopes should be present at similar concentrations in the spleen. three independent studies showed that spleen-derived cd8 / cytotoxic t cells from intraperitoneally in-to compare the ctl precursor frequency for the two epitopes, limiting dilution analyses (lda) using spleen-fected b6 mice recognize the h-2d b -but not the h-2k brestricted epitope in secondary ctl assays. first, berg-derived lymphocytes from b6 mice intraperitoneally infected with mhv-jhm 8 days previously and from naive b6 mice were performed (fig. 3) . both virus-specific memory and effector cells should be present in the spleen at this time (29), although only memory cells may be measured in this secondary ctl assay (30). in these assays, threefold serial dilutions of responder spleen cells from individual mice were plated in a volume of 200 ml in the wells of 96-well round bottom tissue culture plates (costar, cambridge, ma) in complete rpmi media supplemented with 5% rat concanavalin a supernatant containing 50 mm methyl-a-d-mannopyranoside. in addition to immune or naive responder splenocytes, individual wells received 5 1 10 5 irradiated (3000 rad) syngeneic spleen cells which had been coated with peptide (10 was greater than 3 standard deviations above the mean suggested that in vivo priming in the spleen was similarly efficient for both epitopes. these data are most consistent with inefficient in vitro stimulation of the h-2k b -restricted cd8 / t cells. antigen is likely to be processed by different antigen presenting cells (apcs) in vivo and in vitro and at different sites in vivo. the microenvironment may allow adequate presentation of the h-2k b -restricted epitope in vivo but this may not occur in vitro. although some h-2k b -restricted epitope is likely expressed on the cell surface of apcs in vitro, this amount may be inadequate to stimulate in vivo primed cells, in fected syngeneic spleen cells, a step which our study 24, 76-85 (1973) . indicates inadequately stimulates the h-2k b -restricted 5. nagashima, k., wege, h., meyermann, r., and ter meulen, v., acta precursors, leading to a skewed result. third, the data h-2d b -restricted epitope in cytotoxicity assays. 9. yamaguchi, k., goto, n., kyuwa, s., hayami, m., and toyoda, y., j. neuroimmunol. 32, 1-9 (1991). in contrast to these data, use of peptide-coated splefrequencies were measured for the two epitopes and proc. natl. acad. sci. usa key: cord-298847-szezd2vb authors: jacomy, hélène; talbot, pierre j title: vacuolating encephalitis in mice infected by human coronavirus oc43 date: 2003-10-10 journal: virology doi: 10.1016/s0042-6822(03)00323-4 sha: doc_id: 298847 cord_uid: szezd2vb involvement of viruses in human neurodegenerative diseases and the underlying pathologic mechanisms remain generally unclear. human respiratory coronaviruses (hcov) can infect neural cells, persist in human brain, and activate myelin-reactive t cells. as a means of understanding the human infection, we characterized in vivo the neurotropic and neuroinvasive properties of hcov-oc43 through the development of an experimental animal model. virus inoculation of 21-day postnatal c57bl/6 and balb/c mice led to a generalized infection of the whole cns, demonstrating hcov-oc43 neuroinvasiveness and neurovirulence. this acute infection targeted neurons, which underwent vacuolation and degeneration while infected regions presented strong microglial reactivity and inflammatory reactions. damage to the cns was not immunologically mediated and microglial reactivity was instead a consequence of direct virus-mediated neuronal injury. although this acute encephalitis appears generally similar to that induced by murine coronaviruses, an important difference rests in the prominent spongiform-like degeneration that could trigger neuropathology in surviving animals. although the etiology of most neuroautoimmune, neuroinflammatory, and/or neurodegenerative diseases remains unclear, virus infections could directly trigger neurodegeneration or initiate a cns-directed inflammatory process leading to central nervous system (cns) damage, or a combination of both. indeed, parkinson's disease, alzheimer's disease, amyotrophic lateral sclerosis (als), and multiple sclerosis (ms) could actually represent infectious diseases (calne et al., 1986; kristensson, 1992; kirk and zhou, 1996; allen et al., 1996; hayase and tobita, 1997; klein et al., 1999; boucher et al., 2001; jubelt and berger, 2001; giraud et al., 2001; sola et al., 2002) . moreover, psychiatric disorders were also investigated as a possible consequence of viral infections (waltrip et al., 1995; lewis, 2001) . the vertebrate cns was long thought to be inaccessible to cells of the immune system or to viruses. however, the presence of virus in the cns is more frequent than expected and viral detection in the cerebrospinal fluid of patients suggests the ability of viruses to cross the bloodbrain barrier (koskiniemi and vaheri, 1989; georgsson, 1994) . in fact, neuroinvasive viruses can damage the cns and produce neurological disease in sensitive hosts, due to the misdirected immune response of the host (virus-induced immunopathology) and/or viral replication in cells of the brain (virus-induced cytopathology). nevertheless, primary infections of the brain are not common and viruses are the leading cause of encephalitis, which results from either direct infection (acute encephalitis) or the immune response to an infection (postinfectious encephalitis or acute demyelinating encephalomyelitis). in acute encephalitis, viral replication occurs in the brain tissue itself, causing destructive lesions of the gray matter: this was reported after herpes simplex, rabies, or some arbovirus infections (rupprecht et al., 2002; shoji et al., 2002) . therefore, the knowledge of infectious agents involved in neurological diseases and mechanisms underlying the induction of neuropathology by these pathogens will be invaluable for preventing and developing novel clinical interventions. coronaviruses are enveloped positive-stranded rna viruses that infect multiple species of mammals, including man, causing diseases that range from encephalitis to enteritis. human coronaviruses (hcov) are recognized respiratory pathogens responsible for up to 35% of common colds (mcintosh, 1996; myint, 1994) and also involved in nocosomial infections (sizun et al., 2000) . they have occasionally been associated with other pathologies, such as pneumonia, meningitis, and enteritis (riski and hovi, 1980; resta et al., 1985) . moreover, hcov have the ability to replicate and persist in human neural cells (bonavia et al., 1997; arbour et al., 1999a,b) and to have neuroinvasive properties (burks et al., 1980; murray et al., 1992; stewart et al., 1992; arbour et al., 2000) . this has stimulated research on their possible involvement in neurological disorders. of the two known hcov serotypes, designated oc43 and 229e, hcov-oc43 is antigenically related to murine coronaviruses (mhv) . given that, under certain conditions, mhv causes experimental cns inflammatory demyelination that pathologically resembles ms (bailey et al., 1949; lampert et al., 1973; weiner, 1973; wang et al., 1990) , the related human coronavirus represents a logical target for investigation. in the present study, we report the development of a mouse model to characterize in vivo hcov-oc43-mediated neuropathogenesis. we describe the acute disease induced by hcov-oc43 infection, which resulted from neuronal infection and loss. this animal model constitutes a tool to study neuroinvasive and neurovirulence properties of a common cold virus and the mechanisms underlying the development of a diffuse vacuolating meningoencephalitis, an emerging medical problem (shoji et al., 2002; whitley and gnann, 2002) . balb/c and c57bl/6 mice were selected in view of their relative susceptibility to both respiratory and enteric strains of mhv (barthold and smith, 1987) . we tried different inoculation routes to establish the neurotropic and neuroinvasive properties of hcov-oc43 in mice. an intraperitoneal inoculation with a virus dose of 10 5 tcid 50 revealed that hcov-oc43 virus infection could be lethal until 8 days postnatal (dpn) and the same doses were nonlethal at 21 dpn. with an intraoral inoculation of 10 4 -10 5 tcid 50 of virus, we were unable to reveal the presence of virus or virus gene products in any tissue tested (brain, spinal cord, heart, lung, liver, and spleen), even by rt-pcr. mice were susceptible to intranasal (in) inhalation of the hcov-oc43 solution at 10 4 -10 5 tcid 50 . this infection was lethal in 1-week-old c57bl/6 mice. however, 21 dpn mice infected this way did not show clinical signs of pathology, but 4 of the 8 animals were found positive for viral rna by rt-pcr analyses. viral rna was mainly found in the cns but some mice also showed virus rna in the spleen (data not shown). therefore, virus could spread from the periphery to the cns after in inhalation. having shown hcov-oc43 neuroinvasive properties, we chose for the remaining experimentation to use intracerebral inoculation (ic) so as to favor a cns infection. the ic route results in a more reproducible infection, a better control of viral doses introduced into the brain. the correlation between viral infectious dose and 100% mortality in the two strains of mice after inoculation is shown in table 1 . mice became less susceptible with age and were resistant at 35 dpn for c57bl/6 and at 28 dpn for balb/c mice. we then determined the optimal experimental conditions to obtain a sublethal dose that still allowed virus replication and virus-induced cns pathology in 21-day-old mice. viral dose was administered under deep anesthesia and was determined to be 10 l of a virus solution containing 10 tcid 50 for c57bl/6 and 10 5 tcid 50 for balb/c mice aged 21 dpn. under these conditions, inoculated mice developed signs of acute disease characterized by loss of weight, apathy, ruffled fur, humped posture, and wasting (figs. 1a and b) . animals showed atrophy of skeletal muscles and occasionally exhibited paralysis of their forelimbs. during the second week postinfection, some of the animals recovered and clinical signs totally disappeared. for others, pathological signs increased and led to death. the infected animals became anorexic, inactive, and dehydrated, increasing percentages of mortality. we established survival curves for each mouse strain (fig. 1c) . eighty percent of the c57bl/6 mice died within the first 15 days postinfection and only 20% of balb/c mice died during this period, even after receiving a higher viral dose. moreover, mice inoculated with supernatants from cell cultures infected with brain tissue from affected mice developed the same disease, demonstrating that the virus was responsible for pathology. viral rna could be detected in the brain as early as 24 h postinfection, and after 2 to 3 days in the spinal cord. all c57bl/6 mice were positive for hcov-oc43 rna during the first 11 days postinfection and during the first 9 days for balb/c mice ( figs. 2a and b) . a screening of viral repnote. this dose (expressed in tcid 50 ) is in function of mouse age (days postnatal; dpn) at the time of inoculation. lication was performed by rt-pcr in a variety of tissues and results obtained indicated that infection was restricted to the cns during the first 9 days postinfection. after that, in the most affected mice, viral rna was also found in heart, spleen, and lungs, and at lower levels in liver and muscles between the 11th and 13th days postinfection in c57bl/6, suggesting a viremic spread or transneuronal transmission (fig. 3b) . the presence of hcov-oc43 rna was detectable in the brain until 11 days postinfection for 40% of balb/c mice and until 15 days postinfection for 25% of c57bl/6 mice. no viral rna could be found from tissues harvested after these times postinfection. it was also confirmed that the rt-pcr assay designed to specifically detect hcov-oc43 was indeed specific and could not have detected an enzootic mhv strain (fig. 3c) . infectious virus appeared around 3 days postinfection and could be isolated from the cns of c57bl/6 mice during the first 2 weeks postinfection ( fig. 2a) . the highest levels of infectious virions were found between 5 and 9 days postinfection (fig. 2c) . in balb/c mice, virus was detectable at 1 day postinfection and reached the highest titer around 3 days postinfection (figs. 2b and c). the highest infectious titers observed were 10 8 tcid 50 /g for brain and 10 6 tcid 50 /g for spinal cord extracts. no infectivity could be detected at and after 13 days postinfection for c57bl/6 and 9 days postinfection for balb/c mice. viral proteins were found in the brain and spinal cord of c57bl/6 mice between 5 and 11 days postinfection (fig. 3a) and were undetectable after 10 days postinfection. we detected two forms of the n protein, as already noted in 8 dpn hcov-oc43-inoculated mice (jacomy and talbot, 2001) or after mhv-4 infection (talbot et al., 1984) . blood collected at different time points after infection revealed that serum contained antibodies specific for hcov-oc43. humoral immunity started to appear at 1 week postinfection and increased during the first month postinfection, and antiviral antibodies were still present at 4 months postinfection, as shown by indirect immunofluorescence on infected hrt-18 cells (data not shown). no immunofluorescent cells were seen with serum obtain from control mice. histochemical labeling of viral distribution at different times after infection revealed that virus infection initiated by ic inoculation was quickly disseminated throughout the cns. cells positive for viral antigens were first observed at hcov-oc43-infected mice gained weight normally during the first 5 days after infection, after which they all lost weight during the acute phase of the disease. the more affected mice lost more weight more rapidly than less affected mice and died during this period. after 9 days postinfection, mice which survived gained weight to reach the weight of control animals around 21 days postinfection. (c) survival curves of mice after hcov-oc43 infection. balb/c mice received a higher dose than the c57bl/6 mice, 10,000 tcid 50 versus 10 tcid 50 . however, c57bl/6 were less resistant, with 80 versus 20% of death after infection. 3 days postinfection in the gray matter of the brains of c57bl/6 mice. at this time, microglial activation was still undetectable as assessed by mac-2 immunostaining. at 1 week postinfection, hcov-oc43 had spread to all cns regions, predominantly in the entire cerebral cortex, the striatum, the hippocampus, the hypothalamus areas, the colliculus superior, and the brain stem, including the spinal cord (figs. 4 and 5). the cerebellum was frequently spared, but purkinje cells were found positive for virus in some animals. astrogliosis revealed by gfap staining increased and activated microglial cells started to appear along the ventricles (figs. 4f, g, and h). activated microglial cells were not observed in the cns of noninfected control mice at any time during investigation, as monitored by the absence of staining for mac-2, a marker not expressed in nonreactive microglia (walther et al., 2000) . in the spinal cord at 7 days postinfection, an hcov-oc43-specific mab labeled sensory and motor neurons, and microgliosis and astrogliosis were also detected in infected regions (fig. 5) . the progression of the infection was accompanied by identical neuropathologic features in the two strains of mice: neurons exhibited severe signs of pathology, most of them showing necrosis and vacuolation. this started by the development of small and round empty vacuoles in the cytoplasm, which increased in size (figs. 4c, d, and e). these spongiform-like lesions were seen primarily within the neuronal cell bodies, the neuropil being generally unaffected (fig. 6c ). this feature was never observed in noninfected brain (fig. 6a) . ultrastructurally, numerous cells presented cytoplasm disorganization without lysis of the cellular membranes. degenerative changes included cytoplasm rarefaction, dilatation of the rough endoplasmic reticulum (rer), and disaggregation of polyribosomes leading to the appearance of free ribosomes. hematoxylin-eosin staining also revealed the presence of degenerated neurons with picknotic or small densely stained nuclei and eosinophilic cytoplasm (figs. 6c, e, h, and i). at an advanced stage of disease, loss of neurons was pronounced and was particularly evident in ca1 and ca3 hippocampal layers ( fig. 6d -i). histological examination of the brain or spinal cord revealed scattered infiltration of inflammatory cells, starting by mononuclear cell infiltrations (fig. 6b ) and perivascular cuffing. some macrophage-mediated elimination (neufig. 2 . hcov-oc43 infectious virus and rna in the cns of 21 dpn mice. (a) 100% of brains from c57bl/6 mice inoculated ic with 10 tcid 50 of hcov-oc43 were positive for viral rna between 3 and 11 days postinfection. only 25% of the surviving mice were found positive after 15 days postinfection and rna was not found thereafter. infectious virus appeared later and disappeared before elimination of viral rna. between 5 and 11 days postinfection, 100% of brains contained infectious virus. (b) detection of hcov-oc43 rna in the brain of balb/c mice inoculated ic with 10 5 tcid 50 of hcov-oc43 revealed that 100% of these mice were positive until 9 days postinfection. infectious virus was detectable in all mice only during the first 3 days postinfection and gradually fewer mice were found positive. (c) histogram representing the amount of infectious virus detected in five brains from the two strains of mice at different intervals postinfection. the limit of the detection assay was 10 0.5 tcid 50 . ronophagia) was also encountered. in the spinal cord, viral particles observed 7 days postinfection at the electron microscopic level were mostly localized in the cell cytoplasm, closely associated with the golgi apparatus or in extracellular spaces (fig. 7) . viral replication and transneuronal passage occurred in a stepwise fashion that utilized existing cellular processes. when hcov-oc43 replication and spread reached maximal levels, around 9 days postinfection, astrogliosis and microgliosis progressively increased in all infected regions of the cns until the death of the animal (figs. 4g and h). therefore, a correlation between pathological signs of disease observed in mice and morphological injury of the brain was apparent. clearance of mhv from the cns appears to involve t cells (sussman et al., 1989) and age-acquired resistance to virus could be abolished in immunosuppressed animals (zimmer and dales, 1989) . therefore, we examined the effect of immunosuppression on hcov-oc43-mediated neuropathogenesis. the immunosuppressive effects of cyclosporin a (csa) are clearly established (borel et al., 1976) . as csa causes a specific reversible inhibition of immunocompetent lymphocytes (preferentially t cells) and inhibits gene transcription for certain cytokines, in particular il-2 (kupiec-weglinski et al., 1984; elliott et al., 1984; shevach, 1985) , we investigated whether csa could modify the course of the acute hcov-oc43 infection on the development of cns lesions or on viral replication. it is known that csa injected into mice at 50 mg/kg/day induces neurotoxicity (hypocellular and disorganized organs), whereas csa at 12.5 mg/kg/day induces no abnormalities and spread to all organs (boland et al., 1984) . therefore, csa doses were selected to avoid cytotoxic effects and mortality in mice and were in accordance with immunosuppression-inducing doses described in the literature (bolton et al., 1982; pasick et al., 1992) . control mice treated with csa at a daily dose of 20 mg/kg did not show any apparent adverse effects: they gained weight normally and did not present ruffled fur or lethargy. for csa-treated and untreated mice, the kinetics of weight loss was similar after hcov-oc43 infection. nevertheless, immunosuppression by csa slightly precipitated the disease but increased mortality (fig. 8) . this was more pronounced in mice treated with csa at 20 mg/kg/day where 100% of mice died, whereas only 80% of oil-treated mice succumbed to hcov-oc43 infection. infection of mice by hcov-oc43 was dependent on a number of variables, including dose, route of inoculation, age of the host, and its genetic background. indeed, our results show striking susceptibility differences between two strains of mice: balb/c mice were more resistant than c57bl/6. moreover, resistance increased with age in the two strains of mice. this suggests that susceptibility to human coronavirus neuropathogenesis may be linked to genetic factors. our study also confirms that human coronaviruses have neuroinvasive properties in mice, which was first shown in newborn mice (barthold et al., 1990) , and that such neuroinvasion is possible even after maturation of the immune system (king et al., 1992) , which is consistent with their detection in human brain (burks et al., 1980; murray et al., 1992; stewart et al., 1992; arbour et al., 2000) . even though our study does not confirm a specific route of entry into the cns, a transneuronal route already demonstrated for mhv (lavi et al., 1988; barthold et al., 1990; perlman et al., 1990a) constitutes a likely possibility. twenty-one days postnatal mice infected by ic inoculation of hcov-oc43 developed signs of acute disease characterized by apathy, hunched posture, ruffled fur, and tremors, comparable to pathological signs described after mhv infection (kristensson et al., 1986) . following ic inoculapathologic symptoms and mortality even with very high viral doses (lavi et al., 1986) . the clinical signs of pathology after hcov-oc43 ic inoculation coincided with the peak in virus yields observed at approximately 5 to 9 days postinfection for c57bl/6 mice. this indicates that virus replication in the cns apparently played a major role in the establishment of the pathology. infected mice showed extensive inflammatory responses characterized by mononuclear perivascular cuffing, neuronophagia, and a great number of reactive glial cells in the infected regions. to investigate whether infiltration of inflammatory cells contributed to neurodegeneration or if infectious virus was directly responsible for vacuolating lesions and neuronal death, we evaluated the effect of treating animals with cyclosporin a, a powerful immunosuppressant drug. with the dose used (10 or 20 mg/kg/day), csa is known to be distributed extensively throughout the body and not to cause neurotoxicity in mice (boland et al., 1984) . immunosuppression precipitated human coronavirus-induced disease and increased the percentage of acute death (80 vs 100%). under csa treatment, neurons also presented vacuolation and degeneration. therefore, the pathology observed following hcov-oc43 infection was likely not immunologically mediated, unlike that induced by mhv-a59 and mhv-jhm (sussman et al., 1989; wang et al., 1990) , although experiments with immunodeficient mice of the same genetic background will be needed to definitely address this question with hcov-oc43. moreover, macrophage/microglial reactivity was delayed when related to infection and appeared only when the virus was present in most parts of the brain. the inflammatory response and macrophage/microglial cell recruitment seem to be strongly correlated with virus clearance, as was also demonstrated after mhv infection (sussman et al., 1989) . some strains of mhv, including a59 and jhm, are neuroinvasive in rodents, eliciting either an acute encephalitis or a chronic paralytic disease (for review, see perlman, 1998) . unlike the slow neurodegenerative disease caused by mhv (bailey et al., 1949; lampert et al., 1973; weiner, 1973; lavi et al., 1984; wang et al., 1990) , hcov-oc43 resulted in a productive and cytotoxic infection of neuronal cells in the cns, which led to neurodegeneration. group of 10 c57bl/6 mice treated with cyclosporin a at 20 mg/kg/day (oc43/20 mgcsa) and at 10 mg/kg/day (oc43/10 mgcsa) became more susceptible to hcov-oc43 infection, with 100 and 90% of death after infection versus 80% in non-csa-treated animal. infected c57bl/6 mice treated with oil alone (oc43/oil) presented similar survival curves as previously reported, with 80% of death after infection. noninfected hcov-oc43 mice treated with csa at 20 mg/kg/day (control/20 mgcsa) illustrated that csa was not toxic under these conditions. having previously demonstrated a persistent infection of hcov-oc43 in primary murine cns cell cultures and in the cns of mice inoculated at 8dpn (jacomy and talbot, 2001) , and given the observations that mhv antigens or rna were still detectable in the spinal cord several weeks after infection (woyciechowska et al., 1984; perlman et al., 1990b) , we expected to detect a persistent infection in the cns of infected 21 dpn mice. however, rt-pcr analysis revealed that viral rna could not be detected after the second week postinfection, suggesting a nonpersistent infection of hcov-oc43 virus in 21 dpn mice. histological analysis of infected 21 dpn mice showed virus spread throughout the brain and spinal cord, as we had previously described for 8 dpn mice (jacomy and talbot, 2001) . neurons remained the major cellular targets for virus, which was probably disseminated from neurons to neurons by a cell-to-cell transport, as was described after mhv infection (lavi et al., 1988; . nevertheless, even though neurons were susceptible to mhv infection, oligodendrocytes and astrocytes represented the major infected cell types (perlman and ries, 1987; . this may explain why the pathology observed in mice was different after infection with these two antigenically related coronaviruses: mhv can cause demyelination (bailey et al., 1949; lampert et al., 1973; weiner, 1973; wang et al., 1990 ) with a spongy state observed in the white matter (lavi et al., 1984) , whereas hcov-oc43 encephalitis is accompanied by vacuolating degeneration of the gray matter. the latter lesions were not detected in a previous study (pearson and mims, 1983; barthold et al., 1990) , probably because mice were younger and died within 3 to 7 days postinfection. spongiform cellular changes were occasionally reported after mhv infection. for example, vacuolation was observed in the subthalamic-nigral region after ic inoculation of mhv-a59 (fishman et al., 1985) , and foci of vacuolation were observed in the hypothalamus, cerebellar peduncles, and pons regions after in inoculation of mhv-s ( barthold and smith, 1983) . nevertheless, these degenerative changes were not commonly observed after mhv infection and were restricted to small cell populations, even after infection with 300-to 600-fold higher virus doses into c57bl/6 mice. interestingly, the appearance of clear round vacuoles and neuronal death represents a hallmark of cns degeneration observed in prion diseases (prusiner, 1998) . nevertheless, mitochondrial disease (mckelvie et al., 1991) , leigh's disease (kimura et al., 1991; agapitos et al., 1997) , pick's disease (deruaz et al., 1993) , or alzheimer's disease (duffy et al., 1988; budka et al., 1995) also display spongiform cns lesions, which are independent of the prion protein. pathways and causal start points of transmissible spongiform encephalopathies or acute encephalitis remain unknown. occasionally, viral infections of the cns were described to induce spreading spongiosis, such as in human t cell leukemia virus associated myelopathy (htlv-1) or in hiv encephalitis (rhodes, 1987; goldwater and paton, 1989) . in rodents, mutants of vsv virus (rabinowitz et al., 1976) and moloney murine leukemia virus (mo-mulv) (gardner et al., 1973; czub et al., 1994) were shown to experimentally induce spongiosis. histologically, vacuolar degeneration induced by hcov-oc43 was mainly restricted to the neuronal cell bodies, whereas that caused by prion or retrovirus first affected the neuropil. moreover, the inflammatory response was very limited in prion encephalopathy, whereas hcov-oc43 induced extensive brain inflammation. these indicate different mechanisms underlying vacuolation and neuronal death. interestingly, noninflammatory neuropathologies have been considered evidence against a viral etiology. infections by opportunistic pathogens such as respiratory or enteric virus in immuosuppressed patients (hiv, transplantation, and cancer chemotherapy) may also cause cns pathology. it has been reported that immunocompromised patients have increased incidences of malignancies induced by viral infection (penn, 1987) . therefore, severe cases of encephalitis have devastating effects on the brain and spinal cord functions. given our previous observations of hcov-oc43 persistence in human brain (stewart et al., 1992; arbour et al., 2000) , we propose that respiratory pathogens with a neurotropic and neuroinvasive potential could be associated with neurodegenerative disease in susceptible individuals. this animal model of human coronavirus neuropathogenesis may prove helpful in the characterization of coronavirus-induced neurodegeneration in surviving infected animals and of transneuronal virus spread to the cns. moreover, susceptibilities of endothelial cells and leukocytes to viral infection need to be investigated as a possible alternate route of virus entry into the cns. finally, possible explanations for our observations of striking differences in susceptibility to hcov-oc43 infection of two strains of mice remain to be investigated. even though mice are not the natural host for hcov-oc43 infections, they may provide data that, together with studies using human neural cell cultures and post-mortem brain tissue, may contribute to our understanding of the underlying mechanisms and neuropathological consequences of coronavirus infections in humans. the oc43 strain of hcov was originally obtained from the american type culture collection (atcc, rockville, md), plaque-purified, and grown on the human rectal carcinoma cell line hrt-18 as previously described . hcov-oc43 virus stocks (10 6 tcid 50 / ml) were kept at ϫ80°c. to determine the susceptibility of mice to hcov-oc43 infection, two different strains of mhv-seronegative female mice (balb/c and c57bl/6, from jackson laboratories, bar harbor, me, u.s.a.) were inoculated. inoculations were performed on mice at various days postnatal (1, 8, 15, 21, 28, 35 dpn) using 10 l of various dilutions of the initial virus stock and using different inoculation routes: intraperitoneal, intraoral, intranasal, and intracerebral, to investigate hcov-oc43 infection parameters, in particular its neuroinvasive property. the viral dose was administrated ic under deep anesthesia of ketamine-xylamine (ketamine at 200 mg/kg and xylamine at 10 mg/kg). in the present study, we chose to infect 21 dpn mice with an ic inoculation of 10 l containing 10 tcid 50 of hcov-oc43 for c57bl/6 and 10,000 tcid 50 for balb/c mice. twenty mice of each strain were used to establish survival curves. every 2 days postinfection, five animals from two groups of infected mice of each strain were sacrificed and processed for detection of viral rna, viral proteins, and infectious virus. moreover, two infected mice of each strain were perfused every 2 days for histological analysis during the first month postinfection. for each experiment, age-matched control animals, which had received a virus-free solution containing culture medium from the hrt-18 cell line, were used. to confirm that hcov-oc43 was responsible for the observed pathology, we infected hrt-18 cell cultures with brain homogenates, prepared as described below, from infected mice. cell-free supernatants harvested 4 days later were confirmed to contain infectious virus and 10 l was reinoculated ic into other animals. cyclosporin a (sigma chemical co., st. louis, mo) was rehydrated in pure ethanol as specified by the manufacturer. it was then dissolved before use in olive oil to favor free diffusion of hydrophobic cyclosporin molecules through the plasma membrane into the cytoplasm (handschumacher et al., 1984) , and heated for 2 h at 60°c. mice received a subcutaneous injection of 10 or 20 mg/kg/day. this drug was administered 1 day prior to virus inoculation and daily thereafter for 10 days. three groups of 10 female c57bl/6 mice were infected with 10 tcid 50 of hcov-oc43 and 8 control females were inoculated with hrt-18 medium. two groups of hcov-oc43-infected mice received a single daily injection of csa, either 10 or 20 mg/kg/day. four control mice and the third group of hcov-oc43-infected mice received injection of olive oil alone. the four remaining control mice were treated with csa at 20 mg/kg/day, to verify the absence of csa toxicity. brain and spinal cords were dissected, homogenized in 10% (w/v) sterile pbs, centrifuged at 4°c, 20 min at 1000 g; then supernatants were immediately frozen at ϫ80°c and stored until assayed. the extracts were processed for the presence and quantification of infectious virus by an indirect immunohistochemistry assay, as previously described (bonavia et al., 1997) . hcov-oc43-susceptible hrt-18 cells were inoculated with serial logarithmic dilutions of each tissue sample in a 96-well linbro plate (icn biomedical canada ltd., costa mesa, ca). after 4 days of incubation at 33°c in 5% (v/v) co 2 , cells were washed in pbs and fixed with 0.3% (v/v) hydrogen peroxide (h 2 o 2 ) in methanol for 30 min. after washing with pbs, they were incubated for 2 h at 37°c in 1/1000 dilution of an ascites fluid from mouse mab 1-10c.3, directed against the nucleocapsid protein of hcov-oc43 (arbour et al., 1999b) . afterwards, cells were washed in pbs and hrp goat antimouse immunoglobulins (dako, diagnostics canada inc., mississauga, on) were added and incubated for 2 h at 37°c. antibody complexes were detected by incubation in 3.3јdiaminobenzidine tetrahydrochloride solution (dab, sigma), with 0.01% (v/v) h 2 o 2 . mice were perfused by intraventricular injection of 4% (v/v) paraformaldehyde, under deep ketamine-xylazine anesthesia. brains and spinal cords were removed and tissue blocks were left in the fixative for 24 h. coronal sections from brain and segments from cervical and lumbar spinal cord were sectioned at a thickness of 40 m with a lancer vibratome. serial sections were collected in 0.05 m trisbuffered saline (tbs) and were then incubated overnight with primary antibodies, as previously described (jacomy and bosler, 1996) . for viral antigens, we used 1/1000 dilutions of ascites fluids of the 4-e11.3 hybridoma that secretes monoclonal antibodies specific for the nucleocapsid protein of the serologically related hemagglutinating encephalomyelitis virus of pigs (bonavia et al., 1997) . astrocytes were identified with a rabbit anti glial fibrillary acidic protein antibody (gfap, dako) diluted 1/500, microglia/macrophages by an ascites fluid of the rat mac-2 antibody (atcc) diluted 1/1000. then, sections were rinsed and processed with vectastain abc kit (vector laboratories, burlingame, ca). labeling was revealed with 0.03% (w/v) dab solution (sigma) and 0.01% (v/v) h 2 o 2 , which yielded a dark brown product. some sections were counterstained with the classical cresyl violet stain. to further investigate histological changes occurring in mouse brains, half hemispheres from control and infected animals were paraffin-embedded and 10-m sections were stained with hematoxylin-eosin. this was performed by the pathology department, animal resources centre, mcgill university (montréal, québec, canada). samples for electron microscopy were postfixed for 2 h with 2% (v/v) osmium tetraoxide in 0.1 m phosphate buffer at ph 7.5, dehydrated in graded ethanol series, and eponembedded as previously described (jacomy and bosler, 1996) . one-micron sections were stained with toluidine blue and examined by light microscopy. subsequent ultrathin sections were collected on collodion-coated single-slot grids, stained with lead citrate, and examined with transmission electron microscope. blood from infected or control mice were collected at 1, 2, 3, or 4 weeks and at 2, 3, and 4 months postinfection. sera were collected and kept at ϫ20°c until use for the detection of antibodies against hcov-oc43 by indirect immunofluorescent labeling of infected hrt-18 cells. briefly, hrt-18 cells cultured on 12-well slides were infected by hcov-oc43 and fixed 4 days later in cold methanol and then kept at ϫ20°c until needed. at the time of the assays, slides were incubated 1 h at room temperature with serum from control and infected mice, diluted 1/100, 1/500, and 1/1000. after several washes in pbs, slides were incubated 1 h at 37°c with alexa fluor 488 f(abј) 2 fragments of goat antimouse igg (hϩl), at a dilution of 1/15,000 (molecular probes, inc., eugene, or) and observed under a fluorescence microscope. tissues were homogenized in sub buffer, containing 8 m urea, 0.5% (w/v) sds, and 2% (v/v) ␤-mercaptoethanol and then centrifuged for 15 min at 4°c, in a microfuge at 13,000 g and supernatants were collected, as previously described (jacomy et al., 1999) . samples (5 g total protein) were fractionated on a 7.5% polyacrylamide gel (sds-page) and either visualized by coomassic blue staining or transferred to nitrocellulose for western blot analysis. nitrocellulose membranes were preincubated in 5% (w/v) skimmed milk powder in ts buffer (0.05 m tris, ph 7.4, 0.15 m nacl) and then incubated overnight at 4°c with 4e11.3 antiviral mab. after several washes with ts buffer containing 0.05% (v/v) tween 20, membranes were incubated 1 h with peroxidase-conjugated anti-mouse igg diluted 1/1000 (dako). bands were visualized using a western blot chemoluminescent kit (super signal, pierce, rockford, md). tissues were dissected every 2 days postinfection. total rna was extracted by homogenization in trizol (gibco-brl, burlington, ca). for rt-pcr, one pair of hcov-oc43 primers was designed to amplify a region containing 305 nucleotides (primers o1 and o3) of the gene coding for the n protein (arbour et al., 1999b) . the target sequences were specific to hcov-oc43 and did not amplify mhv. the suitability of rna for rt-pcr amplification was verified by an rt-pcr specific for a housekeeping gene encoding glyceraldehyde-3-phosphate dehydrogenase (gapdh) using a pair of gapdh primers amplifying a region containing 833 nucleotides (arbour et al., 1999b) . one pair of mhv primers was also designed to amplify a conserved region of the mhv n protein gene. primers were 5ј-cctctactgtaaaacct-gatatgg-3ј and 5ј-ctaatttagatccaaagaaga-agc-3ј, corresponding to nucleotides 677-700 and 868 -991, respectively. approximately 5 g of rna was reverse transcribed with expand moloney murine leukemia virus reverse transcriptase (gibco-brl) and the cdna products were incubated in 20 pmol of each sense and antisense primers, 2.5 mm mgcl 2 , 1ϫ pcr buffer (10 mm tris-hcl, ph 8.3; 50 mm kcl), and 0.4 mm of each deoxynucleotide triphosphate, heated at 94°c for 5 min and 60°c (hcov-oc43) or 50°c (gapdh and mhv) for 5 min. after the addition of expand high-fidelity pcr system dna polymerase (rtaq, 5000 u/l; amersham pharmacia biotech inc., baie d'urfé, qc), 30 amplification cycles of 2 min at 72°c, 1 min at 95°c, and 2 min at 60°c (hcov-oc43) or 50°c (gapdh and mhv) were performed. ten microliters of this 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a neurotropic coronavirus to spinal cord white matter via neurons and astrocytes t-cellmediated clearance of mouse hepatitis virus strain jhm from the central nervous system western and dot immunoblotting analysis of viral antigens and antibodies: application to murine hepatitis virus galectin-3 is upregulated in microglial cells in response to ischemic brain lesions, but not to facial nerve axotomy borna disease virus and schizophrenia demyelination induced by murine hepatitis virus jhm strain (mhv-4) is immunologically mediated pathogenesis of demyelination induced by mouse hepatitis virus viral encephalitis: familiar infections and emerging pathogens acute and subacute demyelination induced by mouse hepatitis virus strain a59 in c3h mice in vivo and in vitro models of demyelinating diseases xxiv. the infectious process in cyclosporin a treated winstar lewis rats inoculated with jhm virus we thank annie boucher, inrs-institut armand-frappier, for the critical review of the manuscript and francine lambert for excellent technical assistance. we also thank dr. serge dea (who tragically passed away on january 3, 2003), inrs-institut armand-frappier, for the generous gift of the 4-e11.3 antibody, and dr. yves robitaille, mcgill university, for constructive comments on neuropathology. we also thank the mcgill university animal resources centre for their help with some histology. this work was supported by grant mt-9203 from the canadian institutes of health research (institute of infection and immunity). key: cord-290883-r2744fb3 authors: torres, juan m.; sánchez, carlos; suñé, carlos; smerdou, cristian; prevec, ludvik; graham, frank; enjuanes, luis title: induction of antibodies protecting against transmissible gastroenteritis coronavirus (tgev) by recombinant adenovirus expressing tgev spike protein date: 1995-11-30 journal: virology doi: 10.1006/viro.1995.0023 sha: doc_id: 290883 cord_uid: r2744fb3 abstract ten recombinant adenoviruses expressing either fragments of 1135, 1587, or 3329 nt or the full-length spike gene of transmissible gastroenteritis coronavirus (tgev) have been constructed. these recombinants produce s polypeptides with apparent molecular masses of 68, 86, 135, and 200 kda, respectively. expression of the recombinant antigen driven by ad5 promoters was inhibited by the insertion of an exogenous sv-40 promoter. most of the recombinant antigens remain intracytoplasmic in infected cells. all the recombinant-directed expression products contain functional antigenic sites c and b (gebaueret al.,1991,virology183, 225–238). the recombinant antigen of 135 kda and that of 200 kda, which represents the whole spike protein, also contain antigenic sites d and a, which have previously been shown to be the major inducers of tgev-neutralizing antibodies. interestingly, here we show that recombinant s protein fragments expressing only sites c and b also induced tgev-neutralizing antibodies. the chimeric ad5–tgev recombinants elicited lactogenic immunity in hamsters, including the production of tgev-neutralizing antibodies. the antisera induced in swine by the ad5 recombinants expressing the amino-terminal 26% of the spike protein (containing sites c and b) or the full-length spike protein, when mixed with a lethal dose of virus prior to administration to susceptible piglets, delayed or completely prevented the induction of symptoms of disease, respectively. immune response to coronaviruses enjuanes and van der zeijst, 1995) : the spike protein (s) transmissible gastroenteritis coronavirus (tgev) in(buchmeier et al., 1984; cavanagh et al., 1986 ; daniel et fects the enteric and respiratory tissues of newborn pigal., 1993; daniel and talbot, 1990; koolen et al., 1990) , lets resulting in mortality of nearly 100% (saif and wesley, the membrane protein (fleming et al., 1989; . protection of newborn animals from tgev infecal., 1992; welch and saif, 1988) , and the nucleoprotein tion requires the induction of secretory iga in milk. previ-(buchmeier et al., 1984; ; lecomte et ous studies have shown that precursors of mucosal iga al., 1987; nakanaga et al., 1986; talbot et al., 1984; wesplasma cells originate in lymphoepithelial structures in seling et al., 1993) . the study of the induction of protecthe gastrointestinal and respiratory tracts. these precurtive immunity to tgev has focused on s protein because sor cells switch to iga production in gut-or bronchusit is the major inducer of tgev-neutralizing antibodies associated lymphoepithelial tissues and migrate to disjimé nez et al., 1986; laude et al., seminated mucosal effector sites, including gastrointesti-1992) and it mediates binding of tgev to its cellular nal and upper respiratory tracts, as well as to exocrine receptor godet et al., 1994) . a correlatissues such as the mammary gland. recombinant hution between the antigenic and the physical structure of man adenovirus 5 (ad5) has efficiently been used to in-s protein has been established ; duce protection against viral infections (berkner, 1988; jimé nez et al., 1986; suñé et al., 1990) . site a is also graham and prevec, 1992) . we have reported that ad5 involved in the induction of in vivo protection (de diego infects mucosal tissues of swine (torres et al., 1995 (torres et al., ), et al., 1992 , but the precise roles of the different antiindicating that recombinant adenoviruses might be used genic sites in eliciting resistance to tgev are unknown to induce mucosal immunity against tgev. helper-inde(enjuanes and van der zeijst, 1995) . pendent ad5-based vectors with the capacity to express in this paper we describe 10 ad5-tgev recombinants foreign genes of up to 4.9 kb have been developed (bett expressing either full-length tgev spike protein or three et al., 1993) . truncated amino-terminal fragments of this protein. these recombinants induced immune responses in hamsters and swine which neutralized tgev infectivity. in addition, we demonstrate that porcine serum from ad-tion. finally, we show that virus-neutralizing antibodies tains the 3-end of ad5 from the xhoi site at 70 map units (m.u.) with a deletion of the xbai d fragment from are induced in the milk of ad-tgev-immune hamsters. 78.5 to 84.3 m.u. within the ad5 e3 coding region. plasmid pab14 also contains the 3-end of ad5 from map unit 70 materials and methods to 100 with a 2685-nucleotide deletion in the e3 coding eukaryotic cells and viruses region. plasmid pfg173 contains a deletion of essential sequences to the left of e3 in the ad genome that renders the epithelial swine testicle (st) cell line (mcclurkin it unable to produce infectious ad5 (bett et al., 1993; and norman, 1966) and human 293 cells which constitu-hanke et al., 1990; mittal et al., 1993) . tively express the 5-end 11% of the ad5 genome (graham et al., 1977) were used to grow the recombinant construction of recombinant vectors adenoviruses. pur46-mad strain of tgev (sá nchez et al., 1990) was cloned, sequenced, and used as a source the general procedure followed to construct recombiof the s gene . neutralization of nant ad5 viruses expressing tgev s gene fragments tgev was performed by incubating serial 10-fold dilu-(ad-ts) is summarized in fig. 1 . s gene sequences were tions of the virus with a 1/20 dilution of the antibody at flanked by sv-40 pr and polyadenylation sequences 37њ for 30 min, and the virus-antibody mixture was plated when indicated (fig. 2) , by subcloning them into plasmid on st cells as previously described (correa et al., 1988) . psv2x3 or psv2x4. cassettes with s gene sequences the neutralization index (ni) was defined as the log 10 of were inserted into the unique xbai site of the partially the ratio of the pfu after incubating the virus in the deleted e3 gene on plasmid pfg144k3 or pab14, both presence of medium or the indicated antiserum. ni indiof which include the 3-end of ad5. alternatively, s gene ces are determined rather than titers since in the first fragments were removed from the original plasmid or procedure virus-antibody mixtures are evaluated in the from psv2x3-ts vectors without sv-40 pr signal, or withplaque assay without further dilution of the antibody, proout both pr and polyadenylation sequences, using the viding highly reproducible results and information about restriction endonucleases indicated in fig. 1 . in this case, the potency of the antibody (the titer reduction expressed fragment ends were blunted with klenow and t4 dna in logarithmic units rather than the ability of the serum polymerase and cloned into the xbai site of pfg144k3 to neutralize a few pfu). or pab14 plasmids that were blunted and dephosphory-ad5 strain dl309 contains a small deletion from 83 to lated according to standard procedures (maniatis et al., 85 map units and an unknown substitution in the e3 1989) . each of these plasmids is noninfectious by itself, region (jones and shenk, 1979) . pfg140 is an infectious but can generate infectious virus following cotransfection circularized form of ad5 dl309 carrying a 2.2-kb dna of 293 cells along with a plasmid, pfg173, which coninsert (pmx2) encoding ampicillin resistance (apr) and a tains the 5-end of ad5 ( fig. 1 ) (graham and prevec, bacterial origin of replication. plasmid pfg140 was used 1992; hitt et al., 1995 hitt et al., , 1994 . this results in the rescue as positive control for infectious ad5 dna (graham et of genes cloned into the e3 region of viral vectors. coal., 1988) . transfection was performed essentially as described using the calcium phosphate precipitate technique (gra-plasmids and bacteria ham and van der eb, 1973) . after 8 to 15 days, plaques were isolated and expanded, and viral dna was ana-the tgev s gene was cloned into bluescript (stralyzed by hindiii restriction enzyme digestion. viruses tagene) or pya plasmids (smerdou et al., 1995) as prewith the expected dna pattern were plaque purified viously described . escherichia coli three times and the junction of the constructs was se-dh5 or xl1-blue cells (stratagene) were transformed quenced to verify the expected primary structure. recomwith newly constructed plasmids by electroporation binants ad-ts01 and ad-ts02 are identical to recombi(dower et al., 1988) . plasmid dna was prepared by the nants ad-ts5 and ad-ts6, respectively, except that the alkaline lysis method (birnboim and doly, 1979) and purifirst two were constructed using cloning vector pab14 fied by cscl-ethidium bromide density gradient centrifuwith the large deletion on e3 gene, while in the construcgation. s gene fragments or the full-length s gene were tion of the second pair of recombinants plasmid flanked either by sv-40 promoter (pr) alone or by both pfg144k3, with the smaller deletion on e3, was used. pr and polyadenylation sequences, as indicated. s gene fragments were first subcloned into psv2x3 or psv2x4 immunoprecipitation of s antigens expressed by plasmids . the structures of the three recombinant ad-ts key plasmids (pfg144k3, pab14, and pfg173) used in the construction of ad5-tgev recombinants have been subconfluent 293 cells grown in dulbecco's modified eagle medium with 5% horse serum (gibco europe) were reported previously (bett et al., 1993; mittal et al., 1993) . plasmid pfg144k3 was derived from pfg144 (ghosh-infected with ad-ts recombinants at a multiplicity of infection (m.o.i.) of 30 pfu per cell. after 1 hr of virus choudhury et al., 1986) and as essential features con(maniatis et al., 1989) . s gene sequences previously cloned into bluescript(sk 0 ) (promega) or pya (smerdou et al., 1995) plasmids were excised using the indicated restriction endonucleases and subcloned into psv2x3 or psv2x4, in which the s gene sequences were flanked by sv-40 pr, polyadenylation sequences, or both. to generate recombinants ad-ts07, ad-ts05, ad-ts9, and ad-ts06 s gene sequences were cloned directly into plasmid pfg144k3 or pab14. s gene sequences either alone or flanked by sv-40 sequences were subcloned into the xbai site of pfg144k3 or pab14, or excised with the indicated restriction endonucleases, blunted using the klenow polymerase fragment, and cloned into blunted xbai unique site of these vectors. infectious ad-ts recombinants expressing s protein fragments were generated by cotransfecting 293 cells with pfg144k3-ts or pab14-ts (which carry s gene sequences from tgev and pfg173 plasmids). diagrams are not to scale. the origins of dna fragments flanking the s gene are indicated with squares filled with different motifs. numbers below the bar representing the ad5 genome (bottom) indicate map units. mcs, multicloning site; pr, promoter; an, polyadenylation signal; de3, deletion in e3 gene; r.e., restriction endonuclease; ts refers to sequences derived from tgev spike gene. adsorption at 37њ, fresh medium was added and cells dium, and refed with fresh medium containing 50 mci/ml of pro-mix: l-[ 35 s] in vitro methionine/cysteine labeling were incubated for 22 hr at 37њ. medium was then replaced by methionine-and cysteine-free medium con-mix (1 ci/mmol, cod. no. sjq0079, amersham ibé rica). cell monolayers were incubated 1.5 hr, detached with a taining 2% dialyzed serum. cells were incubated for 1 hr at 37њ, washed with methionine-and cysteine-free me-rubber policeman, washed with cold phosphate-buffered saline, ph 7.2 (pbs), collected by centrifugation at 3000 standard) and ad-ts recombinants grown under the same conditions were immunoprecipitated in parallel. rpm for 15 min at 4њ in a microfuge, and lysed in ripa buffer (50 mm tris-hcl buffer, ph 7.5, 150 mm nacl, 1% the same number of infected cells was analyzed for each recombinant. similar relative expression levels were ob-triton x-100, 1% sodium dodecyl sulfate (sds), and 0.2 mm pmsf). viscosity was reduced by mixing the tubes tained in many (ú5) experiments. after protein resolution in polyacrylamide gel electrophoresis and autoradiogra-in a vortex mixer and passing the samples through a 0.6-mm needle 10 times. extracts were centrifuged at phy, the intensity of the immunoprecipitated bands from ad-ts extracts was compared with that of the reference 30,000 g for 30 min at 4њ in a microfuge. labeled proteins were immunoprecipitated with tgev-specific porcine se-[ 35 s]tgev with known protein concentration (determined using bca protein assay reagent, pierce) to estimate rum which had been preadsorbed several times with 293 cells infected with adenovirus ad5 dl309. further the amount of s antigen. absorption of the antiserum did not eliminate the nonspecific bands. antigen-antibody complexes were bound immunofluorescence to protein a-sepharose by overnight incubation at 4њ. sepharose beads were washed three times with ripa st cells at a density of approximately 1.5 1 10 5 cells/ cm 2 in microslide culture chambers (miles scientific) buffer containing 0.2% sds, and the final pellet was resuspended in electrophoresis sample buffer containing were infected with adenovirus ad140 which contains no s gene insert, or with ad-ts recombinants, at a m.o.i. 2.5% sds and 5% 2-mercaptoethanol (laemmli, 1970) . samples were boiled for 3 min, the beads were sedi-of 3 pfu/cell. at 24 hr postinfection, cell monolayers were washed and fixed either with methanol:acetone (1:1) at mented by low-speed centrifugation, and supernatants were analyzed by polyacrylamide gel electrophoresis 020њ for 15 min or with 4% paraformaldehyde in pbs for 20 min at room temperature. cells were washed three and autoradiography. to estimate the amount of protein expressed by each recombinant different dilutions of su-times with pbs and once with 0.3% bovine serum albumin (bsa) in pbs for 10 min at room temperature. the cells crose gradient-purified 35 s-labeled tgev (used as an were incubated with hybridoma supernatants containing highest dilution giving a binding threefold higher than background. a mixture of mabs 1d.b12, 5b.h1, and 1d.g3 (specific for s protein sites b, c, and d, respectively) or with mab detection of the different antigenic sites in the s protein fragments, encoded by recombinant ad-ts, was h2-19 specific for a 70k ad5 antigen. after three additional washings with pbs, cells were covered with a carried out by cria using the antiserum elicited in hamsters by the different recombinants. the binding of 125 i-1:200 dilution of fluoresceinated goat anti-mouse immunoglobulins (cappel laboratories) in 0.3% bsa in pbs, labeled mabs to purified tgev bound to microplates was performed as previously reported (correa et al., 1988) incubated for 40 min at room temperature, washed five times for 10 min each with pbs, and mounted on glyc-with some modifications. briefly, purified tgev (0.1 mg/ well) was plated, remaining binding sites were saturated erol-pbs (9:1). with 5% bsa in pbs, and 125 i-labeled mabs (sp act 1.5 binding of 125 i-labeled mabs to 293 cells infected 1 10 7 cpm/mg; 4 1 10 5 cpm/well) were added and incuwith recombinant ad-ts bated for 2 hr at 37њ in the presence of fivefold dilutions of the competitor antiserum prepared in pbs with 0.1% confluent st cell monolayers plated on 24-well mi-bsa. microplates were washed six times with 0.1% bsa croplates were infected (m.o.i. 40 pfu/cell) with recombiand 0.1% tween-20 in pbs. well bottoms were cut and nant ad-ts viruses. at 24 hr postinfection, cells were bound radioactivity was determined in a gamma counter. washed with pbs and fixed in methanol:acetone (1:1) for the percentage of radioactivity bound was determined 15 min at 020њ or in 4% paraformaldehyde in pbs for 20 in relation to the radioactivity bound in the absence of min at room temperature. cells were washed three times competitor mab. purified homologous mabs were used with pbs and for 2 hr with 0.5% bsa in pbs. aliquots of as positive controls in the cria. 0.25 ml of 125 i-labeled purified mabs (1 1 10 6 cpm/well; 1.5 1 10 7 cpm/mg) (greenwood et al., 1963) protection of swine by immune serum 0.2% bsa were added to each well and incubated for 1 hr at room temperature, and the cell monolayers were the virulent tgev strain pur46-sw11-st2 (1 1 10 7 washed six times with pbs. mab binding was deter-pfu/swine) was mixed with 3 ml of the porcine antiserum mined by collecting the cells in 0.25 ml of 0.5 n naoh induced by recombinants ad-ts8 or ad-ts06, incuand counting the radioactivity in a gamma counter. bated at 37њ for 60 min, and administered using a gastric tube to 2-day-old miniswine born from tgev-seronega-immunization of hamsters and swine tive sows. inoculated animals were fed three times per day with milk formula for newborns (nidina 1, nestlé ) eight-week-old golden syrian hamsters were immucontaining 3 ml of the antiserum. control animals were nized with infectious ad-ts recombinants by three treated following the same procedure but using serum routes: oral (4 1 10 8 pfu in 0.2 ml of pbs), nasal (2 1 induced by wt ad5. virus titers after 1, 2, and 3 days in 10 8 pfu/0.1 ml), and intraperitoneal (1 1 10 9 pfu/0.5 ml). animals challenged with virus treated with control serum the virus was administered at days 0, 32, 60, and 90, and 1, 2, or 5 days postinoculation in animals challenged and orbital plexus puncture bleedings were performed with tgev immune serum-treated virus were determined at days 0, 32, 47, 87, 105, and 115. females with highest in tissue extracts from jejunum and ileum, lungs, mesentiters of tgev-specific antibodies were crossed with nonteric, and mediastinal lymph nodes. tissue homogenizaimmune males, and 8 days later another dose of the tion was performed at 4њ using an omni 2000 homogehomologous ad-ts recombinant was administered. nizer (omni international). twenty-four hours after delivery, hamsters were subcutaneously administered 10 iu of oxytocin. the milk was collected 1 hr later by applying vacuum with a syringe. results milk was diluted fourfold in pbs and stored at 020њ. ad5-tgev recombinants one-month-old swine, from crossing large white and belgium landrace, were immunized three times at 0, 28, ten ad5-tgev recombinants expressing tgev s and 56 days, each time by three routes: oral (1 1 10 9 gene fragments were constructed using vectors with dif-pfu), nasal (1 1 10 9 pfu), and intraperitoneal (1 1 10 9 ferent deletions on e3 gene or combinations of sv-40 pfu per dose). serum was collected 14 days after the promoter and polyadenylation signals. using these relast immunization. combinants s protein fragments of four different sizes were expressed. the recombinants were obtained by radioimmunoassay (ria) and competitive ria (cria) replacing the e3 gene of the ad5 genome with s gene with 125 i-labeled mabs sequences starting from nt 08 and the first 5-end 1135, 1587, 3329, or 4341 nt of the s gene. these recombinants ria was performed using purified tgev as antigen (0.1 mg/well) as previously described (jimé nez et al., code for fragments of 378, 529, 1109, and 1447 amino acids (aa) extended from the amino-terminus (fig. 2) . the 1986). titers in ria were defined as the inverse of the faint band (results not shown). recombinant products with apparent molecular masses of 68 and 135 kda (fig. 3, lanes c and e, respectively) were obtained for recombinant s protein fragments of 378 and 1109 aa, respectively. recombinants ad-ts07 and ad-ts5, both coding for polypeptides of 529 aa, gave a main band of 86 kda and a minor band of 80 kda (lane d), which probably corresponds to an underglycosylated form of the antigen or to a degradation product. the difference between the expected and the apparent molecular mass of the recombinant products suggests that these are heavily glycosylated, as occurs during s protein synthesis after tgev serum using extracts from 293 cells infected with the chimeric ad-ts viruses (fig. 3) . the amount of s protein was based on the comparison of band intensity after last product represents the full-length spike protein. the immunoprecipitation and autoradiography of 35 s-labeled constructs were obtained using either plasmid recombinant antigens and reference sucrose gradient-pfg144k3 or plasmid pab14 (fig. 1) , with deletions of purified 35 s-labeled tgev with known protein concentra-1.88 or 2.69 kb, respectively, in e3 (bett et al., 1993) . tion. both reference virus and recombinant antigens were recombinant plasmids were constructed as summarized labeled and analyzed in parallel using the same experi(fig. 1) . when indicated, the s gene fragments were mental conditions. since the distribution of the methioflanked by pr and polyadenylation signals (fig. 2 ) by clonnine and cysteine in the different fragments was similar, ing them into vector psv2x3 or psv2x4. inserts were no significant correction of band intensity was necessary subcloned into plasmid pfg144k3 or pab14 containing in the analysis. the expression levels ranged from 0.1 the 3-end half of ad5. human 293 cells were cotransto 10 mg of s protein per 10 6 infected cells. maximum fected with one of these plasmids and pfg173, which expression levels (5 to 10 mg/10 6 cells) were obtained contains almost the entire ad5 genome with a lethal for recombinants ad-ts5, ad-ts8, and ad-ts07, interdeletion across the e3 region. fully infectious ad-ts mediate levels (1 to 3 mg/10 6 cells) for ad-ts9, ad-ts2, viruses were recovered following recombination in co-ad-ts05, and ad-ts6, and minimum (around 0.1 mg/ transfected 293 cells. recombinant viruses were plaque 10 6 cells) for recombinants ad-ts01, ad-ts02, and adpurified. the dna from all the recombinants gave the ts06. relative expression levels were highly reproducpattern and sequence expected for each insert by hindiii ible in different experiments. all the recombinants, inrestriction endonuclease analysis and sequencing of cluding those expressing minimum amounts of antigen, dna junctions (results not shown). were also consistently positive in the immunofluores-after infection of 293 cells with ad-ts recombinants, cence and 125 i binding assays and in the induction of s protein antigens remained cell associated. tris buffer tgev-specific antibodies (see below). containing 1% sds was used to solubilize them. the when indicated, the s gene fragment cloned into ad5 estimated size of recombinant s antigen expressed by was flanked by pr and polyadenylation signals (fig. 2) . comparison of the expression levels in constructs with and representative results are shown (fig. 3) . s polypep-s gene fragments of the same size indicated that ad5 tides were detected with a polyclonal tgev-specific porrecombinants made using pfg144k3 plasmids excine serum. good specific immunoprecipitation bands pressed higher levels of antigen than those based on were systematically obtained with all recombinants explasmid pab14, although in some cases (i.e., recombinant ad-ts07 compared with ad-ts5) the level of ex-cept ad-ts01, ad-ts02, and ad-ts06, which gave a pression was similar (results not shown). in recombinants with the same e3 deletion it was also observed that removal of sv-40 pr yielded ad-ts recombinants with higher expression levels (results not shown). to study the cellular location of recombinant s antigen, we used immunofluorescence analysis of st cells infected with four selected recombinants each coding for s fragments of different size: 387, 529, 1109, and 1447 (full-length s protein) amino acids. a bright fluorescent signal was observed in the cytoplasm of methanol-acetone-fixed cells infected with recombinants ad-ts8, ad-ts5, and ad-ts9 (results not shown). highest fluorescence intensity was seen with tgev-infected cells and ts recombinants. when immunofluorescence was performed with a human ad5-specific mab (which binds 72k protein) bright fluorescence was observed on discrete though amino acids 380 to 387 of s protein site d are areas of the nucleus, but not in the cytoplasm (results coded by recombinant ad-ts5, this site was poorly recnot shown), in contrast to the cytoplasmic fluorescence ognized by mab 1d.g3 specific for d site on ad-ts5observed with tgev-specific mabs. infected cells (fig. 4) . the four antigenic sites were an estimation of the relative amount of s antigen loweakly detected in cells infected by recombinant adcated in the cytoplasm or accessible on the surface of ts06, probably due to the low replication level of this ad5-infected st cells was determined by studying the recombinant. binding of 125 i-labeled mab 1d.b12 (site b-specific) to methanol-or paraformaldehyde-fixed cells (results not immunogenicity of the recombinants shown). this mab was selected because it recognizes an epitope present in all ad-ts recombinants. cells in-immune responses elicited by the different recombinants were studied by inoculating hamsters both orona-fected with recombinants ad-ts8, ad-ts5, and ad-ts9 permeabilized with methanol-acetone expressed the sally and intraperitoneally (fig. 5) . seven of the ten recombinants summarized in fig. 2 elicited titers in ria highest amount of s antigen, which ranged between 60 and 66% of the amount expressed on st cells infected higher than 2500 and ni between 1 and 3. the best inducers of tgev-neutralizing antibodies were recombi-with tgev. in cells infected with these recombinants the binding of site b-specific mab to exposed antigen was nants ad-ts8, ad-ts2, and ad-ts06, expressing either the smallest protein fragment or the full-length protein around 10% of the binding to cytoplasmic s antigen of tgev-infected cells. that is, the amount of s antigen (fig. 2) . four recombinants (ad-ts8, ad-ts5, ad-ts9, and detected on the surface of the infected cells was at least sixfold lower than that seen in the cytoplasm. the recom-ad-ts06), each expressing s gene fragments of different lengths (fig. 2) were selected to study the induction of binant products were not detected in the supernatants of infected cells, although the media were not concen-an immune response to sites a, b, and d by cria (fig. 6 ). site c was not included in the study because the trated to detect small antigen amounts. proper folding of the s protein fragments expressed amino acid sequence pnsd recognized by mabs specific for this site is present in pro-by the four selected recombinants was evaluated by determining the amount of 125 i-labeled mab specific for anti-teins of the immunoglobulin superfamily and other serum proteins (correa et al., 1988; i. correa and l. enjuanes, genic sites a, b, c, and d bound to infected st cells (fig. 4) . all recombinants expressed sites c and b. recombi-unpublished results). recombinant ad-ts9 induced an immune response to antigenic sites b, d, and a (fig. 6) . nant ad-ts9, in addition, expressed sites d and a. alfig. 5 . immune response induced by ad-ts recombinants in hamsters. groups of four golden syrian hamsters were immunized at time 0 and at times indicated by arrows (see materials and methods) with the indicated recombinants. sera collected at 0, 32, 47, 87, and, in some cases, at 105 and 115 days postinfection were evaluated by ria and neutralization against tgev. mean serum titers and standard deviation errors are represented for each time point. the titer by ria was defined as the inverse of the highest antibody dilution giving a binding three times higher than the background in the ria assay. the ni was defined as the log 10 of the ratio of the pfu after incubating the virus in the presence of medium or the indicated antiserum. all recombinants induced a strong response to site b and milk was determined between days 1 and 2 during lactation (fig. 7) . the three recombinants induced anti(fig. 6a) which is conformation and glycosylation depenbodies in serum with titers in ria ranging from 5 1 10 3 dent . as expected, site a was only to 1.5 1 10 4 and in milk from 2 1 10 3 to 3 1 10 3 (fig. reconstituted by recombinants ad-ts06 and ad-ts9, 7a). serum and milk antibodies neutralized tgev with expressing the full-length s protein or the 135-kda s nis ranging from 2 to 4 and around 1, respectively (fig. antigen, but not by recombinants which do not include 7b). as expected, recombinants with no insert did not the residues implicated in this site (fig. 6c) . elicit tgev-specific antibodies. while antibody titers in induction of lactogenic immunity by ad-ts sera decreased with insert size, the ni increased, sugrecombinants gesting that antibodies to site a contributed significantly to the neutralization of tgev. induction of immune response in swine by ad-ts8, ad-ts9, and ad-ts06 were crossed with nonrecombinants ad-ts8 and ad-ts06 immune males and administered a third dose of the homologous ad-ts recombinant 10 days before delivery. the ad-ts8 and ad-ts06 recombinants expressing the smallest insert and the full-length spike protein, re-the presence of tgev-specific antibodies in the sera and 5, respectively). to study the potential of these antisera for protection against tgev, sera induced by these recombinants were examined for the ability to prevent tgev infection. virulent tgev (pur46-sw11-st2 strain, 1 1 10 7 pfu/dose) was mixed with the antibody induced by each recombinant, incubated at 37њ for 60 min, and administered to highly susceptible 2-day-old miniswine. virus titers were determined in jejunum and ileum, lungs, mesenteric, and mediastinal lymph nodes at 1, 2, 3, and 5 days postinoculation. the results (fig. 8) indicated that virus titers found in the enteric tissues were between 10 2 and 10 3 -fold lower when virus was premixed with antiserum induced by recombinant ad-ts8 (fig. 8d) , and very low titers (õ5 1 10 2 pfu/g of tissue) of infectious virus were detected in the small intestine of newborn pigs that were administered the antibody elicited by recombinant ad-ts06 (fig. 8f) . in contrast, titers ranging between 7 1 10 3 and 1 1 10 7 pfu/g of tissue were detected in the tissues of control animals to which serum induced by wt ad5, used as a control, was administered (fig. 8b ). in addition, neither mortality nor clinical symptoms were observed in animals treated with serum induced by recombinant ad-ts06 (fig. 8e) , while control animals presented diarrhea 24-30 hr postinfection and died around day 3 postinfection (fig. 8a) . ten ad5-tgev recombinants have been constructed and screened for their ability to express spike protein fragments of tgev. four recombinants expressing the full-length spike protein or truncated fragments spanning different lengths of s protein from the amino-terminus have been selected, and their ability to induce virusneutralizing antibodies was determined. these ad-ts viruses induced lactogenic immunity in hamsters, and the recombinant expressing the full-length s protein elicited antisera that, when mixed with a lethal dose of virus prior to administration to susceptible piglets, prevented the induction of disease symptoms. helper-independent ad5 viruses with a deletion in the e3 gene have been constructed, and the s gene was inserted into the e3 gene. two types of ad5 recombinants interest to determine the comparative levels of expression in these two plasmids. expression levels were always higher using ad5 viruses with the smaller deletion spectively, were selected to study the induction of tgevneutralizing antibodies in swine. although the level of in e3, independent of the insert size, suggesting that removal of the splicing acceptor site after the l4 gene recombinant antigen produced in st cells was high for recombinant ad-ts8 and low for ad-ts06 (figs. 3 and might have reduced e3 gene expression. sequences inserted without an exogenous polyadenylation signal 4), both recombinants induced high titers of tgev-specific antibodies in swine as determined by ria (1 1 10 4 were successfully expressed, indicating that the polyadenylation signal of the e3 gene has probably been used. and 5 1 10 4 , respectively) and by neutralization (ni of 2 in general, recombinants with relatively small inserts low levels of s antigen and, accordingly, of all antigenic sites (a, b, c, and d), probably due to low replication (1135, 1587, and 3329 nt) expressed larger amounts of s polypeptide than those with larger (4470-nt) inserts. levels. nevertheless, antigenic sites a and b were properly folded after infection with ad-ts06 virus since high the recombinants with smaller inserts gave ad5 titers in cell culture between 3 1 10 8 and 1 1 10 9 pfu/ml, while antibody levels against these sites were elicited in hamsters, as detected by cria (fig. 6 ). s protein trimer forma-ad5-ts virus with an insert of 4470 nt consistently gave titers lower than 10 7 pfu/ml. thus, the level of expression tion easily explains the dichotomy between low expression levels and high efficiency in eliciting a high immune in these recombinants correlates well with their level of replication. the three recombinants (ad-ts8, ad-ts5, response. s protein trimers (the native form of the glycoprotein in the virus) probably are more stable and better and ad-ts9) with genome sizes lower than 104% of wt ad5 were stable after 10 passages, while the recombi-represent the peplomer in the native virion. although recombinant ad-ts5 contains the sequences coding for nant with a genome size close to 105% of wt ad5 (ad-ts06) was unstable (results not shown). these results site d core (located in s protein from aa 377 to 390) lenstra et al., 1991; posthumus et are in line with previous work suggesting that the ad5 virion has the ability to package approximately 105% of al., 1990), it was very weakly detected by site d-specific mabs, while sites c and b, also encoded in this recombi-the wt genome length. this value is generally considered to be the maximum working capacity of the system nant, were well represented. site d may have been hidden by incorrect folding of the s protein in this area. (ghosh-choudhury et al., 1986; berkner, 1988; bett et al., 1993) . site a, the major inducer of tgev-neutralizing antibodies, was detected in larger amounts after infection by recom-viruses in which the inserted gene was flanked by an sv-40 pr always showed lower expression levels than binant ad-ts9 (expressing s protein without the membrane anchor domain) than by recombinant ad-ts06 those not flanked by this pr (fig. 3) . this suggests that the sv-40 pr, in the context that has been used in this (which expresses the full-length s protein). this may be a consequence of the higher expression levels provided work, is inhibiting and transcription is probably driven from the nearby ad5 e3 pr. the transcription could also by ad-ts9, since it has been previously shown (godet et al., 1991) that the full-length spike forms trimers and be driven from the major late protein pr that is located far to the left at m.u. 16. similar observations have been reconstitutes site a better than truncated s proteins missing the membrane anchor domain. in fact, one of the two made with other ad5-based vectors containing analogous e3 substitutions (schneider et al., 1989; major inducers of tgev-neutralizing antibodies was ad-ts06 virus, in spite of the low amount of s protein pro-and both et al., 1993) . antigenic sites c, b, d, and a (starting from the amino-duced by this recombinant. seven of ten ad-ts recombinants expressing s frag-terminal end) have been defined on s protein (correa et al., 1988; gebauer et al., 1991) . sequences coding for ments induced tgev-neutralizing antibodies in hamsters. recombinant ad-ts8, expressing a truncated form sites c and b were included in all recombinants and, in fact, s polypeptides with these two sites were detected of s protein spanning 378 aa from the amino-terminus (which includes sites c and b but not site a), induced after infection with all ad-ts viruses. the recombinant coding for the full-length s protein (ad-ts06) expressed virus-neutralizing antibodies. since site c does not infig. 8 . protection of swine with porcine sera elicited by ad-ts recombinants. tgev-specific swine antiserum was elicited by administration of wt ad5 virus, ad-ts8, or ad-ts06 recombinants (see materials and methods). the number of swine surviving after the oral administration of 1 1 10 7 pfu of the virulent strain pur46-sw11-st2 of tgev mixed with antisera induced by (a) wt ad5 or by the recombinants (c) ad-ts8 or (e) ad-ts06 expressing the 1135 amino-terminal nt or the full-length spike protein, respectively, is shown. the recovery of infectious virus was determined 1, 2, and 3 or 5 days postinfection (when the animals either died or were sacrificed) in the indicated tissue homogenates, in animals administered the virulent virus with serum from (b) ad5, (d) ad-ts8, or (f) ad-ts06 immune swine. three groups of five swine were used to follow the survival rate. the infectious virus was followed in three groups of three animals each. mean values have been represented. standard deviations were lower than 25% in all cases and are not shown. duce virus-neutralizing antibodies, site b, or neighboring and van der zeijst, 1995) or, alternatively, other factors similarly to those described in mouse hepatitis virus sys-antigenic domains involved in virus neutralization, have been reconstituted in a functional form. it has been pro-tem (fazakerley et al., 1992; yokomori et al., 1993) . recombinant adenoviruses expressing only the 378 amino-posed that factors mapping in the s segment which has been deleted in the porcine respiratory coronavirus terminal residues of the s polypeptide (which are mostly deleted in prcv strains) provide partial protection (prcv) (from aa 21 to 241) (callebaut et al., 1988; sá nchez et al., 1990) , and more precisely alterations in amino against tgev. these data indicate that the amino-terminal s protein fragment might be relevant to confer enteric acid 219 (or residues close to it) might be involved in the loss of enteric tropism (sá nchez et al., 1992) . these fac-tropism by complementing the binding of n-aminopeptidase (identified as a major tgev receptor) to an s protein tors might be the presence of a second receptor binding site recognized by a putative second receptor (enjuanes domain mapping close to antigenic site a (delmas et al., 1992; godet et al., 1994) . another observation supports with higher titers in swine than in hamsters, although both species were permissive to virus infection. s protein this hypothesis. ad5 vectors have been used to express the amino-terminal 564 aa of prcv s protein, resulting has been previously expressed using e. coli (hu et al., 1984 (hu et al., , 1987 or poxviruses (pulford and britton, 1991) , but in production of tgev-neutralizing antibodies (callebaut et al., 1994) which did not protect against challenge with tgev-neutralizing antibodies were only elicited with recombinant poxviruses. expression of s antigenic site d, virulent tgev (callebaut and pensaert, 1995). by contrast, the ad-ts recombinant eliciting antiserum provid-as a fusion protein on the surface of e. coli led to induction of tgev-neutralizing antibodies when purified re-ing passive protection against challenge with virulent tgev carries s sequences derived from tgev instead combinant antigen was used as immunogen, but not when live vector was administered (bousquet et al., of prcv. the presence of 224 aa (from residue 21 to 244) in recombinant ad-ts06, which are deleted in the 1994). using salmonella typhimurium, site d has been expressed and tgev-neutralizing antibodies have been prcv, might have been critical to achieve the observed protection. this interpretation is in agreement with the elicited in serum and in mucosal areas using live recombinant bacteria (smerdou et al., 1995) but protection ex-partial protection seeing with the antiserum elicited in swine by recombinant ad-ts8, which includes the se-periments using these systems have not been reported. ad5 vectors have a high probability of inducing effective quences deleted in prcv, but at the same time indicates that larger spike protein fragments (as those including mucosal immunity against tgev, since this virus showed tropism for mucosal tissues in pigs, and the animals site a) are needed to elicite full protection. protection by recombinant adenoviruses expressing s infected with this virus experienced neither respiratory nor intestinal disorders (callebaut and pensaert, 1995; protein fragments lacking site a extends the results recently reported (tulboly et al., 1994) on s protein exprescallebaut et al., 1994; torres et al., 1995) . sion using baculoviruses. these authors showed induction of tgev-neutralizing antibodies only with recombi-acknowledgments nants expressing s protein fragments spanning 745 aa we thank granja cantoblanco de animales de laboratorio (hospital or more from the amino-terminus, that is, with s protein general g. marañó n, comunidad de madrid) and laboratorios sobrino cyanamid (olot, girona) for providing inbred and outbred swine, re-fragments including site a, but not with s protein fragantigenic differentiation between transmissible gastroenteri-tgev expressed by recombinant baculovirus tgev coronavirus orf4 encodes a membrane protein that is incorporated callebaut major antigen of porcine respiratory coronavirus virus retaining spike glycopolypeptide s2 but not s1 adenovirus-based expression is unable to induce virus-neutralizing or haemagglutination-inhibiting vectors and recombinant vaccines a new technique for the antigenic structure of the e2 glycoprotein from transmissible gastroassay of infectivity of human adenovirus 5 dna mcderinfection by affinity-purified spike glycoprotein of murine hepatitis mott charactermurine coronavirus spike glycoprotein and evidence that it forms istics of a human cell line transformed by dna from human adenovipart of a complex tridimensional structure the prepara-epitope specificity of protective lactogenic immunity against swine tion of 131 i-labelled human growth hormone of high specific radioactransmissible gastroenteritis virus 5367-herpes simplex virus igg fc receptor induced using recombinant 5375. adenovirus vectors expressing glycoproteins e and i techniques for human adenovirus vector construction and character cell biology: a four major antigenic sites of the coronavirus transmissible gastroen-laboratory handbook high efficiency expression of the surface glycoprotein gp195 of porcine transmissitransformation of e. coli by high voltage electroporation. nucleic ble gastroenteritis virus studies of fazakerley tgev s protein gp195 expressed in e. coli and by a tge-vaccinia the v5a13.1 envelope glycoprotein deletion mutant of mouse hepativirus recombinant monoclonal antibodies to the matrix (e1) glycoprotein (1986). critical epitopes in transmissible gastroenteritis virus neutralof mouse hepatitis virus protect mice from encephalitis. virology 168, ization isolation of adenovirus type 5 host range deletion mutants defective for transformation of rat embryo s missible gastroenteritis virus immunogenic peptide comprising a mouse hepatitis virus a59 b-cell epitope and an influenza virus t-cell epitope protects residues involved in the formation of the antigenic sites of the s against lethal infection human adenovirus cloning vectors based on infectious acid changes in the viral glycoprotein m affect induction of alpha bacterial plasmids processing and antigenicity of entire and anchor-free spike glycoprotein-s of coronavirus lecomte antigenic homology among coronaviruses retype 3-induced acute disease by an anti-nucleoprotein monoclonal antibody glycoprotein of vsv by infectious adenovirus vectors selection of mimotopes from a random sequence expression library by monoclonal antibodies against transmissible gastroentritis coro molecular cloning: tis coronavirus s protein fused to e. coli heat-labile toxin b subunit in attenuated salmonella for oral immunization. submitted for publi-a laboratory manual studies on transmissible gastroenteritis of swine. ii. selected characteristics of a cytopatho-in ''immunochemistry of viruses. ii. the basis for serodiagnosis and vaccines mechanisms of transmissible gastroenteritis coronavirus neutralization rus based vector using the firefly luciferase as a reporter gene protective effect of monoclonal antibodies on lethal mouse hepatitis virus infection hepatitis virus-4 (strain jhm): correlation with biological activities 276, and their use in protection against transmissible gastroenteritis virus immunogenicity of the s protein of transmissible gastroenteritis virus ex-f. l. (1990). a recombinant human adenovirus vaccine against rabies intracellular processing of the porcine coronavirus transmissible gastroenteritis virus spike protein strain of transmissible gastroenteritis virus: comparison of reactivity with virulent and attenuated virus mouse hepatitis ''diseases of swine iowa state univ. press, virus spike and nucleocapsid proteins expressed by adenovirus vector protect mice against a lethal infection a spike protein-dependent cellular factor other than the viral receptor is required for mouse hepatitis virus entry key: cord-312210-3x9s3g8n authors: stoian, ana; rowland, raymond r.r.; petrovan, vlad; sheahan, maureen; samuel, melissa s.; whitworth, kristin m.; wells, kevin d.; zhang, jianqiang; beaton, benjamin; cigan, mark; prather, randall s. title: the use of cells from anpep knockout pigs to evaluate the role of aminopeptidase n (apn) as a receptor for porcine deltacoronavirus (pdcov) date: 2019-12-24 journal: virology doi: 10.1016/j.virol.2019.12.007 sha: doc_id: 312210 cord_uid: 3x9s3g8n the coronaviruses, porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev), and porcine deltacoronavirus (pdcov) represent important sources of neonatal diarrhea on pig farms. the requirement for aminopeptidase n (apn) as a receptor for tgev, but not for pedv, is well established. in this study, the biological relevance of apn as a receptor for pdcov was tested by using crispr/cas9 to knockout the apn gene, anpep, in pigs. porcine alveolar macrophages (pams) from anpep knockout (ko) pigs showed resistance to pdcov infection. however, lung fibroblast-like cells, derived from the anpep ko pam cultures, supported pdcov infection to high levels. the results suggest that apn is a receptor for pdcov in pams but is not necessary for infection of lung-derived fibroblast cells. the infection of the anpep ko pigs with pdcov further confirmed that apn is dispensable as a receptor for pdcov. coronaviruses belong to the family coronaviridae, order nidovirales, and are important pathogens of humans and animals. coronaviruses are divided into four genera, alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. infection of neonatal pigs with the alphacoronaviruses, transmissible gastroenteritis virus (tgev) or porcine epidemic diarrhea virus (pedv), results in mal-absorptive diarrhea, which can lead to dehydration and death (madson et al., 2016; saif et al., 2012) . the first outbreak of pedv on u.s. farms in 2013 resulted in the death of nearly 7 million pigs or about 10% of u.s. pig production for that year (stevenson et al., 2013) . in 2014, a diarrhea-causing porcine deltacoronavirus (pdcov) was isolated from five pig farms in ohio (wang et al., 2014) . the m and n gene sequences were 99% identical to another pdcov, hku15, from china. since 2014, pdcov has rapidly spread throughout pig-producing regions in the u.s. to date, four host proteins, aminopeptidase n (apn), angiotensinconverting enzyme 2 (ace2), dipeptidyl peptidase 4 (dpp4), and carcinoembryonic antigen-related cell adhesion. molecule 1 (ceacam1), have been described to function as coronavirus receptors (delmas et al., 1992; li et al., 2003; raj et al., 2013; dveksler et al., 1991) . porcine apn (papn), a 963 amino acid, type ii membrane metallopeptidase, participates in the removal of nterminal amino acids from protein substrates during digestion. delmas et al. (1994) were the first to characterize an apn peptide sequence, located between amino acids 717 and 813, as required for infection of cells with tgev. the corresponding receptor region on tgev is located on the s1 subunit c-terminal domain (s1-ctd) of the tgev spike (s) protein (godet et al., 1994) . recent studies have focused on the potential role of apn as a receptor for pdcov. the data have yielded three different observations and conclusions. in 2018, wang et al. showed that cell lines, such as vero and bhk-21, were resistant to infection with pdcov and tgev. permissiveness was acquired after transfection of both cell lines with a plasmid expressing a papn cdna. pdcov and tgev growth curves on the transfected cells showed a yield greater than 5 log 10 tcid 50 /ml. the authors concluded that papn is a functional receptor for pdcov. also, in 2018, zhu et al., reported similar results. however, hela cells, which were papn-negative, and resistant to tgev, supported pdcov infection. furthermore, a knockout of papn expression in the porcine ipihttps://doi.org/10.1016/j.virol.2019.12.007 received 10 october 2019; received in revised form 16 december 2019; accepted 17 december 2019 21 cell line was prepared by using crispr to inactivate anpep, the gene which codes for apn. the anpep ko ipi-21 cells were completely resistant to tgev, but retained permissiveness for pdcov. the authors concluded that "papn is likely not a critical functional receptor for pdcov, although it is involved in pdcov infection". the third outcome related to papn and pdcov is found in li et al. (2018) , who evaluated the permissiveness of anpep knockout (ko) st cells to infection with tgev and pdcov. st cells lacking papn were completely resistant to tgev but retained a small capacity to support pdcov infection. the authors concluded that pdcov utilizes papn as a primary receptor for virus attachment, but the presence of a second co-receptor contributes to the permissiveness of cells for infection. furthermore, the pdcov coreceptor can retain function independent of papn. in this study, we investigated the role of papn as a receptor for pdcov by evaluating the permissiveness of different cell populations derived from the lungs of anpep ko and wild-type (wt) pigs. porcine alveolar macrophages from anpep ko pigs were resistant to pdcov and tgev. however, lung fibroblast-like cells, which appeared following the outgrowth of anpep ko pam cultures, were susceptible to pdcov but remained resistance to tgev. furthermore, anpep ko pigs supported pdcov infection. the results support a role for papn as a receptor for pdcov, but the presence of a second, unknown receptor or factor can substitute for papn function. porcine alveolar macrophages (pams) from anpep ko and wt pigs were used to evaluate the permissiveness of cells for infection with pdcov and tgev. as shown in figure panels 1a and 1b, the anpep wt pams were permissive for infection with pdcov and tgev, while no infected tgev or pdcov cells were detected in pams from the anpep ko pigs. the results showed that pams from pigs lacking a functional anpep gene are resistant to tgev and pdcov infection. the long-term culture of pam cultures typically results in the outgrowth of a minor population of lung mesenchymal stem cells (mscs), which exhibit a fibroblast-like morphology (khatri et al., 2015) . by two weeks, the pam cultures were completely overgrown with fibroblastlike cells along with the disappearance of macrophages. the fibroblastlike cells from the wt and ko pigs were passaged at least two times and then infected with pdcov or tgev. the fibroblast-like cells derived from the anpep wt pigs were permissive for both tgev and pdcov ( fig. 1c and d) . however, the anpep ko fibroblast-like cell cultures showed no evidence of tgev infection, but showed several pdcovinfected cells, all possessing a fibroblast-like morphology. these data confirmed the requirement of apn for the permissiveness of the fibroblast cells to tgev; however, the absence of apn had no effect on infection of fibroblast-like cells with pdcov. the permissiveness of anpep wt and ko pams and fibroblast-like cells for tgev and pdcov infection was also evaluated by determining percent virus antigen-positive cells after infection with different mois of virus. the results for wt pams are shown in fig. 2a . an moi = 1 produced 20% and 80% antigen-positive cells for pdcov and tgev, respectively. the corresponding virus dilution endpoints were 0.0001 and 0.1 moi. increasing the moi to 10 increased the percent pdcov the results for virus yields on anpep wt and ko fibroblast-like cells are shown in fig. 3 . at an moi = 1, pdcov grew to near equal levels on fibroblast-like cells derived from the anpep wt and ko pigs; whereas, tgev could only be propagated on fibroblast-like cells derived from the anpep wt pigs. previous work from our lab showed that anpep ko pigs are completely resistant to infection with tgev (whitworth et al., 2018) . we tested the ability of pdcov to infect anpep ko pigs. the results for virus infection are summarized in table 1 . all pigs were positive for pdcov nucleic acid at one day after infection. on the second day, two anpep wt and three ko pigs remained rt-pcr-positive. by three days after infection, only one pig, an anpep ko pig, was positive for pdcov nucleic acid. by four days after infection, all pigs were negative for virus nucleic acid in feces. serum samples obtained at three days after infection were negative for virus nucleic acid (data not shown). at four days after infection, one wt and one ko pigs were removed from the study and necropsied. rt-pcr amplification of pdcov nucleic acid in intestines and mesenteric lymph nodes showed that both pigs were negative for pdcov. the presence of pdcov infection was also assessed by virus-specific neutralizing activity measured in serum at the termination of the study, 14 days after infection. the results, summarized in table 1 , showed that two of the remaining six pigs seroconverted; one wt and one ko pig. clinical signs, such as diarrhea and fever, were not detected in any of the infected wt or ko pigs. the absence of clinical signs is likely a result of the older-age pigs used in the infection study. together, the rt-pcr and serological results showed that anpep ko pigs are not resistant to infection with pdcov. the results from this study showed no pdcov infection of pams obtained from anpep ko pigs (see fig. 1a ), which supports the observations of wang et al. (2018) , who concluded that apn is a receptor for pdcov. in contrast, the pdcov-positive infection results for fibroblast-like cells derived from the anpep ko cultures support the observations of zhu et al. (2018) , who concluded that apn is not a functional receptor for pdcov (see fig. 1c ). when taken together, our data support the observations of li et al. (2018) who concluded that there are apn-dependent and apn-independent receptors for pdcov. we hypothesize that pams possess only the apn receptor, while the lung-derived fibroblast-like cells possess both receptors. however, since the apn-independent receptors for pdcov have not been identified, we currently cannot easily prove this hypothesis and it remains to be elucidated in future studies. ultimately, the importance of papn as a receptor for pdcov is determined by the permissiveness of anpep ko pigs for infection. the results in table 1 , showing positive results for the presence of pdcov nucleic acid in the feces of infected anpep ko pigs confirms the results obtained following the infection of ko fibroblast-like cells. these data support the notion that apn is not required for pdcov infection of pigs. however, the conclusion that anpep ko pigs retain permissiveness for infection with pdcov comes with a few caveats. for example, all fecal samples on day 1 after infection were positive for pdcov nucleic acid and by 3 days, the virus was only detected in one ko pig. intestinal tissues from the two pigs tested, one wt and one ko, were negative for pdcov. the positive results obtained for feces can be explained by the presence of environmental contamination caused by the residual inoculum used for infection. however, support for a productive infection of the ko pigs comes from the seroconversion of one ko pig at 14 days after infection (see table 1 ). a second caveat comes from the transient nature of pdcov infection in the wt and ko pigs used in this study, which made it difficult to determine if apn plays a role in the disease process. previous studies show that neonatal piglets are more susceptible than weaned pigs to enteric virus infection jung et al., 2015) . the relationship of pig age and pdcov infection outcome has not been studied in detail; however, it is speculated that pdcov infection may be age-dependent, similar to pedv, with more severe outcomes in younger piglets. in this study, 28-33 day-old anpep ko and wt pigs were used for pdcov inoculation. in future studies, a more sensitive neonatal pig infection model can be used which may yield a more accurate determination regarding the role of apn in pdcov infection of its natural host. the anpep-edited pigs used in this study were derived by breeding founder animals created using direct zygote injection of crispr/cas9 along with two crispr guides directed at exon 2 of anpep . along with sequencing of the anpep gene, the apn phenotype for each knockout allele was confirmed by the absence of cd13 expression in intestines and by the resistance of anpep ko pigs to infection with tgev (whitworth et al., 2018) . a. stoian, et al. virology 541 (2020) 136-140 the pdcov isolate, south dakota, was used for the infection of cells and pigs (vitosh-sillman et al., 2016) . pdcov stocks were prepared on st cells maintained in mem supplemented with 7% fbs, pen-strep (80 units/ml and 80 μg/ml, respectively), 3 μg/ml fungizone, 25 mm hepes mem, and 0.2 μg/ml l-1-tosylamide-2-phenylethyl chloromethyl ketone (tpck) trypsin, as described in chen et al. (2015) . after 2 h, the media was replaced with mem-fbs with antibiotics. tgev, purdue strain, was obtained from iowa state university. tgev was maintained in the same media without tpck. cells were maintained at 37°c and 5% co 2 . after 48 h, the virus was harvested and titrated on st cells. serial 1:10 dilutions of virus in triplicate were performed on a 96well plate containing st cells. twenty-four h later, the cells were fixed for 10 min in 80% acetone and air-dried. for detection of pdcov, cells were stained with anti-n protein mab sd110-121 (kindly provided by steve lawson at south dakota state university), which was diluted 1:500 in pbs with 5% goat serum (pbs-gs). detection of tgev was performed using anti-fipv3-70 mab (custom monoclonals international, usa), diluted 1:700 in pbs-gs. after 1 h incubation with antibody at 37 o c, the cells were washed with pbs and bound antibody detected with alexafluor488-labeled goat anti-mouse igg (cat. no. a-11001, invitrogen) diluted 1:400 in pbs-gs. after 1 h incubation at 37°c, the plates were washed with pbs, nuclei counterstained with propidium iodide (pi), and cells viewed under a fluorescence microscope. the 50% tissue culture infectious dose (tcid 50 /ml) was calculated according to the method of reed and muench (1938) . the recovery of porcine alveolar macrophages is described in wells et al. (2017) . lungs were removed from euthanized pigs and lavaged by pouring 100 ml of cold pbs into the trachea. pams were sedimented by centrifugation at 1200×g for 10 min at 4°c and cells re-suspended and washed once in cold sterile pbs. the final cell pellet was re-suspended in freezing medium containing 45% rpmi 1640 with antibiotics, 45% fbs, 10% dimethylsulfoxide (dmso) and stored in liquid nitrogen until use. for the infection, approximately 10 4 pams were added to each well of a 96 well plate and incubated overnight at 37 o c in 5% co 2 . the cells were gently washed to remove non-adherent cells. serial 1:10 dilutions of virus in media were added to wells in triplicate. after an overnight incubation, the cells were washed with pbs and fixed for 10 min in 80% acetone. presence of either pdcov or tgev in the infected pams was confirmed as described above. for enrichment of lung mesenchymal stem cells, pams were cultured in rpmi-1640 supplemented with 10% fbs, l-glutamine, and antibiotics. the continuous maintenance of pams for two weeks resulted in the overgrowth of cultures by fibroblast-like cells, which comprise only about 1% of the lung lavage material. the confluent cells were removed by trypsinization and passaged at least 2 times. the infection of fibroblast-like cells was carried out as described for the pam cultures. all experiments involving animals and viruses were performed in accordance with the federation of animal science societies guide for the care and use of agricultural animals in research and teaching, the usda animal welfare act and animal welfare regulations; and after approval by the kansas state university and university of missouri institutional animal care and institutional biosafety committees. three ko piglets were obtained from an anpep −/− sow mated with an anpep −/− boar. the anpep ko genotype of each piglet was confirmed by pcr and dna sequencing. to avoid unnecessary stress, the weaned piglets were transported from the university of missouri rearing facility to kansas state university at three weeks of age. three anpep wt piglets, obtained from a separate mating, were included as positive infection controls. at the time of virus infection, the ko and wt pigs were 28 and 33 days old, respectively. pigs were infected with 6.2 log 10 tcid 50 of pdcov administered as a single oral dose in 20 ml of culture medium. a 5 cm tube was attached to the end of the inoculating syringe to allow the virus to flow down the back of the throat. throughout the study, the infected wt and ko pigs were housed in the same pen to allow for continuous pig-to-pig contact. caretakers and researchers were blind as to the genotype of each pig. fecal swabs were collected daily from each pig beginning one day prior to infection until the end of the study. each swab was placed in a 15 ml conical tube containing 1 ml of mem with pen-strep and fungizone. the tube was vortexed briefly to mix the swab contents, aliquoted into 1.5 ml cryovial storage tubes, and stored at −80°c. blood for serum was collected into serum separator tubes at 0, 3, 7, 10 and 14 days after infection. total rna was extracted from fecal and serum samples using a magmax tm -96 total rna isolation kit (invitrogen tm ) according to the manufacturer's instructions. rt-pcr was performed using a commercial kit, ez-ped/tgenatur/pdcov mpx 1.0 (tetracore) on a cfx-96 realtime pcr system (bio-rad). results were reported as ct. serum samples were serially diluted 1:2, starting at a dilution of 1:16 in 100 μl of mem supplemented with 7% fbs, antibiotics and 0.2 μg/ml tpck trypsin. one hundred microliters of diluted serum were mixed with 100 μl of pdcov south dakota an 800 tcid50 virus in 100 μl media 800 tcid 50 /100 μl on a 96 well plate and incubated 1 h at 37°c. samples were transferred to a 96-well plate of confluent st cells and incubated for 24 h at 37°c, 5% co 2 . infected cells were detected by ifa using an anti-pdcov mab. the neutralizing antibody titer was reported as the highest serum dilution at which 100% of virus infection was inhibited. pathogenicity and pathogenesis of a united states porcine deltacoronavirus cell culture isolate in 5-day-old neonatal piglets aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev determinants essential for the transmissible gastroenteritis virus-receptor interaction reside within a domain of aminopeptidase-n that is distinct from the enzymatic site cloning of the mouse hepatitis virus (mhv) receptor: expression in human and hamster cell lines confers susceptibility to mhv major receptor-binding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein comparative pathogenesis of us porcine epidemic diarrhea virus (pedv) strain pc21a in conventional 9-day-old nursing piglets vs. 26-day-old weaned pigs porcine lung mesenchymal stromal cells possess differentiation and immunoregulatory properties broad receptor engagement of an emerging global coronavirus may potentiate its diverse cross-species transmissibility angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus characterization of porcine epidemic diarrhea virus isolate us/ iowa/18984/2013, infection in 1-day-old cesarean-derived colostrum-deprived piglets dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc a simple method of estimating fifty percent endpoints coronaviruses emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences effect of porcine epidemic diarrhea virus infectious doses on infection outcomes in naive conventional neonatal and weaned pigs experimental infection of conventional nursing pigs and their dams with porcine deltacoronavirus porcine deltacoronavirus engages the transmissible gastroenteritis virus functional receptor porcine aminopeptidase n for infectious cellular entry detection and genetic characterization of deltacoronavirus in pigs replacement of porcine cd163 scavenger receptor cysteine-rich domain 5 with a cd163-like homolog confers resistance of pigs to genotype 1 but not genotype 2 porcine reproductive and respiratory syndrome virus zygote injection of crispr/ cas9 rna successfully modifies the target gene without delaying blastocyst development or altering the sex ratio in pigs resistance to coronavirus infection in amino peptidase n-deficient pigs contribution of porcine aminopeptidase n to porcine deltacoronavirus infection the work was supported by genus plc and food for the 21st century at the university of missouri. the authors acknowledge many people, including undergraduate students who made many of the experiments possible. key: cord-293635-36pmai6s authors: held, katherine s.; chen, benjamin p.; kuziel, william a.; rollins, barrett j.; lane, thomas e. title: differential roles of ccl2 and ccr2 in host defense to coronavirus infection date: 2004-11-24 journal: virology doi: 10.1016/j.virol.2004.09.006 sha: doc_id: 293635 cord_uid: 36pmai6s the cc chemokine ligand 2 (ccl2, monocyte chemoattractant protein-1) is important in coordinating the immune response following microbial infection by regulating t cell polarization as well as leukocyte migration and accumulation within infected tissues. the present study examines the consequences of mouse hepatitis virus (mhv) infection in mice lacking ccl2 (ccl2(−/−)) in order to determine if signaling by this chemokine is relevant in host defense. intracerebral infection of ccl2(−/−) mice with mhv did not result in increased morbidity or mortality as compared to either wild type or ccr2(−/−) mice and ccl2(−/−) mice cleared replicating virus from the brain. in contrast, ccr2(−/−) mice displayed an impaired ability to clear virus from the brain that was accompanied by a reduction in the numbers of antigen-specific t cells as compared to both ccl2(−/−) and wild-type mice. the paucity in t cell accumulation within the central nervous system (cns) of mhv-infected ccr2(−/−) mice was not the result of either a deficiency in antigen-presenting cell (apc) accumulation within draining cervical lymph nodes (cln) or the generation of virus-specific t cells within this compartment. a similar reduction in macrophage infiltration into the cns was observed in both ccl2(−/−) and ccr2(−/−) mice when compared to wild-type mice, indicating that both ccl2 and cc chemokine receptor 2 (ccr2) contribute to macrophage migration and accumulation within the cns following mhv infection. together, these data demonstrate that ccr2, but not ccl2, is important in host defense following viral infection of the cns, and ccr2 ligand(s), other than ccl2, participates in generating a protective response. chemokines are small (7-15 kda) cytokines capable of inducing a diverse array of biological effects that are important in both host defense and disease progression in response to infection. although initially described in induc-ing the directional migration of targeted populations of leukocytes during periods of inflammation, chemokines are now recognized as important signaling molecules in linking innate and adaptive immune responses (baggiolini, 1998; cyster, 1999; luster, 1998 luster, , 2002 ward et al., 1998) . for example, the cc chemokine ligand 2 (ccl2) is capable of regulating the pathobiology of various inflammatory disease models of humans such as asthma, multiple sclerosis, and atherosclerosis (boring et al., 1998; gosling et al., 1999 ; for reviews, see baggiolini, 2001; gerard and rollins, 2001; gu et al., 1999; mahad and ransohoff, 2003) . in addition to its potent chemoattractive effect on monocytes and macrophages, ccl2 also influences th2 polarization in response to certain antigenic challenge (gu et al., 2000; hogaboam et al., 1998; karpus and kennedy, 1997; warmington et al., 1999) . the influence of ccl2 on t cell polarization may be due to the fact that ccl2 is constitutively expressed within secondary lymphoid tissue and would be capable of affecting cellular responses following exposure to antigen (gu et al., 2000) . thus, expression of ccl2 is capable of influencing both innate as well as adaptive immune responses by regulating monocyte and t cell responses, respectively. mouse hepatitis virus (mhv) is a positive-strand rna virus that is a member of the coronaviridae family of viruses. intracranial instillation of mhv into the cns of susceptible mice results in widespread replication of virus in neurons and glia accompanied by a robust inflammatory response consisting of neutrophils, nk cells, t cells, and macrophages (williamson and stohlman, 1990) . t cells are required for reduction of viral burden within the brain and this process is mediated by secretion of ifn-g and perforinmediated lysis of infected cells parra et al., 1999 parra et al., , 2001 pearce et al., 1994; williamson and stohlman, 1990; yamaguchi et al., 1991) . clearance is incomplete and surviving mice will often develop an immune-mediated demyelinating disease characterized by viral persistence in white matter tracts accompanied by lesions of white matter damage (dales and anderson, 1995; fazakerley and buchmeier, 1993; lane and buchmeier, 1997) . instillation of mhv into the cns of mice results in a regulated expression of chemokine genes that is dictated, in part, by viral burden (lane et al., 1998) . in addition, the functional contributions of chemokines and their receptors to either host defense or disease development are dependent on the stage of disease, for example, acute or chronic (chen et al., 2001; lane, 2003a, 2003b; glass et al., 2001; lane et al., 1998 lane et al., , 2000 liu et al., 2000 liu et al., , 2001 trifilo et al., 2003) . analysis of chemokine receptor expression following mhv infection reveals that the cc chemokine receptors 2 (ccr2) and 5 (ccr5) are expressed by endogenous cells of the cns as well as by inflammatory t cells and macrophages, indicating a role for these receptors in regulating both the immune response and disease development (chen et al., 2001; lane, 2003a, 2003b; glass et al., 2001) . indeed, mhv infection of ccr2 à/à mice resulted in a dramatic increase in mortality and enhanced viral recovery from the brain that correlated with reduced t cell and macrophage entry into the cns as compared to viral infection of ccr2 +/+ mice (chen et al., 2001) . the attenuated inflammatory response within the cns observed in mhv-infected ccr2 à/à mice was associated with a muted th1 immune response characterized by an impaired ability to generate antigen-specific t cells (chen et al., 2001) . therefore, these data clearly indicate that ccr2 is important in defense against mhv infection of the cns by promoting a protective th1 response. the present study examines the consequences of mhv infection in mice lacking the major ccr2 signaling ligand ccl2 (ccl2 à/à ) in order to determine if signaling by this chemokine is relevant in the generation of an effective immune response and host defense. this is an important question as ccr2 is currently the only known functional receptor for ccl2, yet these molecules appear to have markedly different effects on immune response to antigenic challenge. ccl2 is important in the development of th2 responses whereas ccr2 promotes a th1 response while muting a th2 response (boring et al., 1997; gu et al., 1997 gu et al., , 1999 gu et al., , 2000 hogaboam et al., 1998; traynor et al., 2000; warmington et al., 1999) . the data presented indicate that both ccl2 and ccr2 influence leukocyte migration into the cns in response to viral infection. however, ccr2 is clearly more influential in directing t cell trafficking into the cns as compared to ccl2, indicating that ligands other than ccl2 are important in signaling through the ccr2 receptor. mice were infected intracranially with 10 pfu of mhv and morbidity and mortality recorded. as shown in fig. 1a , clinical disease in ccr2 à/à mice was more severe compared to both wild type and ccl2 à/à mice starting at day 3 postinfection (pi), and this difference was significant (p = 0.005) from days 4 to 7 pi. moreover, mhv-infected ccr2 à/à mice exhibited a more rapid onset of death compared to the other experimental groups (fig. 1b) . in contrast, the severity of disease in mhv-infected ccl2 à/à mice was reduced early following infection but eventually no difference was detected as compared to wild-type mice. death was initially delayed in infected ccl2 à/à mice as compared to wild-type mice; however, by day 9 pi, there was no difference in mortality between these two groups of mice (fig. 1b) . examination of viral titers within the brains of mice indicated that ccr2 à/à mice had significantly higher titers as compared to wild-type mice at days 5 and 7 pi, and exhibited significantly higher viral titers within the brains at later times pi when compared to both wild type and ccl2 à/à mice (table 1) . both cd4 + and cd8 + t cells are required for optimal clearance of virus from the brains of mhv-infected mice lane et al., 2000; pearce et al., 1994; williamson and stohlman, 1990; yamaguchi et al., 1991) . to assess the contributions of ccr2 and ccl2 to t cell migration and accumulation within the cns of mhvinfected mice, brains were removed at defined times pi and t cell infiltration determined by flow cytometry. the data shown in fig. 2 indicate that cd4 + and cd8 + t cell infiltration into the cns of infected ccl2 à/à and ccr2 à/à is dramatically reduced as compared to wild-type mice. however, comparison of t cell trafficking within the brains of ccl2 à/à and ccr2 à/à reveals differences in the functional contributions of each molecule to t cell migration. examination of total numbers of t cells in the brains of ccl2 à/à mice indicated that cd4 + t cell infiltration into the cns is reduced by 58% (p v 0.01) and 36% at days 5 and 7 pi, respectively, as compared to wild-type mice ( fig. 2a) . cd8 + t cell trafficking into the cns of ccl2 à/à mice is reduced by 65% (p v 0.01) and 55% (p v 0.01) at days 5 and 7, respectively, as compared to wild-type mice (fig. 2b ). cd4 + and cd8 + t cell accumulation within the brains of ccr2 à/à mice is also significantly reduced at days 5 and 7 in comparison to wild-type mice (figs. 2a and b) . a 75% (p b 0.005) and 83% (p b 0.003) reduction in cd4 + t cells present within the brain was observed in ccr2 à/à mice when compared to wild-type mice at days 5 and 7 pi, respectively ( fig. 2a) . similarly, cd8 + t cell recruitment was impaired by 71% (p b 0.005) and 78% (p b 0.005) at days 5 and 7 pi in comparison to t cell levels in wild-type mice (fig. 2b) . although total numbers of t cells were reduced within the brains of ccl2 à/à mice at day 7 compared to wild-type mice, the overall percentage of cd4 + and cd8 + t cells present within the inflammatory infiltrate was remarkably similar, indicating that the frequency of t cells present within the cellular infiltrate examined was comparable between wild type and ccl2 à/à mice (figs. 2c and d). of the total cells isolated from the brains of wildtype mice at day 7 pi, cd4 + and cd8 + t cells comprised 9% and 11%, respectively, while cd4 + and cd8 + t cells accounted for 10% and 9%, respectively, in the brains of infected ccl2 à/à mice at this time (figs. 2c and d) . in marked contrast, only between 0.5% and 3% of the infiltrating cells into the cns of ccr2 à/à mice at day 7 pi were cd4 + and cd8 + t cells (figs. 2c and d) . in addition to examining t cell infiltration within the cns of mhv-infected mice, the ability of macrophages (f480 + cd45 high ) to migrate and accumulate in the brains of mice was also determined. the data shown in fig. 3 indicate that macrophage trafficking into the brains of infected ccl2 à/à and ccr2 à/à mice was reduced as compared to wild-type mice. in contrast to t cell trafficking, there was a similar reduction in the number of macrophages present within the brains in both ccl2 à/à and ccr2 à/à mice, indicating that both ligand and receptor are important in directing these cells into the cns in response to mhv infection. table 1 viral titers within brains of mhv-infected mice days postinfection viral titer log 10 (pfu/g) a n wild type infected 5 5.7 f 0.09 8 7 5.5 f 0.08 22 10-12 3.0 f 0.52 10 ccl2 à/à infected 5 5.9 f 0.09 7 7 5.7 f 0.11 9 10-12 3.1 f 0.33 7 ccr2 à/à infected 5 6.0 f 0.03 b 9 7 6.0 f 0.16 c 9 10-12 4.5 f 0.18 d 4 a all titer data are presented as mean f sem. no virus was detected from sham mice of all groups. b p v 0.002 as compared to wild type at day 5 pi; not significant compared to ccl2 à/à mice. c p v 0.003 as compared to wild type at day 7 pi; not significant compared to ccl2 à/à mice. d p v 0.01 as compared to wild type at days 10-12 pi; p v 0.02 as compared to ccl2 à/à at days 10-12 pi. wild type, ccl2 à/à , and ccr2 à/à mice (all on the c57bl/6 background) were infected ic with 10 pfu of mhv and disease severity recorded. ccr2 à/à mice exhibited an overall increase in the severity of clinical disease progression (a) as compared to wild type and ccl2 à/à mice, and this correlated with a more rapid onset and overall increase in mortality (b). results presented were from five separate experiments. wild-type mice, n = 54; ccr2 à/à mice, n = 28; ccl2 à/à mice, n = 29. data were presented as the mean f sem. clinical disease severity was significantly (*p v 0.005) worse in ccr2 à/à mice when compared to wild type and ccl2 à/à mice. in order to determine the numbers and frequencies of virus-specific t cells within the brains of infected mice, cells were isolated at day 7 pi and viral specificity determined by intracellular ifn-g staining following pulsing of cells with defined t cell epitopes. such analysis revealed a 10-fold reduction (p v 0.05) in the number of cd4 + t cells responding to the immunodominant epitope within the transmembrane (m) protein at residues 133-147 (m133-147) present within the brains of ccr2 à/à mice as compared to wild-type mice ( fig. 2a ) (xue et al., 1995) . in addition, the frequency of m133-147-specific cd4 + t cells in ccr2 à/à mice (1.9%) was also reduced (p v 0.001) in comparison with wild-type mice (3.9%) (fig. 2c ). in contrast, there was no dramatic difference in the overall number or frequency of m133-147-specific cd4 + t cells in the brains of ccl2 à/à mice (3.7%) when compared to wildtype mice (figs. 2a and c). the strain of mhv used for these studies (v5a13.1) contains a deletion within the surface (s) glycoprotein where the immunodominant cd8 + t cell epitope (s510-518) for mhv is located (castro and perlman, 1995; dalziel et al., 1986) . therefore, viral specificity for cd8 + t cells was determined by looking at cells responding to an additional cd8 + t cell epitope fig. 2 . characterization of t cell infiltration into the cns of mhv-infected mice. wild type, ccl2 à/à , and ccr2 à/à mice were infected ic with 10 pfu of mhv and t cell infiltration into the cns determined at days 5 and 7 pi. in order to determine the frequency and numbers of virus-specific t cells present within the brains, mononuclear cells were surface stained for either cd4 or cd8 and ifn-g (intracellular) expression evaluated following stimulation with either the defined cd4 epitope m133-147 or cd8 epitope s598-605. total numbers of infiltrating cd4 + and cd8 + t cells are indicated in the left-hand y-axis while numbers of antigen-specific cd4 + and cd8 + t cells are indicated in right-hand y-axis (a and b, respectively). data are presented as the mean f sem. the frequency of infiltrating cd4 + and cd8 + t cells present within the brains of mhv-infected mice is indicated in the left-hand y-axis while the frequency of antigen-specific cd4 + and cd8 + t cells is indicated in the right-hand y-axis (c and d, respectively). data are presented as the average of frequencies. results presented were from two separate experiments; n = 7 for mhv-infected wild type and ccl2 à/à mice, n = 4 for mhv-infected ccr2 à/à mice. *p v 0.01 as compared to infected wild type at the corresponding time point. **p v 0.005 to infected wild type at the corresponding time point and not significant as compared to that of ccl2 à/à mice. ***p v 0.005 as compared to infected wild type at the corresponding time point and p v 0.05 as compared to infected ccl2 à/à mice. a p v 0.05 as compared to antigen-specific cd4 + t cells from infected wild type at day 7 pi. not significant as compared to that of ccl2 à/à mice. b p v 0.01 as compared to antigen-specific cd8 + t cells from infected wild type at day 7 pi. not significant as compared to that of ccl2 à/à mice. c p v 0.001 as compared to antigen-specific cd4 + t cells from infected wild type at day 7 pi. not significant as compared to that of ccl2 à/à mice. d p v 0.04 as compared to antigen-specific cd8 + t cells from infected wild-type mice at day 7 pi. p v 0.05 as compared to antigen-specific cd8 + t cells from infected ccl2 à/à mice at day 7 pi. present within the s glycoprotein at residues 598-605 (s598-605) (castro and perlman, 1995) . similar to the cd4 + t cell results, there was an approximately 7-fold reduction (p v 0.008) in numbers of cd8 + t cells responding to the s598-605 epitope in ccr2 à/à mice and the frequency of these cells (5.1%) was also reduced (p v 0.04) as compared to wild-type mice (10.4%) (figs. 2b and d) . although numbers of s598-605-specific cd8 + t cells were reduced within the brains of ccl2 à/à mice, this was not significant compared to wild type, and the frequency (11.1%) was actually slightly higher (figs. 2b and d). expression of non-elr cxc chemokine ligands t cells are required for optimal host defense following mhv infection of the cns by reducing the viral burden. the fact that ccr2 à/à mice exhibited significantly higher viral titers within the brains and were not able to reduce the viral load as compared to both mhv-infected ccl2 à/à and wild-type mice suggested the possibility of impaired expression of t cell chemoattractants. we have previously demonstrated that the non-elr cxc chemokine ligands 9 (cxcl9-mig, monokine induced by interferon gamma) and 10 (cxcl10-ip-10, interferon inducible protein 10 kda) contribute in host defense against mhv infection by recruiting t cells into the cns following infection . examination of transcripts at days 5 and 7 pi revealed differential expression patterns for each chemokine. at day 5 pi, low levels of cxcl9 transcripts were detected in all mice examined with infected wild-type mice exhibiting the lowest levels ( figs. 4a and b) . however, by day 7 pi, cxcl9 expression was elevated in wild type, ccl2 à/à , and ccr2 à/à mice were infected with 10 pfu of mhv and macrophage infiltration determined. total cells were isolated from the brains of infected mice and sham (noninfected) mice at days 5 and 7 pi, and f4/80 + cd45 high cells determined by flow cytometry using fitc-f4/80and pe-cd45-conjugated antibodies. numbers presented indicate the total number of dual-positive cells within the gated population. data are presented as the mean f sem. results presented were from two separate experiments; n = 7 for mhv-infected wild type and ccl2 à/à mice, n = 4 for mhv-infected ccr2 à/à mice. *p v 0.004 as compared to infected wild type at day 5 pi. both wild-type and ccl2 à/à mice as compared to ccr2 à/à mice, indicating that the ability to generate substantial levels of cxcl9 transcripts during acute disease was impaired in these mice. although cxcl10 transcript levels were decreased in ccr2 à/à mice at day 5 pi when compared to wild type and ccl2 à/à mice, equivalent levels were observed in all mice by day 7 pi (figs. 4a and b) . the results presented above suggest that ccr2 may either be required for generation of antigen-specific t cells or contributes to migration of antigen-specific t cells to the brain. in order to address these possibilities, draining cervical lymph nodes (clns) were removed from mhvinfected wild type, ccl2 à/à , and ccr2 à/à mice and the frequency of antigen-presenting cell (apcs) (determined by cd11c + antigen expression) was assessed (peters et al., 2000 (peters et al., , 2001 traynor et al., 2002) . examination of cd11c + cells present within the cln at day 2 pi of all strains of mice revealed no increase in the frequency of these cells as compared to sham-infected mice (fig. 5) . by days 5 and 7 pi, all mice exhibited an increase in the percentage of cd11c + cells when compared to controls (fig. 5) . both ccr2 à/à and ccl2 à/à mice displayed an overall lower frequency of cd11c + cells within the cln as compared to wild-type mice at day 7 pi; however, these differences were not significant. we next evaluated the frequency of virusspecific t cells present in the cln at day 5 pi in order to determine if there were differences in the generation of an adaptive immune response. intracellular staining for ifn-g in response to exposure of t cells to defined cd4 and cd8 viral epitopes revealed ccr2 à/à mice had the highest frequency of antigen-specific t cells remaining in the cln at this time, followed by ccl2 à/à mice, with wild-type mice displaying the lowest frequency (fig. 6) . these data clearly indicate that generation of mhv-specific t cells is not impaired in mice lacking ccr2. the data presented in this report clearly indicate that both ccl2 and ccr2 enhance t cell and macrophage trafficking into the cns in response to mhv infection and participate in a protective immune response. however, there are dramatic differences in the level of contribution of each molecule with regards to disease outcome. virusspecific t cells lacking ccr2 were unable to migrate into the brains as efficiently when compared to t cells in wild type or ccl2 à/à mice, and this correlated with increased mortality (fig. 1) and delayed clearance of virus from the brains of ccr2 à/à mice (table 1 ). these findings indicate that ccr2 ligands other than ccl2 exhibit a greater influence in coordinating a protective response as compared to ccl2. in addition to ccl2, the ccr2 ligand ccl7 (monocyte chemoattractant protein-3, mcp-3) is also expressed within the cns of mhv-infected mice and may influence t cell trafficking (lane et al., 1998) . importantly, the data presented support and extend an earlier study by our group indicating that ccr2 signaling contributed to host defense following mhv infection of the cns (chen et al., 2001) , and clearly demonstrate that the major ccr2 ligand ccl2 is not necessary for viral clearance from the brain. in addition, these data demonstrate that lack of ccr2 signaling and disease outcome is not dependent on the mouse strain as earlier studies were performed in 129 â b6 mice whereas the current study utilized c57bl/6 mice (chen et al., 2001) . the dramatic reduction in number and frequency of antigen-specific t cells within the brains of mhv-infected ccr2 à/à suggested that either the ability of t cells to migrate in response to chemokine signals derived from within the cns was impaired or there was a dampened ability to generate antigen-specific t cells. in order to answer these questions, we examined chemokine expression within the brains as well as apc accumulation in draining cln. we have previously demonstrated that the chemokines cxcl10 and ccl5 exert potent chemotactic effects on th1 cells following mhv infection and targeted neutralization of either of these chemokines through use of antibodies or knockout mice results in increased viral titers within the brain accompanied by increased mortality (dufour et al., 2002; lane, 2003a, 2003b; . the data presented in this study clearly indicate that ccr2 also exerts a protective effect, in part, by aiding in t cell migration into the cns. analysis of the chemokine mrna expression profile within the brains of infected ccr2 à/à mice reveals muted expression of cxcl9 transcripts at day 7 pi. this may reflect the fact that t cell infiltration was reduced at this time, and given that ifn-g is important in enhancing cxcl9 gene transcription, this could explain why transcript levels are reduced (mahalingam et al., 2001) . in contrast, expression of cxcl10 was comparable between all three groups of mice at each time point examined. this is not completely surprising in that type i interferons, which are expressed within the brains of mhv-infected mice, can induce expression of cxcl10 (campbell et al., 1999; memet et al., 1991) . based on these data, it is unlikely that the diminished expression of cxcl9 within the brains of infected ccr2 à/à mice accounts for the dramatic decrease in t cell accumulation. rather, this deficiency is more likely the result of some other defect resulting from impaired ccr2 signaling. one possibility revolves around muted expression of other t cell chemokine receptors, such as cxcr3, on t cells lacking ccr2. we have previously demonstrated that mhv infection of either ccl3 à/à or ccr5 à/à mice results in altered expression of chemokine receptors on antigen-specific t cells that results in a pronounced inability of these cells to migrate to sites of infection (glass et al., 2003a; trifilo et al., 2003) . recent studies have demonstrated that distinct populations of dendritic cells exist within secondary lymphoid tissue and are responsive to chemokine signaling (aliberti et al., 2000; luster, 2001 luster, , 2002 mccoll, 2002; serbina et al., 2003) . indeed, ccr2 is expressed on professional apc including macrophages and dendritic cells, and is thought to contribute to defense following microbial challenge by enhancing recruitment as well as production of antimicrobial products such as tnf-a and no by these cells (luster, 2002; mccoll, 2002; serbina et al., 2003) . in addition, the absence of ccr2 signaling results in diminished trafficking and accumulation of dendritic cells within secondary lymphoid tissues following antigenic challenge (peters et al., 2000 (peters et al., , 2001 sato et al., 2000) . the demonstration of a pronounced deficiency in the ability to attract sufficient numbers of antigen-specific t cells to control mhv replication within the brain following infection of ccr2 à/à mice is consistent with earlier studies that suggest an important role for ccr2 in contributing to t cell activation in other models of microbial infection (peters et al., 2000 (peters et al., , 2001 traynor et al., 2002) . the present data, however, show that mice lacking ccr2 or its major ligand ccl2 do not have deficiencies in the generation of mhv-specific cd4 + or cd8 + t cells, although ccr2 à/à mice have a dramatic handicap in trafficking of these t cells from the periphery into the cns. indeed, analysis of draining cln obtained from mhvinfected mice reveals an actual increase in the frequency of virus-specific cd4 + and cd8 + t cells in ccr2 à/à mice when compared to both wild type and ccl2 à/à mice, suggesting an impaired ability to exit this tissue. this is consistent with an earlier study by our laboratory demonstrating that mhv infection of mice lacking ccl3 resulted in the retention of virus-specific cells in the cln, and this was the result of impaired expression of chemokine receptors, including cxcr3 and ccr5 that greatly aids t cells in their ability to migrate to the brain (trifilo et al., 2003) . moreover, we have shown that chemokine expression is important in the generation of effector t cells by enhancing dendritic cell migration and activation. mice lacking ccl3 exhibited a pronounced decrease in dendritic cell trafficking to draining cln, and this was accompanied by diminished expression of t cell activation factors cd40 and mhc class ii in response to mhv infection and this correlated with altered t cell responses (trifilo and lane, 2004) . a similar scenario may exist in mice lacking ccr2 as dendritic cells express this chemokine receptor. lack of ccr2 expression on this population of cells may result in diminished activation accompanied by an impaired ability to impart key signals to antigen-specific t cells that enable them to exit secondary lymphoid tissue and traffic to sites of infection. we are currently examining this possibility. our laboratory has systematically characterized the contributions of select chemokines in regulating immune cell activation, migration, and trafficking in response to instillation of mhv into the cns of susceptible mice. in addition, we have demonstrated important roles for chemokines in linking innate and adaptive immune responses to mhv infection of the cns (trifilo and lane, 2004; trifilo et al., 2003) . these studies have clearly indicated important and nonredundant roles for certain chemokines in these diverse biologic processes. importantly, the data presented highlight the complexity of chemokine signaling with fig. 6 . analysis of mhv-specific t cells within the cln of mhv-infected mice. ccr2 à/à , ccl2 à/à , and wild-type mice were infected ic with 10 pfu of mhv and cln isolated at day 5 pi, and the t cell response to virus was determined. harvested cells from experimental mice were pooled (three mice per group) and stained for either cd4 or cd8 (fitc conjugated), and ifn-g (pe conjugated) expression evaluated following stimulation with the cd4 epitope m133-147 or cd8 epitope s598-605. the percent of responding t cells from mhv-infected mice is shown. data are presented as the mean f sem and representative of three separate experiments. regards to downstream effects on the ability of antigensensitized t cells to migrate to sites of infection. identification of the ccr2 ligand(s) important in imparting information to mhv-specific t cells that enable these cells to traffic into the cns will provide information with regards to how chemokine and chemokine receptors coordinate immune responses following viral infection of the cns. mice ccl2 à/à and ccr2 à/à mice on c57bl/6 background were generated as described previously (kuziel et al., 1997; lu et al., 1998) . control wild-type c57bl/6 mice were purchased from the national cancer institute. all mice were bred and housed under specific pathogen-free conditions in enclosed filter-top cages. mouse hepatitis virus (mhv) stain v5a13.1 [containing a 142 amino acid deletion in the surface (s) glycoprotein] was used in all studies described (dalziel et al., 1986) . agematched (6-8 weeks) mice were anesthetized by inhalation of methoxyflurane (pitman-moor, inc., washington crossing, nj), followed by intracerebral (ic) injection with 10 pfu of mhv-v5a13.1 suspended in 30 al of sterile saline. control (sham) animals were injected with sterile saline alone. mice were sacrificed at scheduled time points, and brains and draining clns were collected. one-half of each brain was used for plaque assay on the dbt astrocytoma cell line to determine viral burden and the remaining halves used for other experimental procedures (chen et al., 2001; hirano et al., 1978; liu et al., 2000) . following infection with virus, mice were evaluated for signs of clinical disease using a previously described scale . scoring was based as follows: 0, no abnormality; 1, limp tail; 2, waddling gait and partial hindlimb weakness; 3, complete hindlimb paralysis; 4, death. mononuclear cells were obtained from brains and cln as previously described (chen et al., 2001; trifilo et al., 2003) . fluorescein isothiocyanate (fitc)-conjugated rat anti-mouse cd4 and cd8 antibodies were used to detect cd4 + and cd8 + t cells (gk1.5 and ly-2, respectively; bd pharmingen, san diego, ca). fitc-conjugated rat antimouse f4/80 (c1:a3-1, serotec, oxford, uk) was used to detect activated macrophage/microglial cells . dual staining with fitc-f4/80 and r-phycoerythrin (pe)-cd45 (30-f11; bd pharmingen) was used to distinguish infiltrating macrophages from residential microglia (ford et al., 1995; sedgwick et al., 1998) . fitc-cd11c (hl3; bd pharmingen) was used to stain dendritic cells (peters et al., 2000) . in all cases, appropriate isotypematched fitc/pe antibodies were used as controls. cells were incubated with antibodies for 1 h at 4 8c, washed, and fixed in 1% paraformaldehyde . data were acquired on a facstar (becton dickinson, mountain view, ca) and analyzed by flowjo software (tree star inc., san carlos, ca). data are presented as the percent positive within the gated population and then numerated based on the number of total live cells per tissue. intracellular cytokine staining was performed using a previously described procedure (chen et al., 2001; wu et al., 2000) . in brief, cells were collected from the brains or cln at scheduled time points and 1 â 10 6 total cells were stimulated with viral peptides (castro and perlman, 1995; xue et al., 1995) . after incubation for 6 h at 37 8c in media containing golgi stop (cytofix/cytoperm kit, bd pharmingen), cells were washed and blocked with pbs containing 10% fbs and a 1:200 dilution of cd16/32 (bd pharmingen). cells were then stained for surface antigens with either fitc-conjugated cd4, cd8, or rat-igg-2b (as control) for 1 h at 4 8c. cells were fixed and permeabilized using cytofix/cytoperm kit (bd pharmingen) and stained for intracellular ifn-g using phycoerythrin (pe)-conjugated anti-ifn-g (1:50; xmg1.2, bd pharmingen) for 1 h at 4 8c. data were acquired on a facstar and analyzed by flowjo software. data are presented as the percentage of positive cells within the gated population. the absolute numbers of antigen-specific cd4 + or cd8 + t lymphocytes were calculated by multiplying the fraction of dual-positive cells by the total number of cells obtained from the tissue. percentage of cd4/cd8 t cells that are virus specific was calculated by dividing the dual-positive population by the cd4/cd8-positive population. to detect chemokine mrna transcripts within the cns of mhv-infected mice, total 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monocyte chemotactic protein-1 and ccr2 in the development of t1 immunity the cc chemokine ligand 3 regulates cd11c(+)cd11b(+)cd8alpha(à) dendritic cell maturation and activation following viral infection of the central nervous system: implications for a role in t cell activation cc chemokine ligand 3 (ccl3) regulates cd8(+)-t-cell effector function and migration following viral infection chemokines and t lymphocytes: more than an attraction effect of c-c chemokine receptor 2 (ccr2) knockout on type-2 (schisotsomal antigen-elicited) pulmonary granuloma formation effective clearance of mouse hepatitis virus from the central nervous system requires both cd4 + and cd8 + t cells cd4 and cd8 t cells have redundant but not identical roles in virus-induced demyelination identification of a cd4 + t cell epitope within the m protein of a neurotropic coronavirus protection of mice from a lethal coronavirus infection in the central nervous system by adoptive transfer of virus-specific t cell clones this work was supported by national institute of health grants 41249 (to t.e.l.) and ca53091 (to b.j.r.), and national multiple sclerosis society grant 3278-a-3 (to t.e.l.). key: cord-291192-wm2eyaam authors: becares, martina; sanchez, carlos m.; sola, isabel; enjuanes, luis; zuñiga, sonia title: antigenic structures stably expressed by recombinant tgev-derived vectors date: 2014-08-09 journal: virology doi: 10.1016/j.virol.2014.07.027 sha: doc_id: 291192 cord_uid: wm2eyaam coronaviruses (covs) are positive-stranded rna viruses with potential as immunization vectors, expressing high levels of heterologous genes and eliciting both secretory and systemic immune responses. nevertheless, its high recombination rate may result in the loss of the full-length foreign gene, limiting their use as vectors. transmissible gastroenteritis virus (tgev) was engineered to express porcine reproductive and respiratory syndrome virus (prrsv) small protein domains, as a strategy to improve heterologous gene stability. after serial passage in tissue cultures, stable expression of small prrsv protein antigenic domains was achieved. therefore, size reduction of the heterologous genes inserted in cov-derived vectors led to the stable expression of antigenic domains. immunization of piglets with these tgev vectors led to partial protection against a challenge with a virulent prrsv strain, as immunized animals showed reduced clinical signs and lung damage. further improvement of tgev-derived vectors will require the engineering of vectors with decreased recombination rate. the order nidovirales comprises enveloped single-stranded, positive-sense rna viruses. the nidovirales order includes the coronaviridae family that contains viruses with the largest known rna genome, of around 30 kb (enjuanes et al., 2008) . coronavirus (covs) infect a wide range of mammalian and avian species. the development of efficient cov reverse genetics systems (almazan et al., 2006 (almazan et al., , 2013 (almazan et al., , 2000 (almazan et al., , 2014 casais et al., 2001; thiel et al., 2001; yount et al., 2003 yount et al., , 2002 makes them promising expression vectors, with several advantages over other viral expression systems. covs replicate in the cytoplasm without a dna intermediary, making integration of the virus genome into the host cell chromosome unlikely (lai and cavanagh, 1997) . in addition, these viruses have the largest rna virus genome and, in principle, have room for the insertion of large foreign genes (enjuanes et al., 2005; masters, 1999) . as covs in general infect both respiratory and enteric mucosal surfaces, they may be used to target the antigen to these areas, stimulating the mucosal immune system to induce a pleiotropic secretory immune response, including lactogenic immunity . in fact, it has been described that a pleiotropic secretory immune response is best induced by the stimulation of gut associated lymphoid tissues (saif, 1996) . moreover, the tropism of covs may be engineered by modifying the spike (s) gene (casais et al., 2003; sanchez et al., 1999) , and non-pathogenic cov strains infecting most species of interest (human, porcine, bovine, canine, feline, and avian) are available and therefore are suitable to develop safe virus vectors (cavanagh et al., 2007; ortego et al., 2002) . in fact, several studies have reported the construction of covderived viral vectors expressing high levels of heterologous proteins, including reporter and viral proteins (bentley et al., 2013; ribes et al., 2011; shen et al., 2009 shen et al., , 2003 . foreign gene expression levels can be regulated by the use of different transcription-regulating sequences (trss) ranging from intermediate to high gene expression levels (alonso et al., 2002a) . in addition, our group has recently identified an optimized transcription-regulating motif, enhancing by 5-fold the mrna levels of a viral gene, which can be used in expression vectors based in cov genomes (mateos-gomez et al., 2011) . additionally, a combination of these trss could be used to drive the expression of two or three heterologous genes from just one infectious cdna (i.e., dicistronic or tricistronic vectors). genetic stability of a heterologous gene within the viral vector is essential for its development as a live immunization vector. in general, the stability of heterologous genes is high for dna viruses and negative rna viruses, in which the low level of recombination contributes to the maintenance of the inserted foreign genes (bukreyev et al., 2006) in contrast, positive rna viruses are highly prone to recombination, both homologous and non-homologous (alejska et al., 2005; figlerowicz et al., 2003) leading to the loss of the inserted genes and avoiding their expression over a long time period. covs are positive rna genomes with high recombination frequency (denison et al., 2011; lai, 1996; sanchez et al., 1992) . genetic instability leading to the loss of heterologous genes has been frequently reported in cov-derived vectors, both in vitro (bentley et al., 2013; cruz et al., 2010; sola et al., 2003) and in vivo (bentley et al., 2013) . in view of the frequent instability of cov-based vectors expressing proteins of large size, we explored whether the reduction of heterologous gene size was a useful strategy to increase insert stability, by reducing the probability of the presence of toxic domains in the inserted gene or protein. in fact, the expression of small protein domains is a common strategy used to reduce toxicity when toxic proteins are expressed in bacteria (edwards et al., 2000; samuelson, 2011) . tgev infects the enteric and respiratory tissues of newborn piglets resulting in a mortality of nearly 100% (saif and wesley, 1992) . interestingly, some non-enteric tgev variants with alterations in the s protein have a tropism restricted to the respiratory tract, and show attenuated phenotype (sanchez et al., 1999) . tgev-derived vectors have been successfully engineered for the expression of green fluorescent protein (gfp). the gfp gene was expressed by replacing the non-essential genes 3a and 3b, leading to very stable (420 passages in tissue culture) high expression levels of the heterologous protein (50 μg/10 6 cells) . recombinant tgev (rtgev) vectors have been engineered for dicistronic expression of heterologous genes, such as porcine reproductive and respiratory syndrome virus (prrsv) gp5 and m proteins (cruz et al., 2010) , or rotavirus vp2 and vp6, in which formation of rotavirus virus like particles (vlps) in the cytoplasm of rtgev infected cells was observed (enjuanes et al., 2007) . tgev has been previously used as an immunization vector to confer partial protection against prrsv infection (cruz et al., 2010) . in addition, an engineered rtgev in which the tropism was modified replacing the s protein by the homologous one from mouse hepatitis virus (mhv) was used to confer protection against rotavirus infections (ribes et al., 2011) . the engineered rtgev expressing rotavirus vp7 protein was then evaluated in the mouse model. the recombinant virus triggered a humoral response via systemic (serum igg and iga) and mucosal (intestinal iga) antibodies. in addition, partial protection against rotavirus-induced diarrhea was observed in 62% of the challenged animals. porcine reproductive and respiratory syndrome (prrs) is the most important infectious disease affecting swineherds worldwide. it is characterized by reproductive failure in sows, as well as severe pneumonia in piglets (lunney et al., 2010) . the causative agent of prrs is prrs virus (prrsv) that is included in arteriviridae family, in the order nidovirales. prrsv is an enveloped, singlestranded positive sense rna virus of approximately 15 kb in length that contains 9 open reading frames (orfs). orf1a and orf1b encode the replicase non-structural proteins, while orfs 2 to 7 encode structural proteins: the small envelope protein (e), the membrane protein (m), nucleocapsid protein (n) and the glycoproteins gp2a, gp3, gp4, gp5, and the recently identified protein 5a (dokland, 2010; firth et al., 2011; johnson et al., 2011) . currently, prrs causes huge economic losses in the swine industry, but commercially available vaccines are only partially effective (charerntantanakul, 2012) . prrsv infection induces a weak innate immune response, probably contributing to the reduced and delayed subsequent humoral and cellular immune responses, and also to virus persistence (kimman et al., 2009 ). this is probably due to the limited interferon alpha (ifn-α) elicited by prrsv (albina et al., 1998; calzada-nova et al., 2010) . the knowledge on prrsv correlates of protection is limited. neutralizing antibodies against prrsv are mainly directed to gp5 protein (kim and yoon, 2008; ostrowski et al., 2002) , although neutralizing antibodies recognizing gp3 and gp4 have also been described following prrsv infection (costers et al., 2010; oleksiewicz et al., 2002; vanhee et al., 2011) . prrsv m protein is a potent inducer of t-cell proliferation in piglets infected with prrsv, and may also play a role in protection (bautista et al., 1999; jeong et al., 2010) . current vaccines against prrsv have a limited efficacy. best results have been obtained using modified live vaccines, although they have several problems such as incomplete protection, virus shedding and possible reversion to virulence (charerntantanakul, 2012) . vector-based vaccines could represent an advantage to stimulate both humoral and cell immune responses against prrsv (cruz et al., 2010) . given the potential of cov-derived vectors, and the requirement of more efficient vaccines against prrsv, the work presented here is focused on the use of tgev as a vector for the expression of prrsv antigenic combinations. the expression of prrsv small domains containing the epitopes relevant for protection would lead to a significant increase in vector stability. previous work from our laboratory has shown that rtgevs coexpressing full-length prrsv gp5 (wild type or modified) and m proteins induced partial protection against prrsv (cruz et al., 2010) . the modest results obtained may be due to the instability of gp5 protein in the rtgev system, resulting in a significant loss of gp5 expression in 8-10 passages in tissue culture. expression of full-length prrsv gp3 or gp4 proteins was also toxic for rtgev leading to the loss of the heterologous gene sequence (m. becares, s. zuñiga and l. enjuanes, unpublished results) . in this work the stability of the expression of small domains of prrsv gp3, gp4 and gp5, previously described as potentially relevant in the induction of protection against prrsv has been studied, in comparison with the expression of full-length proteins, using rtgev vectors. our results showed that reduction of the heterologous genes size inserted in the cov-derived vector is a promising strategy to achieve stable expression. additionally, as prrsv m protein was stable in rtgev, several antigenic structures were engineered using this protein as scaffold for the expression of small antigenic domains, resulting in high stability. furthermore, immunization of piglets with these live attenuated rtgev vectors partially protected against prrsv, with reduction of clinical signs and lung damage as well as a faster viremia decrease. prrsv m protein is a long non-glycosilated membrane protein of around 170 amino acids, which is the most highly conserved structural protein of prrsv (meng et al., 1995) and has been involved in the induction of t-cell response against prrsv (bautista et al., 1999) . a rtgev vector expressing prrsv m protein was generated encoding prrsv m gene in the location previously occupied by non-essential genes 3a and 3b. prrsv m gene expression was driven by the transcription-regulating sequence of gene 3a (trs 3a ) (fig. 1a) . a rtgev-s 7.1 -trs 3a -m was recovered, with a titer of 10 8 pfu/ml, as expected for rtgev viruses. in order to test the stability of prrsv m protein expressed by this vector, cloned viruses were serially passaged in tissue culture and the maintenance of the heterologous gene was evaluated at different passages by the analysis of plaque-purified viral clones. the presence of the heterologous sequence in the viral genome was evaluated by rt-pcr, using specific primers flanking the insertion region. after 8 and 16 serial passages of the rtgev-s 7.1 -trs 3a -m, all the isolated viral clones still contained m gene sequence (fig. 1b) , with the expected size and sequence as revealed by pcr product sequencing. in addition, all the isolated clones expressed m protein mrna (fig. 1b) , confirming m gene stability in rtgev system. moreover, m protein expression was analyzed by immunofluorescence in st cells infected at moi 0.5. m protein was expressed in 96% of the infected cells (fig. 1c) , with expression levels remaining constant through the passages (data not shown). altogether, these results indicated that expression of prrsv m protein in rtgev was fully stable. expression of small domains of prrsv gp5 protein using rtgev netralizing antibodies recognizing gp5 are considered the most relevant for protection, with the epitope responsible for the neutralization located in the ectodomain of gp5 protein (kim and yoon, 2008; ostrowski et al., 2002) . previous studies from our group indicated that the partial protection observed after immunization with rtgevs expressing full length gp5 protein, both wild type or glycosylation mutants, was probably due to the toxicity of this protein in rtgev system, leading to heterologous gene loss with passages (cruz et al., 2010) . as a consequence, we decided to evaluate the expression of small domains containing epitopes potentially relevant in protection, as the reduction of potential toxic domains could increase vector stability. in a first approach, a rtgev co-expressing gp5 ectodomain (gp5ecto) and m protein was engineered. gp5ecto transcription was driven by the trs 3a , and that of m protein by an optimized trs partially derived from gene n trs (trs 22n ) (alonso et al., 2002a) . m protein was included in the rtgev construct because it was fully stable and we previously observed that it increased gp5 stability, probably by forming the heterodimer observed in the native virus (cruz et al., 2010) . in fact, we postulate that the expressed gp5 domains and m protein could form a heterodimer similar to the one observed in the virus, what may be important for its immunogenicity. gp5ecto consisted in the 68 most n-terminal amino acids of the olot91 gp5 protein, which according to bioinformatics predictions cover the ectodomain of the protein. this domain included the protein motifs relevant in protection, such as the immunodominant epitope and the epitope genomic rna (grna) and subgenomic mrna (mrna) encoding prrsv m protein were detected. the arrow indicates the expected size of the corresponding pcr product. numbers on the left indicate the molecular weight markers (mw) size in base pairs. (c) immunofluorescence analysis of st cells infected with the passage 16 rtgev-s 7.1 -trs 3a -m at 8 hpi. a polyclonal antibody specific for tgev and a secondary antibody staining red were used to identify virus-infected cells. expression of prrsv m protein was detected with a monoclonal antibody and a secondary antibody staining green. critical in neutralization as well as the glycosylation sites ( fig. 2a) . to allow gp5ecto protein detection, a hemaglutinin (ha) tag was fused to gp5 protein domain ( fig. 2a ). this tag is small (9 amino acids) and was previously used for cov protein tagging, without showing any toxicity (alvarez et al., 2010) . rtgev-s 7.1 -trs 3a -gp5ecto-trs 22n -m was recovered with titers similar to those of wt virus. the stability of gp5ecto domain with virus passages was analyzed by rt-pcr analysis of isolated clones. after 8 passages in tissue culture, 80% of the isolated clones contained the gp5ecto sequence ( fig. 2b ) and expressed the corresponding mrna (data not shown), representing a modest increase of stability compared with gp5 full-length (60% stable) (fig. 2b) . unfortunately, in both cases, the heterologous gp5 sequences were lost at passage 16 (data not shown), indicating that gp5ecto long-term stability did not represent a sufficient improvement as compared to full-length gp5. similar conclusions were extracted from immunofluorescence analysis of protein expression (data not shown). in a second step, an additional reduction in gp5 size was designed, by eliminating the predicted signal peptide of gp5, whose cleavage is controversial (thaa et al., 2013; wissink et al., 2003) . the resulting 34 amino acid fragment of gp5 protein (gp5fr) was inserted in rtgev, leading to rtgev-s 7.1 -trs 3a -gp5fr-trs 22n -m virus. this small domain contained the gp5 epitope critical in neutralization, glycosylation sites and the cysteine residue involved in the gp5-m heterodimer formation. for gp5fr detection a flag tag was fused at the carboxi-terminus ( fig. 2a ). this flag tag has been successfully used in cov protein tagging (alvarez et al., 2010) . the additional size reduction of the gp5 fragment cloned in rtgev led to an improvement in heterologous gene stability after 8 passages in tissue culture, with 90% of the independent clones containing gp5fr sequence (fig. 2b ) and expressing gp5fr mrna (data not shown). moreover, long-term stability was significantly improved, with up to 50% of the isolated clones stably maintaining gp5fr after 16 passages in tissue culture (data not shown). studies on prrsv immunobiology have revealed that prrsv neutralizing antibodies recognized epitopes within the minor structural glycoproteins gp2a, gp3 and gp4 (costers et al., 2010; oleksiewicz et al., 2002) . rtgevs were engineered expressing these proteins, alone or in various combinations, including the tricistronic expression of gp2a, gp3 and gp4. none of those proteins was stably expressed by rtgev vectors, with prrsv gp3 protein resulting extremely toxic for rtgev system, leading to its expression loss in early stages (m. becares, s. zuñiga and l. enjuanes, unpublished results). the recent identification of antigenic, linear domains in gp3 and gp4 (costers et al., 2010; vanhee et al., 2011) allowed the application of the small domain expression strategy to these proteins. fusion domains including gp3 or gp4 epitopes critical in neutralization, flanked by a few amino acids (table 1) , and preceded by the corresponding signal peptide were designed. these peptidic domains consisting of 55 and 50 amino acids of gp3 (gp3fr) and gp4 (gp4fr), respectively, were fused to the flag tag at their c terminus end (fig. 3a) . the gp3 and gp4 fragments were cloned in tgev genome in the location previously occupied by non-essential genes 3a and 3b and their transcription was driven by the trs 3a . recombinant viruses rtgev-s 7.1 -trs 3a -gp3fr and rtgev-s 7.1 -trs 3a -gp4fr were recovered with titers similar to those of the wt virus. the stability of the recombinant viruses was analyzed after 8 and 16 passages in tissue culture, by studying 10 plaque-purified clones by rt-pcr. all the clones maintained the heterologous gene sequence, and (gp5ecto), and gp5 fragment (gp5fr) that comprises the ectodomain lacking the signal peptide (sp). immunodominant epitope (ide) and epitope critical in neutralization (ecn), n-glycosylation sites (yellow), and the cysteine involved in gp5-m heterodimer formation (red) are also shown. gp5ecto and gp5fr included an ha or flag tag, respectively, for their detection (tg, blue). (b) rt-pcr analysis of ten clones from plaque-purified passage 8 rtgev-s 7.1 -trs 3a -gp5-trs 22n -m (gp5), rtgev-s 7.1 -trs 3a -gp5ecto-trs 22n -m (gp5ecto) and rtgev-s 7.1 -trs 3a -gp5fr-trs 22n -m (gp5fr) viruses. the arrow indicates the expected size of the corresponding pcr product. numbers on the left indicate the molecular weight markers (mw) size in base pairs. lower size bands (indicated by red asterisks) correspond to deletion products from heterologous gene, meaning genomic instability. numbers on the right indicate the overall stability of each construct. expressed the corresponding mrna (fig. 3b ), indicating that both gp3fr and gp4fr were fully stable in the rtgev vector. protein detection using anti-flag antibody failed for gp3fr, gp4fr and gp5fr, both in immunofluorescence and western blot assays (data not shown). altogether, these data revealed that heterologous gene size reduction led to a drastic increase in the stability of rtgev vectors. m protein is the most conserved structural protein among prrsv strains (kapur et al., 1996; murtaugh et al., 1998) and it is the main inducer of virus-specific t-cell response (bautista et al., 1999; jeong et al., 2010) . our results indicated that m protein was fully stable in rtgev (see above). therefore, we postulated that m protein could be used as a scaffold for the expression of small antigenic domains. as a proof of principle, the gp3 epitope critical in neutralization (ecn) domain was selected for expression fused to m protein. two exposed locations into m protein were predicted using tmpred transmembrane topology prediction algorithm (hofmann and stoffel, 1993) : the n-terminus and a loop comprising amino acids 63 to 70. gp3 ecn was inserted at these m protein locations, leading to chimeric structures, gp3ep-ntermm and gp3ep-mloop, respectively (fig. 4a) . these chimeric genes were cloned into rtgev vector, and recombinant viruses rtgev-s 7.1 -trs 3a -gp3ep-ntermm and rtgev-s 7.1 -trs 3a -gp3ep-mloop were rescued with titers similar to those of the parental virus. the stability of the recovered viruses was analyzed. after 8 or 16 passages in tissue culture 10 independent clones were screened by rt-pcr. all the independent clones maintained the heterologous gene sequence (data not shown) after 8 passages, whereas after 16 passages 90% and 100% of rtgev-s 7.1 -trs 3a -gp3ep-ntermm and rtgev-s 7.1 -trs 3a -gp3ep-mloop, respectively, contained the heterologous gene sequence and expressed the corresponding mrna (fig. 4b) . in order to evaluate stability and expression levels of the chimeric proteins, double immunofluorescence was performed on cells infected with passage 16 rtgev-s 7.1 -trs 3a -gp3ep-ntermm and rtgev-s 7.1 -trs 3a -gp3ep-mloop viruses. m protein scaffold was detected in 95% of infected cells in both cases (fig. 4c ). this detection level was similar to that observed in rtgev-s 7.1 -trs 3a -m expressing full-length wt m protein (see above). in contrast, flag epitope was detected in 92% and 63% of the rtgev-s 7.1 -trs 3a -gp3ep-ntermm and rtgev-s 7.1 -trs 3a -gp3ep-mloop infected cells, respectively (fig. 4c) . these results strongly suggested that m protein n-terminus was a more exposed location, and therefore better to present antigens. this data is in agreement with previous observations demonstrating that arterivirus m protein is tolerant to manipulations of its ectodomain (verheije et al., 2002) . in order to evaluate the protection provided by rtgevs expressing prrsv antigens, the rtgevs that showed an increased stability in cell culture were tested in vivo. for that purpose, rtgev-s 7.1 -trs 3a -m, rtgev-s 7.1 -trs 3a -gp5fr-m, rtgev-s 7.1 -trs 3a -gp3fr, rtgev-s 7.1 -trs 3a -gp4fr, and rtgev-s 7.1 -trs 3a -gp3ep-ntermm were selected for in vivo experiments. two groups of twelve days-old piglets were inoculated with 1 â 10 8 pfu/animal of each rtgev-s 7.1 -trs 3a -m, rtgev-s 7.1 -trs 3a -gp5fr-m, rtgev-s 7.1 -trs 3a -gp3fr, rtgev-s 7.1 -trs 3a -gp4fr, and rtgev-s 7.1 -trs 3a -gp3ep-ntermm (immunized group), or rtgev (non-immunized group), respectively, by three routes: oral, nasal and intragrastic. previous data from our group indicated that, in these conditions, the virus in the inoculum reached the target organs (respiratory and digestive tracts) and replicated to high titers (cruz et al., 2011; sanchez et al., 1999) . a boost was performed 2 weeks after inoculation using the same conditions. two weeks later, a challenge was performed with 1 â 10 6 tcid 50 of prrsv olot91-like virulent strain. a control group was inoculated with 1 â 10 8 pfu/animal of rtgev and boosted two weeks later, but not challenged. pigs were monitored for clinical signs, focusing on respiratory symptoms such as tachypnoea, and abdominal breathing. prrsv infection resulted in moderate fever, depression and respiratory signs that persist from days 5 to 25 after challenge (fig. 5a) . the percentage of animals showing respiratory symptoms was significantly higher in the non-immunized group than in the immunized group (fig. 5a) . moreover, the average weight gain, which was reduced in challenged animals, was higher in immunized animals than in non-immunized animals between 21 and 28 days post-challenge (data not shown). lung damage was analyzed by histopathology of lungs from five randomly chosen piglets per group. lungs from challenged piglets exhibited features that are characteristic of prrsv infection such as pneumocyte hypertrophy and hyperplasia, and intra-alveolar accumulation of cell debris (fig. 5b, left panels) . the lungs from immunized animals showed a lower degree of lung damage than those from non-immunized piglets (fig. 5b, right panel) , indicating a certain degree of protection. the lower extent of lung inflammation observed in immunized animals was in agreement with the lower levels of proinflamatory cytokine il-8 observed in vaccinated animals' sera (fig. 5c) . immunized animals showed a moderate increase in il-8 by 3 days post-challenge (31 dpi, as shown in the figure), but levels rapidly returned to normal, while non-vaccinated animals showed a higher elevation of this cytokine, that continued at elevated levels during the experimental infection. altogether, these results suggested that rtgev vectors expressing prrsv antigens conferred partial protection against prrsv infection. to further analyze the protection conferred by rtgevs expressing prrsv antigens, prrsv viremia was analyzed by rt-qpcr at different times post-challenge. similar virus titers in serum were obtained in all challenged animals at the initial stages postchallenge (28-42 dpi) (fig. 6) . interestingly, a significant reduction in virus titer was observed in the immunized group at 21-28 days post-challenge (49-56 dpi). in order to evaluate the potential of rtgevs stably expressing prrsv antigens as inducers of immunity against prrsv, the humoral response was analyzed at different times postinoculation. the antibody response against the tgev vector, prrsv virus and prrsv individual proteins expressed by rtgevs were determined by elisa. all the animals elicited a high humoral immune response against tgev indicating that the vector infected target tissues as expected, even though the piglets presented preexisting anti-tgev antibodies (data not shown). seroconversion against total prrsv was observed in all challenged animals by day 10 after infection with the virulent virus, while for individual gp3, gp4 and m protein it was detected by day 14 after challenge. in all the above-mentioned cases, no differences were observed between immunized and non-immunized animals (data not shown). the humoral response against prrsv n protein followed identical pattern to that obtained for anti-prrsv antibodies (data not shown). these data indicated that anti-prrsv total antibodies response was most likely directed against n protein and did not play a role in protection, in agreement with previous reports showing an early non-neutralizing antibody response obtained after prrsv infection (mateu and diaz, 2008) . interestingly, a higher and faster antibody response against gp5 protein was found from day 42 post-immunization in immunized animals as compared to the non-immunized ones (fig. 7a) . the neutralizing antibody response was evaluated in sera from immunized and non-immunized animals at 14, 21 and 28 days postchallenge. non-immunized animals showed higher levels of prrsv neutralizing antibodies than the immunized ones (fig. 7b ). this data, suggested a less effective prrsv infection in the immunized piglets, supporting partial protection against prrsv infection. in this study rtgev was engineered for the expression of small protein domains relevant in immune response against prrsv. previous results from our group indicated that instability of certain heterologous gene expression by rtgev might represent an important limitation for its use as an immunogenic vector. the expression of small protein domains was used as a strategy to improve heterologous gene stability. stable expression of protein antigenic domains contained in highly unstable full-length proteins was achieved. additionally, as full-length prrsv m protein was stably expressed by rtgev, it was used as a scaffold for the generation of chimeric proteins that exposed other prrsv antigens, resulting in highly stable expression. therefore, size reduction of the heterologous genes inserted in cov-derived vectors resulted in a promising strategy to achieve stable expression. protection experiments showed that rtgev, stably expressing prrsv antigenic structures, elicited partial protection against prrsv, with a reduction of clinical signs and lung damage in immunized piglets. the potential of cov-derived vectors as systems for gene delivery has been limited due to its restricted stability. in general, genetic stability is highly dependent on the nature of the foreign gene, with some inserts maintained at least twenty passages whereas others are lost at passage two (de haan et al., 2005; enjuanes et al., 2007; shen et al., 2009; sola et al., 2003) . in addition, other factors affect genetic stability of recombinant covs, such as the insert size (de haan et al., 2005) , and the genomic location in which it is inserted (bentley et al., 2013) . the maintenance of the inserted genes will also depend on the recombination rate, conditioned both by the insert size and the presence in the foreign sequence of regions showing homology with the virus genome, favoring homologous recombination (wang et al., 2003) . furthermore, other heterologous gene or protein characteristics may affect insert stability, leading to loss of the inserts harmful for the infected cell or virus replication. therefore, it is very difficult to predict the specific insert stability in advance, before a highly effort-consuming process to generate the cov-derived vectors expressing the heterologous antigen has been accomplished. prrsv gp5 and m genes have similar lengths (606 and 522 nucleotides, respectively) and small ectodomains exposed between three transmembrane domains. nevertheless, m protein was fully stable in rtgev vectors, while gp5 protein resulted toxic and its expression was lost after 4-5 passages of the virus in cell culture. interestingly, prrsv hydrophobic m protein resulted highly toxic in other expression systems, including bacteria and insect cells (jeong et al., 2010; plana-durán et al., 1997) . as deletion of the heterologous genes could be due to homologous recombination between the heterologous gene and tgev genome because of sequence identity, this possibility was analyzed. no statistically significant sequence identity was identified between prrsv gp5 sequence and tgev genome. in fact, analysis of the deletions observed in the unstable recovered viral clones did not show a common pattern of recombination. in constrast, random deletions were observed ranging from small deletions affecting trs, to larger ones covering most prrsv gp5 gene sequence (data not shown), that in all cases led to the loss of protein expression. additionally, the gp5 gene, when expressed alone in rtgev vectors, was lost at very early passages while it was stably maintained until passage 8 when it was co-expressed with m protein, most likely due to the formation of gp5-m heterodimers (cruz et al., 2010) . these data suggested that the instability was caused by protein toxicity affecting host cell viability or viral life cycle, rather than a negative effect of the heterologous gene sequences on virus genomic stability. this toxicity would confer selective advantage to those viral clones that did not express gp5 protein. in this work we showed that size reduction of the foreign insert significantly improved heterologous gene stability. even in the case of highly toxic inserts, such as prrsv gp3 protein, size reduction led to 100% stability. therefore, heterologous gene size reduction is a promising strategy to achieve stable expression in tgev-derived vectors and in general in covs. this effect could be due to a decrease of the probability of non-homologous recombination in shorter sequences, or to the elimination of protein domains toxically affecting the host cell or rtgev life cycle. this result is in agreement with previous studies showing that gene size affected foreign gene stability in cov vectors, as the larger firefly luciferase gene resulted less stable than the shorter renilla luciferase gene, when expressed by feline infectious peritonitis virus (fipv) vectors (de haan et al., 2005) . in this paper we used prrsv m protein, which we have shown that displays a high stability in rtgev vectors as a scaffold for the expression of small antigenic domains. this approach may be useful to modify the trafficking and accumulation of small protein domains expressed alone. in fact, protein detection using anti-flag antibody failed for gp3fr, gp4fr and gp5fr, both in immunofluorescence and western blot assays (data not shown), probably due to low accumulation of those peptides inside the cell. interestingly, gp3ep-ntermm was detected at high levels, indicating a higher accumulation in the infected cell of the chimeric protein. long-term stability of cov-derived vectors has not been systematically addressed. few proteins have been reported to be stably expressed by covs. among these, gfp was stable for more than 6 or 20 passages in tissue culture when expressed by mhv or tgev, respectively (sarma et al., 2002; sola et al., 2003) , but it showed instability in ibv-derived vectors (bentley et al., 2013) . prrsv m protein was also stably expressed by rtgev for more than 16 passages (this manuscript). proteins such as luciferase expressed by mhv and ibv-derived vectors (bentley et al., 2013; de haan et al., 2005) , prrsv gp5 protein expressed using tgev virus vectors (cruz et al., 2010) or gus when expressed by tgev minigenomes (alonso et al., 2002b) were lost at early passages. in our experience, using rtgev vectors, only around 25% of the heterologous genes were stably expressed for more than 8 passages in tissue culture [(alonso et al., 2002b; cruz et al., 2010; sola et al., 2003) , and unpublished results]. this instability is a key limiting factor in the use of cov-based vectors for the expression of full-length proteins. to improve the stability and efficacy of cov-derived vectors it would be essential to understand the factors that control the high recombination frequency of covs. to this end, a detailed analysis of cov proteins involved in genetic recombination is needed. several enzymes involved in cov rna synthesis, such as nsp13 (helicase), nsp15 (endonuclease), nsp14 (exonuclease), nsp7 and nsp8 (rna processivity components), or n protein could modulate recombination in covs. the engineering of recombination defective cov mutants by knocking-down one or several genes involved in the recombination process could be the first step to achieve stable expression of large heterologous genes. in this study, three prrsv protein domains from gp3, gp4 and gp5 proteins, recognized by neutralizing antibodies were expressed by rtgev and used as immunogens (costers et al., 2010; plagemann, 2004; vanhee et al., 2011; wissink et al., 2003) , and the humoral response elicited by these rtgevs was measured. after challenge, a faster response against gp5 protein was observed in immunized piglets. in contrast, the response against gp3 and gp4 was similar in immunized and non-immunized piglets. these data suggested that gp5 fragment was immunogenic, while gp3 and gp4 domains were antigenic but had a reduced immunogenicity. immunization of piglets with a combination of rtgev expressing prrsv antigens led to a clear reduction of clinical symptoms after challenge, a lower degree of lung damage and a faster viremia reduction. these results represent an improvement over previous vaccination experiments using rtgev vectors expressing prrsv antigens (cruz et al., 2010) . prrsv correlates of protection remain to be identified (kimman et al., 2009) , what represents an additional limitation for the development of new vaccine candidates. neutralizing antibodies seem relevant for preventing prrsv infection (lopez and osorio, 2004) , but not enough to provide full protection (murtaugh and genzow, 2011) . t-cell responses seem also required in prrsv clearance (mateu and diaz, 2008) . in the present study, immunized animals showed a significant faster recall antibody response against gp5 protein, which is generally considered the main target of neutralizing antibodies (kim and yoon, 2008; ostrowski et al., 2002) . non-immunized animals developed a higher neutralizing response after challenge with a virulent prrsv strain, what is considered as an indication of higher infection, whereas the immunized animals were significantly, although partially, protected against prrsv infection. with the available data, it is not possible to determine whether the observed protection was due to an undetectable neutralizing antibody response before challenge [even commercial available vaccines have been reported to fail in the induction of detectable levels of neutralizing antibodies before challenge (geldhof et al., 2012) ] to immune cell responses [most likely directed to m protein present in the immunization cocktail], or both. the higher gp5 protein specific antibody response was observed from day 10 post-challenge, while significant differences in neutralizing antibodies between immunized and non-immunized animals were observed between 21 and 28 days post-challenge, correlating with the differences in viremia, what suggests that the observed neutralizing response was due to a higher infection of nonimmunized swine. the relative contribution to protection of the humoral and cellular responses has not been determined. when a correlation between protection and induction of specific cytokines was analyzed, il-8 levels were significantly different between immunized and non-immunized piglets, with levels consistently higher in non-immunized animals. the exacerbated il-8 response elicited in non-immunized animals correlated with the higher lung damage observed in these animals. this result was in agreement with previous studies showing that piglets with more severe symptoms, including viremia and lung lesions, had a continuous elevation of il-8 in serum, while in animals with milder symptoms il-8 levels returned to normal by 7 dpi (petry et al., 2007) . higher, but not significant, levels of ifn-α were also observed in immunized animals compared to non-immunized animals (data not shown), at day 3 post-prrsv challenge. this result suggested that immunized animals developed a higher innate immune response, which nevertheless did not seem strong enough to induce a higher adaptative immune response. the construction of rtgevs expressing small antigenic domains has considerably improved the stability of the expression vectors. nevertheless, some of these small antigens may have limited immunogenicity. therefore, the expression of full-length antigens by engineering cov vectors with decreased recombination rate deserves further attention to definitely launch covs as efficacious vaccine vectors for animal and human health. experiments involving animals were performed in strict accordance with eu (2010/63/ue) and spanish (rd 1201/2005 and 32/2007) guidelines. all the protocols were approved by the in site ethical review committee. baby hamster kidney (bhk-21) cells stably transformed with the gene coding for porcine aminopeptidase n (bhk-papn) (delmas et al., 1994) were grown in dulbecco's modified eagle's medium (dmem) supplemented with 5% fetal calf serum (fcs) and g418 (1.5 mg/ml) as a selection agent. recombinant tgev viruses obtained in this work were grown in swine testis (st) cells (mcclurkin and norman, 1966) . tissue culture adapted prrsv olot91 (genbank kc862570) strain was grown and titrated in monkey kidney marc-145 cells (kim et al., 1993) . challenge was performed with a virulent prrsv strain homologous to prrsv olot91 (prrsv-olot91-like). prrsv-olot91-like was propagated in porcine macrophages differentiated from fresh peripheral blood mononuclear cells (pbmcs) as previously described (enjuanes et al., 1976) . briefly, 6 â 10 8 pbmcs isolated from fresh blood by centrifugation were seeded in 90-mmdiameter plates in roswell park memorial institute medium (rpmi) supplemented with 40% heat-inactivated swine serum. after 48 h, non-adherent cells where removed and attached macrophages, that showed 80% of confluence, were infected with 10 5 tcid 50 of the parental prrsv-olot91-like. at 72 h postinfection (hpi), when cytopathic effect was clear, supernatant was collected and centrifuged. virus was titrated in porcine alveolar macrophages (pams) as previously described (duan et al., 1997) . fusion products gp3fr, gp4fr, gp5fr gp3ep-mloop and gp3ep-ntermm were chemically synthesized and purchased from gen-eart (germany). the prrsv olot91 protein sequences forming the fusion products are summarized in table 1 . gp5ecto sequence was amplified by pcr using the forward primer (5 0 -gcaggtcctatgtacccctacgacgtgcccgactacgccatgagatg-ttctcacaaattggggc-3 0 ) and the reverse primer (5 0 -gcgctcagct-caggtctcgactgcccaatcaaaatg-3 0 ), which included ppumi and blpi restriction sites (underlined), respectively. m sequence was amplified using the forward primer (5 0 -gcaggtcctatgggaagcc-tagacgatttttg-3 0 ) and reverse primer (5 0 -gggctaagcttacc-ggccatacttgacgagg-3 0 ), which included ppumi and blpi restriction sites (underlined), respectively. in both cases, plasmid psl-trs 3a -orf5-trs 22n -orf6 (cruz et al., 2010) was used as a template. prrsv sequences, both chemically synthesized or pcr amplified, were digested with restriction endonucleases ppumi and blpi and cloned into the same sites of plasmid psl-tgev-s 7.1 -3ab including tgev genomic sequence from nt 22973 to 25873. prrsv sequences replaced non-essential genes 3a and 3b, leading to intermediate plasmids psl-trs 3a -gp3fr, psl-trs 3a -gp4fr, psl-trs 3a -gp5fr, psl-trs 3a -gp3ep-mloop, psl-trs 3a -gp3fr-ntermm, psl-trs 3a -gp5ecto and psl-trs 3a -m. for the generation of the dicistronic vectors psl-trs 3a -gp5ecto-trs 22n -m and psl-trs 3a -gp5fr-trs 22n -m, the sequence of m protein preceded by the optimized synthetic trs 22n (alonso et al., 2002a) was obtained from psl-trs 3a -orf5-trs 22n -orf6 by digestion with restriction endonuclease blpi and cloned into the same site of psl-trs 3a -gp5ecto and psl-trs 3a -gp5fr. finally, all intermediate plasmids containing prrsv sequences were digested with avrii. the resulting fragments were cloned into the same sites of plasmid pbac-tgev-s 7.1 (c.m. sanchez, m. becares, s. zuñiga and l. enjuanes, unpublished results) . this plasmid was derived from the original pbac-tgev fl (almazan et al., 2000) , containing restriction sites paci and mlui flanking s gene (ortego et al., 2003) . cloning steps led to plasmids pbac-s 7.1 -trs 3a -gp3fr, pbac-s 7.1 -trs 3a -gp4fr, pbac-s 7.1 -trs 3a -m, pbac-s 7.1 -trs 3a -gp3ep-ntermm, pbac-s 7.1 -trs 3a -gp3ep-mloop, pbac-s 7.1 -trs 3a -gp5ecto-trs 22n -m and pbac-s 7.1 -trs 3a -gp5fr-trs 22n -m. all cloning steps were checked by sequencing of the pcr fragments and cloning junctions. transfection and recovery of infectious rtgevs from cdna clones bhk-papn cells were grown to 90% confluence in 35-mmdiameter plates and transfected with 4 μg of the corresponding pbac and 12 μl of lipofectamine 2000 (invitrogen), according to the manufacturer's specifications. after 6 h of incubation at 37 1c, cells were trypsinized and plated over a confluent st monolayer grown in 35-mm-diameter plate. after a 2-day incubation period, the cell supernatants were harvested (passage 0). rtgevs were cloned by three plaque purification steps. rtgevs were grown and titrated as previously described (jimenez et al., 1986) . two clones of each rtgev were serially passaged, independently, in st cells every 24 h. at passage 8 and 16 ten viral clones were plaque purified. rna from recombinant viruses was purified from infected st cells grown to overconfluence on 12-well plates. total intracellular rna was extracted at 18 hpi using the rneasy mini kit (qiagen) according to the manufacturer's recommendations. reverse transcription was performed with high capacity rna-to-cdna™ kit (life technologies) according to the manufacturer's instructions. pcrs were performed to analyze the size and sequence of viral genomic rna (grna), at the locus where the heterologous genes were inserted, and heterologous mrna size and sequence synthesis. the primers used and the expected pcr fragment sizes are shown in table 2 . subconfluent st cells grown on glass coverslips were mock infected or infected at a multiplicity of infection (moi) of 0.5 with each rtgev. at 8 hpi cells were washed with phosphate-buffered saline (pbs), fixed with 4% paraformaldehyde, permeabilized with 0.1% triton x-100 in pbs and blocked in pbs with 10% fcs. monoclonal antibodies specific for flag (flag m2, 1:500, sigma), prrsv m protein (em11e10c7, 1:100, kindly provided by inge-nasa), or a polyclonal rabbit serum specific for tgev (1:1000) were used. bound primary antibody was detected with a alexa fluor 488 or 594-conjugated antibodies specific for mouse or rabbit, respectively (1:500, invitrogen). cell nuclei were stained with 4 0 , 6-diamidino-2-phenylindole (dapi) (1:200, sigma). confocal microscopy was performed using a leica sp5 laser scanning microscope, and images were collected and processed with las af software (leica, wetzlar, germany). the percentage of infected cells expressing prrsv antigens was estimated by the analysis of 10 independent microscopy fields, which represent an average of more than 400 cells. forty-five twelve days-old non-colostrum-deprived piglets, born from prrsv seronegative sows, were inoculated with rtgev by three different routes (oral, gastric and intranasal) following standard procedures (sanchez et al., 1999) . piglets were divided into three 15-animal groups. piglets of group 1 were inoculated with a mix of 1 â 10 8 plaque forming units (pfu)/animal of each of rtgev-s 7.1 -trs 3a -gp3fr, rtgev-s 7.1 -trs 3a -gp4fr, rtgev-s 7.1 -trs 3a -gp5fr-m, rtgev-s 7.1 -trs 3a -m and rtgev-s 7.1 -trs 3a -gp3ep-ntermm. piglets of groups 2 and 3 were inoculated with 1 â 10 8 pfu/animal of rtgev-s 7.1. two weeks after the first immunization, all piglets were boosted in the same conditions, and two weeks later piglets of groups 1 and 2 were challenged with 10 6 tcid 50 of prrsv-olot91-like per animal by intranasal route. infected animals were monitored daily to detect symptoms of disease, and body weights were determined every 7 days. blood samples were taken at days 0, 14, 28, 31, 35, 38, 42, 49 and 56post-first inoculation. five and ten animals per group were euthanized and necropsied at days 35 and 56, respectively. lung macroscopic lesions were evaluated, and lung samples were collected frozen and in 10% buffered-formalin. five piglets per group were randomly chosen for histopathological study. lung representative sections were fixed with 4% paraformaldehyde and stored in 70% ethanol at 4 1c. paraffin embedding, sectioning and hematoxylin-eosin staining were performed by the histology service in the national center of biotechnology (cnb-csic, spain). samples were examined with a zeiss axiophot fluorescence microscope. determination of the lung damage score was obtained from unbiased observation of 50 microscopy fields per animal, scoring from 0 to 3 attending to interstitial, peribronchiolar, and perivascular inflammation (page et al., 2012) . quantification of porcine il-1β, il-10, ifn-α, ifnγ, tnfα, il-4 and il-8 in serum samples was carried out using swine cytokine magnetic 7-plex panel (life technologies, tm), and the luminex 100 is analyzer, according to the manufacturer's instructions. three serum samples, corresponding to the same experimental group and the same date of extraction, were randomly pooled and analyzed in a single well. data were calculated by xponent software using a fiveparameter model derived from the known reference cytokine concentrations supplied by the manufacturer. the sensitivity of this assay allowed the detection of cytokine concentrations with the following limits of detection: il-1β (36.808 pg/ml), il-10 (4.572 pg/ ml), ifn-α (2.661 pg/ml), ifnγ (0.342 pg/ml), tnfα (146.86 pg/ml), il-4 (0.548 pg/ml), il-8 (0.786 pg/ml). viral rna was isolated from 250 μl of serum using magmax™ viral rna isolation kit (life technologies) according to the manufacturer's instructions. prrsv rna quantity was measured by rt-qpcr analysis using a custom taqman assay detecting prrsv n rna (taqman probe 6-fam-acggcttttaatcaaggc-mgb; forward primer 5 0 -ttccctctgcttgcaatcg-3 0 ; reverse primer 5 0 -ggatgaaagcgacgcagttc-3 0 ), and the agpath-id one-step rt-pcr kit (life technologies) according to the manufacturer's instructions. the data were acquired with an abi prism 7500 sequence detection system and analyzed with abi prism 7000 sds version 1.2.3 software (applied biosystems). viremia levels were expressed as the rt-qpcr cycle threshold (ct) values. antibodies induced against tgev and prrsv viruses or prrsv purified proteins were detected by elisa as described before (sambrook and russell, 2001) . prrsv gp3, gp4, gp5, m and n proteins were expressed using the baculovirus-insect cell system. recombinant proteins were purified to near homogeneity by metal chelate affinity chromatography using ni-nta agarose (sigma-aldrich, madrid, spain) as previously described (nogales et al., 2011) . elisas were performed using partially purified tgev (0.2 μg per well) and prrsv (0.05 μg per well) viruses, or prrsv purified proteins gp3 (0.25 μg per well), gp4 (0.5 μg per well), gp5 (0.1 μg per well), m (0.1 μg per well) and n (0.2 μg per well). antigens were bound to 96-well microplates, saturated with 5% bovine serum albumin (bsa) in pbs for 2 h at 37 1c and incubated with serial dilutions of the serum sample in wash buffer (0.1% bsa, 0.05% tween20 in pbs) for 90 min at 37 1c. microplates were washed three times with wash buffer. bound antibodies were detected by incubation with peroxidase-conjugated protein a (biorad) diluted 1:10000 in pbs with 0.1% bsa. elisa was developed with k-blue tmb substrate (neogen, lexington, ky) for 5 min table 2 analysis of rtgevs stability by rt-pcr. expected product size and primers used for the analysis of viral grna and heterologous mrna expressed by rtgevs. expected size (bp) grna a mrna b reverse primer (5 0 -3 0 ) a pcr for grna analysis was performed with the forward primer (5 0 -attacgaaccaattgaaaaagtgc-3 0 ) and the reverse primer (5 0 -ccgcctga-gaaaaggctgcattg-3 0 ) in all cases. b in all cases, forward primer (5 0 -gtgagtgtagcgtggctatatctcttc-3 0 ), complementary to the viral leader sequence was used. c mrna size shown in the table corresponds to gp5fr or gp5ecto. at room temperature. reactions were stopped with 1.5 m h 2 so 4 , and the absorbance was read at 450 nm. the elisa values of the sera were expressed as sample to positive ratio [sp-ratio¼(od of sample à od of negative control)/(od of positive controlà od of negative control)]. serial dilutions of heat-inactivated serum were incubated for 1 h at 37 1c in the presence of 100 pfus of prrsv-olot91 in dmem containing 5% fcs. the mixtures were added to confluent marc-145 cells in 24-well plates. after one hour incubation at 37 1c medium was removed and 1 ml of dmem containing 2% fcs and 0.5% agar was added. after 72 h, cells were fixed with 10% formaldehyde in pbs, stained with a crystal violet solution, and lysis plaques were counted. a positive control serum, obtained from a prrsv infected pig at 56 dpi led to 98-100% of virus neutralization at a sera dilution of 1:16. in contrast, a non-immune control serum led to a neutralization of 1 to 5% in the same experimental conditions. the neutralization index of each serum sample was expressed relative to the one obtained with the negative control serum in the experiment [neutralization index ¼100% à (pfus serum sample/pfus negative control) â 100]. two-tailed, unpaired student t tests were used to analyze difference in mean values between groups. all results were expressed as means 7the standard deviations of the means. chisquare test was used to analyze statistical significance of differences in percentages of immunized and non-immunized groups. p values o0.1 were considered significant (noymer, 2008) . interferon-alpha response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus two types of non-homologous rna recombination in brome mosaic virus construction of a sars-cov infectious cdna clone and a 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full-length infectious cdna of severe acute respiratory syndrome coronavirus systematic assembly of a fulllength infectious cdna of mouse hepatitis virus strain a59 we thank h. nauwynck and m. vanhee for sharing information about prrsv epitopes and ingenasa for kindly providing us with anti-m protein monoclonal anitbodies. we also thank m. gonzález, s. ros and r. fernández for technical assistance.this study was supported by grants from the ministry of science and innovation of spain (bio2010-16705), and the european community's seventh framework programme (fp7/ 2007(fp7/ -2013 key: cord-290282-oxyzndsj authors: ortego, javier; sola, isabel; almazán, fernando; ceriani, juan e; riquelme, cristina; balasch, monica; plana, juan; enjuanes, luis title: transmissible gastroenteritis coronavirus gene 7 is not essential but influences in vivo virus replication and virulence date: 2003-03-30 journal: virology doi: 10.1016/s0042-6822(02)00096-x sha: doc_id: 290282 cord_uid: oxyzndsj transmissible gastroenteritis coronavirus (tgev) contains eight overlapping genes that are expressed from a 3′-coterminal nested set of leader-containing mrnas. to facilitate the genetic manipulation of the viral genome, genes were separated by duplication of transcription regulating sequences (trss) and introduction of unique restriction endonuclease sites at the 5′ end of each gene using an infectious cdna clone. the recombinant tgev (rtgev) replicated in cell culture with similar efficiency to the wild-type virus and stably maintained the modifications introduced into the genome. in contrast, the rtgev replication level in the lungs and gut of infected piglets and virulence were significantly reduced. rtgev in which gene 7 expression was abrogated (rtgev-δ7) were recovered from cdna constructs, indicating that tgev gene 7 was a nonessential gene for virus replication. interestingly, in vivo infections with rtgev-δ7 showed an additional reduction in virus replication in the lung and gut, and in virulence, indicating that tgev gene 7 influences virus pathogenesis. transmissible gastroenteritis coronavirus (tgev) is a member of the coronaviridae family, which, together with the arteriviridae and roniviridae families forms the nidovirales order (cowley and walker, 2002; enjuanes et al., 2000a; mayo, 2002) . tgev replicates in both the villous epithelial cells of the small intestine and the lung cells of newborn piglets, resulting in a mortality of nearly 100% (saif and wesley, 1992) . the tgev genome contains a leader sequence at the 5ј end and a poly(a) tail at the 3ј end. genes are arranged in the order 5ј-rep-s-3a-3b-e-m-n-7-3ј (enjuanes et al., 2000b; penzes et al., 2001) . the 3ј end of the majority of tgev genes overlaps with the 5ј terminus of the next gene (enjuanes et al., 2000b) , complicating insertion of heterologous genes into the viral genome and deletion of different genes to determine whether they are essential. the tgev gene 7, located at the 3ј end of the genome, encodes a 78 amino acid hydrophobic protein that may play a role in membrane-associated replication complexes or in virion assembly (tung et al., 1992) . gene 7 is a groupspecific gene (de haan et al., 2002) with homologous versions in group 1 coronaviruses such as feline infectious peritonitis virus (fcov) and canine enteric coronavirus (ccov) (enjuanes et al., 2000a; lai and cavanagh, 1997) . in contrast, group 2 coronaviruses such as mouse hepatitis virus (mhv), bovine enteric coronavirus (bcov), or the human coronavirus (hcov) oc43, and group 3 coronaviruses such as avian infectious bronchitis virus (ibv), do not have a homologous gene 7 (enjuanes et al., 2000a; lai and cavanagh, 1997) . interestingly, the group 1 coronavirus hcov-229e does not have gene 7 (herold et al., 1993) , which could indicate that this gene is nonessential for coronavirus replication, even in group 1 coronaviruses. to study whether gene 7 is dispensable for tgev replication, its deletion by reverse genetics would be required. we used a genomic tgev cdna clone assembled as a bacterial artificial chromosome (bac) (almazán et al., 2000; gonzález et al., 2002) to separate the overlapping genes by duplicating sequences at the 5ј flank of each gene and by introducing unique restriction endonuclease sites between each gene pair. gene separation allowed the deletion of gene 7, showing that it is nonessential for virus replication. furthermore, we show that the accumulation of modifications in gene domains where the trss are located and insertion of restriction sites led to the generation of a collection of recombinant tgevs (rtgevs) with variable virulence, including a highly attenuated recombinant. these viruses could be the basis for coronavirus vector development. to facilitate genetic manipulation of the viral genome, full-length cdna clones were constructed by separating the contiguous genes and inserting unique restriction sites between each gene pair (fig. 1a ). cdna clones with the wild-type sequence or containing one [pbac-tgev-paci (p), pbac-tgev-mlui (m), pbac-tgev-fsei (f), and pbac-tgev-asci (a)], two [pbac-tgev-fsei-pmei (f-pm), pbac-tgev-pmei-asci (pm-a), and pbac-tgev-paci-mlui (p-m)], three [pbac-tgev-fsei-pmei-asci (f-pm-a)], or five [pbac-tgev-paci-mlui-fsei-pmei-asci (rs)] restriction endonuclease sites (fig. 1b) were transfected into bhk cells expressing the porcine amino peptidase n (papn) (bhk-papn cells). on the third day of transfection, a cytopathic effect was observed in cells transfected with each cdna, but not in mock-treated cells. virus production was amplified by passing the supernatants four times in cultured cells. after the fourth passage, viruses were cloned by three plaque-isolation steps, and their genomes were partially sequenced. all the rtgev viruses conserved the modifications engineered in the cdnas (data not shown), indicating that the orf separation and the insertion of unique endonuclease restriction sites between genes were stably maintained in the rtgev genomes. cloned rtgev viruses containing the unique restriction sites showed similar growth kinetics in cell culture to the parental rtgev-wt after infection at both low (0.05) and high (5) m.o.i. (fig. 2) , indicating that removal of the overlapping region between tgev genes and the insertion of endonuclease restriction sites did not significantly affect the in vitro virus replication. to analyze whether gene 7 was essential for viral growth, recombinant virus genomes with gene 7 deleted were generated from pbac-tgev-rs constructs, contain-ing either the s gene from the tgev strain pur-c11 (sánchez et al., 1999) able to infect both the enteric and the respiratory tracts (pbac-tgev-sc11-rs-⌬7) or the s gene from the strain ptv (sánchez et al., 1999) with an exclusive respiratory tropism (pbac-tgev-sptv-rs-⌬7). the rtgev-⌬7 contained a deletion spanning 21 nucleotides upstream of the orf 7 aug and the first 17 nucleotides of this orf (fig. 3a) . bhk-papn cells were transfected with plasmids including five restriction endonuclease sites and carrying gene 7 (pbac-tgev-sptv-rs and pbac-tgev-sc11-rs), or without this gene (pbac-tgev-sptv-rs-⌬7 and pbac-tgev-sc11-rs-⌬7). virus titers were determined in supernatants throughout four additional passages in cell culture ( fig. 3b and c). viruses were recovered from the cdnas containing the deletion of gene 7, and viral titers increased with passage, basically to the same extent as the viruses with gene 7 (rtgev-sptv-rs and rtgev-sc11-rs). as expected, no virus was recovered from the mock-transfected cultures. after four passages in cell cultures, the recombinant viruses were cloned by three plaque isolation steps. the cytopathic effect and plaque morphology produced by the rtgev-sptv-rs-⌬7 and rtgev-sc11-rs-⌬7 were identical to those of the parental viruses containing the complete genome (data not shown). the isolate rtgev-sc11-rs-⌬7 induced the formation of large-size plaques (3-mm-diameter), whereas the virus rtgev-sptv-rs-⌬7 induced smallsized plaques (1-mm-diameter). the cloned viruses with gene 7 deleted showed standard growth kinetics after infection at an m.o.i. of 5 ( fig. 3d and e). recombinant rtgev-sptv-rs-⌬7 and rtgev-sc11-rs-⌬7 generated the highest virus titers (around 6 ϫ 10 8 and 10 7 pfu/ml, respectively) at 24 h postinfection, similar to those of the parental viruses rtgev-sptv-rs and rtgev-sc11-rs. these data indicated that the protein encoded by gene 7 was not essential for tgev replication in cell culture. to confirm that the subgenomic mrna (sgmrna) 7 was not transcribed, bhk-papn cells were infected with rtgev-sptv-rs or rtgev-sptv-rs-⌬7 viruses. total rna was extracted and analyzed by northern blot with a probe complementary to the 3ј end of the tgev genome (fig. 4a ). the mobility and relative amount of the sgmrnas 2, 3, 4, 5, and 6 were indistinguishable in both viruses. as expected, sgmrna 7 was transcribed in rt-gev-sptv-rs-infected cells, but not in cells infected with rtgev-sptv-rs-⌬7. analysis of viral proteins at 16 h p.i. by western blot showed that the amount of s, n, m, and e proteins was similar in rtgev-sptv-rs-⌬7-infected cells and in cells infected with the parental virus rtgev-sptv-rs, except for protein 7 that was not detected in rtgev-sptv-rs-⌬7 virus-infected cells (fig. 4b) , confirming that the partial deletion of gene 7 prevented the synthesis of protein 7. identical results were obtained with rtgev-sc11-rs and rtgev-sc11-rs-⌬7 (data not shown). in vivo growth of a selected set of rtgevs containing the unique endonuclease restriction sites, and the rtgev-rs-⌬7 viruses, was determined by infecting newborn piglets. the animals were sacrificed at 1, 2, 3, and 4 days p.i. recombinant viruses with a modification including a restriction endonuclease site 5ј upstream of some genes, in general, showed lower titers than the wild-type virus, aloutlined sequences indicate the punctual mutations introduced to generate unique restriction sites. the core sequence (cs) is underlined. the atg start codon is shown in bold. duplicated sequences are indicated by dark boxes. tgev genes are indicated by light boxes. 1b, replicase 1b gene; s, spike gene; e, envelope gene; m, membrane gene; n, nucleoprotein gene. *, gene rep 1b termination codon (tga) and the initiation codon of gene s (atg) partially overlap. the sequence of the 24 and 83 nt located 5ј upstream of genes s and 3a, respectively, are described in the full-length tgev genome sequence previously reported (penzes et al., 2001) . (b) schematic illustration of the full-length tgev cdna without (top bar) or with one (pbac-tgev-p, pbac-tgev-m, pbac-tgev-f, and pbac-tgev-a), two (pbac-tgev-f-pm, pbac-tgev-pm-a, and pbac-tgev-p-m), three (pbac-tgev-f-pm-a), or five (pbac-tgev-rs) restriction endonuclease sites. cmv, cytomegalovirus immediate-early promoter; rep, replicase; an, poly(a) tail of 24 a residues; hdv, hepatitis delta virus ribozyme; bgh, bovine growth hormone termination and polyadenylation sequences. though there was some titer variability over the 4 days postinfection ( fig. 5a and b) . alteration in domains 5ј upstream of two or more genes did not lead to a significant decrease in virus titer in comparison with recombinants with a single modification. interestingly, analysis of viral growth in the gut of infected piglets showed a 100-to 5000-fold reduction of recombinant viruses containing one or more restriction sites in relation to the rtgev-wt virus ( fig. 5d and e) . the rtgev-rs, that included modifications 5ј upstream of five genes and insertion of endonuclease restriction sites between each contiguous gene, showed a titer decrease comparable with that of rtgevs with two (tgev-f-a) or three (tgev-f-pm-a) restriction endonuclease sites. these data show that modification of sequences 5ј upstream of genes affected virus replication in the gut. recombinant viruses were isolated from the gut and sequenced. all the modifi-cations introduced were stably maintained in the tgev genome during in vivo infections (data not shown). the growth in lungs of gene 7 deletion mutants (rtgev-sptv-rs-⌬7 and rtgev-sc11-rs ⌬7) showed around 100fold reduction of virus titers compared with the parental viruses rtgevsptv-rs and rtgev-sc11-rs (fig. 5c ). in contrast, in vivo growth of rtgev-sc11-rs-⌬7 in the gut showed slightly lower replication levels than rtgev-sc11-rs (5 ϫ 10 4 pfu/g) probably because the introduction of modifications at the 5ј upstream of five genes had already caused a significant titer reduction (fig. 5f ). as expected, the respiratory recombinants rtgev-sptv-rs and rtgev-sptv-rs-⌬7 did not grow in the gut since the s gene was derived from the respiratory ptv strain. piglet survival after infection by rtgev viruses was compared with survival after infection with their virulent parental virus tgev-pur46-c11 (tgev-sc11-wt). rtgev viruses with one, two, three, or five unique restriction sites were highly attenuated (they produced mild enteritis and led to 80 to 90% survival at 5 days p.i.), except for rtgev-p and rtgev-m viruses, in which the restriction sites paci and mlui were introduced by point mutations, without introducing trs duplication. in these two recombinant viruses the survival was from 0 to 20%. piglets infected by rtgev-sc11-rs-⌬7 showed 100% survival, although a very mild and transitory enteritis was observed in 50% of the animals. these data indicate that gene 7 deletion further reduced virus virulence. in general, a good correlation be-tween growth of recombinants with a single restriction endonuclease insertion and virulence was not clear. this could be due to differences in the distribution of viral antigens and inflammatory responses in pigs infected with wild-type or each mutant. nevertheless, in viruses with two or more restriction endonuclease sites inserted there was a good correlation between virus titers in the gut and mortality. tgev genomes with all the genes separated by unique endonuclease restriction sites have been engineered. the separation of tgev genes implied the duplication of sequences 5ј upstream of each gene, a sequence domain involved in regulating transcription, and affected in vivo virus growth and virulence. in this article, the first demonstration that tgev gene 7 is nonessential for virus viability is provided. in addition, it has been demonstrated that gene 7 deletion affects tgev replication and attenuates virus virulence in piglets, its natural host. interestingly, the introfig. 5 . in vivo growth kinetics of rtgev viruses. two-to three-day-old non-colostrum-deprived swine were used to study the growth kinetics of rtgev viruses containing one (a and d) or more (b and e) endonuclease restriction sites, or partial deletion of gene 7 (c and f). groups of 12 to 20 piglets were oronasally (2 ϫ 10 8 pfu/pig) and intragastrically (3 ϫ 10 8 pfu/pig) inoculated. virus titers at the indicated number of days postinoculation were determined in the indicated tissue extracts. the whole organs were homogenized to obtain representative samples. error bars represent standard deviations of the mean from four experiments. (g) number of surviving piglets at different days postinoculation. results from a representative experiment of two that gave similar results are shown. recombinant virus titers were compared with that of the wild-type virus by the kruskal-wallis test and, in general, were significant (p ͻ 0.05) between viruses with gene 7 deleted and the wild-type virus. western blot analysis of lysates from bhk-papn cells infected with either rtgev-sptv-rs or rtgev-sptv-rs-⌬7 viruses. cell extracts were obtained at 16 h p.i., resolved by 5 to 20% gradient sds-page, transferred to nitrocellulose membranes, and immunoblotted with monoclonal antibodies specific for s, n, m, and e, and with an antiserum specific for a protein 7 peptide (garwes et al., 1989 ). duction of one or more modifications into the tgev genome has led to the generation of a collection of tgev recombinants with a variable degree of virulence. overlapping of genes has been proposed as a mechanism by which nidoviruses preserve the genetic integrity of vital parts of their genomes (de vries et al., 2000) . nevertheless, in coronavirus we have generated viable and stable rtgev viruses in which genes have been separated by the insertion of unique restriction endonuclease sites and modification of the domains where the transcription-regulating sequences (trss) map. maximum titers of mutant rtgev viruses in cell culture were similar to those obtained for the parental virus, indicating that changes in the sequences between tgev genes had little effect on viral yield. in contrast, in vivo assays showed lower tgev mutant virus replication in the lungs and gut, and attenuation of virulence in viruses in which a trs duplication was introduced between genes. in arteriviruses, mutants that had the overlap between orfs 4 and 5, or between orfs 5 and 6 removed, were also viable (de vries et al., 2000) . taken together, these data demonstrate that gene overlapping is not an obligatory requirement for nidovirales viability. viral attenuation resulting from gene separation was possibly due to modification of the trss affecting mrna transcription levels. in the case of nonsegmented negativestrand rna viruses it has also been shown that sequence alterations, such as restriction endonuclease site or heterologous gene insertion, and gene rearrangement may affect virus replication (wertz et al., 2002) . gene 7 is a group 1 specific gene, absent in genome of coronaviruses from groups 2 and 3 (armstrong et al., 1983; boursnell et al., 1985; kamahora et al., 1989; lapps et al., 1987; skinner and siddell, 1985) . interestingly, no mortality was observed in piglets infected with rtgev-⌬7. since these recombinants still replicate with titers between 10 3 and 10 5 pfu/g of tissue ( fig. 5c and f) , tgev-⌬7 mutants could be good candidates as virus vectors. a relationship between gene 7 and virulence has also been observed in the fcovs. the most 3ј end genes of these viruses are genes 7a and 7b. deletions in fcov orf 7a, which is homologous to the tgev orf 7, have been reported in a natural infection of a cat population and correlated with a decrease in virulence (kennedy et al., 2001) . similarly, mutations in fcov orf 7b occur in vitro and have also been correlated with loss of virus virulence (herrewegh et al., 1995) . therefore, the most 3ј end genes of group 1 coronaviruses seem to influence in general virus pathogenesis. deletion of gene 7 did not affect virus replication in cell culture. therefore, the reduction of in vivo virus replication and virulence was probably due to an effect on virus-host interaction. it has been suggested that coronavirus groupspecific genes, such as gene 7, may affect host immune response (de haan et al., 2002) . it would be of interest to determine whether the immune response to a heterologous gene inserted in recombinant viruses with and without gene 7 is influenced by the presence of this gene. in an attempt to identify gene 7 homologous protein sequences or motifs, a sequence search was performed in the databases using gene 7 sequences without success. the high hydrophobicity of tgev protein 7 (garwes et al., 1989) could facilitate its insertion in membranes providing a role in virus replication, since coronavirus replication complexes have been associated with membranes (dennison and sims, 2001; snijder et al., 2001) . similarly, the tentative location of protein 7 within the nucleus (garwes et al., 1989) could be taken as an indication for a possible interference with the cell cycle, similarly to coronavirus nucleoprotein that seems to interact with cell nucleolus proteins and interfere with cell cycle (hiscox, 2002) . therefore, genes 3a, 3b, and 7 of group 1 coronaviruses are nonessential for replication. similarly, genes 2a/he and 4/5a, and possibly gene e, are dispensable in mhv (de haan et al., 2002; kuo and masters, 2002) . manipulation of transcription-regulating sequences and deletion of nonessential genes, such as gene 7, will facilitate the study of the molecular basis of viral attenuation and provide an attractive approach to generate attenuated rtgevs with high potential as virus vectors. baby hamster kidney cells (bhk-21) stably transformed with the gene coding for the porcine aminopeptidase n (bhk-papn) (delmas et al., 1994) were grown in dmem supplemented with 2% fetal calf serum and geneticin g418 (1.5 mg/ml) as a selection agent. bhk-papn cells were used for all the experiments except for standard virus titrations that were performed in porcine swine testis (st) cells (mcclurkin and norman, 1966) . virus titers were compared by the kruskal-wallis test (motulsky, 1995) and, when significant (p ͻ 0.05), it was indicated. rtgev viruses were generated from pbac-tgev constructs containing the s gene from the virulent tgev strain pur-c11 (sc11) as described (almazán et al., 2000; gonzalez et al. 2002) . viruses containing the s gene from the attenuated strain ptv (sptv) were derived from the corresponding pbac tgev vectors with gene sc11 by replacing this gene by that (sptv) of the respiratory strain. two different approaches were followed to introduce into pbac-tgev unique restriction endonuclease sites separating each gene of tgev genome (fig. 1a) , leading to pbac-tgev-rs. the first approach was the introduction of punctual mutations in the tgev genome to generate the restriction sites paci (between genes rep 1b and s) and mlui (between genes s and 3a) (fig. 1b) . due to overlapping in the tgev genome, the second approach involved the duplication of 13, 22, and 19 nucleotides from the 5ј transcription-regulating sequences of genes m, n, and 7 (trs-m, trs-n, and trs-7) after the restriction sites fsei, pmei, and asci, respectively ( fig. 1a and b) . point mutations, duplications, and insertion of restriction endonuclease sites were generated by overlapping pcr amplification from the tgev genome as described (ortego et al., 2002) . the assembly of the full-length pbac-tgev constructs was performed as previously reported (almazán et al., 2000; gonzález et al., 2002) . to generate the deletion ⌬7, oligonucleotide primers 5ј-asc-7.17-vs (5ј-gaggcgcgcctgctgtatttat-tacag-3ј), including the restriction site asci (underlined) and the deletion of 21 nucleotides from trs-7 plus the first 17 nucleotides of orf 7, and bgh34-rs (5ј-cagatg-gctggcaactagaaggc-3ј) were used to generate a pcr product comprising from nt 28,094 to nt 28,764 of the tgev cdna clone. pcr product was digested with asci and bamhi and cloned into the asci-bamhi-digested pbac-tgev-sc11-rs and pbac-tgev-sptv-rs, generating pbac-tgev-sc11-rs-⌬7 and pbac-tgev-sptv-rs-⌬7, respectively. bhk-papn cells were grown to 60% confluence on 35-mm-diameter plates and transfected with 10 g of cdna plasmid and 15 g of lipofectin (life technologies, gibco) according to the manufacturer's specifications. recovery and amplification of viruses were performed as described (ortego et al., 2002) . cell lysates were analyzed by 5 to 20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page). proteins were transferred to a nitrocellulose membrane and analyzed as described (ortego et al., 2002) , using mabs specific for s (5b.h1), n (3d.c10), m (9d.b4), and e (v27) proteins (ortego et al., 2002) , and a swine polyclonal antibody specific for tgev protein 7 (provided by p. britton, compton, uk). total rna was extracted by using an ultraspec rna isolation system (biotecx) according to the manufacturer's instructions and analyzed by northern blotting as described (ortego et al., 2002) . two-to three-day-old non-colostrum-deprived swine, from crossing large white and belgium landrace, were used to study in vivo growth kinetics of rtgev, as described (sanchez et al., 1999) . piglets were obtained from sows seronegative for tgev, as tested by radioimmunoassay. engineering the largest rna virus genome as an infectious bacterial artificial chromosome sequence of the nucleocapsid gene from murine coronavirus mhv-a59 sequences of the nucleocapsid genes from two strains of avian infectious bronchitis virus the complete genome sequence of gill-associated virus of penaeus monodon prawns indicates a gene organisation unique among nidoviruses the group-specific murine coronavirus genes are not essential, but their deletion, by reverse genetics, is attenuating in the natural host genetic manipulation of equine arteritis virus using full-length cdna clones: separation of overlapping genes and expression of a foreign epitope further characterization of aminopeptidase-n as a receptor for coronaviruses mhv-a59 gene 1 proteins are associated with two distinct membrane populations coronaviridae nidovirales the polypeptide of mr 14000 of porcine transmissible gastroenteritis virus: gene assignment and intracellular location stabilization of a full-length infectious cdna clone of transmissible gastroenteritis coronavirus by the insertion of an intron nucleotide sequence of the human coronavirus 229e rna polymerase locus the molecular genetics of feline coronavirus comparative sequence analysis of the orf7a/7b transcription unit of different biotypes the nucleolus-a gateway to viral infection? sequence analysis of nucleocapsid gene and leader rna of human coronavirus oc43 deletions in the 7a orf of feline coronavirus associated with an epidemic of feline infectious peritonitis genetic evidence for a structural interaction between the carboxy termini of the membrane and nucleocapsid proteins of mouse hepatitis virus the molecular biology of coronaviruses sequence analysis of the bovine coronavirus nucleocapsid and matrix protein genes a summary of taxonomic changes recently approved by ictv studies on transmissible gastroenteritis of swine. ii. selected characteristics of a cytopathogenic virus common to five isolates from transmissible gastroenteritis comparing two paired groups: paired t and wilcoxon tests generation of a replication competent, propagation-deficient virus vector based on the transmissible gastroenteritis coronavirus genome complete genome sequence of transmissible gastroenteritis coronavirus pur46-mad clone and evolution of the purdue virus cluster transmissible gastroenteritis targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence coding sequence of coronavirus mhv-jhm mrna 4 non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the arterivirus replication complex the 9-kda hydrophobic protein encoded at the 3ј end of the porcine transmissible gastroenteritis coronavirus genome is membrane-associated adding genes to the rna genome of vesicular stomatitis virus: positional effects on stability of expression this work was supported by grants from the comisión interministerial de ciencia y tecnología (cicyt), la consejería de educación y cultura de la comunidad de madrid, fort dodge veterinaria, and the european union (frame v, key action 2, control of infectious disease projects qlrt-1999-00002, qlrt-1999-30739, and qlrt-2000. i.s. received postdoctoral fellowships from the community of madrid and the european union (frame v, key action 2, control of infectious disease projects). key: cord-289152-w5ynbewh authors: lee, sang-myeong; kleiboeker, steven b. title: porcine arterivirus activates the nf-κb pathway through iκb degradation date: 2005-11-10 journal: virology doi: 10.1016/j.virol.2005.07.034 sha: doc_id: 289152 cord_uid: w5ynbewh nuclear factor-kappab (nf-κb) is a critical regulator of innate and adaptive immune function as well as cell proliferation and survival. the present study demonstrated for the first time that a virus belonging to the arteriviridae family activates nf-κb in marc-145 cells and alveolar macrophages. in porcine reproductive and respiratory syndrome virus (prrsv)-infected cells, nf-κb activation was characterized by translocation of nf-κb from the cytoplasm to the nucleus, increased dna binding activity, and nf-κb-regulated gene expression. nf-κb activation was increased as prrsv infection progressed and in a viral dose-dependent manner. uv-inactivation of prrsv significantly reduced the level of nf-κb activation. degradation of iκb protein was detected late in prrsv infection, and overexpression of the dominant negative form of iκbα (iκbαdn) significantly suppressed nf-κb activation induced by prrsv. however, iκbαdn did not affect viral replication and viral cytopathic effect. prrsv infection induced oxidative stress in cells by generating reactive oxygen species (ros), and antioxidants inhibited nf-κb dna binding activity in prrsv-infected cells, suggesting ros as a mechanism by which nf-κb was activated by prrsv infection. moreover, nf-κb-dependent expression of matrix metalloproteinase (mmp)-2 and mmp-9 was observed in prrsv-infected cells, an observation which implies that nf-κb activation is a biologically significant aspect of prrsv pathogenesis. the results presented here provide a basis for understanding molecular pathways of pathology and immune evasion associated with disease caused by prrsv. prrsv is an enveloped, positive-stranded rna virus that is a member of the order nidovirales, family arteriviridae, along with lactate dehydrogenase-elevating virus of mice, equine arteritis virus, and simian hemorrhagic fever virus. prrsv causes one of the most economically important diseases of swine which is characterized by severe and sometimes fatal respiratory disease and reproductive failure. infection with prrsv also predisposes pigs to infection by bacterial and viral pathogens such as steptococcus suis, haemophilus parasuis, mycoplasma hyopneumoniae, acti-nobaccillus pleuropneumoniae, salmonella spp., and swine influenza virus (benfield et al., 1992; done and paton, 1995; galina et al., 1994; groschup et al., 1993; kawashima et al., 1996; zeman et al., 1993) . the most consistent pathological lesions caused by prrsv during acute infection are interstitial pneumonia and mild lymphocytic encephalitis (halbur et al., 1995; plagemann, 1996; rossow et al., 1995 rossow et al., , 1996 . tissue macrophages and monocytes are the major target cells during both acute and persistent infection (molitor et al., 1997) , although pneumocytes and epithelial germ cells of the testis have also been shown to be infected (sur et al., 1996 (sur et al., , 1997 . viruses are known to control cellular signal transduction pathways, and the nf-nb pathway is a common target of many viruses. nf-nb is an inducible transcription factor that plays a key role in inflammation, innate immune responses, the regulation of cell proliferation, and cell survival (caamano and hunter, 2002; li and verma, 2002) . activation of nf-nb by viral infection is a key trigger to inducing type i interferon (ifn) transcription and other immune responses, including pro-inflammatory cytokines, chemokines, adhesion molecules, matrix metalloproteinases (mmps), cyclooxygenase 2 (cox2), and inducible nitric oxide synthase (inos) (caamano and hunter, 2002; santoro et al., 2003) . these molecules are involved in initiating adaptive immune responses by recruiting immune cells to the site of infection. furthermore, it was recently demonstrated that temporally activated nf-nb confers an essential innate antiviral response against cytoplasmic rna viruses (human parainfluenza virus type 3 and respiratory syncytial virus) in an ifn-independent manner, showing the importance of nf-nb in the innate antiviral response (bose et al., 2003) . nf-nb exists as a homodimer or heterodimer comprised of one or two of five subunits, rela (p65), p50, relb, c-rel, and p52. the predominant form is a heterodimer composed of p50 and rela subunits . when inactive, nf-nb is sequestered in the cytoplasm by associating with inhibitory proteins of the inb family, including inba, inbh, and inbe, which mask the nuclear localization signal. in response to a wide range of stress signals (e.g., lipopolysaccharide (lps), tumor necrosis factor (tnf), interleukin (il)-1, and virus infection), the inactive nf-nb-inb complex is dissociated via serine phosphorylation by inb kinase (ikk) and degradation of inb in proteasomes. these events lead to the unmasking of the nuclear localization sequence of nf-nb, which then allows nf-nb to enter the nucleus and activate transcription of target genes . while the immune response against prrsv is not fully characterized, experimental work has demonstrated that the adaptive immune response of prrsv-infected pigs is generally ineffective (horter et al., 2002; murtaugh et al., 2002; wills et al., 1997 wills et al., , 2003 . specific evidence of an ineffective adaptive immune response includes a slow neutralizing antibody response, which is typically not detected until 3 weeks p.i. (albina et al., 1998b) and does not reach maximum levels until 10 -18 weeks p.i. (nelson et al., 1994; yoon et al., 1995) . while the importance of a cellmediated response for protection against prrsv is well accepted, the effectiveness of this response during the early phases of disease also appears to be suboptimal (murtaugh et al., 2002) . for example, the t-cell response to prrsv is weak and transient and cannot be re-stimulated for more than 4 weeks post-challenge (molitor et al., 1997; xiao et al., 2004) . additionally, ifn-g responses of prrsvinfected pigs were relatively weak and increased slowly in comparison to pseudorabies-virus-infected pigs (meier et al., 2003) . although the precise mechanisms for the ineffective nature of the adaptive immune response to prrsv are not known, prrsv evasion of the innate immune responses, such as the type i ifn response, may set the stage for subsequent subversion of the adaptive immune response. previous studies demonstrated that prrsv appears to elicit weak innate interferon and cytokine responses compared to other viruses such as swine influenza virus (siv), porcine respiratory coronavirus (prcv), transmissible gastroenteritis (tge) virus, and pseudorabies virus (albina et al., 1998a; meier et al., 2003; van reeth and nauwynck, 2000; van reeth et al., 1999 . because nf-nb is important for regulation of type i ifn and after 48 h, cells were fixed and permeabilized followed by ifa. nuclear translocation of nf-nb was detected by confocal laser microscopy with fitc staining following incubation with a specific antibody recognizing the nf-nb p65 subunit. (b) nuclear translocation of nf-nb detected by western blot of nuclear extracts from marc-145 cells and pam cultures. both cell types were infected with prrsv at moi = 0.1. at 5, 24, 36, 48, and 60 (only in pam) h post-infection, nuclear extracts were prepared and subjected to western blot analysis, as described in materials and methods. cytokines (lenardo et al., 1989; mogensen and paludan, 2001) , we hypothesized that prrsv infection may inhibit nf-nb activation to prevent antiviral responses. the experiments presented herein were designed to determine if prrsv infection modulates nf-nb activation in host cells. after observing nf-nb activation following prrsv infection, the mechanisms by which prrsv mediates nf-nb activation, as well as the role of nf-nb activation in prrsv replication, were studied using a dominant negative form of inba. the present study provides a basis for understanding the molecular pathways of pathology and immune evasion associated with disease caused by prrsv. nuclear translocation, which is one of the key steps during activation of nf-nb, was detected by indirect fluorescent antibody (ifa) staining following infection with prrsv (fig. 1a) . in uninfected marc-145 cells, p65 staining was predominantly cytoplasmic, whereas prrsv infection (multiplicity of infection (moi) = 0.1) for 48 h resulted in nuclear localization of nf-nb p65 staining and increased cytoplasmic staining of nf-nb p65. in fig. 1b , western blot analysis shows that infection with prrsv led to accumulation of nf-nb protein in the nucleus. compared to uninfected control cells, the amount of nf-nb increased as prrsv infection progressed in marc-145 cells and pam cultures. increased nf-nb concentrations in the nucleus were apparent at 24 h p.i. in marc-145 cells and at 36 h p.i. in pam cultures. in these (and subsequent) experiments, cell viability was assessed by trypan blue staining, and cultures were consistently found to be approximately 96% viable in mock-infected cells and 92% in prrsv-infected cells at 48 h p.i. to determine if nuclear translocated nf-nb was capable of binding nb binding motifs following prrsv infection, an nf-nb p65 transcription factor assay was performed using nuclear extracts of marc-145 cells and pam cultures infected with prrsv. marc-145 cells and pam cultures were either mock-infected or infected with prrsv at moi = 0.1, and nuclear extracts were prepared at the indicated times after virus infection. following infection with prrsv, nf-nb p65 dna binding activity increased as prrsv infection progressed. although a slight, though statistically significant decrease was consistently observed at 24 h p.i. in pam cultures (but not marc-145 cells), the predominant effect of prrsv infection in both cell types was an increase in nf-nb dna binding activity. as shown in fig. 2 , significant increases in nf-nb dna binding activity in prrsv-infected cells were measured, especially at 36 and 48 h p.i, compared with that in mock-infected cells. at 48 h p.i., nf-nb p65 dna binding activities increased 5.7-fold in marc-145 cells and 2.2-fold in pam cultures. the nf-nb dna binding activity observed in these assays was ablated by an excess of unlabeled competitor, but not by an excess of unlabeled noncompetitor (data not shown). taken together, these results demonstrated that prrsv induces nuclear translocation of nf-nb followed by increased dna binding activity of nf-nb both in marc-145 cells and pam cultures. prrsv enhances nf-jb-regulated gene expression, and nf-jb activation by prrsv is dependent on viral dose and active viral replication translocation into the nucleus allows nf-nb to stimulate expression of target genes. thus, an nf-nb reporter assay was used to determine if prrsv infection enhanced nf-nb-regulated gene expression. marc-145 cells were transiently transfected with an nf-nb luciferase reporter plasmid (nf-nb-luc), which contains nb binding motifs under the control of a cmv promoter. therefore, luciferase expression is under control of nf-nb activation. after transfection, cells were infected with prrsv for 1 h or left uninfected. nf-nb-regulated luciferase expression was significantly enhanced during prrsv infection at 36 and 48 h p.i. which correlated with an increased level of nf-nb dna binding activity (fig. 3a) . nf-nb activity was 2.53-fold and 6.08-fold higher in prrsv-infected cells at 36 and 48 h p.i. compared to uninfected control cells. these findings show that prrsv infection stimulated nf-nbregulated gene expression late in infection, which means that nf-nb activated by prrsv is transcriptionally active and functional. to determine if there was a relationship between prrsv replication and nf-nb activation, marc-145 cells were infected at various mois and nf-nb activation was monitored by measuring nf-nb-regulated luciferase expression. as shown in fig. 3b , a higher moi resulted in higher levels of nf-nb activation, suggesting that nf-nb activation by prrsv is viral dose-dependent. in some viral infections, binding of the viral particle to a cellular surface receptor is sufficient to trigger signaling cascades that activate nf-nb. to test this possibility, uv-inactivated prrsv was used to determine if prrsv binding to its receptor mediates nf-nb activation. as shown in fig. 3b , uv-inactivation of prrsv decreased nf-nb activation compared to infection with noninactivated (fully infectious) prrsv. nf-nb-regulated gene expression was reduced from 3.34-fold to 1.43-fold at moi = 1, from 1.99-fold to 1.15 at moi = 0.1, and from 1.42-fold to 0.96-fold at moi = 0.01. these data demonstrated that nf-nb levels are elevated primarily as a result of prrsv replication and that uv-inactivation of prrsv significantly decreased this effect. a key step that leads to nf-nb activation in response to many extracellular stimuli is degradation of inb proteins. therefore, it was determined if prrsv-mediated changes in the inb proteins correlated with increased nf-nb activity. protein levels of inba, inbh, and inbe were monitored by western blot analysis following a time course of infection in marc-145 cells and pam cultures. results in fig. 4 show that inba, inbh, and inbe were found in uninfected marc-145 cells and pams and the protein levels were mostly unchanged throughout the time course. in marc-145 cells (fig. 4a ), prrsv infection resulted in a lower concentration of inba at 48 h p.i. compared to uninfected control cells, suggesting proteosomal degradation of inba. this correlated with the highest nf-nb activity at 48 h p.i. among the time points tested. however, inba was still weakly detectable in prrsv-infected cells at late times p.i. inbh and inbe remained relatively constant at all time points in prrsv-infected marc-145 cells. in pam cultures, degradation of inba and inbe was detected after prrsv infection at 48 h and 60 h p.i. as shown in fig. 4b . the degradation of inbh protein was detected at 60 h p.i. the onset of the degradation of inb proteins correlated with the nf-nb activation later in prrsv infection as observed above. for both marc-145 cells and pam cultures, the same blot was also reacted with an actin-specific antibody to confirm that comparable amounts of protein were loaded in each lane. #p < 0.05 compared to mock-infected control, *p < 0.01 compared to mock-infected control. results are representative of at least three independent experiments. (b) marc-145 cells transfected with nf-nb-luc and phrg-tk plasmid were infected with prrsv or uv-inactivated prrsv at moi = 1, 0.1, and 0.01 for 1 h. after washing with pbs, fresh medium was added. at 24 h post-infection, cells were lysed. firefly and renillar luciferase activities were measured by using a dual luciferase reporter assay kit. the luciferase assays were performed in triplicate. firefly luciferase activity was normalized by renillar luciferase activity. results are shown as the relative fold change compared to that of mock-infected cells. the symbol c indicates p < 0.001 for results from infectious virus compared to mock-infected controls. the symbols #, *, and k indicate p values <0.01, <0.001, and <0.05, respectively, for results from uvinactivated virus compared to infectious virus. these results are representative of at least three independent experiments. each bar represents the averaged data from one representative experiment. values are shown as the mean t sd from triplicate wells. to determine if nf-nb activation by prrsv was dependent on inba degradation in marc-145 cells, the nf-nb pathway was blocked by using an adenovirus vector expressing a dominant negative form of inba (ad-inbadn) which lacks both constitutive (barroga et al., 1995) and inducible (brown et al., 1995) phosphorylation sites. thus, inbadn is a potent nf-nb inhibitor. the same adenovirus vector expressing gfp (ad-egfp) instead of inbadn was used as a control. marc-145 cells were infected with ad-egfp or ad-inbadn at various mois (1, 10, or 100) for 16 h and then transfected with pnf-nb luc plasmid and phrg-tk plasmid followed by superinfection with prrsv at moi = 0.1. after 48 h, cells were lysed and analyzed for luciferase activity. overexpression of inbadn significantly suppressed constitutive nf-nb activity compared to that in the ad-egfp, and such inbadn expressing cells failed to activate nf-nb in response to prrsv infection (fig. 5) . it was then determined if nf-nb was required for efficient prrsv replication. cell culture medium was collected at 0, 5, 24, 36, and 48 h p.i. production of infectious progeny virus was determined by serial 10-fold dilutions of viral stocks with 50% tissue culture infectious dose (tcid 50 ) titers calculated by the method of reed and muench (1938) on marc-145 cells. the results representing three independent experiments are shown in fig. 6 . the kinetics of prrsv replication were compared to that in control cells. the results showed that neither ad-egfp (fig. 6a ) nor ad-inbadn (fig. 6b ) affected prrsv replication in marc-145 cells. in addition, a typical prrsv-induced cpe was observed in both ad-egfp-and ad-inbadn-infected cells. therefore, this result showed that blocking the nf-nb pathway by overexpression of inbadn does not alter production of prrsv progeny viruses. intracellular ros production was detected by staining with the hydrogen-peroxide-sensitive fluorescent dye dcfh-da which is cleaved intracellularly by nonspecific 5 . degradation of inba is required in nf-nb activation induced by prrsv. marc-145 cells were infected with ad-egfp or ad-inbadn at different moi for 16 h and then transfected with pnf-nb luc plasmid and phrg-tk plasmid. cells were then mock-infected or infected with prrsv at moi = 0.1. at 48 h p.i., cells were lysed, and lysates were analyzed for firefly and renillar luciferase activities using a dual luciferase reporter assay kit. firefly luciferase activity was normalized by renillar luciferase activity. results are shown as the relative fold change compared to that of mockinfected cells. for all assays, analysis was performed in triplicate, and values are shown as mean t sd. these results are representative of at least three independent experiments. *p < 0.001. esterases to form dcfh. ros in the cells then oxidizes dcfh to form the fluorescent product dcf (sawada et al., 1996) . as shown in fig. 7a , dcf fluorescence was enhanced in cells infected with prrsv at 48 h p.i, suggesting that prrsv induced ros production in marc-145 cells. to determine if ros induction by prrsv contributed to activation of nf-nb, cells were treated with antioxidants and nf-nb binding activity was measured at 48 h p.i. the dna binding activity of nf-nb p65 was markedly reduced in prrsv-infected cells when treated with the antioxidant pdtc or nac as shown in fig. 7b . the highest concentration of pdtc or nac used in this experiment reduced nf-nb activity to less than 30% in prrsv-infected cells. to rule out that the observed effect is simply due to inhibition of viral replication by antioxidants, virus titer was determined at 24 and 48 h p.i. (fig. 7c) . prrsv replication was not significantly affected by either pdtc or nac in all concentration tested. to investigate a possible biological role of nf-nb activation in prrsv pathogenesis, mrna expression of mmp-2 and mmp-9 which are regulated by nf-nb were determined at 48 h p.i. as shown in fig. 8 , mmp-2 and mmp-9 gene expression were significantly enhanced by prrsv infection which increased expression by approximately 13-and 10-fold, respectively. overexpression of inbadn completely blocked mmp-2 and mmp-9 gene expression. therefore, these data indicate that the activation of the nf-nb pathway by prrsv was necessary for enhanced mrna expressions of mmp-2 and mmp-9. virus -host interactions lead to both activation and inhibition of complex cellular pathways, resulting in antiviral responses as well as enhanced viral replication and virulence. despite years of research, little is known about intracellular signaling pathways that play key roles after prrsv infection and the role of these pathways in prrsv pathogenesis. the present study demonstrated for the first time that a virus belonging to the arteriviridae family activates nf-nb in host cells and that potential mechanisms of prrsv-mediated nf-nb activation are derived from the inb protein degradation and ros induction. the major target cells of prrsv in vivo are tissue macrophages such as pams. marc-145 cells are the only continuous cell line that is highly permissive for prrsv infection (kim et al., 1993) , and they are typically used for in vitro experiments of prrsv as well as virus maintenance and attenuation in the laboratory. viruses could have different effects on nf-nb pathways depending on the cell type infected as demonstrated in epstein-barr virus and measles virus infection (devergne et al., 1996; dhib-jalbut et al., 1999; dreyfus et al., 1999; fang et al., 2001; helin et al., 2001) . therefore, in the present study, both marc-145 cells and pam cultures were used to determine if prrsv activates the nf-nb pathway. this study showed that prrsv infection resulted in increased nuclear translocation of nf-nb and increased dna binding activity both in marc-145 cells and pam cultures. in addition, nf-nbdependent luciferase expression was significantly increased in marc-145 cells by prrsv infection. although the nf-nb reporter assay was not successfully performed in pam cultures (due to extremely low transfection efficiencies of pam cultures), results presented here clearly demonstrated that prrsv activates the nf-nb pathway in both its natural target cells, pam, as well as in a continuous cell line, marc-145 cells. viruses have developed various strategies which lead to either activation or inhibition of nf-nb-dependent gene transcription for their benefits (santoro et al., 2003) . the nf-nb pathway can be activated as a protective response of the host to viruses. therefore, some viruses, such as vaccinia virus, african swine fever virus, influenza a virus, and mengovirus, have evolved strategies to block nf-nb activation in order to evade the innate immune response (powell et al., 1996; shisler and jin, 2004; wang et al., 2000; zoll et al., 2002) . the nf-nb pathway can also be activated directly by viruses. viruses including hiv, herpesviruses, hepatitis c virus, encephalomyocarditis virus, reovirus, dengue virus, west nile virus, and herpes simplex virus have evolved strategies to activate nf-nb to exploit nf-nb for optimized replication, or to control host cell proliferation and survival to maximize viral progeny production (connolly et al., 2000; goodkin et al., 2003; jan et al., 2000; santoro et al., 2003; schwarz et al., 1998; waris et al., 2003) . despite the importance of the nf-nb pathway in immune response, it has not been determined if prrsv or other arteriviruses modulate this pathway. previous studies demonstrated that prrsv induced weak type i ifn responses (albina et al., 1998a; lee et al., 2004; miller et al., 2004; van reeth et al., 1999) . therefore, it has been postulated that prrsv inhibited the nf-nb pathway to evade antiviral responses of host cells. however, the present study provides evidence that prrsv actually activates the nf-nb pathway in pams, which are primary target cells in vivo. the synthesis of type i ifn is regulated trascriptionally and post-transcriptionally, and various transcription factors such as the interferon regulatory factor (irf) family as well as nf-nb may be involved (hiscott et al., 2003; taniguchi and takaoka, 2002; wathelet et al., 1998) . therefore, it is possible that prrsv blocks ifn gene expression at transcriptional and/or post-transcription levels but does not inhibit the nf-nb pathway. fig. 8 . prrsv increases mmp-2 and mmp-9 gene expression through an nf-nb-dependent pathway. marc-145 cells were infected with prrsv for 48 h at moi = 0.1, and total rna was extracted and treated with dnase i. quantitative real-time rt-pcr was performed for mmp-2 or mmp-9 specific primers. results are expressed as relative fold changes of mmp-2 or mmp-9 mrna using cyclophilin as an internal control. values are shown as the means t sd from triplicate wells and represent two independent experiments. *p < 0.001 compared to ad-egfp/prrsvinfected cells. a number of studies have suggested that oxidative stress induced by increased generation of ros is involved in the activation of nf-nb (ghosh and karin, 2002; janssen-heininger et al., 2000) . virus infections such as human immunodeficiency virus (hiv) (israel and gougerot-pocidalo, 1997) , cytomegalovirus (cmv) (speir, 2000) , influenza virus (flory et al., 2000) , hepatitis b virus (hbv) , hepatitis c virus (hcv) (gong et al., 2001) , japanese encephalitis virus (lin et al., 2004) , and herpes simplex virus (mogensen et al., 2003) activate the nf-nb pathway through ros production. our study demonstrated that prrsv generated ros, and the involvement of ros in nf-nb activation by prrsv was demonstrated by reduced nf-nb binding activity in the presence of pdtc and nac. it has been shown that oxidative stress induced by ros is associated with viral pathogenesis in case of influenza virus and hiv (peterhans, 1997; schwarz, 1996) . however, the role of ros in prrsv pathogenesis remains to be elucidated. viruses modulate nf-nb activation through various mechanisms. activation of nf-nb is usually mediated by degradation of inba in a proteasome-dependent mechanism after phosphorylation by ikk (hayden and ghosh, 2004) . inba is generally thought to be the major inhibitor of nf-nb activation. nf-nb activation by influenza virus is mediated by oxidative radicals and activation of ikk as a result of overexpression of viral proteins in endoplasmic reticulum (flory et al., 2000) . the tax transactivator oncoprotein of human t-lymphotropic virus-1 activates nf-nb by interacting directly with ikk (o'mahony et al., 2004) . hsv-1 induces persistent translocation of nf-nb by inba degradation (patel et al., 1998) . in this study, western blot analysis of inb protein levels revealed that inba protein was degraded in prrsv-infected cells and the expression of inba-dn eliminated nf-nb activation by prrsv. this finding demonstrates that nf-nb activation by prrsv is mediated at least in part by inba degradation in marc-145 cells and the degradation of inba, inbh, and inbe in pam cultures. this result indicates the possibility that different molecules are involved in nf-nb activation in pam culture infected with prrsv compared to marc-145 cells. however, the precise mechanism through which prrsv influences the inb degradation in both cells is presently unknown. some viruses activate the nf-nb pathway through viral protein-cellular receptor interaction. for instance, hiv gp120 and ebv gp350 activate nf-nb signaling pathway through binding to cd4 and cd21 (bossis et al., 2002; d'addario et al., 1999; sugano et al., 1997) . however, it is unlikely that nf-nb activation by prrsv is triggered solely by viral binding to its cognate cellular receptor because the level of nf-nb activation by prrsv increased as prrsv replication progression was significantly reduced by uv-inactivation of virus. therefore, it is possible that prrsv replication or viral protein expression is a prerequisite for activation of the nf-nb pathway. alternatively, a soluble factor induced by prrsv could be responsible for the delayed nf-nb activation. previous studies have demonstrated a requirement for nf-nb activation in viral replication. influenza virus infection is dependent on an active nf-nb signaling pathway (nimmerjahn et al., 2004) . inhibition of nf-nb activation blocked influenza virus infection of susceptible cells, and cells with low nf-nb activity were poorly susceptible to influenza virus infection (nimmerjahn et al., 2004) . efficient replication of hsv-1 is promoted by nf-nb activation through the inb kinase-inb-p65 pathway (gregory et al., 2004) . however, blocking the nf-nb pathway by overexpression of dominant negative forms of inba did not interfere with prrsv replication, suggesting that activation of the nf-nb pathway is non-essential for efficient viral replication. similarly, other studies with japanese encephalitis virus or cytomegalovirus demonstrated that nf-nb activation is not required for efficient viral replication (benedict et al., 2004; liao et al., 2001) . although nf-nb activation does not play an essential role in prrsv replication in vitro, it does not mean that nf-nb has no contribution to prrsv pathogenesis in vivo. macrophages represent an important source for a variety of soluble immune mediators, including cytokines, chemokines, mmps, and adhesion molecules which often contain nf-nb binding sites in their promoters (caamano and hunter, 2002; kim and koh, 2000; li and verma, 2002; mogensen and paludan, 2001) . the mmps are a group of zinc-and calcium-dependent endopeptidases that degrade an extracellular matrix implicated in tissue remodeling and chronic inflammation. mmps, especially mmp-2 and mmp-9, play a role in immune responses by promoting infiltration of inflammatory cells (kumagai et al., 1999) . a previous study demonstrated that prrsv infection significantly increased mmp-2 and mmp-9 which correlated with the appearance of severe histological lung lesions characterized by massive lymphomononuclear cell infiltration and possible local immunosuppression in prrsv-infected pigs. therefore, it is possible that mmps produced in prrsv-infected cells by nf-nb activation could mediate the influx of new cells of the monocyte/macrophage lineage. it is well established that nf-nb may play a pivotal role in apoptosis of virus-infected cells. the nf-nb activation by viruses could be either an anti-apoptotic response to maximize viral replication by prolonging host cell survival or pro-apoptotic response as a mechanism to increase virus spread (bowie et al., 2004; mi et al., 2001) . in cells infected with viruses such as sindbis virus, reovirus, or dengue virus, apoptosis is facilitated by the activation of nf-nb which was triggered by viral infection (connolly et al., 2000; jan et al., 2000; lin et al., 1995 lin et al., , 1998 . in others, activated nf-nb prevents apoptosis and prolongs cell survival (bowie et al., 2004; goodkin et al., 2003; grimm et al., 2005; su et al., 2001; thomas et al., 2002) . prrsv has been known to induce apoptosis mostly in bystander cells in vivo (sirinarumitr et al., 1998) . it is not known yet whether the activation of nf-nb in prrsv infection plays an anti-apoptotic role or pro-apoptotic role. in summary, the present study demonstrated that prrsv activates nf-nb via inb degradation in marc-145 cells and pam cultures and that nf-nb activation is not required for efficient prrsv replication in vitro. ros induction likely contributes to activation of the nf-nb pathway in prrsvinfected cells. however, the detailed molecular mechanisms and viral components underlying nf-nb activation remain to be elucidated. this study also suggested a possible role of nf-nb activation in prrsv pathogenesis by showing that prrsv increased mmp-2 and mmp-9 mrna expression through the nf-nb pathway. future studies will confirm if prrsv activates the nf-nb pathway in vivo. understanding the role of nf-nb activation following prrsv infection will contribute important information about the molecular pathogenesis of prrsv infection. the marc-145 cell line, which is a clone of the african green monkey kidney cell line ma-104, and the hek-293 cell line, which is derived from human embryonic kidney cells were cultured and maintained in dulbecco's modified eagle medium (dmem) supplemented with 10% fbs, 0.25 ag/ml fungizone, 100 u/ml penicillin, 10 ag/ml streptomycin sulfate, and 5 ag/ml gentamicin (biowhittaker inc., walkersville, md) and then held at 37 -c in a humidified 5% co 2 incubator. the pam cultures were obtained by bronchoalveolar lavage of 2-to 3-week-old domestic piglets from a prrsv negative herd. the lungs were removed from the piglets immediately after euthanizing by electrocution, and rpmi-1640 medium (life technologies, grand island, ny) was introduced through the main stem bronchi. bronchoalveolar lavage fluid was centrifuged at 400 â g for 7 min. after centrifugation, cell pellets were resuspended in rpmi-1640 medium supplemented with 10% fetal bovine serum (fbs), 2 mm l-glutamine, 0.25 ag/ml fungizone, 100 u/ml penicillin, 10 ag/ml streptomycin sulfate, and 5 ag/ml gentamicin (biowhittaker, walkersville, md) and plated at a density of 2 -4 â 10 5 cells/well in a 24-well primaria plate (becton dickinson and company, franklin lakes, nj) . the pam cultures were confirmed to be prrsv-negative by rt-pcr before used in subsequent experiments. pam cultures were incubated for 24 h at 37 -c in a humidified 5% co 2 incubator and washed once with complete rpmi-1640 media before use. all cells were maintained at 37 -c in a humidified 5% co 2 incubator. prrsv isolate 25544 was obtained from clinical cases submitted to the university of missouri's veterinary medicine diagnostic laboratory. virus stocks of prrsv were prepared in marc-145 cells. a low multiplicity of infection (moi < 0.05) was used to prepare viral stocks. for virus infection, cells were initially adsorbed with virus at the indicated moi for 1 h at 37 -c. after 1 h of adsorption, cells were gently washed with medium. at indicated time points post-infection (p.i.), culture media were harvested for the virus titration, and cells were lysed to prepare cellular extracts. adenovirus vectors expressing a dominant negative inba or green fluorescent protein (gfp) were kind gifts from dr. steven l. bachenheimer (university of north carolina, usa). adenovirus stocks were prepared in hek-293 cells. subconfluent monolayers of marc-145 cells in a 24well plate were transfected with 10 ng of prl-tk (promega, madison, wi) and 100 ng of pnf-nbluc (stratagene, la jolla, ca) using the lipofectamine transfection reagent (invitrogen carlsbad, ca). the prl-tk plasmid contains the renilla reniformis (sea pansy) luciferase gene under the transcriptional control of the herpesvirus thymidine kinase promoter and constitutively expresses low levels of renillar luciferase. the pnf-nbluc plasmid contains the firefly luciferase gene under the transcriptional control of a synthetic promoter containing five direct repeats of the nf-nb binding element. after transfection, cells were mock-infected or infected with viruses at the indicated moi. at various time points post-infection, cell monolayers were lysed in 150 al of passive lysis buffer (promega, madison, wi) followed by cell lysate analysis for both luciferase activities by using the dual luciferase reporter assay (promega, madison, wi). luciferase activity was measured as relative light units (rlus) using a luminometer (turner biosystems, inc. sunnyvale, ca). for all assays, experiments were performed in triplicate. for each experimental point, the average of the firefly luciferase activity was divided by the average of sea pansy luciferase activity to correct for differences in transfection efficiencies. the resulting ratios were used to compare the expression of the firefly luciferase gene in virus-infected cells to that present in uninfected (mock) cells. cytoplasmic and nuclear protein extracts from marc-145 cells and pam cultures were prepared with the nuclear extraction kit (active motif, inc., carlsbad, ca) according to the manufacturer's protocol. protein concentration was determined by the bio-rad protein assay (bio-rad, hercules, ca) with bovine serum albumin as a standard. nf-jb p65 transcription factor assay nf-nb binding to nb sites was assessed using the trans-am nf-nb p65 transcription factor assay kit (active motif, inc., carlsbad, ca). in this assay, an oligonucleotide containing the nf-nb consensus site is attached to a 96-well plate. the active form of nf-nb contained in cell extracts specifically binds to this oligonucleotide and can be revealed by incubation with antibodies using enzyme-linked immunosorbent assay technology with absorbance reading. ten microgram of nuclear proteins was analyzed for p65 binding to nb oligonucleotide according to the manufacturer's instructions. the specificity of the assay was monitored by competition with free wild-type nb consensus oligonucleotide or mutated nb consensus oligonucleotide. marc-145 cells were washed three times with pbs, fixed and permeabilized in cold methanol for 10 min at 4 -c, and washed three times with pbs. cells were incubated for 1 h at room temperature (rt) with a 1:500 dilution of primary mouse anti-nf-nb p65 antibody (santa cruz biotechnology, santa cruz, ca). the cells were then washed five times in pbs followed by incubation for 1 h with the anti-mouse igg secondary antibody conjugated with fitc (sigma, st. louis, mo). the cells were washed again with pbs and then examined with an olympus 1x70 microscope fitted with a bio-rad mrc-600 confocal laser (bio-rad, hercules, ca). western blotting was performed by utilizing a standard protocol (davis et al., 1994) . briefly, cytoplasmic or nuclear extracts were diluted (2:1) in 2â sample buffer and boiled for 5 min. twenty micrograms of each extract was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and transferred to a nitrocellulose membrane (amersham biosciences, piscataway, nj). the membrane was washed with phosphate-buffered saline-tween 20 (tpbs), blocked in a solution of tpbs containing 5% nonfat dry milk, and then washed three times. the membrane was then incubated with primary antibody overnight at 4 -c or 1 h at rt, washed three times with tpbs, and incubated with the secondary antibody horseradish peroxidase (hrp) conjugate solution for 1 h at rt. samples were washed three times with tpbs, and then the signal was detected with the chemiluminescent protein detection system (amersham biosciences, piscataway, nj). antibodies used for western blot are anti-nf-nb p65 (santa cruz biotechnology, santa cruz, ca), anti-inba (cell signaling, beverly, ma), anti-inbh (santa cruz biotechnology, santa cruz, ca), anti-inbq (santa cruz biotechnology, santa cruz, ca), anti-rabbit igg-hrp (amersham biosciences, piscataway, nj), and anti-actin (sigma, st. louis, mo). cells were treated with 2v,7v-dichlorofluorescein diacetate (dcfh-da, calbiochem, la jolla, ca) for 15 min and washed twice with pbs. the cells were observed under a fluorescence microscope. the antioxidants used in this study were n-acetyl-lcysteine (nac; calbiochem, la jolla, ca) in pbs and pyrollidine dithiocarbamate (pdtc; sigma, st. louis, mo) in pbs. concentrations which did not show cytotoxicity were used in this study. none of the solvents alone affected nf-nb activation in the concentrations used in this study (data not shown). extraction of rna was performed using trizol (invitrogen, carlsbad, ca), and the nucleospin rna ii kit (bd biosciences inc., palo alto, ca) with dnase i digestion performed directly on the spin column according to the manufacturer's instructions. heterologous competitor rna for quantification of mmp-2, mmp-9, or cyclophilin was synthesized using the respective real-time rt-pcr primer sequences in a methodology previously described (kleiboeker, 2003) . the concentration of purified competitor rna was estimated by measuring the absorbance at 260 nm, and the purity was assessed by determining the ratio of absorbance at 260 nm to the absorbance at 280 nm. samples were considered to be relatively pure and suitable for use as quantification standards if the ratio was !2.0. following purification, the rna was serially diluted in rnase-free dh 2 o and stored as aliquots at à80 -c. the number of molecules of competitor rna/al was estimated based on the rna concentration and the molecular weight of the transcript. amplification of 2 al rna was performed using the qiagen quantitect probe rt-pcr kit (qiagen inc., valencia, ca) with thermocycling, and detection was performed in a stratagene mx4000 (stratagene inc., la jolla, ca). samples were analyzed in triplicate. thermocycling conditions were: 50 -c (30 min), 95 -c (15 min), followed by 40 cycles of denaturation (94 -c, 15 s) and annealing/ extension (60 -c, 60 s). the primers and probe used for 5 exonuclease (taqman) amplification of mmp-9 were 5v-ccaccacaacatcacctattgg-3v (forward), 5v-gaa-ggcgcgggcaaa-3v (reverse), and 6-fam-tccaaaa-ctactcggaagacttgccgc-bhq1-3v (probe). the primers and probe used for 5v exonuclease (taqman) amplification of mmp-2 were 5v-ccgtcgcccatcat-caa-3v (forward), 5v-caggtattgcactgccaactct-3v (reverse), and 6-fam-cgatgtcgcccccaaaacgga-bhq1-3 (probe). amplification of cyclophilin was performed as previously described (miller et al., 2004) and was used for normalization of mmp-2 and mmp-9 transcript quantities. all oligonucleotide primers were used at a final concentration of 0.3 am, and the dual-labeled probes were used at a final concentration of 0.2 am. all oligonucleotide primers and probes were synthesized by integrated dna technologies, inc. (coralville, ia). the relative copy numbers of mmp-2 and mmp-9 compared to cyclophilin were determined as previously described (pfaffl, 2001) . the student's t test was used for the statistical analyses. p values of less than 0.05 were considered statistically significant. interferon-alpha response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus immune responses in pigs infected with porcine reproductive and respiratory syndrome virus (prrsv) constitutive phosphorylation of i kappa b alpha by casein kinase ii neutrality of the canonical nf-kappabdependent pathway for human and murine cytomegalovirus transcription and replication in vitro characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr-2332) temporal activation of nf-kappab regulates an interferon-independent innate antiviral response against cytoplasmic rna viruses nf-kappab activation upon interaction of hiv-1 envelope glycoproteins with cell surface cd4 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virus-specific t-cell and macrophage recruitment in porcine reproductive and respiratory syndrome virus infection in pigs is independent of virus load characterization of the humoral immune response to porcine reproductive and respiratory syndrome (prrs) virus infection laboratory investigation of prrs virus infection in three swine herds the mengovirus leader protein suppresses alpha/beta interferon production by inhibition of the iron/ferritin-mediated activation of nf-kappa b key: cord-299994-1ksfo0pr authors: kanitz, manuel; blanck, sandra; heine, andreas; gulyaeva, anastasia a.; gorbalenya, alexander e.; ziebuhr, john; diederich, wibke e. title: structural basis for catalysis and substrate specificity of a 3c-like cysteine protease from a mosquito mesonivirus date: 2019-05-02 journal: virology doi: 10.1016/j.virol.2019.05.001 sha: doc_id: 299994 cord_uid: 1ksfo0pr cavally virus (cavv) is a mosquito-borne plus-strand rna virus in the family mesoniviridae (order nidovirales). we present x-ray structures for the cavv 3c-like protease (3cl(pro)), as a free enzyme and in complex with a peptide aldehyde inhibitor mimicking the p4-to-p1 residues of a natural substrate. the 3cl(pro) structure (refined to 1.94 å) shows that the protein forms dimers. the monomers are comprised of n-terminal domains i and ii, which adopt a chymotrypsin-like fold, and a c-terminal α-helical domain iii. the catalytic cys-his dyad is assisted by a complex network of interactions involving a water molecule that mediates polar contacts between the catalytic his and a conserved asp located in the domain ii-iii junction and is suitably positioned to stabilize the developing positive charge of the catalytic his in the transition state during catalysis. the study also reveals the structural basis for the distinct p2 asn-specific substrate-binding pocket of mesonivirus 3cl(pro)s. proteases with a two-β-barrel fold prototyped by chymotrypsin form one of the largest clans of proteolytic enzymes, called the pa clan, and are encoded by pro-and eukaryotes as well as many viruses (rawlings et al., 2012) . the cellular chymotrypsin-like enzymes invariantly employ a canonical catalytic ser-his-asp triad (hedstrom, 2002; polgar, 2005) and have evolved, by duplication and diversification, a large spectrum of substrate specificities facilitating their diverse roles in many biological processes. likewise, rna viruses with single-stranded rna genomes of positive polarity (ssrna + viruses) often employ chymotrypsin-like enzymes to control genome expression and remodel host cell functions by targeting different proteins. one of the major monophyletic groups of rna virus chymotrypsinlike enzymes is known as 3c and 3c-like proteases (3c/3cl pro ; see below), which evolved by extensive diversification during virus speciation. in contrast to cellular and other viral homologs, these enzymes uniquely diversified the principal catalytic nucleophile residue to employ either the canonical ser or an unconventional cys (depending on the virus lineage) (bazan and fletterick, 1988; gorbalenya et al., 1986 gorbalenya et al., , 1989a malcolm, 1995) , while (somewhat counterintuitively) sharing a conserved narrow substrate specificity. in those proteases that employ cys as the principal nucleophile, the catalytic asp may be replaced with another residue, either another acidic residue (glu) (matthews et al., 1994; mosimann et al., 1997) or a residue with a side chain that has little or no similarity to that of asp, further reflecting the pronounced sequence divergence of many ssrna + virus proteases from their cellular homologs . probably, these replacements have evolved due to the exceptionally large mutation space that rna viruses of this lineage explored in the course of their evolution (lauber et al., 2013) and have been fixed to meet the distinct requirements for ser and cys residues in mediating the nucleophilic attack during catalysis (mcgrath et al., 1989) . 3c/3cl pro s possess a conserved substrate specificity that is predominantly directed against gln or glu in the p1 and a small residue in the p1' positions of the substrate (gorbalenya et al., 1989b; kräusslich and wimmer, 1988; ziebuhr et al., 2000) (numbering according to the scheme introduced by schechter and berger (1967) ). these substrate residues are accommodated in a protease substrate pocket that generally includes a highly conserved his residue (which differs from the catalytic his) and another residue, often ser or thr, which together define the s1 subsite homologous to that of cellular chymotrypsin-like proteases (bazan and fletterick, 1988; gorbalenya et al., 1989a) . these residues are generally considered valuable targets for the development of broadly acting antiviral drugs (kuo et al., 2009; yang et al., 2005; banerjee et al., 2018; pillaiyar et al., 2016) . viral 3c/3cl pro s are produced as part of large polyproteins that contain the key replicative domains and several other proteins (for reviews see (kräusslich and wimmer, 1988; ziebuhr et al., 2000; dougherty and semler, 1993; ryan and flint, 1997) ). the individual proteins are released by the 3c/3cl pro in both cis-and trans-cleavage reactions, thereby regulating the formation of viral replication complexes in a timely coordinated manner. in addition, some 3c/3cl pro s may mediate the processing of capsid polyproteins to generate the proteins required for virus particle formation as well as cleaving cellular proteins to facilitate virus reproduction. viruses encoding 3c/ 3cl pro s belong to two large monophyletic orders, the picornavirales (which infect uni-and multicellular eukaryotes) and the nidovirales (which infect vertebrates and invertebrates), but also include several other virus families or groups, such as the plant potyviridae and sobemovirus, and the vertebrate caliciviridae and astroviridae, displaying more distant affinities to one of these two virus orders. over the past years, our appreciation of the natural 3c/3cl pro diversity and conservation of specific residues has been steadily improved by comparative genomics, while our understanding of the roles and properties of these proteases in viral replication was largely based on just a few viruses of the above taxa, with a strong focus on viruses that (may) infect humans. specifically, the structural characterization of 3c/ 3cl pro s remained limited to those encoded by several mammalian viruses and one avian virus of the picornaviridae, coronaviridae, arteriviridae, caliciviridae, and astroviridae, and plant potyviridae and sobemovirus (matthews et al., 1994; banerjee et al., 2018; phan et al., 2002; nunn et al., 2005; speroni et al., 2009; gayathri et al., 2006; damalanka et al., 2018; weerawarna et al., 2016; galasiti kankanamalage et al., 2017; takahashi et al., 2013; kim et al., 2012; muhaxhiri et al., 2013; allaire et al., 1994; anand et al., 2002 anand et al., , 2003 bergmann et al., 1997; xue et al., 2008; yang et al., 2003; barrette-ng et al., 2002) . all these proteases have substrate specificities that are critically determined by the p1 position. in this study, we sought to address these knowledge gaps by determining the structure of a 3cl pro encoded by cavally virus, an invertebrate nidovirus. the protease of this virus represents family c107 in the pa(c) subclan of chymotrypsin-like proteases in the merops database (rawlings et al., 2012) and displays a substrate specificity that is mainly directed toward the p2 position of the substrate (blanck et al., 2014; blanck and ziebuhr, 2016) . cavally virus (cavv) is a member of the genus alphamesonivirus in the family mesoniviridae which, together with the families arteriviridae, coronaviridae, and roniviridae as well as five others just approved by ictv (siddell et al., 2019) , forms the order nidovirales (fig. 1) . a distinct feature of previously characterized proteases from this virus order is the presence of an extra c-terminal domain with regulator function. this domain was shown to adopt different folds in arteri-and coronavirus enzymes (representing protease families s32 and c30, respectively) (anand et al., 2002; barrette-ng et al., 2002) while there is currently no structural information on other nidovirus 3cl pro s, such as those of roni-, toro-and bafiniviruses, all of which representing distinct protease families in the merops database (c62, s65, and s75, respectively); toro-and bafiniviruses belong to distinct subfamilies of the newly established family tobaniviridae. here, we present the first crystal structures of a mesonivirus 3cl pro , both for the free enzyme and in complex with a covalently bound inhibitor. the protein structure comprises a two-β-barrel fold that is linked to a large c-terminal helical domain of > 100 residues. the structure analysis identifies critical residues involved in substrate binding and suggests that mesonivirus 3cl pro s employ a catalytic system that depends on (i) cys153 (principal nucleophile), (ii) his48 (general base), and (iii) a water molecule mediating a polar contact between (iv) asp216 and the catalytic his48. a strikingly similar arrangement was previously reported for coronavirus 3cl pro structures, which share very low sequence identity (< 10%) with 3cl pro orthologs from other nidovirus families, further supporting the universal key roles of these residues in the respective enzymes (fig. 2 ). the cavv 3cl pro crystal structure determined at 1.94 å resolution shows two 3cl pro molecules in the asymmetric unit (fig. 3a) . the two molecules, named a (residues 1-314) and b (residues 1-305), form a dimer with an overall contact surface of 1728 å 2 , involving 4 salt bridges and 24 hydrogen bonds as calculated by pdbepisa (krissinel and henrick, 2007) . molecules a and b are quite similar, with a rootmean-square deviation (rmsd) of 0.59 å (as determined for all equivalent c α atoms). each molecule is comprised of three domains, called i, ii, and iii, respectively. domains i and ii have a chymotrypsin-like, two-β-barrel fold that is formed by seven and six β-strands, respectively (β2-β8 and β9-β13 plus β15). the principal catalytic residues, cys153 and his48 (blanck et al., 2014) , are part of strand β9 and 3 10 helix ƞ6, respectively, and are positioned in the center of the active-site cleft located between domains i and ii. domain ii is connected to the c-terminal domain iii, the latter being formed by seven helices (ƞ7 and α2 to α7) (fig. 3a ). due to partially poor electron density, residues 51-55, 191-194, and 214-220 of chain a as well as residues 27-30 and 306-314 of chain b were omitted from the model. the c-terminal part of chain a wraps around chain b and thereby comes in close contact to the active site of chain b. the n-terminal domains i and ii of cavv 3cl pro resemble those of related enzymes from the coronaviridae and arteriviridae and the 3c/ 3cl pro enzymes from other rna + viruses that lack an extra c-terminal domain ( fig. 3b ) (matthews et al., 1994; mosimann et al., 1997; phan et al., 2002; speroni et al., 2009; muhaxhiri et al., 2013; allaire et al., 1994; anand et al., 2002 anand et al., , 2003 bergmann et al., 1997; xue et al., 2008; yang et al., 2003; barrette-ng et al., 2002; zhao et al., 2008) . the s γ atom of the catalytic cys153 in chain b is in h-bond distance to the carbonyl oxygen of phe150 (3.5 å), the carbonyl oxygen of right, presumed primary host of the respective virus species, 3cl pro principal nucleophile residue, and 3cl pro pdb structure availability. information on cavv is highlighted using a gray background. information on nidovirus genomes used for phylogeny reconstruction is provided in the supplemental information. box, sh-alrt branch support is shown using three ranges of values. structure-based msa of mesonivirus and coronavirus 3cl pro s. strictly conserved residues are indicated using red background color, partially conserved residues are indicated in red. secondary structure elements are shown for cavv (pdb id: 5lac, chain b) and tgev (pdb id: 1lvo, chain a) in blue above the alignment. residues that were omitted from the respective structure models are indicated in green. residue numbers given above the alignment refer to the cavv 3cl pro sequence corresponding to the cavv 3cl pro structure (pdb id: 5lac, chain b). crucial cavv 3cl pro residues are indicated below the alignment: catalytic residues -by asterisks; key residues in the s1 subsite of the substrate-binding pocket -by filled circles; the asp residue that interacts with a conserved water molecule in the active site -by a black triangle. pdb ids used to produce the structure-based msa are given in the supplemental information. cys169 (3.3 å), and the n ε of the catalytic his48 (3.7 å), respectively. the n δ of catalytic his48 makes also contact to the carboxylate side chain of asp216 through water molecule 558 with distances of 2.7 å and 2.9 å, respectively, forming an angle of 118.9°(chain b). this water molecule establishes another h-bond to water molecule 548 (2.7 å), which is also hydrogen-bonded to o γ of thr88 (3.0 å) and the side chain oxygen of tyr215 (2.7 å) (fig. 4) . furthermore, asp216 forms a salt bridge to arg47 with h-bond distances of 2.8 and 3.0 å, respectively. in monomer a, the residues of the active site exhibit a higher flexibility compared to the residues of chain b, which is indicated by (i) a slightly more disordered electron density for his48, (ii) an alternative side chain conformation for cys153 (56% conformer a, 44% conformer b), and (iii) the lack of visible asp216. overall structure. the structure of cavv 3cl pro in complex with bz-yynq-h, a peptide aldehyde inhibitor representing the p1-p4 residues of the c-terminal cavv 3cl pro autoprocessing site, contains four rather than two molecules in the asymmetric unit. the molecules form a tetramer (a dimer of dimers) with rmsds of 0.53 å (for dimer a-b) and 0.65 å (for dimer c-d), respectively. rmsd values of all four monomers are low and range between 0.33 and 0.56 å, suggesting that the four monomers adopt the same fold. the total contact areas between monomers a and b and between c and d were calculated to be 1770 å 2 and 1640 å 2 , respectively. dimer a/b is stabilized by four salt bridges and 29 hydrogen bonds whereas, in the contact surface of c/d, three salt bridges and 24 hydrogen bonds are present as determined by pdbepisa (krissinel and henrick, 2007) . with only 494 å 2 , the contact area between the two dimers is significantly smaller and involves three salt bridges and eight hydrogen bonds. in both monomers a and c, arg28 was found to reach into the substrate-binding pocket of another molecule (c and a, respectively), resulting in different interactions between enzyme and ligand in those cases (see below). (in monomer c, the side chain of arg28 was omitted due to missing electron density, but it would interact with molecule a if present.) as expected, the ligand bz-yynq-h has reacted with the sulfhydryl moiety of the catalytic cys153 and the resulting thiohemiacetal is clearly detectable in the active site with well-defined density in the 2mfo-dfc map for monomers a, b, and c (fig. 5a ). also for monomer d, the fo-fc map indicates that the ligand is bound but, in this case, the electron density is relatively poor. we therefore omitted the ligand from the final model. the binding mode of the ligand to the three subsites s1, s2, and s3 is nearly identical for monomers a, b, and c, while slight variations exist among the three monomers with respect to the s4 subsite (fig. 5b) . binding mode. in monomers a, b, and c, the ligand is covalently bound to cys153 with c-s bond lengths of 1.9 å (chain a), 1.8 å (chain b), and 1.8 å (chain c), respectively. the oxyanion hole that stabilizes the noncovalent michaelis complex (otto and schirmeister, 1997) and the transition state includes the amide nitrogens of gly151 (2.9 å to the oxygen of the thiohemiacetal) and cys153 (3.1 å to the oxygen of the thiohemiacetal) in molecule a. similar distances are observed in molecule c for the oxygen atom of the thiohemiacetal (2.8 å to gly151 and 2.9 å to cys153), while the distances in molecule b are much longer (4.4 å to gly 151 and 4.5 å to cys153) due to r-configuration. the ligand is suitably positioned to facilitate the nucleophilic attack of the sulfhydryl moiety at the prochiral carbonyl carbon, resulting in the formation of the (s)-configured thiohemiacetal in molecules a and c. overall, the binding pockets of the monomers have a similar shape, except for two flexible regions, tyr215-val222 and a small region around the oxyanion hole (phe150-cys153). s1 subpocket. in monomer a, the s1 subpocket is formed by his168 and thr148, plus arg28 of chain c, with the main chain carbonyl oxygen of arg forming a hydrogen bond to the n ε of the p1 gln of the ligand with a distance of 2.8 å. as the side chain of arg28(c) was not clearly defined in the electron density, it was not considered further. in contrast, the side chain of arg28 of chain a is well defined in the density of the s1 site of monomer c (fig. 6a ), which is organized in a manner similar to that of monomer a. the side chain closes the s1 site and the main chain carbonyl oxygen of arg28 forms a hydrogen bond to the n ε of the p1 gln (2.8 å). the o ε of the p1 gln establishes h-bonds to the n ε of his168 (c) at a distance of 2.7 å and to the o γ of thr148 (c) at a distance of 2.6 å. in monomer b, the ligand is bound slightly differently (fig. 6b ). the s1 subpocket is formed by his168 (b), thr148 (b), and val149 (b). the polar interactions between ligand and s1 subsite are very similar to those observed for monomers a and c. the binding is stabilized by hbonds between the o ε of the p1 gln to both the o γ of thr148 (b; 2.4 å) and the n ε of his168 (b; 2.8 å). we consider it likely that the observed interactions of arg28 (of chain c and a, respectively) with the s1 subpockets of another monomer (a and c, respectively) result from the crystal packing and thus represent crystallographic artifacts that are unlikely to occur in solution. s2 subpocket. the p2 asn residue in monomer c of the bz-yynq-h ligand points toward the s2 subpocket ( fig. 6c ) formed by asp216 and ser52. polar contacts are established between the n δ of the ligand p2 asn and the carbonyl oxygen of asp216 (chain a: 2.8 å; chain b: 2.9 å; chain c: 2.9 å). in monomers a and b, a second hydrogen bond is formed with the o γ of ser52 (chain a: 2.9 å; chain b: 3.4 å). because of insufficient electron density, the latter interaction could not be confirmed for monomer c. s3 subpocket. the s3 pocket (fig. 6d) is formed by asp173, with a hydrogen bond between o δ of asp173 and the oxygen of the p3 tyrosine (monomer a: 2.7 å; monomer c: 2.5 å) stabilizing the binding to the ligand. in monomer c, an additional hydrogen bond (3.1 å) to n ε of arg28 (a) is formed. a similar binding mode was observed for the p3 tyrosine in molecule b, albeit the side chain oh was not included in the model. s4 subpocket. the s4 pocket (fig. 6e) is formed by leu209, tyr 215, and val222 and flanked by a loop consisting of residues asp216 to asn221. the hydroxyl functionality of the ligand establishes hydrogen bonds to the carbonyl oxygen of tyr215 (monomer a: 2.7 å; monomer c: 2.6 å) and to the carbonyl oxygen of glu218 (chain a: 3.3 å; chain c: 2.3 å). the observed binding mode of the ligand differs in monomer b, where the residues of the loop region adopt a different conformation, resulting in a smaller s4 pocket, which, in turn, leads to a relocation of the p4 tyrosine. for the ligand in monomer b, an intramolecular hydrogen bond to the p2 asn residue is observed (2.5 å). for the n-terminal benzoyl group of the ligand, different conformations were observed for each monomer, resulting in different interactions. while the ligand does not establish any h-bonds in monomer a, h-bonds are formed to the amide nitrogen of val222 (3.6 å) in superposition of the peptide aldehyde bound to chain a (blue), chain b (yellow), and chain c (pink), respectively, each represented as sticks and colorcoded by atom type. the substrate-binding pocket of chain c (light blue) is shown in surface representation. subpockets s1 to s4 are indicated. monomer b and to the carbonyl oxygen atom of thr220 (2.8 å) in monomer c. in this study, we present the first crystal structure of a 3cl pro from a mosquito-borne nidovirus representing the family mesoniviridae in the nidovirales, a profoundly divergent order of rna + viruses. the structure supports and extends previous studies suggesting that nidovirus 3c-like main proteases share a three-domain domain organization involving a chymotrypsin-like fold (domains i and ii) and a c-terminal domain iii (anand et al., 2002; barrette-ng et al., 2002; blanck et al., 2014; blanck and ziebuhr, 2016; nga et al., 2011; zirkel et al., 2011 zirkel et al., , 2013 . domain iii is absent (or much smaller) in the related 3c/3cl pro s of other rna + viruses, while the n-terminal chymotrypsin-like fold (birktoft and blow, 1972; matthews et al., 1967) is conserved in all these enzymes (matthews et al., 1994; mosimann et al., 1997; muhaxhiri et al., 2013; allaire et al., 1994) . despite their conserved structural organization, nidovirus 3cl pro s are remarkably diverse with respect to their catalytic residues, again illustrating the profoundly divergent evolution of the various subfamilies of the order nidovirales. thus for example, arterivirus, torovirus and bafinivirus 3cl pro s employ a canonical ser-his-asp triad (barrette-ng et al., 2002; smits et al., 2006; ulferts et al., 2011) , while coronavirus, mesonivirus, and ronivirus 3cl pro s use a cys-his catalatic dyad that, as shown previously for coronavirus (anand et al., 2002) and, in this study, for mesonivirus 3cl pro , is assisted by a water molecule interacting with an asp residue located in a noncanonical position in the primary structure, c-proximal of the catalytic cys (see below and fig. 2) . database searches for related structures using the dali server (holm and rosenström, 2010) revealed the 3c-like cysteine proteases of the subfamily coronavirinae as the closest structural homologs of the cavv 3cl pro (pdb id: 5lac, chain a). using the hcov-hku1 3cl pro structure (pdb id: 3d23, chain a) , an rmsd (c α ) of 3.8 å (sequence identity 8%; z-score: 15.0) was calculated (239 residues of 299 from 3d23, chain a, were aligned). dimerization and interfaces. similar to what was shown previously for coronavirus 3cl pro s, the cavv 3cl pro forms a tight dimer in the crystal structure. the arrangement of the two molecules in dimer a/ b and c/d, respectively, is similar to the orientation reported for the two protomers that form the coronavirus 3cl pro dimer (anand et al., 2002 yang et al., 2003) . each monomer of the two dimers of the fig. 6 . key interactions of the ligand in the s1 (a), s2 (c), s3 (d), and s4 (e) subpockets, respectively, of monomer c and in the s1 subpocket of monomer b (b) of the cavv 3cl pro (pdb id: 5lak). atoms of the ligand (pink) and protease (chain c in light blue and chain a in green) are represented as sticks and color-coded by atom types. in panel (b), the ligand is shown in yellow and residues of chain b are shown in pink. only residues involved in hydrogen bonding are shown. cavv 3cl pro structure reaches into the other, with its n-terminus (residues 1-16) interacting with residues of the dimer mate, forming 6 hydrogen bonds (a2-b147, a5-b136, a5-b136, a7-b147, a10-b134 (2x)) and 1 salt bridge (a1-b296) in the case of monomer a. for the entire a/b dimer, 20 hydrogen bonds and 5 salt bridges are formed. the large contact interfaces observed for dimers a/b (1764 å 2 ) and c/d (1640 å 2 ) suggest that cavv 3cl pro dimer formation is of functional relevance which remains to be confirmed in further studies, such as those performed for coronavirus 3cl pro s, in which the role of dimerization for trans-cleavage activity was confirmed and characterized in significant detail (for a review, see (xia and kang, 2011) ). in contrast to the a/b and c/d dimers described above, the formation of the tetramer a/b-c/d with an interface area of 494 å 2 (involving three salt bridges and eight hydrogen bonds between monomers a and c) likely represents a crystallographic artifact. substrate-binding pocket. the s1 pockets of cavv 3cl pro and coronavirus 3cl pro s are similar and can accommodate a gln residue as shown here for a peptide aldehyde corresponding to the p1-to-p4 residues of the c-terminal cavv 3cl pro autoprocessing site. the structure shows that, in addition to his168, a conserved thr residue (thr148) is located in the s1 subsite and suitably positioned to establish interactions with gln (or glu), both of which are common in the p1 position of mesonivirus (including cavv) 3cl pro substrates (blanck and ziebuhr, 2016) . interactions between the equivalent thr/his residues conserved in other viral 3c/3cl pro s, including arterivirus nsp4 (barrette-ng et al., 2002; snijder et al., 1996) , and the side chains of p1 glu/gln residues have been shown previously to be critically involved in the binding of gln and glu residues, respectively. interestingly, mesonivirus 3cl pro s do not appear to have a strong preference for gln in the p1 position of substrates (which is a typical feature in coronavirus 3cl pro s) nor do they have a preference for glu over gln as shown for arterivirus nsp4 enzymes (ziebuhr et al., 2000) . a possible explanation for this less pronounced specificity for gln or glu in mesonivirus 3cl pro s may be that his and thr do not establish the same type of interactions with nearby located residues that appear to be required for fine-tuning the p1 specificity for gln and glu, respectively anand et al., 2002; bergmann et al., 1997) . thus, for example, the equivalent his residue in the s1 subsite of coronavirus 3cl pro s was reported to establish interactions with two other residues (phe and tyr) (anand et al., 2002) . these latter interactions are thought to keep the his residue over a wide ph range in the neutral state required for interacting with the p1 gln side chain. compared to other viral 3c/ 3cl pro s, mesonivirus 3cl pro s have a less pronounced specificity toward the p1 residue and tolerate a range of different residues in this position. in addition to gln and glu, lys and several other residues are found at the p1 position of predicted cleavage sites in the replicase polyprotein of cavv and several other mesonivirus 3cl pro s (blanck and ziebuhr, 2016) . interestingly, a replacement of his168 with ala in a bacterial fusion protein construct containing the cavv 3cl pro was previously shown to have differential effects on the cleavage of the n-and cterminal 3cl pro autoprocessing sites (blanck et al., 2014) . while cleavage at the n-terminal autoprocessing site was retained, cleavage of the c-terminal processing site was abolished in a cavv 3cl pro h168a mutant. as the n-terminal autoprocessing site contains a lys residue in the p1 position, while the c-terminal autoprocessing site contains gln in this position, it is tempting to speculate that his168 is required for the cleavage of substrates containing gln (and, likely, glu) in the p1 position, while this his residue may be (largely) dispensable for the cleavage of substrates containing lys in the p1 position. to test this hypothesis and, more generally, establish the structural details of substrate binding for 3cl pro substrates with other residues at the p1 (and other) position(s), additional studies of enzyme/inhibitor complexes with suitable peptidic inhibitors should be performed. also, it may be worth testing if (and to what extent) specific p1 and other residues flanking the scissile bond affect the cleavage efficiency at specific polyprotein cleavage sites, thereby possibly contributing to the timely coordinated release of specific processing products from the viral replicase polyproteins. as indicated above, the p1 gln of molecules a and c was found to (also) interact with the backbone carbonyl oxygen of arg28 from a molecule of the other dimer. we however consider this a crystallographic artifact and, therefore, will not discuss this further. biochemical studies and comparative sequence analyses of mesonivirus replicase polyproteins (blanck et al., 2014; blanck and ziebuhr, 2016) identified the p2 position as a key specificity determinant of mesonivirus 3cl pro s. in cavv and most other members of the genus alphamesonivirus, the p2 position of putative 3cl pro substrates is predominantly occupied by asn. the crystal structure of the 3cl pro /inhibitor complex shows that the asn side chain fits perfectly into the s2 subpocket, with its carboxamide functionality acting as hydrogen bond donor and acceptor in interactions with the main chain carbonyl oxygen of asp216 and the oγ atom of ser52, respectively. the position of the strictly conserved asp216 is stabilized by a salt bridge with the conserved arg47 residue, suggesting that asp216 has a dual functional role: besides its involvement in the coordination of the water molecule that interacts with the catalytic his48 residue, it is part of the s2 pocket, where it interacts with the carboxamide moiety of the asn side chain. in addition to its specificity for asn, the cavv 3cl pro s2 pocket would be suitable (and large enough) to accommodate other residues, such as thr, which is found at the p2 position of one (out of 12) predicted cavv 3cl pro cleavage sites. in contrast, the p2 position of 3cl pro cleavage sites is either predominantly occupied by a leu residue (in coronaviruses) or varies considerably (in arteriviruses). the direct interaction partner of the peptidic ligand in the s3 subpocket is asp173, which forms a hydrogen bond to the phenolic hydroxyl of the tyrosine p3 residue. in this subpocket, also basic residues, such as lys or arg, as well as residues carrying a donor/acceptor function could establish a similar interaction pattern. this view is also supported by the occurrence of a range of other residues in the p3 position of cavv 3cl pro substrates (blanck and ziebuhr, 2016) . compared to the s3 and s1 subsites of coronavirus 3cl pro s, the respective subsites are more spacious in the cavv 3cl pro (see fig. 7 ). the rather hydrophobic s4 subpocket, consisting of ile199, ile207, leu209, and val222, is able to accommodate small hydrophobic residues including leu, which is the most common residue at the p4 position of cavv 3cl pro substrates. however, as the loop formed by residues 216-221 covering the s4 pocket results in a semi-open pocket, larger residues with hydrogen bond donor functionalities, such as tyr, can also be accommodated. in contrast to coronavirus 3cl pro s, which offer only space for smaller residues, such as ser, val or thr, in a fairly closed s4 pocket (ziebuhr et al., 2000; yang et al., 2003 yang et al., , 2005 anand et al., 2002 anand et al., , 2003 , the s4 subsite is larger in the cavv 3cl pro . the main difference between the different chains present in the cavv 3cl pro /inhibitor crystal structure is that, in chain b (but not in chains a and c), a flipped glu218 blocks the access to the s4 pocket, resulting in a deviating position of the benzoyl group which in turn enables an intramolecular hydrogen bond to the p2 substituent (fig. 5) . also, no major differences are observed between the structures of the free enzyme and the enzyme/inhibitor complex. the superposition of 293 cα atoms of 5lac (chain b) and 5lak (chain a) revealed an rmsd of 0.76 å. the only major difference observed upon complex formation is a peptide flip of phe150 enabling a hydrogen bond between the carbonyl oxygen of the inhibitor (gln5) and the amide backbone of residue gly151 (2.9 å). as reported previously, the primary structures of 3c-like cysteine proteases encoded by coronavirinae, roniviridae, and mesoniviridae are poorly conserved except for two strictly conserved sequence signatures, rh and gxcg, that include the catalytic his and cys residues (underlined) (blanck et al., 2014; nga et al., 2011; zirkel et al., 2011 zirkel et al., , 2013 ziebuhr et al., 2003) . our study suggests that this list of highly conserved residues may be extended by an asp residue, asp187 in the sars-cov-3cl pro and asp216 in the cavv 3cl pro , that is shown to be conserved among the 3cl pro s of all known coronavirus and mesonivirus m. kanitz, et al. virology 533 (2019) 21-33 3cl pro s in a similar position of the sequence (fig. 2) . furthermore, previous comparative sequence analyses revealed that an equivalent asp residue may also be conserved in ronivirus 3cl pro s , suggesting a key function for this residue. in the first crystal structure reported for a coronavirus 3cl pro , this asp (asp186 in the tgev 3cl pro sequence) was found to form a hydrogen bond to a water molecule located in a position that, in chymotrypsin and related serine proteases, is occupied by the side chain of the third member of the catalytic triad (typically asp) (anand et al., 2002) . asp186 was observed to form a salt bridge with arg40. the strict conservation of the asp and arg residues in coronavirus 3cl pro s was suggested to indicate an important role of asp (and arg) in maintaining the active-site geometry including the substrate-binding site, while a direct role for asp186 in catalysis was considered unlikely. our database searches revealed the presence of a water molecule that mediates a contact between the conserved asp and the catalytic his residue in 70 out of 80 coronavirus 3cl pro structures available in the protein data bank. in the remaining 10 structures, this water molecule is not visible, most likely because of insufficient resolution of the respective structure. the angle between asp, the conserved water molecule and the catalytic his ranged between 119°and 145°(mean 130.3°) in the coronavirus 3cl pro structures and between 122.8°and 128.4°in the cavv 3cl pro /inhibitor complex structure (chains a-c) and 118.9°in the structure of the free enzyme (chain b), thus representing a nearly perfect angle for a hydrogen-bonded water bridge. in all monomers of the two cavv 3cl pro structures presented in this study, asp216 and arg47 form a salt bridge, with asp216 retaining its capability to act as a hydrogen bond acceptor for the buried water molecule. the conserved water forms a hydrogen bond network with several partners and serves as a kind of relay station. it forms hydrogen bonds to asp216 (see above) and the backbone nitrogen of his48. furthermore, the water molecule interacts with n δ of his48, supporting a role in catalysis by stabilizing the protonated state of the catalytic his during the transition state (fig. 4) . in chains a and c of the structure of the cavv 3cl pro /inhibitor complex (pdb id: 5lak) and in chain b of the structure of the free enzyme (pdb id: 5lac), another conserved water molecule acts as a fourth partner. in coronavirus 3cl pro structures, the position of this second water molecule is occupied by his and gln, respectively, each of them donating a hydrogen bond to the conserved water molecule (sars-cov 3cl pro his164; pdb id: 3sne (zhu et al., 2011) ; mers-cov 3cl pro gln167; pdb id: 4wme (needle et al., 2015) ) (fig. 8) . these observations lead us to suggest that the 3cl pro s of both coronavirinae and mesoniviridae share a conserved catalytic cys-his dyad that is assisted by a water molecule interacting with an asp side chain. in both coronavirus and mesonivirus proteases, the asp residue forms a salt bridge with a conserved arg that immediately precedes the catalytic his in the primary structure (fig. 2) . furthermore, as revealed by the cavv 3cl pro /inhibitor structure (fig. 6) , the main chain carbonyl oxygen of this asp residue contributes also to the s2 subsite of the substrate-binding pocket. taken together, the study provides interesting new insight into the molecular basis of substrate binding and catalysis of nidovirus 3cl pro s. with less than 10% amino acid sequence identity, nidovirus 3cl pro s are extremely diverse and include enzymes that either employ the classical catalytic triad of chymotrypsin-like serine proteases (ser-his-asp) as shown for members of the arteriviridae and (former) torovirinae (barrette-ng et al., 2002; smits et al., 2006; ulferts et al., 2011) or a noncanonical cys-his catalytic dyad assisted by a water molecule that is oriented by a complex network of interactions involving a conserved asp residue located in a region that connects domains ii and iii. also, this work reveals the structural basis for the distinct specificities reported for coronavirus and mesonivirus 3cl pro s, respectively. phylogeny reconstruction. nidovirus phylogeny was reconstructed based on an msa of the conserved core of the rdrp which was generated using the viralis platform (gorbalenya et al., 2010) . representatives of 67 species, 67 out of 88 delineated by demarc and recognized now by ictv (siddell et al., 2019; adams et al., 2016; de groot et al., 2013) , were included in this analysis (supplementary table 1 ). iq tree 1.5.5 (nguyen et al., 2015) with automatically selected lg + f + r7 evolutionary model was used. to estimate branch support, the shimodaira-hasegawa-like approximate likelihood ratio test (sh-alrt) with 1000 replicates was conducted (guindon et al., 2010) . structure-based multiple sequence alignment. the structurebased msa of coronavirus and cavv 3cl pro s was built using the pymol molecular graphics system, version 2.2.3 (schrödinger, llc), command "extra_fit", alignment method "cealign", with only alpha carbon atoms considered. the tgev 3cl pro structure (pdb entry 1lvo, chain a) served as a reference to which other structures ( supplementary fig. 7 . surface representation of mesonivirus and coronavirus 3cl pro substratebinding sites using (a) the structure of the cavv 3cl pro /inhibitor complex (pdb id: 5lak; pink) and (b) the structure of tgev 3cl pro in a complex with a peptidic inhibitor (pdb id: 1p9u; light blue). ligands are represented as sticks and color-coded by atom types. amino acids involved in substrate binding are shown as lines and color-coded by atom types. catalytic residues and the conserved his residue in the s1 subsite are indicated (see text for details). (gorbalenya et al., 2010) . the two msas were aligned by muscle v3.8.31 in the profile mode (edgar, 2004) . the resulting msa was manually adjusted to improve regional sequence and structure similarity, mostly in the c-terminal domain. this refinement took into account another structure-based alignment of the same enzymes which was generated using the protein structure comparison service pdbefold at the european bioinformatics institute (krissinel and henrick, 2004) which superimposes multiple structures simultaneously (krissinel et al., 2005) . secondary structures were retrieved from the dssp database (hekkelman and vriend, 2005; touw et al., 2015) . the resulting alignment was visualized by espript 2.1 (gouet et al., 2003; robert and gouet, 2014) . production and purification of wild-type cavv 3cl pro . wild type cavv 3cl pro was produced as described before (blanck and ziebuhr, 2016) using a slightly modified purification protocol. briefly, a fusion protein, in which the maltose-binding protein (mbp) sequence was fused to the complete 3cl pro coding sequence (corresponding to the cavv pp1a/pp1ab residues 1387 to 1700, genbank nc_015668), was produced in e. coli and purified by amylose affinity chromatography. following cleavage with factor xa, mbp and 3cl pro (the latter containing no extra residues) were separated by anion-exchange chromatography. pooled peak fractions containing cavv 3cl pro were further purified by size-exclusion chromatography in buffer containing 20 mm tris (ph 8.0), 150 mm nacl, 0.1 mm edta, and 1 mm dtt and using an äktapurifier 10 chromatography system equipped with a hiload 16/60 superdex 75 column (ge healthcare). cavv 3cl pro eluted as a single peak after 60 ml. following adjustment of the protein concentration to 7 mg/ml using amicon ® ultra filters (10 kda nmwl), the protein was immediately used for crystallization experiments. production and purification of semet-cavv 3cl pro . the mbp-pp1a-1387-1700 coding sequence (blanck et al., 2014) was subcloned into pet11d (novagen) and selenomethionine-labeled mbp-3cl pro fusion protein was produced using the methionine auxotroph e. coli strain b834(de3). freshly transformed cells were grown in sm medium (2x m9 salts, 0.4% glucose, amino acids except methionine [each at 40 μg/ ml], vitamins [1 μg/ml], adenine, thymine, uracil, guanine [each at 200 μg/ml] and trace elements) containing carbenicillin (75 μg/ml) and methionine (40 μg/ml). at an optical density at 600 nm of 0.6, the cells were harvested by low-speed centrifugation, resuspended in prewarmed sm medium without methionine and incubated for another 4 h at 37°c. then, seleno-l-methionine (40 μg/ml) was added to the medium and the culture was incubated for 30 min at 37°c. next, protein expression was induced with 1 mm isopropyl-β-d-thiogalactopyranoside (iptg) and the culture was incubated overnight at 16°c under vigorous shaking (225 rpm). semet-cavv 3cl pro was purified as described above, concentrated to 7 mg/ml and used in crystallization trials. crystallization. crystallization screens (1152 different conditions) were performed at the marxtal laboratory (philipps university marburg) using cavv 3cl pro (7 mg/ml) or a mixture of cavv 3cl pro and n-benzoyl peptide (bz-yynq-h; thinkpeptides, oxford, uk). the sequence of the peptide was derived from the cavv 3cl pro c-terminus and represents the p4-p3-p2-p1 residues of the c-terminal 3cl pro autoprocessing site. for crystallization of the enzyme/inhibitor complex, the protein solution (7 mg/ml) was pre-incubated with the peptide (1.5 mm) for 2 h to facilitate the formation of the thiohemiacetal with the sulfhydryl group of the catalytic cys153. for the free enzyme, crystals suitable for diffraction experiments were obtained using 0.2 m lithium acetate, 24% polyethylene glycol (peg3350). crystals grew overnight using a mixture of 0.5 μl reservoir solution and 1 μl protein solution in a 15-well hanging-drop plate (qiagen). before flash-cooling in liquid nitrogen, crystals were incubated for 10-15 s in reservoir solution containing 20% glycerol. semet-cavv 3cl pro was crystallized using identical conditions. first crystals of the 3cl pro /inhibitor complex were observed after 6 days at 18°c. the best crystals that were used for subsequent diffraction experiments were obtained by using 0.1 m bicine (ph 8.5) with 20% peg6000 as the reservoir. before flash-cooling, the crystals were incubated for 10-15 s in reservoir solution containing 20% peg400. x-ray diffraction. preliminary diffraction experiments were carried out using a cukɑ-x-ray source from incoatec iμs with a mar345dtb image plate detector, which rendered diffraction up to 2.8 å for the cavv 3cl pro , the cavv 3cl pro /inhibitor complex, and the semet-cavv 3cl pro crystals. high resolution data sets for the cavv 3cl pro and semet-labeled cavv 3cl pro crystals, respectively, were collected at the mx beam line 14.2 at bessy ii (berliner elektronenspeicherring-gesellschaft für synchrotronstrahlung, helmholtz-zentrum berlin für energie und materialien, berlin, germany) (mueller et al., 2015) . measurements were carried out at 100 k and a wavelength of 0.91841 å. data were processed using xdsapp (krug et al., 2012) resulting in a cavv 3cl pro semet dataset diffracting up to a resolution of 1.89 å (see table 1 ). in order to obtain phase information, a mad experiment using crystals of semet-cavv 3cl pro , belonging to the space group p2 1 2 1 2 (unit cell dimensions: a = 94.2 å, b = 111.4 å, c = 58.2 å) with two molecules in the asymmetric unit, was carried out. data were collected at the peak wavelength (0.97971 å) with a significant anomalous signal up to 2.27 å (d''/sig(d'') = 1.083-4.647), at the inflection point (λ = 0.97990 å; 2.77 å; d''/sig(d'') = 1.015-2.574), and at a high energy remote wavelength (λ = 0.91841 å) with a resolution up to 1.89 å. the anomalous signal allowed us to unambiguously determine 12 out of 14 possible semet sites using shelxd (cc max = 58.2) (schneider and sheldrick, 2002) , followed by phasing performed with shelxe (sheldrick, 2002) , all implemented in hkl2map (pape and schneider, 2004 ). an initial electron density map was calculated and a model (581 amino acids; 10 chains; 208 amino acids in the longest chain; score 0.960) was built by arp/warp (langer et al., 2008) . this model was further modified using coot (emsley et al., 2010) and refined using phenix (adams et al., 2010) . a dataset for the co-crystallized cavv 3cl pro /inhibitor complex was collected at beam line bm30-a at the esrf (european synchrotron radiation facility, grenoble, france). measurements were carried out at 100 k at a wavelength of 0.979742 å. the dataset was processed to a resolution of 2.3 å using xds (kabsch, 2010) . the crystals belong to space group p2 1 with cell dimensions a = 72.1 å, b = 110.0 å, c = 98.4 å and β = 105.9°, featuring four molecules in the asymmetric unit. structure determination, model building, and refinement. due to its higher resolution and superior data quality, the semet-cavv 3cl pro dataset was used for further model building and refinement. the refinement of the semet-cavv 3cl pro structure was carried out in phenix using xyz refinement, tls refinement (from the tlsmd-server; 12 groups), individual atomic displacement parameters (adps; isotropic), occupancy refinement, and finally ncs refinement (torsion angles). water molecules were added in the automatic model building of arp/warp. for molecular replacement and first refinement cycles of the 3cl pro /inhibitor complex structure, an automated refinement script based on phenix was used (schiebel et al., 2016) . the structure of the semet-cavv 3cl pro was used as search model. for the refinement process in phenix, xyz refinement, tls refinement (from the tlsmd-server; 22 groups), grouped b-factors, occupancy refinement, and ncs refinement (torsion angles) were carried out. for r-free calculations, a 5% data fraction was used. coot was used to add water molecules and for the fitting of amino acid side chains using σ-weighted 2fo-fc and fo-fc difference electron density maps. restraints for ligands were obtained by using the grade web server (http://grade.globalphasing. org/cgi-bin/grade/server.cgi). average b-values were calculated using m. kanitz, et al. virology 533 (2019) 21-33 moleman (kleywegt et al., 2006) and ramachandran plot statistics were analyzed with procheck (laskowski et al., 1993 ) (see table 2 ). coordinates and structure factors for the cavv 3cl pro and the cavv 3cl pro /inhibitor complex have been deposited in the protein data bank under the pdb ids 5lac and 5lak, respectively. none. the work was supported by the deutsche forschungsgemeinschaft (sfb1021 a01, to jz). the contribution of aag and aeg was funded in part by the eu horizon2020 evag 653316 project and the lumc mobile program (to aeg). we are very grateful to prof. dr. wolfgang buckel, head of the laboratory of microbial biochemistry and max planck fellow of the mpi for terrestrial microbiology, marburg, for helpful discussions regarding mechanistic aspects. we thank the beamline staff of bessy ii (helmholtz-zentrum berlin) and esrf for their outstanding help and support during data collection and acknowledge the generous support by travel grants from the helmholtz-zentrum für materialien und energie, berlin. we also thank ralf pöschke (marxtal, philipps university marburg) and karin schultheiß (medical virology, justus liebig university giessen) for excellent technical assistance, and igor sidorov and dmitry samborskiy for assistance with the viralis platform. phenix: a comprehensive python-based system for macromolecular structure solution picornaviral 3c cysteine proteinases have a fold similar to chymotrypsin-like serine proteinases structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain coronavirus 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east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes muscle: multiple sequence alignment with high accuracy and high throughput features and development of coot structure-based exploration and exploitation of the s4 subsite of norovirus 3cl protease in the design of potent and permeable inhibitors crystal structure of the serine protease domain of sesbania mosaic virus polyprotein and mutational analysis of residues forming the s1-binding pocket viral cysteine proteases poliovirus-encoded proteinase 3c: a possible evolutionary link between cellular serine and cysteine proteinase families cysteine proteases of positive strand rna viruses and chymotrypsin-like serine proteases. a distinct protein superfamily with a common structural fold coronavirus genome: prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis practical application of bioinformatics by the multidisciplinary vizier consortium espript/endscript: extracting and rendering sequence and 3d information from atomic structures of proteins new algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of phyml 3.0 serine protease mechanism and specificity mrs: a fast and compact retrieval system for biological data dali server: conservation mapping in 3d broad-spectrum antivirals against 3c or 3c-like proteases of picornaviruses, noroviruses, and coronaviruses around o viral proteinases secondary-structure matching (ssm), a new tool for fast protein structure alignment in three dimensions multiple alignment of protein structures in three dimensions inference of macromolecular assemblies from crystalline state xdsapp: a graphical user interphase for the convenient processing of diffraction data using xds individual and common inhibitors of coronavirus and picornavirus main proteases automated macromolecular model building for x-ray crystallography using arp/warp version 7 procheck: a program to check the stereochemical quality of protein structures partitioning the genetic diversity of a virus family: approach and evaluation through a case study of picornaviruses mesoniviridae: a proposed new family in the order nidovirales formed by a single species of mosquito-borne viruses the footprint of genome architecture in the largest genome expansion in rna viruses the picornaviral 3c proteinases: cysteine nucleophiles in serine proteinase folds three-dimensional structure of tosyl-alpha-chymotrypsin structure of human rhinovirus 3c protease reveals a trypsin-like polypeptide fold, rna-binding site, and means for cleaving precursor polyprotein crystal structures of two engineered thiol trypsins refined x-ray crystallographic structure of the poliovirus 3c gene product the macromolecular crystallography beamlines at bessy ii of the helmholtz-zentrum berlin: current status and perspectives structural basis of substrate specificity and protease inhibition in norwalk virus structures of the middle east respiratory syndrome coronavirus 3c-like protease reveal insights into substrate specificity discovery of the first insect nidovirus, a missing evolutionary link in the emergence of the largest rna virus genomes iq-tree: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies crystal structure of tobacco etch virus protease shows the protein c terminus bound within the active site cysteine proteases and their inhibitors hkl2map: a graphical user interface for macromolecular phasing with shelx programs structural basis for the substrate specificity of tobacco etch virus protease an overview of severe acute respiratory syndrome-coronavirus (sars-cov) 3cl protease inhibitors: peptidomimetics and small molecule chemotherapy the catalytic triad of serine peptidases merops: the database of proteolytic enzymes, their substrates and inhibitors deciphering key features in protein structures with the new endscript server virus-encoded proteinases of the picornavirus super-group on the size of the active site in proteases. i. papain high-throughput crystallography: reliable and efficient identification of fragment hits substructure solution with shelxd macromolecular phasing with shelxe additional changes to taxonomy ratified in a special vote by the international committee on taxonomy of viruses characterization of a torovirus main proteinase the arterivirus nsp4 protease is the prototype of a novel group of chymotrypsin-like enzymes, the 3c-like serine proteases structural and biochemical analysis of human pathogenic astrovirus serine protease at 2.0 a resolution structural and dynamics characterization of norovirus protease a series of pdb-related databanks for everyday needs characterization of bafinivirus main protease autoprocessing activities structure-based design and synthesis of triazole-based macrocyclic inhibitors of norovirus protease: structural, biochemical, spectroscopic, and antiviral studies activation and maturation of sars-cov main protease structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor design of wide-spectrum inhibitors targeting coronavirus main proteases structure of the main protease from a global infectious human coronavirus. hcov-hku1 peptide aldehyde inhibitors challenge the substrate specificity of the sars-coronavirus main protease virus-encoded proteinases and proteolytic processing in the nidovirales the 3c-like proteinase of an invertebrate nidovirus links coronavirus and potyvirus homologs an insect nidovirus emerging from a primary tropical rainforest identification and characterization of genetically divergent members of the newly established family mesoniviridae supplementary data to this article can be found online at https:// doi.org/10.1016/j.virol.2019.05.001. key: cord-290231-4m9lj0uq authors: guirakhoo, farshad; bolin, richard a.; roehrig, john t. title: the murray valley encephalitis virus prm protein confers acid resistance to virus particles and alters the expression of epitopes within the r2 domain of e glycoprotein date: 1992-12-31 journal: virology doi: 10.1016/0042-6822(92)90267-s sha: doc_id: 290231 cord_uid: 4m9lj0uq abstract to study the role of the precursor to the membrane protein (prm) in flavivirus maturation, we inhibited the proteolytic processing of the murray valley encephalitis (mve) virus prm to membrane protein in infected cells by adding the acidotropic agent ammonium chloride late in the virus replication cycle. viruses purified from supernatants of ammonium chloride-treated cells contained prm protein and were unable to fuse c6/36 mosquito cells from without. when ammonium chloride was removed from the cells, both the processing of prm and the fusion activity of the purified viruses were partially restored. by using monoclonal antibodies (mabs) specific for the envelope (e) glycoprotein of mve virus, we found that at least three epitopes were less accessible to their corresponding antibodies in the prm-containing mve virus particles. amino-terminal sequencing of proteolytic fragments of the e protein which were reactive with sequence-specific peptide antisera or mab enabled us to estimate the site of the e protein interacting with the prm to be within amino acids 200 to 327. since prm-containing viruses were up to 400-fold more resistant to a low ph environment, we conclude that the e-prm interaction might be necessary to protect the e protein from irreversible conformational changes caused by maturation into the acidic vesicles of the exocytic pathway. for enveloped viruses to enter into their target cells, virus-cell membrane fusion must take place to release the nucleocapsid into the cytoplasm. this occurs either directly at the plasma membrane, as with paramyxoviruses and herpesviruses, or in the endosomal vesicles after the viruses are taken up by receptor-mediated endocytosis, as with togaviruses and orthomyxoviruses (for review see marsh and helenius, 1989) . generally the fusion-mediating envelope proteins are activated by proteolytic cleavage of the spike precursor proteins by a host protease late during virus assembly. cleavage activation of the glycoproteins involved in fusion has been demonstrated with the gpe2 of murine coronaviruses (sturman and holmes, 1984; sturman et al., 1985) the ha of influenza viruses (wiley and skehel, 1987 ) the gp160 of retroviruses (mccune et al., 1988; willey et al., 1991) and the pe2 of alphaviruses (lobigs and garoff, 1990; lobigs et a/., 1990; white et al., 1983) . the cleaved glycoprotein (e.g., ha) undergoes an irreversible conformational change in the acidic ph milieu of the endocytic vesicles which exposes a hydrophobic fusion site that mediates the virus-cell membrane fusion. this cleavage of the precursor protein is essential for virus infectivity but not particle formation. a semliki forest virus (sfv) mutant defective in pe2 cleavage expressed pe2 at the cell ' to whom reprint requests should be addressed. surface but did not function in low ph-triggered cell-tocell fusion (lobigs and garoff, 1990; lobigs et al., 1990) . murray valley encephalitis (mve) virus is a member of the flaviviridae, a family of small, positive-strand rna viruses. the mature flavivirions contain three structural proteins: capsid (c), membrane (m), and envelope (e), with molecular masses of 14-15, 7-8, and 50-60 kda, respectively. the e protein is the only structural protein responsible for the induction of neutralizing, hemagglutination-inhibiting, and protective antibodies, it is the receptor-binding protein and is also involved in acid-catalyzed membrane fusion (heinz and roehrig, 1990; guirakhoo et al., 1991) . little is known about the function of the m protein. the m protein is cleaved from its precursor protein (prm) after the consensus sequence r-x-r/k-r shortly before or after virus release westaway, 1987) . this cleavage is associated with a rearrangement of the oligomeric structures on the surface of the virion (wengler and wengler, 1989) . by using monoclonal antibodies (mabs) and protease maps, we previously demonstrated that the e glycoprotein of tick-borne encephalitis (tbe) virus undergoes an irreversible conformational change, predominantly in the epitopes of domain a, at mildly acidic ph . consistent with this, purified tbe virus demonstrated fusion from without (ffwo) of the mosquito cell line c6/36 only after exposure of cell-bound virus to low ph. the ffwo was abolished if the processing of prm to m was interrupted using the acidotropic agent ammonium chloride (guirakhoo et a/., 1991) . in this paper we identify those epitopes on the e protein which are covered or dissociated by the presence of prm, demonstrate that prm-containing viruses are more resistant to low ph, localize the site on the e protein which is involved in the e-prm interaction, and finally hypothesize that the association of e-prm is necessary for the authentic expression of the e protein. production and analysis of the immature virus particles aedes albopictus cells (clone c6/36) were grown on microcarriers (cytodex 2, pharmacia fine chemicals, upsala, sweden)' in dulbecco's minimal essential medium (d-mem) buffered with 25 mni hepes and supplemented with 10% fetal calf serum (fcs, hyclone laboratories, inc., logan, ut). cells were infected with mve virus (strain ord river) at a multiplicity of infection (m.o.i.) of 10 in d-mem with 29/o fcs. ammonium chloride (25 m/m) was added at 48 hr postinfection (p.i.) and the viruses were harvested on day 6. at this time, ammonium chloride-containing medium was replaced with d-mem, 2% fcs, without ammonium chloride, and the viruses were reharvested on day 9 p.i. viruses were precipitated with 8% polyethylene glycol 8000 (fisher scientific, fair lawn, nj), purified by two cycles of glycerol-tartrate gradients (obijeski et al., 1976) , and resuspended in 0.3 ml of d-mem. protein concentrations were determined by the bio-rad protein assay (bio-rad laboratories, richmond, ca). untreated mve viruses (without ammonium chloride) were propagated and purified as described above and were used as positive controls in all experiments. for the acid resistance experiments (see below) mve viruses were also grown in sw1 3 mammalian cells in the presence or absence of ammonium chloride. to quantify the amount of viral structural proteins in each propagation, the purified viruses were subjected to sds-page on 12.5% acryamide gels (laemmli, 1970) and proteins were stained using a rapid silver stain kit (icn, cleveland, oh) according to the manufacturer's protocols. gels were scanned by a microprocessor-controled spectrophotometer (gilford system 2600) and the peaks corresponding to each viral protein were identified and integrated to determine areas under the peaks, and the area ratios of prm to e were calculated. purified viruses were diluted geometrically (from 200 to 6.25 pg/mi) in d-mem containing 1% bsa. thirty microliters of each dilution was added to prechilled c6/ 36 cells and the fusion assay was performed at ph 5.0. the number of nuclei and the number of cells in five microscopic fields (magnification 1 oo-fold) were counted and the fusion index [l -(number of cells/ number of nuclei)] was calculated. capture enzyme-linked immunosorbent assay (elisa) microtiter plates were coated overnight at 4" with a 1:500 dilution of rabbit anti-mve hyperimmune serum. this dilution of capture antibody was determined by box-titration against m-and prm-containing virus seeds, maximizing and standardizing the concentration of captured antigens. wells were blocked with 3% goat serum in pbs, a 1:5 dilution of tissue cultures containing prm-or m-mve virus was added, and plates were incubated for 1 hr at 37". ascites of the various mabs were then titrated on both viruses for 1 hr at 37" starting at 1: 100 or 1: 1000 dilution depending on antibody potency. bounds mabs were detected by goat anti-mouse alkaline phosphatase conjugate. quantitation of bound conjugate was determined by adding sigma 104 substrate and reading absorbance at 405 nm in a titertek mc spectrophotometer after 30 min. the specificity and the biological activity of all mabs except 6b4a-10 have been published elsewhere (hawkes et a/., 1988) . mab 6b4a-10 was prepared against japanese encephalitis virus, strain nakayama, using standard hybridoma techniques. this mab reacted with all members of the mve virus serocomplex, blocked mve virus-mediated hemagglutination, and neutralized mve virus infectivity in vitro. the e glycoprotein epitope defined by 6b4a-10 has been designated e-8. the acid sensitivity of the mve virus epitopes was determined using a modification of the standard elisa protocol. two hundred and fifty microliters of purified mve virus (100 pg total protein) in 0.2 m tris-hci, ph 8.0, was adjusted to ph 6.0 using 120 ~1 of 0.2 m hci and incubated at 25" for 30 min. both acid-treated virus and untreated virus (ph 8.0) samples were diluted to 5 ml with carbonate buffer, ph 9.5. lmmulon ii microtiter plates were coated with 100 ~1 virus per well and incubated overnight at 4". the plates were blocked for 1 hr at 37" with 3% bsa in pbs. protein mabs were diluted geometrically (from 10 pg/well total protein) and added to each well. after 2 hr at 25", the plates were washed and further incubated with a 1:500 dilution of goat antimouse alkaline phosphatase conjugate. bound antibody was detected by addition of sigma 104 substrate, and absorbance was measured at 405 nm after 5 min. although each antibody was tested in a dilution series, only the results with 1.25 gg antibody are reported. analysis of the mab reactivities with reduced and nonreduced mve virus page sample buffer (laemmli, 1970) with or without the reducing agent, 2-mercaptoethanol, was added to 10 pg of purified mve virus. samples were boiled for 3 min at 100" and separated on 12.5% sds gels. proteins were then transferred to nitrocellulose sheet (towbin et a/., 1979) and blocked with 3% goat serum. the reactivity of mabs defining the epitopes e-4b, e-6, or e-8 with reduced or nonreduced forms of mve virus e glycoprotein was assessed using goat anti-mouse alkaline phosphatase conjugate (jackson lmmuno research, west grove, pa). proteolytic digestion of the native mve virus was performed in d-mem, ph 7.4, using predetermined concentrations of either trypsin or v8 protease (sigma chemical co.) and the fragments reacting with mab e-8 were identified by using goat anti-mouse alkaline phosphatase conjugate (see below). for the proteolytic digestion of the predenatured virus, 60 pg of purified mve virus in d-mem was precipitated in 60% tca (end concentration 12%) on an ice bath for 30 min, washed with cold acetone, and resuspended in 50 ~1 0.125 m tris, ph 6.8, containing 0.5% sds and 10% glycerol. after 24 hr incubation at 37" samples were boiled for 3 min and subjected to proteolytic digestion using either chymotrypsin or v-8 proteases (sigma chemical co.) at the protein to enzyme ratios of 4:l and 30: 1, respectively, at 37" for 30 min. after addition of sds and 2-mercaptoethanol (final concentrations 4%) the samples were boiled and separated on 12.5% sds gels. fragments were transferred to nitrocellulose membrane by electroblotting (towbin et al., 1979) . nitrocellulose was then blocked by 3% goat serum in pbs, ph 7.4, for 1 hr. strips were cut and incubated with either mab (defining epitope e-8) or mouse sera which were immunized with e-specific synthetic peptides as follows: mve 02, amino acids 35-50; mve 04, amino acids 122-l 41; mve 06, amino acids 230-251; and mve 17, amino acids 356-376. the sequence and characteristics of these antipeptides have been published previously (roehrig eta/., 1989) . after 1 hr at 37", goat anti-mouse alkaline phosphatase conjugate (jackson lmmuno research) was added and the immune fragments were visualized by adding bcip/nbt substrate (kpi, inc., gaithersburg, md). for the amino-terminal sequencing, fragments were prepared as described above except that electroblotting was performed on polyvinylidene difluoride (pvdf) membrane (immobilon-p, millipore, bedford, ma) using caps transfer buffer (10 mm caps, 0.5 mm dithiothreitol, dtt, and 10% methanol adjusted to ph 1 1 .o with nach). fragments were stained with 0.1% coomassie blue (baker g-250) in 50% methanol and 10% acetic acid. the amino-terminal sequences were determined either on an applied biosystems 473a (foster city, ca) or on a porton instrument 2090 (tarzana, ca) protein sequencer using standard edman chemistry. plaque assay of prm-and m-containing mve viruses after exposure to different ph buffers equal amounts of plaque forming units, pfus (6 x 1 o'/mi for viruses grown in c6/36 cells and 4.5 x 1 o5 for viruses grown in sw13 cells), from the supernatants of ammonium chloride-treated and untreated mve virus-infected cells were incubated in different ph buffers, ranging from 7.5 to 4.5, using 1 m hepes or 1 m mes containing d-mem. after a 15 min-incubation at room temperature, the inoculum was exponentially diluted in ba-1 diluent (hunt eta/., 1991) and examined for infectivity in vero cells using the standard plaque assay. the relationship between prm processing and the fusion activity to understand the role of prm protein in virus maturation and its interaction with the e glycoprotein, we investigated the effect that ammonium chloride had on mve viruses grown in c6/36 mosquito cells. ammonium chloride has been shown to accumulate in the post-golgi acidic vesicles and interrupt the processing of prm to m protein (randolph eta/., 1990) . the unprocessed prm protein was incorporated into the released viruses grown in the presence of ammonium chloride at 6 days p.i. (fig. 1 a, lane 2) . when ammonium chloride was removed, the cells partially recovered, and the normal processing of the prm to m resumed (fig. la, lane 3) . since some unprocessed prm proteins were also found in mve-infected cells in the absence of ammonium chloride ( fig. 1 a, lane l) , we attempted to quantify the amount of prm in each sample. this was achieved by scanning the stained viral proteins following separation by sds-page. by using nonradioactive viruses it was not always possible to clearly demonstrate the presence of the small m protein in minigels. the fact that the ratios of the viral structural proteins in the absence of ammonium chloride remain constant enabled us to evaluate the amount of prm processing indirectly by calculating its ratio to other structural proteins (e or c) which are more abundant in virions. the ratio of the area of each peak corresponding to c or prm proteins to e as well as prm to c protein was calculated. the ratio of c to e protein remained constant in the presence or absence of ammonium chloride (data not shown) whereas the ratio of prm to e protein was significantly increased in the presence of ammonium chloride (from 0.15 to 0.86) and reduced to 0.55 following removal of ammonium chloride (fig. 1a) . similar results were obtained when the ratio of prm to c was calculated (data not shown). these data confirm that partial recovery of prm processing has occurred once the ammonium chloride has been removed. to determine the fusion activity of the viruses obtained from the supernatants of virus-infected ammonium chloride-treated cells, the ffwo assay was performed using monolayers of c6/36 cells as described previously (guirakhoo et a/., 199 1) . no polykaryocyte formation was observed (up to 200 pg/ml of purified virus) using viruses harvested in the presence of ammonium chloride at 6 days p.i.; however, when ammonium chloride was removed, the fusion activity of puri-fied viruses obtained on day 9 was significantly restored ( fig. 1 b) . these results are in agreement with our previous finding with tbe virus which demonstrated a direct relationship between processing of prm and fusion activity of e glycoprotein (guirakhoo eta/., 1991) and further manifest that both phenomena are reversible. epitope analysis of the e-proteins of prm-and mcontaining viruses a capture elisa was performed to analyze the effect that blocking the prm cleavage had on the expression of e glycoprotein epitopes in native virions. the reactivities of mabs defining nine distinct epitopes on the mve e glycoprotein were compared on m-and prmcontaining viruses using supernatants of ammonium chloride-treated or untreated virus-infected c6/36 cells. the antigenicity of the prm-and m-containing virus supernatants was equivalent in end-point elisa antigen titration using a 1:500 dilution of polyclonal rabbit anti-mve virus capture sera and anti-mve virus mouse hyperimmune ascitic fluid (hiaf) as detector. therefore, a 1:5 dilution of each supernatant was used in subsequent epitope mapping with mabs. the equivalent antigenicity was confirmed by end-point elisa titration of the mve hiaf in the standardized capture assay (fig. 2b) . even though these virus supernatants had equal antigenicity, the prm-containing virus supernatant was 1 o-fold less infectious than the m-containing virus supernatant (3.9 x 1 o* vs 2.3 x 10' pfu/ml). the reactivities of three e glycoprotein epitopes were significantly reduced in prm-containing viruses (fig. 2c) . two of these epitopes were subcomplex-and complex-reactive (e-6 and e-8) and one was groupreactive (e-4b), being conserved in all flaviviruses (hawkes et al., 1988) . the reactivities of other mve virus-specific mabs were identical with both viruses (fig. 2a) . structural properties of the epitopes corresponding to these mabs were assessed by comparing the reactivities of mabs after incubation of the purified viruses at low ph to those of untreated viruses (fig. 3) . as can be seen, the antigenic reactivities of all mabs and the hiaf were significantly reduced upon low ph treatment of the virus. of the e-8 mab binding site on the e glycoprotein figure 4a demonstrates the reactivities of the three mabs defining the epitopes e-4b, e-6, and e-8, with the mve virus e glycoprotein under reducing and nonreducing conditions. as can be seen neither the e-4b nor e-6 mabs recognized the e protein in its reduced form (treated with 2-mercaptoethanol), whereas e-8 mab was still reactive after protein reduction. this reactivity was further sustained after the reduction and alkylation of the cysteine residues of the e glycoprotein using dlt/sds followed by iodoacetamide treatment (johnson et a/., 1990) (data not shown). since the reactivity of the e-8 mab was reduced with prm-containing viruses and the fact that it could recognize the e protein in immunoblots in its linear conformation, we attempted to obtain more information about the site involved in prm-e interaction by identifying the e-8 binding site. the proteolytic digestion of the native e protein with trypsin (which cleaves after k or r) or v8 endoproteinase (which cleaves after e or d) did not produce fragments smaller than 30 kda that were reactive with e-8 mab (fig. 4b) . in order to produce smaller fragment reactive with the e-8 mab, the mve virus was denatured by sds prior to protease digestion. using chymotrypsin (which cleaves after f, t, or y) or v8 protease, 20-and 22-kda fragments, fr 20 and fr 22, were identified, respectively. these two fragments, which reacted with e-8 mab, were also recog-nized by mouse antiserum immunized with synthetic peptide mve 06 (figs. 4c and 4d). a 23-kda fragment, fr 23, and a 19-kda fragment, fr 19, were produced using v8 or chymotrypsin, respectively, which were recognized by mve 04 antibodies but not by mve 06 antibodies or e-8 mab. antisera prepared against mve 17 also recognized a v8 fragment of 16 kda fr 16 (fig. 4d ). fr 23, 22, 16, and 14 were sequenced at their amino-terminal ends and the results are shown in ta (dalgarno et a/., 1986) predict only one potential nlinked glycosylation site on the e protein at position n-l 54 and the fact that this site might contain carbohydrate of the complex type (endo h resistant) (winkler because it has been shown with west nile virus that the e and prm glycoproteins remain as heterodimers during transport and maturation through acidic compartments of the exocytic route (wengler and wengler, 1989) , we compared the infectivity of the prm-containing mve virus with that of the m-containing mve virus following virus exposure to acidic conditions. equal pfus of the prm-and m-virus containing supernatants from c6/36 or sw1 3 cells were subjected to environments of different ph values and their resultant infectivity in a plaque assay was examined (fig. 6) . no significant difference in infectivity of either virus preparation was observed after the incubation of viruses at ph values between 7.5 and 6.5. upon incubation at ph 6.0, the infectivity of both m-containing viruses (grown in c6/36 or sw1 3 cells) was drastically abolished; however, this loss of infectivity was less dramatic for prmcontaining viruses than for m-containing viruses (150fold loss versus 1360-fold loss for viruses grown in c6/ 36 cells, fig. 6a tion 199) or fr 16 (starting at position 327), we predict its binding site to be between amino acids 200 and 327 on the e glycoprotein (fig. 5) . low ph sensitivity of prm-and m-containing mve viruses (table 1) have been marked by arrowheads. a fragments were generated by v-8 digestion of sds-denatured purified mve virus, separated on 12.5% sds gels, and transferred to pvdf membranes by western blotting. membranes were stained with coomassie blue and subjected to amino-terminal sequence analysis. ' fragments were generated as described above, transferred to nitrocellulose, and subjected to immune detection using mve antipeptrde antibodies (p2. p4, p6. p17; for sequence and length see materials and methods) or mab e-8 as described under materials and methods, for viruses grown in sw1 3 cells, fig. 6b ). maximal loss of infectivity was achieved upon incubation at ph 4.5; nevertheless, the prm-containing viruses were still 1 ofold (those grown in c6/36 cells) and 400-fold (those grown in sw1 3 cells) more resistant to the low ph environment than the m-containing viruses. the proteolytic cleavage of prm occurs after a dibasic amino acid motif, presumably by a trypsin-like host enzyme similar to those responsible for normal processing of prohormones (fischer and scheller, 1988; steiner e2 al., 1984) . recently, an enzyme designated paired basic amino acid cleaving enzyme (pace) has been cloned and purified from a human liver cell line (wise et al., 1990) . a similar endoprotease was also isolated from the trans-golgi membranes of rat liver cells, which was shown to cleave in vitro the f, protein of new castle disease virus after a dibasic residue motif (sakaguchi et al., 199 1) . we believe that the late maturational processing of prm at the r-x-r/k-r cleavage motif is the mechanism used to activate the flavivirus fusion protein (e glycoprotein). the infectivity of prm-containing viruses (grown in c6/36 cells) for mammalian cells was lo-fold lower than that of the mature mve (m-containing) viruses, although the amount of antigen detected by polyclonal antibodies was similar for both viruses. these results indicate that prm processing is required for full virus infectivity. a similar observation has been made with west nile (wn) virus in which the ratio of physical particles to pfu was about 600 for the fully infectious extracellular (m-containing) viruses and was about 60-fold higher for the cell-associated (prm-containing) viruses (wengler and wengler, 1989) . upon subsequent infection and endocytosis, the e glycoprotein probably undergoes a conformational change in the acidic environment of endocytic vesicles, which results in membrane fusion and release of the nucleocapsid into the cytoplasm. consistent with this hypothesis are our previous findings that the e glycoprotein of tbe virus undergoes irreversible conformational changes at ph less than 6.2. these changes, which predominantly occur in the a-domain epitopes and the i2 site mandl et al., 1989) , are probably necessary to trigger the fusion activity of the e glycoprotein. it has been shown that the fusion activity of the tbe virus can be blocked effectively by three mabs, two of which were specific for epitopes of the a-domain, and one recognized the i2 epitope (guirakhoo et al., 1991) . the a-domain contains an amino acid sequence (g l f g) that is conserved in the fusion sequences of other enveloped viruses. we have also observed with dengue virus that the region containing the conservative fusion sequence is more accessible to antipeptide antibodies following low ph treatment . by using mabs specific for mve virus e glycoprotein in a capture elisa, we demonstrated that if the processing of prm was interrupted at least three of these epitopes on the e glycoprotein were less accessible on the virus particle. the observation that all of the epitopes were acid sensitive indicates that they are probably located in the a-domain. the sensitivity of the hiaf to low ph also suggests that these epitopes are the predominant epitopes recognized following immunization with intact virus. it may be significant that these three epitopes are associated with hemagglutination of red blood cells. at least one of these epitopes (e-8) also elicits virus-neutralizing antibodies. since other functionally important epitopes which induce neutralizing, hemagglutinationinhibiting, and protective antibodies (hawkes et a/., 1988) are still present in the prm-containing viruses (fig. 2 a) , and the fact that these viruses are still capable of binding to and infecting their host cells, we did not expect that the fusion peptide or the receptorbinding sites would be directly involved in the prm-e interactions. this was confirmed when the immunereactive fragments generated by v8 protease were sequenced. it was shown that predominantly three sites on the e glycoprotein were exposed to the proteolytic attack of the v8 enzyme. this cleavage, which occurred on positions 138, 199, and 326, generated four fragments reactive with sequence-specific antipeptide antibody or e-8 mab. similar segments on the e pro-tein of wn virus, which are located in ll and r3, have also been shown to be exposed to proteolytic attack (wengler er a/., 1987) . combining results of the reactivity pattern of the antipeptide antibodies with proteolytic fragments and also information obtained from n-terminal sequence analysis of the immune fragments, we were able to map the binding site of the e-8 mab which was covered or dissociated by the presence of prm. this site which reside between amino acids 200 and 327 on the e protein includes more than 80% of the r2 domain proposed for wn (nowak and wengier, 1987) and mve (roehrig et a/., 1989) viruses. no actual biological function of the r2 domain had been previously identified. it is noteworthy that the synthetic peptide mve 06, which consists of 21 amino acids within the r2 domain (230-251), was cross-reactive with other members of the flaviviruses (roehrig et al., 1989) , indicating the biological importance of this region. several studies have indicated that when influenza a virus and semliki forest virus were incubated at low ph they became irreversibly inactivated (doms et a/., 1985; kielian and helenius 1985; stegman eta/., 1989; white et a/., 1982) . this is probably due to exposure and self-aggregation of the hydrophobic fusion peptide which, therefore, can no longer interact with target membranes (nir et al., 1990) . if the flavivirus e glycoprotein were not protected during transport to the cell surface, the acidic milieu of the post-golgi vesicles could inactivate it. because it has been shown with wn virus that the e and prm proteins remain as heterodimers during exocytosis (wengler and wengler, 1989) we reasoned that the function of the prm is to prevent conformational changes in the e glycoprotein at low ph, thus assuring safe transport of these proteins through post-golgi acidic vesicles during maturation until the virions are released. a similar role has also been proposed for the pe2 of alphaviruses, which forms a stable dimer with the el protein (critical for infection and fusion) in the acidic compartment of the exocytic route (lobigs and garoff, 1990; presley et a/., 1991; wahlberg et al., 1989) . different approaches might be utilized by other viruses to secure the authentic expression of the envelope proteins on the infected cells. it has been shown that the m2 protein of influ-enza virus raises the ph of the post-golgi vesicles by about 0.8 unit, thus preventing the expression of the low ph conformation of the ha molecules on the plasma membranes of the infected cells (ciampor et a/., 1992) . the high resistance of the prm-containing viruses to low ph might indicate that the prm could shield some segments of the e protein which are otherwise protonated in a low ph environment, leading to the exposure of the fusion peptide and consequent loss of infectivity. concomitant with the low ph sensitivity of the m-containing viruses was a loss of epitope reactivity in elisa following incubation at ph 6.0 or below. because prm-containing viruses are still infectious, apparently the prm protein can be cleaved after the virus has undergone endocytosis. using chymotrypsin, trypsin, and thermolysin it was demonstrated that the prm protein of the wn virus is highly sensitive to proteases . in our laboratory we also observed that by using a low concentration of trypsin, the prm protein of the mve virus is cleaved prior to the e protein (data not shown). our results predict that the expression of authentic e glycoprotein requires the coexpression of prm. this would explain why flavivirus vaccine candidates produced from the expression of only e glycoprotein were partially protective against homotypic challenge, whereas those prepared from the expression of both prm and e glycoproteins gave a significant level of protection (bray and lai, 1991) . similar results were observed with japanese encephalitis virus, where high neutralizing and hemagglutination-inhibiting antibodies which correlated with a high level of protection were found in mice inoculated with recombinant vaccine viruses expressing both prm and e but not e pro-tein alone (konishi et al., 1991; yasuda et a/., 1990) . we are currently performing experiments to define further the role of prm in virus infectivity and the events occurring after prm-containing viruses enter their target cells. we are also attempting to determine if a similar area on the e protein of other flaviviruses is involved in the prm interaction. dengue virus premembrane and membrane proteins elicit a protective immune response. l/iro/ogy 185 regulation of ph by the m2 protein of influenza a viruses partial nucleotide sequence of the murray valley encephalitis virus genome: comparison of the encoded polypeptides with yellow fever virus structural and non-structural proteins membrane fusion activity of the influenza virus hemagglutinin: the low ph-induced conformational change prohormone processing and the secretory pathway epitope model of tick-borne encephalitis virus envelope glycoprotein e: analysis of structural properties, role of carbohydrate side chain, and conformational changes occurring at acidic ph fusion activity of flaviviruses: comparison of mature and immature (prm-containing) tick-borne encephalitis virions antigenic structure of the murray valley encephalitis virus e glycoprotein flaviviruses: the basis for serodiagnosis and vaccines synthetic peptides of the e2 glycoprotein of venezuelan equine encephalomyelitis virus. ii. antibody to the amino terminus protects animals by limiting viral replication variants of venezuelan equine encephalitis virus that resist neutralization define a domain of the e2 glycoprotein ph-induced alterations in the fusogenic spike protein of semliki forest virus. /. cell f?iol comparison of protective immunityelicited by recombinant vaccinia viruses that synthesize e or nsl of japanese encephalitis virus cleavage of structural proteins during the assembly of the head of bacteriophage t4 fusion function of the semliki forest virus spike is activated by proteolytlc cleavage of the envelope glycoprotein precursor ~62 spike protein ollgomenzation control of semliki forest virus fusion antigenic structure of the flavivirus envelope protein e at the molecular level, using tick-borne encephalitis virus as a model japanese encephalitis virus-vaccinia recombinants produce particulate forms of the structural membrane proteins and induce high levels of protection against lethal jev infection endoproteolytic cleavage of gpl60 is required for the activation of human immunodeficiency virus fusion of enveloped viruses with cells and liposomes: activity and inactivation. ln analysis of disulfides present in the membrane proteins of the west nile flavivirus structural proteins of la crosse virus proteolytic processing of the sindbis virus membrane protein precursor pe2 is nonessential for growth in vertebrate cells but is required for efficient growth in invertebrate cells acidotropic amines inhibit proteolytic processing of flavivirus prm protein. viral-ogy174 structure of the flavivirus genome synthetic peptides derived from the deduced amino acid sequence of the e-glycoprotein of murray valley encephalitis virus elicit antiviral antibody antibodies to dengue 2 virus e-glycoprotein synthetic peptides identify antigenic conformation ldentiflcatton of endoprotease activity in the rrans golgi membranes of rat liver cells that specifically processes in vitro the fusion glycoprotein precursor of virulent newcastle disease virus membrane fusion activity of influenza virus: effects of gangliosides and negatively charged phospholipids in target liposomes golgiigranule processing of peptide hormone and neuropeptide precursors: a minireview proteolytic cleavage of peplomeric glycoprotein e2 of mhv yields two 90k subunits and activates cell fusion proteolytic cleavage of the e2 glycoprotein of murine coronavirus: activation of cell-fusing activity of virions by trypsin and separation of two different 90k cleavage fragments electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications the heterodimerit association between the membrane proteins of semliki forest virus changes its sensitivity to low ph during virus maturation cell-associated west nile flavivirus is covered with e + pre-m protein heterodimers which are destroyed and reorganized by proteolytic cleavage during virus release analysis of the influence of proteolytic cleavage on the structural organization of the surface of the west nile flavivirus leads to the isolation of a protease-resistant e protein oligomer from the viral surface flavivirus replication strategy hemagglutinin of influenza virus expressed from a cloned gene promotes membrane fusion membrane fusion proteins of enveloped animal viruses the structure and function of the hemagglutinin membrane glycoprotein of influenza virus mutations within the human immunodeficiency virus type 1 gpl60 envelope glycoprotein alter its intracellular transport and processing studies on the glycosylation of flavivirus e proteins and the role of carbohydrate in antigenic structure expression of a human proprotein processing enzyme: correct cleavage of the von wilebrand factor precursor at a paired basic amino acid site induction of protective immunity in animals vaccinated with recombinant vaccinia viruses that express prem and e glycoproteins of japanese encephalitis virus we thank craig miles and cindy bozic of macromolecular resources, colorado state university, and danny jue and tamara hine of the biotechnology core facility, centers for disease control, for amino-terminal sequencing of the enzymatically derived e glycoprotein fragments. key: cord-293790-7hyelm88 authors: guévin, carl; manna, david; bélanger, claudia; konan, kouacou v.; mak, paul; labonté, patrick title: autophagy protein atg5 interacts transiently with the hepatitis c virus rna polymerase (ns5b) early during infection date: 2010-09-01 journal: virology doi: 10.1016/j.virol.2010.05.032 sha: doc_id: 293790 cord_uid: 7hyelm88 autophagy is an important cellular process by which atg5 initiates the formation of double membrane vesicles (dmvs). upon infection, dmvs have been shown to harbor the replicase complex of positive-strand rna viruses such as mhv, poliovirus, and equine arteritis virus. recently, it has been shown that autophagy proteins are proviral factors that favor initiation of hepatitis c virus (hcv) infection. here, we identified atg5 as an interacting protein for the hcv ns5b. atg5/ns5b interaction was confirmed by co-ip and metabolic labeling studies. furthermore, atg5 protein colocalizes with ns4b, a constituent of the membranous web. importantly, immunofluorescence staining demonstrated a strong colocalization of atg5 and ns5b within perinuclear regions of infected cells at 2 days postinfection. however, colocalization was completely lacking at 5 dpi, suggesting that hcv utilizes atg5 as a proviral factor during the onset of viral infection. finally, inhibition of autophagy through atg5 silencing blocks hcv replication. there is compelling evidence that replication of all positivestranded rna viruses requires the formation of virus-induced membrane vesicles and that dmvs are the sites of genome replication for some positive-stranded rna viruses such as pv, mhv, eav, dengue virus, coronavirus, and coxsackievirus lee et al., 2008; pedersen et al., 1999; schlegel et al., 1996; suhy et al., 2000; wong et al., 2008) . in cells, dmvs derived from the endoplasmic reticulum membranes can be produced through the ubiquitous autophagy pathways (klionsky and emr, 2000) . autophagy, which has been well characterized in yeast, is an essential process by which bulk protein degradation and organelle turnover take place (kim and klionsky, 2000; mizushima et al., 1998) . in response to a limited supply of amino acids due to environmental depletion, the autophagy process will generate a new pool of amino acids required for cellular homeostasis. recently, the contribution of an autophagy protein, atg5, to viral replication has been demonstrated (prentice et al., 2004) . indeed, production of mhv particles is profoundly reduced (n99%) in atg5−/− knockout (ko) embryonic stem cells (prentice et al., 2004) . in this system, the expression of atg5 in atg5−/− cells restores virus production. the relationship between atg5 and mhv replication may come from the ability of atg5 to initiate the formation of dmvs, which have been observed in mhv-infected cells mizushima et al., 2001) . as seen for dengue virus-2 (dv2), when autophagy is blocked by atg5-ko mef cells, extracellular dv2 virus titers are reduced by 3fold compared to those from wild-type mef cells (lee et al., 2008) . altered vesicles, called the membranous web, have been observed in cells harboring hcv replicons gosert et al., 2003) . because hcv replicons replicate autonomously in huh-7, it has been proposed that the membranous web that contain the hcv replication complexes represents the genuine site of viral replication moradpour et al., 2003) . in hcv-infected cells, accumulation of lipid droplets, shown to be essential for hcv replication, has been observed in the proximity of the membranous web (miyanari et al., 2007) . given the involvement of autophagy at the site of replication of other positive-stranded rna viruses, are the autophagy proteins or structure involved in hcv replication? in that regard, tanida et al. (2009) recently showed that atg7 silencing decreased the levels of infectious hcvcc by about 40%, whereas intracellular hcv rna and protein levels remained unchanged. at the same time, another group demonstrated that autophagy proteins (atg4b and beclin-1) are required only for the initiation of incoming hcv rna translation/replication ). to identify novel cellular factors that may play an essential role in hcv rna replication, we have previously screened a human liver cdna library for proteins interacting with the hcv ns5b rnadependent rna polymerase (rdrp). here we report that atg5, a protein required for the formation of dmv in embryonic stem cell (mizushima et al., 2001) , specifically interacts with hcv ns5b. we propose that the ns5b/atg5 interaction may be required for the initial onset of hcv replication. using hcv ns5bδ21 protein as a bait in the yeast two-hybrid system, we identified positive clones from a human liver library. the clones that showed the strongest blue color (mel 1 gene activation) on sd (−leu, −trp, −his, −ade/+x α-gal) plates were chosen for further characterization. two of these positive clones matched the sequence of the human eukaryotic initiation factor 4aii (eif4aii), recently identified as an interacting protein for hcv ns5b (kyono et al., 2002) . two more clones corresponded to the embl/genbank/ ddbj accession number y711588a. this gene is highly homologous to the saccharomyces cerevisiae atg5 gene (hammond et al., 1998) . atg5/ns5b interaction was confirmed in the yeast two-hybrid system using the full-length human atg5 gene amplified from a human liver cdna library (clontech) (fig. 1) . as a control, a panel of proteins (rar-β, rar-α, hcv core, and nonstructural protein: ns3 prot , ns3 hel , ns4a, and ns4b) cloned in the gal4 dna binding domain was tested for interaction with atg5. from this panel, only hcv ns3 prot protein gave a weak signal, but the interaction was not characterized further (data not shown). to demonstrate a physical interaction between atg5 and ns5b proteins, we performed co-ip on radio-inert or metabolically labeled yeast cells coexpressing atg5 and ns5b. we used yeast cells in this study because cotransfection of huh-7 cells with c-myc-ns5b and hatagged atg5 resulted in a low level expression of atg5 protein. yeast extracts were immunoprecipitated with monoclonal antibody against either the c-myc or the ha tag, followed by separation on sds-page. as shown in fig. 2a , an immunoreactive band (lane 2) that corresponds to the size of the ns5b protein (68 kda; lane 4) was coimmunoprecipitated with atg5. this band was not present when the extracts were immunoprecipitated with monoclonal anti-5a antibody (lane 1) or without any antibody (protein a/g sepharose beads alone) (lane 3). the bands corresponding to the 50-and 25-kda molecular weight are the heavy and light chains, respectively, of the antibody. conversely, atg5 was coimmunoprecipitated with ns5b when the same extract was incubated with monoclonal anti-c-myc antibody. indeed, a distinct immunoreactive band (lane 5) that corresponds to the size of atg5 (lane 7) was detected. however, this band could not be immunoprecipitated by the monoclonal anti-5a antibody (lane 6). this proteinprotein interaction was further substantiated by the metabolic labeling experiments shown in fig. 2b in which atg5 was constitutively expressed and ns5b expression was under a copper-inducible promoter. as expected, when the labeled extracts were immunoprecipitated with monoclonal anti-ha, a prominent band corresponding to the size of atg5 was detected in yeast extracts under induced (lane 1) and noninduced (lane 2) conditions. however, the band corresponding to ns5b was only detected in lane 1 (induced condition). when the same extracts were immunoprecipitated with monoclonal anti-c-myc antibody, an intense band corresponding to the size of ns5b was apparent under the induced condition (lane 3). as expected, a band corresponding to the size of atg5 was also detected in the extracts under the induced condition (lane 3) but not under the noninduced condition (lane 4). taken together, these observations indicate that atg5 and ns5b interact. to map the interaction domains between ns5b and atg5, n-and c-terminally truncated mutants of both proteins were assessed by a yeast two-hybrid system. for that purpose, the truncated fragments were inserted into a pgbkt7 or pgadt7 plasmid. as illustrated in fig. 3 , all constructs containing the c-terminal end of the ns5b displayed interaction with atg5, while truncation of the ns5b cterminus completely abrogated such interaction. to confirm the data obtained by yeast two-hybrid screening, co-ip of the putative fig. 1 . the ns5bδ21 protein interacts with the full-length human atg5 in yeast twohybrid assay. ns5b and atg5 fused to the gal4 dna binding and activation domains, respectively, were double-transformed into ah109 cells. four independent colonies of recombinant ah109 cells were allowed to grow for a few days on −leu, −trp sd medium (left panel), after which they were replica-plated onto −leu, −trp, −his, −ade + x-αgal plates (right panel). a. soluble yeast extracts containing ns5bδ21 (n-terminal c-myc tag) and atg5 (n-terminal ha tag) were incubated with different monoclonal antibodies and the immunoprecipitates were pulled down using protein a/g beads. precipitated proteins were revealed by western blot using anti-c-myc (left panel) or anti-ha (right panel) monoclonal antibodies. note that ip of atg5 using anti-ha monoclonal antibody coprecipitated ns5bδ21 (lane 2). anti-hcv ns5a (lane 1) or no antibody (beads only, lane 3) did not precipitate ns5bδ21. soluble extract loaded on the gel was used as the size marker for ns5bδ21. ip of ns5bδ21 using antic-myc antibody precipitated atg5 (lane 5) but not anti-hcv ns5a antibody (lane 6). soluble extract loaded on the gel was used as the size marker for atg5 (lane 7). b. soluble [ 35 s]metlabeled protein extract was immunoprecipitated using anti-ha (lanes 1 and 2) or anti-c-myc (lanes 3 and 4). extracts were incubated in the presence (lanes 1 and 3) or absence (lanes 2 and 4) of cupric sulfate, which induced ns5bδ21 expression. precipitated bands were analyzed by sds-page followed by autoradiography. interacting domains was performed. both techniques were able to identify the c-terminal end (aa 450-570) of ns5b as the interacting domain for atg5 (fig. 3) . the ns5b may have several amino acids involved in this interaction since the fragments 1-501, 500-535, and 536-570 independently interacted with atg5. thus, the binding domain of ns5b to atg5 corresponds to the back surface of the thumb domain which has been proposed to be a premium site for proteinprotein interaction due to the presence of a highly conserved patch of basic amino acids (bressanelli et al., 1999) . the interaction observed between atg5 and the hcv ns5b rdrp suggests that atg5 might be involved in hcv replication. in cells replicating hcv rna, ns5b has been localized to the membranous web gosert et al., 2003) . therefore, we analyzed the localization of atg5 and ns4b (a well-recognized marker of the membranous web) in genome-length c5b replicon cells (aligo et al., 2009; konan et al., 2003) . the results presented in fig. 4a indicate that fig. 3 . mapping of the ns5bδ21 and atg5 binding domains. results are indicated in the column on the right and were obtained by yeast two-hybrid assay (first two columns) or by co-ip (third column). deletion mutants of ns5b were assessed for interaction with full-length atg5. the binding area covered the n-terminal amino acids 450-570 as indicated by yeast two-hybrid assay and co-ip. note that none of the deletion mutants of ns5bδ21 self-activated in the yeast two-hybrid screen, as shown in the second column. nd indicates not done. ns4b indeed colocalized with the endogenous atg5 protein. it is known that atg5 and its conjugated form, atg5-apg12, are mostly cytoplasmic in mouse embryonic stem cells but that their association with the membrane increases upon starvation (mizushima et al., 2001) . thus, we looked at the subcellular membrane association of atg5 in replicon cells. as expected, both ns5b and atg5 proteins were excluded from the cytoplasmic fraction (fig. 4b) . indeed, both proteins appeared to reside in the microsomal/mitochondrial fractions, suggesting that atg5 is directed to membranes in replicon cells. note that the nuclear fractions contain both proteins most likely through contamination from the single-step purification protocol of the nuclear fraction. previously, we and others have unsuccessfully attempted to identify a hcv protein involved in the modulation of the autophagic response that occurs upon hcv infection (ait-goughoulte et al., 2008; ). it has recently been suggested that autophagy is required only early in infection . therefore, we evaluated the colocalization of atg5 and ns5b in infected huh7 cells at 2 days postinfection (2 dpi). the selection of 2 dpi, as our earliest time point, was based on the slow replication of jfh1 in huh7 cells for the first few days of infection zhong et al., 2005) . indeed, at 1 dpi, hcv core, ns5b, and ns5a were undetectable by immunofluorescence staining (data not shown). therefore, infected huh7 cells were transfected with pegfp-atg5 and analyzed at 2 or 5 dpi for the presence of ns5b and atg5 (fig. 5a) . to our surprise, a strong colocalization of gfp-atg5 and ns5b was evident in approximately 80% of infected cells at 2 dpi, but completely disappeared at 5 dpi. this result suggests that atg5-ns5b interaction occurs only during the initial onset of hcv replication and may explain why this interaction has not been detected previously. atg5 silencing is known to disrupt autophagy (matsushita et al., 2007; mizushima et al., 2001) . thus, we used atg5 sirna to evaluate the importance of atg5-ns5b interaction on hcv replication. as controls, a scramble sirna was used. the results indicate that silencing atg5 up to 2 dpi results in undetectable hcv core protein (fig. 5b) and in a marked reduction in intracellular viral replication as observed by qrt-pcr (fig. 5c ). this results suggest that atg5 is required for proper viral replication and this requirement is likely through atg5-ns5b interaction. recent reports suggest a role for autophagic proteins in hcv replication and/or secretion (ait-goughoulte et al., 2008; tanida et al., 2009 ). however, these reports are conflicting and no consensus has yet been reached. here we provide for the first time a link between a hcv protein, ns5b, and colocalization of atg5 and ns5b was observed at 2 dpi (magnified area in b) but not at 5 dpi (magnified area in c). b. silencing atg5 reduced viral replication in huh7 cells. huh7 cells were transfected with sirna targeting atg5 or with a scramble sirna as control. the cells were then infected for 2 days and analyzed for the presence of hcv core protein by western blot. as expected, scramble sirna had no effect on hcv replication, whereas atg5 greatly reduced hcv protein expression. c. quantification of intracellular hcv genome from samples in panel b. mock, mock-infected cells. the autophagy machinery. the specific interaction observed between atg5 and ns5b was through the thumb domain of the polymerase, a region with numerous basic amino acids that could favor proteinprotein interaction. using huh7 cells harboring hcv replicon, we showed that atg5 is associated with the membrane and colocalizes with the membranous web constituent, ns4b. we then used hcvcc to better define the subcellular distribution of atg5 and ns5b during the course of viral replication. interestingly, strong colocalization between the two proteins was only seen early in infection and was completely absent late in infection (fig. 5a) . this result may imply that the atg5-ns5b interaction is required for the onset of the viral replication. since the primary known function of atg5 is the formation of the crescent shape dmv, one could argue that hcv requires membrane import during the early stages of viral infection. indeed, atg5 is involved in other positive-strand rna virus replication, probably through the formation of dmv (khakpoor et al., 2009; lee et al., 2008; prentice et al., 2004) . however, we were unsuccessful in visualizing these crescent shaped vesicles or autophagosomes in hcv-infected cells. although unconjugated atg5 can be found on the crescent-shaped autophagosome precursor (mizushima et al., 2001) , maturation into the autophagosome requires the conjugate atg5-apg12 as well as a series of specific interactions with autophagy proteins (george et al., 2000; kim et al., 2001; mizushima et al., 2001) . because viruses such as mhv and perhaps hcv may utilize atg5 to initiate dmv formation but may not require further maturation of the dmv into autophagosomes, the function of apg12 in virus-induced dmvs remains to be determined. replicase proteins of positive-stranded rna viruses are localized in virus-induced membrane vesicles. in hcv replicon-harboring cells, a membranous structure that contains both viral proteins and rna, called the membranous web, has been identified . it has been shown that the formation of the membranous web can be induced by ns4b alone (konan et al., 2003) . another report has shown physical interaction between ns5b (or ns5a) and the snarelike protein, hvap-33 (tu et al., 1999) , leading to the localization of the hcv replicase complex on lipid rafts (aizaki et al., 2006; gao et al., 2004) . despite these findings, the role of the host factors in the formation and function of the hcv replication complex needs to be better defined. in that regard, we propose that autophagic proteins, and perhaps the resulting membranes, are indispensable during the onset of hcv replication. yeast strains for yeast two-hybrid screening were obtained from clontech (mountain view, ca, usa) as components of the pretransformed matchmaker cdna libraries and the matchmaker two-hybrid system 3. s. cerevisiae y187 (matα), which contained the pretransformed human cdna library (complexity n2-4 × 10 6 independent clones) cloned into the gal4 activation domain vector (pact2) was allowed to mate with s. cerevisiae ah109 (mata), which had been transformed with a gal4 dna-binding domain vector (pgbkt7) containing hcv ns5b as a bait. to construct the bait, the hcv ns5b gene lacking the region encoding the c-terminal 21 amino acid residues was amplified by pcr using the hcv-bk (genotype 1b) genomic cdna as a template and ns5b-h1 and ns5b-r1700 as primers (table s1 ). the resulting ns5bδ21 gene containing unique ecori and bamhi sites at the n-and c-termini, respectively, was cloned into the pgbkt7 expression plasmid to generate an in-frame fusion protein with a gal4 dna binding domain. the resulting plasmid, pgbkt7-5bδ21 was sequenced and subsequently used to transform the ah109 yeast strain. following mating, the diploid yeast strain (y187-ah109-ns5b) was selected on sd medium in the absence of leucine, tryptophan, and histidine (−lth). eight hundred potential positive yeast clones were obtained from two million screenable clones and were replica-plated onto x-α gal indicator plates in the absence of leucine, tryptophan, histidine, and adenine. three hundred blue colonies (positive for x-α gal as a result of mel1 gene activation) were selected by prototrophy for histidine and adenine. these clones were retested for positive interaction, and pact2/cdna plasmids were isolated from 20 strong positive clones as reflected by the intensity of the blue color formed on x-α gal indicator plates. these plasmids were retransformed into yeast strains carrying the bait construct, pgbkt7-5bδ21 or pgbkt7, to confirm true interactions. clones that gave a positive signal when cotransformed with pgbkt7-5bδ21 were chosen for dna sequencing. the dna sequences of the positive pact2/cdna clones were translated and compared with a nonredundant sequence database using the blast program through the national center for biotechnology information network service. clustal_x program was used to analyze statistically significant matches. to obtain the full-length coding region of the hatg5 gene (825 bp), the primers atg5-h1 and atg5-r800 (table s1) were prepared for pcr amplification using a human liver cdna library (clontech) as a template. pcr products with unique ndei and bamhi at the n-and c-termini, respectively, were cloned into the pgadt7 vector. the sequence of the resulting plasmid was confirmed by dna sequencing. the integrity of the hatg5 and ns5bδ21 genes inserted into the yeast plasmids (pgbkt7 and pgadt7) was confirmed in vitro by expressing the two proteins using the tnt® t7 coupled reticulocite lysate system (promega, madison, wi, usa) as described by the manufacturer. final confirmation of hapg5/ns5bδ21 interactions by yeast two-hybrid experiments in ah109 cells was carried out as described previously. for co-ip, n-terminal tag ha-hapg5 and c-myc-ns5bδ21 proteins were expressed in the bj2168 yeast strain. briefly, the hapg5 and ns5bδ21 genes were amplified using the tag-ha/apg5-r800-trp and tag-myc/ns5b-r1700-leu primer sets, respectively (table s1 ). the pcr products were then cloned into the yep c (ns5b) and yeptdh (hapg5) plasmids using unique restriction sites (table s1 ). the resulting expression plasmids, yep c -ns5bδ21 (copper-inducible) and yeptdh-hapg5, were used to transform a protease-deficient yeast strain (bj2168). the double transformant yeast strain, yep c -ns5bδ21/yeptdh-hapg5 was grown in sd medium depleted of tryptophan and leucine (−lt). for the induction of c-myc-ns5bδ21, cupric sulfate (1 μm) was added to the −lt medium and incubated overnight at 30°c. yeast extracts containing soluble hapg5 and ns5bδ21 proteins were prepared according to the protocol of mizushima et al. (2001) . briefly, yeast cells were washed and resuspended in ice-cold tes buffer (50 mm tris, 5 mm edta, and 150 mm nacl, ph 7.5). yeast cell walls were disrupted with acid-washed glass beads by vortexing vigorously for 10 minutes. after centrifugation at 3000 × g for 5 minutes, the supernatant (cytoplasmic and microsomal fractions) was mixed with 0.1 volume of 10% np40 and incubated for 15 minutes before centrifugation at 10,000 × g for 15 minutes. aliquots of the resulting supernatant (500-600 μg of protein) were incubated with or without 1 μl of monoclonal anti-myc antibody (9e10: santa cruz, ca, usa) or anti-ha antibody (f6: santa cruz) for 2 hours. a protein a/g sepharose bead mixture (pierce, rockford, il, usa) (10 μl) was added, and samples were incubated for an additional 2 hours. the sepharose beads were washed three times with tes buffer, and the bound proteins were eluted with 30 μl of laemmli buffer. samples (20 μl) were analyzed by sds-page and immunoblotting. proteins were detected by monoclonal anti-c-myc (9e10) or anti-ha (f2) and visualized by enhanced chemiluminescence (ecl; amersham pharmacia biotech; baie d'urfé, qc, canada). alternatively, co-ip was performed using metabolically labeled cell extract. in brief, bj2168 yeast cells carrying the yep c -ns5bδ21 and yeptdh-hapg5 plasmids were grown in sd (−lt) medium overnight at 30°c. when the cells reached mid-log phase, they were washed and incubated for 1 hour in sd (−lt) medium depleted in methionine. subsequently, 10 μm of cupric sulfate and 35 s-labeled methionine (50 μci) were added to the culture, and incubated for 3 hours. after washing the cells twice with sd medium, yeast extracts were prepared for co-ip study as described previously. immunoprecipitated proteins were separated on sds-page followed by autoradiography. the negative control included yeast extracts prepared from the yeast strain without cupric sulfate induction (i.e., expressing only ha-hapg5) and subjected to the same analysis. deletion mutants of ns5bδ21 were generated by pcr using the primers indicated in table s1 . pcr fragments were inserted in pgbkt7 or pgadt7 and the interactions were analyzed by yeast two-hybrid assay. to identify the interaction domains, we performed co-ip of the deletion mutants expressed in the bj2168 yeast strain using the yep c and yeptdh plasmids. transcribed full-length jfh1 rna (megascript, ambion, streetville, ontario, canada), and viral stocks were produced by infection of huh7 cells at a multiplicity of infection (moi) of 0.01, as described previously (guevin et al., 2009) . for subcellular fractionation, the hapg5 gene was cloned into the pegfp-c1 plasmid (clontech). the resulting pegfp-hapg5 plasmid was then transfected into clone-a and naïve huh7 cells using lipofectamine as suggested by the manufacturer (invitrogen; burlington, ontario, canada). the engineering and characterization of the clone-a cells, which constitutively expressed the hcv nonstructural proteins (ns3, 4a, 4b, 5a, and 5b), have been reported elsewhere (howe et al., 2006) . at 48 hours after transfection, cells were trypsinized and washed twice with pbs. after washing, 2 × 10 7 cells were homogenized in a hypotonic buffer containing 10 mm tris-hcl, ph 7.5, and 2 mm mgcl 2 , followed by centrifugation at 1000 × g for 5 min to yield the nuclear fraction. the supernatant was then centrifuged at 14,000 × g for 40 min to pellet the microsomal/ mitochondrial (mit/mic) fraction. the nuclear and the mit/mic pellets were resuspended in the same volume as the final supernatant using the hypotonic buffer, and 20 μl of each extract was resolved on sds-page and immunoblotting using either a rabbit polyclonal antiserum directed against the hcv ns5bδ21 or a mouse monoclonal anti-gfp (gfp-20; sigma). proteins were visualized by enhanced chemiluminescence (ecl; amersham pharmacia biotech). for indirect immunofluorescence, cells were transfected with the pegfp-hatg5 and infected with hcvcc jfh1 at a moi of 0.01. at 24 hours after transfection, the cells were trypsinized and grown on glass coverslips for another 24 hours. the coverslips were then fixed in pbs containing 4% formaldehyde for 10 min, washed three times in pbs, and incubated for 1 h at 4°c in blocking buffer (pbs, 3% bovine serum albumin, 0.1% triton x-100). after three washes in pbs, the coverslips were incubated with a rabbit polyclonal antibody directed against the hcv ns5b protein (generously provided by dr takaji wakita, national institute of infectious diseases, tokyo, japan) (dilution 1:200) in blocking buffer for 1 hour at room temperature (rt). the coverslips were then washed three times in pbs and incubated for 1 hour at rt with alexa fluor 488 or 568-conjugated secondary antibody goat anti-mouse or anti-rabbit igg (jackson immunoresearch laboratories inc., west grove, pa, usa) (dilution 1:500). coverslips were washed four times in pbs and mounted on glass slides with prolong™ antifade (molecular probes), and cells were examined with a laser scanning confocal biorad radiance 2000 microscope. sirna duplexes targeting human were purchased from ambion (sirna atg5 no. am16708a and scramble sirna no. 4611g). sirna duplexes (150 pmol) were transfected into 1 × 10 5 huh7 cells using the rnaimax transfection reagent (invitrogen) and infected 6 hours later with hcv jfh-1. protein knockdown was usually analyzed 48 hours after transfection. cells were washed three times in phosphate-buffered saline and lysed in ripa buffer (50 mm tris-hcl, ph 8.0, 1% (vol./vol.) nonidet p40, 0.5% sodium deoxycholate, 150 mm nacl and 0.1% (vol./vol.) sds) with a complete protease inhibitor mixture (roche applied science). after sds-page electrophoresis, protein samples were transferred to an immuno-blot pvdf membrane for protein blotting (bio-rad) for 45 min. nonspecific binding sites were blocked for 1 hour in pbs-5% skimmed milk, and the membrane was stained for 1 hour with the primary antibodies. the antibodies used were hcv polyclonal anti-core (obtained from dr denis leclerc, laval university, canada) (dilution 1:1000) and anti-atg5 (fl-275) polyclonal antibody (santa cruz biotechnology, ca, usa) (dilution 1:1000). after incubating with the primary antibody, the membranes were washed four times in pbs-0.1% tween-20. bound antibodies were detected by incubation for 45 min with a goat anti-rabbit hrp antibody (jackson immunoresearch) (dilution 1:10,000). the signals were developed with supersignal™ west pico chemiluminescent substrate (pierce). total cellular rna was prepared from sirna-transfected cells by using the rneasy mini kit (qiagen). the cdna were prepared from 250 ng of total cellular rna. briefly, rnas were incubated 3 min at 70°c then cooled on ice for 2 min before the addition of 4 μl of rt-buffer 5x (invitrogen), 2 μl of dtt (0.1 m), 1 μl of random primer p (dn6) (100 ng/μl), 1 μl of dntp (20 mm), 20 u of rnasin, and 100 u of mmlv reverse transcriptase. samples were incubated for 10 min at 25°c and 1 h at 37°c. to inactivate the mmlv, samples were incubated 15 min at 70°c, and cdnas were diluted to a final volume of 200 μl with rnase-free water. primers used for amplification were 5′utr-r: 5′-gagtgggttta tccaagaaag-3′ and 5′utr-f: 5′-tctgcggaaccggtgagt-3′. the mixture consists of 2.5 μl of cdna in a final volume of 25 μl of the reaction mixture containing 8.6 μl h 2 o, 0.5 μl of probe fam-utr (12.5 μm) ccggaattgccgggaagactg, and 0.25 μl (90 μm) of each hcv primers. for the internal control, the 18s ribosomal rna kit was used as suggested by the manufacturer (applied biosystem). the mixture was completed with 12.5 μl of the taqman universal master mix 2x (applied biosystem), and the amplification was performed as suggested by the manufacturer in a rotor-gene rg-3000 (corbet research). hepatitis c virus genotype 1a growth and induction of autophagy polypyrimidine-tract-binding protein is a component of the hcv rna replication complex and necessary for rna synthesis formation and function of hepatitis c virus replication complexes require residues in the carboxy-terminal domain of ns4b protein crystal structure of the rna-dependent rna polymerase of hepatitis c virus autophagy proteins promote hepatitis c virus replication the autophagy machinery is required to initiate hepatitis c virus replication expression of hepatitis c virus proteins induces distinct membrane alterations including a candidate viral replication complex interactions between viral nonstructural proteins and host protein hvap-33 mediate the formation of hepatitis c virus rna replication complex on lipid raft apg5p functions in the sequestration step in the cytoplasm-to-vacuole targeting and macroautophagy pathways rna replication of mouse hepatitis virus takes place at double-membrane vesicles identification of the hepatitis c virus rna replication complex in huh-7 cells harboring subgenomic replicons novel hcv replication mouse model using human hepatocellular carcinoma xenografts homology between a human apoptosis specific protein and the product of apg5, a gene involved in autophagy in yeast molecular mechanism of a thumb domain hepatitis c virus nonnucleoside rna-dependent rna polymerase inhibitor a role for autophagolysosomes in dengue virus 3 production in hepg2 cells autophagy, cytoplasm-to-vacuole targeting pathway, and pexophagy in yeast and mammalian cells membrane recruitment of aut7p in the autophagy and cytoplasm to vacuole targeting pathways requires aut1p, aut2p, and the autophagy conjugation complex cell biology-autophagy as a regulated pathway of cellular degradation nonstructural protein precursor ns4a/b from hepatitis c virus alters function and ultrastructure of host secretory apparatus human eukaryotic initiation factor 4aii associates with hepatitis c virus ns5b protein in vitro autophagic machinery activated by dengue virus enhances virus replication structure of atg5.atg16, a complex essential for autophagy the lipid droplet is an important organelle for hepatitis c virus production a protein conjugation system essential for autophagy dissection of autophagosome formation using apg5-deficient mouse embryonic stem cells membrane association of hepatitis c virus nonstructural proteins and identification of the membrane alteration that harbors the viral replication complex open reading frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulumderived double-membrane vesicles which carry the viral replication complex coronavirus replication complex formation utilizes components of cellular autophagy cellular origin and ultrastructure of membranes induced during poliovirus infection remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles knockdown of autophagy-related gene decreases the production of infectious hepatitis c virus particles hepatitis c virus rna polymerase and ns5a complex with a snare-like protein production of infectious hepatitis c virus in tissue culture from a cloned viral genome autophagosome supports coxsackievirus b3 replication in host cells robust hepatitis c virus infection in vitro we are grateful to takaji wakita and denis leclerc for reagents and nathalie fournier for technical assistance. this work was supported by nserc of canada (grant no. 312225-05). c. g. and c. b. were supported by a fellowship from the armand-frappier foundation (canada). supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.virol.2010.05.032. key: cord-307396-u6v6bxwj authors: liao, y.; yuan, q.; torres, j.; tam, j.p.; liu, d.x. title: biochemical and functional characterization of the membrane association and membrane permeabilizing activity of the severe acute respiratory syndrome coronavirus envelope protein date: 2006-06-05 journal: virology doi: 10.1016/j.virol.2006.01.028 sha: doc_id: 307396 cord_uid: u6v6bxwj a diverse group of cytolytic animal viruses encodes small, hydrophobic proteins to modify host cell membrane permeability to ions and small molecules during their infection cycles. in this study, we show that expression of the sars-cov e protein in mammalian cells alters the membrane permeability of these cells. immunofluorescent staining and cell fractionation studies demonstrate that this protein is an integral membrane protein. it is mainly localized to the er and the golgi apparatus. the protein can be translocated to the cell surface and is partially associated with lipid rafts. further biochemical characterization of the protein reveals that it is posttranslationally modified by palmitoylation on all three cysteine residues. systematic mutagenesis studies confirm that the membrane permeabilizing activity of the sars-cov e protein is associated with its transmembrane domain. the causative agent of severe acute respiratory syndrome (sars) was identified to be a novel coronavirus (sars-cov), an enveloped virus with a single strand, positive-sense rna genome of 29.7 kb in length (rota et al., 2003) . in sars-covinfected cells, a 3′-coterminal nested set of nine mrna species, including the genome-length mrna, mrna 1, and eight subgenomic mrnas (mrna 2-mrna 9), is produced . four structural proteins, spike (s), membrane (m), envelope (e), and nucleocapsid (n), arranged in the order 5′-s-e-m-n-3′, are encoded by subgenomic mrna 2, 4, 5, and 9, respectively. in addition, 3a protein, a recently identified minor structural protein, is encoded by the first orf of subgenomic mrna3 (ito et al., 2005; yuan et al., 2005) . a group of small, highly hydrophobic viral proteins, termed viroporin, has been identified in diverse viral systems. these include the hcv p7 protein (pavlovic et al., 2003) , human immunodeficiency virus type 1(hiv-1) vpu (gonzalez and carrasco, 1998; schubert et al., 1996) , influenza a virus m2 (pinto et al., 1992) , hepatitis a virus 2b (jecht et al., 1998) , semliki forest virus 6k (sanz et al., 1994) , picornavirus 2b (agirre et al., 2002; aldabe et al., 1996) , chlorella virus pbcv-1 kcv (mehmel et al., 2003) , and avian reovirus p10 protein (bodelon et al., 2002) . these proteins contain at least one transmembrane domain that interacts with and expands the lipid bilayer. the transmembrane domain could form hydrophilic pores in the membrane by oligomerization (carrasco et al., 1995; gonzalez and carrasco, 2003) . the hydrophilic channels would allow low molecular weight hydrophilic molecules to across the membrane barrier, leading to the disruption of membrane potential, collapse of ionic gradients, and release of essential compounds from the cell. alterations in ion concentration would promote translation of viral versus cellular mrnas, as translation of mrnas from many cytolytic animal viruses is fairly resistant to high sodium concentrations virology 349 (2006) 364 -275 www.elsevier.com/locate/yviro (carrasco et al., 1995; gonzalez and carrasco, 2003) . progressive membrane damage during viral replication cycles would also result in cell lysis, promote viral budding and release, and facilitate virus spread to surrounding cells. therefore, disruption of the function of viroporins could abrogate viral infectivity, rendering these proteins as suitable targets for the development of antiviral drugs. coronavirus e protein is a minor structural protein (liu and inglis, 1991; yu et al., 1994) . it plays essential roles in virion assembly, budding, morphogenesis, and regulation of other cellular functions machamer, 2001, 2002; fischer et al., 1998; lim and liu, 2001) . in a recent study, we demonstrated that the sars-cov e protein could obviously enhance the membrane permeability of bacterial cells to onitrophenyl-β-d-galactopyranoside and hygromycin b, suggesting that the protein may function as a viroporin (liao et al., 2004) . similar observations were recently reported on mouse hepatitis virus e protein (madan et al., 2005) . in a separate study, the transmembrane domain of the protein was indeed shown to be able to form a cation channel on artificial membrane (wilson et al., 2004) . molecular simulation and in vitro oligomerization studies indicate that this domain could form stable pentamers (torres et al., 2005) . in this study, we show that the expression of sars-cov e protein alters membrane permeability of mammalian cells. this membrane permeabilizing activity is associated with the transmembrane domain. unlike in bacterial cells, mutations of the three cysteine residues alone do not obviously affect the membrane permeabilizing activity of the protein. further biochemical characterization of the e protein shows that it is an integral membrane protein, and is posttranslationally modified by palmitoylation on all three cysteine residues. to test if the sars-cov e protein could affect the membrane permeability of mammalian cells, the flag-tagged e protein was expressed in hela cells. at 12 h posttransfection, cells were treated with two different concentrations of hygromycin b for 30 min, and then radiolabeled with [ 35 s] methionine-cysteine for 3 h. cell extracts were prepared and the expression of e protein was detected by immunoprecipitation with anti-flag antibody under mild washing conditions. as shown in fig. 1 , extracts prepared from cells without treatment with hygromycin b showed the detection of the e protein and some other cellular proteins. in cells treated with 1 and 2 mm of hygromycin b, the expression of the e protein was reduced to 6 and 2%, respectively (fig. 1) . however, in cells transfected with the sars-cov n protein, a similar amount of the n protein was detected in cells both treated and untreated with hygromycin b (fig. 1) . the expression of the n protein was marginally reduced to 90 and 85%, respectively (fig. 1) . these results confirm that expression of e protein in mammalian cells alters the membrane permeability of these cells to hygromycin b. effects of mutations of the three cysteine residues on the membrane permeabilizing activity of the sars-cov e protein sars-cov e protein contains three cysteine residues at amino acid positions 40, 43, and 44, respectively. these residues are located 3-7 amino acids downstream of the c-terminal residue of the transmembrane domain (fig. 2) . the first and third cysteine residues, at amino acid positions 40 and 44, respectively, were previously shown to play certain roles in oligomerization of the e protein (liao et al., 2004) . they may also be involved in the e protein-induced alteration of membrane permeability in bacterial cells (liao et al., 2004) . to systematically test the effects of these residues on the expression, posttranslational modification, folding, oligomerization, and the membrane-permeabilizing activities of e protein, seven mutants, c40-a, c43-a, c44-a, c40/44-a, c40/43-a, c43/44-a, and c40/43/44-a, with mutations of the three cysteine residues to alanine either individually or in combination of two or three, were made by site-directed mutagenesis (fig. 2) . western blotting analysis of cells expressing wild type and most mutant constructs showed specific detection of three species migrating at the range of molecular masses from 14 to 18 kda under reducing conditions and representing three isoforms of the e protein (fig. 3a) . these isoforms may be derived from posttranslational modification of the protein. the apparent molecular masses of these isoforms on sds-page are significantly larger than the calculated molecular mass of approximately 10 kda for the flag-tagged e protein. in the membrane permeability assay shown in fig. 3b , 0.5 and 1 mm of hygromycin b were used. the use of lower concentrations of hygromycin b is to ensure the detection of subtle changes on membrane permeability induced by the mutant constructs. meanwhile, sars-cov n protein was cotransfected into hela cell together with wild type and mutant e proteins to aid assessment of the inhibitory effect of protein synthesis by hygromycin b. expression of wild type and mutant e protein showed that similar levels of inhibition of protein synthesis by hygromycin b were obtained (fig. 3b ). when 0.5 and 1 mm of hygromycin b were added to the culture medium, wild type and mutant e constructs render similar levels of inhibition to the expression of both n and e proteins (fig. 3b) . these results suggest that, contrary to the previous results observed in bacterial cells, these cysteine residues do not render significant effects on the membrane permeabilizing activity of the e protein. the reason for this discrepancy is uncertain, but it may reflect differences in posttranslational modifications, membrane association, subcellular localization, and translocation of the e protein in prokaryotic and eukaryotic cells. hela cells expressing the flag-tagged wild type e (lanes 1, 2, and 3) and seven cysteine to alanine mutation constructs (lanes 4-24) were treated with 0, 0.5, and 1 mm of hygromycin b for 30 min at 12 h posttransfection, and radiolabeled with [ 35 s] methionine-cysteine for 3 h. cell lysates were prepared and the expression of e protein was detected by immunoprecipitation with anti-flag antibody under mild washing conditions. sars-cov n protein was coexpressed with wild type and mutant e protein, and the expression of n protein was detected by immunoprecipitation with polyclonal anti-n antibodies. polypeptides were separated by sds-page and visualized by autoradiography. the percentages of e and n proteins detected in the presence of hygromycin b were determined by densitometry and indicated at the bottom. numbers on the left indicate molecular masses in kilodaltons. effects of mutations introduced into the transmembrane domain on the membrane permeabilizing activity of the sars-cov e protein sars-cov e protein contains an unusually long putative transmembrane domain of 29 amino acid residues with a high leucine/isoleucine/valine content (55.17%) (arbely et al., 2004) . recent molecular simulation and biochemical evidence showed that this domain may be involved in the formation of ion channel by oligomerization (torres et al., 2005) . mutations of the putative transmembrane domain were therefore carried out to study its functions in membrane association and permeabilizing activity of the e protein. as shown in fig. 2 , four mutants, em1, em2, em3, and em4, were initially made by mutation of 3-7 leucine/valine residues to charged amino acid residues in the transmembrane domain. two more mutants, em5 and em6, were subsequently made. em5, which contains mutation of n15 to e, was constructed based on the molecular simulation studies showing that this residue may be essential for oligomerization of the protein ( fig. 2 ) (torres et al., 2005) . em6 was made by combination of the em4 and c40/43/44-a ( fig. 2 ). expression of these mutants showed the detection of polypeptides with apparent molecular masses ranging from 10 to 18 kda (fig. 4a) . interestingly, mutations introduced into em2, em3, em4, and em6 significantly change the migration rate of the corresponding mutant e protein on sds-page. the apparent molecular mass of these mutants is approximately 10 kda, which is consistent with the predicted molecular weight for the flag-tagged e protein (fig. 4a ). the fact that substitutions of the hydrophobic amino acid residues in the transmembrane domain of the e protein with charged amino acids significantly alter the migrating properties of the e protein in sds-page may reflect the changes in overall conformation and membrane association of these mutants compared to wild type e protein. in the hygromycin b permeability assays, cells transfected with em1, em2, and em5 constructs showed a similar degree of inhibition on protein synthesis as in cells expressing wild type e protein (fig 4b) . in cells expressing em3 and em4, much less inhibition of protein synthesis by hygromycin b was observed compared to cells expressing wild type e protein (fig. 4b) . no obvious inhibition of protein synthesis was observed in cells expressing em6 and n protein (fig. 4b) . these results confirm that the transmembrane domain is essential for the membrane permeabilizing activity of the protein, and further suggest that dramatic mutations of the transmembrane domain are required to disrupt this function. the combination of mutations in the transmembrane domain and the three cysteine residues abolishes the membrane permeabilizing activity of e protein, suggesting that these cysteine residues and may play certain roles in the membrane association and permeabilizing activity of the e protein. hela cells expressing the flag-tagged wild type e (lanes 1, 2, and 3) and six mutant e constructs (lanes 4-21), respectively, were treated with 0, 0.5, and 1 mm of hygromycin b for 30 min at 12 h posttransfection, and radiolabeled with [ 35 s] methionine-cysteine for 3 h. cell lysates were prepared and the expression of e protein was detected by immunoprecipitation with anti-flag antibody under mild washing conditions. sars-cov n protein was coexpressed with wild type and mutant e protein, and the expression of n protein was detected by immunoprecipitation with polyclonal anti-n antibodies. polypeptides were separated by sds-page and visualized by autoradiography. the percentages of e and n proteins detected in the presence of hygromycin b were determined by densitometry and indicated at the bottom. numbers on the left indicate molecular masses in kilodaltons. to characterize the membrane association property of the sars-cov e protein, hela cells expressing the flag-tagged e protein were fractionated into membrane and cytosol fractions, and the presence of the e protein in each fraction was analyzed by western blot. as shown in fig. 5 , the protein was almost exclusively located in the membrane fraction. western blot analysis of the same fractions with anti-gm130 antibody (abcam) showed the detection of an unknown host protein of approximately 60 kda that is exclusively located in the membrane fraction (fig. 5) . similarly, fractionation of hela cells expressing the flag-tagged ibv e protein also showed exclusive detection of the protein in the membrane fraction (fig. 5) . the membrane fraction was then treated with either 1% triton x-100, 100 mm na 2 co 3 ph 11 (high ph), or 1 m kcl (high salt), and centrifuged to separate the soluble contents (s) from the pellets (p). treatment of the membrane fraction with 1 m kcl showed that both sars-cov and ibv e proteins were solely detected in the pellets (fig. 5) . treatment of the same membrane pellets with 1% triton x-100 and high ph led to the detection of the e proteins in both the supernatants and the pellets (fig. 5) . as an integral membrane protein control, anti-gm130 antibodies detected the protein exclusively in the supernatants after treatment of the membrane fraction with triton x-100 (fig. 5) . in samples treated with both high ph and high salt, the protein was detected in the pellets only (fig. 5) , confirming that the procedures and conditions used to fractionate the cell lysates and to treat the membrane fractions are appropriate. oligomerization of viroporin is thought to be critical for the formation and expansion of the hydrophilic pore in the lipid bilayers. to determine the oligomerization status of the sars-cov e protein, the e protein with a his-tag at the c-terminus was expressed in insect cells using a baculovirus expression system and purified by ni-nta purification system. the purified e protein was concentrated and subjected to crosslinking with three different concentrations of glutaraldehyde, a short self-polymerizing reagent that reacts with lysine, tyrosine, histidine, and tryptophan. cross-linking with glutaraldehyde showed the detection of dimer, trimer, tetramer, pentamer, and other higher-order oligomers/aggregates of the e protein under either non-reducing (fig. 6 , lanes 1-3) or reducing (fig. 6 , lanes 4-6) conditions. it was noted that more higher-order oligomers/aggregates were detected under nonreducing conditions when higher concentrations of the crosslinking reagent were used (fig. 6, lanes 1-3) . these results indicate that both interchain disulfide bond formation and hydrophobic interaction are contributing to the oligomerization of the e protein. more detailed characterization of the oligomerization status of the sars-cov e protein was hampered by the low expression efficiency of the protein in the system. the e protein from coronavirus mhv and ibv was previously shown to undergo modification by palmitoylation hela cells expressing the flag-tagged sars-cov and ibv e proteins, respectively, were harvested at 12 h posttransfection, broken by 20 stokes with a dounce cell homogenizer, and fractionated into cytosol (c) and membrane (m) fractions after removal of cell debris and nuclei. the membrane fraction was treated with 1% triton x-100, 100 mm na 2 co 3 (ph 11), and 1 m kcl, respectively, and further fractionated into soluble (s) and pellet (p) fractions. polypeptides were separated by sds-page and analyzed by western blot using either anti-flag antibody or anti-gm130 antibody (abcam). numbers on the left indicate molecular masses in kilodaltons. fig. 6 . oligomerization of sars-cov e protein. the his-tagged e protein expressed in sf9 insect cells was purified using ni-nta purification system (qiagen), and incubated with three different concentrations of glutaraldehyde (0.1, 0.25, and 0.5 mm) for 1 h at room temperature. the reaction was quenched by adding 100 mm glycine. polypeptides were separated on sds-15% polyacrylamide gel in the presence or absence of 1% β-mercaptoethanol, and analyzed by western blot with anti-his antibody. different oligomers of the e protein are indicated on the right. numbers on the left indicate molecular masses in kilodaltons. (11, 43) . to verify if sars-cov e protein is palmitoylated, two independent experiments were performed. first, treatment of the e protein with 1m hydroxylamine showed the reduced detection of the more slowly migrating isoforms (fig. 7a, lanes 1 and 2) . as a control, treatment of the ibv e protein with the same reagent abolished the detection of upper bands (fig. 7a, lanes 3 and 4) . second, the three cysteine residues in combinations of two or all three were mutated to alanine (fig. 2) . wild type and mutant e proteins were then expressed in hela cells and labeled with [ 3 h] palmitic acid or [ 35 s] methionine-cysteine. as shown in fig. 7b , wild type and all mutant e proteins were efficiently labeled with [ 35 s] methionine-cysteine (fig. 7b, upper panel) . in cells labeled with [ 3 h] palmitate, wild type and the three mutants with mutations of different combinations of two cysteine residues (c40/44-a, c40/43-a and c43/44-a) were efficiently detected (fig. 7b, lower panel, lanes 1-4) . however, the construct with mutation of all three cysteine residues (c40/43/44-a) was not labeled (fig. 7b , lower panel, lane 5). as a positive control, the ibv e protein was also efficiently labeled by [ 3 h] palmitate (fig. 7b, lower panel, lane 6) . these results confirm that sars-cov e protein is modified by palmitoylation at all three cysteine residues. mutational analysis of the subcellular localization and membrane association property of sars-cov e protein to further analyze the membrane association properties of the e protein, its subcellular localization was studied by indirect immunofluorescence. hela cells overexpressing the flag-tagged e protein were fixed with 4% paraformaldehyde at 12 h postinfection and stained with anti-flag monoclonal antibody (fig. 8a) . in cells permeabilized with 0.2% triton x-100, the flag-tagged e protein is mainly localized to the perinuclear regions of the cells (fig. 8a, panel a) . the staining patterns largely overlap with calnexin, an er resident protein (panels b and c). it was also noted that some granules and punctated staining patterns are not well merged with the calnexin staining patterns. they may represent aggregates of the e protein. the exact subcellular localization of a coronavirus e protein is an issue of debate in the current literature (corse and machamer, 2003) . although clear er localization of the coronavirus ibv e protein was observed at early time points in a time course experiment using an overexpression system (lim and liu, 2001) , no such localization patterns were observed as reported by corse and machamer (2003) . to clarify that the above observed er localization pattern may be due to the high expression level of the protein in hela cells using the vaccinia/t7 system, the subcellular localization of the sars-cov e protein in another cell type with lower expression efficiency of the protein was carried out. as shown in fig. 8a , expression of the flag-tagged sars-cov in bhk cells stably expressing the t7 rna polymerase (buchholz et al., 1999) showed that the protein exhibits typical golgi localization patterns (panels d-f). expression of the untagged sars-cov e protein in the same cell type also shows very similar golgi localization patterns as the flag-tagged protein (fig. 8a , panels g-i). these results suggest that the predominant er localization patterns in hela cells observed above may be due to the cell type used and the very high expression levels of the protein in individual cells with the vaccinia/t7 expression system. the subcellular localization of wild type and six mutants, em1, em2, em3, em4, em5, and em6, was then studied in bhk cells. the localization patterns of em1, em2, and em5 were similar to wild type e protein, showing predominant golgi localization patterns (fig. 8b) . in cells expressing em3 and em4, more diffuse staining patterns throughout the cytoplasm were observed (fig. 8b) . it suggests that these mutations may change the membrane association properties of the protein, leading to the alteration of the subcellular localization of the protein. in cells expressing em6, a diffuse localization pattern was observed (fig. 8b) . the membrane association properties of wild type and mutant e proteins were further confirmed by fractionation of hela cells expressing e protein into membrane and cytosol fractions, and the presence of e protein in each fraction was analyzed by western blot. as shown in fig. 8c, 95 .28% of wild type e protein was detected in the membrane fraction. the percentages of the mutant e protein detected in the similarly prepared membrane fraction were 93.67% for em1, 89.85% for em2, 62.60% for em3, 58.58%for em4, 93.64% for em5, and 55.46% for em6 (fig. 8c) . to test if the e protein may be associated with lipid rafts, the low-density, detergent-insoluble membrane fraction was isolated from hela cells overexpressing the sars-cov e protein. as shown in fig. 9 , the majority of the e protein was detected at the bottom fractions (lanes 9-11). however, a certain proportion of e protein associated with lipid rafts was detected in fractions 3, 4, and 5 (fig. 9, lanes 3-5) . as a marker for lipid rafts, the gm1 was detected in fractions 4 and 5 (fig. 9, lanes 3 and 4) . all coronaviruses encode a small hydrophobic envelope protein with essential functions in virion assembly, budding, and morphogenesis machamer, 2001, 2002; fischer et al., 1998; lim and liu, 2001) . in a previous study, the sars-cov e protein was shown to obviously enhance the membrane permeability of bacterial cells to onitrophenyl-β-d-galactopyranoside and hygromycin b (liao et al., 2004) . in this study, we show that this protein can also alter membrane permeability of mammalian cells to the general translation inhibitor, hygromycin b. evidence present further demonstrates that the sars-cov e protein is an integral membrane protein, and its membrane-permeabilizing activity is associated with the transmembrane domain. the sars-cov e protein contains a putative long transmembrane domain of 29 amino acid residues (arbely et al., 2004) . in a recent report, arbely et al. (2004) showed that the protein might have a highly unusual topology, consisting of a short transmembrane helical hairpin that forms an inversion about a previously unidentified pseudo-center of symmetry. this hairpin structure could deform lipid bilayers and cause tabulation (arbely et al., 2004) . the full-length and n-terminal 40 amino acid region were shown to be able to form cationselective ion channels on artificial lipid bilayers (wilson et al., 2004) . by using molecular simulation and synthetic peptide approaches, it was shown that the transmembrane domain of e protein could form ion channels by homooligomerization into stable dimers, trimers, and pentamers (torres et al., 2005) . the mutagenesis data present in this study showed that introduction of radical mutations to the transmembrane domain is required to block the membrane-permeabilizing activity of the protein. mutations introduced into the transmembrane domain of sars-cov e protein in em2 as well as in em3 and em4 drastically change the migration properties of the e protein in sds-page (fig. 4a) . it suggests that these mutations would have significantly altered the overall folding, hydrophobicity, and membrane association properties of the e protein. however, this mutant shows very similar properties in subcellular localization, membrane association, and membrane-permeabilizing activity as wild type e protein. the high tolerance of e protein to such dramatic mutations indicates that maintenance of these properties would be essential for the functionality of e protein in coronavirus life cycles. this possibility would warrant more systematic studies by using an infectious clone system. the first and third cys residues of the e protein were previously shown to play important roles in oligomerization and modification of membrane permeability in bacterial cells (liao et al., 2004) . the protein could form homodimers and trimers by interchain disulfide bonds in both bacterial and mammalian cells (liao et al., 2004) . in this study, mutation of all the three cysteine residues did not obviously affect the membrane-permeabilizing activity of the e protein. instead, these residues could affect the membrane association of the protein as they were shown to be modified by palmitoylation. examples of palmitoylation of viral and cellular proteins on multiple cysteine residues include influenza virus ha protein, members of seven transmembrane domains containing gprotein-coupled receptors (ccr5 and endothelin b receptor, etc.), and other cellular proteins (bijlmakers and marsh, 2003) . lipid modification by palmitic acid has been reported for a number of viral envelope proteins, including the hemagglutinin (ha) and m2 protein of influenza virus (melkonian et al., 1999; schroeder et al., 2004; veit et al., 1991; zhang et al., 2000) , gp160 of hiv-1 (rousso et al., 2000) , and env protein of murine leukemia virus (li et al., 2002) . the e protein of coronavirus mouse hepatitis virus and ibv was also reported to be palmitoylated (corse and machamer, 2001) . it has been noted that palmitoylation of viral envelope proteins usually takes place on cysteine residues located within the transmembrane domain or in the cytoplasmic tail close to this domain (bhattacharya et al., 2004; hausmann et al., 1998; schmidt et al., 1988; veit et al., 1989) . this thioester linkage of fatty acids to viral envelope proteins is a posttranslational event that takes place in the cis or medial golgi after exit from the er and after oligomerization but prior to acquisition of endo h (endo-β-n-acetylglucosaminidase h) resistance (bonatti et al., 1989; veit and schmidt, 1993) . palmitoylation of viral proteins plays a considerable role in virus infectivity, virion assembly, and release. for example, palmitoylation of the ha protein of influenza virus enhances its association with lipid rafts (melkonian et al., 1999; zhang et al., 2000) ; palmitoylation of the hiv-1 envelope glycoprotein is critical for viral infectivity (rousso et al., 2000) ; palmitoylation of the murine leukemia virus envelope protein is critical for its association with lipid rafts and cell surface expression (li et al., 2002) ; palmitoylation of the rous sarcoma virus transmembrane glycoprotein is required for protein stability and virus infectivity (ochsenbauer-jambor et al., 2001) . the observed palmitoylation of the sars-cov e protein may play certain roles in its cell surface expression and association with lipid rafts. in this study, we show that radical mutations introduced into the transmembrane domain of the sars-cov e protein could neither totally block the membrane-permeabilizing activity nor completely disrupt the membrane association properties of the protein, unless the three cysteine residues were simultaneously mutated. this may reflect the relatively low sensitivity of the hygromycin b assays used to detect the membrane-permeabilizing activity of the protein. alternatively, it suggests that the palmitoylated mutant e protein may be still tightly associated with cellular membrane, as palmitoylation of the protein would help target the protein to the cellular membrane. this membrane association could, in turn, cause membrane damage, leading to the increased membrane permeability to hygromycin b. subcellular fractionation and biochemical characterization studies demonstrated that, similar to the ibv e protein, the sars-cov e protein behaves as an integral membrane protein. however, it was consistently observed that, under the experimental conditions used, certain proportions of both sars-cov and ibv e proteins associated with cellular membranes were resistant to 1% triton x-100, while some of the membrane-associated proteins can be released from the membrane pellets by treatment with high ph. two possibilities are considered. first, as observed in this study, sars-cov e protein is partially associated with lipid rafts. this may render the protein resistant to the treatment by triton x-100. second, immunofluorescent staining of cells expressing the e protein showed the detection of the protein with punctated staining patterns at both cell surface and intracellular structures. it would suggest that the protein may form aggregates. these aggregates fig. 9 . association of sars-cov e protein with lipid rafts. hela cells expressing the flag-tagged sars e protein were lysed with 1% triton, and centrifuged to remove insoluble material and nuclei. the supernatants were fractionated by ultracentrifugation with a sucrose gradient, and 11 fractions were collected. the presence of the sars-cov e protein in each fraction was analyzed by western blot using anti-flag antibody, and the presence of gm1 was determined by dot blot. numbers on the left indicate molecular masses in kilodaltons. may be cofractionated with the membrane-associated e protein and can be released by high ph but are resistant to the detergent treatment. cell surface expression is a prerequisite for sars-cov e protein to alter the membrane permeability of the cells. over the course of this study, it was consistently observed that sars-cov e protein exhibits much weaker cell surface staining than the ibv e protein even in cells overexpressing the protein (data not shown). this may reflect the unique topology of the sars-cov e protein on cellular membranes. this possibility is currently under investigation. amplification of respective template dnas with appropriate primers was performed with pfu dna polymerase (strategene) with 2 mm mgcl 2 . the pcr conditions were 35 cycles of 94°c for 45 s, 46-58°c for 45 s, and 72°c for 30 s. the annealing temperature and extension time were subjected to adjustments according to the melting temperatures of the primers used and the lengths of the pcr fragments synthesized. site-directed mutagenesis was carried out with two rounds of pcr and two pairs of primers (liu et al., 1997) . hela cells were grown at 37°c in 5% co 2 and maintained in glasgow's modified eagle's medium supplemented with 10% fetal calf serum. sars-cov e and mutants were placed under the control of a t7 promoter and transiently expressed in mammalian cells using a vaccinia virus-t7 expression system. briefly, 60-80% confluent monolayers of hela cells grown on dishes (falcon) were infected with 10 plaque-forming units/cell of a recombinant vaccinia virus (vtf7-3) that expresses t7 rna polymerase. two hours later, cells were transfected with plasmid dna mixed with effectene according to the instructions of the manufacturer (qiagen). cells were harvested at 12 to 24 h posttransfection. total proteins extracted from hela cells were lysed with 2× sds loading buffer in the presence of 200 mm dtt plus 10 mm of iodoacetamide and subjected to sds-page. proteins were transferred to pvdf membrane (stratagene) and blocked overnight at 4°c in blocking buffer (5% fat free milk powder in pbst buffer). the membrane was incubated with 1:2000 diluted primary antibodies in blocking buffer for 2 h at room temperature. after washing three times with pbst, the membrane was incubated with 1:2000 diluted anti-mouse or anti-rabbit igg antibodies conjugated with horseradish peroxidase (dako) in blocking buffer for 1 h at room temperature. after washing for three times with pbst, the polypeptides were detected with a chemiluminescence detection kit (ecl, amersham biosciences) according to the instructions of the manufacturer. permeability of the plasma membrane of cells expressing sars-cov e or mutants to hygromycin b was determined as described below. briefly, hela cells in 100 mm dish were transfected with plasmids described as above, the cells were pretreated with different concentrations of hygromycin b (sigma) for 30 min in methionine-cysteine free medium at 12 h posttransfection, and then 25 μci/ml of [ 35 s] methionine-cysteine (amersham) was added to the culture medium. the cells were incubated at 37°c for 3 h in the presence or absence of hygromycin b, harvested, and lysed in 1× radioimmune precipitation assay buffer (ripa) containing 1.0 mm phenylmethylsulfonyl fluoride and 10 μg/ml each of aprotinin and leupeptin (roche applied science). the cell extracts were clarified for 10 min at 13,000 rpm at 4°c, and the proteins were immunoprecipitated with appropriate antibodies for 1 h at 4°c, incubated with 20 μl of protein a-agarose overnight at 4°c, and washed three times with ripa. the proteins were analyzed by 15% sds-page. for palmitoylation assay, hela cells in 60mm dish were labeled with 1 mci/ml of [9, 10-3 h] palmitic acid (50ci/mmol; amersham) for 10 h at 4 h posttransfection. a duplicate dish of cells was labeled with 25 μci/ml of [ 35 s] methionine-cysteine (amersham) for 10 h at 4 h posttransfection. cells were harvested and proteins were immunoprecipitated as described above. a cdna fragment covering the sars-cov e protein with a his-tag at the c-terminus was cloned to the transfer vector pvl1392 (pharmingen). a recombinant baculovirus expressing the his-tagged sars-cov e protein was generated by cotransfection of the pvl1392-e-his construct together with baculogold dna (pharmingen) into sf9 cells according to the instruction of the manufacturer. fresh sf9 cells were infected with the recombinant virus and harvested at 72 h postinfection. the his-tagged e protein was purified using ni-nta purification system (qiagen) according to the instruction of the manufacturer. the his-tagged e protein was incubated with 0.1, 0.25, and 0.5 mm glutaraldehyde at room temperature for 1 h. the reaction was quenched by adding 100 mm glycine. polypeptides were separated on sds-15% polyacrylamide gel in the presence or absence of 1% β-mercaptoethanol, and analyzed by western blot with anti-his antibody. hela cells expressing e protein or various mutants were fixed with 4.0% paraformaldehyde for 10 min at 16 h posttransfection, washed three times with 1× pbs, permeabilized with 0.2% triton x-100 for 10 min at room temperature, and washed three times with 1× pbs. rabbit anti-e antibodies were used to detect e protein and mutants, and mouse anti-calnexin monoclonal antibodies (abcam) were used to detect calnexin, an er marker. mouse anti-flag monoclonal antibodies were also used to detect flag-tagged e protein and mutants. hela cells were resuspended in hypotonic buffer (1 mm tris-hcl ph 7.4, 0.1 mm edta, 15 mm nacl) containing 2 μg of leupeptin per ml and 0.4 mm phenylmethylsulfonyl fluoride and broken by 20 strokes with a dounce cell homogenizer. cell debris and nuclei were removed by centrifugation at 1500 × g for 10 min at 4°c. the cytosol fraction and membrane fraction (postnuclear fraction) were separated by ultracentrifugation through a 6% sucrose cushion at 150,000×g for 30 min at 4°c. membrane pellets were resuspended in hypotonic buffer, treated with 1% triton x-100, 100 mm na 2 co 3 or 1 m kcl for 30 min, and further fractionated into supernatant (s) and pellet (p) fractions by ultracentrifugation at 150,000 × g for 30 min at 4°c. hela cells expressing the flag-tagged sars-c0v e from two 10 cm dishes were washed twice with ice-cold pbs and lysed on ice for 30 min in 1 ml of 1% triton x-100 tne lysis buffer (25 mm tris-hcl ph 7.5, 150 mm nacl, 5 mm edta) supplemented with protease inhibitor cocktail (roche). the cell lysates were homogenized with 25 strokes using a dounce homogenizer and centrifuged at 3000 × g at 4°c for 5 min to remove insoluble materials and nuclei. the supernatants were mixed with 1 ml of 80% sucrose in lysis buffer, placed at the bottom of a ultracentrifuge tube, overlaid with 6 ml of 30% and 3 ml of 5% sucrose in tne lysis buffer, and ultracentrifuged at 38,000 rpm at 4°c in a sw41 rotor (beckman) for 18 h. after centrifugation, 11 fractions (1 ml each) were collected from the top to the bottom and analyzed immediately by western blot or stored at −80°c. plasmid pflage was constructed by cloning an ecorv-and ecori-digested pcr fragment into ecorv-and ecori-digested pflag. the flag tag is fused to the n-terminal end of the e protein. the two primers used are: 5′-cgggatatcctact-cattcgttt cggaa-3′ and 5′-ccggaattcttagac-cagaagatcaggaac-3′. mutations were introduced into the e gene by two rounds of pcr. the pcr amplified fragments were cloned into ecorv-and ecori-digested pflag. all plasmids and the introduced mutations were confirmed by automated dna sequencing. viroporin-mediated membrane permeabilization membrane permeabilization by poliovirus proteins 2b and 2bc a highly unusual palindromic transmembrane helical hairpin formed by sars coronavirus e protein human immunodeficiency virus type 1 envelope glycoproteins that lack cytoplasmic domain cysteines: impact on association with membrane lipid rafts and incorporation onto budding virus particles the on-off story of protein palmitoylation modification of late membrane permeability in avian reovirus-infected cells palmitoylation of viral membrane glycoproteins takes place after exit from the endoplasmic reticulum generation of bovine respiratory syncytial virus (brsv) from cdna: brsv ns2 is not essential for virus replication in tissue culture, and the human rsv leader region acts as a functional brsv genome promoter modification of membrane permeability by animal viruses infectious bronchitis virus e protein is targeted to the golgi complex and directs release of virus-like particles the cytoplasmic tail of infectious bronchitis virus e protein directs golgi targeting the cytoplasmic tails of infectious bronchitis virus e and m proteins mediate their interaction analysis of constructed e gene mutants of mouse hepatitis virus confirms a pivotal role for e protein in coronavirus assembly the human immunodeficiency virus type 1 vpu protein enhances membrane permeability adenovirus death protein, a transmembrane protein encoded in the e3 region, is palmitoylated at the cytoplasmic tail severe acute respiratory syndrome coronavirus 3a protein is a viral structural protein membrane permeability induced by hepatitis a virus proteins 2b and 2bc and proteolytic processing of hav 2bc palmitoylation of the murine leukemia virus envelope protein is critical for lipid raft association and surface expression expression of sars-coronavirus envelope protein in escherichia coli cells alters membrane permeability the missing link in coronavirus assembly association of the infectious bronchitis virus3c protein with the virion envelope proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein: identification of a 10-kilodalton polypeptide and determination of its cleavage sites viroporin activity of murine hepatitis virus e protein possible function for virus encoded k+ channel kcv in the replication of chlorella virus pbcv-1 role of lipid modifications in targeting proteins to detergent-resistant membrane rafts. many raft proteins are acylated, while few are prenylated palmitoylation of the rous sarcoma virus transmembrane glycoprotein is required for protein stability and virus infectivity the hepatitis c virus forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives influenza virus m2 protein has ion channel activity palmitoylation of the hiv-1 envelope glycoprotein is critical for viral infectivity semliki forest virus 6k protein modifies membrane permeability after inducible expression in escherichia coli cells chemical identification of cysteine as palmitoylation site in a transmembrane protein (semliki forest virus e1) the influenza virus ion channel and maturation cofactor m2 is a cholesterol-binding protein identification of an ion channel activity of the vpu transmembrane domain and its involvement in the regulation of virus release from hiv-1-infected cells mechanisms and enzymes involved in sars coronavirus genome expression the transmembrane oligomers of coronavirus protein e timing of palmitoylation of influenza virus hemagglutinin different palmitoylation of paramyxovirus glycoproteins the m2 protein of influenza a virus is acylated sars coronavirus e protein forms cation-selective ion channels mouse hepatitis virus gene 5b protein is a new virion envelope protein subcellular localization and membrane association of sars-cov 3a protein influenza virus assembly and lipid raft microdomains: a role for the cytoplasmic tails of the spike glycoproteins this work was supported by the agency for science technology and research, singapore and a grant the biomedical research council (bmrc 03/1/22/17/220). key: cord-318400-l9kwxsq7 authors: chhabra, rajesh; kuchipudi, suresh v; chantrey, julian; ganapathy, kannan title: pathogenicity and tissue tropism of infectious bronchitis virus is associated with elevated apoptosis and innate immune responses date: 2016-01-15 journal: virology doi: 10.1016/j.virol.2015.11.011 sha: doc_id: 318400 cord_uid: l9kwxsq7 to establish a characteristic host response to predict the pathogenicity and tissue tropism of infectious bronchitis viruses (ibv), we investigated innate immune responses (iir) and apoptosis in chicken embryo kidney cells (cekc) and tracheal organ cultures (toc) infected with three ibv strains. results showed nephropathogenic ibv strains 885 and qx induced greater apoptosis in cekc than m41, which induced greater apoptosis in tocs compared to 885 and qx. elevated iir is associated with tissue tropism of different ibv strains. compared to m41, 885 and qx caused greater induction of toll like receptor 3 (tlr3), melanoma differentiation associated protein 5 (mda5) and interferon beta (ifn-β) in cekc. in contrast, m41 infection caused greater expression of these genes than 885 or qx in tocs. in summary, greater levels of apoptosis and elevated levels of tlr3, mda5 and ifn-β expression are associated with increased pathogenicity of ibv strains in renal and tracheal tissues. infectious bronchitis (ib) is an acute and highly contagious disease caused by a gamma coronavirus that affects chickens of all ages and is characterized by lesions in respiratory and urogenital organs (cavanagh, 2007; dolz et al., 2006) . avian infectious bronchitis virus (ibv) continues to cause serious economic losses to global chicken production. along with highly pathogenic avian influenza (hpai) and velogenic newcastle disease (nd), ib is the most economically important viral respiratory disease affecting poultry industry worldwide (cook et al., 2012) . vaccination has been considered as the most reliable approach for ibv control (meeusen et al., 2007) however current vaccines have proved to be inadequate due to constant emergence of new variant viruses (de wit, 2000; de wit et al., 2011) . concurrent circulation of both classic and variant ibvs has been identified in most parts of the world, raising major challenges to the current ibv prevention and control strategies. current ibv isolates present high antigenic diversity (hofstad, 1975) , and emergent strains that differ in antigenic properties, tissue tropism and pathogenicity are continuously being reported across the world (gelb et al., 1991; jackwood, 2012; shaw et al., 1996; zanella et al., 2000) . while all ibv strains appear to initially infect chickens via the respiratory tract, vireamia enables spread to secondary sites for further replication and persistence. an example is ibv strain m41, which replicates primarily in the respiratory tract and subsequently spreads and replicates in a range of other tissues. in contrast, strains of ibv such as strain qx and is/885/00 are primarily nephropathogenic (benyeda et al., 2009; meir et al., 2004) . is/885/00, referred to as a nephropathogenic infectious bronchitis virus (nibv), was first isolated from a severe outbreak of renal disease in several broiler farms in israel (meir et al., 2004) and has been detected in many other middle east countries (abdel-moneim et al., 2012; awad et al., 2014; mahmood et al., 2011) . ibv qx was first isolated in china from chickens with proventriculitis (yudong et al., 1998) but was later found to cause renal, (ganapathy et al., 2012; liu and kong, 2004; terregino et al., 2008; worthington et al., 2008) , respiratory and reproductive lesions (ducatez et al., 2009; terregino et al., 2008) in chicken flocks in europe, asia, africa and middle east (beato et al., 2005; domanska-blicharz et al., 2006; gough et al., 2008; jackwood, 2012) . differences in tissue tropism and thus differences in the pathogenicity of ibv strains have been hypothesized to be associated with differences in the binding properties of their spike proteins (casais et al., 2003; wickramasinghe et al., 2011) . while the ability to bind to susceptible host cells is the first step in the viral life cycle, host innate immune responses could also be a major contributing factor to the pathological outcome of ibv infection. variant ibvs continually emerge and the host determinants of ibv pathogenicity are not yet fully understood. early cellular and innate immune responses of virus infected cells in vitro could act as useful indicators for predicting the pathological outcome of viral infection in vivo. for example, the three different genotypes of newcastle disease viruses (ndv) produce distinct host response patterns in chicken spleenocytes, which is useful to differentiate the ndv genotypes (hu et al., 2012) . in order to establish the characteristic host response to predict the tissue tropism and pathogenicity of ibvs, we investigated apoptosis and innate immune responses in chicken embryo kidney (cek) cells and tracheal organ cultures (tocs) following infection with is/885/00-like, qx-like and m41 ibv strains. cek cells infected with ibv strains 885, qx or m41 were subjected to immuno-cytochemical staining of viral nucleoprotein (np) at 6 h post-infection (hpi). the dose (moi 1.0) used resulted in a similar level of infection in cek cells across all the three virus strains (fig. 1 ). cek cells and tocs were infected with ibv strains 885, qx, m41 or mock infected, cell metabolic activity and percentage of apoptotic cells were evaluated at 24 and 48 hpi by mtt assay and annexin v binding assay, respectively. a significant (po0.05) reduction in cell metabolic activity was found in ibv infected cek cells, compared with mock infected cells, both at 24 and 48 hpi ( fig. 2a) . a significantly (po0.05) greater level of apoptosis was also found in ibv infected cek cells at 24 and 48 h after virus infection (fig. 2b) . total apoptotic cells in ibv or mock infected tocs were evaluated by tunel assay (fig. 3) . it was found that ibv infection resulted in significant increase in total apoptotic cells in tocs when compared with mock infected controls at 24 and 48 hpi (fig. 3c) . notably, infection of cek cells with ibv strains 885 and qx resulted in significantly (po0.05) greater level of cell death, as shown by reduced metabolic activity ( fig. 2a ) and increased apoptosis (fig. 2b ) when compared to m41 infected cells both at 24 and 48 hpi. in contrast, significantly higher levels of apoptosis were observed in m41 infected tocs compared with those infected with 885 or qx (fig. 2c) . infection with ibv strains 885 or qx resulted in significantly (po0.05) lower infectious virus production from cek cells at 24 and 48 hpi when compared with m41 infected cells (fig. 2d ). in sharp contrast, significantly (po0.05) lower infectious virus production was observed from m41 infected tocs compared with 885 or qx infected tocs (fig. 2e ). cek cells were infected with ibv strains 885, qx, m41 or mock infected at a moi of 1.0, and expression of lgp2, mda, tlr1, tlr2, tlr3 and tlr7 were analysed at 9 and 24 hpi. it was observed that expression of lgp2 (fig. 4a ) and mda5 (fig. 4b ) was significantly upregulated at 9 hpi but down-regulated at 24 hpi in ibv infected cek cells compared with mock infected cells (po0.05). however, at 9 hpi, infection with ibv strains 885 and qx resulted in significantly (po0.05) greater mda5 expression than m41 in cek cells (fig. 4b ). there was no significant change in expression level of tlr1 (fig. 4c ). tlr2 (fig. 4d ) was observed at 9 hpi (po0.05), but the expression of these genes was significantly (po0.05) down-regulated at 24 hpi in ibv infected cek cells, compared to mock infected cells ( fig. 4c and d) . a significant (po0.05) up-regulation of tlr3 expression was detected at 9 hpi but compared with mock infected cells, was either unchanged or significantly (po0.05) down-regulated at 24 hpi in ibv infected cek cells (fig. 4e) . interestingly, ibv strains 885 and qx infection resulted in significantly greater tlr3 expression than m41 infection in cek cells at 9 hpi (fig. 4e ). in summary, infection of cek cells with ibv strains 885 and qx, compared with m41, resulted in significantly greater tlr3 and mda5 expression in the early stage of infection (9 hpi). notably, infection of cek cells with ibvs 885 and qx resulted in significantly (p o0.05) greater ifn-β expression than m41 at 9 hpi ( fig. 5b ). however, ifn-β expression was significantly (p o0.05) down-regulated at 24 hpi in all ibv infected cek cells compared with mock infected cells (fig. 5b ). the significant (p o0.05) up-regulation of il-6 ( fig. 5c ) expression was observed at 9 hpi in 885 and qx infected cek cells, but was not significantly (p o0.05) affected in m41 infected cells. there was no significant (p o0.05) change at 9 hpi but a significant (p o0.05) down-regulation at 24 hpi in the levels of ifn-α ( we investigated apoptosis and innate immune responses in cek cells and tocs following infection with ibv strains 885, qx and m41. infection of cek cells with ibv strains 885 and qx that predominantly cause renal lesions in chickens resulted in significantly greater levels of apoptosis when compared to the m41 strain that causes mainly respiratory lesions. similarly, infection of tocs with m41 resulted in greater apoptosis than with the other two nephropathogenic strains. a variety of viruses have been shown to induce apoptosis in infected host cells (clarke and tyler, 2009; shen and shenk, 1995; teodoro and branton, 1997) . ibvs are known to induce apoptosis in infected cells and b-cell lymphoma 2 (bcl-2) family proteins modulate ibv-induced apoptosis (li et al., 2007; liu et al., 2001; zhong et al., 2012a zhong et al., , 2012b . the modulation of bcl-2 family proteins during ibv infection has also been postulated to be under the regulation of signalling pathways such as endoplasmic reticulum (er) stress and mitogen-activated protein kinase/extracellular signal-regulated kinase (mapk/erk) pathways (fung and liu, 2014; zhong et al., 2012a) . furthermore, ibv infection results in a growth-inhibitory effect by inducing cell cycle arrest at s and g 2 /m phases (li et al., 2007) , both of which results in apoptosis. our findings suggest a strong association between apoptosis in cek cells or tocs to the ability of ibv strains to cause renal or respiratory lesions respectively in chickens. nephropathogenic ibv strains 885 and qx resulted in significantly greater up-regulation of innate immune sensing genes namely tlr3 and mda5 along with greater ifn-β mrna levels in cek cells at 9 h of infection when compared to m41 infection. in contrast, these genes were significantly up-regulated in m41 infected tocs than those infected with 885 or qx at 9 hpi. concurrent with our findings, a previous study observed a significant increase of tlr3 mrna expression in both the tracheas and lungs of ibv infected chickens, when compared to uninfected controls (kameka et al., 2014) . we did not identify any particular change in regulation pattern of other innate immune genes, namely tlr1 and tlr2. tlr3 and tlr7 are well known for recognition of rna virus encoded pathogen associated molecular patterns (pamps) (akira, 2001) . tlr7 was not significantly affected by ibv infection in cek cells. the end products of the tlr3 signalling pathway are the production of anti-viral type i interferon (ifn)-α and -β, whereas tlr7 activation results in the production of pro-inflammatory cytokines (guillot et al., 2005) . concurrent with tlr3 activation, we found a significant up-regulation of ifnβ, but not ifnα mrna expression, at 9 h after ibv infection in cek cells. likewise, the lack of tlr7 induction correlated with no change in the expression of pro-inflammatory cytokines, namely il-1β, il-8 and nlrp3 in ibv infected cek cells. in contrast to our findings, a recent study found that ifn-β transcription was not found in cek cells until 12 h after infection with ibv m41 at moi of 0.1 (kint et al., 2015) . this difference may have been due to variation in the moi used and origin of cells. notably, expressions of all the innate immune genes tested were down-regulated at 24 h after ibv infection in cek cells. this could be a consequence of host shut-off induced by ibv virus replication. corona viruses (covs) are known to down-regulate host gene expression and increased host mrna degradation in order to suppress host innate immune responses (kamitani et al., 2006; tohya et al., 2009) . corona virus nucelocapsid (n) protein has been shown to interfere with the 2 0 ,5 0 -oligoadenylate synthetase/rnasel (2 0 -5 0 oas) activation, which is responsible for type i ifn induction and can also inhibit the production of various pro-inflammatory cytokines and chemokines via global translational shutdown (ye et al., 2007; zhong et al., 2012b) . however, mda5 and ifn-β expression was also up-regulated in tocs at 24 h after ibv infection suggesting a sustained immune response in tocs compared with cek cells. we found a similar virus replication pattern in ceks and tocs with a comparable virus output. higher levels of apoptosis and ifn-β expression is also associated with significantly lower infectious virus particle production from both cek cells and tocs. this could be due to increased antiviral state induced by ifn-β and/or increased apoptosis. apoptosis of cultured cells have previously been shown to be associated with reduced production of infectious influenza virus in duck cells (kuchipudi et al., 2012a) . several in vitro and ex vivo studies have suggested a key role of tlr3 in viral detection (alexopoulou et al., 2001; bowie and haga, 2005; finberg and kurt-jones, 2004; tabeta et al., 2004) . however, recent evidence suggests tlr3 contributes to pathogenic effects of viral infections. tlr3 mediates west nile virus entry into the brain, causing lethal encephalitis (wang et al., 2004 ) and contributes to a detrimental inflammatory response to influenza virus infection in mice, resulting in acute pneumonia (le goffic et al., 2006) . a recent study has shown that mda5, but not tlr3, is involved in the sensing of ibv (kint et al., 2015) . however, higher mrna levels of mda5 and tlr3 correlated with the in vivo pathogenicity of the ibv strains used in this study, such that the nephropathogenic and tracheotropic ibvs induced much higher levels in cek cells and tocs, respectively. it is possible that mda5 activation is a protective antiviral host response against the virus whereas tlr3 contributes to some detrimental effects from viral infection. the precise role of mda5 and tlr3 in host defence against ibv infection and/or disease pathogenesis is not yet fully understood and hence, warrants further in-depth studies. we found that lgp2 expression was significantly increased at 9 h but down-regulated at 24 h after ibv infection in cek cells. lgp2 functions as a negative regulator by interfering with the recognition of viral rna by rig-i and mda5 (yoneyama et al., 2005) . mda5 is ifn-inducible (kang et al., 2004) , so once an ifn response is triggered, this innate antiviral loop initiates autoamplification of mda5 until the natural inhibitor lgp2 is induced (yoneyama et al., 2005) . however, we found that the expression of lgp2, mda5 and ifn-β mrna levels were down-regulated at 24 h after ibv infection, possibly due to the virus induced host gene shutoff. in agreement with our findings, a recent study also reported a down-regulation of tlr3, il-1β and ifn-γ expression in ibv infected chicken tracheas at 12 hpi (kameka et al., 2014) . in this study, we infected cells at moi of 1.0; however, infection at a lower moi could be helpful to profile the effect of lgp2 on mda5 and ifn-β regulation in ibv infected cells. there have been conflicting reports on the difference in virulence among ibv strains using this in vitro system. cook et al. (1976) did not find marked differences when compared three strains of ibv on the basis of their effect on tracheal cilia. raj and jones (1996) also reported little difference among several ibv strains using measurement of ciliary activity as a criterion for damage to the tracheal epithelium. however, abd el rahman et al. (2009) has revealed that the strains differed in their efficiency to infect the tracheal epithelium. infection by beaudette and qx resulted in a larger number of infected cells compared to itlay-02 and 4/91 strains, when tracheal organ cultures (tocs) were infected with the same amount of virus (abd el rahman et al., 2009) . in summary, our study found that greater levels of apoptosis and elevated expression of tlr3, mda5 and ifn-β is associated with increased pathogenicity of ibvs in kidney (cek cells) and respiratory (tocs) tissues. the findings of this study using cek cells and tocs raise a strong possibility that host innate immune response could aid generation of a predictive prognosis for the tissue tropism of novel ibv strains. however, further studies are needed to confirm the association between pathogenicity and host response in vivo and to establish the mechanisms underlying such responses. relative mrna expression was determined by real-time pcr normalised to 18s rrna. graph values are the mean of three biological replicates with error bars as standard error, and are expressed as fold change relative to the mocked-infected group. different letters indicate that the differences between groups at that time point are significant (po 0.05). monolayers of primary chicken kidney (cek) cells were prepared from kidneys of specific-pathogen-free (spf) chicken embryos after 18 day incubation (lohmann, germany). kidneys removed aseptically were collected in minimum essential medium (mem), supplemented with 0.5% fetal bovine serum (sigma) and 1% antibiotics (penicillin and streptomycin). the kidneys were minced into small pieces using sterile scissors, followed by trypsinization (0.25% trypsin) for 30 min at 37°c and filtered through a 100 mm mesh to remove tissue debris. after centrifugation at 1500 g for 10 min, cell pellet was re-suspended in the mem medium and seeded at 1 â 10 5 cells/cm 2 in cell culture plates. we used a traditional ibv strain m41, and two variant ibv strains namely is/885/00-like (885) (awad et al., 2013) and qx-like strain kg3p (ganapathy et al., 2012) were used. is/885/00 causes mortality, poor weight gain and severe renal damage, while qx causes renal and reproductive problems. allantoic fluid containing these ibv strains were propagated in tocs prepared from 19 to 20-day old spf chicken embryos (cook et al., 1976) . for the cek cell infection study, all ibv strains were titrated on cek cells using an immuno-cytochemical focus assay described previously (kuchipudi et al., 2012a) . in brief, serial 2-fold dilutions of a known volume of toc supernatants were used to infect cek cells in 96-well culture plates (cellstar, grenier bio-one, uk). cells were washed after a 2 h incubation with virus, followed by a further 4 h incubation, and then fixed in 1:1 acetone:methanol for 10 min. cells were subjected to viral nucleoprotein detection by anti-ibv nucleoprotein monoclonal antibody (prionics, uk) followed by visualization with envisionþsystem-hrp (dab; dako, ely, uk). cells expressing viral nucleoprotein (np) were counted, and the mean number of positive cells in five fields was used to calculate focus-forming units of virus per microlitre of inoculum. for toc infection study viruses were titrated in chicken embryo tocs and expressed in median ciliostatic doses (cd 50 )/ml (cook et al., 1976) . in brief, for each dilution of virus, three toc rings with 100% of the cilia beating were used. tocs were inoculated with viruses (0.1 ml) in serial dilutions before being incubated at 37.0°c in a rotating incubator (lab thermal equipment, greenfield, nr oldham) at 8 rpm. the tocs were observed for ciliary activity post-infection, a reduction in at least 5% mean activity compared with mock infected were considered as positive for ibv infection (cook et al., 1976) and further confirmed by rt-pcr followed by sequencing . cek cells were seeded on cell culture plates (cellstar, grenier bio-one, uk) at a density of 1 â 10 5 cells/cm 2 . after incubation at 37°c for 48 h, 80-90% confluent monolayers were washed three times with phosphate-buffered saline (pbs), and then either infected with different ibv strains at multiplicity of infection (moi) 1.0 or mock infected with cell culture medium. triplicate wells were used for each virus or mock infection for each time point. cells were analyzed at 24 and 48 h post-infection (hpi) to determine the effect of virus infection on cell metabolic activity and induction of apoptosis. cell culture supernatant was titrated for infectious virus in toc expressed as cd 50 /ml (cook et al., 1976) . at 9 and 24 hpi cell pellets were collected in rlt buffer (qiagen, uk) for total rna extraction and cell metabolic activity was analysed at 24 and 48 hpi using celltiter 96, a nonradioactive cell proliferation assay (promega, madison, wi, usa), according to the manufacturer's instructions. tocs were infected 5 days after preparation to allow the early inflammatory responses of the tissue to subside (reemers et al., 2009) . tocs were infected with ibv strains using a dose of 4 log 10 cd 50 per individual ring, or mock-infected with toc medium without virus. three rings were used for each virus or mock infection for each time point. the infection dose of 4 log 10 cd 50 was chosen based on a preliminary study wherein this dose caused 100% cilliostasis in toc by day 3 after infection. samples of toc supernatant or complete rings were collected at 9, 24 and 48 hpi. toc rings were analysed for apoptotic cells using in situ terminal deoxynucleotidyl transferase dutp nick end labelling (tunel) assay, and supernatants were titrated for quantification of infectious virus at 24 and 48 hpi. total rna extracted from toc rings collected at 9 and 24 hpi were used for the quantification of host gene expression analysis. at 24 and 48 h after virus or mock infection, cek cells in 24well tissue culture plates were analysed to quantify the percent of apoptotic cells. briefly, cells were washed in pbs and removed from the tissue culture plate by trypsinization, followed by inactivation with growth medium containing serum. the cells were then washed, resuspended in binding buffer (clontec, ca), and subsequently analysed by staining with annexin v-fitc and pi according to the manufacturer's instructions (apoalert s , annexin v-fitc apoptosis kit, clontech, ca) using flow cytometer (bd accuri c6, san jose, ca). fluorescence was quantified using cflow software (bd accuri). the single-dye positive controls were prepared by staining representative cells with fitc and pi dye individually and used for estimation of proper colour compensation. unstained cell samples were used as a negative control to adjust the threshold. apoptotic cells were detected and quantified by the tunel assay using the peroxidase (pod) in situ cell death detection kit (roche diagnostics, indianapolis, in, usa) according to the manufacturer's instructions. a total of five sections were analyzed for each time point. briefly, cells were fixed in paraformaldehyde and permeabilized with 0.1% triton x-100 at room temperature. after equilibration, specimens were overlaid with 50 μl tunel reaction mixture or 50 μl label solution of the no-enzyme control. after initial incubation for 1 h at 37°c in a dark humidified atmosphere, reactions were then developed using peroxidase substrate kit dab (vector laboratories, burlingame, usa). sections were counterstained with haematoxylin (merck, germany) and mounted with dpx (dibutyl phthalate, polystyrene granules and xylene) aqueous mounting medium (vwr international, leuvan). for each sample, the total number of positive cells/400 â microscopic field was calculated. supernatants from virus infected cek cells and tocs were titrated for infectious virus and expressed as cd 50 /ml (cook et al., 1976) . in brief, tocs were inoculated with 0.1 ml of supernatant in serial dilutions, and three rings with 100% of the cilia beating were used for each dilution and incubated at 37.0°c in a rotating incubator (lab thermal equipment, greenfield, nr oldham) at lowest rotation speed. the tocs were observed for ciliary activity; those exhibiting less than 5% mean activity were recorded as ibv positive. total rna from cell pellets in rlt buffer (qiagen, uk) and toc ring was extracted using rneasy plus mini-qiashredder kit (qiagen, uk) following the manufacturer's instructions. rna concentration was quantified by nanodrop s nd-1000 (nanodrop, wilmington, de/ nanodrop nd-1000; peqlab). the cdna was generated from 1 mg of rna using the superscript iii first-strand cdna synthesis system (life technologies) with random primers as per the manufacturer's recommendations. the generated cdna samples further diluted to 1:50 in nuclease-free water were used in quantitative reverse transcription pcr (qrt-pcr) analysis. qrt-pcr was performed on the lightcycler s 480 (roche, uk), using lightcycler 480 sybr green i master mix (roche, uk). primers were designed with primer express version 2.0 (applied biosystems), based on previously reported sequences (table 1) . all primers were provided by eurofins genomics (edersberg, germany). the cycling conditions were 10 min at 95°c, 45 cycles of 10 s at 95°c, 10 s at 60°c annealing temperature and 10 s at 72°c, finally followed by a melting curve analysis to ensure the specificity of the sybr green pcr. qrt-pcr data was normalised using a relative standard curve method to 18s ribosomal rna (18srrna) expression (kuchipudi et al., 2012b) and the data were presented as fold difference in gene expression of virus versus mock infected samples. the data were analysed using one-way anova, followed by the post-hoc lsd multiple comparison test using graphpad prism version 6 software. differences between groups at that time point were considered significant at p o0.05. table 1 primers used in the study. primer sequences: sense (s) and anti-sense (as) 18s rrna (18s ribosomal rna ) (kuchipudi et al., 2012b) (s) tgtgccgctagaggtgaaatt (as) tggcaaatgctttcgcttt lgp2 (laboratory of genetics and physiology 2) g.k., s.v.k, and r.c conceived and designed the study. g.k supervised r.c who carried out all the experiments. s.v.k and r.c analyzed the data and wrote the manuscript with editorial contributions from all other authors. comparative analysis of the sialic acid binding activity and the tropism for the respiratory epithelium of four different strains of avian infectious bronchitis virus emergence of a novel genotype of avian infectious bronchitis virus in egypt toll-like receptors and innate immunity recognition of doublestranded rna and activation of nf-kappab by toll-like receptor 3 detection of variant infectious bronchitis viruses in broiler flocks in libya immunopathogenesis of infectious bronchitis virus related to is/1494/06 and is/885 in specific pathogen-free chicks evidence of circulation of a chinese strain of infectious bronchitis virus (qxibv) in 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recent progress in studies of arterivirus-and coronavirus-host interactions rajesh chhabra is a commonwealth scholar, funded by the uk government (incs-2012-180). key: cord-291306-g9qmmugg authors: vey, martin; orlich, michaela; adler, sabine; klenk, hans-dieter; rott, rudolf; garten, wolfgang title: hemagglutinin activation of pathogenic avian influenza viruses of serotype h7 requires the protease recognition motif r-x-k/r-r date: 1992-05-31 journal: virology doi: 10.1016/0042-6822(92)90775-k sha: doc_id: 291306 cord_uid: g9qmmugg abstract the hemagglutinin of influenza virus a/fpv/rostock/34 (h7) was altered at its multibasic cleavage site by site-directed mutagenesis and assayed for proteolytic activation after expression in cv-1 cells. the results indicated that the cellular protease responsible for activation recognizes the tetrapeptide motif r-x-k/r-r that must be presented in the correct sequence position. studies on plaque variants of influenza virus a/fowinictoria/75 (h7n7) showed that alteration of the consensus sequence resulted in a loss of pathogenicity for chickens. it has been well established that the susceptibility of the hemagglutinin to proteolytic activation by argininespecific host proteases is essential for the pathogenicity of influenza viruses (i). the hemagglutinins of mammalian and apathogenic avian strains, which cause local infection, are cleaved only in a restricted number of cell types. on the other hand, with the pathogenic avian strains causing systemic infection, hemagglutinin is activated by proteases present in a broad range of different host cells. the key factor that determines the differential cleavability is the structure of the cleavage site of the hemagglutinin. mammalian and apathogenic avian strains have a single arginine at this site, and plasmin (2) a factor x-like protease from allantoic fluid of chicken eggs (3) and bacterial proteases (4) have been identified as enzymes activating this type of hemagglutinin. the ubiquitous proteases responsible for activation of the hemagglutinin of the pathogenic strains have been less well characterized. they are calcium-dependent and have a neutral ph optimum (5) and they can be inhibited by specific pep-tidy1 chloroalkyl ketones (6). it has long been known that the hemagglutinins activated by these enzymes have multiple lysine and arginine residues at theircleavage sites, and it has been shown that most of these basic amino acids are critical for cleavage activation (7). the exact sequence required for enzyme recognition, however, was not fully understood. in the present study we show that the hemagglutinin cleavage sites of the pathogenic strains have the consensus sequence r-x-k/r-r, and we also present evidence that ' part of this work was presented at the vlllth international congress of virology in berlin 1990. ' to whom reprint requests should be addressed. conserved proline residues in their neighborhood are not important for cleavability. comparison of the published hemagglutinin sequences of the pathogenic avian influenza-a-viruses of serotype h7 reveals a number of conserved amino acids upstream of the cleavage site, notably a series of arginine and lysine residues in positions -1 to -6 and two proline residues in positions -7 or -8 and in position -10 (table 1) . to determine whether these conserved regions are important for the cleavability of the h7 hemagglutinin, we subjected a cdna clone of the hemagglutinin of influenza virus aifpv/rostock/34 to site-directed mutagenesis at the cleavage site and from the panel of mutants obtained, we have selected two groups. in the first one, the arginine and lysine residues have been exchanged by each other or have been replaced by noncharged amino acids. this group comprised mutants 21, 13, 22,7, 23, 1307, ks, 4, and 10 . in the second group, comprising mutants pro,,,-ala, pro,,,-ala, and pro-*, the proline residues in positions -8 and -10 have been exchanged (table 1) . to avoid distortions that might be imposed on the protein by insertions or deletions (8) only mutants with substitutions have been used. to see whether the fpv hemagglutinin mutants were processed by host cell proteases, they were expressed in cv-1 cells using an sv40 vector and analyzed by page after radiolabeling and immunoprecipitation (fig. 1) . only mutants 21 and ks were cleaved by the endogenous enzyme, as was the case with wild type. however, all hemagglutinin mutants were cleaved by trypsin added to the medium indicating that they were exposed at the cell surface and that lack of cleavage was not the result of a transport defect. due to release into the medium which was observed with wild type as well as all mutants (unpublished results), ha, was sometimes underrepresented when compared to ha,. as summarized in table 1 , the following conclusions can be drawn from the data described so far. the results obtained with mutants 7, 13. 21, 22, and 23 indicate that the motif arg-,-x-,-lys/arg-,-arg-, is required for proteases present in cv-1 or equivalent cells to recognize the fpv hemagglutinin as an appropriate substrate. arginine is obligatory in position -1, since mutants 7 and 22 which have lysine in this position, are not cleaved. lysine present mostly in position -2 can be replaced by arginine as is the case with a/turkey/ england/63, but not by nonbasic amino acids, such as asparagine in mutant 13 or glutamic acid in mutants v4 and v5 of a/fowl/victoria/75 (see below). the amino acid in position -3 may be basic, acidic, or neutral as indicated by mutant 21 and the hemagglutinin of another fpv isolate that have glutamic acid in this position. the amino acid in position -4 of the fpv hemagglutinin has to be arginine; if lysine is present in this position, cleavage is very inefficient as demonstrated by mutant 1307. the lysine residues in positions -5 and -6 are not essential, since they have been replaced in fpv mutant ks and in mutants sc32 and sc35 of strain a/seal/mass/l/80 (9) by threonine or are missing in strain a/turkey/england/63 without altering cleavability. table 1 shows also that mutant 10 which has retained the tetrapeptide motif in a shifted position is no longer cleaved in cv-1 cells. this observation demonstrates that accessibility of the cleavage site is also essential for activation. the recognition motif is present in this mutant in a slightly shifted position and has therefore probably lost its correct steric presentation for the cellular protease. other factors modulating accessibility of the cleavage site by steric hindrance have also been reported. thus, interference of a carbohydrate side chain with cleavability has been observed with subtypes h5 (10, 1 i) and h3 (12) . furthermore, the finding that a hemagglutinin with a single arginine at the cleavage site became highly cleavable after in-sertion of a bulging peptide suggests also a mechanism of steric alteration (8). finally, the results obtained on mutants pro,,,-ala, pro,,,-ala, and pro*-indicate that the proline residues in positions -8 and -10 are not essential for cleavability (table 1) . since proline is known to have distinct effects on peptide folding and since the proline residues in positions -8 and particularly in position -10 are conserved, this observation was unexpected. it is also remarkable in the light of the finding that the f protein of sendai virus acquires high cleavability when a proline residue is introduced at position -2 next to lysine or arginine at position -1 of the cleavage site (13) . it will be interesting to see whether or not the enzymes recognizing the motifs r-x-k/r-r and p-k/r which both appear to be ubiquitous belong to the same type of proteases. it was then of interest to find out if the mutant hemagglutinins were able to fuse after cleavage. hemagglutinin was therefore expressed in cv-1 cells, treated with trypsin if not cleaved by the endogenous protease, and examined for its capacity to induce cell fusion after exposure to low ph. except for mutants 4 and 10, cell fusion was observed with wild type and most mutants, regardless as to whether they were activated by tr-ypsin or the cv-1 cell enzyme (fig. 2) . the inability of mutants 4 and 10 to induce fusion demonstrates that hemagglutinin is not activated if it is cleaved between positions -2 and -1. compatible with previous studies by us and others showing that cleavage between positions +1 and +2 (14) and amino acid exchanges in the amino terminal sequence of ha, (15) do not result in activation, this observation underlines the concept that the fusion peptide has a highly specific structure. the studies employing site-directed mutagenesis on hemagglutinin cdna have been extended by an analysis of influenza virus variants with altered hemagglutinin cleavability. in previous work, pathogenic variants with increased hemagglutinin cleavability could be obtained, when apathogenic avian influenza virus strains were adapted to non-permissive host cells (11, 9, 16) . we have now derived from the pathogenic strain al fowl/victoria/75 (h7n7) a number of variants with restricted hemagglutinin cleavability. this was accomplished by selecting plaque variants that were able to undergo multiple replication cycles in mdck cells and in cec only in the presence of trypsin (fig. 3a) . whereas chickens died 4-6 days after infection with wild-type virus, birds infected with the plaque variants survived the observation period of 14 days without showing disease symptoms. thus, the plaque variants had lost pathogenicity for chickens. as had to be expected from these observations, the mutants were resistant to the endogenous protease present in cec, mutations were screened by g-or a-track analysis, respectively. ha genes containing the desired mutations were then analyzed using the dideoxynucleotide chain-termination sequencing technique (33. of the 26 different mutants obtained, 10 were selected for vectorial expression in cv-1 cells. except for the desired mutations, they contained no other amino acid exchanges. the mutated ha genes were excised from the phage replicative form dna with bglll and ligated into the compatible barnhi site of the simian virus 40 expression vector pa1 lsvl3. subconfluent cv-1 cells, which were grown in dulbecco's modified eagle's medium (dmem) in the presence of 10% fetal calf serum, were cotransfected with the sv40-ha recombinant genomes and the sv40 helper genomes using either deae-dextran or lipofectin (brl, berlin, germany). 56 hr after infection with fpv-ha-recombinant sv40, cv-1 cells were incubated in dmem without methionine for 60 min. [35s]methionine (100 pci/ml) was added to the cell cultures for 15 min at 37". the radioactive labeling was chased by replacement of the radioactive medium by dmem containing 10 mm i-methionine at 37" for 60 min before the cells were solubllized by adding ripa buffer at 0". the hemagglutinin was immunoprecipitated from the cell lysates by standard procedures using an anti-fpv serum raised in rabbits, separated by sds-page under reducing conditions, and analyzed by fluorography. of the two ha-bands seen in many samples, the faster migrating one is endoglucosaminidase h-sensitive (data not shown) and, thus, represents hemagglutinin with immature carbohydrate side chains. whereas wild-type hemagglutinin was readily cleaved in these cells (fig. 3b) . rna sequence analysis of the victoria hemagglutinin and its variants demonstrated that, besides a silent mutation in variant v3, only mutations had occurred that resulted in amino acid exchanges at the cleavage site. these was the exchange of arginine at position -4 for lysine with variants vl , v2, and v3 and the exchange of lysine at position -2 for glutamic acid with variants v4 and v5 (table 1) . these observations further substantiate the requirement of the consensus sequence r-x-k/r-r for high cleavability and they show that alteration of this motif results in a decrease of pathogenicity. this sequence is not only found with all pathogenic h7 viruses, but also with most pathogenic h5 strains (7). furthermore h3 hemagglutinin acquires high cleavability after insertion of this motif by site directed mutagenesis (15, 12) . the only exception are pathogenic a/chick/pennsylvania/83 isolates which have the unusual tetrapeptide k-k-k-r at the cleavage site (7). it thus appears that in this particular case the hemagglutinin structure allows a lysine in position -4. the motif r-x-k/r-r is also found in the f protein of paramyxoviwith altered hemagglutinin cleavability. apathogenic variants were prepared by infecting cec with egg-grown wild-type virus at a m.o.i. of l-0.001 pfu/cell in the presence of trypsin (5 rglml). progeny virus was subjected to a plaque passage in cec. three days after incubation in the absence of trypsin, visible plaques were marked, and a second overlay containing 10 pg/ml trypsin was added to the dishes. additional plaques were picked 3 days later and subjected to another round of growth in cec cultures followed by plaque passage with and without trypsin as before. this procedure was repeated several times, until generation of plaques depended completely on the presence of trypsin. five variants designated vl -v5, were obtained in this way. ruses (17) and with other highly cleavable glycoproteins present in togaviruses (18) , coronaviruses (19) , retroviruses (20) , and cytomegalovirus (2 1). deviations from this motif are not compatible with high cleavability, as shown with rous sarcoma virus (22) , hiv (23), sv5 (24, and ndv (25) . the characteristic basic motif is not restricted to viral glycoproteins. it is also present in a variety of cellular proteins which are most likely activated by the same type of enzymes. examples of such precursor proteins are insulin receptor (26) , insulin-like growth factor i receptor (27) , von willebrand factor (28) , and uvomorulin (29) . all of these proteins do not show sequence homology to the influenza virus hemagglutinin downstream the cleavage site (positions +1 , +2, +3, etc.) thus, it appears that this region, although highly conserved among the different hemagglutinin serotypes, is not important for the consensus sequence of the cleavage site. the ubiquitous protease responsible for the activation of the hemagglutinins containing several basic amino acids has not been identified yet. in previous studies it has been shown to be a ca*+-dependent membrane-bound enzyme with a neutral ph optimum (5). in these properties it resembles the kex2 protease from yeast that cleaves pro-a factor and pro-killer toxin at lys-arg and arg-arg sites (30) . an analogue found in mammalian tissues is furin, a human precursor protein-processing endoprotease with sequence homology and shared topology to the kex2 protease (31) . furin is localized in the golgi compartment, is also ca2+-dependent and membrane-associated. its substrate specificity is also characterized by recognition of the basic tetrapeptide motif described above (32) . whether furin is the protease activating viral proteins of the fpv hemagglutinin type is the subject of current studies. advances in virus research proc. nat/. acad. sci proc. nat/. acad. sci. usa we thank dr. j. h. hegemann for preparation of the oligonucleotides. this work was supported by the deutsche forschungsgemeinschaft (sfb 286; ro 202-7-l) and by the fonds derchemischen industrie. key: cord-309919-sm5o0g1c authors: eichwald, catherine; ackermann, mathias; fraefel, cornel title: mammalian orthoreovirus core protein μ2 reorganizes host microtubule-organizing center components. date: 2020-08-04 journal: virology doi: 10.1016/j.virol.2020.07.008 sha: doc_id: 309919 cord_uid: sm5o0g1c filamentous mammalian orthoreovirus (mrv) viral factories (vfs) are membrane-less cytosolic inclusions in which virus transcription, replication of dsrna genome segments, and packaging of virus progeny into newly synthesized virus cores take place. in infected cells, the mrv μ2 protein forms punctae in the enlarged region of the filamentous vfs that are co-localized with γ-tubulin and resistant to nocodazole treatment, and permitted microtubule (mt)-extension, features common to mt-organizing centers (mtocs). using a previously established reconstituted vf model, we addressed the functions of mt-components and mtocs concerning their roles in the formation of filamentous vfs. indeed, the mtoc markers γ-tubulin and centrin were redistributed within the vf-like structures (vfls) in a μ2-dependent manner. moreover, the mt-nucleation centers significantly increased in numbers, and γ-tubulin was pulled-down in a binding assay when co-expressed with histidine-tagged-μ2 and μns. thus, μ2, by interaction with γ-tubulin, can modulate mtocs localization and function according to viral needs. here, specific µ2 punctae observed in filamentous vfs are investigated concerning their 89 ability to co-localize with other reovirus proteins and host elements. our study shows that µ2 90 punctae in vfs co-localize with γ-tubulin, are resistant to nocodazole, and permit mt 91 emergence, common features for mtocs. moreover, using the vfls model, we found that 92 specific µ2/µns ratios that support filamentous morphology relocalize γ-tubulin and centrin to 93 foci within the vfls. such association is obliterated upon mt overexpression. 94 immunofluorescence microscopy of reovirus t1l infected cells at 12 hpi, revealed µ2 97 punctae inside the filamentous vfs (fig 1a) . the punctae co-localized neither with other viral 98 proteins (µns, σns, λ2, σ3, µ1) (fig 1b-e (fig 2d and e) . co-staining for µ2 and α-tubulin, however, 100 showed bundles of mts extending from the punctae, suggesting that the punctae may have a role 101 as mtocs (fig. 2c) . indeed, co-staining for µ2 and γ-tubulin, a conventional marker for 102 centrosomes and other mtocs (roostalu and surrey, 2017) , showed µ2 and γ-tubulin co-103 localizing in the punctae as denoted by immunofluorescence photomicrograph and profile 104 intensities of the linear region of interest (lroi) (fig 2b) . importantly, nocodazole treatment, 105 which is a well-known mt-depolymerizing agent, failed to disrupt the punctae, which remained 106 positive for both µ2 and γ-tubulin (fig 3a and b) . our results show that γ-tubulin localization is 107 intensified in µ2 punctae upon nocodazole treatment (fig 3d) , consistent with the fact that 108 mtocs are nocodazole resistant (rogalski and singer, 1984) . reovirus protein µns is mainly 109 dispersed from punctae when cells are treated with nocodazole (fig 3a) , suggesting a mild or no 110 j o u r n a l p r e -p r o o f role in µ2 punctae formation. as expected, mt bundles depolymerized upon nocodazole 111 treatment (fig 3c) mainou et al., 2013) . nocodazole was then removed from the medium, allowing mts to re-117 polymerize for 0, 5, 15, 30, or 60 min before methanol-fixation (fig 4a) . immediately after 118 nocodazole removal (i.e., at 0 min), µ2-punctae were observed in the vfs while filamentous µ2 119 and mts were not. however, within only 15 min after nocodazole removal, polymerizing mts 120 with associated µ2 were observed extending from the punctae (fig 4b, c, and d) . 5 and 6 , first, fourth, and fifth rows) mainly localized in mtocs. in 134 contrast, γ-tubulin and centrin localized to defined punctae in vfls reconstituted with µ2/µns 135 transfection ratios of 2:1 and 2:2 ( figures 5 and 6, second and third rows) . to evaluate if γ-136 tubulin and centrin punctae increment in number and redistribute to vflss generated with 137 different µ2/µns ratios, we first quantified cells with more than two γ-tubulin punctae. as 138 denoted in figure 7a , a significantly higher percentage of cells containing >2 mtocs is 139 observed at 2:1 and 2:2 transfection ratios of µ2(t1l)/µns when compared to the other ratios. the tric chaperonin 395 controls reovirus replication through outer-capsid folding microtubule nucleating 397 gamma-tusc assembles structures with 13-fold microtubule-like symmetry chlamydomonas alpha-tubulin is posttranslationally 400 modified by acetylation on the epsilon-amino group of a lysine identification of an acetylation site of chlamydomonas alpha-402 tubulin a mechanism for reorientation of 405 cortical microtubule arrays driven by microtubule severing microtubule-organizing centres: a re-evaluation src kinase mediates productive endocytic sorting of 409 reovirus during cell entry 411 reovirus cell entry requires functional microtubules 413 analysis of microtubule dynamic instability using a plus-end growth marker localization of 416 mammalian orthoreovirus proteins to cytoplasmic factory-like structures via nonoverlapping 417 regions of microns reovirus sigma ns 419 protein localizes to inclusions through an association requiring the mu ns amino terminus human papillomavirus type 16 e7 422 oncoprotein associates with the centrosomal component gamma-tubulin core protein mu2 is a second determinant of nucleoside 425 triphosphatase activities by reovirus cores reovirus core protein 427 mu2 determines the filamentous morphology of viral inclusion bodies by interacting with 428 and stabilizing microtubules differing effects of herpes simplex virus 1 and 430 pseudorabies virus infections on centrosomal function microtubules containing acetylated alpha-tubulin in 432 mammalian cells in culture the hcmv assembly compartment is a dynamic golgi-derived mtoc 435 that controls nuclear rotation and virus spread cytochemical, fluorescent-antibody and electron 437 microscopic studies on the growth of reovirus (echo 10) in tissue culture associations of elements of the golgi apparatus with 440 microtubules microtubule nucleation: beyond the template a mechanistic view on the evolutionary origin for centrin-based control of 444 centriole duplication microtubule-organizing centers: from the centrosome to 446 non-centrosomal sites genome packaging of reovirus is 449 mediated by the scaffolding property of the microtubule network luminal localization of α-tubulin 452 k40 acetylation by cryo-em analysis of fab-labeled microtubules katanin disrupts the 454 microtubule lattice and increases polymer number in c. elegans meiosis an itam in a nonenveloped virus regulates activation of 458 nf-κb, induction of beta interferon, and viral spread molecular basis of viral neurotropism: 460 experimental reovirus infection the centrosome, a multitalented renaissance organelle monoclonal antibodies to reovirus 464 reveal structure/function relationships between capsid proteins and genetics of susceptibility 465 to antibody action heterologous expression of 467 antigenic peptides in bacillus subtilis biofilms the sequences of reovirus serotype 3 genome 469 segments m1 and m3 encoding the minor protein mu 2 and the major nonstructural protein 470 mu ns, respectively virus factories and mini-organelles generated 472 for virus replication nucleation of microtubule assembly 474 by a gamma-tubulin-containing ring complex reovirus mu2 protein 476 inhibits interferon signaling through a novel mechanism involving nuclear accumulation of 477 interferon regulatory factor 9 key: cord-319179-gqaxf7mz authors: denison, m.; perlman, s. title: identification of putative polymerase gene product in cells infected with murine coronavirus a59 date: 1987-04-30 journal: virology doi: 10.1016/0042-6822(87)90303-5 sha: doc_id: 319179 cord_uid: gqaxf7mz abstract the virion rna of mouse hepatitis virus, strain a59 (mhv-a59) is believed to be the mrna for the viral rna-dependent rna polymerase. the cell-free translation of virion rna results in the synthesis of two predominant products p220 and p28 (m. r. denison and s. perlman, 1986, j. virol. 60, 12–18). p28 is a basic protein and is readily detected by two-dimensional gel electrophoresis. when infected cells and isolated virions were assayed for this protein by two-dimensional gel electrophoresis, p28 could be detected in infected cells labeled at late times after infection, but not at early times or in purified virions. p28 represents the first protein product of the putative coronavirus polymerase gene to be identified in infected cells. the virion rna of mouse hepatitis virus, strain a59 (mhv-a59) is believed to be the mrna for the viral rnadependent rna polymerase. the ceil-free translation of virion rna results in the synthesis of two predominant products ~220 and p28 (m. r. denison and s. perlman, 1986,j. viral. 60, 12-l 8) . p28 is a basic protein and is readily detected by two-dimensional gel electrophoresis. when infected cells and isolated virions were assayed for this protein by twodimensional gel electrophoresis, p28 could be detected in infected cells labeled at late times after infection, but not at early times or in purified virions. p28 represents the first protein product of the putative coronavirus polymerase gene to be identified in infected cells, o 1987 academic press. hc. the genome of mouse hepatitis virus (mhv), a member of the coronavirus family, is a polyadenylated positive-strand rna (2, 3). the mhv virion does not contain its own rna-dependent rna polymerase, and the first event in the replication of this virus, after adsorption, penetration, and uncoating must be the synthesis of a polymerase. virus-specific polymerase activity has been detected at both early and late times in the infectious cycle (4, 5). the polymerase detected at early times p.i. synthesizes rna complementary to virion rna, whereas the late appearing polymerase synthesizes subgenomic and genomic rna of the same polarity as virion rna (positive-strand rna) (6). the viral rna polymerase is postulated to be a translation product of virion rna (2, 7). the study of the viral polymerase in intact cells is hampered by the small amount of polymerase present, and no proteins with polymerase activity have yet been isolated. to study the synthesis of the putative viral polymerase under more favorable conditions, we translated virion rna in cell-free lysates from rabbit reticulocytes (1). we showed that the virion rna is translated in vitro into several large peptides of molecular weight greater than 200,000, with a predominant product of 220,000 (~220) in agreement with other workers (7) and also identified a 28,000-da protein which was the n-terminal portion of the larger precursor proteins. in the presence of protease inhibitors such as leupeptin, the synthesis of ~220 and p28 was diminished, and a new protein with mol wt 250,000 became evident. as the first step in determining if such proteolytic processing occurs in ' to whom requests for reprints should be addressed. infected cells, we assayed infected cells for the presence of ~28. our results showed that this protein was present at late times p.i. and was not present in isolated virions. mhv-a59, originally obtained from dr. susan weiss, was grown in balb/c 17cl-1 cells and titered in l-2 cells. both were grown in monolayer cultures in dulbecco's minimum essential medium (dmem) with 10% fetal calf serum. when mhv virion rna is translated in a cell-free rabbit reticulocyte lysate, two major products ~220 and p28 are apparent after 60 min of incubation (i). neither product appears if translation is performed in the presence of either znclp or leupeptin, both inhibitors of serine and thiol proteases. to determine if either ~220 or p28 was present in infected tissue culture cells, we labeled cells with [35s] methionine and analyzed the products at different times after infection. mhv does not efficiently shut off host cell protein synthesis until very late times after infection, making the analysis of virus-specific proteins difficult. consequently, we analyzed infected cell lysates by two-dimensional gel electrophoresis in order to maximize our ability to detect either ~220 or ~28. in preliminary experiments, we determined that ~220 synthesized in the cell-free lysate did not enter the first dimension of two-dimensional gels whether isoelectric focusing (ief) or nonequilibrium ph gradient electrophoresis (nephge) was used, probably because of its large size. on the other hand, we found that p28 could readily be detected using nephge gels in the first dimension, but could not be detected if ief gels were used (fig. 1 ). this suggested that p28 was a basic protein and presumably did not enter the ief gel for this reason. p28 is not synthesized in the presence of leupeptin when analyzed by one-dimensional sdspolyacrylamide gel electrophoresis and, as expected, the p28 which we have identified by two-dimensional gel electrophoresis was similarly not synthesized in the presence of this inhibitor. in the next set of experiments, uninfected and infected cell lysates were analyzed for ~28 by two-dimensional gel electrophoresis. p28 was detected in l-2 cells labeled between 5 and 8 hr p.i., the time of maximal viral rna and protein synthesis, but could not be detected in either uninfected cells or infected cells labeled prior to 5 hr p.i. the results of representative experiments are shown in figs. 2 and 3 . p28 was not detected in uninfected cells (figs. 2c and 3c) or in infected cells labeled for the period l-5 hr p.i. (figs. 2a and 3a) . when p28 labeled in cell-free lysates was added to infected cell lysates labeled l-5 hr p.i. prior to electrophoresis, it was easily detected (figs. 2b and 3b), confirming its apparent absence in cells labeled at early times after infection. on the other hand, when cells were labeled 7-7.5 hr p.i., a protein migrating with the identical mobility as the cell-free product was readily detected in infected cells (figs. 2d and 3d ). the nucleocapsid protein n and the transmembrane protein el were also apparent in infected cells labeled at this time (fig. 2d) . to determine if p28 was a minor protein actually present in virions, infected 17cl-1 cells were labeled with [35s] methionine and virus was purified as described in fig. 4 . a portion of the labeled virus preparation was analyzed directly by two-dimensional gel electrophoresis whereas a second part was mixed with the [35s] methionine-labeled cell-free products prior to analysis (fig. 4) . no p28 could be detected associated with purified virions. although proteolytic processing is an important component of the replication of many viruses (8), the glycoprotein e2 is the only structural protein known to undergo cleavage in coronavirus-infected cells (2, 3). virion rna is translated in a rabbit reticulocyte cell-free system into a series of related large proteins which are processed into smaller products, including a 28,000-da basic protein (1, 7) . the presence of p28 in infected cells suggests that the same proteolytic processing occurs in infected cells as occurred in the cell-free system and that the viral polymerase is translated initially as a large precursor protein which is processed into several smaller proteins. the functions of p28 and the other translation products of virion rna are not known at present. the fact that p28 is a basic protein which is most abundant in infected cells at late times after infection suggests that it may be involved in the processing of either genomic or subgenomic rna. coronaviruses replicate by a unique mechanism in which each positive-strand rna the corresponding position where p28 would be located if it were present in these samples, molecule is joined to a leader rna by a mechanism that does not involve splicing (9, 70). p28 may be involved in some aspect of the synthesis or processing of leader rna, such as the decision as to whether genomic rna will function as mrna or previrion rna. the cell-free translation and processing of mhv virion rna resembles that of the b component of cowpea mosaic virus (cpmv) (i 1, 12). in both cases, a primary translation product with molecular weight greater than 200,000 da is observed, with rapid processing to proteins with approximate molecular weight of 170,000 and 32,000 (cpmv) or 200,000 and 28,000 (mhv). both the 170k and 32k cpmv proteins are present in infected cells, and the 32k protein, which has protease activity (13) is believed to be involved in the processing of capsid proteins. by analogy with cpmv, p28 may be a viral protease, although the precursor to the viral polymerase would be the only substrate for such an enzyme in mhv-infected cells. p28 is the first translation product of mhv virion rna to be identified in infected cells and its study should prove useful in the study of the mhv polymerase structure and function. the cells were placed in dmem lacking methionine with 2% dialyzed fetal calf serum, and 30 min later virus was concentrated from the final sucrose gradient by centrifugation for 3 hr at 30,000 rpm in the beckman sw56 rotor. the pellet was prepared for analysis by two-dimensional gel electrophoresis as described in fig. 1. (a) virion proteins. (b) mixture of [36s]methionine-labeled virion proteins and we thank dr. peter rubenstein for help with the two-dimensional 19 laemmli helpful discussions. this research was supported in part by nih biomedical research gel electrophoresis of proteins: a practical approach key: cord-319403-5qyc0wsz authors: miura, tanya a.; wang, jieru; holmes, kathryn v.; mason, robert j. title: rat coronaviruses infect rat alveolar type i epithelial cells and induce expression of cxc chemokines date: 2007-12-01 journal: virology doi: 10.1016/j.virol.2007.07.030 sha: doc_id: 319403 cord_uid: 5qyc0wsz we analyzed the ability of two rat coronavirus (rcov) strains, sialodacryoadenitis virus (sdav) and parker’s rcov (rcov-p), to infect rat alveolar type i cells and induce chemokine expression. primary rat alveolar type ii cells were transdifferentiated into the type i cell phenotype. type i cells were productively infected with sdav and rcov-p, and both live virus and uv-inactivated virus induced mrna and protein expression of three cxc chemokines: cinc-2, cinc-3, and lix, which are neutrophil chemoattractants. dual immunolabeling of type i cells for viral antigen and cxc chemokines showed that chemokines were expressed primarily by uninfected cells. virus-induced chemokine expression was reduced by the il-1 receptor antagonist, suggesting that il-1 produced by infected cells induces uninfected cells to express chemokines. primary cultures of alveolar epithelial cells are an important model for the early events in viral infection that lead to pulmonary inflammation. the large surface area of the alveolar epithelium makes it an important interface between respiratory pathogens and the host. alveolar type i cells, which make up 95% of this surface area, are terminally differentiated and function in gas exchange and fluid homeostasis (williams, 2003) . alveolar type ii cells are dividing cells that transdifferentiate in vivo into the type i cell phenotype during the repair of damaged alveolar epithelium. type ii cells also produce surfactant proteins and lipids, which keep the alveoli expanded and function in innate defense of the lung (evans et al., 1975; mason, 2006) . epithelial cells that line the respiratory tract initiate pulmonary inflammation in response to pollutants, allergens, or infectious agents (martin et al., 1997) . bronchial epithelial cells and alveolar type ii cells express and secrete proinflammatory cytokines and chemokines in response to infection with respiratory viruses including respiratory syncytial virus, influenza a virus, and the sars-associated coronavirus (sars-cov) yen et al., 2006; zhang et al., 2001) . the role of alveolar type i cells in immune responses to viral infection is largely unknown. coronaviruses are an important cause of respiratory infections of animals, humans, and birds. five coronaviruses are known to cause respiratory disease in humans. human coronaviruses (hcovs) 229e and oc43 are etiologic agents of the common cold and can cause more serious lower respiratory tract disease in elderly or immunocompromised patients (birch et al., 2005; pene et al., 2003) . infection with sars-cov was associated with an epidemic of severe acute respiratory syndrome in 2002-2003 that had a case fatality rate of ∼10% (ksiazek et al., 2003; tsui et al., 2003) . hcov-nl63 and hcov-hku1 are recently identified coronaviruses that are associated with both upper and lower respiratory tract diseases in children and adults kuypers et al., 2007; pyrc et al., 2007; van der hoek et al., 2004; woo et al., 2005) . coronaviruses also cause respiratory diseases in rats, dogs, cows, pigs, and poultry (weiss and navas-martin, 2005) . several strains of rat coronavirus (rcov) have been isolated from the lungs of rats with clinical or subclinical infections (compton et al., 1999; kojima and okaniwa, 1991; parker et al., 1970) . the two prototype strains of rcov have different tissue tropisms and disease associations in rats. sialodacryoadenitis virus (sdav) causes disease primarily in the upper respiratory tract, salivary and lacrimal glands, and eyes (bhatt et al., 1972; jacoby et al., 1975) . sdav has also been isolated from the lower respiratory tract and can cause mild interstitial pneumonia in young rats (wojcinski and percy, 1986) . in contrast, parker's rcov (rcov-p) has only been isolated from the respiratory tract and causes fatal pneumonia in suckling rats (bhatt and jacoby, 1977; parker et al., 1970) . in adult rats inoculated intranasally with rcov-p, virus replicates in the upper and lower respiratory tracts. neutrophils are observed in the nasal cavity, trachea, and alveoli on days 2-5, followed by infiltration of mononuclear cells in the lung on day 5 after infection. rcov-p infection of adult rats causes interstitial pneumonia and focal edema in the alveoli, which resolves by day 8 after infection (bhatt and jacoby, 1977) . as with rcov-p, upon intranasal inoculation of adult rats with sdav, neutrophils are the primary infiltrating cell type in the respiratory epithelium early in infection (jacoby et al., 1975; wojcinski and percy, 1986) . rcov is an important model in which to study the pathogenesis of pulmonary infection caused by a coronavirus in its natural host. here we examine the role of alveolar type i cells in the innate immune response to rat coronavirus infection. primary cultures of rat alveolar epithelial cells are a wellcharacterized model of differentiated lung epithelium, which have not been studied previously with respect to viral infection . alveolar type ii cells isolated from rat lung can be cultured in vitro to either maintain the type ii cell phenotype or transdifferentiate into the type i cell phenotype (dobbs et al., 1985 (dobbs et al., , 1988 mason et al., 2002; nishina et al., 2005) . because alveolar type i cells are difficult to isolate at a high yield and purity, transdifferentiated type ii cells are used as a model to study the characteristics and functions of type i cells in vitro (borok et al., 1998a; dobbs et al., 1985; williams, 2003) . after 3-5 days in culture on plastic in the absence of rat serum and keratinocyte growth factor (kgf), rat alveolar type ii cells change in morphology from a cuboidal to a flattened shape, and have decreased expression of surfactant proteins and lamellar bodies and increased expression of the type i cell markers, t1α, aquaporin v, and caveolin-1 (borok et al., 1998a (borok et al., , 1998b danto et al., 1995; manzer et al., 2006; mason et al., 2002) . in this study, cultures of rat alveolar type i cells were evaluated for susceptibility to rcov infection, and for expression and secretion of proinflammatory cytokines and chemokines in response to rcov inoculation. to our knowledge, this is the first study to evaluate the in vitro response of rat alveolar epithelial cells to infection with a rat virus. two strains of rcov, rcov-p and sdav, caused productive infection of alveolar type i cells. infection of type i cell cultures with these coronaviruses increased expression and secretion of cxc chemokines, cinc-2, cinc-3, and lix, which are important neutrophil chemoattractants. secretion of these chemokines was also induced by uv-inactivated viruses, showing that viral replication was not required for the induction of chemokine expression. dual immunolabeling of viral antigen and cxc chemokines showed that expression of cxc chemokines was primarily from uninfected cells in these cultures. virus-induced chemokine expression was reduced by il-1 receptor antagonist, but not soluble tnf receptor, indicating that il-1 produced by infected cells may induce chemokine expression from the uninfected type i cells. these data suggest that alveolar type i cells can secrete chemokines in response to viral infection, which may lead to infiltration of neutrophils into the lung. rat coronavirus strains rcov-p and sdav infect primary cultures of rat alveolar type i cells type i cells infected with rcov-p showed mild cytopathic effects including cell rounding and vacuolation at 24 h after inoculation ( fig. 1 ). type i cells infected with sdav had more pronounced cytopathic effects including syncytia formation and cell detachment. no cytopathic effects were observed in mockinoculated type i cell cultures. at 24 h after virus inoculation, viral nucleocapsid protein was detected in type i cells inoculated with rcov-p or sdav, but not in mock-inoculated cells. culture media from type i cells inoculated with rcov-p or sdav were collected at various times after inoculation and titrated by plaque assay. rcov-p and sdav replicated with similar kinetics in alveolar type i cells (fig. 2) . infectious virus was first detected in the supernatant medium by 12 h after inoculation, and reached a maximum titer of 10 5 -10 6 pfu/ml at 24 h. culture media from type i cells collected 6 h or 24 h after inoculation with sucrose density gradient purified rcov-p or sdav were assayed for the presence of 19 rat cytokine and chemokine proteins by protein macroarray analysis (fig. 3) . at 6 h after inoculation, both rcov-p-and sdav-inoculated type i cells showed increased secretion of the chemokines cinc-2, cinc-3 (cxcl-2/3, mip-2), fractalkine (cx 3 cl-1), gm-csf, ifn-γ, lix (cxcl-5), leptin, and mip-3α (ccl20), compared to mock-inoculated cells. by 24 h after inoculation, cinc 2, cinc-3, fractalkine, lix, mip-3α, and tnf-α were increased in cultures that were inoculated with each of these viruses. infection of type i cells with sdav increased the expression of several other cytokines, including il-1α, il-1β, il-4, il-6, and il-10, compared to mock or rcov-p inoculated cultures. because cinc-2, cinc-3, and lix are closely related cxc chemokines that are expressed by rat alveolar epithelial cells in response to lps or cytokines (jeyaseelan et al., 2005; manzer et al., 2006; nishina et al., 2005; vanderbilt et al., 2003) , we further quantified their expression and secretion in rcovinfected type i cells. the expression of cxc chemokines, cinc-2, cinc-3, and lix, was confirmed by quantitative rt-pcr and elisa (fig. 4) . infection of type i cells with rcov-p or sdav induced expression of mrna encoding cinc-2α, cinc-3, and lix at 6 h and 24 h after inoculation (fig. 4a ). infection with rcov-p or sdav also induced secretion of cinc-2, cinc-3, and lix proteins by 6 h after inoculation (fig. 4b) . secretion of the cxc proteins increased further at 24 h and 48 h after inoculation. the elisa for cinc-2 did not differentiate between cinc-2α and cinc-2β, whereas the probe used in rt-pcr was specific for cinc-2α. rat alveolar type i cells do not express cinc-2β constitutively or in response to cytokines or ozone (nishina et al., 2005; wang et al., 2006) . to determine whether viral replication was required to induce cxc chemokine expression, we inactivated the infectivity of rcov-p and sdav by uv irradiation. inactivation of virus infectivity was confirmed by plaque assay of the uvtreated viral inoculum or medium from type i cells 24 h after exposure to uv-treated virus, in comparison to untreated virus. the titers of uv-treated rcov-p and sdav were below the limit of detection of our plaque assay (11.1 pfu/ml). alveolar type i cells exposed to uv-treated virus for 24 h did not express viral nucleocapsid protein ( fig. 1 ), but nevertheless induced secretion of cinc-2, cinc-3, and lix compared to mockinoculated type i cells (fig. 5 ). there was no difference in the amount of cxc chemokines secreted from type i cells treated with uv-inactivated virus compared to cells treated with infectious virus (p b 0.05). to determine whether the cxc chemokine response to infectious or uv-inactivated rcov-p or sdav was specific for the rat coronaviruses, we inoculated rat alveolar type i cells with a related group 2a coronavirus, mouse hepatitis virus (mhv) strain a59 that had been purified by sucrose density gradient centrifugation. no viral antigen was detected by immunolabeling at 24 h after inoculation of type i cells with mhv-a59, and the expression of cxc chemokines cinc-2, cinc-3, and lix was not increased compared to mock-inoculated cells (data not shown). to determine whether the increase in cxc chemokine expression in alveolar type i cell cultures was limited to virusinfected cells, at 16 h after virus inoculation type i cell cultures were treated with brefeldin a for 6 h before dual immunolabeling of viral nucleocapsid protein and cxc chemokines (fig. 6 ). brefeldin a inhibits export of secreted proteins from the distal golgi to the cell surface (miller et al., 1992) . as expected from the elisa data, cultures of type i cells that had been inoculated with rcov-p or sdav had increased immunolabeling with cinc-2, cinc-3, and lix antibodies compared to mock inoculated cultures. cinc-2 and lix were much more brightly labeled than cinc-3 (data not shown), corresponding to the concentrations of protein detected by elisa (fig. 4b) . the majority of cells that were positive for cxc chemokine expression did not express viral nucleocapsid protein. therefore, the elisa data showing increased chemokine secretion in rcov-infected type i cell cultures is mostly from uninfected cells. these data suggest that rcovs induce cxc chemokine expression in type i cell cultures through an indirect mechanism. signaling through the il-1 receptor, but not the tnf receptor, promotes sdav-induced cxc chemokine expression in rat alveolar type i cells to test the hypothesis that a paracrine factor secreted from infected cells induces cxc chemokine expression in uninfected cells in the culture, we analyzed the ability of antagonists for the il-1 and tnf receptors to block the cxc chemokine response. the il-1 receptor antagonist (il-1ra) binds strongly to the il-1 type i receptor, thereby preventing signal transduction by il-1α and il-1β (dinarello, 1996) . the tnf soluble receptor protein (tnfsrp) binds to both soluble and membrane-bound forms of tnf-α, thereby inhibiting their activity (dinarello, 2005) . treatment of sdav-inoculated cells with il-1ra decreased the level of cinc-2, cinc-3, and lix secretion when it was added to type i cells prior to inoculation and maintained in the medium throughout infection with sdav (fig. 7) . the presence of tnfsrp had no effect on the chemokine secretion during sdav infection of type i cells. when both il-1ra and tnfsrp were present during sdav infection, the levels of cinc-2, cinc-3, and lix expression were the same as levels from cells that were treated with il-1ra alone. these data suggest that il-1 that is secreted from infected alveolar type i cells induces expression of cxc chemokines in uninfected cells in the culture. although type i cells make up 95% of the surface area of the alveolar epithelium, their role in the pathogenesis of respiratory virus infections and in innate immunity is largely unknown. in this study, we evaluated the ability of rat coronaviruses to infect rat alveolar type i cells in vitro. despite differences in tissue tropism and disease association in vivo, two strains of rcov were able to infect primary alveolar epithelial cells in vitro. we showed that infection of alveolar type i cells with either rcov-p or sdav induced expression of chemokines, cinc-2, cinc-3, lix, mip-3α, and fractalkine at both 6 h and 24 h after inoculation. our protein array analysis also identified cytokines that are increased in sdav-infected, but not rcov-p-infected, alveolar type i cell cultures. this differential cytokine response may play a role in the different pathogenesis of rcov-p and sdav infections in vivo. fractalkine, the sole member of the cx 3 c chemokine family, is predominately a membrane-bound chemokine and functions in the recruitment and adhesion of cytotoxic lymphocytes to endothelial cell surfaces (stievano et al., 2004) . mip-3α, which is chemotactic for dendritic cells, b cells, and memory t cells, is expressed by human bronchial epithelial cells in response to inflammatory mediators il-1β and tnf-α (starner et al., 2003) . previous studies showed that alveolar type ii cells express cxc chemokines both constitutively and in response to treatment with cytokines, lps, or acid and infection with pseudomonas aeruginosa ( 2005; mason, 2006; nishina et al., 2005; vanderbilt et al., 2003) . our study contributes to the growing body of data that supports a role for alveolar type i cells in chemokine expression wang et al., 2006) . cxc chemokines are important mediators of neutrophil chemotaxis and immunopathology associated with respiratory virus infections (londhe et al., 2005; tumpey et al., 2005; turner, 1990; yen et al., 2006; zhang et al., 2001) . therefore, we further quantified the expression of cxc family members cinc-2, cinc-3, and lix made by rat alveolar type i cells in response to rcov infection by quantitative rt-pcr and elisa. levels of cinc-2, cinc-3, and lix mrnas and secreted proteins were increased in rcovinfected type i cell cultures. cinc-2, cinc-3, and lix are closely related cxc-elr + chemokines that are similar to il-8/ cxcl8 in humans (shibata et al., 1995; smith et al., 2002) . cinc-2, cinc-3, and lix specifically bind and signal through cxcr2, a seven transmembrane domain receptor expressed on rat type ii alveolar cells, endothelial cells, and neutrophils (chandrasekar et al., 2003; dunstan et al., 1996; shibata et al., 2000; vanderbilt et al., 2003) . neutrophil chemotaxis and activation are the primary functions of cxcr2 signaling. however, cxcr2 also mediates cellular proliferation and morphogenesis, angiogenesis, and wound healing (devalaraja et al., 2000) . cxc chemokines secreted from infected alveolar epithelial cells could potentially signal through cxcr2 on type ii pneumocytes to stimulate type ii cell proliferation and transdifferentiation into the type i cell phenotype, resulting in repair of damaged epithelium. expression of cxcr2 and secretion of cxc chemokines are upregulated in alveolar type ii cells by lung injury (vanderbilt et al., 2003) . in addition, cinc-1 signaling through cxcr2 on type ii cells promotes alveolar development in newborn rats that are exposed to hyperoxic conditions (auten et al., 2001) . cxc chemokine signaling through cxcr2 on alveolar type ii cells and endothelial cells may also promote retraction of these cells, which would promote increased migration of neutrophils and other inflammatory cells into the alveoli (schraufstatter et al., 2001) . although cinc-2, cinc-3, and lix signal through the same receptor, they may have some distinct functions during viral infection. for example, although cinc-2 and cinc-3 have similar neutrophil chemotactic activity, cinc-3 induces greater calcium mobilization than cinc-2, resulting in increased neutrophil activation (shibata et al., 2000) . further studies are needed to elucidate the roles of cinc-2, cinc-3, and lix in the innate immune response to rcov infection in vivo. the production of cxc chemokines by alveolar epithelial cells during viral infection is probably responsible for the recruitment of neutrophils to the infected alveoli, an early event in a variety of respiratory viral infections. respiratory viruses, including influenza a virus, rhinovirus, and respiratory syncytial virus (rsv), induce expression of cxc chemokines, which results in an influx of neutrophils into the respiratory tract (abu-harb et al., 1999; tumpey et al., 2005; turner, 1990; wang et al., 1998; zhang et al., 2001; zhu et al., 1997) . infiltration of neutrophils into lung and other tissues occurs soon after infection with coronaviruses including rat coronaviruses, mouse hepatitis virus strains 1 and jhm, and sars-cov, and has been implicated in both immunopathology and virus clearance (bhatt and jacoby, 1977; de albuquerque et al., 2006; haagmans et al., 2004; iacono et al., 2006; tsui et al., 2003; yen et al., 2006; zhou et al., 2003) . after intranasal inoculation of adult rats with rcov-p, neutrophils are found in the upper respiratory tract on day 2 and in the lung on day 5, corresponding to the presence of viral antigen and histological lesions (bhatt and jacoby, 1977) . neutrophils are also the earliest infiltrating cell type in the respiratory epithelium after intranasal inoculation with sdav (jacoby et al., 1975; wojcinski and percy, 1986) . the role of neutrophils in rcov clearance and immunopathology is not yet known. primary cultures of alveolar epithelial cells are an important model in fig. 5 . uv-inactivated viruses induce secretion of chemokines from alveolar type i cells. type i cells were inoculated with rcov-p or sdav, mockinoculated, or treated with uv-inactivated rcov-p or sdav (see fig. 1 ). media were harvested 24 h after treatment and chemokine secretion was analyzed by elisa. asterisks ( * ) indicate significant difference from mock-inoculated cells, p b 0.05. data are average values of duplicate samples from 3 experiments. which to study the early events in viral infection that lead to pulmonary inflammation. like infection with rcov-p or sdav, other uv-inactivated viruses also induce expression of cxc chemokines. uv-inactivated murine coronavirus, mhv-4 variant v5a13.1, induces expression of cxc and cc chemokine genes in primary mouse astrocyte cultures and in the cns of infected mice during acute and chronic phases of cns disease (lane et al., 1998) . uvinactivated rsvor soluble viral glycoproteins (f and g proteins) induce expression of il-8 in a549 cells (tirado et al., 2005) . our data show that a component of purified virions that was not destroyed by uv light was responsible for inducing chemokine expression. the viral hemagglutinin esterase glycoprotein (he) was not required for the induction of cxc chemokines because sdav virions do not contain he (gagneten et al., 1996; miura and holmes, unpublished observation) . perhaps interaction of the viral spike glycoprotein with as yet unknown cellular receptor(s) triggers chemokine expression, as sars-cov spike protein does in a549 cells (chang et al., 2004) . alternatively, other viral structural proteins may induce expression of chemokines (law et al., 2007; tirado et al., 2005) . for several rna viruses, dsrna replicative intermediates induce chemokine expression (hewson et al., 2005; imaizumi et al., 2004; londhe et al., 2005) . however, this is unlikely to be the case for rcov because viral replication was not required for chemokine expression. viral infection can induce chemokine expression either by a direct or indirect mechanism (melchjorsen et al., 2003) . in our rcov-infected type i cell cultures, dual immunolabeling of viral antigen and cxc chemokines demonstrated that the cxc fig. 6 . virus infection induces expression of chemokines from uninfected type i cells in the culture. type i cells were treated with brefeldin a 16 h after inoculation with sdav and fixed 6 h later. fixed cells were immunolabeled for expression of viral nucleocapsid protein (red) and cinc-2 or lix (green). nuclei were stained blue with dapi. representative fields from duplicate experiments are shown. fig. 7 . there is decreased expression of cinc-2, cinc-3, and lix in sdav-infected type i cells that have been treated with il-1ra. type i cells were treated with il-1ra, tnfsrp, or both proteins and inoculated with sdav. after 1 h, the inoculum was removed, cells were rinsed and refed with medium with or without the indicated receptor antagonists. after 18 h the media were analyzed for cxc chemokines by elisa. asterisks (*) indicate a statistically significant difference between sdavinfected cells without receptor antagonist treatment and sdav-infected cells with receptor antagonist treatment (p b 0.05). data are representative of duplicate experiments with triplicate samples. chemokines were expressed principally in cells that did not express viral nucleocapsid protein. this suggests that rcov infection of alveolar type i cells can indirectly induce expression of chemokines in uninfected cells. infected cells may release a soluble factor such as il-1 or tnf-α, which could stimulate nearby cells to express and secrete cxc chemokines nishina et al., 2005) . to test this hypothesis, we inoculated type i cells with sdav in the presence of antagonists for the il-1 and/or tnf receptors, il-1ra and tnfsrp, respectively. treatment with il-1ra, but not tnfsrp, decreased cxc chemokine secretion from sdavinfected cultures (fig. 7) . thus, signaling through the il-1 receptor contributes to the induction of cxc chemokine expression in sdav-infected type i cell cultures. il-1ra blocks signaling of both il-1α and il-1β through the il-1 receptor (dinarello, 1996) . il-1 is important in the induction of cxc chemokines in lung cells in vitro and in vivo. both il-1α and il-1β induce expression and secretion of cxc chemokines in primary cultures of rat alveolar type i and type ii cells nishina et al., 2005; unpublished observations). furthermore, il-1 receptor knockout mice have a decreased neutrophil response to influenza infection, likely due to decreased il-1-dependent expression of cxc chemokines (schmitz et al., 2005) . a potential limitation of this study is that we isolated type ii cells and transdifferentiated them into a type i-like cell phenotype in vitro wang et al., 2006) . while transdifferentiated type i cells express t1α and many other type i cell markers, are morphologically like type i cells, and secrete mip-2 and mcp-1 in response to lps and il-1, they may not express all of the genes expressed in freshly isolated type i cells (gonzalez et al., 2005; manzer et al., 2006; wang et al., 2006) . although we think that it is unlikely, it is possible that type i cells in vivo will respond differently to viral infection. animal models to evaluate the mechanisms of viral induction of chemokines in the lung are valuable in studying viral pathogenesis and designing treatments for viral diseases. the expression of chemokines in sars patients has been correlated with increased disease severity (tang et al., 2005) . sars pathogenesis has been studied in rodent models: aged mice, sars-cov adapted to cause disease in rodents, or transgenic mice that express the sars receptor, human angiotensin converting enzyme 2 (mccray et al., 2007; nagata et al., 2007; roberts et al., 2007 roberts et al., , 2005 tseng et al., 2007) . we are studying rat coronaviruses as a model for respiratory coronavirus infection in the natural host of the virus. the study of other rodent viruses in their natural hosts has advanced the understanding of other respiratory viral pathogens, such as sendai virus and pneumonia virus of mice as models for human parainfluenza and rsv respectively (faisca and desmecht, 2007; rosenberg et al., 2005) . recently the mhv-1 strain of murine coronavirus was shown to induce severe respiratory disease in mice and was proposed as a model for sars-cov (de albuquerque et al., 2006) . primary cultures of rat alveolar epithelial cells will be a valuable model for dissecting the viruscell interactions and innate immune responses that are important in the critical early steps in pulmonary infection of a virus in its natural host. isolation and culture of alveolar epithelial cells alveolar type ii cells were isolated from 6-8-week-old, specific pathogen-free male sprague-dawley rats (harlan sprague dawley, indianapolis, in) as previously described (dobbs and mason, 1979; manzer et al., 2006) . briefly, type ii cells were dissociated from rat lung with porcine pancreatic elastase (roche molecular biochemicals, indianapolis, in) and were partially purified on discontinuous optiprep (axis-shield poc as, oslo, norway) density gradients. cells were plated on plastic tissue culture dishes in dmem supplemented with 5% fbs, 2 mm glutamine, 2.5 ug/ml amphotericin b, 100 ug/ml streptomycin, and 100 ug/ml penicillin g, and incubated at 37°c and 10% co 2 . culture of type ii cells on plastic in dmem containing 5% fbs for 3-5 days results in trans-differentiation into the type i cell phenotype, characterized by loss of expression of surfactant proteins, gain of expression of t1α and aquaporin v, and changes in morphology and lectin-binding specificities (borok et al., 1998a (borok et al., , 1998b danto et al., 1995; dobbs et al., 1985; manzer et al., 2006; wang et al., 2006) . rcov-p and sdav were obtained from dr. d. percy (university of guelph, guelph, ontario) (percy et al., 1991) . viruses were propagated in a subclone of murine l929 cells, l2p-41.a (gagneten et al., 1996) , purified by sucrose density gradient ultracentrifugation as previously described, and stored in tms buffer (25 mm tris, 25 mm maleic acid, 100 mm nacl, ph 6.5) with 5% glycerol (sturman et al., 1980) . viral titers were determined by plaque assay on l2p-41.a cells (described below). purified virus was diluted in dmem to achieve equivalent doses of rcov-p and sdav for inoculation. for experiments with uv-inactivated viruses, the diluted viruses were exposed on ice to uv irradiation in a stratalinker uv crosslinker (stratagene, la jolla, ca) at a cumulative dose of 120 mj/cm 2 . inactivation of virus was demonstrated by plaque assay of the uv-irradiated virus and medium from type i cells 24 h after inoculation with the uv-irradiated virus, as well as by immunofluorescent detection of viral antigens in type i cells 24 h after inoculation with the uv-irradiated virus, using a cross-reactive mab to the mouse hepatitis virus nucleocapsid protein (described below). these analyses confirmed that the infectivity of the rcovs was completely eliminated by this uv treatment. uv-irradiated viral inocula were shown to be free of bacterial contamination by inoculation of blood agar and incubation at 37°c. primary alveolar epithelial cells were cultured as described above for 5 days to obtain a type i cell phenotype, then inoculated with rcov-p, sdav, uv-inactivated rcov-p or sdav, or mock inoculated. after a 1 h adsorption period, the cells were washed twice with dmem, refed with dmem/5% fbs and antibiotics, and incubated at 37°c and 10% co 2 . for mock inoculation of alveolar type i cells, tms buffer with 5% glycerol was diluted in dmem at the same ratio as the virus inoculum. twenty-four hours after inoculation, type i cells on coverslips were fixed in methanol/acetic acid (3:1). viral nucleocapsid antigen was detected using a monoclonal antibody to mouse hepatitis virus nucleocapsid protein (kindly provided by dr. j. leibowitz, texas a&m university, college station, tx) that cross-reacts with rcov nucleocapsid protein, followed by a fitc-conjugated goat anti-mouse secondary antibody (jackson immunoresearch laboratories, inc., west grove, pa). at intervals after inoculation, media from inoculated cultures were collected for viral titration, cytokine protein arrays and elisas. total cellular rna was extracted for quantification of chemokine mrna as described below. for experiments with receptor antagonists, il-1ra (10 ug/ml), tnfsrp (10 ug/ml), neither antagonist, or both il-1ra and tnfsrp were added to type i cells 30 min prior to inoculation with sdav. the concentration of antagonist was maintained in the culture throughout the 18 h infection. il-1ra and tnfsrp were kindly provided by dr. charles dinarello (university of colorado health sciences center, denver, co). stocks of purified virus or medium from virus infected cultures was serially diluted in dmem without fbs and used to inoculate triplicate wells of near confluent l2p-41.a cells. after a 1 h adsorption period at 37°c, the inoculum was removed and the cells were overlaid with mem, 4% fbs, antibiotics, and 0.5% seakem le agarose (cambrex, rockland, me). plaques were stained after 72 h incubation at 37°c, with the agarose overlay medium containing 6% neutral red (sigma-aldrich). type i cells were inoculated with rcov-p or sdav, or were mock-inoculated as described above. media from type i cells were collected 24 h after inoculation, centrifuged to remove cellular debris, and incubated with membranes spotted with antibodies specific for 19 rat cytokines and chemokines (raybiotech, inc., norcross, ga). the membranes were processed according to the manufacturer's recommendations. the membranes were exposed to x-ray film and the film was scanned for densitometry analysis using kodak molecular imaging software (eastman kodak company, rochester, ny). densitometry data were normalized to internal positive controls on each membrane and graphed as relative units. total rna was isolated from alveolar epithelial cells using trizol reagent according to the manufacturer's specifications (invitrogen corp., carlsbad, ca). reverse transcription and taqman pcr (applied bioscience) were performed on an abi prism 7700 sequence detection system as previously described , using published primer pairs and fluorogenic probes for cinc-2α , cinc-3 (nishina et al., 2005) , and lix (jeyaseelan et al., 2005) . the mrna expression levels of cinc-2, cinc-3, and lix were normalized to the level of gapdh mrna for each sample, using a commercially available rat gapdh kit (applied bioscience). media from rcov-inoculated or mock-inoculated alveolar type i cells were harvested 6, 24, and 48 h after inoculation for the measurement of cinc-2, cinc-3, and lix by elisa. elisa assays were developed by elisatech (aurora, co) using standards and antibodies from r&d systems (minneapolis, mn). the cinc-2β antibody (r&d systems) used in the elisa shows significant cross-reactivity to cinc-2α, hence the product of this elisa is referred to as cinc-2. chemokine levels were quantified by comparison of values with that of a standard curve for each chemokine. the reported values are averages ± standard error of the mean of at least three independent experiments with two replicate wells per experiment. the chemokine concentrations were compared between samples using one-way anova. p values were computed using the newman-keuls multiple comparison test. type i cells were inoculated with rcov-p or sdav, or were mock-inoculated. after 18 h, a protein transport inhibitor that contained brefeldin a (bd golgiplug, bd biosciences) was added to the culture medium as recommended by the manufacturer. six hours later, the cells were fixed with 2% paraformaldehyde and permeabilized with 0.2% triton x-100 (sigma aldrich). viral nucleocapsid protein was detected using a monoclonal antibody to mhv nucleocapsid (described above) followed by an alexafluor 594-conjugated anti-mouse secondary antibody (invitrogen). cxc chemokines were detected with goat antibodies (r&d systems) to rat lix (af543), cinc-3 (af525), or cinc-2α (af516) followed by an alexafluor 488-conjugated anti-goat secondary antibody (invitrogen). il-8 and neutrophil elastase levels in the respiratory tract of infants with rsv bronchiolitis anti-neutrophil chemokine 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vitro: role of nf-kappab this work was support by nih grants ai-059576 and hl-029891. the authors would like to thank julian leibowitz, dean percy, and charles dinarello for reagents; rizwan manzer, dennis voelker, emily travanty, and samuel dominguez for helpful discussions; and karen edeen, anna castano, and christian pontillo for expert technical support. key: cord-322062-nnefbeo6 authors: tam, albert w.; smith, matthew m.; guerra, martha e.; huang, chiao-chain; bradley, daniel w.; fry, kirk e.; reyes, gregory r. title: hepatitis e virus (hev): molecular cloning and sequencing of the full-length viral genome date: 1991-11-30 journal: virology doi: 10.1016/0042-6822(91)90760-9 sha: doc_id: 322062 cord_uid: nnefbeo6 abstract we have recently described the cloning of a portion of the hepatitis e virus (hev) and confirmed its etiologic association with enterically transmitted (waterborne, epidemic) non-a, non-b hepatitis. the virus consists of a single-stranded, positive-sense rna genome of approximately 7.5 kb, with a polyadenylated 3' end. we now report on the cloning and nucleotide sequencing of an overlapping, contiguous set of cdna clones representing the entire genome of the hev burma strain [hev(b)]. the largest open reading frame extends approximately 5 kb from the fend and contains the rna-directed rna polymerase and nucleoside triphosphate binding motifs. the second major open reading frame (orf2) begins 37 by downstream of the first and extends approximately 2 kb to the termination codon present 65 by from the 3' terminal stretch of poly(a) residues. orf2 contains a consensus signal peptide sequence at its amino terminus and a capsid-like region with a high content of basic amino acids similar to that seen with other virus capsid proteins. a third open reading frame partially overlaps the first and second and encompasses only 369 bp. in addition to the 7.5-kb full-length genomic transcript, two subgenomic polyadenylated messages of approximately 3.7 and 2.0 kb were detected in infected liver using a probe from the 3' third of the genome. the genomic organization of the virus is consistent with the fend encoding nonstructural and the 3' end encoding the viral structural gene(s). the expression strategy of the virus involves the use of three different open reading frames and at least three different transcripts. hev was previously determined to be a nonenveloped particle with a diameter of 27–34 nm. these findings on the genetic organization and expression strategy of hev suggest that it is the prototype human pathogen for a new class of rna virus or perhaps a separate genus within the caliciviridae family viral hepatitis results from infection with one of at least four very different viral agents. available serological tests allow the diagnosis of acute hepatitis due to infection with hepatitis a virus (hav) and hepatitis b virus (hbv). hbv is required for propagation of the delta agent, or hepatitis d virus (hdv); this co-infection results in a high proportion of cases progressing to chronic active hepatitis. the clinical and diagnostic exclusion of hav and hbv led to the recognition of other viral hepatitides that were formerly grouped together as non-a, non-b hepatitis (nanbh) (prince et a/., 1974; feinstone et a/., 1975; tabor, 1985) . nanbh is caused by more than one viral agent and can be transmitted by either parenteral or fecal/oral routes (bradley, 1990a; reyes and baroudy, 1991) . the cloning of a blood-borne agent, termed hepatitis c virus (hcv) by us and others led to the development of a specific assay for circulating antibody to hcv (choo etal., 1989; kuo et al., 1989; kubo eta/, 1989; maeno et al., 1990; reyes et a/., 1991 d) . this assay predomi-nantly detects infections at the chronic stage, but has facilitated the identification of hcv as the cause of up to 90% of parenterally transmitted nanbh. a second epidemiologically distinct form of nanbh was shown to occur in both epidemic and sporadic patterns in developing countries and is referred to as enterically transmitted non-a, non-b hepatitis (et-nanbh) due to its water-borne mode of virus transmission and presumed enteric route of infection (khuroo, 1980; wong et a/., 1980) . et-nanbh has been documented in india, pakistan, burma, ussr, costa rica, mexico, and countries in africa, where epidemic outbreaks can generally be traced to fecal contamination of drinking water (bradley and maynard, 1986; bradley, 1990b) . the causative viral agent was previously shown to passage successfully in cynomolgus macaques (cyno) and tamarins with typical liver enzyme elevations and recovery of morphologically similar 27-to 34-nm viruslike particles from the feces of clinical specimens and experimental animals (balayan et al,, 1983; anjaparidze et al., 1986; bradley et al., 1987; arankalle et al., 1988) . we recently reported the isolation of a partial cdna clone from the virus responsible for et-nanbh, and have termed the newly identified agent the hepatitis e virus (hev) (reyes et a/., 1990) . the clone was from a burma isolate of hev and hybridized with cdna made from five other distinct geographic isolates. these molecular epidemiological findings are consistent with the available serologic data based on the use of immune electron microscopy and immunofluorescence blocking studies that indicate a single major agent is responsible for the majority of et-nanbh seen worldwide (purcell and ticehurst, 1988; bradley et al., 1988a; krawczynski and bradley, 1989) . we now report on the molecular cloning and sequencing of the complete hev (burma; b) viral genome together with the deduced amino acid sequences of viral-encoded proteins general perspectives on the genetic organization of the virus, as deduced from sequence and open reading frame analyses, indicate that hev bears some similarity to the caliciviridae but may represent a new class of nonenveloped rna virus. rna purification. total cellular rna was isolated from normal and hev(b)-infected cyno livers by the guanidinium-lici precipitation method (cathala et al., 1983) and pofy(a)+ rna was selected by one round of oligo(dt) cellulose chromatography (aviv and leder, 1972) . cdna library construction and screening. synthesis and screening of the infectious bile cdna library had previously been described (reyes et al., 1990) . oligo(dt)-, random hexamer-, and hev sequence-specific oligomer-primed (primer a, see fig. 1 ) cdna were synthesized using a commercially available cdna synthesis kit (boehringer-mannheim biochemicals, indianapolis, in), ligated to ecorl linker-adapters and cloned into x gtl0 (stratagene, san diego, ca). g-tailed cdna was made essentially as described before (tam et al., 1989) . briefly, first strand cdna primed with hev sequence-specific primer c (see fig. 1 ) was tailed with dgtp using terminal deoxynucleotidyl transferase. the modified cdna was then amplified in a polymerase chain reaction (pcr) (saiki et al., 1985; mullis and faloona, 1987) employing the same synthetic hev primer and an oligo(dc) primer, both of which contained an ecorl cloning site at the 5' end. all four cdna libraries were screened with appropriate synthetic oligomer probes (applied biosystems, foster city, ca) described under results. hybridizations were generally performed in duplicate (using 32p kinased probes) at 42" in 30% formamide, 5x ssc, 5x denhardt's (0.1% ficoll, 0.1% polyvinylpyrrolidone, 0.1% bsa), 50 mm sodium phosphate, ph 7.0, and 50 @g/ml salmon sperm dna. after an overnight hybridization, filters were washed three times with 0.2x ssc and 0.1% sds at 37-42" depending on the length of the oligomer probe. primer extension analysis. primer extension studies were carried out using oligonucleotide primers kinased to a specific activity greater than 3 x 10' cprn/pg with [t-~~p]atp (icn radiochemicals, irvine, ca) essentially as described (mcknight et a/., 1981) . extension products were separated on a 6% pofyacrylamide-8 m urea sequencing gel that was subsequentty dried and autoradiographed. the sequences forthe hev primers used in these studies are: primer a: 5'-cccgataagcagcctcaagcctc-3' primer b: 5'-ccgcgtacacactaaccccccggc-caataat-tcacgctgg-3' primer c: 5'-caagctggcgaggttgcattagg-3' primer d: 5'-acagcaticgccagggcagagtt-3' northern blot analysis. four micrograms of hev(b)infected cyno liver poly(a)+ rna was electrophoresed on a 1.2% agarose gel containing 2.2 m formaldehyde and transferred onto a nitrocellulose filter. the filter was hybridized under high stringency conditions with a radiolabeled betg-1 ecorl fragment insert (5 x lo* wmhg). dna nucleotide sequencing. dna sequencing was performed by the dideoxynucleotide method @anger et a/., 1977) using 7-deaza-dgtp (pharmacia, piscataway, nj). all sequencing reactions were carried out on both strands using bluescript plasmid (stratagene, san diego, ca) subclones obtained from hev xgtlo phage clones. appropriate overlapping subfragments were exploited wherever possible, or adjoining dissimilarend subclones were employed for unambiguous orientation. sequencing primers were commercially available or synthesized based on derived hev sequences. 7-deaza-dgtp eliminated areas of compression due to the high g + c content of the viral genome (see results) . computer analyses of nucleotide and amino acid sequences. computer programs for manipulation of nucleic acid and protein sequences were obtained from lntelligenetics (mountain view, ca). a partial hev cdna clone, et1 .l, was isolated by differential screening of a cdna library constructed from infectious bile collected from a third-passage cyno inoculated with subpassaged fecal suspensions originally derived from burma patients with weff-defined et-nanbh (reyes et al., 1990) . bife was chosen as the rna source for cdna synthesis because it contained relatively large numbers of virus particles when hev cdna clones were identified from libraries made from randomly primed cyno bile (solid square), or from cyno liver after priming by oligo-dt (solid circle), random sequence hexamers (open circle) and hev-sequence specific oligonucleotides (open square). the designations given to the various clones are indicated together with their sizes and relative position and overlap along the -7.5 kb genome. a and b represent synthetic oligonucleotides used for the generation and screening, respectively, of specifically primed cdna libraries, the anchor pcr strategy using g-tailing and pcr (primer c) was used in the synthesis of primer extension libraries for the extreme 5' end. the procedure yielded numerous clones by hybridization with primer d, of which bet-expcr2 is a representative example. primer extension studies confirmed the 5' extent of the viral genome (see fig. 2 ). the bet1 clone contained a long stretch of poly(a) residues at its 3' end indicating its position at the 3' terminus of the viral genome. compared with fecal preparations. it was also expected that the lower sequence complexity would enhance the sensitivity of the differential (plus/minus) screening protocol used for clone identification. et1 .l contained a 1.3-kb ecorl fragment that was exogenous to both human and cyno genomic dna and specifically hybridized to cdna derived only from infected sources (reyes et al., 1990) . oligonucleotides based on the end sequences of et1 .1 were used as hybridization probes to rescreen the original bile-derived cdna library. the largest identified clone, betg-1, contained a 2.6-kb ecorl insert. restriction mapping revealed that the original et1 .l clone was contained within the larger betg-1 ( fig. 1; fry et a/., 1991) . the same end-probe strategy was used with oligonucleotides derived from betg-1 to screen oligo(dt)primed and random hexamer-primed hev(b)-infected cyno liver cdna libraries. a collection of overlapping clones was identified from both libraries (fig. 1) . one of the oligo(dt)-primed clones, bet1 contained two ecorl fragments that comprised 2.4 kb in total length. the authenticity of the ecorl site was strengthened by its presence in another clone, bet4, isolated from the random-primed cdna library. a long poly(a) stretch of -150-200 adenosine residues was located at the 3' end of bet1 confirming the original observation that genomic rna could be selected on oligo-dt cellulose . this result indicated that the 3' end of the viral genome was present in the bet1 clone. the 5' end of the viral genome was isolated from a cdna library made by primer extension using a synthetic 23-bp oligonucleotide complementary to the 5' end of clone bet8 (primer a, see fig. 1 ). one of two positive clones identified by an oligonucleotide probe (primer b), located 5'to the specific primer, was clone bet-spl , this clone contained a single large insert of 2.6 kb. with the acquisition of bet-spl, the composite cdna map (omitting overlaps) spanned approximately 7.4 kb from the 5' end of bet-spl to the polyadenylated 3'end of clone bet1 ; in good agreement with the maximum length of hev rna as detected on northern blots. the 5' end of bet-spl was therefore believed to be in close proximity to the putative 5' end of the viral genome. primer extension studies using poly(a)-selected rna from infected cyno liver were performed in order to firmly establish the distance from the existing 5'end of bet-spl to the end of the genome (fig. 2) . two specific oligonucleotide primers (primers c and d, see fig. 1 ) were synthesized 143 and 72 bp from the 5'end of bet-spl and used to prime cdna synthesis after 32p labeling their 5' ends with polynucleotide kinase. the resulting extension products for each synthesis reaction were, respectively, 50 and 51 bp longer than the expected product, thereby suggesting that the 5' end of bet-spl was about 50 nucleotides from the 5' end of the virus (fig. 2) . after several failed attempts at cloning the remaining 5' end sequences by oligonucleotide hybridization of specifically primed cdna libraries, an alternative expansion/enrichment procedure of pcr amplification of specifically primed g-tailed cdna was applied (tam et al., 1989 ). an atiquot of the amplified material was fcorl digested, electrophoresed, blotted, and probed with a 5' internal hev oligomer (primer d). this hybridization study confirmed the amplification of the &aired hev extension products (data not shown). this same dna, after preparative gel etectrophoresis, was recovered and ligated into xgtl 0. the specific priming procedure (followed by pcr amplification) resulted in a high percentage (over 10%) of hev-positive recombinants in the enriched library. bet-expcr2 is a representative clone from over 50 analyzed; all of these were 50 bp in length and therefore in agreement with the primer extension experiment. the isolation of bet-expcr2 completed the hev genomic cdna cloning. the entire nucleotide and deduced amino acid sequence of hev are presented in fig. 3 . the nucleotide composition of the hev genomic rna is 17% a, 32% c, 26% g, and 25% u, conferring an overall g -i-c content of 58%. sequence homology to any nucleotide sequences contained in the genbank database could not be detected when the hev sequence was searched in either the forward or reverse orientation. only two regions were identified that had homology with previously described nonstructural gene elements present in other positive strand rna viruses (see below; . using the cod rny sequence analysis program, the -7.2-kb of hev sequence, exclusive of the 3' poly(a) tract, was analyzed for the presence of open reading frames (orf) in the six possible translation frames (fig. 4) . the identification of the rna-dependent rna polymerase in the original et1 .l clone (reyes et al., 1990) and strand-specific probe hybridization (reyes et al., 199 1 b) established the positive-sense orientation of the hev genome. a representation of the potential orfs and stop codons in the three positive-polarity frames is presented in fig. 4a . two large potential orfs were found in the first and second reading frames. orfl begins at the 5' end of the viral genome after 27 bp of apparent noncoding sequence at the 5' end, and then extends 5079 bp before termination at nucleotide position 5107. the second major orf (orf2) begins at nucleotide position 5147 and extends 1980 bp before terminating 65 bp upstream of the poly(a) tail. the termination of orfl and the transition into orf2 was confirmed by sequencing the region in question five times using two different hev sequencespecific primers. the sequence of a second clone in this region yielded the same results. furthermore, cdna clones isolated directly from infected human (huang et al., 1991 , data not shown). a third positive-polarity reading frame of 369 bp (orf3) overlaps both orfl and orf2 and was found by independent experiments to encode an immunoreactive epitope recognized by sera from hev-infected humans and animals (yarbough et af., 1991; reyes et a/., 1991b) . no orfs greater than 590 bp were identified by computer search of the negative-polarity rna strand (fig. 4b) . the nucleotide frequencies at each codon position were also analyzed and a comparison was made with two other hepatotropic positive-strand rnaviruses (ta-ble 1). the overall frequencies for hev and hcv are similar (-58% g + c), but differ markedly from that of hav (37% g + c). the relatively high g + c content results in a higher overall frequency of codons containing g + c throughout the hev coding sequence. this contrasts with the cg dinucleotide discrimination in the second and third position that has been noted in human coding sequences (nussinov, 1981) . there appears to be a slight selection for codons ending in c, which is also seen with hcv; however, the discrimination against codons ending in a is far more apparent (-9%) and is a unique fea?ure of hev when compared to hav and hcv. the third position discrimination a lluiulllluul u1111jul1uului111u lll1uuuul1 11111 jui i 11 b iu uuuu~iu 1~11 p i uj l iuiu i i uii i u uuulii ~ui.~uu~~ computer generated open reading frame analysis of the entire hev nucleotide sequence is presented in both the forward (a) and reverse directions (b). the positions of all termination codons are depicted by arrows. the three forward orfs are numbered 1, 2, and 3 and those on the opposite strand similarly labeled. the forward (positive-sense) orientation was defined by strand-specific hybridization of genomic rna (reyes et al.. 1991 b) and the identification of consensus sequence motifs related to nonstructural gene products in orfl (kamer and argos, 1984) . the horizontal line running through the various orfs indicates that orf with the highest probability of encoding protein as predicted by the algorithm devised by shepherd based on the rny codon analysis in all three orfs (shepherd, 198 1) . the hydrophilic@ plot of orf2 is presented in (c). the dotted line plotted at the -5 value on they-axis represents the midpoint with hydrophobic domains above and hydrophilic domains below the dotted line. note the large hydrophobic region at the beginning of the sequence that marks the putative signal sequence highlighted in fig. 3. against a is shared by the structural orf region of another positive-strand rna virus, rubella virus, where the frequency of a is only 7% (frey and marr, 1988) . hev and hcv are also similar in their apparent preference for g in the first coding position (35 and 33%, respectively). the computer translation of the partial nucleotide sequence from clone et1 .1 led to the detection of a conserved amino acid motif recognized in all positivestrand rna viruses (reyes er a/., 1990; fry et a/., 199 1). the canonical gly-asp-asp (gdd) tripeptide (amino acids 1550-l 552, identified by asterisks in fig. 3) is believed to encode a portion of the rna-dependent rna polymerase (rdrp) gene critical to viral replication (kamer and argos, 1984) . translation of the complete orfl revealed a second region 5' to the rdrp gene bearing similarity to another nonstructural gene product (fry et a/., 199 1) . two well-conserved sequence motifs have been found in association with purine nucleoside triphosphate (ntp)-binding activity (gentry, 1985; strauss and strauss, 1988) . the first, site a (g/axxxxgks/t), is represented in the hev sequence by gvpgsgks at amino acid position 975-982 (underlined in fig. 3) . a version of the second ntpbinding motif, site b (dead), occurs approximately 46 amino acids downstream (3') from site a and is represented in hev by the partially conserved amino acid sequence deap at position 1029-l 032 (underlined in fig. 3 ). the latter site is believed to interact with the mg+2 cation of the mg-ntp complex for rna-or dna-dependent ntpase activity. a superfamily of helicases involved in replication, recombination, and dna repair has been described with consensus features similar to those described here for ntp-binding (gorbalenya eta/., 1989). these nonstructural genesimilarities are seen in other geographically distinct isolates of hev and may indicate a putative helicase function for this region. the localization of an ntp-binding domain and the rdrp gene to orfl is consistent with a genomic organization where the nonstructural genes are expressed from the 5' end of the viral genome. translation of the second major open reading frame, orf2, indicated a novel polypeptide not present in the pir protein database. the hydropathicity plot of the sequence indicated a large hydrophobic domain at the amino terminus of orf2 followed by a hydrophilic electropositive peak (fig. 4~ ). the hydrophobic region marks a typical signal sequence (amino acids 5 to 22) and contains a potential cleavage site (paippp) as predicted by the lntelligenetics eukaryotic secretory signal sequence program. in orf2, between residues 22 and 322, nearly 10% of the amino acids are arginine conferring a high isoelectric point (pl = 10.35) to the first half of the orf2 polypeptide. the basic charge of capsid proteins is believed to indicate their involvement in the encapsidation of the genomic transcript by effectively neutralizing the electronegatively charged rna (dalgarno ef al,, 1983; rice et a/., 1985) . the mechanism of capsid assembly in hev, and the exact nature of the membrane targeting (if any) of the orf2 polypeptide, will require further study. such studies will be facil-itated by the availability of an appropriate in vitro propagation system for hev and immunospecific anti-hev reagents. the utilization of orf2 was substantiated by the independent isolation of a cdna clone by immunoscreening of a hgtl 1 cdna expression library made from the hev (mexico) isolate (yarbough et a/., 1991) . that xgt 11 clone mapped to the 3' end of orf2. these same experiments identified a second cdna epitope clone that was localized to orf3: the third positive-polarity open reading frame that overlaps both orfl and orf2. the fact that sera from acutely infected humans and animals detected hev antigens encoded by orf2 and orf3 confirmed their expression and established that the virus utilized all three positive-polarity reading frames. the presence of a consensus s&al sequence motif in orf2, together with the immunodominant seroreactivity of an identified epitupe (yarbough et a/., 1991) suggested that the viral structural protein(s) were encoded by this region of the genome. the mechanism by which orf2 and orf3 are expressed was suggested by a northern mot hybridization using the betg-1 clone as probe ( fig. 5 ). in addition to the previously identified poly(a) transcript of -7.5 kb, the probe also hybridized to subgenomic messages of 2.0 and 3.7 kb present in the infected cyno liver. it is of note that et1 .l did not originally identify these subgenomic messages (reyes et al., 1990) and other northern blot studies using probes located 5' to et1 .l also did not hybridize to these viral-specific transcripts (data not shown). these same subgenomic messages were identified in poly(a)-selected r#a from hev(m)-infected cyno liver when the epitope-encoding clones were used as probes (yarbough et al., 1991) . the orf2 epitope is located at the extreme 3' end of that reading frame (yarbough ez a/., 1991) , therefore indicating that these messages may be co-terminal with the 3' end of the viral genomic transcript. it is posfig. 5 . northern blot analysis of hev (burma)-infected cyno liver rna. three hev transcripts were detected using the 2.6-kb ecorl insert from betg-1 as probe. numbers to the left represent the sizes of the three hybridizing rna species as determined relative to rna size markers. hev cdna probes were negative against similarly preoared rna from uninfected liver (data not shown). sible that these polyadenylated subgenomic messages are used in the expression of orf2 and orf3. et-nanbh has been well-documented in both sporadic and epidemic outbreaks throughout the developing world. hepatitis e virus has been established as the major causative agent of et-nanbh by the association of hev-specific sequences with human specimens derived from six geographically diverse epidemics and also through the detection of these same sequences in various specimens derived from experimentally infected animals (reyes et al., 1990) . hev viral particles recovered from infected patients are similar to those recovered from infected primates. the virus contains a single-strand, positive-sense rna genome of approximately 7.5 kb. the nucleotide sequence described here comprises 7 194 bases excluding the poly(a) tail. if the 3' stretch of adenosine residues (at least 150-200 nucleotides) is included, the determined sequence agrees well with the genome size originally estimated by northern hybridization studies (reyes et al., 1990) . open reading frame analysis of the nucleotide sequence revealed two major positive-polarity orfs. a portion of orfl appears to encode the rdrp gene of the virus. the highly conserved amino acid residues, including the invariant gdd tripeptide found in all posi-tive-strand animal and plant rna viruses, can be located in the deduced amino acid sequence (reyes et a/., 1990; . additional evidence for the encoded polyprotein having a function in viral replication is provided by the presence of conserved motifs involved in purine ntpase activity found in a variety of cellular and viral helicases (geider and hoffman-berling, 1981) . these helicases promote the unwinding of dna, rna, or dna-rna duplexes required for genome replication, recombination, repair, and transcription. the deduced amino acid sequence of orf2 suggests that it encodes a capsid-like peptide following the canonical signal sequence at its 5'end. orf2 would appear to be the major orf encoding the viral structural protein(s). an identified immunoreactive epitope in orf3 indicates that the virus utilizes all three positive-polarity frames for encoding viral proteins (yarbough et al., 199 1; reyes et a/., 199 1 c) . this pattern of gene expression employed by hev has not been described in the various families of single-stranded positive-sense, nonenveloped rna viruses affecting humans or animals. among the enveloped rna viruses, the structural proteins of rubella virus and certain alphaviruses are found in a different reading frame from those encoding the nonstructural proteins and are also expressed from a subgenomic 3'end transcript (ou eta/., 1982) . the presence of hev-specific subgenomic rnas localized to the 3' one-third of the genome suggests that these may be the transcripts from which these 3' end orfs are expressed and is indicative of a unique expression strategy among nonenveloped positive-sense rna viruses infecting humans. the mechanism by which these subgenomic transcripts are generated is unknown. the differential abundance of the various messages (i.e., 7.6 kb > 2 kb > 3.7 kb; see fig. 5 ) does, however, suggest active transcriptional regulation rather than genomic rna fragmentation as the means by which these subgenomic messages are generated. this would in turn imply the existence of an internal rna initiation sequence and expression from the anti-genomic strand. experiments are currently in progress to map the 5'ends of these subgenomic transcripts. we at this time, however, cannot exclude other mechanisms of expression for orf2 and orf3 including frameshifting or internal translation initiation, although there is little evidence for the latter among other positive-sense rna viruses (march and haenni, 1987) . it is also possible that complex rna splicing could account for these subgenomic messages although there is evidence that northern hybridization probes from the extreme 5' end failed to detect hybridization to the 3.7-and 2.0-kb messages (a. w. tam, unreported) . 6 . hev genomic organization: the proposed organization of the hev genome is presented with the nonstructural genes encoded by the 5' orfl and the structural genes located at the 3' end of the genome (orf2 and possibly orf3). the genomic organization, nature of the virus particle (enveloped or nonenveloped), presence of subgenomic messages, and the presence of a 3'terminal poly(a) addition is compared for the various positive-sense, single-stranded rna virus families. the relative locations of the various virus sequence motifs is &so indicated, including the: hel, putative helicase motif or ntp binding domain; pol, rna-directed rna polymerase; ss, signal sequence; ire, immunoreactive epitope. s, structural gene coding region; ns, nonstructural gene coding region. it is postulated from the proposed genomic organization of hev, as presented in fig. 6 , that the nonstructural viral proteins are translated from the full-length genomic rna. the 5' nonstructural/3 structural genomic organization of hev is similar to that found in the alphavirus, rubivirus, and coronavirus families (see fig. 6 ). there is an absence of any significant homology with these enveloped viruses at both the nucleotide and amino acid levels (excluding the canonical amino acid residues noted above for the nonstructural gene products). immune electron microscopy has clearly established that the virions of hev are 27-to 34-nm nonenveloped viral particles and are therefore clearly distinguished from these enveloped viruses. picornaviruses are small nonenveloped, single-stranded, positive-sense, polyadenylated rna viruses. the various members of the picornaviridae, however, exhibit vastly different genomic organization (siddell, 1987) . hev has been shown to be unrelated antigenically and biophysically to picornaviruses (arankalle, 1988) . it was previously hypothesized that hev is calicivirus-like based on the biophysical characterization of viral particles (bradley and balayan, 1988; bradley et a/., 1988a,b) . recently the nucleotide sequence of a large portion of the nonstructural gene region of feline calicivirus (fcv) has become available for comparison to hev (neill, 1990) . although having a similar overall genomic organization to that of hev with 5'-nonstructural and 3'-structural genes, it is clear that fcv shares a higher degree of similarity with picornaviruses in the recognized nonstructural gene motifs. the proposed gene order for the nonstructural polypeptides in fcv is 2c (ntp-binding), 3c (cysteine protease), followed by the 3d gene (rdrp). the distance between the ntp-binding site motif a and the gdd triplet of the rorp is 1100 amino acids in fcv compared to rhe 568 amino acids in hev. in addition there is no evidence in the hev sequence for an intervening cysteine prateaselike region as recognized in fcv. these finding8 would further suggests that hev represents either the prototype member of an as yet unclassified novel virus family or perhaps a separate genus within the calciviridae. it is too early, however, to propose 8 dlfinitive classification of hev beyond the hypothesis presented here based on the proposed genetic organization and expression strategy of the virus. fecal-oral transmitted non-a, non-b hepatitis induced in monkeys aetiological association of a virus-like particle with enterically transmitted non-a, non-b hepatitis purification of bioiogi&fy active globin messenger rna by chromatography on al&o-thymidyfic acidcellulose evidence for a virus in non-a, non-6 hepatitis transmitted via the fecal-oral route hepatitis non-a, non-b viruses become identified as hepatitis c and e viruses enterically-transmitted non-a. non-b hepatitis virus of enterically transmitted non-a, non-b hepatitis etiology and natural history of post-transfusion and enterically-transmitted non-a, non-b hepatitis enterically transmitted non-a, non-b hepatitis: serial passage of disease in cynomolgus macaques and tamarins and recovery of disease-associated 27-to 34-nm viruslike particles enterically transmitted non-a, non-b hepatitis: etiology of disease and laboratory studies in nonhuman primates aetiological agent of enterically transmitted non-a, non-b hepatitis a method for isolation of intact translationally active ribonucleic acid isolation of cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome ross river virus 26s rna: complete nucleotide sequence and deduced sequence of the encoded structural proteins transfusion-associated hepatitis not due to viral hepatitis type a or b sequence of the region coding for virion proteins c and e2 and the carboxy terminus of the nonstructural proteins of rubella virus: comparison with alphaviruses hepatitis e virus (hev): strain variation in the nonstructural gene region encoding consensus motifs for an rna-dependent rna polymerase and an atpigtp binding site, ~%%%%, i ress locating a nucleotide bi ding site in the thymidine kinase of vaccinia virus and of herpes mplex virus by scoring triply aligned protein sequences. hoc two related supedemilies of putative helicases involved in replication, recombination, repair and expression of dna and rna genomes hepatitis e virus: cloning of a mexican strain and comparison to the burmese strain primary structural comparison of rna-dependent polymerases from plant, animal, and bacterial viruses study of an epidemic of non-a, non-b hepatitis: possibility of another human hepatitis virus distinct from posttransfusion non-a, non-b type the detection and classification of membrane-spanning proteins enterically transmitted non-a, non-b hepatitis: identification of virus-associated antigen in experimentally infected cynomolgus macaques a cdna fragment of hepatitis c virus isolated from an implicated donor of post-transfusion non-a, non-b hepatitis in japan an assay for circulating antibodies to a major etiologic virus of human non-a, non-b hepatitis a cdna clone closely associated with non-a, non-b hepatitis analysis of transcriptional regulatory signals of the hsv thymidine kinase gene: identification of an upstream control region organization of plant virus genomes that comprise a single rna molecule specific synthesis of dna in vitro via a polymerase catalysed chain reaction. ln nucleotide sequence of a region of the feline calicivirus genome which encodes picornavirus-like rna-dependent rna polymerase, cysteine protease and 2c polypeptides eukaryotic dinucleotide preference rules and their implications for degenerate codon usage sequence studies of several alphavlrus genomic rnas in the region containing the start of the subgenomic rna long-incubation post-transfusion hepatitis without serological evidence of exposure to hepatitis b virus enterically transmitted non-a, non-b hepatitis: epidemiology and clinical characteristics molecular biology of non-a, non-b hepatitis agents: the hepatitis c and hepatitis e viruses molecular cloning of the hepatitis e virus. /n "viral hepatitis and liver disease hepatitis e virus (hev): epitope mapping and detection of strain variation hepatitis e virus (hev): the novel agent responsible for enterically transmitted non-a, non-b hepatitis isolation of a cdna from the virus responsible for enterically transmitted non-a, non-b hepatitis nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution enzymatic amplification of b-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia dna sequencing with chain terminating inhibitors. froc method to determine the reading frame of a protein from the purinelpyrimidine genome sequence and its possible evolutionary justification. hoc the organization and expression of coronavirus genomes evolution of rna viruses the three viruses of non-a, non-b hepatitis construction of cdna libraries from small numbers of cells using sequence independent primers. nucleic acids ffes epidemic and endemic hepatitis in india: evidence for a non-a, non-b hepatitis virus aetiology hepatitis e virus: identification of type common epitopes, j viral we thank dr. michael lovett for his review and comments on this manuscript and appreciate the expert assistance of r. cuevas, j. fernandez, and the genelabs visual arts department. key: cord-307354-dkwcheu0 authors: abernathy, emma; glaunsinger, britt title: emerging roles for rna degradation in viral replication and antiviral defense date: 2015-05-31 journal: virology doi: 10.1016/j.virol.2015.02.007 sha: doc_id: 307354 cord_uid: dkwcheu0 abstract viral replication significantly alters the gene expression landscape of infected cells. many of these changes are driven by viral manipulation of host transcription or translation machinery. several mammalian viruses encode factors that broadly dampen gene expression by directly targeting messenger rna (mrna). here, we highlight how these factors promote mrna degradation to globally regulate both host and viral gene expression. although these viral factors are not homologous and use distinct mechanisms to target mrna, many of them display striking parallels in their strategies for executing rna degradation and invoke key features of cellular rna quality control pathways. in some cases, there is a lack of selectivity for degradation of host versus viral mrna, indicating that the purposes of virus-induced mrna degradation extend beyond redirecting cellular resources towards viral gene expression. in addition, several antiviral pathways use rna degradation as a viral restriction mechanism, and we will summarize new findings related to how these host-encoded ribonucleases target and destroy viral rna. a recurring theme in many virus-host interactions is the attempt to restrict gene expression. for the cell, such restriction is used as an antiviral mechanism. for the virus, dampening gene expression can be used to liberate cellular resources, escape immune detection, and regulate viral transcript abundance. this review will focus on how this virus-host battle plays out at a terminal stage of the gene expression cascadethat of messenger rna (mrna) degradationas research over the last several years has revealed how regulating mrna demise plays important and unexpected roles in the lifecycles of diverse viruses. we will highlight how unrelated viruses have evolved remarkably similar strategies to promote mrna degradation, even though the degree and nature of selectivity often differ. also notable is the apparent viral mimicry of some cellular rna degradation pathways, which themselves have emerging antiviral roles. rates of individual mrna degradation in a cell vary widely, and are regulated by a large cohort of rna binding proteins that control translation, localization, and access to the decay machinery. however, nearly all mrnas are protected by a 5 0 7-methyl-guanosine (7mg) cap and a 3 0 poly(a) tail, features that physically protect the mrna ends from exonucleolytic decay, and also serve to recruit translation initiation machinery. circularization of mrna during translation through interactions between cap-binding and poly(a) tail binding proteins adds additional protection from cellular decay enzymes. degradation of mrnas at the end of their translational life, termed basal decay, occurs in several stages but initiates with gradual shortening of the poly(a) tail, termed deadenylation, by cellular decay factors including the ccr4-not complex and poly(a)-specific ribonuclease (parn). poly(a) tail length is a determinate of mrna stability and translational competence, and thus is tightly controlled (eckmann et al., 2011) . deadenylation triggers removal of the 7mg cap by the decapping complex dcp1/2 and its activators. these events expose the mrna to rapid exonucleolytic degradation, primarily from the 5 0 end by xrn1, but also from the 3 0 end by the exosome and dis3l2 ( fig. 1 ) (gallouzi and wilusz, 2013) . the fact that basal decay proceeds from the mrna ends allows for tight control of mrna degradation, as removal of the poly(a) tail and cap is regulated, rate-limiting, and in some cases may even be reversible (schoenberg and maquat, 2009; weill et al., 2012) . however, the subsequent exonucleolytic decay of the message body is rapid and irreversible. to maintain transcriptome fidelity cells also need to immediately destroy cytoplasmic mrnas recognized as aberrant. in such cases, the strategy for degradation differs fundamentally from that of basal decay, in that mrnas are usually cleaved internally by an endonuclease rather than gradually trimmed from either end. the best-characterized cellular mrna quality control (qc) pathway is nonsense-mediated decay (nmd), which identifies mrnas with premature termination codons (ptc) (fig. 1) (popp and maquat, 2013) . numerous cellular factors comprise the nmd machinery, but the central nmd regulator is upf1, whose activation leads to translational repression and accelerated degradation of the ptc-containing mrna. during nmd in mammals, this rapid mrna degradation is triggered by endonucleolytic cleavage of the mrna by the smg6 endonuclease at the site of the ptc, followed by degradation of the cleaved fragments by components of the basal mrna decay machinery such as xrn1 (lykke-andersen et al., 2014; schweingruber et al., 2013) . other rna qc pathways similarly recognize aberrant translation events such as stalled or non-terminating ribosomes indicative of rna errors and lead to inactivation of the mrna in question through endonucleolytic cleavage (inada, 2013) . all viruses known to drive widespread mrna degradation do so by causing internal endonucleolytic cleavages or by directly removing the mrna 5 0 cap structure (fig. 1) . regardless of the precise mechanisms used, these strategies have in common one salient feature: they bypass the rate-limiting and regulated steps of deadenylation and cellular decapping, much like the cellular rna qc pathways. this ensures both immediate translational inactivation and exposure of the mrna ends to the processive cellular exonucleases. however, unlike the tightly regulated cellular qc endonucleases, during infection a large proportion of the cytoplasmic mrna population is targeted for cleavage. this allows the viruses to broadly restrict gene expression, as mrnas they target are degraded much more rapidly than they would be if they entered the basal decay pathway. furthermore, akin to cellular pathways like nmd, viruses that cleave mrnas often usurp xrn1 to complete the degradation process (gaglia et al., 2012) . four classes of viruses have been shown to cause endonucleolytic cleavage of mrnas for the purpose of restricting gene expression ( table 1 ). the alpha-herpesviruses, gamma-herpesviruses, and influenza a viruses encode non-homologous endonucleases that cleave mrnas directly. sars coronavirus (sars cov) does not encode an rna cleaving enzyme, but nonetheless activates an as yet unknown cellular endonuclease to cleave mrnas. in each examined case, viral specificity for mrnas (as opposed to other types of rna) is conferred by the act of translation or recognition of mrna features associated with translational competence, similar to cellular rna qc pathways kamitani et al., 2009; read, 2013) . alpha-herpesviruses such as herpes simplex-1 (hsv-1) express a fen1-like nuclease termed virion host shutoff protein (vhs) that is directed to mrnas through interactions with the translation fig. 1 . overview of cellular and viral decay pathways. basal decay begins with the rate-limiting step of deadenylation, followed by decapping and exonucleolytic degradation of the mrna body. quality control decay pathways such as nmd recognize aberrant mrnas during translation, including the presence of premature termination codons (ptc), and induce endonucleolytic cleavage, whereupon the fragments are degraded by exonucleases. virus-induced decay also bypass early steps of the basal decay pathway and involves internal cleavage of mrnas, usually in a translation-linked manner, which is followed by degradation by host exonucleases. initiation factors eif4h and eif4ai/ii (feng et al., 2005; page and read, 2010) . if this interaction is disrupted but the catalytic endonuclease activity remains intact, no host shutoff occurs, indicating that recruitment of vhs to the pool of translating mrnas is crucial to its ability to dampen gene expression (feng et al., 2005; sarma et al., 2008; shiflett and read, 2013) . in vitro, vhs lacks specificity, cleaving mrnas and non-mrnas indiscriminately, as well as anywhere along the rna (read, 2013) . however, in cells or in the presence of cell extracts, vhs preferentially cuts mrnas at unstructured sites within the 5 0 utr or near the start codon of capped mrnas (karr and read, 1999; shiflett and read, 2013) . cut sites also cluster downstream of the encephalomyocarditis virus (emcv) internal ribosome entry site (ires), which recruits the vhs-targeting translation factors eif4ai/ii, but not near the more minimal cricket paralysis virus (crpv) ires that recruits the ribosome in the absence of eif4f (shiflett and read, 2013) . further support for the hypothesis that vhs accesses its cleavage sites during translation initiation comes from experiments showing that specific cleavage sites can be repressed or enhanced by mutating the target mrna start codon or enhancing its kozak consensus context, respectively (read, 2013; shiflett and read, 2013) . however, the observation that an mrna with a capproximal hairpin structure that prevents 40s recruitment remains fully susceptible to vhs cleavage argues against an absolute requirement for ribosomal scanning (gaglia et al., 2012) . one possibility is that assembly of the eif4f complex on the mrna cap induces local rna structure remodeling that creates vhs accessible sites, but that more directed cleavages occur near the start codon during the process of 40s scanning. after vhs-induced cleavage, the resulting 3 0 mrna fragments are degraded by the cellular xrn1 exonuclease (gaglia et al., 2012) . gamma-herpesviruses encode a viral endonuclease that, although not homologous to alpha-herpesvirus vhs, also broadly targets cytoplasmic mrnas for cleavage and subsequent degradation. this protein, termed sox in kaposi's sarcoma-associated herpesvirus (kshv), musox in murine gamma-herpesvirus 68 (mhv68), and bglf5 in epstein-barr virus (ebv), is a member of the pd(d/e)xk restriction endonuclease superfamily. the sox ortholog in hsv-1 was originally shown to have dnase activity involved in viral dna genome replication (wilkinson and weller, 2003) , a function it presumably retains in all herpesviruses in addition to the gamma-herpesvirus-specific mrna degradation activity. both the dna and rna cleavage activities of the protein require the same catalytic core region (bagneris et al., 2011; glaunsinger et al., 2005) . although sox specifically targets translationally competent mrnas, active translation is not a requirement for target recognition and the molecular features that direct sox to mrnas remain unknown. sox-induced mrna cleavage occurs at one or more specific, but as-yet poorly sequence defined rna elements (z50 nt) that can be present anywhere along the length of a target gaglia et al., 2012) . although a sox targeting element can confer a new cleavage event if moved to a different location on an mrna, it is incapable of directing cleavage by sox if introduced into noncoding rnas transcribed by rna polymerase i or iii (gaglia et al., 2012) . similar to vhs, recombinant sox and bglf5 display relaxed rna targeting specificity in vitro (bagneris et al., 2011; buisson et al., 2009) , indicating that additional mrna-specific features must be required for sox recruitment in cells. single function mutants of sox and musox that are defective for mrna cleavage but retain their dnase activity have mutations that map to regions outside the catalytic core on the protein surface (bagneris et al., 2011; covarrubias et al., 2009; glaunsinger et al., 2005) . furthermore, the crystal structure of bglf5 revealed the presence of a flexible "bridge" domain that crosses the active site and contains residues involved in host shutoff (buisson et al., 2009; horst et al., 2012) . these noncatalytic regions may therefore function in targeting the gammaherpesvirus sox orthologs to translationally competent mrnas, perhaps through interactions with specific mrna binding proteins. similar to vhs, sox-cleaved mrnas subsequently enter the cellular mrna decay pathway and are degraded by xrn1 gaglia et al., 2012) . the pa-x protein of influenza a virus (iav) is a recently discovered mrna endonuclease involved in restricting host gene expression (jagger et al., 2012) . it is expressed by a ribosome frameshifting event during translation of the pa subunit of the viral rna-dependent rna polymerase (rdrp), itself an endonuclease that is responsible for cap snatching in the nucleus. like the gamma-herpesvirus sox orthologs (and many endonucleases involved in cap snatching), pa-x is a member of the pd(d/e)xk nuclease family. pa-x retains the n-terminal pa endonuclease domain but contains a distinct c-terminus that augments the cellular mrna degradation activity of the protein via unknown mechanisms (desmet et al., 2013; jagger et al., 2012) . one possibility is that c-terminal sequences are involved in directing pa-x to its mrna targets. in this regard, it would be interesting to determine whether pa-x targeting is linked to translation and feeds into the cellular xrn1 decay pathway, as has been shown for other viral mrna restriction factors. sars cov expresses a host shutoff factor, nsp1, that binds the 40s ribosome, simultaneously inducing cleavage of mrnas and inactivating the ribosome (huang et al., 2011; kamitani et al., 2009) . by binding the 40s ribosome, nsp1 is recruited to all translationally competent mrnas, allowing for broad targeting of cellular transcripts. nsp1 itself does not possess detectable intrinsic nuclease activity, suggesting that nsp1-induced mrna cleavage may instead occur through activation of a cellular rna surveillance pathway (almeida et al., 2007; huang et al., 2011) . candidate pathways include those involved in monitoring translational efficiency, given that the nsp1-40s interaction leads to ribosome inactivation in addition to mrna cleavage (narayanan and makino, 2013) . for example, the no-go decay pathway degrades mrnas with stalled ribosomes, albeit using a currently unknown endonuclease (harigaya and parker, 2010) . as has been observed for cellular qc pathways like nmd as well as the herpesviral endonucleases, degradation of the cleaved mrnas in nsp1-expressing cells is executed by xrn1 (gaglia et al., 2012) . poxviruses and african swine fever virus (asfv) are the only viruses known to encode decapping enzymes. similar to cellular shors et al., 1999; souliere et al., 2009) . the asfv decapping enzyme g5r also contains a nudix domain essential for decapping. both the vacv and asfv decapping enzymes are inhibited in the presence of excess uncapped rnas, but only the vacv d10 enzyme is also inhibited by cap analogs (parrish et al., 2009 ). this suggests that g5r recognizes its substrates by binding the rna body rather than the cap, whereas vacv d10 binds both the methylated cap and the rna body (parrish et al., 2009) . extensive site-directed mutagenesis of vacv d10 identified eight amino acids in the catalytic core of d10 important for decapping activity and showed that d10 recognizes the cap in a manner distinct from other characterized cap binding proteins (souliere et al., 2010) . it is unknown why poxviruses expresses two functional decapping enzymes, although the reason may be linked to the fact that d9 is expressed early during infection while d10 is expressed later, after dna replication. additionally, there are some differences between the two enzymes, including the observations that d9 requires longer rna substrates than d10, and d9 mutants have less pronounced phenotypes than d10 mutants moss, 2006, 2007) . therefore, the kinetic and functional requirements for decapping may vary as vacv infection progresses. as decapping renders the 5 0 end of an mrna unprotected, it is likely that d9 and d10 cleaved mrnas are digested by xrn1, similar to the cleavage products induced by vhs, sox, and nsp1. interestingly, a recent rnai screen suggested a positive role for xrn1 in vacv replication (sivan et al., 2013) , perhaps indicating that xrn1-mediated rna degradation plays an important role in the viral lifecycle. it is often presumed that restriction of cellular gene expression during infection serves in part to divert resources for the selective enhancement of viral gene expression. however, in each of the above documented examples there is not a clear escape mechanism to broadly protect viral mrnas from inactivation. instead, these viruses may benefit from reduced transcript levels during infection, either because mrna inactivation helps them regulate their gene expression kinetics or other aspects of the viral lifecycle. during vacv infection, the decapping enzymes d9 and d10 fail to discriminate between viral and cellular mrna. targeting viral transcripts is proposed to help facilitate transitions between the classes of gene expression, as d10 mutants exhibit delayed onset of early and late viral gene expression (liu et al., 2014; parrish and moss, 2006) . similarly, alpha-and gamma-herpesviral mrnas are inherently susceptible to endonucleolytic cleavage. during hsv-1 infection, vhs plays an important role in mediating the effective transition between the expression of immediate-early (î±), early (î²), and late (î³) genes (read, 2013) . there are some discrepancies in the field as to exactly which viral mrnas are susceptible to vhs-mediated degradation during infection. some data suggest that only î± mrnas are targeted (shu et al., 2013; taddeo et al., 2013) , while data from other groups indicate that î±, î², and even some î³ mrnas are susceptible to degradation by vhs (kwong and frenkel, 1987; read, 1987, 1989) . regardless of the extent of viral mrna degradation, targeting of viral mrnas by vhs helps facilitate the transition between viral gene classes as infection progresses. furthermore, during infection with a vhs null virus, î³ mrnas are excluded from polysomes due to 'translational overload', whereby the capacity of the translation machinery becomes overwhelmed due to an excess of mrnas produced earlier in infection . this confirms the long-held hypothesis that host shutoff is a means of liberating translational machinery for viral use-with the twist that both host and viral transcripts must be degraded to ensure efficient translation of î³ mrnas. further contributing to the robust accumulation of î³ proteins is the inactivation of vhs later during infection by the virion proteins vp16, vp22, and ul47 (read, 2013; shu et al., 2013) . all three are packaged into the viral particle along with vhs, and it has been suggested that sequestering vhs in this complex represents an early stage in virion assembly and protects mrnas from cleavage late in infection. thus, despite widespread viral mrna susceptibility, vhs targeting of mrnas appears temporally controlled. the sox homologs in mhv68 (musox) and ebv (bglf5) have also been shown to target viral mrnas for cleavage (abernathy et al., 2014; horst et al., 2012) . unlike vhs, however, there is no indication that sox or its orthologs are inactivated as infection progresses. during mhv68 infection, musox broadly targets viral mrnas from all three kinetic classes, which generally leads to corresponding decreases in viral protein levels in each class (abernathy et al., 2014) . thus, unlike hsv-1 infection, the targeting of viral mrnas during gamma-herpesvirus infection is not a mechanism to redirect the translation machinery towards viral genes. this also suggests that translation factors do not become limiting during mhv68 infection. selective inactivation of the mrna degradation activity of musox results in altered protein composition of progeny virions, which ultimately impacts subsequent rounds of infection by favoring lytic cycle entry over latency. the mutant also exhibits replication defects in multiple cell types (abernathy et al., 2014; richner et al., 2011) . deletion of bglf5 during ebv infection also results in accumulation of several viral proteins, as well as causes nuclear egress defects (feederle et al., 2009 ). however, because bglf5 has dual roles in viral genome maturation and mrna degradation and the bglf5 mutant virus lacks both functions, it is not possible to ascribe the above phenotypes solely to a defect in host shutoff. nonetheless, these data support the hypothesis that degradation of viral mrna during gamma-herpesvirus infection plays important roles in regulating gene expression and subsequent viral particle composition. unlike herpesviral and poxviral mrnas, sars cov transcripts are categorically resistant to nsp1-induced cleavage and degradation. this protection is due to the presence of a protective 5 0 leader sequence present on all viral mrnas, although the mechanism of protection remains unclear (huang et al., 2011) . however, while cov mrnas escape endonucleolytic cleavage, they do not escape nsp1-induced ribosome inactivation, raising the issue of what advantage is conferred by the protective sequence (huang et al., 2011; lokugamage et al., 2012) . one likely possibility is that ribosome inactivation is not complete, and consequently viral gene expression is not as severely impacted as cellular gene expression. whether this represents a mechanism to fine tune viral protein synthesis in a manner important for the viral lifecycle in vivo remains an interesting question for future investigation. degradation of mrna has recently been shown to be highly interconnected with many other cellular processes including transcription, mrna export, and translation (braun and young, 2014; huch and nissan, 2014) . it is thus likely that the broad virus-induced mrna decay described above will result in changes to other rna processes as well. one example of this is altered mrna 3 0 end processing in the nucleus that occurs as a consequence of enhanced mrna decay in the cytoplasm (fig. 2) . poly(a) binding protein (pabpc) normally binds to poly(a) tails of mrnas in the cytoplasm, where it contributes to the regulation of mrna stability and enhances translation. however, pabpc becomes strongly relocalized to the nucleus in cells expressing sox, musox, bglf5, vhs, pa-x or nsp1 (arias et al., 2009; khaperskyy et al., 2014; kumar and glaunsinger, 2010; lee and glaunsinger, 2009; park et al., 2014) . nuclear import occurs because within its rna binding domains, pabpc harbors noncanonical nuclear localization signals (nls) that are masked when it is bound to poly(a) tails in the cytoplasm. however, during accelerated mrna degradation by these viral proteins, pabpc is released from poly(a) tails, exposing its nls for interaction with the nuclear import machinery . such aberrant accumulation of pabpc in the nucleus causes hyperadenylation of nascent transcripts by cellular poly(a) polymerase ii (kumar and glaunsinger, 2010; lee and glaunsinger, 2009 ). these hyperadenylated mrnas are retained in the nucleus, presumably because they are recognized as aberrant by the nuclear rna qc machinery. this process thus contributes to the overall magnitude of host shutoff, as the cytoplasm cannot be efficiently repopulated with newly transcribed mrnas. accelerated cytoplasmic decay may also lead to inhibition of stress granule (sg) formation. sgs are storage sites for translationally stalled mrnas, and form in response to translational arrest that often occurs during viral infection (valiente-echeverria et al., 2012) . many viruses have evolved mechanisms to block their formation, presumably to ensure continued translation of viral proteins. viral nucleases can contribute to sg dispersal, presumably through the bulk reduction of mrnas needed to nucleate sg formation. for example, along with several other iav-encoded proteins, the endonuclease pa-x was recently identified as a potent inhibitor of sgs (khaperskyy et al., 2014) . pa-x-mediated sg inhibition coincides with pabpc relocalization, hinting at a link between host shutoff and sg dynamics. similarly, the vhs nuclease of hsv-2 is required for the sg disruption that occurs during hsv-2 infection (finnen et al., 2014) . however, vhs has also been implicated in translational enhancement of viral late genes (a role separable from its rnase activity) (dauber et al., 2011 , making it difficult to ascribe the sg dispersal phenotype solely to mrna depletion. nonetheless, viral mrna-targeting nucleases provide a unique system to dissect the link between mrna decay and sg assembly. widespread dampening of gene expression during infection is presumed to contribute to viral immune evasion, both by inhibiting expression of cellular immune regulatory genes and by reducing the abundance of viral antigens available for detection. indeed, viruses containing mutations in hsv-1 vhs, mhv68 musox, coronavirus nsp1, and vacv d10 exhibit more severe phenotypes in a mouse model of infection than in cultured cells (liu et al., 2014; richner et al., 2011; smiley, 2004; zust et al., 2007) , suggesting mrna degradation contributes to virulence. activation of the innate immune response leads to expression of hundreds of genes involved in establishing an antiviral state. vhs suppresses the expression of several of these genes including tetherin and viperin, which would normally act to restrict hsv-1 infection (shen et al., 2014; zenner et al., 2013) , as well as many pro-inflammatory cytokines (suzutani et al., 2000) . some of the differences in the in vivo infectivity of wt versus the vhs mutant hsv-1 are alleviated in interferon receptor defective (ifnar ko) mice, suggesting that vhs-induced suppression of the innate immune response contributes to viral fitness (leib et al., 1999; smiley, 2004) . selective inactivation of the musox mrna degradation activity leads to a severe attenuation of mhv68 in b cells during the phase of peak latency establishment (richner et al., 2011) . this could be due to improper immune evasion and/or cell-type specific replication defects, as the musox mutant virus replicates to wt titers in the mouse lung but traffics inefficiently to b cells and displays cell type specific replication defects in cultured cells (abernathy et al., 2014; richner et al., 2011) . similar to the ability of vhs to degrade immune modulatory mrnas, ebv bglf5 also reduces expression of immune molecules, in particular hla i and ii (rowe et al., 2007; zuo et al., 2008) . however, this activity is redundant with other ebv proteins that specifically combat hla processing and transport and thus appears to have only a small effect on cd8ã¾ t cell recognition (quinn et al., 2014) . whether cd8ã¾ t cell recognition or innate immune signaling are influenced by mrna degradation during in vivo infection with other gamma-herpesviruses remains to be determined. both vacv decapping mutants and cov nsp1 mutants also display altered virulence phenotypes, although further research is needed to determine the extent to which these are directly linked to mrna degradation. mice infected with vacv d10 stop and catalytic mutants show less weight loss and mortality compared to a wt infection, and these mutant viruses replicate to lower titers in all organs (liu et al., 2014) . although there is not in vivo data for nsp1 of sars cov, the nsp1 protein of the coronavirus mouse hepatitis virus (mhv) retains the mrna degradation function, as well as several additional roles in inhibiting immune signaling pathways. these activities align well with the observation that an mhv nsp1 deletion virus is severely attenuated in wt mice, but is completely rescued in ifnar ko mice (zust et al., 2007) . determining the extent to which nsp1-induced virulence links to its host shutoff activity will require the use of single function nsp1 mutants selectively defective for mrna cleavage or immune pathway impairment. in this regard, the recent characterization of a panel of sars cov nsp1 mutants that exhibit selective functional defects should help determine the contribution of mrna degradation to the nsp1 virulence phenotypes (jauregui et al., 2013) . the use of rna-targeting nucleases is also a component of many of the cellular antiviral defense pathways, some of which play dual roles in regulating normal cellular metabolism and viral restriction (fig. 3) . for example, in addition to its well-established role in eliminating ptc-containing mrna, the nmd pathway has recently been shown to function in the restriction of positive strand (ã¾) rna viruses in plants and in mammalian cells (balistreri et al., 2014; garcia et al., 2014) . a genetic screen uncovered the central nmd effector upf1 as a cellular restriction factor of the plant (ã¾) rna viruses potato virus x (pvx) and turnip crinkle virus (tcv) (garcia et al., 2014) . nmd-based restriction is hypothesized to act upon the input genomic viral rna undergoing initial rounds of translation, and thus might function before the onset of rnai, the major antiviral pathway in plants. one of the known activators of nmd is an unusually long 3 0 utr on the target mrna, as this can be associated with less efficient translation termination (inada, 2013; kervestin and jacobson, 2012) . in this regard, many (ã¾ ) rna plant viruses encode subgenomic (sg) rnas, which creates the appearance of a long 3 0 utr on the genomic mrna and select sgrnas. indeed, these long 3 0 utrs are required for degradation of pvx and tcv rnas via nmd, confirming that intrinsic features of viral rnas render them susceptible to cellular qc pathways (garcia et al., 2014) . in mammalian cells, depleting nmd factors upf1, smg5, or smg7 leads to increased replication of the alphaviruses semliki forest virus (sfv) and sindbis virus (sinv), suggesting that nmd may target viral genomic rna for degradation (balistreri et al., 2014) . unexpectedly, although like pvx and tcv these viruses have long 3 0 utrs, deletion of the long 3 0 utr of sfv does not alter the restriction by upf1 (balistreri et al., 2014) . furthermore, viral restriction does not require the nmd endonuclease smg6. thus, the mechanism of decay and the viral rna feature(s) that trigger virus-induced activation of nmd in mammalian cells remain to be elucidated. however, a broader role for nmd in controlling mammalian viruses is supported by the fact that multiple retroviruses have evolved mechanisms to restrict nmd. this can occur through inhibitory interactions with nmd components or through viral rna sequences that protect against nmd (mocquet et al., 2012; withers and beemon, 2010) . in plants and insects, rna interference (rnai) is the primary antiviral defense mechanism. the rnai pathway restricts gene expression by processing the long double stranded rnas frequently generated during viral replication into short interfering rnas (sir-nas), which guide endonucleolytic cleavage of complementary target mrnas. although mammalian cells possess the rnai machinery, in most cases rnai does not appear to play a significant antiviral role, and has instead been supplanted by the protein-based interferon response (cullen, 2014) . however, recent data reveal that in select cell types such as es cells, rnai indeed functions in an antiviral capacity maillard et al., 2013) . one hypothesis is that an antiviral role for rnai is retained in these cells because they lack a fully functional interferon response. furthermore, mice express an oocyte-specific n-terminally truncated isoform of the nuclease responsible for generating mature forms of the effector small rnas (mirnas or sirnas), termed dcr1 (flemr et al., 2013) . dcr1 has increased sirna-processing activity relative to the full-length dcr nuclease, perhaps explaining why undifferentiated cells contain rnaibased antiviral activity (cullen, 2014; flemr et al., 2013) . because dcr1 is not expressed in primates, whether similar rnai-based antiviral activity is active in human es cells remains an open question. the interferon (ifn) pathway is the primary effector of the mammalian innate immune response, and its activation can induce the expression of proteins that drive either selective destruction of viral rna or more indiscriminate destruction of viral and cellular rna. an example of the former is the zinc-finger antiviral protein (zap), which binds specifically to viral rnas that contain a zap response element (zre). upon binding to viral rna, zap recruits cellular rna decay machinery, including the deadenylase parn, the rna 3 0 -5 0directed exonuclease complex called the exosome, and the dcp1/2 decapping enzymes via their p72 helicase co-factor (zhu et al., 2011 3 . cellular nucleases with antiviral roles. (a) viral rna of ( ã¾ ) rna viruses can be recognized by cellular qc pathways like nmd, in some cases due to long 3 0 utrs which are inherent to subgenomic rnas (sgrna). this leads to their degradation by smg6 and perhaps other nucleases. (b) rnai cleaves viral dsrna, which is loaded into a rna induced silencing complex (risc) that targets viral rna for endonucleolytic cleavage by ago. (c) ifn-activated mrna degradation pathways include zap and rnase l. zap binds viral rna at specific response elements (zre) and recruits cellular decay factors, including deadenylase parn, de-capping enzyme dcp1, and the 3 0 -5 0 exosome. ifn also induces 2-5a synthase (oas) to synthesize the rnase l activator 2-5a, leading to rnase l dimerization and cleavage of viral and cellular rnas. including hiv, filoviruses, sindbis virus, and mhv68 (bick et al., 2003; muller et al., 2007; xuan et al., 2013; zhu et al., 2011) . zap contains four ccch-type zinc fingers in its n-terminal domain that specifically bind rna and recruit decay factors (zhu et al., 2011; zhu and gao, 2008) . as with many other mrna decay pathways, zap-induced degradation is preceded by inhibition of mrna translation. zap restricts translation of its target mrnas by interacting with the eif4a helicase in a manner that disrupts the ability of eif4a to associate with eif4g (zhu et al., 2012) . translational repression appears selective for zre-containing transcripts, suggesting that zap only binds eif4a associated with its target mrnas (zhu et al., 2012) . unlike the zre-specific targeting of zap, the ifn-activated cellular endonuclease rnase l cleaves a much broader spectrum of rnas. rnase l is inactive as a monomer, but becomes active upon binding 2 0 -5 0 oligoadenylates (2-5a) that are produced by another ifninduced protein, 2-5a synthase (oas). binding of 2-5a allosterically activates rnase l by inducing its dimerization, whereupon it cleaves both viral and cellular rnas, usually at a 5 0 -unn-3 0 consensus (bhattacharyya, 2014; han et al., 2014) . that said, two features of rnase l might cause it to favor viral over cellular mrnas. first, the uu/ua dinucleotides that often make up the rnasel cleavage site are relatively rare in the coding regions of cellular mrnas, possibly as an evolutionary trend to avoid rnase l cleavage (al-saif and khabar, 2012) . second, cellular rnas contain a variety of nucleoside modifications, some of which confer increased resistance to rnase l (anderson et al., 2011) . not surprisingly, many viruses have evolved mechanisms to counteract the activation of rnase l, including blocking ifn induction and directly disrupting 2-5a production (bhattacharyya, 2014; silverman and weiss, 2014) . the expanding number of viruses shown to exert control over the cytoplasmic mrna population through the activity of virally encoded endonucleases or by activating cellular nucleases highlights the importance of this process in diverse viral lifecycles. although we have highlighted select examples of viral endonucleases that promote mrna decay, many other viruses impact rna fate by inactivating mrna degradation enzymes, hijacking or competing with the cellular decay machinery, and relocalizing cellular proteins that control mrna stability (moon and wilusz, 2013) . furthermore, as is frequently the case in virology, the study of this virus-host interplay is sure to offer new insights into the regulation of cellular rna decay pathways. the field is now beginning to uncover how cellular rna degradation enzymes with central roles in basal and qc-associated rna decay are also key contributors to the antiviral response. yet, in some cases the precise players or their regulation may differ from their previously characterized roles in the context of uninfected cells. in this regard, revealing how viral rnas are recognized and marked for degradation by pathways such as nmd remains an important endeavor. this should simultaneously provide insight into cellular rna features that impact qc surveillance, especially given the numerous parallels between mrna degradation by viruses and cellular qc pathways. furthermore, additional research is required to define the importance of rnai in the mammalian antiviral response, including the cell context in which it operates as well as whether it plays antiviral roles in primates. much remains to be discovered about the mechanisms underlying mrna targeting by viral endonucleases as well. questions surrounding the precise roles of translation factors in recruiting or activating nucleases, what sequence elements and context confer cleavage, as well as how active translation impacts targeting all remain active areas of research. finally, although the data all point to important roles for virus-induced mrna degradation in replication and immune evasion in vivo, very little is known about the relative importance of regulating host versus viral mrna abundance in these processes. ongoing and future research should provide answers to these questions, as well as reveal the impact of virus-induced mrna degradation on a diversity of other cellular processes. gammaherpesviral gene expression and virion composition are broadly controlled by accelerated mrna degradation uu/ua dinucleotide frequency reduction in coding regions results in increased mrna stability and protein expression novel beta-barrel fold in 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infection and is orchestrated exclusively from the cytoplasm viruses and rna interference: issues and controversies the herpes simplex virus 1 vhs protein enhances translation of viral true late mrnas and virus production in a cell type-dependent manner the herpes simplex virus 1 virion host shutoff protein enhances translation of viral late mrnas by preventing mrna overload identification of the n-terminal domain of the influenza virus pa responsible for the suppression of host protein synthesis control of poly(a) tail length the epstein-barr virus alkaline exonuclease bglf5 serves pleiotropic functions in virus replication mrna decay during herpes simplex virus (hsv) infections: protein-protein interactions involving the hsv virion host shutoff protein and translation factors eif4h and eif4a the herpes simplex virus 2 virion-associated ribonuclease vhs interferes with stress granule formation a retrotransposon-driven dicer isoform directs endogenous small interfering rna production in mouse oocytes a common strategy for host rna degradation by divergent viruses a distinctively novel exoribonuclease that really likes u nonsense-mediated decay serves as a general viral restriction mechanism in plants the exonuclease and host shutoff functions of the sox protein of kaposi's sarcoma-associated herpesvirus are genetically separable structure of human rnase l reveals the basis for regulated rna decay in the ifn response no-go decay: a quality control mechanism for rna in translation the "bridge" in the epstein-barr virus alkaline exonuclease protein bglf5 contributes to shutoff activity during productive infection sars coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mrnas: viral mrnas are resistant to nsp1-induced rna cleavage interrelations between translation and general mrna degradation in yeast quality control systems for aberrant mrnas induced by aberrant translation elongation and termination an overlapping protein-coding region in influenza a virus segment 3 modulates the host response identification of residues of sars-cov nsp1 that differentially affect inhibition of gene expression and antiviral signaling a twopronged strategy to suppress host protein synthesis by sars coronavirus nsp1 protein the virion host shutoff function of herpes simplex virus degrades the 5 0 end of a target mrna before the 3 0 end nmd: a multifaceted response to premature translational termination influenza a virus host shutoff disables antiviral stress-induced translation arrest nuclear import of cytoplasmic poly(a) binding protein restricts gene expression via hyperadenylation and nuclear retention of mrna importin alpha-mediated nuclear import of cytoplasmic poly(a) binding protein occurs as a direct consequence of cytoplasmic mrna depletion herpes simplex virus-infected cells contain a function(s) that destabilizes both host and viral mrnas aberrant herpesvirus-induced polyadenylation correlates with cellular messenger rna destruction interferons regulate the phenotype of wild-type and mutant herpes simplex viruses in vivo rna interference functions as an antiviral immunity mechanism in mammals the d10 decapping enzyme of vaccinia virus contributes to decay of cellular and viral mrnas and to virulence in mice severe acute respiratory syndrome coronavirus protein nsp1 is a novel eukaryotic translation inhibitor that represses multiple steps of translation initiation human nonsense-mediated rna decay initiates widely by endonucleolysis and targets snorna host genes antiviral rna interference in mammalian cells the human tlymphotropic virus type 1 tax protein inhibits nonsense-mediated mrna decay by interacting with int6/eif3e and upf1 cytoplasmic viruses: rage against the (cellular rna decay) machine inhibition of filovirus replication by the zinc finger antiviral protein interplay between viruses and host mrna degradation a mutant of herpes simplex virus type 1 exhibits increased stability of immediate-early (alpha) mrnas control of mrna stability by the virion host shutoff function of herpes simplex virus the virion host shutoff endonuclease (ul41) of herpes simplex virus interacts with the cellular cap-binding complex eif4f nuclear translocation and regulation of intranuclear distribution of cytoplasmic poly(a)-binding protein are distinct processes mediated by two epstein barr virus proteins the african swine fever virus g5r protein possesses mrna decapping activity characterization of a vaccinia virus mutant with a deletion of the d10r gene encoding a putative negative regulator of gene expression characterization of a second vaccinia virus mrnadecapping enzyme conserved in poxviruses vaccinia virus d10 protein has mrna decapping activity, providing a mechanism for control of host and viral gene expression organizing principles of mammalian nonsensemediated mrna decay cooperation between epstein-barr virus immune evasion proteins spreads protection from cd8ã¾ t cell recognition across all three phases of the lytic cycle virus-encoded endonucleases: expected and novel functions global mrna degradation during lytic gammaherpesvirus infection contributes to establishment of viral latency host shutoff during productive epstein-barr virus infection is mediated by bglf5 and may contribute to immune evasion small interfering rnas that deplete the cellular translation factor eif4h impede mrna degradation by the virion host shutoff protein of herpes simplex virus re-capping the message nonsense-mediated mrna decay-mechanisms of substrate mrna recognition and degradation in mammalian cells herpes simplex virus 1 counteracts viperin via its virion host shutoff protein ul41 mrna decay during herpes simplex virus (hsv) infections: mutations that affect translation of an mrna influence the sites at which it is cleaved by the hsv virion host shutoff (vhs) protein down regulation of gene expression by the vaccinia virus d10 protein the nuclear-cytoplasmic shuttling of virion host shutoff rnase is enabled by pul47 and an embedded nuclear export signal and defines the sites of degradation of au-rich and stable cellular mrnas viral phosphodiesterases that antagonize doublestranded rna signaling to rnase l by degrading 2-5a human genome-wide rnai screen reveals a role for nuclear pore proteins in poxvirus morphogenesis herpes simplex virus virion host shutoff protein: immune evasion mediated by a viral rnase? characterization of the vaccinia virus d10 decapping enzyme provides evidence for a two-metal-ion mechanism insights into the molecular determinants involved in cap recognition by the vaccinia virus d10 decapping enzyme the role of the ul41 gene of herpes simplex virus type 1 in evasion of non-specific host defence mechanisms during primary infection the herpes simplex virus host shutoff rnase degrades cellular and viral mrnas made before infection but not viral mrna made after infection viral modulation of stress granules translational control by changes in poly(a) tail length: recycling mrnas the role of dna recombination in herpes simplex virus dna replication structural features in the rous sarcoma virus rna stability element are necessary for sensing the correct termination codon zap inhibits murine gammaherpesvirus 68 orf64 expression and is antagonized by rta herpes simplex virus 1 counteracts tetherin restriction via its virion host shutoff activity zinc-finger antiviral protein inhibits hiv-1 infection by selectively targeting multiply spliced viral mrnas for degradation zap-mediated mrna degradation translational repression precedes and is required for zap-mediated mrna decay the dnase of gammaherpesviruses impairs recognition by virus-specific cd8 ã¾ t cells through an additional host shutoff function coronavirus non-structural protein 1 is a major pathogenicity factor: implications for the rational design of coronavirus vaccines key: cord-294990-jdjbjkcp authors: thuy, nguyen thanh; huy, tran quang; nga, phan thi; morita, kouichi; dunia, irene; benedetti, lucio title: a new nidovirus (namdinh virus ndiv): its ultrastructural characterization in the c6/36 mosquito cell line date: 2013-07-25 journal: virology doi: 10.1016/j.virol.2013.06.030 sha: doc_id: 294990 cord_uid: jdjbjkcp we describe the ultrastructure of the namdinh virus (ndiv), a new member of the order nidovirales grown in the c6/36 mosquito cell line. uninfected and ndiv-infected cells were investigated by electron microscopy 24–48 h after infection. the results show that the viral nucleocapsid-like particles form clusters concentrated in the vacuoles, the endoplasmic reticulum, and are scattered in the cytoplasm. mature virions of ndiv were released as budding particles on the cell surface where viral components appear to lie beneath and along the plasma membrane. free homogeneous virus particles were obtained by ultracentrifugation on sucrose gradients of culture fluids. the size of the round-shaped particles with a complete internal structure was 80 nm in diameter. this is the first study to provide information on the morphogenesis and ultrastructure of the first insect nidovirus ndiv, a missing evolutionary link in the emergence of the viruses with the largest rna genomes. namdinh virus (ndiv), named after a vietnamese province, was isolated from mosquitoes and is yet to be linked to any pathology. recently, molecular biology methods were used to identify this mosquito-borne virus as possessing a 20.192 kb genome, the largest genome among non-segmented single-stranded rna viruses of insects, and its characteristics have defined ndiv as the prototype of a new member of the order nidovirales (nga et al., 2011) . members of the nidovirales have a wide range of hosts including crustaceans, fishes, birds and a variety of mammals (siddell et al., 2005) . they included the coronaviridae, arteriviridae and roniviridae families that are known to re-arrange host cell membranes for the purpose of replication and assembly. however, many details of these processes remain obscure, and knowledge of the ultrastructural characterization and the various steps in the replication process of these viruses in cell culture is still very limited. using improved cryo-fixation methods, electron tomography and immunogold electron microscopy (em), (2012) showed recently that the arteriviral rna is present within double-membrane vesicles, but no obvious communication with the cytoplasm was detectable. these authors also investigated the structure of the nidovirus-induced membrane modifications and found that these viruses transform the endoplasmic reticulum (er) into a reticulo-vesicular network that integrates numerous inter-connected double-membrane vesicles (knoops, 2011) . here we have applied em methods to investigate the morphological and ultrastructural features of ndiv grown in c6/36 mosquito cells via ultrathin sections, negative staining and immunogold labeling. the results provide useful information regarding ndiv, the first insect nidovirus identified in mosquitoes. c6/36 mosquito cells possess typical round shape with the nucleus remaining on one side of the cell throughout 3 days of observation ( fig. 1a) . at higher magnifications, it is easy to recognize numerous mitochondria, vacuoles ( fig. 1a and b) , and the er (fig. 1c) in the cytoplasm. by em, the control culture did not appear contaminated with other mosquito viruses. twenty-four hours after infection, the shape of the cells did not differ from that of control cells and em observations did not reveal intact virions outside the cells. viral nucleocapsid-like particles appeared accumulated in cell vacuoles (fig. 2a) as well as in the er which showed swollen cisternae (sc) (fig. 2b) in comparison with the er in normal cells (fig. 1c) . individual nucleocapsid-like particles appeared round-shaped, homogenous in size and with an electron-dense interior containing the viral genetic material. the average diameter of the viral nucleocapsid-like particles was 50 nm. vesicular structures containing electron-dense material were also detected in the cytoplasm and identified as putative viral nucleocapsid inclusions. these vesicles possessed doublemembranes ( fig. 2c and d) . after 48 h of infection, the mosquito cells had changed more dramatically. by em observation, the cytopathic effect appeared clearly as cell swelling and cell aggregation (data not shown). abundant ndiv nucleocapsid-like particles appeared as clusters or free in the cytoplasm; no viral nucleocapsid-like particle was identified in mitochondria ( fig. 3 ) or in the nucleus (data not shown). in many cells, 48 h post-infection, the numerous aggregates of viral nucleocapsid-like particles appeared to migrate to the cell surface ( fig. 3 ). at this time point, budding particles were detectable in some cells as protrusions from the plasma membrane; viral nucleocapsid-like particles appeared close to the plasma membrane where putative proteins of the viral envelope had associated (fig. 4) . in the mature period (48 h after infection; budding process), the ndiv clearly appeared at the level of plasma membrane protrusions: the viral envelope consisted in a trilaminar membrane including the host membrane. at this final stage (just before cell lysis), the virus particles appeared ready to bud ( fig. 4b and c) as complete virions (black arrowheads) or as incomplete virions (white arrowheads). the viral nucleocapside-like particles detected in the cytoplasm (white arrows) and accumulated in the er vesicles (ves) of 48 h ndiv-infected c6/36 cells, appear immunoglod-labeled on ultrathin sections with anti-ndiv polyclonal antibodies, as shown in fig. 5 . no viral nucleocapsid-like particle was found in mitochondria (mi). we have been successful in visualizing the virus particles in concentrated fluids by negative staining and immunogold em. the virions appeared round-shaped with complete (black arrowheads) and incomplete (white arrowheads) virus particles. the diameter of the virus particles was about 60 nm for the incomplete particles and about 80 nm for the complete particles ( fig. 6a ). we have measured the size of the smallest and biggest particles of more than 25 particles each per picture, with three pictures. the envelope layer measured 12 nm in width and appeared rather smooth. higher magnifications of virion images revealed short spikes of ndiv, measuring 3-4 nm in length (framed image in fig. 6a ). immunogold labeling of non-fixed isolated virions using the polyclonal antibodies indicated a specific antigenic reaction against the viral envelope (fig. 6b ). ndiv is the first insect nidovirus described, a missing evolutionary link in the emergence of viruses with the largest rna genomes, and it was identified as the prototype of a new member of the order nidovirales (nga et al., 2011) . this study describes the morphogenesis and ultrastructural characterization of ndiv by ultrathin sections, negative staining and immunogold em. ndiv replication appeared to occur in the cytoplasm of the host cells as is the case for other single-stranded rna viruses (denison, 2008) . viral nucleocapsid-like particles also accumulated in cell vacuoles and in the er which showed swollen cisternae. other nidoviruses replicate their genomes in the cytoplasm in association with intracellular membrane rearrangements as single-or doublemembrane vesicles (knoops et al., 2012; knoops, 2011) . however, the exact sites of rna synthesis and the relationships between these membrane vesicles and the cytoplasm remain undefined. in this study, the nucleocapsid-like particle of ndiv was identified in the host cell cytoplasm as possessing a round shape and an inner core observed in several types of vesicles and in the er and its viral nature was confirmed by immunogold labeling on thin sections. in contrast to members of the coronaviridae family such as sars-cov, ndiv particles are formed on membranes of the "budding compartment", a term used to describe the continuous membranous system from the er to the golgi complex. in our study, replication of ndiv in the cytoplasm of c6/36 cells closely follows the er and vesicular localization, and the viral nucleocapsid-like particle accumulates in endoplasmic vesicles as does the replication complex for coronaviruses. certain doublemembrane vesicles also contain dark granular material interspersed among viral nucleocapsid-like particles (goldsmith et al., 2004; knoops et al., 2008; le et al., 2011) , while virions bud from the cytosol. however, we could not identify any opening or pore in these vesicles or in the er that would allow movement of the viral rna from the vesicles or er compartments to the cytosol. replication of ndiv in c6/36 mosquito cells was followed by a maturation period on the plasma membrane during which viral nucleocapsid-like particles were inserted into "protrusions" of this membrane to export the complete virions and the incomplete virions lacking the nucleocapsid. assembly and budding of ndiv have characteristics in common with those of other members of several rna virus families such as the togaviridae, rhabdoviridae, paramyxoviridae, orthomyxoviridae and retroviridae (ahmad et al., 2011; haenni and diaz-ferrao, 2011; hunter, 2001; rota et al., 2003; zhong et al., 2013) . assembly of ndiv at the plasma membrane obviates the need for the assembled particle to navigate to an additional compartment of the secretory pathway because the virions are released directly into the external milieu of the cell. for most enveloped viruses, the location within the cell where envelopment takes place is determined by targeting of the viral glycoprotein(s) at that site (hunter, 2001) . however, the proteins of ndiv have only been identified by molecular biology techniques (nga et al., 2011) , so that the biological functions of these proteins are not fully understood. furthermore, viral nucleocapsid-like particles may be morphologically confused with other structures in the cytoplasm such as coated vesicles, multivesicular bodies, perichromatin granules and glycocalyceal bodies. immunogold em of ultrathin sections confirmed that these viral nucleocapsid-like particles belong to ndiv and appear in the cytoplasm and endoplasmic compartments of the host cells, whereas no viral structures were found in the mitochondria or the nucleus. ndiv isolated from c6/36 cells and highlighted by negative staining presented a homogenous spherical shape. yet ndiv differs from cavally virus, a novel insect nidovirus characterized in 2011 as an enveloped spherical cov-like virion with a diameter of 120 nm, and "club-shaped" surface projections (zirkel et al., 2011) . ndiv is an enveloped virus, whose projections or spikes on the membrane of the viral envelope remain very short, 3-4 nm long and that can identify and bind to receptor sites on the host cell membrane. in addition to the negative staining method, the ndiv was examined by immunogold labeling that expanded the range of applicability of the negative staining technique (beesley and betts, 1985; nguyen et al., 2001) . immunogold em makes it possible to detect and locate proteins of antigens in viruses or cells and can help connect a visible structure with a specific in situ localization site and establish the distribution of a molecule at high resolution (beesley and betts, 1985; de paul et al., 2012; hyatt and eaton, 1993; nguyen et al., 2001; kanak et al., 2002) . immunogold em on unfixed particles of ndiv revealed labeling of the envelope protein(s) by the presence of 10 nm gold particles around isolated virions. in our experiments, ndivs are labeled with an average of 15 gold particles per virion using polyclonal antibodies prepared against the entire viral particle. this value probably reflects the topographic distribution of the viral proteins in non-fixed virus particles, and the accessibility of specific epitope constituents. however, its resolution is limited by the steric hindrance generated by the packing density of the antigenic sites associated with different constituents of the viral envelope. ndiv possesses four virion proteins (p2a, p2b, p3, and p4) as demonstrated previously but the number of antigenic sites of ndiv is still unknown (nga et al., 2011) . with respect to nidoviruses, information concerning the viral envelope proteins of members of the arteriviridae and coronaviridae families is available (dubois-dalcq et al., 1984; pedersen et al., 1999) , but information on the ndiv envelope proteins as well as on the biological functions of these proteins during replication in the host cell requires further studies with the support of specific monoclonal antibodies against the ndiv proteins. using em methods, this study reports valuable information on ultrastructural aspects of ndiv grown in c6/36 mosquito cells for 24-48 h after infection. the results demonstrate that ndiv possesses a homogenous spherical shape, with a diameter of 60-80 nm. by em analyses, we show that replication and assembly of ndiv was only detected in the cytoplasm of the host cells in which viral nucleocapsid-like particles appeared in the cytoplasm and in the endoplasmic compartments such as vacuoles, er, and vesicles, but not in mitochondria. viral particles were released 48 h post-infection via budding at the surface of the plasma membrane. ndiv obtained from the national institute of hygiene and epidemiology, hanoi, vietnam, was identified in four mosquito pools, two from culex vishnui and two from culex tritaeniorhynchus, collected in two provinces of vietnam. mosquito handling for virus isolation and propagation in cell culture was described previously (nga et al., 2011) . virus stock titers were determined via the plaque assay using c6/36 mosquito cells, and stocks were stored at à 80 1c. c6/36 cells were infected at a low multiplicity of infection of 0.1, with the virus obtained from limiting dilution endpoints of early-passage supernatants (zirkel et al., 2011) . ultrathin sections c6/36 mosquito cells grown at 28 1c were infected with 2 â 10 4 pfu/ml of ndiv as described previously (nga et al., 2011) . twenty-four and 48 h after infection, most of the medium and floated cells were discarded, the cells were detached from the culture flask with a cell scraper, and the cell suspension was centrifuged at 2000 g for 5 min. the pellet was re-suspended in phosphate buffered saline (pbs) ph 7.2 without ca ++ and mg ++ , and the suspension was spun at 2000 g for an additional 5 min. the cell pellet was re-suspended and fixed in 2.5% glutaraldehyde in 0.1 m cacodylate buffer ph 7.2 and kept overnight at 4 1c. the sample was then rinsed carefully several times in 0.1 m cacodylate buffer ph 7.2; the final pellet was fixed with 1% oso 4 in the same buffer for 60 min and subsequently dehydrated in graded ethanol (50-100%), washed in propylene oxide and infiltrated for 6 h in a 1:1 mixture of propylene oxide and epoxidic resin (epon). the cells were finally embedded in epon 812. ultrathin 70 nm thick sections were obtained with an ultramicrotome (ultracut uc6, leica) and stained with uranyl acetate and lead citrate. c6/36 cells infected for 48 h with ndiv were detached as described above, washed with pbs and the final pellet was fixed in 3% paraformaldehyde in pbs, ph 7.4, for 1 h. after dehydration in graded ethanol (50-100%) the cells were embedded in lr white resin (ems). after polymerization of the resin for 48 h at 37 1c, the ultrathin 70 nm thick sections were collected on nickel mesh grids and processed for immunolabeling. in the present study, three flasks each containing ndivinfected c6/36 cells prepared in the same conditions and time points after infection were collected; em observations were performed on ultrathin sections and at least 10-15 sections were examined per sample per time point. the virus was concentrated from 48 h-infected cell culture fluid, by centrifugation at 12,000 g for 30 min at 4 1c, after which 6.6% polyethylene glycol 6000 and 2.2% nacl were added to the supernatant. after stirring for 1 h at 4 1c and centrifugation at 12,000 g for 1 h, the supernatant was discarded. the viruscontaining pellet was re-suspended in saline-tris-edta buffer ph 7.4, sedimented at 250,000 g for 1 h and resuspended a second time. purified viral material was used for antibody preparation, and em studies. for negative staining, 30 ml droplets of ndiv suspensions were put on carbon-coated grids for 10 min to pick up the virus sample; the grids were then fixed in 1% glutaraldehyde for 5 min, washed several times in 0.1% ammonium acetate and stained with 1% uranyl acetate for 5 min. for immunogold labeling of the ndiv suspension, the method used was that of nguyen et al. (2001) . droplets of ndiv suspension (30 ml) were placed on a clean parafilm surface and collodium carbon-coated grids were placed on top of the droplets for 10 min to pick up the virus sample. the grids were washed with pbs and allowed to float on a droplet of pbs with 2% bovine serum albumin (bsa) for 30 min to block non-specific antigenic sites. the grids were then made to react for 30 min with anti-ndiv rabbit antibodies diluted in pbs-0.5% bsa. after careful washing with pbs-0.2% bsa several times for 10 min, the grids were incubated with protein a conjugated to 10 nm gold particles for 30 min. this step was followed by several washes with pbs as above and the grids were fixed with 0.1% glutaraldehyde for 5 min. they were then washed thoroughly with 0.1% ammonium acetate and negative stained with 1% uranyl acetate for 5 min. the procedure of immunogold em on ultrathin sections was performed similarly (hyatt and eaton, 1993) . control experiments testing the specificity of the immunolabeling were carried out by incubating some of the samples directly with gold-labeled protein a, without previous incubation with specific antibodies. other control experiments were carried out by incubating some samples with non-specific antibodies. the grids were not allowed to dry during the procedure and always floated on the surface of the drops. all steps were performed at room temperature. the samples were observed through a transmission em jem1010-jeol, operating at 80 kv. polyclonal antibodies were prepared using the entire isolated viral particles as antigen; the dose injected into rabbits was 50 mg ndiv particles diluted in 0.5 ml pbs and mixed with freund complete adjuvant, in a volume ratio of 1:1 on days 1, 7, 14, and 21 (four booster injections). the dose injected was increased to 150 mg ndiv particles diluted in 0.5 ml pbs mix with titermax classic adjuvant, in a 1:1 ratio for the fifth booster injection on day 35. two weeks after the fifth injection, the igg titer of the antibodies against ndiv was determined by the indirect elisa method and the iggs were separated; blood was taken from the rabbit heart, kept at room temperature for 30 min, and then at 4 1c overnight. the blood was centrifuged at 2500 rpm for 15 min, and the serum was separated and kept in aliquots at à 20 1c. hiv-1 replication and gene expression occur at higher levels in neonatal blood naive and memory t-lymphocytes compared with adult blood cells virus diagnostic: a novel use for the protein a-gold probe immunoelectron microscopy: a reliable tool for the analysis of cellular processes seeking membranes: positive-strand rna virus replication complexes assembly of enveloped rna viruses viruses from their first description to epidemics virus assembly immuno gold electron microscopy in virus diagnosis and research optimization of immunogold labeling tem: an elisa-based method for evaluation of blocking agents for quantitative ditection of antigen nidovirus replication structures: hijacking membranes to support viral rna synthesis sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum ultrastructural characterization of arterivirus replication structures: reshaping the endoplasmic reticulum to accommodate viral rna synthesis structural and immunocytochemical features of sars-cov-associated coronavirus grown in cell culture discovery of the first insect nidovirus, a missing evolutionary link in the emergence of the largest rna virus genomes immunocytochemical characterization of viruses and antigenic macromolecules in viral vaccines open reading frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulumderived double-membrane vesicles which carry the viral replication complex characterization of a novel coronavirus associated with severe acute respiratory syndrome coronaviruses, toroviruses, arteriviruses cell-to-cell transmission of viruses an insect nidovirus emerging from a primary tropical rainforest we gratefully acknowledge our colleague anne-lise haenni (institut jacques monod), for assistance, advice, helpful discussions and constant support. this work was supported by ministry of health of vietnam and by grants from the ministerial project (no. 4865/qd-byt). key: cord-288669-46tkedw7 authors: lee, changhee; yoo, dongwan title: the small envelope protein of porcine reproductive and respiratory syndrome virus possesses ion channel protein-like properties date: 2006-11-10 journal: virology doi: 10.1016/j.virol.2006.07.013 sha: doc_id: 288669 cord_uid: 46tkedw7 the small envelope (e) protein of porcine reproductive and respiratory syndrome virus (prrsv) is a hydrophobic 73 amino acid protein encoded in the internal open reading frame (orf) of the bicistronic mrna2. as a first step towards understanding the biological role of e protein during prrsv replication, e gene expression was blocked in a full-length infectious clone by mutating the atg translational initiation to gtg, such that the full-length mutant genomic clone was unable to synthesize the e protein. dna transfection of prrsv-susceptible cells with the e gene knocked-out genomic clone showed the absence of virus infectivity. p129-δe-transfected cells however produced virion particles in the culture supernatant, and these particles contained viral genomic rna, demonstrating that the e protein is essential for prrsv infection but dispensable for virion assembly. electron microscopy suggests that the p129-δe virions assembled in the absence of e had a similar appearance to the wild-type particles. strand-specific rt-pcr demonstrated that the e protein-negative, non-infectious p129-δe virus particles were able to enter cells but further steps of replication were interrupted. the entry of prrsv has been suggested to be via receptor-mediated endocytosis, and lysomotropic basic compounds and known ion-channel blocking agents both inhibited prrsv replication effectively during the uncoating process. the expression of e protein in escherichia coli-mediated cell growth arrests and increased the membrane permeability. cross-linking experiments in cells infected with prrsv or transfected with e gene showed that the e protein was able to form homo-oligomers. taken together, our data suggest that the prrsv e protein is likely an ion-channel protein embedded in the viral envelope and facilitates uncoating of virus and release of the genome in the cytoplasm. porcine reproductive and respiratory syndrome virus (prrsv) has plagued the global swine industry leading to significant economic losses for pig production worldwide (nuemann et al., 2005) . prrsv is a member of the family arteriviridae, which includes equine arteritis virus (eav), lactate dehydrogenase-elevating virus (ldv) of mice and simian hemorrhagic fever virus (shfv) (meulenberg et al., 1993; snijder and meulenberg, 1998) . along with the family coronaviridae, arteriviruses are grouped in the new order of nidovirales (snijder et al., 2005) . despite the similar virion morphology and genome organization, prrsv is divided into two genotypes, the european genotype (lelystad virus; lv) and the north american genotype, based on their antigenic and genetic characteristics (meng et al., 1995; nelsen et al., 1999; nelson et al., 1993; wootton et al., 2000) . prrsv is a small, enveloped virus possessing a singlestranded positive-sense rna of ∼ 15 kb in size with a 5′ cap and a 3′ polyadenylated tail (meulenberg et al., 1993; sagripanti et al., 1986; snijder and meulenberg, 1998; wootton et al., 2000) . the prrsv genome consists of the 5′ untranslated region (utr) , nine open reading frames (orf1a, orf1b, orf2a, orf2b and orfs 3 through 7) and the 3′ utr (meulenberg et al., 1993; snijder and meulenberg, 1998; wootton et al., 2000) . two large orfs la and lb occupy the 5′ two-thirds of the genome and encode 13 non-structural proteins, which are suggested to be involved in the genome replication and transcription (bautista et virology 355 (2006) 30 -43 www.elsevier.com/locate/yviro al., 2002; van dinten et al., 1999) . the remaining orfs, 2a through 7 in the 3′ terminal 3 kb region, encode six membraneassociated proteins (gp2, e, gp3, gp4, gp5 and m) present in the envelope and nucleocapsid (n) protein (meulenberg et al., 1995; wootton et al., 2000) . mature virions are spherical, enveloped particles with a diameter of 50-65 nm and contain a 20-to 30-nm isometric core structure enclosing the genomic rna (benfield et al., 1992; dea et al., 1995) . the small envelope (e) protein is a newly identified structural component in arteriviruses. the prrsv e protein, also known as 2b protein, is translated from the internal orf (orf2b) starting from the + 6 nucleotide position in mrna2 (fig. 1a) . the e protein is 73 and 70 amino acids for the north american and european type of prrsv, respectively. the e protein is highly hydrophobic but contains a cluster of basic amino acids in the hydrophilic c-terminal region. the e protein is non-glycosylated and intracellular membrane-associated wu et al., 2001) . in prrsv-infected pigs, the e protein induces specific antibody (wu et al., 2001) . recent studies with a european prrsv isolate showed that the e protein is incorporated into the virions in association with gp2-gp3-gp4 heterotrimers, suggesting a critical role for the heteromultimeric complex in the virus entry process (wissink et al., 2005) . although the e protein of north american genotype prrsv contains two cysteine residues at positions 49 and 54, a study has shown that e is unable to form disulfide-linked homodimers . in that study, cysteine residues of the e protein were shown to be non-essential for virus multiplication. the function and significance of e in prrsv replication remain to be determined. in the present study, we investigated the role of e protein during prrsv replication. an infectious cdna clone was used to generate an e gene-knockout mutant prrsv, and we report here that the e protein is essential for virus infectivity but dispensable for virus particle formation. the e protein-negative, non-infectious virus particles were able to enter cells but unable to continue the further steps of replication. furthermore, two ion channel blockers were shown to greatly affect prrsvreplication at early stages of infection, suggesting that ion channel activity was essential for virus uncoating. we also found that expression of the e protein enhanced membrane permeability of hygromycin b in bacterial cells. cross-linking studies revealed that the e protein associated with itself into higher-order structures, including dimers, trimers and tetramers. our study suggests that the prrsv e protein may function as a viroporin in the virion envelope that facilitates uncoating of the virus in order to release the genomic rna into the cytoplasm for subsequent replication. to determine the biological significance of e protein during prrsv infection, an e gene-knockout mutant virus was fig. 1. (a) the partial genome organization of prrsv. genomic locations of gp2 and e genes and the e gene-knockout are illustrated. (b) absence of infectivity of the e gene-knockout full-length clone for prrsv, p129-δe. marc-145 cells were transfected with the full-length cdna genomic clone of p129-wt or p129-δe and incubated for 5 days. prrsv-specific cpes were monitored daily and photographed 4 days post-transfection (upper panels). for immunofluorescence, cells were fixed with cold methanol at 2 days post-transfection and incubated with the n-specific mab sdow-17 (lower panels) (magnification 20×). (c) double staining for n (green) and nsp2/3 (red) proteins for p129-wt (upper panels) or p129-δe (lower panels). marc-145 cells transfected with p129-wt or p129-δe plasmid dna were fixed at 2 days post-transfection and co-stained with nsp2/3-specific rabbit antiserum and n-specific mab sdow17. yellow indicates merged images where both n and nsp2/3 are co-localized. generated using an infectious cdna clone. the start codon of orf2b (e gene) was modified to abolish the e protein expression. with the shuttle plasmid by site-directed mutagenesis, 'atg' for translation initiation of orf2b was changed to 'gtg' at genomic positions 12,062 to 12,064. this mutation did not alter the amino acid sequence in orf2a encoding gp2 protein. the a12062g modification was subsequently introduced into the full-length genomic cdna clone by subcloning the eco47iii-bsrgi fragment obtained from the shuttle plasmid. transformants were screened by restriction patterns using xmai to determine the ligated clone, followed by nucleotides sequencing to verify the specific modification in the full-length genome. the resulting e gene-knockout genomic clone was designated p129-δe. the infectivity of the e gene-knockout clone p129-δe was examined by transfection of marc-145 cells. cells were transfected with p129-wt or p129-δe, and the appearance of cpe was monitored daily. p129-wt induced visible cpe at 3 days post-transfection and showed n-specific staining in clusters of cells, indicating the infection and spread of virus to neighboring cells (fig. 1b , left panels). in contrast, transfection of p129-δe did not produce any visible cpe for up to at least 7 days post-transfection, suggesting the lack of infectivity. a few single cells exhibited n-specific fluorescence, and these cells represent individually transfected cells with p129-δe (fig. 1b) . to rule out a possibility that an additional mutation might have been introduced during construction of p129-δe, which may have resulted in the loss of infectivity, six individual mutant clones were independently generated. in these clones, no mutation was identified for the orf2 gene, and upon transfection of cells, all six p129-δe mutant clones were noninfectious (data not shown). this confirmed the conclusion that p129-δe was non-viable and the absence of infectivity was due to the lack of e protein expression, demonstrating the essential role of e for prrsv replication. the transcription ability of p129-δe was examined by double staining of transfected cells using the alexa greenlabeled sdow17 mab specific for n and the texas red-labeled rabbit antiserum specific for nsp2/3 non-structural proteins (fig. 1c) . in cells transfected with p129-wt dna, clusters of cells were stained by both antibodies, indicating both n (green) and nsp2/3 (red) expressions (fig. 1c , upper panels). merging of the two images showed yellow regions where the two proteins colocalized. for p129-δe, dual-staining of n (green) and nsp2/3 (red) was also observed but limited to transfected cells only, and no evidence for the spread of infection was obtained (fig. 1c , lower panels). the dual staining demonstrated the expression of nsp2/3 and n proteins in transfected cells, indicating the synthesis of both non-structural and structural proteins, which in turn suggests that the prrsv genome replicated and mrna transcription occurred upon transfection of p129-δe dna. the results demonstrate that e protein expression is required for prrsv infectivity, but genome replication and transcription may occur without e protein. it leads us to hypothesize that either virion assembly or virus entry is interrupted in the absence of e protein expression. we next examined whether the infectivity of p129-δe could be restored by provision of the e protein in trans. the e gene was expressed in bhk-t7 cells and the expression was confirmed by immunofluorescence staining with e-specific antiserum ( fig. 2a) . bhk-t7 cells were also suitable for transfection of the full-length cdna clone as n-specific staining was detected in many cells (figs. 2b and e) . to determine whether p129-δe in the e gene-transfected cells leads to the production of infectious virus particles, bhk-t7 cells were co-transfected with p129-δe and e gene. the culture supernatant was harvested at 48 h post-transfection from bhk-t7 cells and transferred to marc-145 cells. at 24 h postinoculation, marc-145 cells were fixed, and virus infection was examined by immunofluorescent staining with n-specific mab. marc-145 cells inoculated with a culture fluid from cells cotransfected with p129-δe and e gene showed bright n-specific fluorescent signal, indicating that the p129-δe replication may be rescued by trans-complementation of the e protein (fig. 2c ). in contrast, in cells inoculated with the supernatant from co-transfection of p129-δe and n gene (fig. 2f) , or of p129-δe and an empty plasmid (data not shown), no staining was observed. the staining of marc-145 cells inoculated with the supernatant from bhk cells co-transfected with p129-δe and n gene was however limited to single cells and no infectivity was produced for up to 7 days post-inoculation (fig. 2c) , indicating abortive infection of the p129-δe mutant virus in non-complementing marc-145 cells. attempts to pass the passage-1 culture supernatant to marc-145 resulted in no virus replication, confirming that the p129-δe virus particles generated by the provision of e in trans was only capable of a single round of replication. these data demonstrated that the inability of p129-δe to produce infectious virus was due to the absence of e protein in the virions. because p129-δe did not induce infectivity from transfection, it was of interest to examine whether prrsv particles were not produced in the absence of e protein. due to a low transfection efficiency in marc-145 cells, bhk-21 cells were used to achieve higher transfection efficiency. when stained with n-specific mab at 2 days post-transfection, numerous cells showed n-specific fluorescence (data not shown), indicating high levels of transfection in bhk-21 cells, which may be sufficient for the study of particle formation. bhk-21 cells were transfected with p129-wt or p129-δe, and at 30 h post-transfection, the transfected cells were radiolabeled for 1 day with [ 35 s]methionine/cysteine. the cells were harvested and lysed with ripa buffer. the culture supernatants were collected separately and centrifuged through a 20% (wt/vol) sucrose cushion, and the resulting pellets were dissolved in ripa buffer. the lysates prepared from cells or supernatants were immunoprecipitated with a mixture of individual antibody specific for e, n, or m and resolved by sds-page. as shown in fig. 3a , intracellular major viral-specific proteins were detectable in all lysates (lower panel), confirming the ability of p129-δe for transcription. similarly, in the culture supernatants, the three major virion proteins gp5, m and n were clearly identified (upper panel), showing that, without e protein, virus particles can be produced and released. it was not possible to detect the e protein in the cell lysates or supernatants from p129-wt-transfected cells, and this is probably due to the low abundance of e protein in bhk-21 cells as e is a minor protein (wu et al., 2005) . this result suggests that the prrsv e protein is not required for particle formation. because the virus particles formed in the absence of e protein were non-infectious, these particles were examined for the presence of viral genome. the culture supernatants, prepared from bhk-21 cells transfected with p129-wt or p129-δe, were first incubated with both rnase a and dnase i to remove any possible contamination of viral rna and the transfected dna from cells. the digestion was carried out in the presence or absence of detergents, followed by proteinase k treatment to inactivate the nucleases. rna was extracted from samples and treated again with dnase i, and the e gene region of prrsv was rt-pcr-amplified and sequenced. in cell culture media mixed with the full-length cdna clone as a negative control, no pcr fragment was amplified in the presence or absence of detergents (fig. 3b , lanes 2 and 3), indicating the appropriateness of dnase i treatment. although no amplification was identified for controls ( fig. 3b , lanes 5 and 7), a 260-bp product was specifically amplified for both samples of p129-wt and p129-δe treated with the nucleases but in the absence of detergents (fig. 3b , lanes 4 and 6). sequencing of the 260-bp product confirmed the stable incorporation of the 'a12062g' mutation at the start codon of the e gene in the genome from p129-δe particles. these studies demonstrate that particles formed in the absence of e protein contained the p129-δe genomic rna, which in turn ]methionine/cysteine for 24 h at 37°c, and the supernatants and cells were separately collected. cell lysates were prepared and subjected to immunoprecipitation with n-specific mab (lower panel). the pellets were prepared from culture supernatants by ultracentrifugation, lysed in ripa buffer and used for immunoprecipitation with a mixture of anti-m, anti-n and anti-e-specific antibodies, followed by sds-15% page under reducing conditions (upper panel). lane 1, mock transfected; lane 2, p129-wt transfected; lane 3, p129-δe transfected. (b) incorporation of genomic rna in p129-δe virus particles. culture supernatants were pelleted by ultracentrifugation and the pellets were treated with nucleases in the presence (+) or absence (−) of sds and triton x-100, followed by proteinase k treatment. rt-pcr was conducted for e gene amplification followed by electrophoresis in 1.5% agarose gel. lane 1, molecular weight marker; lanes 2 and 3, culture supernatant spiked with the p129-δe full-length plasmid; lanes 4 and 5, supernatant from p129-wt-transfected cells; lanes 6 and 7, culture supernatant from p129-δe-transfected cells. (c) electron microscopy of the culture supernatant from p129-δe-transfected cells. particles in the culture supernatants released from bhk-21 cells transfected with p129-wt (upper panel) or p129-δe (lower panel) genomic clones were concentrated by ultracentrifugation through a 20% (wt/vol) sucrose cushion. pellets were negatively stained by sodium phosphotungstate and visualized by electron microscopy. scale bar, 100 nm. suggests that the lack of infectivity of p129-δe was not due to the improper packaging of the genome. to obtain further evidence for particle formation in the absence of e, electron microscopy (em) was conducted and the microscopic appearance of p129-δe particles was compared to that of p129-wt virions. at 2 days post-transfection, the culture supernatant from bhk-21 cells was harvested and pelleted by ultracentrifugation through a 20% (wt/vol) sucrose cushion, followed by electron microscopy. prrsv particles were identified in the supernatant of cells transfected with either p129-wt (fig. 3c , upper panel) or p129-δe (fig. 3c , lower panel). no significant morphologic differences between p129-wt and p129-δe particles were noted. each virion for p129-wt or p129-δe was a roughly spherical enveloped particle of 50-60 nm in diameter, with a densely stained core. the em study confirmed that prrsv particles may be formed in the absence of e protein and also showed that the e proteinnegative particles had similar appearance and size to wild-type prrsv particles. to determine if the lack of infectivity of p129-δe particles was due to a low amount of virus produced from bhk-21 cells, the supernatants collected from bhk-21 cells were blindly passaged twice in marc-145 cells to amplify the infectivity. although extensive cpe was readily evident in marc-145 cells inoculated with either passage-1 or passage-2 of p129-wt supernatant, no cpe was detectable with passage-1 or passage-2 of p129-δe supernatant, even after 5 days postinoculation (data not shown). to further determine infectivity in marc-145 cells inoculated with p129-δe virus, time course immunofluorescence was carried out with the n-specific antibody. as shown in fig. 4a , distinct staining of n was first observed at 12 h post-infection in marc-145 cells inoculated with p129-wt supernatant, and then in many clusters of cells by 48 h post-infection, showing the spread of infectivity to neighboring cells (upper panels). in contrast, cells inoculated with passage-1 of p129-δe showed no specific staining throughout the experiments, further indicating the lack of infectivity (lower panels). because neither cpe nor n-specific staining was detectable from serial passages of p129-δe virus, rt-pcr was conducted from cells and culture supernatants inoculated with passage-1 or passage-2 (fig. 4b ). to eliminate a possible carry-over contamination of the transfected dna, the rna preparations were treated with rnase-free dnase i prior to the e gene amplification. a 260-bp product was amplified from both culture media and cells that were inoculated with passage-1 or passage-2 p129-wt virus (fig. 4b, lanes 2, 5, 8 and 11 ). the 260-bp product was also obtainable from cells inoculated with passage-1 p129-δe virus (fig. 4b, lane 3) , but no specific product was amplified from the supernatant (fig. 4b, lane 6) , nor from either supernatants or cells inoculated with passage-2 p129-δe virus (fig. 4b, lanes 9 and 12) . the amplified fragment was sequenced, and the sequencing results confirmed the 'a12062g' mutation at the start codon of orf2b (data not shown). these results suggest that the e protein-negative, noninfectious p129-δe virus particles entered marc-145 cells, but further steps beyond entry were interrupted, resulting in the absence of infectivity. to investigate whether the p129-δe virus genome underwent replication following entry, strand-specific rt-pcr was performed from cells inoculated with passage-1 p129-δe virus. rt-pcr from p129-wt-inoculated cells yielded a specific product of the expected size for both positive-and negativestrand genomes and also for positive-and negative-strand mrna7 (fig. 4c , lanes 3 in all panels). in contrast, only a minimal amount was amplified for the positive-strand genome from cells inoculated with passage-1 p129-δe virus (fig. 4c , panel i, lane 4). this product was likely derived from the incoming genomic rna of p129-δe virus. these results indicate that p129-δe virus was unable to replicate, suggesting that the lack of rna replication of p129-δe virus was likely due to an interruption during virus uncoating, a stage prior to genome replication. the e-protein-negative prrsv particles were shown to contain the viral genome and able to enter cells but unable to release the viral rna for initiation of genome replication. this suggests that prrs virions may contain ion channels and the e protein may function as an ion channel protein embedded in the viral envelope. it is postulated that prrsv replication may be suppressed by ion channel blocking agents if ion channel activity is an essential requirement for prrsv infection. this aspect was investigated in marc-145 cells using amantadine and verapamil. amantadine is known as a proton channel blocker and verapamil is the calcium channel blocking agent. because it has been reported that an acidic environment is required for prrsv infection (kreutz and ackermann, 1996) , basic lysosomotropic agents including ammonium chloride and chloroquine were also included in the study to examine their inhibitory effects on prrsv replication. strand-specific rt-pcr experiments were carried out and demonstrated that prrsv-infected marc-145 cells produced reduced levels of positive-sense genomic rna at 2 days post-infection in the presence of the drugs chloroquine, amantadine and verapamil (fig. 5a, upper panel) . a relatively moderate level of suppression was observed in ammonium chloride-treated cells (lane 4). negative-sense genomic rna was not detectable in cells treated with any of the four drugs until 2 days post-infection (middle and lower panels, lanes 4-7), showing that the initiation of viral rna synthesis was inhibited by the ion channel blockers. furthermore, the number of prrsv-infected cells determined by the n protein staining was significantly reduced by individual drugs in comparison to untreated cells (fig. 5b , upper panels). prrsv-specific cpe was visible but significantly delayed by the ion channel blockers, indicating the negative effects on virus production (lower panels). the yields of virus production were determined by plaque assays from the supernatants in the presence of each drug. the virus titer in untreated cells reached 3 × 10 6 pfu/ml by 3 days post-infection (fig. 5c ). in contrast, fig. 4 . the e protein is essential for prrsv replication. (a) immunofluorescence of n protein in p129-δe inoculated cells. the 'passage-1' virus was prepared as culture supernatant harvested from bhk-21 cells transfected with p129-wt or p129-δe clone. marc-145 cells were inoculated with 'passage-1' and fixed at the indicated times post-inoculation, followed by staining with n-specific mab. (b) detection of viral rna in culture supernatants and in marc-145 cells inoculated with 'passage-1' or 'passage-2' virus. total rna was extracted and treated with dnase i followed by rt-pcr for e gene. (c) strand-specific detection of viral rna in cells by rt-pcr. marc-145 cells were inoculated with 'passage-1' p129-wt or 'passage-1' p129-δe, and total cellular rna was extracted at 2 days post-inoculation. the rna was treated by dnase i and rt-pcr was conducted to amplify the region as illustrated in the figure. numbers in parenthesis indicate the 5′ most nucleotide position in each primer with respect to the prrsv genome. expected sizes of amplified products are indicated on the right of the gel. there was no detectable virus production at 1 day post-infection in the presence of amantadine or verapamil, and the titers of virus production in the presence of these drugs reached only to a maximum of 5 to 8 × 10 1 pfu/ml by 3 days post-infection (fig. 5c) . these results showed that the treatment of infected cells with ion channel blockers greatly reduced the growth rate of prrsv. however, when marc-145 cells were treated with the drugs at 30 min post-infection, no significant inhibitory effect of virus production was observed (data not shown). therefore, it seems that the ion channel blockers effectively interfered with virus uncoating, a step preceding genome replication and consequently affected prrsv production. it is interesting to note that the inhibitory effect by amantadine are due to the blocking of pores that are necessary for post-internalization during uncoating of the virus (wang et al., 1993) . because our data suggest that ion channel activity may be involved during uncoating and that the prrsv e protein is likely responsible for this event, the e protein was assumed to function as a viroporin. we examined whether the e protein possessed general features commonly shared by other viroporins (ion channel proteins). viroporins generally contain the properties of membrane permeability alteration and oligomerization (liao et al., 2004; maldarelli et al., 1993; paterson et al., 2003; sakaguchi et al., 1997; torres et al., 2005) , and so these two common properties were examined for the e protein. the effect of e expression on bacterial growth was first examined in an inducible protein expression system. when the inner bacterial membrane is intact, intracellular lysozymes cannot reach the cell wall. however, permeabilized membranes allow the lysozymes to gain access to the peptidoglycan, leading to cell lysis. this approach has been shown to be suitable as a permeabilization test of the inner bacterial membrane for viral proteins (bodelón et al., 2002; ciccaglione et al., 1998; liao et al., 2004; madan et al., 2005) . the prrsv e protein gene was first cloned in the gst-fusion vector and expressed in escherichia coli. the growth rates of transformed cells were analyzed spectrophotometrically. a drastic arrest of cell growth was observed in the e gene-transformed cells upon iptg induction, whereas bacteria carrying the empty plasmid or the prrsv n gene had no effects on their growth during the 300-min period following induction (fig. 6a ). this finding suggests that the expression of e affected the cell growth negatively by altering the membrane permeability to intracellular lysozyme. to further determine if the growth rate of cells expressing e was the consequence of permeability modification, a hygromycin b permeability assay was carried out. hygromycin b is normally impermeable to the membrane barrier during a short period of time at low concentration, but it can readily penetrate the permeabilized membrane to cause strong inhibition of intracellular protein synthesis. the hygromycin b permeability assay therefore is widely used to study changes of membrane permeability as well as to identify proteins that can form pores in lipid membranes (aldabe et al., 1996; arroyo et al., 1995; bodelón et al., 2002; chang et al., 1999; ciccaglione et al., 1998; de jong et al., 2003; doedens and kirkegaard, 1995; han and harty, 2004; liao et al., 2004; madan et al., 2005) . at 1 h induction for e protein expression, hygromycin b was added to the culture media. the cultures were further incubated for 15 min and metabolically labeled with [ 35 s]methionine/cysteine for 15 min, followed by sds-page of cell lysates. the coomassie blue staining showed that equal amounts of proteins were loaded on the gel (fig. 6b, left panel) . autoradiography indicated that hygromycin b entered cells that expressed the e protein, and protein synthesis was completely blocked in those cells (fig. 6b, right panel, compare lanes 5 and 6, lanes 7 and 8) . for cells expressing the gst or prrsv n protein, hygromycin b did not inhibit protein synthesis (compare lanes 1 and 2, lanes 3 and 4) . these results indicate that the prrsv e protein enhanced membrane permeability in bacterial cells. a second common property of viroporins is oligomerization. the prrsv e protein contains two well-conserved cysteine residues; however, the e protein was shown not to form a disulfide-linked homodimer . the absence of covalently linked homodimers of e was confirmed using the recombinant e protein expressed in hela cells by vtf7-3 vaccinia virus (fig. 7a) . although prrsv n-n dimers of 30 kda were readily detected in cells expressing the n protein (lane 1), no band corresponding to the predicted dimeric form of e was identified in non-reducing conditions (lane 3), concluding that the prrsv e protein does not undergo cysteine-linked homodimerization. because the e protein may form a non-covalent association with itself as shown by gst-pull down assay , the potential of e to form oligomers was further investigated by cross-linking experiments in the presence and absence of other viral constituents. radiolabeled prrsvinfected cells were treated with the membrane-permeable cross-linking reagent dsp, and the cell lysates were immunoprecipitated by n-specific mab or e-specific antiserum followed by sds-page under non-reducing conditions (fig. 7b) . the prrsv n protein formed a number of higher-order oligomers (lanes 1, 2) , as reported previously (wootton and yoo, 2003) . when the e protein in virus-infected cells was subjected to cross-linking, numerous multimeric forms of e protein were identified (lane 3). the e protein multimerization also was examined in the absence of other prrsv proteins in hela cells expressing e protein by t7 vaccinia virus, and again higher-order multimeric forms of the e protein were observed (lane 4), indicating that oligomerization of e is independent of other viral proteins. altogether, our data indicate that the prrsv e protein exists as non-covalently linked oligomers in virus-infected cells and suggest that the multimerization may be the physical basis for viroporin formation of prrsv e. the present study was conducted to investigate the role of prrsv e protein during virus infection. a reverse genetics approach was used to modify the translation initiation of orf2b, so that the modified genome was unable to express the e protein. our experiments show that the absence of e protein expression does not affect genome replication transcription but impairs the production of infectious virus. these data indicate that the prrsv e protein is an essential structural component for infectivity. the e protein however appears not to be an essential component for particle assembly nor genomic encapsidation. the virion particles devoid of e still contained the viral genome and had a similar appearance to that of wildtype virions. for infection, virus particles must proceed through a multiple-step cycle of entry and uncoating, replication and transcription and assembly and release. according to our findings, the e protein does not exert an important function at later events in the infection process such as genome replication, particle assembly and release of virus. we have observed that the e protein-negative, non-infectious virion particles are able to enter cells but subsequent steps of replication are inhibited. this suggests that the particles lacking the e protein most likely remain in the endosome and the viral genome is not released, which is the step between virus entry and genome replication. the uncoating is a critical step for virus infection, during which the lipid envelope is shed and the viral capsid is disassembled to release the genome to initiate a replication cycle in the cytoplasm. for viruses entering cells by receptor-mediated endocytosis, the uncoating process occurs in the endosome where an acidic environment triggers fusion between the viral membrane and the endosomal membrane (reviewed by smith and helenius, 2004) . prrsv enters cells through receptormediated microfilament-dependent endocytosis in which a low ph is required to trigger fusion for proper uncoating by an unknown mechanism (kreutz and ackermann, 1996; nauwynck et al., 1999) . it is possible that an acidic ph in the endosome may lead to conformational changes of viral protein (s) to expose hidden fusogenic domains that facilitate the fusion between viral and endosomal membranes. however, no direct fusion of prrsv with the cell membrane, and no arterivirus membrane proteins possessing the fusogenic property have been reported. it is therefore tempting to speculate that prrsv may contain a viral ion channel protein to promote the uncoating process in the endosome during the early stage of infection. in the present study, we have shown the inhibitory effect of ion channel blockers on prrsv replication, indicating that prrsv may indeed possess virus-coded ion channels whose activity is essential for proper uncoating for infection. virus-coded ion channels, or viroporins, consist of small, highly hydrophobic proteins generally composed of 60-120 amino acids. the insertion of these proteins into membranes creates typical hydrophilic pores or selective ion channels with (a) hydrophobic transmembrane domain(s) facing the lipid bilayer, leading to an alteration of membrane permeability to ions or other small molecules (for a review, see fischer and sansom, 2002; gonzalez and carrasco, 2003) . expression of these membrane-active proteins may cause cellular membrane leakiness further resulting in the development of cytopathic changes to facilitate particle release late in infection, or may be required to promote virus uncoating at an early stage of infection (reviewed by carrasco, 1995) . the prrsv e protein structurally resembles a number of viroporins found in other rna viruses, in that it consists of 70-73 amino acid residues and contains central hydrophobic sequences with a cluster of basic amino acids at the cterminus. the e protein localizes mainly in perinuclear regions including the er and the golgi complex and associates with intracellular membranes (wu et al., 2001) . interestingly, coronaviruses, another family member of the order nidovirales, also code for a small hydrophobic membrane, designated e protein, which may play a crucial role during virus morphogenesis (fischer et al., 1998; liu and inglis, 1991) . in recent studies, the coronavirus e protein has been shown to modify membrane permeability (liao et al., 2004; madan et al., 2005) as well as to form cation-selective ion channels in an artificial membrane (wilson et al., 2004) . our data in the current study also demonstrate the alteration of membrane permeability by the prrsv e protein and the inhibition of bacterial growth by the increase of hygromycin b penetration into bacterial cells. however, we were not able to show similar results in mammalian cells by infection or e gene transfection, which may be due to the different intracellular localization of e in mammalian cells. in prrsv-infected cells, the e protein appears to remain in the er and golgi complex, where it likely participates in assembly of infectious progeny virus, rather than traveling to the plasma membrane. bacterial cells do not possess such intracellular organelles and the expressed e protein may accumulate at the inner bacterial membrane, leading to membrane perturbation, and thus enhancing membrane permeabilization. failure to observe the direct alteration of membrane permeability by e in mammalian cells implies that the role of e is not linked to membrane disorganization and cell lysis in facilitating virus release. the cross-linking studies show that the e protein can form homo-oligomers, including dimers, trimers and tetramers by non-covalent interactions. all information obtained in the present study support the hypothesis that the prrsv e protein contains a potential for a pore-forming activity and thus may function as an ion channel for virus uncoating. the functional and structural features of the prrsv e protein resemble the influenza a virus m2 protein that is best characterized as an ion-channel protein. the m2 protein forms a homo-tetramer in the viral membrane and functions as an ion channel. the m2 channel allows the translocation of protons from the acidic environment of the endosome to the inner space of virions and alters the ph in the virion. as a consequence, the m1 protein in the virion is dissociated from the viral ribonucleoprotein complex, which promotes the ribonucleoprotein complex traveling to the nucleus where the influenza replication takes place (pinto et al., 1992; sakaguchi et al., 1997) . it is noteworthy that prrsv replication could be inhibited by amantadine, an antiviral drug for influenza virus (kreutz and ackermann, 1996; fig. 5) . in summary, we propose a model for prrsv uncoating based on our findings. in this model, e proteins form pores (ion channels) in the viral envelope. upon internalization by receptor-mediated endocytosis through small clathrin-like coated vesicles, the virion particles are transported to the endosome. there, the e-protein ion channels in the viral membrane undergo conformational changes upon exposure to low ph in the endosome, and allow ions to enter the virion, which triggers the disassembly of inner capsid and the release of viral genome in the cytoplasm, such that further steps of genome replication and infection cycle can proceed. marc-145 (kim et al., 1993) , hela and bhk-21 cells were grown in dulbecco's modified eagle medium (dmem) supplemented with 8% fetal bovine serum (fbs; invitrogen), penicillin (100 u/ml) and streptomycin (50 μg/ml). bhk-t7 cells stably expressing the bacteriophage t7 rna polymerase (generously provided by c.y. kang, university of western ontario, london, ontario, canada) were grown in dmem supplemented with 5% fbs, 100 u/ml of penicillin, 50 μg/ml of streptomycin and 500 μg/ml of g418 (geneticin; invitrogen). the cells were maintained at 37°c with 5% co 2 . stocks of prrsv (strain pa8) and recombinant vaccinia virus expressing t7 rna polymerase (vtf7-3; fuerst et al., 1986) were prepared in marc-145 or hela cells, respectively, as described previously (wootton et al., 2001) . the n protein-specific monoclonal antibody (mab) sdow17, the e protein-specific anti-peptide rabbit antiserum and non-structural proteins 2 and 3-specific polyclonal rabbit antiserum are described elsewhere nelson et al., 1993; wootton et al., 2001) . e. coli strains xl1-blue (stratagene) and dh5α were used as hosts for site-directed mutagenesis and general cloning, respectively. cdna cloning of the prrsv n or e gene to produce pcite-n; pgex-n or pcite-e; pgex-e, respectively, is described elsewhere (wootton and yoo, 2003; . to modify the translational initiation codon of prrsv orf2b (e), pcr-based site-directed mutagenesis was first conducted to mutate the atg start codon of the e gene to gtg at genomic nucleotide positions 12,062 to 12,064 using the shuttle vector ptb-shuttle-prrsv-3997 with the following primer pairs; for a12062g mutation, e-ko-fwd (5′-gaattgaaatgaagtggggtctatac-3′: nucleotide positions 12,049 to 12,074) and e-ko-rev (5′-gtata-gaccccacttcatttcaattc-3′: nucleotide positions 12,049 to 12,074), where lowercase letters indicate mutated nucleotides. the a12062g mutation was translationally silent with respect to orf2a encoding the gp2 protein. pcr-based mutagenesis and screening of mutants were performed as described previously (wootton et al., 2001) . the shuttle plasmid carrying the a12062g mutation was digested with eco47iii and bsrgi, and a 908-bp fragment was purified. the wild-type full-length genomic cdna clone was digested with eco47iii and bsrgi, and the 908-bp eco47iii-bsrgi fragment was replaced with the corresponding fragment obtained from the shuttle plasmid. the ligated full-length plasmid dna was screened by xmai digestion, and based on the xmai digestion pattern, positive clones were selected. dna manipulation and cloning were performed according to standard procedures (sambrook and russell, 2001) . the selected clones were sequenced to confirm the presence of the a12062g mutation in the full-length genomic cdna clone. the resulting plasmid was designated pcmv-s-p129-δe. marc-145 cells or bhk-21 cells were seeded on microscope coverslips placed in 35-mm-diameter dishes and grown overnight to 70% confluence. the cells were transfected with 2 μg of plasmid dna using lipofectin (invitrogen) according to the manufacturer's instructions. at 48 h post-transfection, cell monolayers were washed twice in pbs and fixed immediately with cold methanol for 10 min at − 20°c. for time course experiments, marc-145 cells inoculated with purified passage-1 viruses prepared in the transfected bhk-21 cells were fixed at various time points after infection. cells were blocked using 1% bovine serum albumin (bsa) in pbs for 30 min at room temperature (rt) and then incubated with n-specific mab sdow17 for 2 h. after washing five times in pbs, the cells were incubated for 1 h at room temperature with goat antimouse secondary antibody conjugated with alexa green dye (molecular probes). for dual immunofluorescence, cells were co-stained with nsp2/3-specific rabbit antiserum and n-specific mab sdow17, followed by incubation with goat anti-rabbit antibody conjugated with texas red (molecular probes) and goat anti-mouse antibody conjugated with alexa green. the coverslips were washed five times in pbs and mounted on microscope glass slides in mounting buffer (60% glycerol and 0.1% sodium azide in pbs). cell staining was visualized using a fluorescent microscope (model ax70; olympus). bhk cells were seeded in 100 mm-diameter dishes and grown to 70% confluence. cells were transfected for 20 h with 10 μg of the full-length cdna plasmid using lipofectin. the transfected cells were continued for incubation at 37°c in dmem supplemented with 8% fbs for 10 h. at 30 h posttransfection, the cells were starved for 30 min in methioninedeficient mem (invitrogen) and metabolically labeled for 24 h with 100 μci/ml of easytag express protein labeling mix ([ 35 s]methionine and [ 35 s]cysteine, specific activity, 407 mbq/ ml) (perkin-elmer). after a 1-day labeling period, the cells were washed twice with cold pbs and lysed with ripa buffer (1% triton x-100, 1% sodium deoxycholate, 150 mm nacl, 50 mm tris-hcl [ph 7.4], 10 mm edta, 0.1% sds) containing 1 mm phenylmethylsulfonyl fluoride (pmsf). to prepare radiolabeled viral particle samples, the culture supernatant was harvested, and the cell debris was removed by a low-speed centrifugation at 3500 rpm (model 5415; eppendorf) for 10 min at rt. the virus particles were purified through a 20% sucrose cushion (wt/vol) prepared in te buffer (10 mm tris hcl [ph 8], 1 mm edta) at 40,000 rpm for 2 h at 4°c in an sw41 rotor (model xl-90; beckman). the resulting pellets were resuspended in 50 μl of ripa buffer containing 1 mm pmsf. for immunoprecipitation, the dissolved pellets or cell lysates equivalent to 1 in 15 of a 100-mm-diameter dish were adjusted with ripa buffer to a final volume of 100 μl and incubated for 2 h at rt with a mixture of e-specific rabbit antiserum, nspecific mab sdow17, and m-specific rabbit antiserum. the immune complexes were adsorbed to 7 mg of protein-a sepharose cl-4b beads (amersham biosciences) for 16 h at 4°c. the beads were collected by centrifugation at 6000 rpm for 5 min, washed twice with ripa buffer and once with wash buffer (50 mm tris-hcl [ph 7.4], 150 mm nacl). the beads were resuspended in 20 μl of sds-page sample buffer (10 mm tris-hcl [ph 6.8], 25% glycerol, 10% sds, 0.12% [wt/vol] bromophenol blue) with 10% β-mercaptoethanol (βme), boiled for 5 min and analyzed by 15% sdspolyacrylamide gel electrophoresis (page). gels were dried on filter paper and radiographic images were obtained using a phosphorimager (model phosphorimager si; molecular dynamics). virus preparation from full-length cdna clones bhk-21 cells were transfected with the full-length cdna plasmid as described above. after washing of cells with dmem at 20 h post-transfection, the transfected cells were further maintained in dmem supplemented with 8% fbs for 2 days and the culture supernatants were harvested at 2 days posttransfection. the viral particles were purified as described above and the pellets were suspended in dmem. the resulting virus suspension was designated 'passage-1'. the passage-1 virus was used to inoculate fresh marc-145 cells and the 5-day harvest was designated 'passage-2'. the 'passage-3' virus was prepared in the same way as for passage-2. each passage virus was aliquoted and stored at − 80°c until use. culture supernatants of bhk-21 cells transfected with the full-length cdna plasmid were harvested at 2 days posttransfection as described above. the culture fluids were centrifuged at 3500 rpm (model 5415; eppendorf) for 10 min to remove cellular debris. the cleared supernatants were purified as described above, and the pellets were suspended in 50 μl of pbs and subsequently stored at 4°c until use. twenty microliters of the virion suspension was mounted on a formarcoated copper grid. the grids were placed at rt for 5 min, and excess liquid was removed by wicking with filter paper. for negative staining, 20 μl of 2% sodium phosphotungstate (ph 6.8) was dropped onto the grids and incubated at rt for 30 s. the samples were viewed with a transmission electron microscope (model h-7600 120 kev pc-tem; hitachi) operating at 75 kev. viral rna was extracted from either supernatants or lysates of infected cells using the qiaamp viral rna mini-kit (qiagen). to remove any contaminated dna in the rna preparations, samples were treated with 1 u of rq dnase i (promega) at 37°c for 30 min in 50 mm tris-hcl [ph 7.5] and 1 mm mgcl 2 . for detection of viral rna in the virions, samples were prepared as previously described (wieringa et al., 2004) with some modifications. the viral particles in the culture supernatant from bhk-21 cells transfected with the full-length cdna plasmid were pelleted by as described above. the pellet was resuspended in tnm buffer (20 mm tris-hcl [ph 7.5], 50 mm nacl, 5 mm mgcl 2 ). seventy microliters of each sample suspension were added with 2 μl of rq dnase i (1 u/μl; promega) and 3.6 μl of rnase a (10 mg/ml; sigma), and the mixture was incubated for 1 h at 37°c in the presence or absence of detergents (1 μl of triton x-100, and 3.5 μl of 10% sds). after incubation for 1 h at 37°c, the nucleases were inactivated by the addition of 5 μl of proteinase k (20 mg/ml; qiagen) and incubation for 30 min at 50°c. rna was isolated from each sample by using a qiaamp viral rna mini-kit and subsequently, treated one more time with 1 unit of rq dnase i. after dnase i treatment, rna was re-extracted with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) mixture and precipitated at − 80°c for 1 h by adding 0.1 volume of 2.5 m sodium acetate (ph 5.2) and 2 volumes of ethanol. the pellets were washed once with 70% ethanol and dissolved in ultrapure dnase/rnase-free distilled water (invitrogen). the resulting rna samples were used for first-strand cdna synthesis by moloney murine leukemia virus (m-mlv) reverse transcriptase (invitrogen) using the reverse primer orf2b-rev (5′-tcataagatcttctgtaattgctc-3′). the e gene was amplified by taq dna polymerase (invitrogen) using mrna2-fwd (5′-ccgtcattgaac-caacttta-3′) and orf2b-rev. for strand-specific rt-pcr, the specific primer pairs (table 1) were used to amplify dna fragments representing positive-sense genomic rna, negative-sense genomic rna, positive-sense subgenomic mrna7 or negative-sense subgenomic mrna7. pcr was conducted under the following conditions; initial denaturation at 95°c for 5 min, 35 cycles of denaturation at 95°c for 30 s, annealing at 56°c for 30 s and extension at 72°c for 1 min, followed by a final extension at 72°c for 10 min. pcr products were analyzed by 0.8% or 1.5% agarose gel electrophoresis depending on size of the fragment. amplified products were purified using the pcr purification kit (qiagen) and sequenced. stock solutions of ammonium chloride, chloroquine, amantadine and verapamil (sigma) were prepared in water at concentrations of 50 mm, 2 mm, 10 mm and 1 mm, respectively. marc-145 cells grown in 6-well plates were preincubated with different concentrations of the reagents for 30 min and subsequently infected with prrsv at a multiplicity of infection (moi) of 1 for 1 h at 37°c in the presence or absence of the drugs. the virus inoculum was removed and the cells washed three times with mem. the inoculated cells were then incubated in fresh medium in the presence or absence of the reagents and monitored daily for the appearance of cpe. culture supernatants were harvested daily from cells for 3 days and virus titer in the supernatant was determined by plaque assay. at 1 day or 2 days post-infection, marc-145 cells infected with prrsv in presence or absence of the drugs were fixed with cold methanol and subjected to an immunofluorescence assay as described above. the effect of ion channel blockers on prrsv replication was also determined by strand-specific rt-pcr. total cellular rna was extracted from mock-infected or prrsv-infected cells in presence or absence of the drugs using trizol (invitrogen) and dna fragments representing positivesense or negative-sense viral genomic rna were rt-pcr amplified using orf7-specific primers (lee et al., 2006) or negative-sense-specific primers (table 1 ), respectively, as described above. an inducible e. coli expression system was used to express the prrsv e protein fused with glutathione s-transfererase (gst). plasmids were transformed into e. coli strain dh5α to express gst fusion proteins. a single colony was grown in luria bertani (lb) media containing 100 μg/ml ampicillin overnight. one hundred ml of lb media containing 100 μg/ml ampicillin was inoculated with 1/100 of overnight culture. when the absorbance of cultures reached at an od 600 of 0.6, 1 mm isopropylthio-β-d-galactoside (iptg) was added to the media to induce protein synthesis. at indicated times after induction, the densities of bacterial cultures were determined by measuring the light scattering at 600 nm. permeability of the plasma membrane of bacterial cells expressing the prrsv e protein to hygromycin b was determined as described previously (liao et al., 2004) . briefly, bacterial cultures were incubated in the presence or absence of 1 mm of hygromycin b for 15 min at 1 h after iptg induction, and aliquots of l ml were labeled with for 15 min with 4 μci/ml of [ 35 s]methionine/cysteine. the labeled bacterial cells were then harvested and lysed in equal volumes of sds-page sample buffer. proteins were resolved on a 12% sds-page gel and visualized by coomassie blue staining or autoradiography. the prrsv n or e protein was independently expressed in hela cells using the t7-based vaccinia virus vtf7-3. hela cells seeded on 60-mm-diameter dishes were grown to 90% confluence and infected for 1 h at 37°c with vtf7-3 at an moi of 10. following infection, fresh medium was added and incubation continued for an additional 1 h. the cells were washed in opti-mem and transfected with 1.5 μg of a plasmid for 16 h using the reagent lipofectin. the transfected cells were then starved for 30 min in methionine-deficient mem and incubated for 5 h with 100 μci/ml of [ 35 s]methionine/cysteine. to prepare radiolabeled prrsv-infected cells, marc-145 cells seeded on a 100-mm-diameter dish were infected with prrsv containing 1 mm pmsf. the resultant cell lysates were then used in immunoprecipitation with 1 μl of the n-specific mab sdow 17 or 3 μl of the e-specific rabbit antiserum followed by sds-17% page analysis and autoradiography as described above. membrane permeabilization by poliovirus proteins 2b and 2bc membrane permeabilization by different regions of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41 functional properties of the predicted helicase of porcine reproductive and respiratory syndrome virus characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr-2332) modification of late membrane permeability in avian reovirus-infected cells 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oligomeric integral membrane protein expressed at the cell surface influenza virus m2 protein has ion channel activity the cap structure of simian hemorrhagic fever virion rna the active oligomeric state of the minimalistic influenza virus m2 ion channel is a tetramer molecular cloning: a laboratory manual how viruses enter animal cells the molecular biology of arteriviruses identification of a novel structural protein of arteriviruses topley and wilson's microbiology and microbial infections: virology volume the transmembrane oligomers of coronavirus protein e proteolytic processing of the open reading frame 1b-encoded part of arterivirus replicase is mediated by nsp4 serine protease and is essential for virus replication ion channel activity of influenza a virus m2 protein: characterization of the amantadine block structural protein requirements in equine arteritis virus assembly sars coronavirus e protein forms cation-selective ion channels envelope protein requirements for the assembly of infectious virions of porcine reproductive and respiratory syndrome virus homo-oligomerization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein and the role of disulfide linkages full-length sequence of a canadian porcine reproductive and respiratory syndrome virus (prrsv) isolate antigenic importance of the carboxy-terminal beta-strand of the porcine reproductive and respiratory syndrome virus nucleocapsid protein a 10-kda structural protein of porcine reproductive and respiratory syndrome virus encoded by orf2b the 2b protein as a minor structural component of prrsv this study was supported by funding to dy by nserc canada, ontario pork, omaf and the usda nri prrs cap program of the usa. the authors are grateful to pfizer animal health usa for providing the infectious cdna clone for this study. key: cord-292751-tk1oggi9 authors: hosseini, elahe seyed; kashani, narjes riahi; nikzad, hossein; azadbakht, javid; bafrani, hassan hassani; kashani, hamed haddad title: the novel coronavirus disease-2019 (covid-19): mechanism of action, detection and recent therapeutic strategies date: 2020-09-24 journal: virology doi: 10.1016/j.virol.2020.08.011 sha: doc_id: 292751 cord_uid: tk1oggi9 novel coronavirus sars-cov-2, designated as covid-19 by the world health organization (who) on the february 11, 2020, is one of the highly pathogenic β‐coronaviruses which infects human. early diagnosis of covid-19 is the most critical step to treat infection. the diagnostic tools are generally molecular methods, serology and viral culture. recently crispr-based method has been investigated to diagnose and treat coronavirus infection. the emergence of 2019-ncov during the influenza season, has led to the extensive use of antibiotics and neuraminidase enzyme inhibitors, taken orally and intravenously. currently, antiviral inhibitors of sars and mers spike proteins, neuraminidase inhibitors, anti-inflammatory drugs and ek1 peptide are the available therapeutic options for sars-cov-2 infected individuals. in addition, chloroquine, which was previously used for malarial and autoimmune disease, has shown efficacy in the 2019-ncov infection treatment. in severe hypoxaemia, a combination of antibiotics, α-interferon, lopinavir and mechanical ventilation can effectively mitigate the symptoms. comprehensive knowledge on the innate and adaptive immune responses, will make it possible to propose potent antiviral drugs with their effective therapeutic measures for the prevention of viral infection. this therapeutic strategy will help patients worldwide to protect themselves against severe and fatal viral infections, that potentially can evolve and develop drug resistance, and to reduce mortality rates. recent pharmaceutical strategies to treat coronavirus 12-15 improve immune system to fight with covid19 15-18 abbreviations [18] [19] introduction over the last two decades, three coronaviruses have periodically crossed animal species such as bats, transmitted to human populations, and caused an ever-increasing outbreak of a large-scale pandemic [1, 2] . the previously reported viral zoonotic pathogens include sars-cov (severe acute respiratory syndrome coronavirus) and mers (middle east respiratory syndrome coronavirus) [3, 4] , that can cause severe respiratory disease in human [5, 6] . sars-cov-2, a novel coronavirus (which causes covid19) , has fast spread like a pandemic since its outbreak in wuhan, china, in december 2019 [7] . it causes an acute and deadly disease with a 2% j o u r n a l p r e -p r o o f mortality rate. however, this novel coronavirus is usually associated with a mild to severe respiratory disease in humans [1, 8, 9] . this virus has the ability of jumping between species, and causing a variety of diseases as a strange and complex pathogen [10] . due to the frequent interaction between humans and animals, a virus is a common source of zoonotic infection. covid-19, due to its human-to-human transmission, has become a health emergency of global concern [7, 11] . currently, we have no sufficient evidence to propose that a specific wildlife animal is the virus origin. a proper study of the viral source, evolution, mode of zoonotic transmission and infectivity, would help to prevent further infections. [3, 4] . coronaviruses (covs) belong to the nidovirales order, coronaviridae family, which comprises of two subfamilies, namely orthocoronavirinae and letovirinae (international committee on taxonomy of viruses) [12, 13] . covs are genotypically classified into four genera: alpha coronaviruses (a), beta coronaviruses (b), gamma coronaviruses (g), and delta coronaviruses (d), according to their phylogenetic and genomic data. further, β-coronavirus is subdivided into four viral lineages of a to d [14, 15] . coronavirus is an enveloped and non-segmented virus, which has a large positive-sense single-stranded rna virus genome (27-32 kb) , capped and polyadenylated [16] . coronavirus also has crown-shape spikes projecting from its surface (80160 nm in size), from which its name derived [17] . the cov spike (s) glycoprotein attaches to cellular receptors on the host cell and mediates viral entry resulting in interspecies transmission and pathogenesis [15, 18] figure 2 ). it seems that the high sequence identity in sars-cov-2 and pangolin-cov may be due to coincidental convergent evolution [32] . phylogenetic analysis of rbd region of the spike protein, has also led to a further presumption that the identity of sars-cov-2 rbd to pangolins-cov rbd might be result of accidental mutations followed by natural selection, and/or recombination events in pangolins [32] (figure 2 ). most common clinical symptoms of covid-19 disease are dry cough, fever and shortness of breath in the majority of patients. some patients also experience other signs such as sore throat, headache, myalgia, fatigue and diarrhea [33, 34] . in the initial phase of the disease, patients can j o u r n a l p r e -p r o o f be afebrile, only presenting with chills and respiratory symptoms. although most cases appear to be mild, all patients have new pulmonary signs as ground-glass lung opacity on chest x-ray [17, 35] . the symptoms in patients with mild pneumonia are fever, cough, sore throat, tiredness, headache or myalgia [36] . they do not obviously show any of the serious symptoms or complications. some patients were reported to have upper respiratory infection (uri), bilateral patchy opacity in lung [7] , decreased white blood cell or lymphocyte number [37] and increased alt, ast, ldh, ck-mb, crp and esr in these stages of infection [38] . patients with severe pneumonia, suffer from acute respiratory distress syndrome (ards) and refractory hypoxemia. ncov-2019 can cause severe pulmonary infection, respiratory failure, along with organ damage and dysfunction. in case of extra-pulmonary system dysfunctions, such as derangements in hematologic and digestive system, the risk of sepsis and septic shock will be serious, resulting in considerable increase in fatality rate. the findings showed that the disease is mild in the majority of patients (81%) and only a few of them develop severe pneumonia, pulmonary edema, ards, or different organ damages with case morality rate of 2.3%. in children, infection generally presents with much milder clinical symptoms or even asymptomatic, compared with adult. according to previous studies, pregnant women do not seem to have a severe disease, while older patients are at a high risk of developing critical illness [33, 36] . the case fatality rate (cfr) increased in 50 % of patients older than 80 with a history of chronic diseases, such as high blood pressure, diabetes, heart diseases, respiratory diseases, cerebrovascular diseases, endocrine system disorders, digestive system disorders and cancers . in most of cases, the cause of death is respiratory failure, septic shock or several organ failure [33] . in fact, increased c-reactive protein (crp) is an important factor of impaired immunity, characterized by lymphopenia. so, sars-cov-2 is more probably to affect older people with chronic disease due to their poorer immune function [39] . covid-19 has also been found to infect more males (average age of 55.5 years) than females [39] . the less susceptibility of females to viral infections is likely associated with the protective role of x chromosome and sex hormones, which result in stronger immune response to virus [40] . ct imaging findings of patients with covid-19 revealed that most of cases had ground-glass opacities, which may manifest as crazy paving pattern, organizing pneumonia and architectural distortion. on x-rays or chest ct imaging of the examined patients, j o u r n a l p r e -p r o o f early diagnosis is the most important step to manage and treat covid-19. the diagnostic tools are generally molecular methods, serology and viral culture. initial laboratory investigations of hospitalized patients consist of a complete blood count, coagulation testing and serum biochemical test such as creatine kinase (ck), lactate dehydrogenase, procalcitonin, and electrolytes [29, 41] . based on laboratory tests, most patients showed a significant decrease in total number of lymphocytes, suggesting that lymphocytes (particularly t lymphocytes) are likely target of sars-cov-2. in the covid infection, virus particles begin to spread through the respiratory tract and infect the surrounding uninfected cells. this leads to initiate a cytokine storm and consequently trigger a series of sever immune responses. this process results in some changes in immune cells, particularly lymphocytes, and then leads to immune system dysfunction [29] . hence, the decreased number of the circulating lymphocytes could be considered as a diagnostic marker for sars-cov-2 infection and its severity [33] . previous studies reported that there is a correlation between elevated level of pro-inflammatory cytokines like il1b, il6, il12, ifnγ, ip10, and mcp1, and cytokines such as ifnγ, tnfα, il15, and il17, in sars-cov and mers-cov infection respectively, with pulmonary inflammation and lung injury. notably, the high value of cytokines like il1b, ifnγ, ip10, and mcp1, may activate t helper 1 cells (th1) response. this cytokine storm is probably associated with disease severity. however, in sars-cov2 infection, enhanced secretion of t helper 2 (th2) and cytokines like il4 and il10, reduce peripheral white blood cells and immune cells such as lymphocytes, probably leading to suppression of the inflammatory response and immune system function followed by serious lung damage, which differs from sars-cov infection [41] . these findings suggest that sever and uncontrolled inflammatory response have a more damaging effect on covid-19 induced lung injury than viral pathogenicity. therefore, in sars-cov-2 pneumonia, it is vital to control cytokines or chemokines to detect the impact of 2019 coronavirus on their production in the critical phase of the disease [29, 41] . rt-pcr (reverse-transcription polymerase chain reaction) or real-time pcr and genome sequencing for respiratory or blood specimens are the next methods to confirm covid-19 infection (table 1) . together with its time-consuming process and the problems associated to performance of rt-pcr kit are the main obstacles to control this epidemic [49] . chest ct, compared to rt-pcr, is a fast, sensitive, easy to perform and more accurate and reliable tool for screening and diagnosis of covid-19 [50] . chest ct can also show pulmonary abnormalities in covid-19 patients with early negative rt-pcr results [51, 52] . in the primary stage of pneumonia itself, ct images can demonstrate several small ground-glass opacity as well as some interstitial changes [9] , remarkable in the lung periphery [7] [53] . crispr-cas13-based sherlock system consists of two rna guides, which are combined with a cas13 protein, and form a sherlock system to recognize the presence of covid-19 viral rna. at first the team used synthetic fragments of sars-cov-2 rna as a pattern for designing two rna guides, which are able to bind to their complementary sequences in covid-19 rna. in order to visual readout, they used a paper strip (as a paper strip in pregnancy test) for dipping into a prepared sample. then, appearance of a line on the paper strip indicates the existence of virus in the sample [54, 55] . also, another research group has recently proposed a rna-targeting crispr system to target rna genome of sars-cov-2 in the laboratory, to limit its ability to reproduce [56] . this crispr cov-2 patients, particularly to target their lung, which is main infected organ [80] . before therapeutic application of crispr/cas13d system to patients, it is necessary to determine the safety and efficacy of this system in clearance of 2019-ncov and other viruses in animals. if researchers find this therapeutic strategy secure and beneficial, then it would be applied to kill the viruses that have the potential to evolve and also develop drug resistance [58] . since the emergence of 2019-ncov, due to its rapid spread and being a serious threat to human health, researchers have made great efforts to understand the pathogenetic characteristics of this virus to develop effective drugs. due to appearance of the 2019-ncov during the influenza season, orally and intravenously antibiotics and neuraminidase inhibitors such as oseltamivir having been widely used as an experimental treatment for 2019-ncov in china [59, 60] . however, there is no reliable evidence that shows oseltamivir is an effective treatment in 2019-ncov [60] . at present, there is no antiviral drug assumed to provide protection against covid-19 infection; also it will take long time to develop and a vaccine and gets approval for it. [9] . nowadays, griffithsin, as an inhibitor of sars and mers spike, remdesivir, favipiravir and ribavirin (nucleoside analogues), lopinavir/ritonavir (protease enzyme inhibitors) [61] , oseltamivir (neuraminidase inhibitors), anti-inflammatory drugs and ek1 peptide [62] , the clinical potential to be applied against the 2019-ncov infection [67, 68] . chloroquine shows its inhibitory effects against 2019-ncov through increasing endosomal ph required for virus cell fusion, also affecting the glycosylation of cellular receptors of sars-cov [69] . chloroquine is a cheap and safe anti-viral medicine, which is orally administrated and widely distributes all over body especially in the lungs [70] . also, remdesivir, which has a structure chemically similar to hiv reverse-transcriptase inhibitors, is currently in the phase of clinical trials for 2019-ncov [58] . the spike (s) protein is a promising target for the development of antivirals drugs, due to its major role in the virus-receptor interaction. griffithsin, a medicine targeting the oligosaccharides on the surface of different spike proteins [71] , has also been tested in phase i trials for the treatment of hiv and sars-cov [72] . however, the efficacy of spike inhibitors for the treatment of 2019-ncov should be re-evaluated [71] . there are multiple mechanisms of action for nucleoside analogues (adenine or guanine derivative), including lethal mutagenesis, inhibition of rna biosynthesis, or rna chain premature termination [72] . they inhibit synthesis of viral rna in human coronaviruses by targeting the rna-dependent rna polymerase [73] . favipiravir (t-705), a guanine analogue, is an antiviral drug targeting the viral rna-dependent rna polymerase of rna viruses such as ebola, influenza, yellow fever, j o u r n a l p r e -p r o o f chikungunya, norovirus and enterovirus. it has been recently proposed to be effective also against coronaviruses, in vitro [73] . using fabiravir and ribavirin combined with 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nasal, mouth, lung and colon tissues, and tends to be co-expressed with hla-drb1 in the four tissues repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection the financial support for the current research was provided by research deputy of kashan university of medical sciences, kashan, iran. not applicable esh, hhk and nrk provided direction and guidance throughout the preparation of this manuscript. hhb, hn and hhk conducted the literature and drafted the manuscript. other authors reviewed the manuscript and made significant revisions on the drafts. all authors read and approved the final version. the authors declared that they have no competing interests. not applicable. key: cord-293248-8vtd9e4n authors: day, j. michael; zsak, laszlo title: determination and analysis of the full-length chicken parvovirus genome date: 2010-03-30 journal: virology doi: 10.1016/j.virol.2009.12.027 sha: doc_id: 293248 cord_uid: 8vtd9e4n viral enteric disease in poultry is an ongoing problem in many parts of the world. many enteric viruses have been identified in turkeys and chickens, including avian astroviruses, rotaviruses, reoviruses, and coronaviruses. through the application of a molecular screening method targeting particle-associated nucleic acid (pan), we recently described the detection and partial characterization of a novel enteric parvovirus in chickens. subsequent surveys of intestinal homogenates from turkeys and chickens in the united states revealed widespread occurrence of parvovirus in poultry. here we report the first full genome sequence of a novel chicken parvovirus, chpv abu-p1. chpv abu-p1 genome organization, predicted amino acid sequence, and phylogenetic relationships with other described parvoviruses are discussed. enteric disease syndromes are a continual economic burden for the poultry industry throughout the world. the major recognized enteric syndromes in poultry are poult enteritis complex (pec) and poult enteritis mortality syndrome (pems) in turkeys and runting-stunting syndrome (rss) in broiler chickens (barnes and guy, 2003; barnes et al., 2000; goodwin et al., 1993) . poultry enteric disease is marked by diarrhea, stunting, and increased time to market; the more severe forms of enteric disease are characterized by immune dysfunction and increased mortality. numerous viruses have been detected in or isolated from the intestinal tract of poultry (both diseased and healthy), and have subsequently been implicated in poultry enteric disease. these viruses include the avian reoviruses, rotaviruses, astroviruses and coronaviruses; despite these diagnostic efforts and viral descriptions, the role these viruses play in the enteric syndromes are still poorly understood (guy, 1998; pantin-jackwood et al., 2008a; pantin-jackwood et al., 2007a; pantin-jackwood et al., 2008b; pantin-jackwood et al., 2007b; reynolds et al., 1987; spackman et al., 2005) . we recently reported the use of a molecular screening protocol to amplify and identify novel enteric viruses in the intestinal tract of poultry showing characteristic enteric disease signs (zsak et al., 2008) . this random amplification of viral particle-associated nucleic acid identified numerous parvovirus dna sequences directly from the intestinal homogenates of chickens with signs of enteric disease. initial analysis of this chicken-origin parvovirus (chpv) revealed that it is a novel member of the parvovirinae subfamily within the parvoviridae, and led to the development of a specific molecular diagnostic test targeting the chpv non-structural (ns) gene (zsak et al., 2009) . parvoviruses have been described that infect a wide range of invertebrates and vertebrates, and a thorough knowledge of the parvovirus genome and encoded proteins is essential to an understanding of the evolution of parvoviruses in various hosts, including tissue tropism during viral pathogenesis (hueffer and parrish, 2003) . parvoviruses are well known for causing enteric disease in mammals, with the canine parvoviruses recently emerging as important pathogens in the 1970s (decaro et al., 2009; truyen, 1999) . parvoviruses have been known for years as the causative agent of derzsy's disease in young geese, and are also found in muscovy ducks, where they cause multiple clinical signs ranging from enteritis to hepatitis and infectious myocarditis (gough, 2008) . parvoviruses have been previously described in chickens based upon their morphology via electron microscopy and upon their genome size kisary et al., 1984) , and enteric disease signs have been attributed to parvovirus-like particles detected in turkey intestinal tracts (trampel et al., 1983) . a recent diagnostic survey of enteric samples collected from united states turkey and chicken flocks revealed widespread presence of parvovirus in u.s. poultry (zsak et al., 2009) . this is the first indepth molecular characterization and analysis of the full-length genome sequence of chpv. the analysis includes comparisons to other members of the parvovirinae that infect mammals and birds, including two novel turkey-origin parvoviruses (tupv) recently sequenced using a similar molecular approach. virology 399 (2010) 59-64 the chpv abu-p1 genome the fully assembled chpv abu-p1 genome is 5257 nucleotides (nt) in length and has a basic organization similar to previously described members of the parvoviridae (fig. 1) . the genome is flanked on the 5′ and 3′ ends by 206 nt direct repeat sequences, each of which contains 39 nt inverted repeats presumed to form a hairpin structure similar to the structures common in the parvoviruses (fig. 2) (farkas et al., 2004; muzyczka and berns, 2001; sukhumsirichart et al., 2006) . the 206 nt direct repeats are identical and are found in the same orientation 5′ to 3′ in the genome. the genome contains 32.36% a, 21.95% g, 24.08% t, and 21.61% c, with an a + t content of 56.44% and a c + g content of 43.56%. the overall genomic organization of chpv abu-p1 is similar to other parvoviruses, with two major predicted open reading frames (orfs). the 5′ orf is 2085 nt long, and a blastx search using the putative amino acid sequence revealed a protein similar to the parvoviridae non-structural (ns) proteins ns1, with the greatest similarity to human bocavirus ns1. the major 3′ orf is 2028 nt long, and encodes a protein similar to the parvoviridae capsid protein vp1, with the greatest similarity to avian adeno-associated virus, a member of the dependovirus genus. the chpv genome also contains a small (306 nt) orf located between the major 5′ and 3′ orfs. this orf shows no homology to known protein coding regions in the databases. the chpv ns1 orf is preceded upstream by the 39 bp inverted repeat sequence. the ns1 start codon is in a strong kozak context (aagatgg) and is preceded directly upstream by a tata box, an extended caat box, and a putative inr-box (hernandez, 1993; rupp et al., 1990; smale and kadonaga, 2003) . ns1 also contains a wellconserved phosphate-binding loop (p-loop) motif (gxxxxgkt…ee), distinguishing it as a member of the atp and gtp-binding superfamily of proteins (saraste et al., 1990 ). at the amino acid level, the chpv ns1 protein exhibited from 45.2 to 53.9% amino acid similarity (17.1 to 19.0% identity) with ns1 from other parvovirus isolates. the ns1 similarity increased to 89.3 and 100% (99.3 to 83.7% identity) when chpv was compared with two turkey-origin parvoviruses (tupv), tupv 260 and tupv 1078 respectively. the lower identity noted when comparing the chpv and tupv 1078 ns1 coding sequences can be partially explained by the presence of a stop codon beginning at position 1882 in the tupv 1078 ns1 coding region. the predicted vp1 orf begins at position 2998 in the chpv genome. the predicted vp1 start codon (atg) overlaps with the stop codon (aat) of the predicted 306 nt orf located between the chpv ns1 and vp1 orfs. vp1 is preceded upstream by a tata box and a polyadenylation signal (aataaa) follows the orf; this poly-a signal immediately precedes the 39 nt inverted repeat at the 5′ end of the genome. one of two downstream atg codons that begin 417 and 420 nt downstream to the start of the putative vp1 orf may serve as the start codon for a vp2 capsid protein that shares a stop codon (taa) with vp1. the second of these atg codons has the stronger kozak consensus sequence (atgatgg) compared to the translation initiation context of the first codon (gaaatga) and remains in frame (kozak, 1987) . a third start codon with a favorable translation initiation context (gacatgg) is present at position 3067 in the chpv genome, and may represent the beginning of a putative vp3 orf as has been described in the goose and muscovy duck parvoviruses (zadori et al., 1995) . the chpv vp1 protein shares from 46.6 to 59.0% amino acid similarity (10.1 to 24.5% identity) with vp1 from other parvoviruses. the vp1 similarity increases to 100 and 95.1% (99.7 and 79.2% identity) when compared to vp1 from tupv 260 and tupv 1078, respectively. clustalw was used to align the complete chpv genome coding region with complete parvovirus sequences from mammals and birds available in the databases, and with two recently sequenced tupv genome coding regions. the neighbor-joining tree was produced using mega4. the turkey and chicken sequences clustered together, and were clearly separate from the other members of the parvovirinae (fig. 3) . a previous phylogenetic analysis using a conserved region of the chpv and tupv ns1 gene produced a similar tree (zsak et al., 2008) , and a phylogenetic analysis of the chpv and tupv vp1 gene produced an identical tree when compared to the full-length parvovirus coding region (data not shown). this is the first analysis of the complete nucleotide sequence from the chpv abu-p1 strain. small viral particles (15 to about 25 nm) resembling parvovirus have been identified for years in the intestinal tracts of poultry (kisary et al., 1984; trampel et al., 1983; woolcock and shivaprasad, 2008) . the use of a particle-associated nucleic acid (pan) sequencing technique has recently allowed the partial molecular characterization of this novel chpv (zsak et al., 2008) , and led to the identification and sequencing of two additional avian parvoviruses from turkeys: tupv 260, originally detected in the intestinal tract of a turkey from california, and tupv 1078, originally detected in the pooled intestinal tracts from a north carolina turkey farm. all three of these poultry isolates are very similar to each other, yet each differs significantly from other members of the parvovirinae. the high amino acid identities noted among the turkey and chicken isolates suggests that they diverged at sometime in the recent past from a common ancestor. it is known from studies of another member of the parvovirinae, canine parvovirus (cpv), that the parvoviruses, despite their singlestranded dna genome, have a mutation rate that approaches that of rna viruses, which allows rapid evolution and host adaptation (hoelzer et al., 2008; shackelton et al., 2005) . interestingly, chpv and tupv do not group phylogenetically with the parvoviruses that infect geese (gopv) and muscovy ducks (mdpv), parvoviruses that are closely related to adeno-associated virus 2 (aav-2), a member of the dependovirus genus (fig. 3) (zadori et al., 1995; zsak et al., 2008) . further, the chpv ns protein shares only 19.1% amino acid identity with the mdpv ns protein. the itr sequences located at each end of the chpv genome (fig. 2 ) are each 206 nt long, somewhat shorter than those found in the prototypical human parvovirus b19 (383 nt) but longer than those found in some other autonomously replicating parvoviruses, namely the murine parvoviruses that have terminal palindromes of 115 and about 200 nt long in the same genome (astell et al., 1979; deiss et al., 1990) . the terminal palindromes found at the 5′ and 3′ ends of most autonomously replicating parvoviruses bear no nucleotide identity with one another. interestingly, this is not the case with chpv, which has identical palindrome sequences at its 5′ and 3′ end (fig. 2) , a trait it shares with parvovirus b19 (deiss et al., 1990; muzyczka and berns, 2001) . it would be interesting to determine if the 5′ end of the chpv genome exists in an alternative orientation, as is the case with b19, mdpv and gopv, in which the 5′ sequence can be found in one of two "flip" or "flop" orientations, with one orientation being the inverted complement of the other (deiss et al., 1990; zadori et al., 1995) . the chpv ns1 deduced amino acid sequence contains highly conserved motifs important for the initiation of parvovirus replication (zsak et al., 2008) , including a well-conserved phosphate-binding loop ("p-loop") motif specifically involved in the binding of nucleoside triphosphates by this protein (saraste et al., 1990) . this motif is required for pathogenesis in the human parvovirus b19 and is found in many parvovirus isolates (momoeda et al., 1994) . the 5′ major orf of chpv appears to encode the viral capsid proteins vp1, vp2, and vp3. these three proteins together comprise the parvovirus virion (muzyczka and berns, 2001) and are responsible for the production of neutralizing antibodies during an infection (saikawa et al., 1993) . the present analysis does not include members of the densovirinae subfamily, which infect insects and other invertebrates (muzyczka and berns, 2001) . chpv and the closely related tupvs were compared across their entire coding region with representative members of the parvovirinae subfamily, which includes the dependovirus, bocavirus, erythrovirus, amdovirus and parvovirus genera (fig. 3) . it is clear from the multiple sequence alignments and phylogenetic analysis that chpv, along with the closely related tupvs, represents a distinct member of the parvovirinae subfamily and should be recognized as the prototypical member of a novel genus within the parvovirinae. assembly of the prototypical chicken parvovirus (chpv abu-p1) genome the chicken parvovirus strain chosen for this analysis was originally isolated in hungary from the intestines of chickens showing sings of a stunting syndrome (kisary et al., 1984) , and this original cesium chloride gradient-purified parvovirus (provided by j. kisary) was propagated in specific pathogen free (spf) chickens as described previously (kisary, 1985; zsak et al., 2009) . a sequenceindependent polymerase chain reaction (pcr) protocol was employed to amplify particle-associated nucleic acid (pan) present in chpv abu-p1-positive chicken intestinal homogenates, and has been described elsewhere in detail (zsak et al., 2008) . the randomly amplified pan was ligated into the topo-ta cloning vector (invitrogen) and the ligation mix was used to transform competent top-10 e. coli cells (invitrogen). using this approach, a total of 768 clones were identified and sequenced using the m13 forward and reverse primers on an ab-3730 automated dna sequencer. the sequenced clones were used as query sequences to search the genbank non-redundant nucleotide (nr/nt) database using the blastn algorithm and the non-redundant (nr) protein database using the blastx algorithm. four contigs with an average of eightfold coverage and lengths of 1417 nt, 933 nt, 1069 nt, and 739 nt were assembled from 27, 24, 19, and 21 clones, respectively. these contigs had no significant nucleotide similarity to database sequences, but the deduced amino acid sequence from each contig fig. 3 . a phylogenetic tree prepared using the full coding sequence of the indicated parvoviruses. representative genera within the parvovirinae are indicated. the evolutionary relationships were inferred using the neighbor-joining method. phylogenetic analyses were conducted using mega4. genbank accession numbers for chpv, gu214704; tupv 1078, gu214705; and tupv 260, gu214706. revealed significant similarity to the members of the family parvoviridae in the database. pcr primers were designed using the four assembled contigs and were subsequently used to close gaps of 58, 443, and 205 nt between the contigs and assemble the complete chpv abu-p1 genome. the right terminal region of contig 4 contained a portion of the right inverted terminal repeat (itr). primers designed using this partial itr used in combination with forward and reverse primers within contig 1 (left terminal region of the genome) and contig 4 resulted in the cloning of the itr regions at both the right and left genomic termini. the chpv and tupv genomes and orfs were aligned with each other and with selected parvovirus sequences available in the databases using clustalw (thompson et al., 1994) . searches for conserved domains and transcription factor binding sites within the chpv genome were performed with genequest (dnastar/laser-gene8) and the conserved domain database (cdd) search service 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viruses associated with poult enteritis in turkeys grown in california analysis of the complete nucleotide sequences of goose and muscovy duck parvoviruses indicates common ancestral origin with adeno-associated virus 2 development of a polymerase chain reaction procedure for detection of chicken and turkey parvoviruses partial genome sequence analysis of parvoviruses associated with enteric disease in poultry thanks to fenglan li for excellent technical assistance and to the south atlantic area sequencing facility for outstanding support. key: cord-317537-wgu5cd0y authors: lu, hsiang-chia; chen, cheng-en; tsai, meng-hsiun; wang, hsiang-iu; su, hong-ji; yeh, hsin-hung title: cymbidium mosaic potexvirus isolate-dependent host movement systems reveal two movement control determinants and the coat protein is the dominant date: 2009-05-25 journal: virology doi: 10.1016/j.virol.2009.02.049 sha: doc_id: 317537 cord_uid: wgu5cd0y little is known about how plant viruses of a single species exhibit different movement behavior in different host species. two cymbidium mosaic potexvirus (cymmv) isolates, m1 and m2, were studied. both can infect phalaenopsis orchids, but only m1 can systemically infect nicotiana benthamiana plants. protoplast inoculation and whole-mount in situ hybridization revealed that both isolates can replicate in n. benthamiana; however, m2 was restricted to the initially infected cells. genome shuffling between m1 and m2 revealed that two control modes are involved in cymmv host dependent movement. the m1 coat protein (cp) plays a dominant role in controlling cymmv movement between cells, because all chimeric cymmv viruses containing the m1 cp systemically infected n. benthamiana plants. without the m1 cp, one chimeric virus containing the combination of the m1 triple gene block proteins (tgbps), the m2 5′ rna (1–4333), and the m2 cp effectively moved in n. benthamiana plants. further complementation analysis revealed that m1 tgbp1 and tgbp3 are co-required to complement the movement of the chimeric viruses in n. benthamiana. the amino acids within the cp, tgbp1 and tgbp3 which are required or important for cymmv m2 movement in n. benthamiana plants were mapped. the required amino acids within the cp map to the predicted rna binding domain. rna–protein binding assays revealed that m1 cp has higher rna binding affinity than does m2 cp. yeast two-hybrid assays to detect all possible interactions of m1 tgbps and cp, and only tgbp1 and cp self-interactions were observed. viruses have a defined, restricted host range, which varies among different viruses, or sometimes even different isolates of the same virus species. for example, cucumber mosaic virus (cmv) has a host range of more than 1000 plant species in 85 families, including monocotyledonous and dicotyledonous plants, and by contrast, barley stripe mosaic virus (bsmv) has a confined natural host to barley (ictvdb: the universal virus database of the international committee on taxonomy of viruses [http://www.ictvdb.iacr.ac.uk/ictv/fr-index. htm.]; (timian, 1974) . disease may occur when a particular virus adapts and/or shifts to a new host, for which there are many examples in plants and even humans (e.g., severe acute respiratory syndrome). successful infection of a plant host requires that plant viruses achieve (i) replication in the initially infected cells, (ii) movement through plasmodesmata to adjacent cells from the initial infected site and (iii) long-distance spread in a host through the vascular tissue. plant viruses possess only limited genetic information; hence, to complete their infection cycles, interactions between virus proteins and necessary host factors is critical. for a virus to successfully infect different plant hosts, the limited number of virus-encoded proteins must have the ability to recognize and interact with factors in different hosts for a successful infection. for some plant viruses, host plants restrict viral cell-to-cell movement rather than viral replication, which results in a subliminal infection (cheo, 1970; sulzinski and zaitlin, 1982) . in other cases, viruses may move from cell-to-cell, but the plant prevents viral long-distance spread (fujita et al., 1996; saenz et al., 2002; wang et al., 1998) . failure to accomplish any of the stages can be thought of as resistance. in our previous studies, we collected several isolates of cymbidium mosaic virus (cymmv) for construction of virus-induced gene silencing vectors for orchid functional genomic studies (lu et al., 2007) . cymmv is a prevalent orchid virus and belongs to the genus potexvirus in the family flexiviridae (adams et al., 2005b) . the recorded natural host range is largely limited to species belonging to orchidaceae (ictvdb: the universal virus database of the international committee on taxonomy of viruses [http://www.ictvdb.iacr. ac.uk/ictv/fr-index.htm.]; (adams et al., 2005a; zettler et al., 1990) . interestingly, we found two distinct cymmv isolates, m1 and m2, both of which can infect phalaenopsis orchids, but only m1 can systemically infect nicotiana benthamiana plants. because both isolates can systemically infect orchids, this suggests that viral-encoded replication and movement proteins are functional in orchids. however, because only m1 can systemically infect n. benthamiana plants this suggests that m1-encoded proteins could interact successfully with factors in both plant species to allow a systemic infection, but m2-encoded proteins do not. m1 and m2 share very high nucleotide sequence identity (97%), which suggests that subtle differences contribute to the differences in host reaction. viruses in the genus potexvirus have monopartite, positive-sense single-strand rna genomes (adams et al., 2005a) . studies of potexviruses have contributed importantly to our understanding of the complexity of the mechanisms of plant virus movement within plants (lucas, 2006; verchot-lubicz, 2005; verchot-lubicz et al., 2007) . comprehensive studies of potexviruses has revealed that triple gene block proteins (tgbps) and coat proteins (cp) work cooperatively to promote viral movement between cells and long-distance in their plant hosts (bayne et al., 2005; beck et al., 1991; chapman et al., 1992; fedorkin et al., 2001; forster et al., 1992; ju et al., 2007; krishnamurthy et al., 2003; lin et al., 2006; lough et al., 1998 lough et al., , 2000 mitra et al., 2003; tamai and meshi, 2001; verchot-lubicz, 2005 ). the current model suggests that tgbp1 increases the plasmodesmatal size-exclusion limit and moves into neighboring cells to suppress rna silencing (bayne et al., 2005; howard et al., 2004) ; both tgbp1 and cp bind viral rna (vrna) forming viral ribonucleoprotein complexes (vrnp; (karpova et al., 2006; lough et al., 2000 lough et al., , 2001 ; the complex is then delivered by tgbp2 induced vesicles (containing tgbp3) to plasmodesmata, and through plasmodesmata to neighboring cells; later, after the free form of tgbp1 binds membrane-associated tgbp2 and/or tgbp3, then tgbp1 (on the vrnp complex) binds with the membrane-associated tgbp1 complex (tgbp1 with tgbp2 or tgbp3) to form a membrane-bound complex and begin new rounds of replication (verchot-lubicz, 2005) . interactions between tgbp1 and cp (karpova et al., 2006; lough et al. 2000) , and tgbp1 self-interactions (samuels et al., 2007) have been reported, and those between tgbp1 and tgbp2, and between tgbp1 and tgbp3 have been suggested (karpova et al., 2006; krishnamurthy et al., 2002; lough et al., 2000; verchot-lubicz, 2005; yang et al., 2000) . interestingly, trans-complementation of tgbps between potexvirus species was not successful, and it was suggested that species-specific interactions among potexvirus movement proteins are obligatory for cell-to-cell movement (lin et al., 2006) . however, trans-complementation of movement defective potexviruses by movement proteins derived from tomato mosaic tobamovirus, crucifer tobamovirus and red clover necrotic mosaic dianthovirus have been reported (morozov et al., 1997) . although the movement of plant viruses, including potexviruses, has been extensively studied, little is known about how plant viruses of a single species exhibit different movement behavior in different host species. in this study, we aimed to use our cymmv-m1, -m2 and n. benthamiana system to study the molecular basis of cymmv isolatedependent host movement determinants, and to resolve the mechanism involved in cymmv translocation. our studies not only help to identify the molecular determinants of cymmv isolate-dependent host movement, but other phenomena that are not easily resolved by studies using a single isolate. finally, our efforts revealed two control behaviors, dominant and matching modes, involved in cymmv host dependent movement, and showed that the binding strength between the cymmv cp and genomic rna is correlated with the dominant control mode. the construction of the infectious clone of the isolates m1 (pcymmv-m1, fig. 1 ) and its derived expression vector (pcymmv-m1-gfp, fig. 1 ) engineered with the green fluorescent protein (gfp) was previously described (lu et al., 2007) . we followed the same strategy to construct the m2 infectious clone (pcymmv-m2, fig. 1 ) and its derived expression vector (pcymmv-m2-gfp, fig. 1 ). phalaenopsis amabilis var. formosa and nicotiana benthamiana plants were inoculated with transcripts from pcymmv-m1 and pcymmv-m2, and nucleic acids were extracted from the inoculated and upper, noninoculated leaves of pcymmv-m1 and pcymmv-m2-inoculated plants 14 days post-inoculation. similar to our previous findings, both pcymmv-m1 and pcymmv-m2 systemically infected phalaenopsis orchids (fig. 1b) , but only pcymmv-m1 systemically infected n. benthamiana (fig. 1c) . to test whether cymmv-m2 can replicate in n. benthamiana, protoplasts were inoculated with transcripts from pcymmv-m1, pcymmv-m2, pcymmv-m1-gfp and pcymmv-m2-gfp. illumination of gfp was observed from pcymmv-m1-gfp and pcymmv-m2-gfp rectangles represent open reading frames encoded by cymmv genomic rna. rnadependent rna polymerase (rdrp), triple gene block orfs 1, 2, and 3, capsid protein (cp) and green fluorescent protein (gfp). the mutated sequences in pcymmv-r-are indicated by bold letters. scale bar, in nucleotides, is shown at the bottom. p. amabilis var. formosa (b) and n. benthamiana (c) inoculated with buffer (h), pcymmv-rtranscripts (r-), pcymmv-m1 transcripts (m1) and pcymmv-m2 transcripts (m2), with cymmv-infected phalaenopsis used as a positive control (d). total nucleic acids were extracted from cymmv-inoculated and upper leaves distal from the inoculation point, and cymmv was detected by rt-pcr. pcymmv-r-is a replication-incompetent clone used as a negative control (see materials and methods) . numbers at the left correspond to positions of marker dnas (m) (sizes in 1000 base pairs). infected protoplasts at 14 h post-inoculation (fig. 2) . northern blot hybridization also indicated that pcymmv-m1 and pcymmv-m2 replicated in n. benthamiana protoplasts (fig. 3i ). to next analyze why m2 failed to accomplish systemic infection in n. benthamiana plants, more plants were inoculated with transcripts from pcymmv-m1-gfp and pcymmv-m2-gfp. because of limited expression of gfp of both pcymmv-m1-gfp and pcymmv-m2-gfp in plants of n. benthamiana, we therefore inoculated n. benthamiana with transcripts of pcymmv-m1 and pcymmv-m2 and assayed by whole-mount in situ hybridization 14 days post-inoculation. pcymmv-m1 moved from the inoculation foci and infected a broader area of leaves (fig. 2b) , whereas pcymmv-m2 was limited to the initial inoculated cells and did not spread from cell to cell, thus resulting only in subliminal infections (fig. 2b ). genome shuffling between cymmv-m1 and -m2 for complementation analysis pvx tgbps 1-3 and cp proteins are known to be required for virus cell-to-cell movement (bayne et al., 2005; chapman et al., 1992; krishnamurthy et al., 2002 krishnamurthy et al., , 2003 lough et al., 2000; morozov and solovyev, 2003; tamai and meshi, 2001; verchot-lubicz, 2005) . to determine if specific m1 gene(s) can complement m2 for virus movement, we constructed a series of m1 and m2 chimeric viruses (fig. 3) . n. benthamiana protoplasts and plants were inoculated with transcripts derived from each construct. northern blot hybridization revealed that each virus replicated in n. benthamiana protoplasts (fig. 3i) , and statistics analysis (anova) of real-time rt-pcr quantification of average relative percentage (from 3 independent experiments) of cymmv rna from cymmv clone-infected protoplasts at 24 h post-inoculation revealed no significant difference in percentage between these clones (p = 0.88). systemic infections were monitored by performing rt-pcr on leaves distal from the inoculation site at 2 weeks post-inoculation (figs. 3a-h). these analyses showed that if the cymmv construct contained the m1 cp, the virus systemically infected n. benthamiana plants (figs. 3a, c, e, h). one notable exception was the pcymmv-m2/ tgb-m1. this construct contained the m2 cp and 5′ rna sequence (1-4333; include the whole rna-dependent rna polymerase encoding region), but also the m1 triple gene block orfs (fig. 3f ). to identify whether tgbps 1-3 were all necessary to complement the systemic infection of pcymmv-m2/tgb-m1, we performed genome shuffling between m1 and m2 tgbps 1-3, and constructed chimeric viruses (figs. 4a-h). n. benthamiana protoplasts and plants were inoculated with transcripts derived from each construct. all constructs were replication competent in protoplasts as assayed by northern blot hybridization (fig. 4i) , and statistics analysis (anova) of real-time rt-pcr quantification of average relative percentage (from 3 independent experiments) of cymmv rna from cymmv clone-infected protoplasts at 24 h postinoculation revealed no significant difference in percentage between these clones (p = 0.91). systemic infection, as detected by rt-pcr at 2 weeks postinoculation (figs. 4a-h), indicated that only chimeric viruses containing the m1 tgbp1 and tgbp3 moved systemically (fig. 4g ). because these results were complicated, we used the karnaugh maps (harrison, 1969 ) tabular logical calculation to explore possible explanations (data not shown). the results of these analyses are summarized in fig. 5 . the analysis revealed that two control modes, which we termed dominant and matching control modes, are involved in cymmv movement. the m1 cp plays a dominant role in controlling the trafficking of the infection agents within plants. without the m1 cp, another control mode, which required correct matching of the particular movement accessory components including the m2 5′ rna (1-4333), m1 tgb p1, m1tgbp3, and m2 cp was also identified. our studies indicated that the m1 cp plays a major role in determining cymmv movement in n. benthamiana. because m1 and m2 share a very high nucleotide sequence identity (97%), we attempted to identify the individual, important amino acids of the cp that allowed for the cymmv systemic infection of n. benthamiana plants. sequence alignment of the m1 and m2 cps revealed only four amino acid differences (figs. 6a, b). site-directed mutagenesis was then performed to construct a series of cymmv-m2 cp mutants containing specific m1 cp amino acids (figs. 6c-i). all constructs were replication competent in protoplasts as assayed by northern blot hybridization (fig. 6j) , and statistics analysis (anova) of realtime rt-pcr quantification of average relative percentage (from 3 independent experiments) of cymmv rna from cymmv cloneinfected protoplasts at 24 h post-inoculation revealed no significant difference in percentage between these clones (p = 0.83). transcripts derived from each construct were used to inoculate n. benthamiana plants. these analyses showed that the cp amino acid g82a and l89p changes were both required for m2 to systemically infect n. benthamiana plants (figs. 6c, h). interestingly, these amino acids are located within the previously predicted rna binding domain of the cp (fig. 7a ). it has been previously reported that the cp is involved in forming ribonucleoprotein complexes and is an important step for potexvirus movement (karpova et al., 2006; lough et al., 2000 lough et al., , 2001 . our results indicated that the important amino acids of the cp that allowed for the cymmv systemic infection are located within the previously predicted rna binding domain ( fig. 7a; 1) . thus, it might be that the binding affinity between cps and rnas of cymmv m1 and m2 may play roles in cymmv movement, and therefore we attempted to analyze and compare their binding affinities. because the whole genomic rna of cymmv is 6227 nucleotides in length, which made conventional gel-shift assays difficult, we performed modified rna-protein pull-down assays for our analyses. our data indicated that cymmv m1 cp has a higher binding affinity as compared to cymmv m2 cp (fig. 7d, lanes 1-4) . we then also performed cp and cymmv rna binding assays on cymmv m2 cp with the amino acid changes g82a and l89p (derived from clone pcymmv-m2-cp-gl/ap; fig. 6h ). the results indicated that with these amino acid changes (g82a and l89p), the binding affinity was higher as compared to wild-type cymmv-m2 cp (fig 7d, lanes 3 ,4,5 and 6). our analysis also indicated that both m1 tgbp1 and tgbp3 were co-required for cymmv m2 chimeric viruses to move systemically in n. benthamiana plants (fig. 4g) , and was consistent with previous suggestions indicating interactions between potexvirus tgbp1 and tgbp3 (lucas, 2006; verchot-lubicz, 2005; verchot-lubicz et al., 2007) . therefore, we attempted to determine if specific interactions occurred within and between cymmv tgbps and cp by use of the genomic rna (g), tgbp, and cp subgenomic rna are indicated. the pcymmv-r-used as a negative control is illustrated in fig. 1 . the average percentage of relative real-time rt-pcr quantification (from 3 independent experiments) of cymmv rna from cymmv clone-infected protoplasts at 24 h post-inoculation is indicated below the gels. the accumulation of pcymmv-m1 was set at 100% for relative quantification. numbers at the left correspond to positions of marker rnas (sizes in 1000 nucleotides) analyzed in the same gel. the primers cymmv f3783 and cymmv cpr used in construction all chimeric viruses are indicated. other primers used in construction individual chimeric viruses are in supplementary table s1 . numbers at the left correspond to positions of marker rnas (sizes in 1000 nucleotides) analyzed in the same gel. yeast two-hybrid assay. the results were similar to those previously reported in pvx tgbps and cp (samuels et al., 2007) ; where only self interaction of tgbp1 and cp were identified (table 1) . sequence alignment analysis revealed 4 and 2 amino acid differences within the tgbp1 and tgbp3 between cymmv-m1 and cymmv-m2, respectively (figs. 8a, b). loss-of-function assays were performed by using site-directed mutagenesis to substitute tgbp1 and tgbp3 with the 4 and 2 amino acids derived from m2 tgbp1 and tgbp3, respectively (figs. 8c-h). n. benthamiana plants were inoculated with transcripts derived from each construct, and systemic infection was analyzed by rt-pcr (fig. 8) . the chimeric viruses with either amino acids y44h and h94r changes in m1 tgbp1 failed to move systemically (figs. 8c, d). although the chimeric viruses with either amino acids l8q or a71s changes in the m1 tgbp3 did move systemically, the ratio of systemically infected plants was much reduced (figs. 8g, h). to further characterize whether y 44 and/or h 94 in tgbp1 are required for m2 to systemically infect n. benthamiana plants, sitedirected mutagenesis was conducted on pcymmv-m2/tgb3-m1 (fig. 4e ) to construct m2 clones substituted with h44y and r94h at tgbp1, either individually or simultaneously (figs. 8i-k). both y and h at tgbp1 positions 44 and 94 were required for m2 (pcymmv-m2/tgb3-m1) to systemically infect n. benthamiana plants (fig. 8k) . mutants having only one of these changes failed to move systemically (figs. 8i, j). similarly, site-directed mutagenesis was conducted on pcymmv-m2/tgb1-m1 (fig. 4c ) and pcymmv-m2-tgb3-m1-tgb1 hr/yh (fig. 8k) to construct clones substituted with q8l and/ or s71a at tgbp3 (fig. 8l-o) . both q8l and/or s71a in m2 tgbp3 were important but not required for m2 to systemically infect n. benthamiana. mutants having only one of these changes moved systemically, but the ratio of systemically infected plants was much reduced (figs. 8g-o). the y 44 (position 44) and h 94 in tgbp1 are located within the previous predicted ntp/helicase domain but not in the identified conserved motifs (kadare and haenni, 1997; kalinina et al., 2002; leshchiner et al., 2006) , and l 8 and a 71 in tgbp3 are also not located in the previously identified transmembrane domain ( fig. 9b;21) . all constructs used in fig. 8 were replication competent in protoplasts as assayed by northern blot hybridization (fig. 8p) , and statistics analysis (anova) of real-time rt-pcr quantification of the average relative percentage (from 3 independent experiments) of cymmv rna from cymmv clone-infected protoplasts at 24 h postinoculation revealed no significant difference in percentage between these clones (p = 0.92). our studies comparing cymmv isolate-dependent host movement provides new and important information on the molecular basis of how viruses of the same species can move in different host plants. we also demonstrate complex aspects of potexvirus movement not easily identified by studies using a single isolate. the more detailed resolution of these phenomena, as we demonstrate in this paper, will further help to dissect the mechanisms of potexvirus movement in plants. our data indicated that m1-encoded cp plays a major role for cymmv in systemic infection of n. benthamiana plants (fig. 5) . in all our cymmv clones, if the constructs contained the m1 cp, the virus systemically infected n. benthamiana plants (figs. 3a, c, e, h). interestingly, our data also showed that if cp was not properly functional (e.g., m2 cp), alterations in tgbps (e.g., m1 tgbp1 and 3) may compensate for the function of cp and allow cymmv to systemically infect n. benthamiana plants (fig. 3f ). in addition, when comparing pcymmv-m2/tgb-m1 and pcymmv-m1/cp-m2 (figs. 3f and g), only pcymmv-m2/tgb-m1 was movement competent. the tgbps and cp are the same in both chimeric viruses. these data indicate that another control mode which required correct matching of the particular movement accessory components (m2 5′ rna, tgbp1 and tgbp3 and m2 cp) also allowed the systemic infection of cymmv in n. benthamiana ( fig. 3f and fig. 5 ). thus, our data suggested two control modes are involved in cymmv movement in n. benthamiana. the nature of the cymmv vrnp that moves between cells remain a subject for study , and two forms, one is the linear vrnp formed by tgbp1, vrna and cp (lough et al., 2000) and another is virion or altered virion with a single-tailed particle comprising rna, cp and tgbp1 (cruz et al., 1998; karpova et al., 2006) . however, no matter what the proposed form of vrnp that moves between cells, all data suggest that potexvirus cp and vrna are involved in forming the vrnp which traffics to the plasmodesmata. our analysis indicated that the cymmv cp-vrna binding affinity likely plays an important role in cymmv movement in n. benthamiana plants. therefore, it is possible that the binding between cp-vrna may affect the nature of vrnp; thus affecting the movement of cymmv in different hosts. our data also suggested that different combinations of cp-vrna required different degrees of help from tgbps for movement (figs. 3,4) . when the binding between cp-vrna is higher (fig. 7 d) , the need for tgbps in cymmv movement is more flexible (figs. 3a, c, e, h) ; however, when the binding between cp-vrna is weaker, the need for tgbps in cymmv movement is critical and only certain combinations of cymmv movement accessory components allowed for cymmv movement (figs. 3b, f, 4g). it has been previously reported that in systemic hosts of pvx, the movement of tgbps are host dependent (krishnamurthy et al., 2002; yang et al., 2000) . because in different hosts the cellular conditions and morphology are different, it is possible that potexviruses may use different strategies to adapt to different hosts. adjusting movement accessory components could be an efficient way to generate the diversity of functions in response to changes of cellular conditions and provide flexibility for virus movement in different hosts, and thus could be an advantage for viruses with modular designed movement proteins (morozov and solovyev, 2003) . although interactions between potexvirus tgbp1 and tgbp2 and between tgbp1 and tgbp3 have been suggested (karpova et al., 2006; krishnamurthy et al., 2002; lough et al., 2000; verchot-lubicz, 2005; yang et al., 2000) , the interactions have yet to be proven. interestingly, our data, showed that cymmv m1-encoded tgbp1 and tgbp3 are corequired for m2 systemic infection of n. benthamiana (fig. 4g) ; which support the hypothesis for interactions between tgbp1 and tgbp3. however, the results of our yeast two-hybrid analysis were similar to those that have been recently reported showing that tgbp1 was selfassociated, but showed no interactions between tgbp1 and tgbp2 or tgbp3 (samuels et al., 2007) . thus, the interactions may not occur physically, or the interactions could be transient and not easily detected. within the potexvirus tgbp1, the ntpase/helicase domains have been predicated, and 7 conserved regions including two canonical motifs of ntpase, dey and gks, were identified ( fig. 9 ; kadare, and haenni, 1997; kalinina et al., 2002; leshchiner et al., 2006) . although, currently the roles of ntpase/helicase domains in potexvirus movement have not been well established , mutations eliminating the dey motif affects the protein subcellular targeting and mutations eliminating the dey and gks motifs inhibit the ability of tgbp1 to increase plasmodesmata size-exclusion limits as well as virus movement (angell et al., 1996; lough et al., 1998; morozov et al., 1999) . the amino acids identified in cymmv tgbp1 that are required for cymmv-m2 movement in n. benthamiana are within the ntpase/helicase domain, but not in the conserved motifs (fig. 9) . the amino acids are also not in the positions within the ntpase/helicase domain previously reported as essential for bamboo mosaic potexvirus movement (lin et al., 2004) . within the potexvirus tgbp3, a transmembrane domain has been previously reported. pvx fig. 1 . the pcymmv-r-used as a negative control is illustrated in fig. 1 . the average percentage of relative real-time rt-pcr quantification (from 3 independent experiments) of cymmv rna from cymmv clone-infected protoplasts at 24 h post-inoculation is indicated below the gels. the accumulation of pcymmv-m1 was set at 100% for relative quantification. numbers at the left correspond to positions of marker rnas (sizes in 1000 nucleotides) analyzed in the same gel. with substitution mutations within the transmembrane domain is restricted to a single cell, and pvx with mutations outside the transmembrane domain show reduced movement between cells and may not move systemically . the amino acids identified in tgbp3 which are important for cymmv-m2 movement in n. benthamiana are not located in the transmembrane domain ( fig. 9 ; krishnamurthy et al., 2003) . because all amino acids identified in cymmv tgbp1 and tgbp3 are not located in the conserved motifs previously identified among potexviruses, nor within the amino acids reported as essential for potexvirus movement, we speculate that changes in these regions may only slightly affect but not abolish the function (as we see in the case of cymmv-m2 cp) of tgbp1 and tgbp3, and the differential function required for virus movement is host dependent. this may explain why m1 and m2 can both systemically infect phalaenopsis orchid, but only m1 can systemically infect n. benthamiana. (thompson et al., 1997) . the previously identified conserved positive charged amino acids are indicated by stars (abouhaidar and lai, 1989) it is worth noting that correct matching of the particular movement accessory components also includes the cymmv 5′ rna sequence and/or the rna-dependent rna polymerase (rdrp). the potexvirus rdrp has not been reported to be required for virus movement, however it has been suggested that the 5′ untranslated region of the potexvirus genomic rna plays a role in viral cell-to-cell movement (lough et al., 2006) . both cymmv-m1 and -m2 replicated well in n. benthamiana protoplasts or in phalaenopsis orchids. it seems more likely that rna rather than the rdrp plays a role in cymmv movement. however, recently it was reported that the tmv replicase and movement protein function together in altering plasmodesmeta (guenoune-gelbart et al., 2008) . therefore, we cannot rule out the possibility that the rdrp plays a role in controlling cymmv movement in n. benthamiana plants. rna was extracted from plants as described for northern blot analysis and rt-pcr (tian et al., 1996) . t7 rna polymerase and hpaidigested pcymmv-m1 plasmids (cymmv probe; corresponding to the 590 nt of cymmv at its 3′ end) were used to generate the negativesense dig-labeled probes (roche applied science; mannheim, germany). northern blot hybridization was performed as described , and hybridization signals were detected by use of the chemiluminescent substrate cdp star (roche applied science) and exposing blots to fuji medical x-ray film (fuji, tokyo, japan). the construction of pcymmv-m1 and pcymmv-m1-green fluorescent protein (fig. 1a) was as described (lu et al., 2007) . the same approaches were used to construct pcymmv-m2 and pcymmv-m2-gfp. both pcymmv-m1 and -m2 had been completely sequenced. pcymmv-r-is a spontaneous mutation clone obtained during the cloning of pcymmv-m1. pcymmv-r-contains a mutation in the rnadependent rna polymerase (rdrp) region, which causes pretermination of the rdrp (fig. 1a) . the 5′ mutated rdrp region of the pcymmv-r-has been sequenced. the infectious clones pcymmv-m1 and pcymmv-m2 (fig. 1a) were digested with naei and nhei individually. two digested fragments derived from each reaction were gel purified by use of a gel extraction kit (qiagen; hilden, germany); the small fragments derived from pcymmv-m1 and -m2 were exchanged and ligated by use of t4 dna ligase (promega; madison, wi, usa) to construct pcymmv-m1/rdrp-m2 and pcymmv-m2/rdrp-m1 (figs. 3c and d) . the gel purification and ligation conditions followed the manufacturer's manual. the construction of pcymmv-m1/tgb-m2, pcymmv-m1/tgb-m1, pcymmv-m1/cp-m2 and pcymmv-m1/cp-m1 clones (figs. 3e-h) was similar to that of pcymmv-m1/rdrp-m2 and pcymmv-m2/rdrp-m1, except the restriction enzymes nhei and hpai were used to construct pcymmv-m1/tgb-m2 and pcymmv-m2/tgb-m1, and the restriction enzymes hpai and saci was used to construct pcymmv-m1/ cp-m2 and pcymmv-m2/cp-m1. all recombinant clones were sequenced in the modified region to ensure that the clones were correct. pcymmv-m1 and pcymmv-m2 were used as the initial template, and the primer pairs cymmv f3783/cymmv-tgb1 r and cymmv-tgb1 f/cymmv cpr (supplementary table s1 ) were used, respectively in the pcr reactions to amplify the two overlapping fragments. the amplified fragments were gel purified and mixed together at a ratio of 1:1, then used in another pcr reaction. the pcr reaction cycles were 94°c for 5 min for 1 cycle, then 94°c for 30 s, 55°c for 30 s and 72°c for 2 min for a total of 5 cycles. then the primer pair cymmv f3783/cymmv cpr was added for another 30 cycles. the pcr cycles were 94°c for 5 min for 1 cycle, then 94°c for 30 s, 55°c for 30 s and 72°c for 2 min for a total of 30 cycles. the amplified products were digested with nhei and hpai (fig. 3) , and then separated on a 1% agarose gel to purify the 1.3-kb fragment. the pcymmv-m2 was digested with nhei and hpai, then separated on 1% agarose gel to purify the 8-kb fragment. both digested fragments were ligated to yield a construct pcymmv-m2/tgb1-m1 (fig. 4c) . the construction of other recombinant tgb clones (figs. 4d-h) was essentially the same as that for pcymmv-m2/tgb1-m1, except that different primer pairs and templates were used. the primer cymmv f3783/tgb1 and cymmv cpr were used in construction of all tgb recombinant clones. the clone name indicates the origin of the tgb. for example, pcymmv-m2/ tgb1-m1 indicates that the backbone of the clone is pcymmv-m2, but the tgbp1 is derived from pcymmv-m1. for construction of pcymmv-m2/tgb2-m1, an intermediate clone, pcymmv-m1/tgb1-m2 was first constructed and used for a template to amplify the m2 tgbp1 and m1 tgbp2. the amino acid sequences in an overlapping region between tgbp1 and tgbp2 and between tgbp2 and tgbp3 are identical in both pcymmv-m1 and -m2. all primers are shown in supplementary table s1. all recombinant clones were sequenced in the modified region to ensure that the clones were correct. each of the mutated clones derived from pcymmv-m1/m2 with amino acid substitution(s) in the cp, tgbp1 and tgbp3 (figs. 6, 8) was constructed by site-directed mutagenesis as described (lu et al., 2007) . pcymmv-m1 was used as the initial template, and the primer pairs cymmv f3783/tgb1 y44h r and tgb1 y44h f/cymmv cpr were used in the first pcr reactions. the amplified fragments were gel purified and mixed together at a ratio of 1:1, then used in another pcr reaction. the pcr reaction cycles were as described above. the amplified products were digested with nhei and hpai (fig. 3) , and then separated on a 1% agarose gel to purify the 1.3-kb fragment. the pcymmv-m2 was digested with nhei and hpai, then separated on a 1% agarose gel to purify the 8-kb fragment. both digested fragments were ligated to construct tgbp1 +++ +++ +++ tgbp1 tgbp2 tgbp3 tgbp1 tgbp3 +++ − na cp cp +++ +++ +++ a "⁎", plasmid expression ad domain (pgadt7) and bd domain (pgbkt7) are added. b "+", yeast colonies that grew on sd medium (lacking trp and leu). "−", yeast colonies that did not grow on sd medium (lacking trp and leu). c "+", yeast colonies that grew on sd medium (lacking trp, leu, his and ade). "−", yeast colonies that did not grow on sd medium (lacking trp, leu, his and ade). d "+", yeast colonies that were blue. "na" indicates yeast colonies did not grow and no blue histochemical stain was observed. e plasmids containing t-antigen-ad and murine p53-bd were provided by the manufactory and used as positive controls. pcymmv-m1-tgb1 y/h containing a single substitution (y/h) at amino acid position 44 of tgbp1 of pcymmv-m1 (fig. 8c ). the construction of other tgb site-directed mutagenesis clones (fig. 8 ) was essentially the same as that for pcymmv-m1-tgb1 y/h, except that different primer pairs and templates were used. the primer cymmv f3783/tgb1 and cymmv cpr were used in construction of all mutant clones. the remaining primers are in supplementary table s1 . the mutated clones with names beginning with pcymmv-m1-or -m2-indicate that the initial template was pcymmv-m1 or pcymmv-m2, respectively. modifications of nucleotide sequence in an overlapping region between tgbp1 and tgbp2 (one nucleotide substitution) and between tgbp2 and tgbp3 (two nucleotides substitution) do not cause amino acid sequence changes in tgbp2. all mutant clones were sequenced in the modified region to ensure that the mutations were correct. capped transcripts corresponding to the wild-type virus and the constructed vectors of cymmv were synthesized by use of the mmessage mmachine t3 high yield capped rna transcription kit (ambion, inc., austin, tx). in total, 5 μg of pcymmv-m1 and its derivative plasmids were digested with spei and in vitro transcription was conducted according to manufacturer's instruction. protoplasts prepared from nicotiana benthamiana plants and rna transfection were as previously described (satyanarayana et al., 2002) , except that 10 μg of each transcript and 2 × 10 5 cells were used for each inoculation, and after inoculation, protoplasts were incubated at 26°c. the inoculated protoplasts were collected as previously described . aliquots containing approximately 1 × 10 5 cells were collected by centrifugation (1300 ×g) at different times post-inoculation, and rna was isolated by use of trizol reagent (invitrogen, san diego, ca) according to the manufacturer's recommendations. the rna was dissolved in 30 μl depc-treated water, and 5 μg rna (equivalent to 5 × 10 4 cells) was used for northern hybridization analysis. an amount of 5 μg transcripts was dissolved in inoculation buffer (0.05 m nah 2 po 4 /na 2 hpo 4 ph 7.0) and rubbed to carborundumdusted phalaenopsis var. formosa (6 leaf stage) or n. benthamiana (5 leaf stage) by hands wearing latex gloves. the inoculated leaves were washed with excess distilled water, and the viruses were detected 2 weeks post-inoculation. all plants were kept in an insect-proof and fig. 9 . the amino acid sequence alignment of tgbp1 ntp/helicase domain and tgbp3 transmembrane domain of potexvirus. the amino acid sequence alignments were conducted by use of clustal x 1.83 (thompson et al., 1997) . (a) the conserved motifs of the potexvirus ntpase/helicase domain were previously predicted (kalinina et al., 2002) . the 7 predicted ntpase/helicase motifs of potexvirus tgbp1 are shown, and two canonical motifs of ntpase, dey and gks, are indicated. the arrows indicate the amino acid positions important for pcymmv-m2 to systemically infect n. benthamiana. (b) the potexvirus tgbp3 transmembrane domain was previously identified . the cymmv transmembrane domain predicted by das program (http://www.sbc.su.se/~miklos/das/maindas.html) is indicated by a thick black line. the arrows indicate the amino acids important for pcymmv-m2 to systemically infect n. benthamiana. the viruses, abbreviation and accession number used in alignments of tgbp1 ntp/helicase and tgbp3 transmembrane domains are described below. cymbidium mosaic virus (cymmv, accession number ay571289); cymbidium mosaic virus (cymmv, accession number ay571289); foxtail mosaic virus (fmv, accession number nc_001483); watermelon spotted wilt virus (bsmv, accession number nc_003481); cassava common mosaic virus (cscmv, accession number nc_001658); papaya mosaic virus (pmv, accession number nc_001748); cactus virus x (cvx, accession number nc_002815); potato virus x (pvx, accession number nc_001455); white clover mosaic virus (wc1mv, accession number x06728). thermal-controlled (25°c-28°c) greenhouse and with 12 h of light (5.22 μmol/s − 1 m − 2 ) for two weeks. p. amabilis var. formosa (10 cm in height) was purchased from the taiwan sugar research institute (tainan, taiwan). each set of inoculations (figs. 3, 4, 6 and 8) was repeated at least three times. rt-pcr was used to amplify the modified region (primer pairs used for rt-pcr were described in supplementary table 1 ) of each mutated cymmv from two randomly selected plants of each inoculation set followed by sequencing, all detected progeny viruses still maintained the original modification. rna extracted from cymmv-infected plants was used as a template for synthesis of cdnas by moloney murine leukaemia virus (mmlv) reverse transcriptase following the manufacturer's instructions (promega, inc., madison, wi, usa). the pcr amplification conditions were as described (rubio et al., 2000) . the cdnas were pcr amplified in a mixture containing 1.5 mm mgcl 2, 1 mm of each of the 4 dntps, 2.5 u of taq dna polymerase (promega, inc., madison, usa), and 50 ng of each oligonucleotide. the pcr cycles were 94°c for 4 min for 1 cycle, then 94°c for 30 s, 55°c for 30 s and 72°c for 1 min for 30 cycles, then an extension at 72°c for 10 min. pcymmv-m1 and pcymmv-m2 were used as the initial template, and the primer pairs cymmv cp-bamhi f and cymmv cp-bamhi r (supplementary table s1 ) were used in the pcr reactions to amplify the two cymmv cp fragments. the fragments were gel purified by use of a gel extraction kit (qiagen) and digested with bamhi. the fragment derived from pcymmv-m1 and pcymmv-m2 was incubated with the bamhi-digested expression vector pgex-2t and ligated by use of t4 dna ligase (promega) to construct pgex-2t-2:m1cp and pgex-2t-2:m2cp. both clones had been sequenced completely. a total of 5 ml of overnight cultures of escherichia coli xl1-blue transformed with pgex-2t-2:m1cp, pgex-2t-2:m2cp and pgex-2t, were diluted 1:100 with 50 ml luria-bertani medium (lb) containing ampicillin (50 μg/ml), and grown until they reached the od 600 = 1.0 at 37°c. 200 ml lb medium containing ampicillin (50 μg/ml) was added, and bacteria were grown until they reached the od 600 = 0.5 at 37°c. then the fusion protein expression was induced with 1 mm isopropylβ-d-thiogalactopyranoside (iptg) for 2 h at 37°c. after centrifugation at 5000 ×g for 10 min at 4°c, the pellet was suspended in a 10 ml phosphate buffer saline (pbs) (135 mm nacl, 2.7 mm kcl, 1.5 mm kh 2 po 4, and 8 mm k 2 hpo 4 , ph 7.2) and incubated in 1% triton x-100 for 60 min at 4°c then disrupted by sonication. cell debris was removed by centrifugation for 5 min at 4°c at 13,000 ×g. glutathione sepharose beads were added and incubated for 4 h at 4°c. after centrifugation at 3000 ×g for 5 min at 4°c, the pellet was suspended in 1 ml pbs and the wash was repeated 4 times. finally, 500 μl elution buffer (10 mm reduced glutathione; 50 mm tris-hcl ph 8.0) was added, samples were centrifuged at 500 ×g for 5 min at 4°c, the suspension was collected and analyzed in 12% sds-page gels (laemmli, 1970) . the pcymmv-m1 and pcymmv-m2 rna transcripts were synthesized by use of the mmessage mmachine t3 high yield capped rna transcription kit (ambion). the biotinylated diribonucleotide, pucbiotin (midland certified reagents, inc. midland, texas, usa) was ligated to transcripts with t4 rna ligase. the biotin-labeled cymmv rna was incubated with glutathione-s-transferase (gst) fused to cymmv cp in 100 μl incubation buffer (10 mm tris/hcl and 150 mm nacl, 1mm edta, 5 mm mgcl 2 , 1 mm dithiothreitol, ph 7.6) at 20°c for 2 h. glutathione sepharose beads were added and incubated at 4°c for 4 h. after centrifugation at 3000 ×g for 5 min at 4°c, the supernatant was removed and the pellet was re-suspended in incubation buffer of different concentrations (150, 300, 400 and 1000 mm) of sodium chloride. after centrifugation at 3000 ×g at 4°c for 5 min, supernatant (defined here as s) and pellets were collected. the pellets were resuspended in high salt incubation buffer (1 m of sodium chloride) to separate the cymmv cp-rna complex from glutathione sepharose beads. after centrifugation at 3000 ×g at 4°c for 5 min, the supernatant (defined here as p) were collected. the collected s and p were passed through hybond-nx membrane (amersham ge healthcare, inc., piscataway, nj, usa) using a slot-blot apparatus (schleicher and schuell, inc., keene, nh, usa). the membranes were baked at 80°c for 30 min. the signals were detected first using antibody against biotin and by use of a lightshift chemiluminescent emsa kit (pierce, inc., rockford, il) followed the manufacturer's protocol for chemical illumination. fluorescence signals were captured by use of a biospectrum ac imaging system (uvp, llc., upland, ca, usa). whole-mount rna analysis was as described with modification (zachgo et al., 2000) . sample fixing was extended to 2 h rt in a glass vial in pbs containing 0.1% tween 20, 0.08 m egta, 10% dmso and 5% paraformaldehyde. cymmv cp probe (see above) was used for hybridization. pcymmv-m1 was used as the initial template, and the primer pairs tgb1f/tgb1r, tgb2f/tgb2r, tgb3f/tgb 3r, cpf/cpr (supplementary table s1 ) were used in pcr reactions to amplify the tgbp1, tgbp2, tgbp3 and cp fragments. the fragments were gel purified, incubated with the smai digested pgdat7 and pgbkt7 (clontech, mountain view, ca), and ligated by use of t4 dna ligase (promega) to construct ptgb1-ad, ptgb2-ad, ptgb3-ad, pcp-ad, ptgb1-bd, ptgb2-bd, ptgb3-bd and pcp-bd. all clones had been sequenced completely. yeast two-hybrid assays were done by use of the matchmaker yeast two-hybrid system 3 (clontech) following the manufacturer's instructions. yeast was transformed with bait vectors and prey vectors and selected on sd plates lacking leu and trp. after 2 days of growth at 30°c, the yeast colonies were transferred to two different selection plates containing sd medium lacking leu, trp, and sd medium (containing x-α-gal) lacking leu, trp and his. statistical analysis involved anova or dunnett's t test with use of minitab14 (minitab inc., state college, pa, usa). nucleotide sequence of the 3′-terminal region of clover yellow mosaic virus rna virus taxonomy: eighth report of the international committee on taxonomy of virus fauquet, virus taxonomy : eight report of the international committee on taxonomy of viruses cell-to-cell movement of potato virus x is associated with a change in the size-exclusion limit of plasmodesmata in trichome cells of nicotiana clevelandii cell-to-cell movement of potato potexvirus x is dependent on suppression of rna silencing triple gene block proteins of white clover mosaic potexvirus are required for transport mutational analysis of the coat protein gene of potato virus x: effects on virion morphology and viral pathogenicity subliminal infection of cotton by tobacco mosaic virus cell-to-cell and phloem-mediated transport of potato virus x. the role of virions cell-to-cell movement of potato virus x involves distinct functions of the coat protein the coat protein of white clover mosaic potexvirus has a role in facilitating cell-to-cell transport in plants a single codon change in a conserved motif of a bromovirus movement protein gene confers compatibility with a new host tobacco mosaic virus (tmv) replicase and movement protein function synergistically in facilitating tmv spread by lateral diffusion in the plasmodesmal desmotubule of nicotiana benthamiana optimize switching circuits using karnaugh maps potato virus x tgbp1 induces plasmodesmata gating and moves between cells in several host species whereas cp moves only in n. benthamiana leaves mutations in the central domain of potato virus x tgbp2 eliminate granular vesicles and virus cell-to-cell trafficking virus-encoded rna helicases rna helicase activity of the plant virus movement proteins encoded by the first gene of the triple gene block potato virus x rnamediated assembly of single-tailed ternary 'coat protein-rna-movement protein' complexes in vitro transcripts from cloned cdnas of the lettuce infectious yellows closterovirus bipartite genomic rnas are competent for replication in nicotiana benthamiana protoplasts cell-to-cell movement of the pvx 12k, 8k, or coat proteins may depend on the host, leaf developmental stage, and the pvx 25k protein the potato virus x tgbp3 protein associates with the er network for virus cell-to-cell movement cleavage of structural proteins during the assembly of the head of bacteriophage t4 a minimal region in the ntpase/helicase domain of the tgbp1 plant virus movement protein is responsible for atpase activity and cooperative rna binding arg-16 and arg-21 in the nterminal region of the triple-gene-block protein 1 of bamboo mosaic virus are essential for virus movement movement of potexviruses requires species-specific interactions among the cognate triple gene block proteins, as revealed by a trans-complementation assay based on the bamboo mosaic virus satellite rna-mediated expression system molecular dissection of the mechanism by which potexvirus triple gene block proteins mediate cell-to-cell transport of infectious rna cell-to-cell movement of potexviruses: evidence for a ribonucleoprotein complex involving the coat protein and first triple gene block protein trans-complementation of longdistance movement of white clover mosaic virus triple gene block (tgb) mutants: phloem-associated movement of tgbp1 functional analysis of the 5′ untranslated region of potexvirus rna reveals a role in viral replication and cell-to-cell movement strategies for functional validation of genes involved in reproductive stages of orchids plant viral movement proteins: agents for cell-to-cell trafficking of viral genomes the potato virus x tgbp2 protein association with the endoplasmic reticulum plays a role in but is not sufficient for viral cell-to-cell movement triple gene block: modular design of a multifunctional machine for plant virus movement complementation of a potato virus x mutant mediated by bombardment of plant tissues with cloned viral movement protein genes evidence for two nonoverlapping functional domains in the potato virus x 25k movement protein a heterogeneous population of defective rnas is associated with lettuce infectious yellows virus host-specific involvement of the hc protein in the long-distance movement of potyviruses subcellular targeting and interactions among the potato virus x tgb proteins the p23 protein of citrus tristeza virus controls asymmetrical rna accumulation tobacco mosaic virus replication in resistant and susceptible plants: in some resistant species virus is confined to a small number of initially infected cells cell-to-cell movement of potato virus x: the role of p12 and p8 encoded by the second and third open reading frames of the triple gene block the clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools generation of cdnas specific to lettuce infectious yellows closterovirus and other whiteflytransmitted viruses by rt-pcr and degenerate oligonucleotide primers corresponding to the closterovirus gene encoding the heat shock protein 70 homolog the range of symbiosis of barley and barley stripe mosaic virus a new cell-to-cell transport model for potexviruses molecular biology of potexviruses: recent advances mutations in viral movement protein alter systemic infection and identify an intercellular barrier to entry into the phloem long-distance transport system cell-to-cell movement of the 25k protein of potato virus x is regulated by three other viral proteins in situ analysis of rna and protein expression in whole mounts facilitates detection of floral gene expression dynamics viruses of orchids and their control supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.virol.2009.02.049. key: cord-297712-yy4g5npi authors: zhu, xinyu; fang, liurong; wang, dang; yang, yuting; chen, jiyao; ye, xu; foda, mohamed frahat; xiao, shaobo title: porcine deltacoronavirus nsp5 inhibits interferon-β production through the cleavage of nemo date: 2016-12-13 journal: virology doi: 10.1016/j.virol.2016.12.005 sha: doc_id: 297712 cord_uid: yy4g5npi porcine deltacoronavirus (pdcov) causes acute enteric disease and mortality in seronegative neonatal piglets. previously we have demonstrated that pdcov infection suppresses the production of interferon-beta (ifn-β), while the detailed mechanisms are poorly understood. here, we demonstrate that nonstructural protein 5 (nsp5) of pdcov, the 3c-like protease, significantly inhibits sendai virus (sev)-induced ifn-β production by targeting the nf-κb essential modulator (nemo), confirmed by the diminished function of nemo cleaved by pdcov. the pdcov nsp5 cleavage site in the nemo protein was identified as glutamine 231, and was identical to the porcine epidemic diarrhea virus nsp5 cleavage site, revealing the likelihood of a common target in nemo for coronaviruses. furthermore, this cleavage impaired the ability of nemo to activate the ifn response and downstream signaling. taken together, our findings reveal pdcov nsp5 to be a newly identified ifn antagonist and enhance the understanding of immune evasion by deltacoronaviruses. porcine deltacoronavirus (pdcov), a new swine enteropathogenic coronavirus, belongs to the genus deltacoronavirus in the family coronaviridae (woo et al., 2009 (woo et al., , 2012 . it is an enveloped virus with a single-stranded, positive-sense rna genome of nearly 25 kb, initially detected in pigs in hong kong, china in 2012 (woo et al., 2012) . the clinical significance of pdcov has been highlighted since outbreaks of this virus, which causes severe diarrhea and mortality of piglets, occurred in multiple states of the united states in 2014 homwong et al., 2016; hu et al., 2015 hu et al., , 2016 jung et al., 2015; ma et al., 2015; marthaler et al., 2014; thachil et al., 2015) . subsequently, reports of pdcov in china, south korea and canada have caused considerable attention to be paid to the strategy employed by this emerging coronavirus to manipulate the host immune response (dong et al., 2015; lee et al., 2016) . the interferons (ifns) are vital proteins in innate immune signaling, playing a key role in the initial stages of virus invasion. through the recognition of pathogen-associated molecular patterns by pattern recognition receptors, such as cytoplasmic rig-i and mda5, adapter molecules such as ips-1 can be recruited and subsequently transfer the signal to the ikkα, ikkβ, ikkγ (also called nf-κb essential modulator (nemo)), tbk1 and ikk-ε. as an essential adapter, nemo is responsible for the recruitment of tbk1 and ikkε and the activation of ikk complex, leading to the triggering and transportation of nf-κb and irf3 to the nucleus, both of which induce downstream ifn-β production (kawai and akira, 2006; loo and gale, 2011; ramos and gale, 2011; seth et al., 2005; yoneyama and fujita, 2009) . notably, recent studies have demonstrated that several viral proteins encoded by coronaviruses (cov), such as severe acute respiratory syndrome (sars) cov, middle east respiratory syndrome cov and mouse hepatitis virus, can modulate innate antiviral signaling lui et al., 2016; thornbrough et al., 2016) . relying on its proteinase activity, cov non-structural protein 5 (nsp5), also called 3c-like protease or main protease, is responsible for processing the viral polyprotein to produce most of the non-structural proteins during viral replication (lai and cavanagh, 1997; masters, 2006; perlman and netland, 2009; ziebuhr et al., 2000) . the 3c-like protease of the arterivirus porcine reproductive and respiratory syndrome virus (prrsv), and the 3 c proteases of foot-and-mouth disease virus (fmdv) and hepatitis a virus (hav), both of which belong to the picornaviridae family, are reported to impair innate immune signaling (huang et al., 2014; wang et al., 2012 wang et al., , 2014 . a study published by our lab also demonstrated that nsp5, the 3c-like protease of porcine epidemic diarrhea virus (pedv), which is classified into the alphacoronavirus family, antagonizes ifn-β production by cleavage of nemo . recently, our research has shown that infection with pdcov inhibits rig-i-mediated ifn signaling, although the specific inhibition mechanism remains poorly understood . as such, we were extremely interested to discover whether nsp5 of pdcov could disrupt type i ifn signaling. in this study, we reveal that nsp5 of pdcov antagonizes the type i ifn signaling pathway through the cleavage of nemo, a critical constituent of the ikk complex, thus representing a newly identified mechanism by which pdcov evades the innate immune response. human embryonic kidney cells (hek-293t) and porcine kidney cells (pk-15) were obtained from the china center for type culture collection. llc-pk1 cells for pdcov infection were purchased from the atcc (atcc number cl-101) and cultured at 37°c in 5% co2 in dulbecco's modified eagle's medium (invitrogen, usa) supplemented with 10% fetal bovine serum. pdcov strain chn-hn-2014 (genbank number kt336560) isolated from a suckling piglet with severe diarrhea in china in 2014, was the object of this research . sendai virus (sev) was acquired from the center of virus resource and information at the wuhan institute of virology. the utilized luciferase reporter plasmids of ifn-β-luc, nf-κb-luc and irf3-luc have been previously described (wang et al., 2008 (wang et al., , 2010 . the rig-i, ips1, mda5, nemo, and the activated mutant of nemo (nemo-k277a) expression plasmids were constructed as previously described (wang et al., 2008 (wang et al., , 2010 . nemo and its mutants were cloned into the plasmid pcaggs-flag with an nterminal flag tag (wang et al., , 2014 . pdcov nsp5 was amplified and cloned with a c-terminal hemagglutinin (ha) tag into the expression plasmid pcaggs-ha-c. reporter and various expression plasmids were transfected into hek-293t cells or pk-15 cells in 24-well plates. twenty-four hours after transfection, cells were stimulated for 16 h with sev. firefly luciferase and renilla luciferase activities of lysed cells were verified with a luciferase reporter assay system (promega, madison, wi), and normalized to prl-tk (promega). trizol reagent (invitrogen) and avian myeloblastosis virus reverse transcriptase (takara, japan) were utilized for rna extraction and reverse transcription of cdna. each quantitative real-time pcr (qpcr) experiment, evaluated by sybr green, was performed three times and normalized with glyceraldehyde-3-phosphate dehydrogenase (gapdh). primers used for qpcr are detailed in table 1 . in western blot analyses, 30 h after transfection, the cells were treated with lysis buffer (beyotime, china) in 60-mm dishes. the lysates were separated by sds-page and transferred to polyvinylidene difluoride membranes (millipore, usa). following this, an anti-flag antibody (macgene, china), was applied to analyze the expression of proteins such as rig-i, mda5, ips-1, and nemo. pdcov nsp5 and its mutant were tested with an anti-ha antibody (mbl, japan) in western blot analyses. the endogenous nemo in pdcov-infected cells was tested with an anti-nemo polyclonal antibody (abclonal, china). the expression of pdcov n-protein was assessed with an anti-pdcov n-protein monoclonal antibody . the anti-β-actin mouse monoclonal antibody (beyotime, china) was applied to distinguish the expression of β-actin and to determine equal loading of each sample. coronavirus nsp5 plays an indispensable role in viral replication and immunoregulation. to characterize pdcov nsp5 with regards to type i ifn signaling, we constructed an expression vector encoding the nsp5 of pdcov and determined its impact on sev-induced ifn-β synthesis. firstly, the cytotoxicity of pdcov nsp5 in transiently transfected pk-15 and hek-293t cells was evaluated using the methylthiazolyldiphenyl-tetrazolium bromide (mtt) assay. as shown in fig. 1a and fig. 1b , no detectable cytotoxicity could be observed in cells transfected with nsp5 expression plasmid equal to or less than 1.0 μg in 24-well cell culture plates. further, the data presented in fig. 1c and d revealed that nsp5 exhibited strong inhibition of sevinduced ifn-β promoter activity in both cell types. the sev-induced ifn-β protein in the supernatant was also strongly decreased under the ectopic expression of pdcov nsp5 in hek-293t cells (fig. 1e) . moreover, nsp5 inhibited the activity of both irf3 and nf-κbdependent promoters in a dose-dependent manner ( fig. 1f and g). these data revealed the antagonistic role of pdcov nsp5 in type i ifn signaling. as the nsp5 of cov contains the catalytic residue cys144, a point mutation here could disrupt protease activity (anand et al., 2003; hsu et al., 2005; ye et al., 2016) . consistent with this, sequence alignment showed that cys144 (numbering based on pdcov nsp5) residues are highly conserved among other cov subfamilies ( fig. 2a) . conversely to the activity of wild-type pdcov nsp5, the activity of sev-induced ifnβ promoter was strongly restored upon the overexpression of nsp5 c144a (fig. 2b) , implying that the protease activity of pdcov nsp5 participated in ifn-β antagonism. to verify at which point in the signaling cascade nsp5 facilitated its inhibitory role, we assessed several crucial molecules in the mda5/ rig-i signaling pathway, which play an important role in induction of ifn-β production (yoneyama and fujita, 2007) , including rig-i, mda5, ips-1, nemo, and tbk1. similar to previous studies, overexpression of these crucial molecules significantly activated the ifn-β promoter compared with cells transfected with the empty vector control (siu et al., 2014; wang et al., 2012 wang et al., , 2016 . however, pdcov sequences (5′ to 3′) notably impaired the activation of the ifn-β promoter upon stimulation by rig-i, mda5, ips-1 and nemo. in contrast, tbk1induced activation of the ifn-β promoter was not affected by nsp5 (fig. 3a) , suggesting that pdcov nsp5 inhibits rig-i/mda5 signaling by targeting nemo or other upstream proteins. due to the indispensable role of pdcov nsp5 protease activity in ifn-β antagonism, we speculated that pdcov nsp5 cleaved nemo or an upstream molecule to impair type i ifn signaling. thus, rig-i, mda5, ips-1 or nemo were co-transfected with nsp5 into hek-293t cells. western blotting showed a smaller band (~24 kda) in the nemo/pdcov nsp5 coexpression samples, while no similar cleavage products were detected with rig-i, mda5 or ips-1 co-transfections (fig. 3b) . furthermore, the cleavage of nemo increased gradually with increasing transfection dose of pdcov nsp5 (fig. 3c ) and the cleaved product could not be observed following transfection with the nsp5 c144a mutant lacking protease activity (fig. 3d) . these data indicate that pdcov nsp5, relying on its protease activity, targets the essential molecule nemo for cleavage. in order to evaluate the real effect of this cleavage on endogenous nemo protein in viral infection, llc-pk1 cells were infected with pdcov at a multiplicity of infection of 0.1 and harvested at 6 h, 12 h, and 24 h post-infection. the results in fig. 3e indicate that endogenous nemo was clearly reduced within 6 h of infection and the reduction in nemo correlated with the duration of pdcov infection. however, no difference was detected in nemo mrna levels between mock-and pdcov-infected cells at any time during infection (fig. 3f) . the above results indicate that degradation of nemo occurs only in pdcov infection, without affecting transcription. with expression plasmids encoding pdcov nsp5 or its protease-defective mutant c144a, for analyzing ifn-β promoter activity followed by stimulation and harvest as described in fig. 1d . ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001. virology 502 (2017) 33-38 the specific preference for substrate cleavage by cov nsp5 has been previously reported as glutamine (q) at the p1 position (chuck et al., 2010 (chuck et al., , 2011 . as such, we examined the p1 position residue of nemo recognized by pdcov nsp5. considering the 24 kda molecular weight of the cleaved product (n-terminal), a series of nemo mutants were constructed between amino acids 210 and 263 (fig. 4a) . the wt or mutated nemo proteins were co-transfected into hek-293t cells with pdcov nsp5. the results indicated that q218a, q236a/q239a or q259a mutations were cleaved by pdcov nsp5 normally, while q229a or q231a mutations was resistant to cleavage (fig. 4b ). as previously found, the common substrate preference of cov nsp5 consists of leucine (l) at the p2 position and glutamine, lysine (k) or isoleucine (i), all of which contain a long side-chain and positive charge, at the p3 position (chuck et al., 2010 (chuck et al., , 2011 wang et al., 2016) . we speculated that if q231 is the p1 residue, then the recognition and cleavage of q231 by pdcov nsp5 could be affected by a q229a mutation (fig. 4c) . therefore, two q229 mutants at the speculated p3 position were substituted with lysine or arginine (r) which also has a long sidechain and positive charge. evidently, q229k and q229r mutation can be cleaved to produce the same cleavage product as wt nemo (fig. 4d) , revealing that q231, but not q229, is the p1 position residue cleaved by pdcov nsp5. docking studies between cov nsp5 and substrate showed that through van der waals interactions, the side chain of p3 residues could interact with the side chain of glutamic acid at residue 164 (xue et al., 2008) . the homology model for pedv nsp5 and the nemo peptide substrate also illustrated that a long side chain in the p3 position strongly supported nsp5-substrate recognition . these studies provide important clues for the pdcov nsp5-nemo interaction and give a possible explanation for the finding that mutant q229a, but not q229k or q229r, significantly impaired cleavage by pdcov nsp5. to assess the influence of nemo cleavage by pdcov nsp5 in type i ifn signaling, a constitutively active nemo mutant, nemo-k277a, was used, leading to more efficient activation of the ifn-β promoter compared with wt nemo (bloor et al., 2008; wang et al., 2016 wang et al., , 2014 . the k277a mutation has the same cleavage site as wt nemo, with the pdcov nsp5 p1 recognition site also being q231 (fig. 5a) . hence, the ifn-β promoter activated by nemo-k277a, was suppressed by pdcov nsp5 in a dose-dependent manner and this suppression was lost upon the expression of the protease-defective nsp5 c144a mutant ( fig. 5b and c) . to evaluate the activity of the cleavage fragment in activation of the ifn response, nemo-k277a (1-231aa), nemo-k277a (232-419aa) or nemo-k277a (full) expression plasmids were transfected into pk-15 cells. quantitative pcr showed that mrna levels of immune-related molecules, such as isg54, isg56, mx1, oas1, and rantes, were not successfully induced by the cleaved fragments of 1-231 or 232-419 (fig. 5d) . a previous study of pedv nsp5 showed that cleavage fragments of nemo did not activate ifn-β, irf3 or nf-κbdependent promoters . as the cleavage site q231 is identical for both pedv and pdcov nsp5, these results together demonstrate that pdcov nsp5-induced cleavage fragments lack the capacity to activate the type i ifn response and downstream signaling, revealing that the role of nemo in type i ifn signaling can be significantly inhibited following cleavage by pdcov nsp5. so far, evidence presented by recent studies has shown that the 3c and 3c-like proteases of different viruses cleave nemo at multiple residues, such as q383 and q304 targeted by fmdv and hav 3c proteases, e349 targeted by prrsv 3c-like proteases and q231 targeted by pedv 3c-like protease, leading to disruption of ifn-β production (huang et al., 2014; wang et al., 2012 wang et al., , 2016 wang et al., , 2014 and implying that nemo is a common target easily hijacked by viral proteases. in particular, the conserved q231 residue recognized by both pdcov nsp5 and pedv nsp5 prompted us to speculate that the same ifn antagonistic mechanism may be common to members of the coronaviridae family. it is worth investigating whether nsp5 from different cov genera, including alpha-, beta-, gamma-, and deltacoronaviruses, retain the ability to cleave nemo and comparing this property between the different cov genera. in summary, our research demonstrated that pdcov nsp5 has the capacity to impede generation of ifn-β. as a new ifn antagonist encoded by a deltacoronavirus, pdcov nsp5 induced the cleavage of nemo at the conserved residue q231, leading to suppression of the type i ifn signaling pathway. our research provides new insights into strategies and tactics employed by pdcov in host innate immune evasion. after 32 h, pk-15 cells were analyzed for isg54, isg56, mx1, oas1, rantes mrna levels in 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levels foot-and-mouth disease virus 3c protease cleaves nemo to impair innate immune signaling porcine epidemic diarrhea virus 3c-like protease regulates its interferon antagonism by cleaving nemo hepatitis a virus 3c protease cleaves nemo to impair induction of beta interferon coronavirus diversity, phylogeny and interspecies jumping discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design structural basis for the dimerization and substrate recognition specificity of porcine epidemic diarrhea virus 3c-like protease rig-i family rna helicases: cytoplasmic sensor for antiviral innate immunity rna recognition and signal transduction by rig-i-like receptors virus-encoded proteinases and proteolytic processing in the nidovirales key: cord-321162-pgd34ewv authors: holmes, kathryn v.; doller, elizabeth w.; sturman, lawrence s. title: tunicamycin resistant glycosylation of a coronavirus glycoprotein: demonstration of a novel type of viral glycoprotein date: 1981-12-31 journal: virology doi: 10.1016/0042-6822(81)90115-x sha: doc_id: 321162 cord_uid: pgd34ewv abstract tunicamycin has different effects on the glycosylation of the two envelope glycoproteins of mouse hepatitis virus (mhv), a coronavirus. unlike envelope glycoproteins of other viruses, the transmembrane glycoprotein el is glycosylated normally in the presence of tunicamycin. this suggests that glycosylation of el does not involve transfer of core oligosaccharides from dolichol pyrophosphate intermediates to asparagine residues, but may occur by 0-linked glycosylation of serine or threonine residues. synthesis of the peplomeric glycoprotein e2 is not readily detectable in the presence of tunicamycin. inhibition of n-linked glycosylation of e2 by tunicamycin either prevents synthesis or facilitates degradation of the protein moiety of e2. radiolabeling with carbohydrate precursors and borate gel electrophoresis of glycopeptides show that different oligcsaccharide side chains are attached to el and e2. the two coronavirus envelope glycoproteins thus appear to be glycosylated by different mechanisms. in tunicamycin-treated cells, noninfectious virions lacking peplomers are formed at intracytoplasmic membranes and released from the cells. these virions contain normal amounts of nucleocapsid protein and glycosylated el, but lack e2. thus the transmembrane glycoprotein el is the only viral glycoprotein required for the formation of the viral envelope or for virus maturation and release. the peplomeric glycoprotein e2 appears to be required for attachment to virus receptors on the plasma membrane. the coronavirus envelope envelope glycoprotein e1 appears to be a novel type of viral glycoprotein which is post-translationally glycosylated by a tunicamycin-resistant process that yields oligosaccharide side chains different from those of n-linked glycoproteins. these findings suggest that el may be particularly useful as a model for studying the biosynthesis, glycosylation, and intracellular transport of 0-linked glycoproteins. of the synthesis, glycosylation, and intracellular transport of glycoproteins is essential to understanding the structure and function of cell membranes and the role of oligosaccharides in glycoprotein processing and secretion. because glycosylation and transport of viral envelope glycoproteins depend upon cellular processes, the g glycoprotein of vesicular stomatitis virus has been used as an excellent model for glycosylation and transport of n-linked glycoproteins (roth-1 to whom reprint requests should be addressed. man and lodish, 1977; rothman et d, 1978; morrison et al, 19'78; gibson et al, 1979) . oligosaccharides may also be olinked to serine or threonine residues of the polypeptide chain by a process which is less well understood (sharon and lis, 1981) . o-linked oligosaccharides are predominant in many cell surface glycoproteins such as glycophorin (tomita and marchesi, 1975) and in secreted glycoproteins such as submaxillary mucins (slomiany and slomiany, 1978) . although tunicamycin inhibits glycosylation of n-linked glycoproteins (takatsuki et al, 1971 ; lehle and tanner, 1976; schwarz et al, 1976; schwarz et al, 1979; elbein, 1979; schwarz and datema, 1980) , no drug to inhibit o-linked glycosylation has yet been identified (schwarz et al., 1979; sharon and lis, 1981) . all viral envelope glycoproteins studied to date have been of the n-linked type of glycoproteins (leavitt et ok, 19'7'7; morrison et cd, 19'78; schwarz et al, 1979; nakamura and compans, 1978a; cash et d, 1980; pizer et al, 1980; klenk and rott, 1980; choppin and scheid, 1980; ghosh, 1980; stallcup and fields, 1981) . we now present evidence that a coronavirus glycoprotein may be glycosylated by a different mechanism. this glycoprotein may serve as a useful model for the study of o-linked glycoproteins. coronaviruses are enveloped viruses containing -5.8 x lo6 daltons of positive sense, single-stranded polyadenylated rna (tyrrell et cd, 1978; wege et cd, 1978; lai and stohlman, 1978; macnaughton et d, 1978) . these viruses cause a variety of respiratory, enteric, or neurological diseases in animals and man (andrewes et & 1978) . the virions of the a59 strain of mouse hepatitis virus contain three structural polypeptides: a phosphorylated nucleocapsid protein n, and two glycoproteins el and e2 which have several interesting properties (sturman, 1977; sturman and holmes, 1977; sturman et cd, 1980; sturman, 1981) . the glycoprotein e2 forms the large petal-shaped peplomers characteristic of the coronavirus envelope. e2 is a 18ok-dalton glycoprotein which can be cleaved by trypsin to yield two 90k components. the glycoprotein el appears to be a transmembrane molecule with three domains: a glycosylated domain projects from the envelope, a second domain lies within the membrane, and a third domain appears to interact with the nucleocapsid inside the viral envelope. unlike most proteins, when el is boiled in the presence of sds and mercaptoethanol it aggregates into dimers, trimers, and tetramers. the intracellular distribution of el is also unusual. labeling with monospecific fluorescent antibody against isolated el or e2 (sturman et cd, 1980) showed that el remains restricted to the perinuclear area of the cell while e2, like most other viral glycoproteins, migrates rapidly via intracellular membranes to the plasma membrane (doller and holmes, 1980) . in the present study we have used the antibiotic tunicamycin to study the synthesis and glycosylation of the coronavirus mhv. tunicamycin, an analog of udp-nacetylglucosamine, interferes with the formation of dolichol pyrophosphate-hracetylglucosamine which acts as a carrier for n-glycosidic linkage of core oligosaccharides to asparagine residues on glycoproteins. tunicamycin interferes with the cotranslational glycosylation of glycoproteins (takatsuki et al, 1971; lehle and tanner, 1976; schwarz et al, 1979) . we have demonstrated that tunicamycin inhibits formation of the e2 glycoprotein of mhv, but does not prevent synthesis or glycosylation of the transmembrane glycoprotein el, formation of virions, or release of virions from cells. our evidence indicates that the unique el glycoprotein of the coronavirus may be an o-linked glycoprotein, and thus could be a particularly useful model for studying the synthesis, glycosylation, and intracellular transport of o-linked glycoproteins in mammalian cells. virus propagation and putificatim the a59 strain of mouse hepatitis virus (mhv) was grown in the spontaneously transformed 17 cl-l line of balb/c 3t3 mouse fibroblasts as previously described (sturman and takemoto, 1972; sturman, 1977) and assayed by plaque titration in 17 cl 1 cells. to prepare radiolabled virus, cells in 150-mm2 plastic flasks were inoculated with 1.0 ml of mhv at a multiplicity of 0.3 to 3 pfu/cell in dulbecco's modified eagles' minimal essential medium, high glucose (dmem; gibco, grand island, n. y.), and incubated for 1 hr at 37". the inoculum was removed and cells were refed with 30 ml emem + 10% dialyzed fetal bovine serum (dfbs) containing 20 &i/ml of l -rh]amino acid mixture (new england nuclear) and incubated for 24 hr. virus released into the supernatant medium was harvested and purified by a modification of the method described previously (sturman et al, 1980) , using discontinous and continuous sucrose density gradients in tms buffer (containing 0.05 m tris-maleate and 0.1 m nacl, ph 6.0) and omitting the polyethylene glycol precipitation step. l4zbeling and electrophoresis of intracellular viral pol~peptides. to study synthesis, processing, and release of viral specific polypeptides, pulse-chase experiments were performed. confluent monolayers of 1'7 cl 1 cells in 60-mm petri dishes (falcon, inc.) were preincubated for 18 hr at 37" in l-leucine deficient emem (leu-def mem) with 10% dfbs and either mock infected with 0.5 ml/plate of leu-def mem with 10% dfbs or inoculated with 3 to 5 pfu of mhv/cell in 0.5 ml/plate of leudef mem with 10% dfbs. after incubation for 1 hr at 37", the inocula were removed, the cells were refed with leu-def mem with 10% dfbs, with or without 0.5 pg/ml of tunicamycin (eli lilly, indianapolis, ind.), and the cultures were held at 37". four hours prior to pulse labeling, 5 pg/ml of actinomycin d was added to the medium. at 8 and 10 hr after virus inoculation infected and control cells were pulse labeled for 15 min with 20 or 40 &i/ ml of rhll-3, 4, 5-leucine (new england nuclear, inc.). the labeled medium was removed, cells were washed and refed with dmem containing a lo-fold excess of unlabeled l-leucine, 10% fbs, 0.5 pg/ml tunicamycin. at intervals after the pulse, labeled intracellular polypeptides were extracted. cells were washed twice in pbs and solubilized with 1.0 ml/plate of 1% nonidet p40 (np40, accurate chemical corp.) in pbs. nuclei and debris were removed by centrifugation at 1800 q for 10 min at 4". radiolabeled polypeptides were analyzed directly by polyacrylamide gel electrophoresis (page) or were immunoprecipitated with rabbit antiserum against purified, detergent-disrupted virions in the presence of staphylococcal protein a prior to analysis by page (sturman et al, 1980) . samples for sds-page slab gels were heated at 37" for 30 min with an equal volume of sample treatment mixture composed of 6m urea, 4% sds, and 0.05% bromphenol blue in 0.0625m tris-chloride, ph 6.7. sds-page in cylindrical gels was performed using a high ph discontinuous buffer system and fractionation of gels with a gilson automatic linear gel fractionator as previously described (sturman and holmes, 1977) . five to twenty percent polyacrylamide gradient slab gels were prepared and fluorographed as previously described (sturman et al 1980) . anal@s of glycopeptides. the glycoproteins el and e2 were isolated from sds-page of gradient-purified mhv which had been grown for 24 hr in medium containing 3 &i/ml [3hlglucosamine (new england nuclear, inc.). isolated el and e2 were eluted from the gels, digested with 15 pg of self-digested pronase (sigma) per milliliter for 30 hr at 60", concentrated by lyophilization, and analyzed as borate esters by page in tris-borate buffer at ph 8.3 according to the method of weitzman et al. (1979) . electron microscopy. concentrated, gradient-purified virions were prepared for electron microscopy using 2% phosphotungstic acid (pta) at ph 7.2 on carboncoated, formvar-covered, 400-mesh copper grids. electron microscopy of cells was done by fixation with 1% glutaraldehyde followed by postfixation with 1% osmium tetroxide, dehydration in a graded series of ethyl alcohol solutions and propylene oxide, and embedding in epon 812 resin as previously described (compans et al, 1966) . sections were stained with lead citrate and uranyl acetate and examined in a jeol 1oocx electron microscope. the synthesis and processing of coronavirus-specific polypeptides were analyzed by pulse-labeling techniques in cells treated for 4 hr with actinomycin d to reduce cellular protein synthesis. without tunicamycin, a 15-min pulse label with rh]leucine 8 hr after virus inoculation showed synthesis of the three structural polypeptides el, n, and e2 ( fig. 1 , channels l-8). no high-molecular-weight polyproteins were detected. thus each structural polypeptide appears to be translated inedpendently, as also shown by in vitro translation studies with isolated mhv mrnas (siddell et c& 1980; rottier et cd, 1981) . during successive chase periods, no shift in the molecular weight of the nucleocapsid protein n or the peplomeric glycoprotein e2 was observed. since the e2 glycoprotein is known to be extensively glycosylated (sturman, 1977; sturman and holmes, 197'7) , this suggests either that glycosylation of e2 is a cotranslational event and/or that the glycosylated and nonglycosylated e2 are not resolved in this region of the gradient slab gel. pulsechase experiments with labeling at 6,8, or 10 hr after infection show that el is synthesized as a 20k species and then chased up to a broad band of up to 23k (not apparent in fig. 1 , channels 7 and 8 due to overexposure). double labeling studies of intracellular viral polypeptides demonstrated that the 20k form of el is not glycosylated whereas the 23k species can be labeled with phlglucosamine (data not shown). antibody against el purified from np40-disrupted virions by sucrose density gradient sedimentation immunoprecipitates the broad band of el from 20k to 23k (sturman et cd, 1980) . similar pulse-chase radiolabeling studies were performed on cells infected with mhv, treated with 0.5 pg/ml of tunicamycin, and labeled with [8h]leucine ( fig. 1 , channels 9-16). synthesis of the nonglycosylated nucleocapsid protein n was not affected by tunicamycin. synthesis of the 180k e2 glycoprotein was not detectable in the presence of tunicamycin. thus, tunicamycin either inhibits synthesis of e2 or facilitates rapid degradation of newly synthesized e2. tunicamycin has been shown to interfere with synthesis or detection of several other glycoproteins which are cotranslationally glycosylated via n-linked glycosidic bonds (lehle and tanner, 1976; leavitt et al. 1977; olden et al. 1978; gibson et al, 1979; stallcup and fields, 1981) . the three polypeptides of molecular weight 70 to 90k seen in channels 9-16 are cellular polypeptides since they also appear with equal intensity and kinetics in uninfected cells treated with tunicamycin. in the presence of tunicamycin the rate of synthesis of the membrane glycoprotein el is reduced (fig. 1, channels 9 and 10) in comparison to the control (fig. 1, channels 1 and 2) . however, it is clear that el is synthesized as a 20k polypeptide (fig. 1, channels 9 and 10) which is chased into the glycosylated 23k form (fig. 1, virions have not been observed budding from the plasma membrane. formation of virions was not inhibited by 0.5 pg/ml of tunicamycin (fig. 2b) . numerous virions were observed in dilated cisternae of the rer and in smooth-walled vesicles of tunicamycin-treated cells. although no virions were adsorbed to the plasma membrane (fig. 2b) , virions were released from tunicamycin-treated cells. although 0.5 rg/ml of tunicamycin reduced the 24-hr yield of infectious virus looo-fold, large quantities of virions could be purified from the medium by sucrose density gradient ultracentrifugation. the number of virions released from tunicamycin-treated cultures was only about &fold less than from untreated cultures, as estimated by electron microscopy. the virions from tunicamycin-treated cells lacked the characteristic large peplomers of coronaviruses (fig. 3) . the absence of peplomers correlated with the inability of the virions to attach to receptors on the cell surface (fig. 2b) or to initiate infection. the structural proteins of virions purified from tunicamycin-treated or control cultures were compared (fig. 4) . the normal virus contained el, n, and three forms of the e2 glycoprotein: the native 180k form, the 90k cleavage products, and a high-molecular-weight aggregated form (fig. 4a ). in contrast, virus grown with tunicamycin contained el and n in normal amounts but completely lacked the peplomeric glycoprotein e2 (fig. 4b) . these biochemical data thus confirm the ultrastructural observations on the absence of peplomers on virions from tunicamycintreated cells. the el membrane glycoprotein in the virus grown with tunicamycin appeared to be glycosylated normally, as shown by the ratio of glucosamine to methionine labels in the el peaks in fig. 4a and b. this confirms the observation made in the pulse-chase experiments (fig. 1 ) that glycosylation of el is resistant to inhibition by tunicamycin. in fig. 4 it is also apparent that the ratio of glucosamine to methionine label was not constant across the broad peak of el. more extensive glycosylation corresponded with decreased electrophoretic mobility. the oligosaccharide chains of n-and olinked glycoproteins differ markedly in size, diversity, and carbohydrate composition (sharon and lis, 1981) . useful information about the oligosaccharides can be obtained by analyzing the glycopeptides isolated from glycoproteins by page in tris-borate buffer (weitzman et ak, 1979) . at alkaline ph, neutral sugars form negatively charged complexes with borate. the number of borate ions which react with glycopeptides is a function of the composition, sequence, and linkages of the carbohydrates. glycopeptides of dissimilar carbohydrate composition and length exhibit different electrophoretic mobilities. although this separation of glycopeptides is not based on their molecular weights, in general, larger oligosaccha-rides bind more borate than smaller ones, and therefore migrate faster in the gel. differences in the peptide components appear to have little, if any, effect on the electrophoretic mobilities of the glycopeptides. thus glycopeptides from two different n-linked glycoproteins would be expected to migrate rather similarly whereas glycopeptides from n-and o-linked glycoproteins would differ markedly in electrophoretic mobility and distribution. we have used this technique to compare the glycopeptides derived by pronase digestion of isolated el and e2. figure 5 shows that the borate complexes of el and e2 glycopeptides exhibited markedly different electrophoretic patterns. the glycopeptides of el exhibited significantly greater mobility than those of e2. therefore, the borate-glycopeptide complexes of ~i-ijglucosamine-labeled glycoproteins el and e2 of mhv were separated by sds-page, eluted from the gels, and digested with pronase for 30 hr at 30". the glycopeptides were lyophilized and analyzed by electrophoresis on 10% polyacrylamide gels in tris-borate buffer, ph 8.3. profiles of [shlglucosamine-labeled glycopeptides from e2 (a) and el (b) are shown. the anode is to the right. el were significantly more negatively charged than those of e2. figure 5 also shows that fewer glycopeptide components were resolved from el than from e2. this suggests that el may have less diversity of oligosaccharide side chains than e2. these data suggest that the oligosaccharides of el differ markedly from those of e2, and support the hypothesis that the oligosaccharides of el and e2 may be derived by different mechanisms of glycosylation. discussion inhibits glycosylation of the structural glycoproteins of alphaviruses (leavitt et d, 1977) , bunyaviruses (cash et al, 1980) , herpes viruses (pizer et al, 1980) , myxoviruses (nakamura and compans, 1978a; klenk and rott, 19x) ), paramyxoviruses (stallcup and fields, 1981) , rhabdoviruses (morrison et a& 19'78; gibson et a& 1979; klenk and rott, 1980) , and retroviruses (schwarz et al, 1976; diggelman, 1979) , suggesting that all of these viruses contain n-linked glycoproteins which are glycosylated by the transfer of core oligosaccharides from a dolichol pyrophosphate carrier to asparagine residues on the polypeptide. use of tunicamycin has often permitted the identification of the nonglycosylated protein moiety of a viral glycoprotein (morrison et al, 1978 , gibson et a& 1979 nakamura and compans, 1978a; diggelman, 1979) . in some virus strains, however, synthesis of the nonglycosylated polypeptide in the presence of tunicamycin is difficult to detect because complete translation of the glycoprotein mrna may be dependent on cotranslational addition of n-linked oligosaccharide chains, or because the nonglycosylated polypeptide may be highly susceptible to degradation by host cell proteases, or because the nonglycosylated polypeptide may be insoluble (schwarz et al, 1976; leavitt et al, 1977; gibson et d, 1979; diggelman, 1979; pizer et al, 1980; stallcup and fields, 1981) . the coronavirus mhv contains two structural glycoproteins which have been isolated and partially characterized (sturman et ak, 1980) . in 17 cl 1 cells infected with the a59 strain of mhv, tunicamycin specifically interfered with the synthesis of the peplomeric glycoprotein e2 (fig. 1) . neither glycosylated nor nonglycosylated forms of e2 were detected either directly or by immunoprecipitation. this suggests that e2, like other viral structural glycoproteins, may be an n-linked glycoprotein. the transmembrane glycoprotein el or mhv is so far unique among viral structural glycoproteins in that it is glycosylated in the presence of tunicamycin. glycosylation of el appears to be a posttranslational event (fig. 1 ) and the shift from the nonglycosylated 20k form to the glycosylated 23k form is not inhibited by tunicamycin. this provides indirect evidence that el is not an n-linked glycoprotein but may be an o-linked glycoprotein. this hypothesis is also supported by direct evidence concerning the carbohydrate composition and the oligosaccharide side chains of el. early studies on the incorporation of radiolabeled sugars into el and e2 showed that both el and e2 were labeled with [shlglucosamine, but only e2 was labeled with ['hlfucose (sturman and holmes, 1977) . recent studies by niemann and klenk (1981) have identified additional differences between the carbohydrate composition of el and e2. el, like cellular o-linked glycoproteins (thomas and winzler, 1969; spiro and bhoyroo, 1974; slomiany and slomiany, 1978; sharon and lis, 1981) , contains little mannose, no fucose, and possesses a high proportion of n-acetyl galactosamine; whereas e2, like many other n-linked glycoproteins (nakamura and compans, 197813; prehm et al, 1979; weitzman et ul, 1978; burke and keegstra, 1979) , contains both mannose and fucose but no n-acetyl galactosamine. oligosaccharides of el but not e2 are removed by p-elimination (h. niemann and h.-d. klenk, personal communication) . although the size, linkages, and sequences of sugars of the individual oligosaccharide chains of the two mhv glycoproteins have not yet been determined, analysis of the oligopeptides of el and e2 by tris-borate page has shown that the two glycoproteins have distinct oligosaccharide side chains (fig. 5) . this technique affords resolution comparable to or better than bio-gel p6 gel filtration columns. the electrophoretic mobilities of the glycopeptides of e2 were similar to those of glycopeptides from n-linked glycoproteins of sindbis virus or ovalbumin which have been previously described (weitzman et al, 1979) . in contrast, the glycopeptides of el were more negatively charged than those of e2. thus, we have shown that el and e2 differ in carbohydrate composition, electrophoretie patterns of glycopeptides, and response to tunicamycin. these data suggest that e2 is an n-linked glycoprotein and el may be an o-linked glycoprotein. the amino acid sequence and the locations and number of oligosaccharide side chains on the mhv glycoproteins are not yet known. however, all of the oligosaccharide moieties of el appear to be located holmes, doller, and sturman near one end of the polypeptide chain. treatment of mhv virions with bromelain or pronase removed e2 and detached a 5k glycosylated polypeptide from el, leaving an 18k nonglycosylated polypeptide embedded within the viral envelope (sturman and holmes, 1977; sturman, 1981) . it is not yet known whether the glycosylated portion of el which extends outside the viral membrane contains the amino terminal or carboxy terminal region of el. however, this region appears to contain multiple oligosaccharide side chains, since on page el migrates either as a broad, diffuse band of 20k to 23k or, under other conditions, as three or more discrete bands. the role of glycosylation in the functions of el is not yet known. glycosylation may affect the conformation of el or its orientation in the viral envelope. however, glycosylation of el may not be essential for virion formation since both nonglycosylated and glycosylated el are incorporated into a59 virions (data not shown) and the el-like membrane proteins found in some other coronaviruses are apparently not glycosylated (garwes, 1979) . the novel o-linked post-translational glycosylation of el may be associated with its restricted intracellular migration. el is restricted to the perinuclear area of infected cells, in contrast to e2 which migrates rapidly to the plasma membrane (doller and holmes, 1980) . possibly the oligosaccharides of el act as signals for movement from the rer to other membranes. oligosaccharides are important recognition signals for enzymes destined for lysosomes (hasilik, 1980) and glycoproteins destined for endocytosis (baenziger and fiete, 1980; stahl and schlesinger, 1980) . the differential effects of tunicamycin on the synthesis and glycosylation of the two coronavirus envelope glycoproteins has permitted tentative assignment of functions to these glycoproteins. in mhvinfected cells treated with tunicamycin, virions which lack the e2 glycoprotein, and hence the peplomers, bud from intracytoplasmic membranes and are released from the cell (figs. 2, 3, and 4) . thus the transmembrane glycoprotein el appears to be the only envelope glycoprotein required for virus budding. indeed, the location of el on intracytoplasmic membranes may determine the budding site of the coronavirus. budding may occur where the viral nucleocapsid recognizes intracellular membranes altered by the addition of el. the e2 glycoprotein appears-to be essential for virus attachment to receptors on the plasma membrane. virus lacking peplomers showed a markedly decreased infectivity. no virions were observed adsorbed to the plasma membrane of tunicamycin-treated cells although numerous virions lacking e2 were present in the medium (figs. 2 and 3) . many cellular glycoproteins such as fetuin, cornea1 proteoglycan, glycophorin, thyroglobulin, and immunoglobulins contain both asparagine-linked and serine-or threonine-linked oligosaccharides (sharon and lis, 1981) . further detailed study may reveal some o-linked oligosaccharides on viral glycoproteins now believed to contain only n-linked oligosaccharides. indeed, the vaccinia virus hemagglutinin, a nonstructural glycoprotein, has both 0-and n-linked oligosaccharide chains (shida and dales, 1981) . to date, however, no structural viral glycoprotein except the el glycoprotein of mhv has been identified which might contain only o-linked carbohydrates. it is unlikely that glycosylation of el is a virus-encoded process. since mhv has a limited amount of genetic material and codes for only six species of mrna (siddell et al, 1980; rottier et a& 1981) , it is far more likely that glycosylation of el occurs via cellular processes. production of the el glycoprotein may be a good model system for the study of tunicamytin-resistant glycosylation. in mhv-infected cells treated with actinomycin d to inhibit synthesis of cellular glycoproteins and with tunicamycin to inhibit synthesis of the n-linked viral glycoprotein e2, el is the major glycoprotein synthesized. this glycoprotein can be purified in large amounts from virions released from tunicamycin-treated cells. thus, the coronavirus glycoprotein el may be a useful model for analysis of the synthesis, gly-cosylation, and intracellular transport of o-linked glycoproteins. viruses of vertebrates galactoee and n-acetylgalactosamine specific endocytosis of glyeopeptides by isolated rat hepatocytes carbohydrate structure of sindbis virus glycoprotein e2 from virus grown in hamster and chicken cells the effect of glycosylation inhibitors on the maturation and intracellular polypeptide synthesis induced by snowshoe hare the role of viral glycoproteins in adsorption, penetration and pathogenicity of viruses an electron microscopic study of moderate and virulent virus-cell interactions of the parainfluenza virus sv5. virdogy 30,411~422 different intracellular transportation of the envelope glycoproteins el and e2 of the coronavirus mhv the role of lipid-linked saccharides in the biosynthesis of complex carbohydrates viral enteritis in humans and animals synthesis and maturation of glycoproteins of enveloped animal viruses the nonglycosylated glycoprotein of vesicular stomatitis virus is temperature-sensitive and undergoes intracellular aggregation at elevated temperatures cotranslational and posttranslational processing of viral glycoproteins rna of mouse hepatitis virus impaired intracellular migration and altered solubility of nonglycosylated glycoproteins of vesicular stomatitis virus and sindbis virus the specific site of tunicamycin inhibition in the formation of dolichol-bound n-acetylglucosamine derivatives the genomes of three coronaviruses assembly of viral membranes: maturation of the vesicular stomatitis glycoprotein in the presence of tunicamycin effects of glucosamine, 2-deoxy-d-glucose and tunicamytin on glycosylation, sulfation and assembly of influenza viral proteins glycopeptide components of influenza viral glycopeptides the biochemistry and biology of coronaviruses role of carbohydrates in protein secretion and turnover: effects of tunicamycin on the major cell surface glycoprotein of chick embryo fibroblasts effect of tunicamycin on herpes simplex virus glycoproteins and infectious virus production the carbohydrate structure of the glycoproteins of the paramyxovirus sv5 grown in bovine kidney cells glycosylation of a membrane protein is restricted to the growing polypeptide chain but is not necessary for insertion as a transmembrane protein synchronized transmembrane insertion and glycoaylation of a nascent membrane protein translation of three mouse hepatitis virus strain a59 subgenomic rnas in xenopus ti oocytes inhibitors of protein glycosylation suppression of glycoprotein formation of semliki forest, influenza and avian sarcoma virus by tunicamycin inhibition of glycosylation of viral glycoproteins glycoproteins; research booming on long-ignored, ubiquitous compounds biogenesis of vaccinia: carbohydrate of the hemagglutinin molecule coronavirus jhm: cell free synthesis of structural protein p60 structures of the acidic oligosaccharides isolated from rat sublingual glycoprotein structure of the 0-glycosidically linked carbohydrate units of fetuin receptor-mediated pinocytosis of mannose/n-acetylglucosamine-terminated glycoproteins and lysosomal enzymes by macrophages the replication of measles virus in the presence of tunicamycin characterization of a coronavirus. 1. structural proteins: effects of preparative conditions on the migration of protein in polyacrylamide gels the biochemistry and biology of coronaviruses characterization of a coronavirus. ii. glycoproteins of the viral envelope; tryptic peptide analysis isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid enhanced growth of a murine coronavirus in transformed mouse cells structural studies on human erythrocyte glycoproteins amino-acid sequence and oligosaccharide attachment sites of human erythrocyte glycophorin genomic rna of the murine coronavirus analysis of glycopeptides as borate complexes by polyacrylamide gel electrophoresis we thank margaret kerchief, barbara o'neill, cynthia ricard, gale schmidt, and eugene downing for excellent technical assistance. we are grateful to dr. h.-d. klenk and h. niemann for making their data available to us in advance of its publication. this research was supported in part by research grant r67403 from the uniformed services university of the health sciences. the opinions expressed are the private views of the authors and should not be construed as official or necessarily reflecting the views of the uniformed services university school of medicine or the department of defense. this work was presented in part at a conference on the biochemistry and biology of coronaviruses in wiirzburg, west germany, in october, 1989. key: cord-315069-xo4mbxei authors: knorr, d. a.; mullin, r. h.; hearne, p. q.; morris, t. j. title: de novo generation of defective interfering rnas of tomato bushy stunt virus by high multiplicity passage date: 1991-03-31 journal: virology doi: 10.1016/0042-6822(91)90484-s sha: doc_id: 315069 cord_uid: xo4mbxei abstract defective interfering (di) rnas were generated de novo in each of 12 independent isolates of tomato bushy stunt virus (tbsv) upon serial passage at high multiplicities of infection (m.o.i.) in plants, but not in any of 4 additional isolates after 11 serial passages at low m.o.i. the di rnas were detected in rna isolated from virus particles and in 2.3 m licl-soluble rna fractions isolated from inoculated leaves. symptom attenuation leading to persistent infections was closely correlated with the passage in which dis first developed. comparisons of nucleotide sequences of 10 cdna clones from 2 di rna populations and with a previously characterized tbsv di rna revealed the same four regions of sequence from the tbsv genome were strictly conserved in each of the di rnas: the virus 5′ leader sequence of 168 bases; a region of approximately 200–250 bases from the viral polymerase gene; approximately 70 bases from the 3′ terminus of the viral pl9 and p22 genes; and approximately 130 bases from the 3′terminal noncoding region. conservation of the sequence motif present in all of the dis suggests that there might be a common mechanism of di formation as well as selection pressure to maintain sequences essential for replication and encapsidation. defective interfering particles (dls) are deletion mutants that interfere with replication of the virus from which they were derived (for reviews, see; holland, 1990; schlesinger, 1988 ; barrett and dimmock, 1986) . dls have been reported for virtually every family of animal viruses and are thought to occur universally (perrault, 1981) . the dls associated with rna viruses of animals have proved particularly useful for identifying sequence elements involved in viral functions such as encapsidation and replication (levis et a/., 1986; weiss et a/., 1989; schlesinger, 1988) and they have also been implicated as important components in driving viral evolution (steinhauer and holland, 1987; de polo et a/., 1987) . although most frequently studied in cell culture, there is increasing evidence that dls play a role in natural infections in modulating disease symptoms and in establishing virus persistence (barrett and dimmock, 1986 ). however, due to complications presented by the immune and interferon systems in animal hosts, this role has been difficult to establish unambiguously (huang. 1988) . sequence data from this article have been deposited with the embugenbank data libraries under accession nos. m59041~ m59050. ' to whom requests for reprints should be directed. in comparison to studies with animal viruses, relatively few plant viruses with defective genomes have been documented . among these, di rnas which meet the criteria of huang and baltimore (1970) have been described for only two related groups of plant viruses, the tombusviruses and carmoviruses (see morns and knorr, 1990 , for a review). biological and molecular characterization of a novel rna species associated with the cherry strain of tomato bushy stunt virus (tbsv) provided the first definitive demonstration of di rnas associated with a plant virus infection (hillman et a/., 1987) . it was also shown that the lethal necrosis syndrome normally caused by tbsv in solanaceous hosts was prevented by the di rna and persistent infections resulted. the di rna (termed di 1) was shown to be a collinear deletion mutant of tbsv that was dependent on the parent virus for both replication and encapsidation. tbsv di rnas were shown to reduce virus accumulation in infected plants (hillman et al., 1987) and to inhibit genomic rna replication in protoplasts (jones e? al., 1990) . subsequently, di rnas were found associated with two other tombusviruses, cymbidium ringspot virus (burgyan et a/., 1989) and cucumber necrosis virus (d. rochon, personal communication; y. chang and j. morris, unpublished) . among the carmoviruses, which are structurally related to tombusviruses (morris and carrington, 1988) turnip crinkle virus (tcv) has been shown to support di rnas (li eta/., 1989) . it is interesting that in addition to di rnas, tcv also supports a number of small satellite rnas, and at least one chimerit rna with regions derived from both satellite and tcv genomic sequences. the origin of di rnas has been attributed in some cases to template "hopping" or "switching" by the viral rna polymerase which produces deletions in the viral genome (lazzarini, 1981) . in general, dls can arise in cell cultures when a fully competent virus is passaged serially at high multiplicities of infection (m.0.i.). in such experiments, preexisting dls would appear as species of uniform size after serial passage of independent isolates, while de nova generation of dls from the helper virus genome would result in the appearance of di species with different sizes (holland, 1990) . accordingly, we investigated the de nova generation of tbsv di rnas by analogous high m.o.i. passage experiments performed in planta. our results indicate that tbsv di rnas arise spontaneously upon passage, and that the de nova appearance of di rnas is closely associated with attenuation of symptoms induced by the virus. we have also characterized biologically active di rnas from two independent populations generated during passage. comparisons of sequences revealed that di rnas retained four conserved regions from the tbsv genome. detailed studies of the biology, molecular cloning, and genome characterization of the cherry strain of tbsv used in this study have been previously reported (hillman et al., 1985 hearne eta/., 1990) . the di rna-free culture of the tbsv-cherry strain was isolated by passage of a single local lesion from nicotiana glutinosa to the systemic host n. clevelandi from which a tbsv stock preparation was purified, stored in 50% glycerol at -2o", and used for all subsequent inoculations. this stock was determined to be free of di rnas by both biological and northern blot analysis as described by hillman (1986) . a serial passage experiment was initiated from 16 independent local lesion isolates established from difree tbsv inoculated onto n. glutinosa. eight isolates were passaged sequentially in n. clevelandi and eight in n. benthamiana. in each case, six of the independent isolates inoculated to each host species were passaged without dilution (high m.o.i.) and two were passaged at a sap dilution of l/200 (low m.o.i. tific, springfield, nj] ). for low m.o.i. passes, 10 ~1 of sap was diluted to 2 ml with inoculation buffer. plants were maintained at a constant temperature of 2 1 o and passages were performed at 7-day intervals for 11 weeks. tissue was harvested from inoculated leaves at the time of passage and stored at -80" until analyzed. observations on symptom severity and plant survival were recorded at regular intervals throughout the passage experiment. total rna was extracted from 0.3-g samples of inoculated leaves and fractionated in 2.3 m lici-soluble (contains di rna and viral-specific dsrna) and -insoluble (total ssrna) fractions as described previously (hillman et al. 1985 (hillman et al. , 1987 . the lici-soluble rna fractions were analyzed for the presence of di rnas by electrophoresis on nondenaturing 5% polyacrylamide gels in tbe buffer (0.04 m tris-borate, 1 mn/l edta, ph 8.3) and stained with ethidium bromide. virus particles were purified from separate 1.0-g samples of the same tissue by two cycles of precipitation in 8% polyethylene glycol, 0.2 m nacl (hillman et al,, 1985) and encapsidated rnas then were extracted with phenol. virion rnas and licl-insoluble fractions were analyzed on 1.5% agarose gels or denaturing 4.5% polyacrylamide gels containing 7 m urea in tbe buffer, after denaturation of the samples in 50% formamide and 5% formaldehyde. rna was transferred by electroblotting onto nylon membranes (zeta-probe, bio-rad) as described by the manufacturer, and northern analysis was performed as described (hillman et al., 1987) . cdna cloning and polymerase chain reaction amplification purified virion and di rnas, fractionated on sucrose density gradients, were used as templates for synthesizing double-stranded (ds) cdnas essentially as described by dawson et al. (1986) . approximately 5 pg of di rna was denatured with methyl mercuric hydroxide, annealed with a synthetic oligonucleotide complementary to the 3'terminal 19 nucleotides of tbsv, and then treated with 20 units of amv reverse transcriptase (promega). second-strand reactions of 100 ~1, containing the first-strand rna:cdna hybrids, 10 units of dna polymerase i (brl), 500 pm each dntp, and 20 ng of rnase a in 1 x dna polymerase buffer (50 mm potassium phosphate, ph 7.5, 10 mm mgci,, 1 mm p-mercaptoethanol), were incubated for 12 hr at 13". the polymerase chain reaction (pcr) was used to remove homopolymer tails from di rna clones made from virion rnas, prepare cdnas made from lici-soluble rnas for cloning, and generate templates for in vitro rna transcription. first-strand cdna synthesis was initiated using a primer complementary to the 3' terminal 28 nt of tbsv. samples of the rna:cdna hybrids then were used in pcr reactions containing the 5' oligonucleotide primer 5'ggggccctaatacgactc-actataggaaattctccaggatttctcgacc, which contains a bacteriophage t7 rna polymerase promoter sequence fused to the 5'terminal24 nt of tbsv, plus additional 3' primer. alternatively, pcr amplifications were performed using a different 3' proximal primer, 5'gcatgcccgggctgcatttctgc, which contained smal and sphl restriction sites to facilitate cloning. conditions for pcr were as follows: 90 ~1 of 1 x taq polymerase reaction buffer (10 mm tris-hci, ph 8.3; 50 mm kci; 1.5 mm mgci,; 0.1% gelatin; 200 pm each dntp) containing approximately 10 ng of pstl-digested plasmid dna, and 50 pm of each oligonucleotide primer were overlaid with mineral oil, boiled for 5 min, quenched on ice, and then treated with taq polymerase (cetus/perkin-elmer, 2.5 u in 10 ~1 of 1 x reaction buffer). temperature cycling (25 set at 95', 50 set at 50", 2 min at 72") was carried out for 25 cycles using an automatic thermal cycler (cetus/perkin-elmer or mj research, inc.). yields were approximately 5 pg for each pcr amplification of cdna. cloning the di cdnas was accomplished by adding polycytidine tails to non-pcr-amplified templates using terminal deoxynucleotidyltransferase (brl) followed by ligation to oligo(dg)-tailed puc9 dna (boehringer/mannheim). alternatively, pcr-amplified cdnas were restricted with apal and sphl and then inserted into pdk41 (hearne et al., 1990) . di cdnas were used to transform fscherichia colistrain dh5a f (brl) (hanahan, 1983) . templates for sequencing were prepared either by asymmetric pcr amplification of cloned cdnas according to gyllensten (1988) or by subcloning the inserts into pzf19u (mead et al., 1986 ) and preparing single-stranded dna as outlined in sambrook et al. (1989) . sequencing reactions were performed using taq polymerase and reagents as recommended by the supplier (stratagene). a series of standard sequencing primers and custom di-complementary primers allowed determination of the sequence of each clone on both strands. to test the biological activity of the cloned di rna sequences, pcr-amplified inserts were transcribed with t7 rna polymerase as described by janda et al. (1987) and used to inoculate plants together with di-free tbsv rna or virions as described previously (hearne et al., 1990) . to test the hypothesis that tbsv di rnas arise spontaneously during virus infection, we performed a passage experiment similar to those reported using animal virus systems. sixteen independent di-free tbsv isolates, each derived from a single local lesion of the di-free tbsv stock culture, were passaged serially on two systemic plant hosts, n. clevelandii and n. benthamiana. on each host species, six isolates were passed at high m.o.i. and two control isolates were passed at low m.o.i. in these hosts, di-free tbsv induces lethal necrosis within 7-l 0 days postinoculation (d.p.i.), except in the presence of di rnas which are associated with attenuation and the establishment of persistent infections (hillman et a/., 1987) . for each local lesion isolate, detection of di rnas and appearance of symptom attenuation were noted and the results are summarized in table 1 . none of the isolates passed at low m.o.i. developed attenuated symptoms, but by the eleventh passage, each of the isolates passed at high m.o.i. were inducing attenuated symptoms. the passage at which symptom attenuation appeared in each of the high m.o.i. isolates was not uniform, occurring as early as passage 3 for two isolates (c6 and b7), and as late as passage 11 for others (bl 1 and b12). as summarized in table 1 , in each case where symptom attenuation was apparent, low molecular weight (lmw) rnas of approximately 400-600 bases were detected in the lici-soluble fraction of the cellular rna extracts. examples of these are presented for the different isolates at passage 11 in figs. 1 c and 1 d. the small rnas which developed in each of the high m.o.i. isolates hybridized to both 5'-and 3'-proximal tbsv genomic sequences, but generally were larger (-600 bases) than the previously characterized di 1 rna species (-400 bases). in 9 of 12 high m.o.i. isolates, the passage at which di rnas were detected either coincided with or just preceeded the passage at which symptom attenuation was first observed. however, in three isolates (c2, b8, and b9) di rnas were detected several passes prior to symptom attenuation, suggesting that all defective rnas that were generated were not equally able to interfere with virus symptoms. b passage number identifies the number of serial passages on the designated host plants after the initial local lesion transfer at which attenuated symptoms were observed or dls were detected. n designates inoculations that resulted in lethal necrosis at 7 d.p.i. and in which di rnas could not be detected. the experiment was terminated after passage 11 at which time symptoms in each of the high m.o.i. isolates had become attenuated. gel analysis of ssrnas isolated from virus purified from plants at passage 11 and of the lici-soluble rna fraction from each of the high and low m.o.i. isolates passaged in ai. c/eve/a&ii (figs. 1 a and 1 c) or n. benthamiana (figs. 1 b and 1 d) then was performed. it is evident from the analysis of virion rnas on the high-resolution polyactylamide gels (figs. 1a and 1 b) that each of the 12 high m.o.i. isolates contained 1 or 2 major di rna species of a discrete size that were not detectable in any of the 4 low m.o.i. isolates. additional analyses of the lici-soluble fraction isolated from the same infected plants of each isolate (figs. 1 c and 1 d) showed a complex population of low molecular weight rnas in each of the high m.o.i. lines in addition to the prominent di species corresponding in size to the di rna present the virion rna fraction. northern analyses were performed on all of these samples to confirm the tbsv-specific nature of the low molecular weight di-like species. an analysis of lici-fractionated rnas from tissue and virion rnas which is typical and representative for 1 high m.o.i. di rna-containing isolate (blo, passage 11) and 1 difree low m.o.i. isolate (b15, passage 1 1) is given in fig. 2 . the results clearly demonstrate that the low m.o.i. line is completely free of detectable di rna. furthermore, the di rna species purified from virions in the high m.o.i. line is the same size as the major di rna species present in the 2.3 n/l lici-soluble fraction isolated from the same tissue, suggesting both that the rna is highly structured and that it is encapsidated. these results are consistent with the properties of previously characterized di rnas found to be associated with this strain of tbsv (hillman et al., 1987) . the di rnas from two representative isolates passed at high m.o.i. (c6, passage 11, and blo, passage 11) were sequenced to compare them with each other and with the smaller dll species characterized previously (hillman et al., 1987) . the encapsidated di rnas from isolates c6 and blo migrated during electrophoresis as apparently homogeneous populations with an estimated size of approximately 600 bases. complete sequences for four cdna clones generated from c6 lici-soluble rnas and six cdna clones generated from blo vinon rnas were determined and are presented in fig. 3 . although derived independently, the sequence alignments show that the di rnas from the c6 and blo tbsv isolates are strikingly similar. each of the di rnas appears to have been formed by deletion of three large internal regions from the tbsv genome, leaving four regions of sequence with highly conserved boundaries: (region i) the tbsv 5' leader sequence including the initiator methionine codon; (region ii) a block of approximately 200 to 250 bases from the putative polymerase gene; (region iii) approximately 70 bases from the 3' terminus of the viral p19 and p22 open reading frames (orfs); and (region iv) the terminus of the 3' nontranslated region (see fig. 3 ). in each of the clones, patterns of small deletions and nucleotide replacements were identified, indicating that each sequence represented an independent di rna. nevertheless, the entire sequence of each of the di rna clones clearly was derived from the tbsv genome without rearrangement. biological activity of the cloned di cdnas was assessed by coinoculating plants with in vitro rna transcripts and di-free virus. for each sample, a di rna of expected size accumulated to high titer in the presence of tbsv, but not when transcripts were inoculated without virus (fig. 4) . as shown previously with cloned tbsv, a dinucleotide cap was not required for infectivity of the in vitro transcripts of cloned di rnas. the individual cloned di rnas attenuated symptoms to the same degree as the parental, noncloned blo di rna population. interestingly, the homogeneous di rna inoculum derived from cloned cdnas (fig. 4 , lane 2) gave rise to a complex pattern of smaller rna speties in the infected plants, suggesting that di rnas may be capable of sequence evolution upon reintroduction into plants. in vitro rna transcripts derived from cdna clones of c6 and blo di rnas have also been found to replicate in protoplasts in the presence of di-free tbsv (y. chang and j. morris, unpublished) . it should be noted that the strategy used to clone the tbsv di rnas selected molecules capable of hybridizing to the 5' and 3' termini of the virus genome. although this strategy precluded cloning possible di rnas that did not contain these termini, this was not of great concern since each of the cloned di rnas corresponded precisely in size to the predominant species of di rna encapsidated in the population and each was also biologically active when reinoculated to plants as rna transcript. in this study, di rnas were generated in all tbsv isolates passed at high m.o.i., but in none of the iso-lates passed at low m.o.i. we interpret these results as strong evidence that each di rna population arose spontaneously from the viral genome during passage and not from a preexisting template. this conclusion is supported by the demonstration that di rnas appeared in each virus isolate at different times during serial passage and were also of different sizes, these observations are consistent with the generally accepted proof for de nova generation of dls in many well-studied animal virus infections (holland, 1990) . subsequent to completing these experiments, we were able to corroborate this conclusion by observing that di rnas similar in size to those reported here appeared after high m.o.i. passage of tbsv inocula derived from in vitro transcripts of tbsv cdna clones. a thorough characterization of those di rnas and conditions conducive to their generation will be published elsewhere. our results with tbsv parallel reports demonstrating de nova generation of dls in animal viruses (holland, vrrion rnas (vrna) were extracted from virions purified from the same inoculated leaves. samples were electrophoresed in a 1.5% agarose-tbe gel (top), stained with ethidium bromide, electroblotted onto a nylon membrane, and probed with ptbsv-28 (hearne et al., 1990) . locations of single-stranded genomic rna(g) and the two subgenomic rnas 1 (sgl) and 2 (sg2) along with their replicative forms (dssgl and dssg2) are indicated at the right. ). an important difference, however, is that our studies utilized the intact virus host species rather than cultured cells. the difficulties of examining the role of dls in modulating virus disease and in establishing persistent infections are well known (huang, 1988; barrett and dimmock, 1986) . a plausible alternative would be to pursue such studies using plant virus systems. the strong correlation between symptom attenuation and appearance of dls supports our previous findings that inhibition of virus replication by di rnas also lessens disease severity (jones et a/., 1990; hillman et al., 1987) . however, we also observed that in three isolates (c2, b8, and b9) symptom attenuation was delayed by several passes after the initial detection of dls, and in many of the isolates the dominant di rna species became smaller after its initial appearance. although the overall effects of individual di rna species upon virus replication was not determined, it seems likely that some dls may differ in their ability to interfere with tbsv. a more thorough characterization of the evolution and biological activities of different di rna species will be reported elsewhere (d. knorr and j. morris, in preparation) . the sequences retained by the di rnas suggest possible mechanisms for their formation. both the processivity of the viral replicase and local structure of the viral template have been implicated in the formation of di rnas in other viruses. makino et a/. (1988) have suggested that dlsse of the coronavirus mhva59 was formed by viral polymerase "hopping" within regions of secondary structure in the genomic rna. earlier work by lazzarini et a/. (198 1) identified a hexanucleotide sequence on the vesicular stomatitis virus rna at which initiation by the viral polymerase led to formation of di rnas. in the tbsv, the hexanucleotide motif 5'-apuagaa-3' occurs at or near the endpoints of regions ii and iii in the di rnas. it is possible that polymerase dissociation and reinitiation take place in these regions and may account for the large deletions generated between these regions. in cloning tbsv genomic sequences we encountered several regions of "strong stop" signals. these polymerase-destabilizing regions in the rna could also contribute to the formation of di rnas. interestingly, the most highly conserved deletion in the di rnas, occurring between regions ill and iv, does not contain a sequence duplication, but rather an inverted repeat of the sequence aggtta. the schematic diagram in fig. 5 shows the relationships between the tbsv genome and the di rnas isolates of tbsv were cloned after the eleventh passage in nicofiana benthamiana and nicotiana c/eve/and;;, respectively. sequences of six independent di rna clones from the blo isolate and four from the c6 di rna were aligned with the consensus sequence from dll and the four conserved regrons from the tbsv genome. a dash (-) indicates the nucleotide at that position is the same as the tbsv genomic sequence shown below and only nucleotides which differ from the genome are shown, except that the start and stop codons for drfferent viral genes are indicated. a dramond (6) or a space indicates no nucleotide is present at the corresponding position. the endpoints of cdna clones made by tailing, prior to pcr amplification to modify the termini, are indicated by brackets u. and the size of each di is given in parentheses. conserved regions i-iv, as drscussed in the text, are indentified below each block of sequence. the apuagaa hexamer sequences and the aggtta inverted repeats are underlined. sequenced to date. conservation of the same four remonly found in other dls that have been characterized. gions of tbsv sequences in the di rna clones sug-it is possible that internal regions function nonspecifigests that the retained elements are required for rna cally, such as to augment packaging or to separate replication and possibly encapsidation. it is not surprisdifferent functional domains, but if this is the case we ing that dls maintain viral 5' and 3' termini because would not expect the consistency present in the di these regions are likely to be involved in polymerase rna sequences. it is more likely that internal regions binding and the initiation of replication. possible funcfunction more specifically, perhaps as sites of recognitions for the two internal domains (regions ii and ill) are tion for transacting factors important for the initiation less obvious; nevertheless, internal regions are comor regulation of replication. the replicase of bacteriothe mechanism of di-mediated symptom attenuation in tbsv infections is not understood, although recent experiments using protoplasts clearly show that di rnas replicate at the expense of the helper virus (jones et al., 1990) . it seems unlikely that competition for available coat protein during encapsidation is an important factor because proportionally less di than genomic rna is encapsidated. it is also unlikely that a specific di-encoded protein is responsible for attenuation because there are no conserved orfs of significant size which could be translated without internal initiation and all previous attempts to translate di rnas in vitro have been negative (b. hillman, personal communication). consequently, the most likely mechanism leading to symptom attenuation is reducedviral replication resulting from competition between the viral genome and di rnas for replicase. it seems possible that selection pressure resulting in symptom attenuation would favor replicative fitness of the di rnas through fig. 4. infectivity of rna transcripts from cloned tbsv blo di rnas. the di sequences from independent cdna clones of the blo di rnas were amplified by pcr. transcribed in vitro and inoculated onto nicofiene clevelendii plants together with tbsv di-free genomic rna. rna was isolated from inoculated leaves at 7 d.p.i. and the 2.3 m liccsoluble fraction was separated in a 5% polyacrylamide gel under nondenaturing conditions (top), togetherwith di-free tbsv (di-), a sample product of the t7 transcriptions, a pcr-amplified product, and the lici-soluble rna from a noninoculated plant (healthy). below is a northern blot of the same rnas probed with a tbsv di-specific sequence. because no denaturant was employed, only single-stranded tbsv-specific rnas hybridized with the probe. (blumenthal, 1979) and an internal region is also required for maintenance of brome mosaic virus rna 3 . internal sequences in dls associated with vesicular stomatitis virus appear to provide stability and may contribute to the overall persistence of viral infection (o'hara eta/., 1984) . another possible function for the internal domains is in promoting the initiation of encapsidation. it has been suggested for tcv that several internal regions may be involved in nucleating virus assembly (wei et a/,, 1990) . of significance in that study was the identification of a primary binding site near the amber readthrough codon of the viral replicase, a location similar to that of region ii in the tbsv di rnas. judging by relative levels of di versus genomic rnas in purified virion preparations (see, fig. 2 ) di 1 is packaged less efficiently than bl 0, and also contains a smaller, more dissected, region ii. it is possible that biological properties such as infection persistence are related to the ability of tbsv di rnas to become encapsidated, hence the strong 5 . diagram of the relationship between the tbsv genome, regions present in the sequenced tbsv blo and c6 di rna clones, and dll a map of the tbsv genome is presented at the top with the five gene coding regions identified (~33, p92, ~41, and pl9/22) and represented as different shaded blocks. the same shading pattern is used below to represent each di sequence in expanded form, with lines to indicate the derivation of each of four conserved regions from the tbsv genome (i-iv). major deletions forming the four regions are shown as bent lines and straight lines connect the smaller deleted sequences occurring within a region. each di rna contained the entire 5' leader sequence as well as the 3' terminal -130 nucleotides (regions i and iv). the largest difference between the different sequences were the size and internal deletions within reconservation of these sequences in tbsv dls. gion ii. selection of optimal polymerase binding sequences. in addition, the small size and structural stability of di rnas might also contribute to their ability to be maintained. whatever mechanisms are involved, it is clear that selection pressures strongly favor maintenance of di rnas that function to down-regulate virus replication and thereby allow host survival and virus persistence. defective interfering viruses and infections of animals rna replication: function and structure of q@-repiicase adefective interfering rna molecule in cymbidium ringspot virus infections cdna cloning of the complete genome of tobacco mosaic virus and production of infectious transcripts continuing coevolution of virus and defective interfering particles and of genome sequence during undiluted passages: virus mutants exhibiting nearly complete resistance to formerly dominant defective interfering particles lntercistronic as well as terminal sequences are required for efficient amplification of brome mosaic virus rna3 generation of single-stranded dna by the polymerase chain reaction and its application to the direct sequencing of the hladqa locus studies on transformation of f. co/i with plasmids the complete genome structure and infectious rna synthesized from clones of tomato bushy stunt virus genome organization, replication, and defectlve rnas of tomato bushy stunt virus a defectlve interfering rna that contains a mosaic of a plant virus genome organization of the tomato bushy stunt virus genome: characterization of the coat protein gene and the 3'terminus effects of low-molecular weight rna and temperature on tomato bushy stunt virus symptom expression defective viral genomes defective viral particles and viral disease processes modulation of viral disease processes by defective interfering particles high efficiency t7 polymerase synthesis of infectious rna from cloned brome mosaic virus cdna and effects of 5' extensions on transcript infectivity defective-lnterfenng rnas and elevated temperatures inhibit replication of tomato bushy stunt virus in inoculated protoplasts the ongrns of defective interfering particles of the negative strand rna viruses deletion mapping of sindbis virus di rnas derived from cdnas defines the sequences essential for replication and packaging turnip crinkle virus defective interfering rnas intensify viral symptoms and are generated de novo primary structure and translation of a defective interfering rna of murine coronavirus singlestranded dna 'blue' t7 promoter plasmids. a versitile tandem promoter system for cloning and protein engmeering carnation mottle virus and viruses with similar properties. in "the plant viruses. polyhedral vlrions with monopartite rna genomes defective lnterfenng rnas of a plant virus defecttve interfering viruses associated with plant virus infections vesicular stomatitis virus defective lnterfenng particles can contain extensive genomlc sequence rearrangements and base substitutions origin and replication of defective lnterfenng particles molecular cloning: a laboratory manual the generation and amplification of defective interfering rnas rapid evolution of rna viruses structure and assembly of turnip cnnckle virus vi: identification of coat protein btndlng sites on the rna evidence for speclflclty in the encapsidation of sindbls vkus rnas key: cord-294260-g410mavp authors: sztuba-solińska, joanna; stollar, victor; bujarski, jozef j. title: subgenomic messenger rnas: mastering regulation of (+)-strand rna virus life cycle date: 2011-04-10 journal: virology doi: 10.1016/j.virol.2011.02.007 sha: doc_id: 294260 cord_uid: g410mavp many (+)-strand rna viruses use subgenomic (sg) rnas as messengers for protein expression, or to regulate their viral life cycle. three different mechanisms have been described for the synthesis of sg rnas. the first mechanism involves internal initiation on a (−)-strand rna template and requires an internal sgp promoter. the second mechanism makes a prematurely terminated (−)-strand rna which is used as template to make the sg rna. the third mechanism uses discontinuous rna synthesis while making the (−)-strand rna templates. most sg rnas are translated into structural proteins or proteins related to pathogenesis: however other sg rnas regulate the transition between translation and replication, function as riboregulators of replication or translation, or support rna–rna recombination. in this review we discuss these functions of sg rnas and how they influence viral replication, translation and recombination. synthesis of subgenomic (sg) messenger rnas (mrnas) by (+)-strand rna viruses allows the differential expression of specific viral genes, both quantitatively and temporally. sg rnas as considered in this review, have the following properties: (i) they are made in infected cells but do not interfere with the normal course of viral replication; (ii) the sg rna sequences are shorter than their cognate genomic rnas; (iii) their sequences are usually co-terminal with the 3′ genomic sequence but sometimes are co-terminal with the 5′ sequences. yet other viruses make sg rnas which contain a 5′ co-terminal leader joined to a 3′ co-terminal sequence; (iv) typically, whether a messenger sg rna contains only one orf, or multiple orfs, with some rare exceptions (dorokhov et al., 2006) , only the 5′ orf is translated (pasternak et al., 2006; yang et al., 2009) . although most sg rnas function as messengers and are translated, other sg rnas, generally those with 5′ co-terminal sequences, have other functions. the production of sg rnas was initially reported in studies of brome mosaic virus (bmv) and was followed by the discovery of sg rna in tobacco mosaic virus (tmv)-infected leaves and later in alpha-, carmo-and sobemo-families (strauss and strauss, 1994; rico and hernandez, 2009; mcgavin and macfarlane, 2009 ). indeed, among (+) strand rna viruses of plants, only viruses of the potyviridae and comoviridae as well as the sequiviruses of the sequiviridae family do not use this strategy (zaccomer et al., 1995) . our goal in this review is to describe the major mechanisms by which sg rnas are generated and to discuss their roles in the life cycle of (+) strand rna viruses. since the last reviews discussing that subject white, 2002; miller and white, 2006) , these rnas have been shown to function not only as mrnas, but also as riboregulators of replication and transcription; as well, sg rnas may participate in genome rearrangements that help maintain genomic integrity and possibly the acquisition of non-self sequences. although replicon rnas made in the laboratory to study the replication of hepatitis c virus (hcv) and other flaviviruses have also been referred to as subgenomic rnas, since they are not seen in a normal infection, they will not be considered in this review. 3′ end, and one or more internal or subgenomic promoters (sgps). to synthesize a sg rna, the viral rdrp recognizes and binds to the sgp and initiates transcription . this mechanism was first found with bmv, a tripartite plant virus. the (−) strand of dicistronic bmv rna 3 serves as template for transcription of the 3′ co-terminal sg rna4 due to de novo internal initiation from the sgp. the bmv sgp has been mapped to the sequence from nt −95 to +16 ( fig. 2a ). its modular composition includes an au-rich enhancing region, a poly (u) tract, a core region, the +1 c transcription initiation site, and a downstream sequence (wierzchoslawski et al., 2004) . the enhancing region (nts −95 to −20) greatly increases the amount of sg rna 4. the core region (nts −19 to −1) contains a stem-loop (sl) structure which is responsible for rdrp binding. adkins and kao (1998) have suggested an induced fit mechanism whereby the rdrp recognizes key nucleotides, after which nucleotide base-pairing occurs and the sl structure forms. another model proposes that the sl structure binds to the rdrp complex, after which the key nucleotides stabilize the hairpin (haasnoot et al., 2000 (haasnoot et al., , 2002 . regardless of the mechanism, internal initiation appears to be a multistep process, which involves both host and viral proteins (hertz and huang, 1995a,b; adkins and kao, 1998; haasnoot et al., 2002; fig. 1 . schematic representation of different mechanisms for subgenomic rna synthesis. (a) internal initiation model , (b) premature termination model showing termination either during (−) and (+)-strand synthesis (white, 2002) , (c) leader-primed transcription model, (d) discontinuous template synthesis (pasternak et al., 2006) . genomic and sg rna (+)-strands are depicted as horizontal yellow boxes, the (−)-strands are depicted as green boxes, the ovals represent rdrp enzymes capable of starting/stopping at the internal initiation sgp promoters that are depicted as blue sgp boxes. the leader trs (l-trs) and the body trs (b-trs) are represented by dark green and brown boxes, respectively. refer to the text for details. sivakumaran et al., 2004) . other (+)-strand rna viruses use sgps with a similar organization haasnoot et al., 2000; morales et al., 2004; olsthoorn et al., 2004) . tmv has a single-stranded non-segmented rna genome and represents another example of a virus that synthesizes sg rnas via internal initiation. three of these sg rnas are co-terminal with the 3′ terminus of the genomic rna; the smallest sg rna has only one orf, and encodes cp. the middle-sized sg rna has two orfs (for mp and cp), but only the 5′ orf, that for mp, is expressed. the third and largest sg rna, referred as the i 1, has been detected in tmv-infected tobacco tissue. it contains in addition to orfs for mp and cp, a 5′ orf for a 54-kda protein with a sequence identical to that of the readthrough region of the 183-kda replicase. interestingly, rna structures that are able to regulate the activity of tmv promoters can be far removed from the initiation site. culver et al. (1993) found that the location of the tmv sgp in relation to the 3′ utr is a crucial factor in enhancing expression. later, szecsi et al. (1999) and shivprasad et al. (1999) associated the positive effect of the 3′ utr with the three pseudoknots contained therein; these were found to redistribute rdrp activity localizing most of it to the nearest sgp promoter. the sgp domain is shown as a solid, black box located between the 3a and cp orfs on bmv (−) rna3. the arrow indicates the initiation site and the direction of subgenomic rna4 synthesis. the bottom expansion shows the nucleotide sequence of the sgp promoter (−95 to +16). the sgp subdomains are indicated as follows: the enhancer including the poly u tract (nt −95 to −20), the core region (nt −19 to −1) including the hairpin, the initiation +1 cytidilate, and the downstream portion. (b) tmv sgp promoters are indicated by the black rectangles: the first promoter is located between 183-kda rdrp protein orf and mp orfs, and the second one between mp and cp orfs. the arrow above the g rna indicates the position of the amber read-through codon, the putative initiation site for i 1 sg rna expressing 54-kda protein of unknown function. the arrow below (−) g rna indicates the initiation site for one of the sg rnas. the bottom expansion shows the nucleotide sequence of the sgp promoter (−95 to +40). the promoter core (−35 to +10),the two hairpin structures, sl1 and sl2, are indicated,as well as the initiation +1 cytidilate, and the downstream portion (grdzelishvili et al., 2000) . (c) the sgp promoter of sv (−) strand rna is shown in the top line as a solid, black box. the arrow on (−) g rna diagram indicates the initiation site and the direction of sg rna synthesis. the bottom expansion shows the nucleotide sequence of the sgp promoter (−98 to +14). the sequence between −19 to +5 represents the minimal sequence with promoter activity. the sequence from −40 to +14 was shown to enhance the promoter activity. mutations at the marked nucleotide positions: −81, −75, −55, down regulate the sgp promoter activity. both rna sequence and secondary structures are generally critical for the activity of an sgp (koev et al., 1999; haasnoot et al., 2000) . in the case of the tmv promoter for the middle-sized sg rna (fig. 2b ), computer analysis predicted two stem-loop structures (sl1 and sl2) upstream of the transcription start site. the sl1 secondary structure, rather than its sequence, was critical for promoter activity. on the (van marle et al., 1999) ; only the loop and the top of the stem of the predicted hairpin structure are presented (b) linear representation of the tbsv rna genome showing its coding organization. the relative positions of interacting rna elements involved in sg rna transcription are shown above the genome and are indicated by arrows. initiation sites for sg rna transcription are labeled sg1 and sg2, and corresponding structures of the two sg rnas are represented by bold arrows below the genome. below, the long-distance rna-rna interactions that regulate sg rna transcription in tbsv are shown in detail. relevant sequences of the tbsv genome are presented with corresponding genomic coordinates. the as1/rs1 base-pairing interaction is essential for the efficient transcription of sg rna1 (choi and white, 2002) , while the as2/rs2 and de-a/ ce-a base-pairing interactions promote sg rna2 transcription (choi et al., 2001; lin and white, 2004 ); (c) representation of genomic rna1 of flock house virus (fhv). interaction between the distal subgenomic control element (dsce) and the proximal subgenomic control element (psce) is required for rna3 synthesis. the internal replication element (intre) and 3′ replication element (3′ re) are required for rna1 replication. below, potential base pairing of the dsce to regions proximal to the subgenomic region start site. gray boxes mark psce residues that potentially base pair to the dsce. putative helices 1 and 2 are bracketed. arrow, rna3 start site at nt 2721 (lindenbach et al., 2002) ; (d) schematic of the rcnmv genome showing the relative positions of the in trans interacting rna elements: the loop portion of a stem-loop in rna2 (termed trans-activator or ta) and a complementary sequence in rna1 (termed ta binding site or tabs) located just upstream from the initiation site for sg mrna transcription (guenther et al., 2004) ; (e) representation of clsv genome and the proposed as2 and rs2 interaction during regulation of sg rna2 synthesis (xu and white, 2008) ; (f) overview of the bydv genome and the in cisinteraction between genomic 5′-utr stem-loop (bcl) and the genomic 3′-bte, and the in trans interaction between the genomic 3′ bte and the 5′ utr of sg rna1 . numbers depict the nucleotide positions in the viral genomic rna. the nucleotides participating in the long-range base-pairing are joined by lines. other hand, removal of most of the sl2 region increased accumulation of the sg rna four-fold (grdzelishvili et al., 2000) . also, the three sgp promoters of barley yellow dwarf virus (bydv) fold into different size stem-loops downstream of their respective initiation sites . in the case of promoters of barley stripe mosaic virus (bsmv) sg rnas β1, β2, and γ, the conservation of both the sequences and the secondary structures appear to be important for their activity (johnson et al., 2003) . here the substantial differences between sg rnas β1, β2, and γ promoter sequences were postulated to explain competition for the viral rdrp, coordination of the temporal expression and abundance of the proteins, and constitutive expression of the γ b protein. viruses producing multiple sg rnas often contain homologous sequences within their sgp promoters . also, certain elements in genomic promoters for (−) strand synthesis can share similarities with elements in the internal promoters; see for example, a stem-loop c (slc) in bmv (haasnoot et al., 2002) and the triloop hairpin (hpe) in alfalfa mosaic virus (amv) (olsthoorn et al., 2004) . initiation from the 3′ end of the (−) sense genomic bmv rna obeys different rules than does the initiation of sg rna (stawicki and kao, 1999) , likely due to additional factors (diez et al., 2000) that can adjust the properties of the rdrp (adkins and kao, 1998; ranjith-kumar et al., 2003; haasnoot et al., 2002; sivakumaran et al., 2004) . in the case of sindbis virus (sv) (the prototype of the genus alphavirus, family togaviridae), the minimal sequence of the (−) strand rna needed for the sgp activity extends from nt −19 to nt + 5 but a sequence extending from −98 to +14 increases promoter activity at least 6 folds. most of this increase could be accounted for by strongly conserved sequences extending from − 40 to −20, and from +6 to +14 (wielgosz et al., 2001) (fig. 2c ). three classes of mutations down regulate the synthesis of the sv sg rna: (i) promoter mutations, (ii) nsp2 protein mutations, and (iii) a single mutation in nsp3. sv makes four nonstructural proteins; nsp1 is responsible for capping and methylation of the g and sg rnas; nsp2 has both a protease activity and an rna helicase activity; the function of nsp3 is not known, and nsp4 is the viral rdrp. promoter mutations which affect the synthesis of sv sg rna have been described by huang (1995a, 1995b) and lin et al. (2002) . these effects are often cell-dependent, suggesting a role for cellular factors in the synthesis of sg rna. mutations in nsp2 of sv and sfv, which mapped to its protease domain, decreased protease activity and thus slowed cleavage of the nonstructural polyproteins, a step necessary for the efficient synthesis of sg rna. also, a single insertional mutation in nsp3 lowered the level of the sv sg rna synthesis, but the mechanism responsible is not known (lastarza et al., 1994) . by incubating a labeled sgp sequence with an sv rna transcriptase/replicase complex, distinct protein sites on nsp4 were found to be responsible for the recognition of the sgp and g promoters stollar, 2004, 2007) . it was shown that arg to ala changes at positions 331 or 332 knocked out the in vitro synthesis of sg but not g rna (li and stollar, 2007) . conversely, by changing the arg residue at 545, 546, or 547 to ala, the synthesis of g rna but not sg rna was knocked out (li et al., 2010) . viruses in the family caliciviridae also make sg rnas by internal initiation on the (-)-strand (morales et al., 2004) . in at least one case, these sg rnas are packaged into viral particles (neill, 2002) . in a second mechanism for making a sg mrna, the rdrp complex, instead of copying the (+) strand rna genome into a full length (−) strand rna, terminates "prematurely" at a specific stop signal, and synthesizes a shortened (−) strand of sg rna, which then serves as a template for the synthesis of a sg (+) strand rna (fig. 1b) . these sg (+) strand rnas, like those made by internal initiation, have sequences which are co-terminal with the 3′ sequence of the viral genome. premature termination (pt) can, however, also occur during synthesis of (+) strand rna (white, 2002) , thus generating 5′ coterminal sg rnas ( fig. 1b) (wierzchoslawski et al., 2006) . premature termination (pt) supporting formation of sg rnas is seen with various rna viruses including toroviruses (van vliet et al., 2002) , roniviruses (cowley et al., 2002) , betanodaviruses (iwamoto et al., (guenther et al., 2004) , closteroviruses , and nodaviruses (lindenbach et al., 2002) . flock house virus (fhv), the prototype virus of nodaviridae, uses pt to make a sg rna. fhv has a bipartite (+) strand rna genome but both rnasare packaged into a single viral particle. rna 1 encodes a replicase protein a, while rna 2 encodes protein α, the precursor of the capsid proteins. fhv infection also gives rise to a 387 nt sg rna (rna 3), the sequence of which is 3′ co-terminal with that of rna 1. rna 3 has two overlapping orfs, b1 and b2. the b1 orf encodes the c-terminal 102 amino acids of the a protein. no function has been associated with this polypeptide. the b2 protein is a suppressor of rna interference, both in plants and cultured insect cells. rnas 1, 2, and 3all have a 5′ cap, but none are polyadenylated. in studies of fhv rna replication in yeast (lindenbach et al., 2002) , two cis-acting elements were found in rna 1 (fig. 3c) : (i) a proximal subgenomic control element (psce) extends from nt 2282 to nt 2777 (the start site for the synthesis of sg rna 3 is at nt 2721), and (ii) a distal subgenomic control element (dsce) from nt1229 to nt 1239. base pairing between the nt sequence 1229 to 1239 in the dsce and both a 6 nt sequence and a 4 nt sequence (almost 200 nt apart) in the psce is required for the synthesis of rna 3. for the most efficient synthesis of rna 3, the psce sequence between nt 2302 and 2777 was needed; however some synthesis was seen with the 5′ limit of the psce at nt 2518. thus, the region from nt 2302 to 2518 serves as an enhancer. disruption of psce/dsce base pairing increased the amount of rna 1 made, but then neither (+) nor (−) strand rna 3 was made. apparently, the long distance interaction between dsce and psce gives rise to a secondary or tertiary structure that results in premature termination when (−) strand rna 1 is made. in addition, work by eckerle et al. (2003) , strongly suggests that the (+) and (−) strands of rna 3 participate in a full-fledged rna 3 replication even in the absence of rna 1. furthermore, as with the synthesis of rna 1 and rna 2, the synthesis of (+) strand fhv rna 3 great exceeds that of (−) strand rna. finally, in this system, rna 3 is required for the synthesis of rna 2, and once rna 2 is made, it suppresses the synthesis of rna 3. tomato bushy stunt virus (tbsv) uses a similar strategy to make sg rnas (fig. 3b ) (white and nagy, 2004; zhang et al., 2004) . while making (−) strand rna, the rdrp complex terminates at the promoter regions for (+) sense sg rnas, generating the subvirallength (−) sense 3′ sg rnas (white, 2002) , which then act as templates to amplify the (+) sense 3′ sg rna. substitution of the transcription-initiating nucleotides for sg rna 1 or 2 inhibits the accumulation of these rnas, but not of the corresponding (−) sense sg rna templates (white, 2002) . additionally, tbsv rdrp can be considered as the essential catalytic complex regulating viral replication and sg rna transcription. wu and white (2007) have shown that both processes can be effectively uncoupled in vivo by deletion of up to five c-terminal rdrp residues. subsequently, the replicase c-terminus was proposed to function at an early step of the pt transcriptional pathway mediating (i) efficient production of minus-strand templates for sg rna production; (ii) accurate termination of minus strands, and (iii) efficient utilization of the sgp promoter (wu and white, 2007) . the rdrp termination depends on a multicomponent rna attenuation signal that includes: (i) long distance rna-rna interactions (fig. 3c) , (ii) the spacer segments between the 5′ receptor sequences (rs) and transcriptional initiation sites and (iii) the corresponding downstream sequences predicted to contain transcriptional promoter elements (lin and white, 2004; lin et al., 2007) . the long-distance base pairings between 5′ receptor sequences (rs1 and rs2) in sgp promoters of rna1 and 2, and the activator sequences (as1 and as2) in the coding region of p92, as well as between the 5′ distal element (de) of sgp promoter of rna2 and the complementary downstream core element a (ce), form a physical barrier during (−) strand synthesis (fig. 3b) (wang et al., 2008) . the first two of these interactions span over 1 and 2 kb, respectively, and the distance between these various structures affects the termination efficiency (lin et al., 2007) . similar cis-acting regulations of the 3′ co-terminal sg rnas production operate in cucumber leaf spot virus (clsv) (fig. 3e ) white, 2008, 2009) , pothos latent aureusvirus (polv) and potato virus x (pvx, potexvirus) (kim and hemenway, 1999) . the pvx sg rna production is regulated in cis by conserved octanucleotide sequences located upstream of the two pvx sgp promoters, as well as by complementary elements in the genomic 5′ utr (kim and hemenway, 1999) . on the other hand, red clover necrotic mosaic virus (rcnmv) exploits an in trans bimolecular interaction to stall and dislodge the replicase complex during (-)-strand synthesis (fig. 3d ) (guenther et al., 2004; tatsuta et al., 2005) . rcnmv possesses a bipartite genome with sg rna being synthesized from rna1. the synthesis of the sg rna requires a trans-activation by an rna hairpin structure located in rna2 (see below) (guenther et al., 2004) . specifically, this activation involves the loop portion of an sl structure in rna2 (trans-activator or ta) which base-pairs with a complementary sequence in rna1 (ta binding site or tabs) that is located upstream from the sg rna transcription initiation site (fig. 3d ) (guenther et al., 2004) . it has been suggested that protein factors may bind to and stabilize the bimolecular rna-rna contact. interestingly, the disruption of the rna-rna interaction does not affect replication of rcnmv genomic components (tatsuta et al., 2005) . thus, intermolecular communication secures the switching between replication and transcription, resolving the problem of their mutual interference. also, tcv has recently been found to use a premature termination mechanism for making sg rna2 (bearing cp orf) . analyses revealed that (−) strand sg rna2 accumulation can be uncoupled from that of its (+) strand counterpart. an extended sl rna structure positioned 5′ to the initiation site for sg rna2 was found to mediate pt mechanism by functioning in the (+) strand of the viral genome. as with other viruses that use a pt mechanism, the high degree of identity between the sgp promoter for (+) strand genome and that for sg rna2 transcription, support the idea that tcv uses a pt mechanism for sg rna2 transcription. as noted above, in addition to making the 3′ co-terminal sg rna, rna 4, bmv also makes a 5′ co-terminal sg rna, rna 3a. in this case, the rdrp likely pauses during (+) strand synthesis at the internal oligo u tract resulting in formation of the 5′ co-terminal 3′ polyadenylated (+) sg rna3a (fig. 1a ) (wierzchoslawski et al., 2006) . the binding of another rdrp molecule to the near-by sgp promoter core hairpin which initiates the sg rna4 transcription might pose an additional obstacle to the progression of the rdrp during (+) strand synthesis (wierzchoslawski et al., 2006; sztuba-solińska and bujarski, 2008) . other examples of elements in sgps that act as road blocks for the progressing rdrp have been reported for citrus leaf blotch virus (clbv) (vives et al., 2002) , grapevine vitivirus a (gav) (galiakparov et al., 2003) , citrus tatter leaf virus (ctlv) (tatineni et al., 2009) , and citrus tristeza virus (ctv) (che et al., 2001) . ctlv contains two overlapping 5′ orfs, the expression of which requires the production of two 5′ (+) strand co-terminal sg rnas, which result from premature termination. the 3′-ends of both sg rnas terminate at two sl structures: sl1 and sl2, with the transcription initiation site for the 3′ sg rnas located in the loop region of sl2 (tatineni et al., 2009) . likewise, ctv terminates one of its 5′ sg rnas (referred as low-molecular-weight tristeza lmt1) at the two sl structures located upstream of the internal initiation site for the 3′ sg rna (gowda et al., 2003) . discontinuous transcription (dt) as a mechanism for the synthesis of 3′ co-terminal sg rnas is exemplified by viruses in the families coronaviridae (prototype virus: murine hepatitis virus or mhv) and arteriviridae (prototype virus: equine arteritis virus or eav) of the order nidovirales (pasternak et al., 2001; sawicki and sawicki, 2005) . a special feature of these (+)-strand rna viruses is that they encode a nested set of sg rnas which vary in size, but all of which have a 3′ sequence co-terminal with the 3′ end of the g rna. these sg rnas encode the structural proteins and in the case of the coronaviruses, also several proteins accessory to the replicase proteins. except for the smallest sg rnas, all contain multiple open orfs, but in each case only the 5′ orf is translated. thus, although most of these sg rnas are structurally polycistronic, functionally they are monocistronic. also of note, in contrast to the sg rnas made by internal initiation or by premature termination (see above), those generated by mhv or eav contain, in addition to coding sequences derived from the 3′ portion of the g rna, a short sequence that is identical to a 5′ leader sequence of the g rna. the explanation for how in a sg rna a 5′ leader is joined to a 3′ terminal sequence remained controversial for some years. models were proposed that suggested discontinuous synthesis of rna transcripts, but this then raised the question as to whether the discontinuous synthesis occurred during (+) or (−) strand syntheses (pasternak et al., 2006 ). the first model, known as the leader-primed transcription model (fig. 1c) , proposed that transcription would start by copying the 3′ end of the g rna (−) strand rna thereby giving rise to a 5′ leader sequence; it followed that the discontinuous step would occur during the synthesis of (+) strand rna. however, the finding of multiple species of (−) strand rnas, the sizes of which corresponded to the sizes of the sg rnas, provided strong support for the proposal by sawicki and sawicki (1995) that discontinuous synthesis of sg rna occurred at the level of (−) strand rna synthesis (fig. 1d) . these (−) strand rnas would then serve as the templates for the sg rnas. this is now the generally accepted model. the key to this model is the finding of transcription-regulatory sequences (trss) in the viral genome. the trss containthree sequence blocks: the core sequence (cs) and the 5′-trs and 3′-trs flanking sequences (fig. 4) (alonso et al., 2002) . the cs includes six nucleotides that are highly conserved in each trs of any given coronavirus. the most frequently used core sequences of coronaviruses, i.e. those belonging to group 1 (hexamer 5′-cuaaac-3′) and group 2 (heptamer 5′-ucuaaac-3′) share homology, while the cs of coronavirus belonging to group 3 (e.g. infectious bronchitis virus coronavirus [ibv]) has the most divergent sequence (5′-cuuaacaa-3′). the 5′ and 3′ flanking regions, which are partially conserved in the different genes of related viruses, influence the activity of the cs (alonso et al., 2002) . the trs at the 3′ end of the 5′ leader sequence is referred to as the leader trs (l-trs), whereas trss located at the 5′ end of each gene downstream of the genes coding for the replicase proteins are referred to as body trss (b-trss). these findings suggested that the trs elements are involved in discontinuous transcription. according to the current model (fig. 1d) , following the synthesis of the mhv replicase proteins, pp1a and pp1ab, synthesis of (−) strand rna begins. in some cases, e.g. for purposes of replication, the rdrp complex makes a complete (−) strand copy of the genome which serves as template for the synthesis of full-length (+) strand g rna. in other cases, i.e. for making sg rnas, (−) strand rna synthesis begins, but stalls after copying one of the body trss in the viral genome. this (−) strand trs at the 3′ end of the nascent rna then base pairs with the (+) strand trs at the 3′ end of the leader sequence, facilitating translocation of the nascent (−) strand rna to the 5′ end of the genome where the rdrp complex copies the leader sequence to the end of the genome (sawicki et al., 2001; pasternak et al., 2001; zúñiga et al., 2004) . the various (−) strand sg rnas made in this fashion serve as templates for the synthesis of the same-length (+) strand sg mrnas and/or the production of shorter internally nested sg rnas (wu and brian, 2010) . the latter mechanism would likely contribute to the greater abundance of the 3′ co-terminal sg rnas. the leader and body trss do not show complete sequence conservation. for instance, with murine hepatitis virus (mhv) di rnas, it was found that there were 2-4 copies of the l-trs with the core sequence 5′ ucuaa, whereas the different b-trss had a core sequence centering around 5′ aaucuaaac. more important, it has been demonstrated that formation of a duplex between the (+)strand l-trs sequence in the genome and the (-)-strand copy of a b-trs is required for synthesis of the sg rna having that trs at its 5′ end (figs. 1d and 3a) . the relative contributions of the l-trs and the b-trs to the different 5'-trs in the sg rna are variable. in some cases, the entire sequence of the junction trs in the sg rna is derived from the b-trs; in other instances, both the l-and the b-trs contribute to the junction trs. in an interesting experiment, it was found that when cells were infected with two different strains of mhv, many of the sg rnas had a leader sequence of the co-infecting virus, indicating that the b-trs of one strain can base pair with the l-trs of the second strain (pasternak et al., 2006) . how is it determined at which b-trs the synthesis of the (−) strand rna stalls, leading to duplex formation with an l-trs? the level of sg rna production by both arteriviruses and coronaviruses was shown to depend upon the efficiency of interactions between l-trs and b-trs (pasternak et al., 2001 (pasternak et al., , 2003 zúñiga et al., 2004; sola et al., 2005) . furthermore, it has been proposed that the l-b trs junctions occur at multiple sites, with a preference for 3′ proximal nucleotides within the b-trs (van der most et al., 1994) . with transmissible gastroenteritis coronavirus (tgev), it has been shown that sequences flanking the core trs influence transcription of the sg rnas (curtis et al., 2004; sola et al., 2005) . among nidovirales, the 3′ end of the g rna is critical for sg rna transcription. in mhv, the 300 nt 3′ utr promotes transcription (lin et al., 1996) , but only the 3′ 55 nts are required for (−) strand rna synthesis. it was also shown that mutations disrupting the u-turn motif of the 5′ utr stem-loop 2 (sl2) affected sg rna synthesis, suggesting that sl2 mediates specific interactions with viral and/or cellular proteins involved in the synthesis of sg rnas (liu et al., 2007) . in general, the 5′ utrs of coronavirus rnas fold into similar secondary structures containing three to four sl structures that include a highly conserved 5 nt hairpin loop sl2 with a u-turn motif; sl1 and sl2 are close to each other (shown in liu et al., 2007) . the polypyrimidine-tract binding (ptb) protein hnrnpi may play a role in the regulation of sg rna transcription due to its ability to interact with short pyrimidine-rich tracts, e.g. ucu, or cucu. since the 5′ utr sl2 contains ucuaa repeats, ptb binding might assist circularization of the viral genome and aid template switching during discontinuous sg rna synthesis oberstrass et al., 2005) . related to the discontinuous mechanism is the question of how the synthesis of sg rnas is regulated, and how the ratios of the different sg rnas to each other are maintained. to address this problem, studies were carried out on the nsp1 protein of both eav and mhv (tijms et al., 2001; donaldson et al., 2007) . this protein contains a zinc-binding domain that assists in rna-protein interactions and regulates the replication/transcription balance (tijms et al., 2001) . the eav nsp1 proteininteracts with p100-binding polypeptide (p100bp) which cofunctions with the rna polymerase ii transcription factor c-myb. it has been speculated that the nsp1-p100bp interaction is important for sg rna synthesis, either directly or by recruiting another protein to the viral rdrp complex. alternatively, nsp1 might modulate transcription in the infected cell, explaining its targeting to the nucleus (tijms and snijder, 2003) . similarly, a deficiency in nucleocapsid protein (n) of human coronavirus (hcov-229e) impaired rna replication, but not transcription, demonstrating that n protein regulates the equilibrium between these two processes (schelle et al., 2005) . also, it has been proposed that a long range interaction regulates tgev sg rna transcription (moreno et al., 2008b) . here, a 9-nucleotide (nt) sequence located 449 nt upstream of the n gene trs core sequence (cs-n) interacts with a complementary sequence immediately upstream of cs-n. the complementarity between these two 9-nt elements in tgev was functionally relevant in the transcriptional activation of the n gene. moreover, a positive correlation between the predicted stability of the base-pairing interaction and the accumulation levels of sg rna n was observed (moreno et al., 2008b) . most of the sg rnas generated by (+)-strand rna viruses function as mrnas. this is consistent with their composition, i.e. the presence of a 5′ cap (van vliet et al., 2002) and a 3′ polya tail (e.g. wierzchoslawski et al., 2006) . some sg rnas lack the 5′ cap but contain an ires structure. for instance, the uncapped sg rnas in tmv strain u1 (grdzelishvili et al., 2000) and in crucifer-infecting tobamovirus (crtmv) (dorokhov et al., 2006) harbor an ires, enabling ribosomes to initiate translation at a distant 5′-site. with other viruses, e.g. turnip crinkle virus (tcv), or barley yellow dwarf virus (bydv), the 3′ utr cap-independent translation elements (cites) are important for launching translation of sg rnas. in some cases a cite acts cooperatively with an ires. among eight distinct structural classes of cites described to date, all contain an sl structure which base-pairs to a 5′ utr sequence. the 3′ location of cites favors the translation of sg rnas over that of genomic rnas (qu and morris, 2000; scheets and redinbaugh, 2006; shen et al., 2006) . although the 5′-utr of potato leafroll virus (plrv) sg rna lacks a 3′ translational enhancer (juszczuk et al., 2000) , an ires signal for translation of replication-associated protein 1 (rap1) has been identified internally within the plrv rna genome 1500 nt downstream of its 5′ end (jaag et al., 2003) . the presence of ca-rich motifs, which increase the length but reduce the secondary structure of the sg rna 5′utr boost the protein expression by the sg rnas of tbsv, carnation mottle virus (cmv), cardamine chlorotic fleck virus (ccfv) (skotniki et al., 1993) , and tcv (qu and morris, 2000) . one of the first demonstrations that an sg rna could control viral rna replication was shown with fhv rna3, a sg rna derived from rna1 (see above) (eckerle and ball, 2002) . rna1 mutants deficient in the sg rna3 synthesis failed to make rna2. however, when rna3 was supplied in trans, synthesis of rna 2 resumed (eckerle and ball, 2002; eckerle et al., 2003) . precisely how rna3 exerts its effect is not understood. however, the rna3-dependent replication signal has been mapped to the rna2 3′ end . the influence of an sg rna on viral replication is also seen with bydv (shen and miller, 2004) . bydv makes three 3' co-terminal sg rnas by internal initiation. sg rna3 has no coding capacity, and its function is not known. translation of bydv g rna and sg rna1 is mediated by bte: barley yellow dwarf (like) translation elementin the 5′end of the 3′ utr (fig. 3f) (shen et al., 2006; rakotondrafara et al., 2006) . btes as cap-independent translation elements can be characterized by two structural features: (i) a conserved 17 nt sequence, that includes a stem loop (sl-i) with a gn rna loop motif, and (ii) a loop (not in sl-i) that can base pair to a loop in the 5′utr of the rna (kneller et al., 2006) . it has been proposed that the premature addition of sg rna2, which contains the 5′ utr bte, regulates bydv replication by inhibiting translation of the viral polymerase from genomic rna (g rna). thus, the g rna ceases to function as a messenger and instead functions as a template for replication (shen and miller, 2004) . a role in separation of replication from translation has been proposed for the 5′ co-terminal sg rnas of ctv, bmv and ctlv (gowda et al., 2003; wierzchoslawski et al., 2006; tatineni et al., 2009) . the 5′ sg rna can serve as a template for expression of viral protein, e.g. movement protein in bmv, which sets the genomic rna free for more efficient replication, recombination and packaging (gowda et al., 2003) . as with bydv sg rna 2, the early addition of the 5′ co-terminal sg rna3a reduced bmv rna replication in barley protoplasts (j. sztuba-solińska and j.j. bujarski, unpublished) . in contrast, the in planta experiments showed a dose-dependent bell-shaped response in which high concentrations of sg rna3a reduced virus yield. we speculate that the translation of minute amounts of initial sg rna3a leads to an excess of mp which facilitates the viral spread. at higher doses the sg rna3a may, however, act as a molecular decoy that sequesters translational factors, reducing translation of replicase proteins (j. sztuba-solińska and j.j. bujarski, unpublished). rna recombination can salvage damaged or mutated viral rnas and can contribute to genome variability (cheng and nagy, 2003; chetverin et al., 2005) . the exchangeable subgenomic components can facilitate the production of rearranged viral rna genomes. for instance, the transcription mechanism in nidovirales, involving leader-body trs duplex formation together with distinct cis-acting signals that affect the nascent strand transfer during sg rnas production, resembles the copychoice rna recombination (figs. 1c and d) (van marle et al., 1999; pasternak et al., 2006) . the high recombination frequencies in coronaviruses and arteriviruses have been associated with the highly structured 3′ utrs in the genomes of these viruses (molenkamp et al., 2000; pasternak et al., 2000 pasternak et al., , 2001 . here, the base-pairing between the donor and acceptor molecules at consensus trs motifs plays a role in the production of nidoviral sg rnas, suggestive of similarity-assisted recombination (yuan et al., 2004; pasternak et al., 2006) . apparently, these viruses frequently use a transcription strategy for recombination. the integrity of non-segmented genomes of closteroviridae is supported by sg rna-assisted recombination. in the case of ctv defective interfering (di) rnas the junction sites coincide with the sg rna transcription initiation sites . this suggested that ctv di rnas emerged by recombination of sg rna with a 5′ region of the g rna ( bar-joseph et al., 1997) . the proposed model emphasizes the role of intergenic au-rich sequence located between two sl structures, between orf10 and orf11 that might induce the sg rna11 premature termination followed by template switching bar-joseph et al., 1997) . a similar mechanism might explain the acquisition by viruses in the family closteroviridae of non-self-sequences, either from co-infecting virus or the host (cuellar et al., 2008) . it is likely that norovirus (nov) recombinant isolates originated from sg rna-mediated rearrangements (bull et al., 2005) . here, the high frequency recombination could result from rdrp stalling at the sl structure of the orf1/orf2 overlap (rohayem et al., 2005) , forcing the enzyme to hop across to either (-)-strand sg rna or g rna species. as a result, the recombinant norovirus isolates can acquire new orf2 and orf3 sequences (bull et al., 2005) . the multipartite viruses in the family bromoviridae also utilize sg rnas for the modular swap of their genomes. the recently described bmv 5′ co-terminal sg rna3a can prime recombination events on the (−) and (+) rna3 strands (wierzchoslawski et al., 2006; sztuba-solińska et al., 2011) . these and the following studies revealed the presence of several recombination hot-spots within rna3 including the 5′ utr, the upstream encapsidation signal (packaging element called pe), the b-box motif and the intergenic polyu track (sztuba-solińska et al., 2011; sztuba-solińska and bujarski, unpublished results) . it was proposed that the rdrp associated with other protein factors, e.g. coat protein, might bridge over the rna3 and sg rna3a molecules in cis and/or in trans at highly structured rna elements such as b box-like motif, pe element, or 3′ tls. this can facilitate template switching during (−) and (+) strand synthesis. apparently, sg rnas can act as building components that contribute to genomic rearrangements of complete viral genes. the activity of transcriptional regulatory elements, their general structure and sequence context, as well as their interactions with protein factors, all affect the production of sg rna. it is not known, however, how these structures cause stalling/detachment/reattachment of an actively copying rdrp, or how they affect the timing of sg rna synthesis. since rdrps frequently encounter base-paired regions while copying a template (ng et al., 2008) , explaining how rna structures affect the progress of an rdrp would greatly improve our understanding of regulation of sg rna synthesis. more work with in vitro systems is needed to shed light on both cis-and trans-acting regulatory signals and their cognate factors that affect the production of sg rna (nagy and pogany, 2000; li et al., 2005a) . one problem is that the replication complexes are membrane bound (mackenzie, 2005; denison, 2008) . however, by using cell-free extracts from evacuolated plant protoplasts komoda et al. (2003) and okamoto et al. (2008) have carried out preliminary studies on replication, transcription and translation mechanisms with tomato mosaic virus (tomv), bmv, tcv, and rcnmv. one question concerning sg rnas that remains to be answered concerns our understanding of how the ratio of genomic to sg rna synthesis is maintained. besides the relative strength of the promoters the concentrations of the various ntps plays a role in determining this ratio (li et al., 2008 (li et al., , 2010 . a related question is whether there is competition between the synthesis of g rna and sg rna. with respect to viruses with multiple sg rnas, why are some rnas made in larger amounts than others? much also remains to be learned about the particular mechanisms of sg rnas synthesis and why certain sg rnas are more robust messengers than other sg rnas or than the g rna. a better understanding of sg rnas and their functions may provide us with new targets for antiviral therapy, specifically concerning important diseases such as sars, caused by a coronavirus. the idea that sg rnas may be a tempting target for antiviral therapy is supported by the identification of plant-derived compounds (picard et al., 2005; that inhibit plant and animal viruses, e.g. in alphaviruses, at nanomolar concentrations. these compounds appear to act by preferential inhibition of synthesis of sg rnas. although the exact mechanism is not known, one possibility is that they bind to sites on the rdrp that recognize the sgps. also, the ability to exogenously regulate sg rnas transcription, e.g., by using a small-molecule ligand and aptamer technology (wang et al., 2008) , offers a promising way to modulate the timing and the levels of sg rna transcription which, in turn, would provide a means to control viral protein expression. this approach has already found use in regulating the function of the higher-order component of the attenuation signal allowing the control oftbsv sg rna transcription (wang and white, 2007; wang et al., 2008) and it offers further promising ways for the analysis and modulation of viral processes at either the level of sg rna transcription or translation. since sgps have been identified as recombination hot spots (suzuki et al., 2003; wierzchoslawski et al., 2004; coyne et al., 2006) , the understanding of the mechanism of sg rna transcription may provide insights into the origin and evolution of viruses. the continual emergence of new recombinant strains, e.g. hcov-nl63 and hcovhku1 isolated in the wake of sars (van der hoek et al., 2004) , and sars-like coronaviruses isolated from animal reservoirs (li, et al., 2005b) , raises the question of the role of sg rnas in the reshuffling of genome sequences of corona-and other viruses. clearly, by incorporating foreign sequences into their genomes, certain viruses acquire new surprising properties (pasternak et al., 2006; moreno et al., 2008a) . for example, the closterovirus sg rna-expressed hsp70h, that facilitates viral assembly and cellular movement, has likely been captured from the host (prokhnevsky et al., 2005) . the coronavirus sg rna-expressed hemagglutinin-esterase (he), acting as a receptor-binding fusion protein, likely originated from orthomyxoviruses (zeng et al., 2008) . viruses that generate sg rnas are increasingly being used as expression vectors. for example, with alphaviruses a foreign gene can be placed under the control of the sgp promoter, replacing the genes encoding the structural protein (rayner et al., 2002; rausalu et al., 2009) . rnas expressed from this promoter are translated in large amounts, producing proteins which can be used in vaccines, or as therapeutics. also, by incorporating an rna sequence encoding an antibody chain that recognizes a ligand on a cell surface, into an sg rna, one can produce proteins targeted to a specific cell type. subgenomic rna promoters dictate the mode of recognition by bromoviral rna-dependent rna polymerases the cis-acting replication signal at the 3′ end of 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virus rna replication rubus chlorotic mottle virus, a new sobemovirus infecting raspberry and bramble synthesis of subgenomic rnas by positive strand rna viruses control of plant virus gene expression and replication by long-distance rna-rna interactions efficient homologous rna recombination and requirement for an open reading frame during replication of equine arteritis virus defective interfering rnas synthesis in vitro of rabbit hemorrhagic disease virus subgenomic rna by internal initiation on (−)-sense genomic rna citrus tristeza virus: a pathogen that changed the course of the citrus industry identification of a coronavirus transcription enhancer partial purification and characterization of cucumber necrosis virus and tomato bushystunt virus rna-dependent rna polymerases: similarities and differences in template usage between tombusvirus and carmovirus rna-dependent rna polymerases the subgenomic rna of feline calicivirus is packaged into viral particles during infection 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subgenomic rna3a derived from rna3 of brome mosaic bromovirus subgenomic messenger rna amplification in coronaviruses uncoupling rna virus replication from transcription via the polymerase: functional and evolutionary insights evidence for a premature termination mechanism of subgenomic mrna transcription in a carmovirus subgenomic mrna transcription in an aureusvirus: downregulation of transcription and evolution of regulatory rna elements rna-based regulation of transcription and translation of aureus virus subgenomic mrna1 involvement of a subgenomic mrna in the generation of a variable population of defective citrus tristeza virus molecules translational control of the subgenomic rnas of severe acute respiratory syndrome coronavirus characterization of heteroclite subgenomic rnas associated with prrsv infection the remarkable variety of plant rna virus genomes structure of coronavirus hemagglutinin-esterase offers insight into corona and influenza virus evolution repression and derepression of minus-strand synthesis in a plus-strand rna virus replicon sequence motifs involved in the regulation of discontinuous coronavirus subgenomic rna synthesis this work was supported by grants from national science foundation (mcb-0920617) and national institutes of health (g1a62203) and by the plant molecular biology center at northern illinois university to jjb, and by grants from the national institutes of health (ai49273) and (ai070728) to vs. key: cord-301755-fxfsr9bj authors: wang, f.-i.; fleming, john o.; lai, michael m.c. title: sequence analysis of the spike protein gene of murine coronavirus variants: study of genetic sites affecting neuropathogenicity date: 1992-02-29 journal: virology doi: 10.1016/0042-6822(92)90041-m sha: doc_id: 301755 cord_uid: fxfsr9bj abstract mouse hepatitis virus (mhv), a coronavirus, causes encephalitis and demyelination in susceptible rodents. previous investigations have shown that the mhv spike (s) protein is a critical determinant of viral tropism and pathogenicity in mice and rats. to understand the molecular basis of mhv neuropathogenesis, we studied the spike protein gene sequences of several neutralization-resistant variants of the jhm strain of mhv, which were selected with monoclonal antibodies (mabs) specific for the s protein. we found that variant 2.2-v-1, which was selected with mab j.2.2 and primarily caused demyelination, had a single point mutation at nucleotide (nt) 3340, as compared to the parental jhm virus, which predominantly caused encephalitis. this site was in the s2 subunit of the s protein. in contrast, variant 7.2-v-1, which was selected with mab j.7.2 and primarily caused encephalitis, had two point mutations at nt 1766 and 1950, which were in the s1 subunit. finally, the double mutant 2.2/7.2-v-2, which was selected with both mabs j.2.2 and j.7.2, and was attenuated with respect to both virulence and the ability to cause demyelination, had a deletion spanning from nt 1523 to 1624 in the s1 and a point mutation at nt 3340 in the s2. we conclude that at least two regions of the s protein contribute to neuropathogenicity of mhv. we have also isolated a partial revertant of 2.2-v-1, which was partially resistant to mab 1.2.2 but retained the same neuropathogenicity as the variant 2.2-v-1. this revenant retained the mutation at nt 3340, but had a second-site mutation at nt 1994, further confirming that nt 3340 contributed to the pathogenic phenotype of mhv. by comparing these results with mhv variants isolated in other laboratories, which had mutations in other sites on the s gene and yet retained the demyelinating ability, we suggest that the ability of jhm viruses to induce demyelination is determined by the interaction of multiple sites on the s gene, rather than the characteristics of a single, unique site. our study also revealed the possible presence of microheterogeneity of s gene sequence, particularly in the s1 region, in these viruses. the sequence microheterogeneity may also contribute to the differences in their biological properties. mouse hepatitis virus (mhv) is a prototypic member of the coronaviridae. the virus is enveloped and contains a single-stranded, positive-sensed, and nonsegmented rna genome of 31 kb in length (lai and stohlman, 1978; pachuk et al., 1989; lee er al., 1991) . the virus particle contains three envelope glycoproteins (s, m, and he) and an internal nucleocapsid protein (n). the s (spike) protein of 180 kda forms peplomers on the virion surface and is responsible for the viral attachment to cellular receptors, the elicitation of neutralizing antibodies, and the induction of cell-to-cell fusion (collins eta/., 1982; sturman eta/., 1985) . alterations in the s protein resulted in changes in viral pathogenicity, as demonstrated by neutralization-escape mutants se-' current address: departments of neurology and medical microbiology and immunology, school of medicine, university of wisconsin at madison, madison, wi 53792. ' to whom correspondence and reprint requests should be addressed. q 1992 academic press, inc. lected with monoclonal antibodies (mabs) specific for the s protein (fleming et a/., 1986 (fleming et a/., , 1987 dalziel et al., 1986; wege et a/., 1988) . furthermore, passive immunization with s-specific monoclonal antibodies also altered viral neuropathogenicity (buchmeier et a/., 1984) . the s protein is cleaved into two 90-kda glycoprotein subunits (sl and s2). its cleavage is carried out by a cellular protease and is probably required for the virusinduced cell fusion (storz et a/., 1981; yoshikura and tajima, 1981; sturman et al., 1985) . the s protein is translated from mrna 3, which represents the transcript of the 4.2-kb gene (s gene), located at roughly 6-10 kb from the 3'-end of the rna genome (lai, 1990) . the complete sequence of the s gene has been reported for three strains of mhv, namely, a59 (luytjes et a/., 1987) jhm (schmidt et a/., 1987) , and mhv-4 (equivalent to jhm used in our laboratory) (parker et a/., 1989 ). it appears that the s gene sequences of different mhv strains differ significantly, with various degrees of deletion and substitutions. infection of the central nervous system (cns) of rodents by mhv, particularly the jhm strain, has been used as a model system for studying human demyelinating diseases, such as multiple sclerosis. parental jhm strain shows tropism for both neurons and glial cells, causing both encephalitis and chronic demyelination (weiner, 1973) . the tropism of mhv is mainly determined by the properties of the s protein, since it is the outermost component of the viral envelope, which interacts with the cellular receptors, and it is also the immunodominant component which interacts with the host immune system (sturman and holmes, 1983) . the role of the other structural proteins in viral pathogenesis is less well understood; passive immunization with mabs specific for n and m (membrane) proteins also changed viral pathogenicity (fleming et a/., 1989; nakanaga et a/., 1986) . previously, we have isolated several neutralizationresistant variants of jhm using mabs specific for the s protein (fleming et a/., 1983) . these variants showed strikingly different capabilities from that of the parental jhm virus in causing demyelination or encephalitis in rodents (fleming eta/., 1986 (fleming eta/., , 1987 . while the parental virus caused encephalitis, the variants selected with one neutralizing mab (j.2.2) had lower virulence and caused demyelination, and the variants selected with two mabs (j.2.2 and 1.7.2) caused neither encephalitis nor demyelination (fleming et al., 1986 (fleming et al., , 1987 . based on these studies, we hypothesized that mab j.2.2 identified a specific encephalitis-determining site and mab j.7.2 identified a specificdemyelination-determining site on the s protein. alternatively, we considered the possibility that a single linear determinant might not be the necessary and sufficient cause of jhm-induced demyelination, and that demyelination would depend on several regions of s or even on other viral genes (fleming et a/., 1987) . our present sequence analysis indicates that the latter hypothesis is correct; that is, the neuropathogenicity of jhm depends on the characteristics of at least two sites on the s gene. complex interaction of different regions of s protein likely influences viral pathogenesis by changing the degree of viral attenuation in viva. the viruses used in this study included several jhm variants which were selected for their resistance to neutralizing mabs specific for the s protein (fleming et a/., 1983) . their virulence and neuropathogenicity in mice are listed in table 1 . the parental virus jhm-dl was a large-plaque isolate of wild-type jhm strain and caused marked encephalitis with chronic demyelination in survivors (stohlman et al., 1982) . the 2.2-v-l note. mab, monoclonal antibody; ld,,, 50% lethal dose in c57/ b6 mice on day 7 after intracerebral inoculation; pfu, plaque forming units; e, predominantly encephalitis; d. predominantly demyelination. (fleming et al., 1986 (fleming et al., , 1987 . variant was selected from jhm-dlwith mab j.2.2, and caused marked demyelination with little encephalitis (fleming et al., 1986) . the 2.2/7.2-v-2 variant was selected from 2.2-v-l with another mab j.7.2 and was nonpathogenic, causing only minimal neuropathology (fleming eta/., 1987) . the 7.2-v-l variant was selected directly from jhm-dl with j.7.2 and remained encephalitic (fleming et a/., 1986 ). an astrocytoma cell line (dbt cell) derived from mice (hirano et a/., 1974) was used throughout the experiment. all of these viruses were initially plaque-purified three times before propagation as virus stock. early passage viruses (before passage 3) were used for preparing large amounts of virus for biochemical studies. the viruses of the same passage level were previously used for pathogenicity studies (fleming et a/., 1986 (fleming et a/., , 1987 . variant viruses were grown in the presence of the respective mab used for their selection. viruses were mixed with an equal amount of mab tissue culture supernatants and incubated for 45 to 60 min at 37" before inoculating onto confluent dbt cell monolayer. after virus adsorption for 1 hr at 37", dulbecco's minimal essential medium containing 1% fetal calf serum was added. the supernatants from infected cell cultures were harvested when the cytopathic effects reached 90-l 00%. the harvested viruses were examined for their resistance to the selecting mabs to ensure that the viruses used for biochemical studies maintained the original phenotype. the jhm-dl virus was grown in the same way except that no antibodies were used. viruses were purified according to a procedure modified from makino et a/. (1983) . briefly, the supernatants were centrifuged at 8000 rpm for 30 min to clear cell debris and were precipitated with 50% saturated am-monium sulfate for at least 1.5 hr at 4", followed by centrifugation at 8000 rpm for 20 min. the pellets were suspended in nte (0.1 m naci, 0.01 mtris-hci, ph 7.4, and 1 mm edta), and the suspensions were centrifuged at 2000 rpm for 10 min to remove undissolved debris. the supernatants were then subjected to two rounds of centrifugation in discontinuous sucrose gradient consisting of 20, 30, 50, and 60% sucrose in nte at 26,000 rpm for 3.5 hr at 4" in a beckman sw28 rotor. viruses at the 30-500/o interface were harvested and diluted with nte and pelleted by centrifugation at 40,000 rpm for 90 min in a beckman sw41 rotor. the virus pellets were resuspended in nte. isolation of partial revertants of neutralizationescape variant 2.2-v-l variant 2.2-v-l was passaged in dbt cells for 10 times in the absence of any antibodies. these viruses were plaque-assayed, and plaques were randomly picked and further purified by three rounds of plaque purification. several plaques were examined for their sensitivity to mab j.2.2. large pools of revertants were prepared and purified following the same procedure as described above. these plaque-purified revertants were designated as vl 0. cdna cloning for viral rna extraction, the virus suspension in nte was mixed with an equal volume of proteinase k buffer (2% sds, 0.025 m edta, 0.2 m tris-hci, ph 7.5, and 0.3 m naci) and proteinase k (200 pg/mi), and incubated for 45 min at 37". rna was extracted twice with phenol/chloroform (1 :l) and precipitated with ethanol. the pelleted rna was resuspended in distilled h,o, and stored at -70". for cdna synthesis, viral rna was first denatured with 10 mm methylmercury at room temperature for 10 min and then incubated at 42" for 45 to 60 min in 50 ~1 pm of dntps, l-3 units of taq polymerase (cetus), and 0.1-l pm of synthetic oligonucleotide primers (first and second primers). the reaction mixture was overlaid with 60 ~1 of mineral oil and subjected to 20-40 cycles of amplification, each cycle consisting of 94" for 1 min, 37-42" for l-2 min, and 72" for 2-3 min. these pcr conditions were adjusted slightly for different reactions, depending on the size of target sequence to be amplified and the pair of primers used. the pcr product was resolved on a 1 oh agarose gel in 1x tbe buffer (0.089 mltris-hci, ph 8.3, 0.089 m boric acid, and 2 mm edta) and stained with ethidium bromide. the specific product band was cut out, and the dna was extracted from the gel with phenol and chloroform, precipitated with ethanol, and the pellet suspended in distilled h,o. the pcr product was treated with 10 units of polynucleotide kinase (boehringer-manheim) in buffer containing 0.07 m tris-hci, ph 8.0, 0.01 m mgci,, 5 mm dlt, 0.1 mm spermidine, and 1 mm atp and incubated at 42" for 45 min. after phenol/chloroform extraction and ethanol precipitation, the pcr product was blunt-ended with 1 unit of t4 dna polymerase (boehringer-manheim) at 37" for 30 min in buffer containing 0.05 mtris-hci, ph 7.5, 7 mm mgci,, 1 mm dlt, 0.25 mm of dntps, followed by a booster reaction with an additional 0.5 unit of t4 dna polymerase and 0.05 mm of dntps at 37" for another 30 min. after phenol/chloroform extraction, the pcr product was ligated using 1 unit of t4 dna ligase (boehringer-manheim) into the smal site of plasmid ptz18u (u.s. biochemicals). the ligation buffer contained 0.05 m tris-hci, ph 7.5, 0.01 m mgci,, 2 mm dtt, and 1 mm atp. the ligation mixture was used to transform competent cell dh5a (brl) following the manufacturer's direction. dna sequencing dna sequencing was performed using doublestranded dnaof plasmid ptz18u. dideoxychain termination sequencing was performed following the procedure outlined in the sequenase version 2.0 dna sequencing kit from u.s. biochemical. the reaction product was analyzed in 69/o polyacrylamide gel containing 7 m urea. the whole s gene, approximately 4.2 kb in length, was cloned into 2 to 3 overlapping fragments after amplification by pcr using appropriate synthetic primers. since we were dealing with several viral isolates with presumably only minor differences in s gene, crosscontamination was a major concern. to avoid this problem, whenever possible, different pairs of primers were used in the cloning of different viral rnas so that distinguishable gene fragments were obtained for each viral isolate. in cases where alternative primers were not available, new aliquots of reagents were used for each isolate. another concern was that the enzymes used, namely, reverse transcriptase, taq polymerase, and sequenase may generate errors. to avoid this problem, clones from different cdna synthesis events, different pcr events, and multiple clones were sequenced. for each isolate, the full-length sequence was initially obtained from two different dna clones. differences in sequence identified by this initial sequencing were further studied by sequencing more clones. only consistent changes were considered as bona fide mutations. some of the sequence variations were confirmed by direct rna sequencing using purified virion rna (banner et a/., 1990) . the complete sequences of s gene of the parental jhm-dl and their neutralization-escape variants are summarized schematically in fig. 1 . the s gene of jhm-dl is nearly identical to that of mhv-4 (parker et a/., 1989) with only 6 nucleotide differences (3 amino acid differences) ( table 2 ). it has a coding capacity of 1376 amino acids. identical to those in other mhvs (schmidt et al., 1987; luytjes et al., 1987) , the predicted proteolytic cleavage site is between amino acid 769 (arg) and 770 (ser). the s gene of 2.2-v-1, which was derived from jhm-dl by selection with mab j.2.2, appeared to consist of two populations. all of the clones contained a nucleotide difference between jhm-dl and 2.2-v-1, which was located at nucleotide (nt) 3340, changing the codon from ctc to tx (leu to phe). besides this mutation, a minority of clones contained four additional mutations at the n-terminal note. mhv-4 sequence was from parker et al. (1989) . note. sequence variations in 2.2-v-l were found consistently in two cdna clones, which were derived from different reverse transcription events and polymerase chain reactions. in contrast, sequence variations in jhm-dl were found randomly in different clones. frequency indicates the number of clones showing the alternative nucleotide out of the total number of clones that were sequenced. end of the s gene (table 3 ). these were located at nt 731, 901, 950, and 1439, changing the codon from ltt to tct (phe to ser), from ccg to tcg (pro to ser), from gga to gaa (gly to glu), and from ccg to ctg (pro to leu), respectively. these mutations were not likely to be due to enzyme errors, because these mutations were observed on two clones derived from different reverse transcription and pcr events. sequencing of multiple jhm-dl-derived clones also showed heterogeneity at several different nucleotide positions (table 3) , which were different from the sequence variations observed in 2.2-v-l. since the only consistent difference between jhm-dl and 2.2-v-l was nt 3340, this mutation was most likely responsible for the resistance of 2.2-v-l to mab j.2.2. however, the contribution of sequence microheterogeneity (table 3) to the phenotype of viruses could not be ruled out. it should be noted that most of the minor sequence variations were in the n-terminal half, which was the more divergent part of the s gene (de groot et a/., 1987). the variant 2.2/7.2-v-2 was derived from 2.2-v-l by selection with mab j.7.2 (fleming et a/., 1987) . sequence analysis showed that 2.2/7.2-v-2 had a 102nucleotide deletion at the n-terminal half between nt 1523 and 1624, corresponding to a 34-amino acid deletion. accompanying the deletion, the two amino acids arginine and tyrosine at the junction of deletion were converted into histidine. as expected, nt 3340 in this variant remained as ttc, similar to that in 2.2-v-l. also, similar to 2.2-v-1, a small number of clones contained several additional point mutations at the up-stream and downstream regions of the deletion (data not shown). these sequence variations were different from those observed in the variant 2.2-v-l. since the 102-nucleotide deletion was the only consistent change observed in all clones of 2.2/7.2-v-2, it was most likely responsible for the phenotype differences. to confirm that the deletion in the n-terminal half of s was responsible for the viral resistance to mab j.7.2, we sequenced the s gene of 7.2-v-1, which was derived directly from jhm-dl by selection with mab j.7.2 (fleming et a/., 1986) . to our surprise, 7.2-v-l did not have a deletion. instead, there were two point mutations consistently detected downstream of the 2.2/ 7.2-v-2 deletion site, one at nt 1766, changing the codon from tca to tta (ser to leu) and the other one at nt 1950, changing the codon from aag to aat (lys to asn). all of these changes were localized around the hypetvariable region (de groot et al., 1987; parker et a/., 1989; banner et a/., 1990) of the s protein. these results suggested that the mutations, including deletions and point mutations, in this region were most likely responsible for the viral resistance to mab j.7.2. nt 3340 of 7.2-v-l remained as ctc, similar to that in jhm-dl, consistent with the fact that 7.2-v-l was directly derived from jhm-dl. to determine whether the mutation at nt 3340 or the microheterogeneity in sl was responsible for the viral resistance to mab j.2.2 and for the pathogenicity of the virus, we attempted to isolate revertants of 2.2-v-l. variant 2.2-v-l was passaged in dbt cells in the absence of any mab, and viruses were randomly selected from plaques. since the parental jhm-dl grew better than the neutralization-resistant variants (unpublished observations), we reasoned that the revertants would become a predominant population during serial viral passages in the absence of selection pressure. indeed, most of the plaque-purified viruses isolated this way were partially sensitive to mab j.2.2 (data not shown). their titers were reduced by mab j.2.2 by approximately 2.7 log,,,. however, none of the viruses examined (n = 7) completely reverted to the level seen in parental jhm-dl, whose titer was reduced by mab j.2.2 by approximately 4 loglo (fleming et al., 1986 (fleming et al., , 1987 . sequence analysis of multiple clones from all seven partial revertants showed that all of them have retained the sequence of 2.2-v-l at nt 3340, which was the only consistent difference observed between jhm-dl and 2.2-v-l. one of these revertants was chosen for complete sequencing; this virus had a consistent change at a second site at nt 1994, changing the codon from aat (asn) to agt (ser). since none of the revertants completely restored the phenotype to that of the parental jhm-dl, the most logical explanation was that different regions of the s protein may cooperate to contribute to the resistance of the virus to neutralization by mab j.2.2. the finding that nt 3340 mutation was retained in all of the partial revet-tants suggested that this site had a major contribution to the resistance to mab j.2.2. preliminary viral pathogenicity study by intracerebral inoculation in mice revealed that vlo had an ld,, of approximately 1 o3 pfu, similar to that of 2.2-v-1, but in contrast to jhm-dl, which had an ld,, of 4 pfu (table 1) (fleming et a/., 1986) . similar to 2.2-v-1, the partially revertant virus primarily caused demyelination with little encephalitis. these results suggested that the second site mutation in the sl region did not alter the pathogenicity of the virus and that the mutation at nt 3340 was most responsible for the neuropathogenicity of the jhm viruses. our sequence analysis of mab-escape mutants of jhm indicated that a mutant selected with mab j.2.2 had a point mutation in the s2 region of the spike protein. by contrast, when mab j.7.2 was used to select mutants, point mutations or a deletion in sl within nt 1500-l 950 were observed. previous attempts at mapping the binding sites of these mabs have failed (weismiller eta/., 1990). our study suggested that mab j.7.2 would bind to sl while mab j.2.2 would bind to s2. these findings are consistent with a recent study measuring the direct binding of the mhv-specific mab, which also indicated that j.2.2 bound to s2 (p. talbot, personal communication). the binding site of j.2.2 is close to other 52 neutralizing epitopes identified in other laboratories (luytjes et al., 1989; routledge et al., 1991) . interestingly, the mutation at nt 3340, which changed the coded amino acid from leu to phe affected one of the three amino acids which are critical for the fusion function of the s protein (gallagher et al., 1991) . however, the variant 2.2-v-l induced fusion when grown on dbt cells (data not shown); this was expected since the loss of the fusion activity required the simultaneous mutations of all the three amino acids (gallagher er al., 199 1) . nevertheless, these findings show that this amino acid plays an important role in both fusion activity and neuropathogenicity of mhv. the region of sl with deletion or point mutations in 2.2/7.2-v-2 or 7.2-v-l mutants corresponds to a previously identified hypervariable site on sl (parker et a/., 1989; banner et a/., 1990) . this site underwent frequent deletions or point mutations upon viral passages in tissue culture or animals (dalziel eta/., 1986; banner et a/., 1990; gallagher et a/., 1990) . although the precise sites of deletion or point mutations were different among these variants, they were in the same general area, i.e., near the c-terminus of sl previously, buchmeier's laboratory also has reported different types of mutation among the neutralization-escape mhv-4 variants, which were derived by use of a monoclonal antibody different from the one used in our study (parker er a/., 1989; gallagher et al., 1990) . all of these variants have either deletion or point mutations in this area of sl these results together suggested that this region represented an immunodominant and highly variable site, although the binding site of mab j.7.2 has not been directly determined. taken together with previous studies, our analysis indicates that at least two sites on s play critical roles in mhv neuropathogenesis: (1) the hypervariable sl region within nt 1500-l 950 and (2) a second region on s2 around nt 3340. preservation of the parental (wildtype) sequences at both of these regions appears to be required for the ability of virus to cause fatal encephalitis, since mutations at either site reduced neurovirulence. the mutation in s2 (2.2-v-l) converted the parental jhm virus, which was highly encephalitic, into a highly demyelinating virus, and an additional mutation (deletion) in sl (2.2/7.2-v-2) led to an essentially non-demyelinating virus (fleming et al., 1987) (table 1) . this result was consistent with the interpretation that the site on sl was tightly associated with demyelination, while the site on s2 was responsible for encephalitis. however, this interpretation is unlikely, since other neutralization-resistant mutants with deletions in the same domain of sl still caused demyelination (dalziel et al., 1986; gallagher et a/., 1990) . thus, we hypothesize that the virtual inability of 2.217.2-v-2 to cause demyelination or encephalitis (fleming et a/., 1987) was due to viral attenuation that occurred when both the sl and s2 determinants were lost or mutated. such an interpretation is consistent with the finding that the mutation in s2 of the variant 2.2-v-l and the mutation in sl of other mutants (dalziel et al., 1986) all led to a similar demyelinating phenotype. this is also consistent with our current understanding of the biological activities of the s protein. the s protein forms peplomers which project from the surface of the mhv virion, and is thus in an ideal position to influence pathogenesis by altering viral attachment to cellular receptors, by interaction with the immune system, by fusion of cellular membranes (collins et a/., 1982; sturman et a/., 1985) and perhaps by other mechanisms, such as influencing viral stability, uncoating, or spread. after intracerebral inoculation, parental jhm-dl rapidly gains entry to glia and neurons throughout the cns and thus is able to cause fatal panencephalitis (weiner, 1973; knobler et a/., 1981) . by contrast, mab-selected jhm mutants (2.2-v-1 and 2.2/7.2-v-2) are less able to spread to neurons, and virulence is thus drastically reduced (fleming et a/,, 1986) . however, these viruses are able to infect glial cells of the white matter of the brain and spinal cord. presumably the requirements of viral entry or replication in glial cells are less stringent than those for neuronal infection. this differential entry may reflect the presence of different receptors utilized by wild-type virus versus selected variants. in addition, the susceptibility of different neural cells to jhm is known to be affected by the developmental stages of different cell species and host factors (pasick and dales, 1991) . in the case of infection by jhm mutants, the immune response clears virus from the white matter by 12 days postinoculation but has the deleterious consequence of inducing myelin loss (wang et al., 1990) . thus, the apparent increase in ability to cause demyelination shown by these and similar variants (dalziel et a/., 1986; wege et a/., 1988) is probably due to the unmasking of potentialities of the parental virus which are usually hidden by its virulence. the ability of mutations at different sites to affect the same biological properties of the s protein was further demonstrated by the sequence analysis of a partial revertant of 2.2-v-l. the second site mutation which partially restored the mab sensitivity of this virus was localized on sl, in contrast to the original mutation on s2. this study suggested that two sterically separated sites (one in the globular portion, and the other in the heptad repeat region of the stalk portion according to the computer modeling [de groot et al., 19871) on the two different s subunits could interact with each other. this effect has also been illustrated by the antibody sensitivity of 2.2/7.2-v-2 and 2.2-v-l. the former was roughly one loglo more resistant than the latter to mab j.2.2 (fleming eta/., 1987) , suggesting that the second mutation further altered the conformation at the first site (in s2). interestingly, the partial revertant of 2.2-v-l retained the same pathogenic properties of the parent variant 2.2-v-l. furthermore, 7.2-v-l variant retained the ability to cause encephalitis (table 1) . these results suggest that the antibody binding and pathogenic properties of the s protein can be dissociated. our study also suggested the possible presence of sequence microheterogeneity in the mhvs (table 3 and data not shown). all of these viruses were initially plaque-purified but had been amplified by limited passages to obtain sufficient virus titer before molecular cloning and sequencing. some of these sequence variations were likely the results of pcr and cloning errors. however, several nucleotide variations were consis-tently noted in repeated pcr products. furthermore, the sequence microheterogeneity was located mostly in the sl hypervariable region (banner et a/., 1990; de groot et a/., 1987) , suggesting that the observed sequence variations represented variations in the rna. since the plaque-purified viruses need to be propagated for either biochemical or pathogenicity studies, this sequence microheterogeneity is likely an inherent property of any virus population. the possible contribution of the sequence microheterogeneity to viral neuropathogenicity is not clear at the present time. in conclusion, our studies suggest that at least two regions of the s gene play critical roles in jhm neuropathogenesis. these regions are near nt 1500-l 950 (sl segment) and nt 3340 (s2 segment). antigenic studies indicate that these sites may be closely linked on the s protein and may influence each other by spatial proximity or steric effects (fleming et al., 1986) . viruses with the parental jhm sequences may infect all cell types and regions of the cns, resulting in fatal panencephalitis. when either site is altered, viral infection is limited to glial cells of the white matter, and subsequent immune attack (wang et al., 1990) or virus-induced cytolysis will lead to demyelination. when both sites are affected, the virus shows very limited distribution and presumably is contained by the immune system at an early stage of pathogenesis. it is probable that other s domains may also contribute to the viral pathogenicity. in addition, although the s gene is considered to be the most important in influencing pathogenicity, other viral genes such as hemagglutinin-esterase (he) (la monica et a/., 1991, yokomori et al., 1991) and those localized to the 3' portion of the genome (lavi et a/., 1990) may also, to some extent, contribute to viral pathogenicity. a clustering of rna recombination sites adjacent to a hypervariable region of the peplomer gene of murine coronavirus murine hepatitis virus-4 (strain jhm) induced neurologic disease is modulated in viva by monoclonal antibody. i/iro/ogy monoclonal antibodies to murine hepatitis virus-4 (strain jhm) define the viral glycoprotein responsible for attachment and cell-cell fusion site-specific alteration of murine hepatitis virus type 4 peplomer glycoprotein e2 results in reduced neurovirulence evidence for a coiled-coil structure in the spike proteins of coronaviruses. 1 monoclonal antibodies to the matrix (el) glycoprotein of mouse hepatitis virus protect mice from encephalitis antigenic relationships of murine coronaviruses: analysis using monoclonal antibodies to jhm (mhv-4) virus pathogenicity of antigenic variants of murine coronavirus jhm selected with monoclonal antibodies experimental demyelination induced by coronavirus jhm (mhv-4): molecular identification of a viral determinant of paralytic disease alteration of the ph dependence of coronavirus-induced cell fusion: effect of mutations in the spike glycoprotein neutralization-resistant variants of a neurotropic coronavirus are generated by deletions within the amino-terminal half of the spike glycoprotein. 1. viral replication and plaque formation of mouse hepatitis virus (mhv-2) in mouse cell line dbt culture mouse hepatitis virus type 4 (jhm strain)-induced fatal central nervous system disease. i. genetic control and the murine neuron as the susceptible site of disease coronavirus: organization, replication and expression of genome rna of mouse hepatitis virus localization of extensive deletions in the structural genes of two neurotropic variants of murine coronavirus jhm determinants of coronavirus mhv pathogenesis are localized to 3' portions of the genome as determined by ribonucleic acid-ribonucleic acid recombination the complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and rna polymerase amino acid sequence of a conserved neutralizing epitope of murine coronavirus primary structure of the glycoprotein e2 of coronavirus mhv-a59 and identification of the trypsin cleavage site characterization of small plaque mutants of mouse hepatitis virus protective effect of monoclonal antibodies on lethal mouse hepatitis virus infection in mice molecular cloning of the gene encoding the putative polymerase of mouse hepatitis coronavirus strain a59 sequence analysis reveals extensive polymorphism and evidence of deletions within the e2 glycoprotein gene of several strains of murine hepatitis virus infection by coronavirus jhm of rat neurons and oligodendrocyte-type-2-astrocyte lineage cells during distinct developmental stages. i. viral analysis of murine coronavirus surface glycoprotein functions by using monoclonal antibodies primer-directed enzymatic amplification of dna with a thermostable dna polymerase nucleotide sequence of the gene encoding the surface projection glycoprotein of coronavirus mhv-jhm murine coronaviruses: isolation and characterization of two plaque morphology variants of the jhm neurotropit strain enhancement of plaque formation and cell fusion of an enteropathogenic coronavirus by trypsin treatment the molecular biology of coronaviruses proteolytic cleavage of the e2 glycoprotein of murine coronavirus: activation of cell fusing activity of virions by trypsin and separation of two different 90k cleavage fragments demyelination induced by murine hepatitis virus jhm strain (mhv-4) is immunologically mediated the peplomer protein e2 of coronavirus jhm as a determinant of neurovirulence: definition of critical epitopes by variant analysis pathogenesis of demyelination induced by a mouse hepatitis virus (jhm virus). acfa neural monoclonal antibodies to the peplomer glycoprotein of coronavlrus mouse hepatitis virus identify two subunits and detect a conformational change in the subunit released under mild alkaline conditions heterogeneity of gene expression of the hemagglutinin-esterase (he) protein of murine coronaviruses role of protease in mouse hepatitis virus-induced cell fusion we thank drs. y-c. chao and c-z. lee for technical advice. we also thank daphne shimoda for the manuscript preparation. this work was supported by u.s. public health services grants ns1816 from the national institutes of health and rg 2283-a-2 of the national multiple sclerosis society. f.i.w. is a recipient of postdoctoral training fellowship from nih. m.m.c.l. is an investigator of howard hughes medical institute. key: cord-299122-djfj4262 authors: yu, hua; jiang, li-fang; fang, dan-yun; yan, hui-jun; zhou, jing-jiao; zhou, jun-mei; liang, yu; gao, yang; zhao, wei; long, bei-guo title: selection of sars-coronavirus-specific b cell epitopes by phage peptide library screening and evaluation of the immunological effect of epitope-based peptides on mice() date: 2007-03-15 journal: virology doi: 10.1016/j.virol.2006.09.016 sha: doc_id: 299122 cord_uid: djfj4262 antibodies to sars-coronavirus (sars-cov)-specific b cell epitopes might recognize the pathogen and interrupt its adherence to and penetration of host cells. hence, these epitopes could be useful for diagnosis and as vaccine constituents. using the phage-displayed peptide library screening method and purified fab fragments of immunoglobulin g (igg fab) from normal human sera and convalescent sera from sars-cov-infected patients as targets, 11 b cell epitopes of sars-cov spike glycoprotein (s protein) and membrane protein (m protein) were screened. after a bioinformatics tool was used to analyze these epitopes, four epitope-based s protein dodecapeptides corresponding to the predominant epitopes were chosen for synthesis. their antigenic specificities and immunogenicities were studied in vitro and in vivo. flow cytometry and elispot analysis of lymphocytes as well as a serologic analysis of antibody showed that these peptides could trigger a rapid, highly effective, and relatively safe immune response in balb/c mice. these findings might aid development of sars diagnostics and vaccines. moreover, the role of s and m proteins as important surface antigens is confirmed. selection of sars-coronavirus-specific b cell epitopes by phage peptide library screening and evaluation of the immunological effect of epitope-based peptides on mice ☆ between 2002 and 2003, severe acute respiratory syndrome (sars), a new virus infectious disease with high fatality rate, spread to 32 countries. a novel coronavirus designated "sars-coronavirus (sars-cov)" was responsible for the serious infectious disease (drosten et al., 2003; fouchier et al., 2003; ksiazek et al., 2003; peiris et al., 2003; poutanen et al., 2003) . although the global outbreak of sars was controlled and the virus had been isolated from wild animals (layan palm civets and raccoon dogs), the actual natural reservoir for sars-cov is still unknown and its reemergence in the future remains a possibility. to better prevent future sars epidemics, protective vaccines and reliable diagnostic reagents must be developed to control this life-threatening disease. b cell epitopes are defined as regions on the surface of the native antigen that are recognized and bind to b cell receptors or specific antibodies. these epitopes are the focus of immunological research as well as the targets of development of vaccine and diagnostic reagent (vanniasinkam et al., 2001; viudes et al., 2001) . therefore, in the present study, we screened and identified specific b cell epitopes of sars-cov using phagedisplayed peptide library, fab fragments from anti-sars-cov immunoglobulin g (igg) and normal human igg as targets, and an improved biopanning procedure. the immune responses induced by the four epitope-based peptides were also evaluated with animal experiments. the product of serum treatment, anti-sars-cov igg fab (45 kda) was shown by elisa and western blotting to bind specifically to sars-cov antigen. the immunoscreening with the two igg fab targets enriched the phage population binding to anti-sars-cov igg fab. of 60 selected phage clones, 43 (72%) immunopositive clones had significant enhancement of binding activity to sars-cov-specific antibodies but not to normal human serum (fig. 1) . the homology between peptide sequences of a series of epitopes inserted into target phages and the primary sequence of the structural protein of sars-cov was compared ( table 1 ). as seen in table 1 , the double selection process resulted in 12 different peptides, which could be divided into three groups: (1) nine peptides with homology to nine different regions of the sars-cov s protein; (2) two peptides with homology to two regions of the m protein; and (3) one peptide without any homology to the sars-cov structural proteins. some peptides showed a striking resemblance (as its peptide sequence shvplatsrtla contained six matches to the m protein sequence) and were isolated more than once (shvplatsrtla was sequenced five times, group 2 in table 1 ). the majority of epitopes were isolated after both screens. after general evaluation of these epitopes with bioinformation software, four predominant epitopes of sars-cov s protein were selected and the corresponding epitope-based peptides (map1, 91 ateksnvvrgwv 102 ; map2, 424 ntrnidatstgn 435 ; map3: 458 pfspdgkpctpp 469 ; and map4, 1065 hegkayfpregv 1076 [according to aat74874]) were synthesized. to prove that the phage-borne peptides were the real epitopes of sars-cov, a peptide competitiveinhibition assay was performed to determine whether the synthetic peptide and the corresponding phage clone competed for the same antibody-binding site. in competitive-inhibition assay, the peptides dose-dependently blocked the binding of the corresponding phage clones to the targets (fig. 2) . moreover, the four peptides reacted with sars-cov-specific serum antibody and did not cross-react with anti-hcov-229e and hcov-oc43 sera or healthy donor sera (fig. 3 ). the antibody responses elicited by the epitope-based peptides were determined using elisa. titers of the antisera from challenged mice are shown in fig. 4 . every epitopebased peptide stimulated antibody production, even after just primary immunization. a second immunization substantially increased antibody titer within a week. the specific antibody was still detectable on day 42 with high titer suggesting that a strong and relatively long-term humoral immune response was induced by each of the four b cell epitopes. these anti-peptide antibodies reacted with sars-cov antigen specifically but failed to react with hcov-229e and hcov-oc43 antigens (fig. 5) . the effects of these peptides on splenic lymphocyte subsets isolated from challenged mice were analyzed using two-color flow cytometry. on day 14 or day 21 after primary immunization, the relative numbers of cd3 − cd19 + b cells increased significantly and that of cd3 + t cells decreased relative to control group levels (table 2) . although both the number of cd4 + t cells and cd8 + t cells decreased simultaneously, the decrease was greater in the latter than the former. consequently, fig. 1 . identification of specific phage clones reactive with anti-sars-cov serum by elisa. 1-30 represent the elisa results reacted between different phage clones selected by immunoscreen and patient serum mixture/normal human serum mixture. 31 represent the results between wild type phages as negative control and serum samples above. after four rounds of biopanning twice, 43 phage clones from 60 selected phage clones were significantly reactive with anti-sars-cov serum, but not with normal human serum. a and b represent respectively the immune reactivity of phage clones picked at random from the first and the second time that phage cloning was carried out. the negative control reacted with neither patient serum mixture nor normal human serum mixture. the ratio of cd4 + t cell/cd8 + t cell was a little higher. however, the increase in cd4 + t cell/cd8 + t cell ratio was similar in the control group and all map-immunized groups, except the map2-immunized group on day 14. it suggested that these epitope-based peptides did not imbalance the immunological system. subsequently, with the activation of t cells, the t cell/b cell ratio progressively returned to a normal level in all groups except the map2-immunized group at a low level. the results demonstrated that humoral immune response was predominant and was rapidly triggered by the epitope-based peptide, especially by map2. since the expression of surface molecules, cd44 and lselectin, on cd4 + t cells was used to distinguish memory cells ( high cd44, and low l-selectin), we monitored these phenotype changes using three-color flow cytometric analysis. the cd4 + t lymphocytes of epitope-based peptide-immunized mice (day 0, just before the third immunization) showed the classical immunological memory phenotype ( high cd44, and low lselectin) (fig. 6 ). with the re-injection of the specific peptide, cd62l was continuously up-regulated (but its level was always lower than that of the pbs control). slight down-regulation of cd44 on cd4 + t cells stimulated by map1 and map4 on day 1 was observed; and then the level of cd44 returned to that on day 0, but subsequently fluctuated and remained higher than that of the pbs control. cd44 was continuously up-regulated on cd4 + t cells from map2 and map3 challenged mice. the frequency of il-4-or ifn-γ-producing cells at the single cell level was determined by elispot assay. as shown in fig. 7 , 7 days after primary immunization with map2 and map4, the number of il-4-producing cells from spleens increased significantly (36-88 spots in 2 × 10 5 cells). fourteen days after primary immunization with map1 and map3, the number of il-4-producing cells remained elevated (31-43 spots in 2 × 10 5 cells). moreover, the number of il-4-producing cells in mice boosted with any of the peptides increased 2-to 3-fold, whereas the number of ifn-γ-producing cells remained at low levels (10-20 spots in 2 × 10 5 cells). these data demonstrate that the four b cell epitopes were more effective enhancers of humoral immunity than cellular immunity. the results of antigenspecific lymphocyte cell proliferation assay indicated that each map dose-dependently stimulated a proliferative response and map3 was the most effective inducer of lymphocyte proliferation (table 3) . splenic lymphocytes from mice on day 42 still exhibited significant proliferative responses to specific antigen, demonstrating that the four epitope-based peptides induced long-term immune responses (data not shown). phage-displayed peptide libraries have a wide variety of uses (cortese et al., 1995; petersen et al., 1995; pereboeva et al., note. each screen consisted of four rounds of panning with affinity-purified target antibodies. the numbers on either side of the sequence denote amino acid sequence numbers. letters in bold denote matches of phage sequences with the sars-cov sequence and italicized bold letters denote conservative substitutions. the numbers on the right indicate the number of times the corresponding peptide was isolated and the numbers in parenthesis refer to the screen at which the peptide was isolated, i.e., the first screen, the second screen or both. a the corresponding phage clone was chosen for competitive-inhibition assay. 1998; wu et al., 2001) . this technique has been used mostly to screen with monoclonal antibodies, and little to screen with purified polyclonal antibody preparations (folgori et al., 1994; kay et al., 1993; roberts et al., 1993; liu et al., 2004) . we were successful in screening with polyclonal antisera using this technique, probably because of the newly improved biopanning we adopted and the targets we chose. anti-sars-cov serum contains mostly sars-cov antibodies and few antibodies to other pathogens. in conventional biopanning processes with anti-sars-cov sera as the only targets, nonspecific phage clones that do not bind to sars-cov-specific immunoglobulins but instead to antibodies specific to other pathogens might be amplified. in addition, some researchers reported that nonspecific clones not binding to the target but to the plastic of plate could also be chosen in several conventional biopanning procedures (adey et al., 1995) . we improved the biopanning procedure to avoid these possibilities and ensure specificity of selection. anti-sars-cov igg fab (as the simplest fragments with specificity of antibodies) facilitated the antigen specificity of our biopanning procedure. by repeating the four-round biopanning, we were able to select eleven sars-cov epitopes. they formed a subset of the linear epitopes. moreover, we found another peptide sequence displayed by an immunopositive phage clone without homology to any primary sequence of sars-cov proteins. the structure of epitopes recognized and bound by b cell receptor (bcr) was shown to be linear or spatial by the classical experiments of michael sela 30 years ago (sela, 1969) . phagedisplayed peptide libraries are generated by shotgun cloning of random oligonucleotides into the 5′ ends of either the piii or pviii genes of filamentous phage. the peptides encoded by inserted nucleotides are displayed on the phage surface and have independent spatial structure (cwirla et al., 1990; luzzago et al., 1993) . so the non-homologous peptide sequence might reflect the presence of a discontinuous epitope of sars-cov. in addition, sars-cov is antigenically crossreactive with other viruses, so the non-homologous peptide sequence might be also an epitope of a different virus. certainly, this finding must be studied further. among the linear epitopes, nine (81%) were considered to be s protein epitopes (five epitopes in s1 protein and four in s2 protein) and two (18%), m protein epitopes. the decrement tendency of numbers of epitopes of s1 protein, s2 protein, and m protein connected with the location of these proteins in the virion. most (60%) of the epitopes were located in regions where predicted epitopes were known to exist, and a minority (24%) of epitopes were located about 6−13 amino acids away from the predicted epitope regions (yuxian et al., 2005; yanbo et al., 2003 yanbo et al., , 2004 . and two were located in regions where the virus binds to the host cell receptor (ace-ii) . s protein is responsible for inducing host immune responses and virus neutralization by antibodies (holmes, 2003; navas-martin and weiss, 2003) , and neutralizing antibodies can protect mice or primates from sars-cov infection (bisht et al., 2004; buchholz et al., 2004; bukreyev et al., 2004; gao et al., 2003; yang et al., 2004) . our research provided an indirect proof that the s protein of sars-cov is an important surface antigen stimulus of the host immune response. the m proteins of other coronaviruses are immunogenic (enjuanes et al., 1995) .the virion structure of sars-cov, b cell epitope characteristics, and our b cell epitope results suggest that m protein of sars-cov also plays an important role in inducing antibody production. we sought to characterize the antigenicity and immunogenicity of the sars-cov epitope-based peptides to facilitate the development of effective sars diagnostics and vaccines. recent studies have demonstrated that sars-cov and other coronaviruses are antigenically cross-reactive (ksiazek et al., 2003; woo et al., 2004) . the four s protein epitope-based peptides were proven sars-cov specificity, and antibodies from mice induced by them also reacted specifically with sars-cov. though further study of their specificity and sensitivity is needed, these epitopes should be considered potential immunoreactive epitopes for diagnostic reagents. the immunological effect of epitope-based peptides was studied. the lymphocyte proliferation assay showed the four epitope-based peptides stimulated the immune system. secretion of il-4 (a b cell growth and differentiation factor) might reflect the state of activation and proliferation of b cells and transition to antibody-producing plasma cells. the increase was dramatically greater in the number of il-4-producing cells than in the number of ifn-γ-producing cells after immunization with these epitope-based peptides, confirming that humoral immunity was predominant. so, the epitopes we selected are real b cell epitopes. the specific antibody induced by the four epitopes persisted at least 6 weeks, which was confirmed by monitoring igg titer. although specific igg could be detected after primary immunization, persistent high titer igg required revaccination. it indicated the involvement of humoral immune memory. within 1 week after revaccination, b cell numbers peaked, which indirectly proved that antigen-specific memory b cells could be induced by these epitope peptides. it suggested that lymphocytes were from the mice immunized with the four different peptides on 14 days, 21 days, and 35 days after primary immunization (n = 5). results are means ± sd. a p value < 0.01 compared with the control group. b p value < 0.05 compared with the control group. fig. 6 . three-color flow cytometric analysis of surface markers (cd44 and cd62l) expression on cd4 + t cells isolated from the spleens of immune mice before (on day 0) and after (on days 1, 2, 3, 4, and 5) the third immunization with map1-4. gates were set on the cd4 + t cells. the experiment was repeated three times with the same overall results. the values within each box indicate the relative cell number. these epitopes were potential for development of sars vaccine. map2 and map4 induced rapid increase in the number of il-4-producing cells to enhance the humoral immune indicated their potential uses for emergent prophylaxis of sars; map1 and map3 induced relatively slow but persistent increase in that to enhance humoral immune indicated their potential uses for long-term prophylaxis of sars. since activated th (cd4 + t) cells are also needed for effective humoral immunity, th cells will be activated rapidly with the help of immune memory. in the mice, cd62l (bradley et al., 1992; yin et al., 1991) and cd44 (budd et al., 1987; butterfield et al., 1989 ) are used to distinguish naive from memory cells, and naive from activated t cells (gerberick et al., 1997) . in our study, in epitope-based peptide-immunized mice, the classical phenotype of immunologic memory (cd44 high , and l-selectin low ) was present on the surface of cd4 + t lymphocytes just before the third immunization (day 0), suggesting that these epitope-based peptides could enhance the cd4 + t memory cell response and thereby increase immune surveillance, defense, and capacity to carry out their auxiliary functions when specific antigen reappeared. because excessive or depressed immune responses can result in immunologic derangement and adversely affect the body, it is important to know whether epitope can keep steady state of the immune system especial for its using as constituent of vaccine. so we monitored the change in lymphocyte subsets numbers of splenic lymphocytes. happily, the safety of the four b cell epitopes was confirmed. our study provided several sars-cov epitopes along with their immunological characteristics. these four epitopes had the ability to induce rapid, strong, and long-term humoral immunity, and they were also safe and effective at least in mice. therefore, the four epitopes might have potential use as constituent of sars vaccine. moreover, our limited studies on antigenic specificity of the four epitopes suggested their potential use as diagnostic biomarkers. in addition, s and m proteins were important surface antigens of sars-cov confirmed by our results of immunoselection of b cell epitopes. anti-sars-cov serum samples were collected from 8 convalescent-phase sars patients (the diagnostic criteria for sars-cov infection followed the clinical description of sars released by the world health organization) within a month after recuperation, tested positive for sars-cov antibody using a commercially available enzyme-linked immunosorbent assay (elisa) kit (huada company, peking, china). antibody titers varied from 1:320 to 1:1280. normal human serum samples (sars-cov seronegative) were pooled from seven blood donors. the 229e and oc43 standard strains of human coronaviruses (hcov-229e, hcov-oc43) and their specific antisera of murine origin were obtained from the guangdong center for disease control (cdc). four epitope-based dodecapeptides were synthesized in the form of an eight-branch multiple antigen peptide (map) construct (purity > 85%; huacheng company, xi'an, china) and the coincidence of the actual molecular mass with the theoretical molecular mass was confirmed by mass spectrography analysis. female 6-to 7-week-old balb/c mice were purchased from the centre of experimental animals sun yat-sen university, guangzhou, china. the animal experiment ethics committee of sun yat-sen university approved the protocols of all experiments performed in this study. all animals were handled according to the guidelines of the chinese council on animal care. immunoglobulin g (igg) from convalescent and normal human serum was purified respectively using an affinity column, protein a sepharose cl-4b (dingguo company, peking china). fab fragment of igg (igg fab) was prepared by digestion of intact igg with caroid, and then the digest was rechromatographed using another protein a sepharose cl-4b column. the purity of the targets was checked by sds-page and their antigenicity was determined using elisa and western blotting. human igg and igg fab (sigma chemical, st. louis, mo, usa) were used as references, and horseradish peroxidase (hrp)-labeled goat antihuman igg and igg fab (sigma) were used as detector antibodies in these identifications. the binding activity to sars-covantigen of the target molecules was determined using elisa. to enrich phage clones specific for anti-sars-cov igg fab, we improved the conventional biopanning procedure. the first round of biopanning was performed on immobilized anti-sars0.3 ± 0.1 0.4 ± 0.1 0.6 ± 0.1 8.5 ± 1.2 fa control 0.8 ± 0.3 1.9 ± 0.4 3.0 ± 0.3 12.5 ± 0.8 a bal b/c mice were injected twice with 100 μg of epitope-based peptides in the form of map constructs. mice receiving 300 μl of pbs or cfa served as negative controls. on day 7 after final immunization, splenocytes for the lymphocyte proliferation assay were pooled from immunized mice. b values are from one representative experiment out of five performed and are presented as the mean stimulation index (of quadruplicate wells) ± sd. cov igg fab to ensure all phage clones binding to it were included in the pool of the first screen. in other words, 4 × 10 10 phages of the original library (new england biolabs, ipswich, ma, usa) were immunoscreened with anti-sars-cov igg fab for 1 h at room temperature. unbound phages were removed by washing and the bound phages were eluted with glycine-hcl (0.2 m, ph 2.2, and 1 mg ml − 1 bsa) and neutralized with tris-hcl (1 m, ph 9.0). after titration on lb/iptg/x-gal (promega, madison, wi, usa) plates and amplification, 4 × 10 10 firstround selected phages were immunoscreened with normal igg fab and the unbound phages were collected to remove phages not specific for anti-sars-cov igg fab in the second biopanning. the protocols for the third and fourth immunoscreen were identical to that of the first round except 2 × 10 11 phages particles were used as input and the tween 20 concentration was increased for washing to 0.5% (v/v). the four rounds of biopanning were repeated once. thirty blue plaques from each fourth-round titration plate were picked at random, amplified in host bacteria (e. coli er2738), and precipitated with polyethylene glycol (peg)/ nacl. elisa was performed on 96-well plates coated respectively with 10 μg ml − 1 anti-sars-cov serum mixture and normal human serum mixture. after blocking, 10 12 phage particles of each clone per well were incubated for 2 h. then, the plates were washed at least six times with tbst (0.5%). hrpconjugated anti-m13 antibody (pharmacia, biotech, piscataway, nj, usa), diluted in bsa blocking buffer (1:5000), was added to the plates and incubated for 1 h. after washing and reacting with substrate 2, 2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (abts) for color development for 20 min in the dark, the absorbance at 405 nm was measured in a microplate reader (multiskan mk3, thermo labsystems, shanghai, china). the equal amount of wild type phages was used as a negative control simultaneously. immunopositive phage clones were preserved as target clones for further study. single-stranded dna (prepared from each target clone as template) and the − 96 giii primer were used for sequencing. sequences of dna inserted into target phage clones were translated into amino acid sequences and compared with that of structural proteins of sars-cov (aas48453, aas48454, aas48455, aas48456, aau07933, aat74874, etc.) which were retrieved from the genebank site (www.ncbi.nlm.nih. gov/) using standard protein-protein blast and clustal w multiple sequence alignment public software. we analyzed several properties of these epitopes including hydrophilicity, antigenicity, accessibility, flexibility, polarity, and secondary structure and chose four predominant epitopes (dodecapeptides) to synthesize for further study. competitive-inhibition assay. in competitive-inhibition experiments, coating with anti-sars-cov serum, blocking, and washing were performed as above. different amounts of the four synthetic peptides and their corresponding phage clones were added simultaneously and incubated for 1 h at 37°c. then the same steps as above were followed and absorbance was measured. the inhibition percentage was calculated as follows: inhibition% = [(od 405 without competitor − od 405 with competitor)/od 405 without competitor] × 100%. binding assay. to assess the specificity of the epitope-based peptides, their ability to bind to normal human, anti-sars-cov, anti-hcov-299e, and anti-hcov-oc43 sera were examined by elisa. each of the above antisera was incubated (1 h, 37°c) in elisa wells coated with 10 μg ml − 1 epitope-based peptides. after being washed, the wells were incubated with corresponding hrp-conjugated second antibody for 1 h, then ophenylenediamine (opd) to develop the color, and finally, optical density (od) values at 492 nm were measured. for monitoring specific antibody titer, cytokine levels, and lymphocyte proliferation, mice were randomly divided into six groups (one group for each of the four peptides, one for the pbs control, and one for the freund adjuvant (fa) control, n = 5). mice were immunized subcutaneously with 100 μg of peptide as immunogen in the presence of freund complete adjuvant and boosted with freshly prepared emulsion of the immunogen and freund incomplete adjuvant at 2-week intervals. generation of immunological memory in mice was by a second immunization and was followed by an 8-week rest period. for the study of the cd4 + t cell recall response, mice with immunological memory were immunized with 100 μg of peptide without adjuvant (akbar et al., 1993) for a third time. blood samples were obtained from the lateral tail vein of mice on days 7, 14, 21, 28, and 42 after primary immunization. antibody titer was measured using synthetic peptides as coating antigens for an igg elisa. hcov-229e and hcov-oc43 viral suspension with a titer of 5 × 10 6 tcid 50 ml − 1 was used to prepare the virus antigen. antibody specificity was determined from the igg elisa reactivity of sera with the antigens, sars-cov, hcov-229e, and hcov-oc43, respectively. spleen was cut into pieces and carefully forced through a metal mesh. for flow cytometric analysis, splenocyte suspensions were treated with 1× rbc lysis buffer (ebioscience, san diego, ca, usa) to lyse erythrocytes, then washed and suspended up to concentration of 2 × 10 7 cells ml − 1 in staining buffer (d-pbs ph 7.0/0.2% nan 3 /5% calf serum). for the enzyme-linked immunospot (elispot) and lymphocyte proliferation assays, lymphocytes were separated from the splenocyte suspensions by density gradient centrifugation and suspended (2 × 10 6 cells ml − 1 in rpmi 1640). all lymphocyte samples were prepared and used freshly. fifty microliters of suspended lymphocytes prepared as above, which were harvested, respectively, from twice-immunized mice on days 14, 21, and 35 after the first immunization and from boosted memory mice on days 0, 1, 2, 3, 4, and 5 after the third immunization, were incubated with a panel of antimouse antibodies against the surface molecules fluorescein isothiocyanate (fitc)-conjugated cd3e, phycoerythrin (pe)conjugated cd19, cd4, cd8a, allophycocyanin (apc)conjugated cd44, and cd62l (l-selectin, ebioscience). after incubation at 4°c for 20 min in the dark, cells were washed three times before being suspended in 4% paraformaldehyde and analyzed using a bd facs aria™ instrument (becton dickinson, franklin lakes, nj, usa). antibody-isotyping control flow cytometric analyses were carried out essentially in the same manner. the data were analyzed using bd facsdiva software. assessment of peptide-specific il-4-or ifn-γ-producing cells using the elispot technique il-4-or ifn-γ-producing cells were assessed by specific elispot kit according to the manufacturer's protocol (u-cytech, utrecht, the netherlands). briefly, 100 μl of lymphocytes from immunized mice on days 7, 14, 28, and 42 after primary immunization were incubated with corresponding peptides or pha at a final concentration of 5 μg ml − 1 in wells coated with captured interleukin-4 (il-4) antibody or interferonγ (ifn-γ) antibody after the wells were washed and blocked. after proper incubation at 37°c with 5% co 2 , the cells were removed by washing, and the secreted cytokine was detected with biotin-labeled anti-murine il-4 or ifn-γ monoclonal antibody. the spots were counted with an automatic reader. one hundred microliters of prepared lymphocytes was cultured in quadruplicate in 96-well plates in the presence of 100 μl of the corresponding peptides at final concentration of 2.5 μg, 5 μg, and 10 μg ml − 1 , respectively, and the control pha was at 10 μg ml − 1 . after incubation (37°c, 68 h), 100 μl of supernatant was removed and 10 μl of 3-(4, 5 dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide (mtt, 50 μg ml − 1 ) was added. after further incubation for 4 h, the lymphocytes were lysed (100 μl of dimethylsulfoxide [dmso] , room temperature for 20 min) for developing color and the od values at 570 nm were measured. student t test was used for statistical analysis. characterization of phage that bind plastic from phage-displayed random peptide libraries a possible role for bc1-2 in regulating t-cell memory a 'balancing act' between cell death and survival severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice long-term cd4 + memory t cells from the spleen lack mel-14, the lymph node homing receptor contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity distinction of virgin and memory t lymphocytes. stable acquisition of the pgp-1 glycoprotein concomitant with antigenic stimulation mucosal immunization of african green monkeys (cercopithecus aethiops) with an attenuated parainfluenza virus expressing the sars coronavirus spike protein for the prevention of sars a subset of memory cd4+ helper t lymphocytes identified by expression of pgp-1 identification of biologically active peptides using random libraries displayed on phage peptides on phage: a vast library of peptides for identifying ligands identification of a novel coronavirus in patients with severe acute respiratory syndrome development of protection against coronavirus induced diseases mimicking of discontinuous epitopes by phage-displayed peptides: ii. selection of clones recognised by a protective monoclonal antibody against the bordetella pertussis toxin from phage peptide libraries a general strategy to identify mimotopes of pathological antigens using only random peptide libraries and human sera aetiology: koch's postulates fulfilled for sars virus effects of a sars-associated coronavirus vaccine in monkeys selective modulation of t cell memory markers cd62l and cd44 on murine draining lymph node cells following allergen and irritant treatment sars-associated coronavirus an m13 phage library displaying random 38-amino-acid peptides as a source of novel sequences with affinity to selected targets a novel coronavirus associated with severe acute respiratory syndrome disease-specific b cell epitopes for serum antibodies from patients with severe acute respiratory syndrome (sars) and serologic detection of sars antibodies by epitope-based peptide antigens mimicking of discontinuous epitopes by phage-displayed peptides: i. epitope mapping of human h ferritin using a phage library of constrained peptides sars: lessons learned from other coronavirus coronavirus as a possible cause of severe acute respiratory syndrome identification of antigenic sites on three hepatitis c virus proteins using phage-displayed peptide libraries mapping of linear epitopes recognized by monoclonal antibodies with genefragment phage display libraries identification of severe acute respiratory syndrome in canada antibody as a surrogate receptor in the screening of a phage display library antigenicity: some molecular aspects b-cell epitope mapping of the vapa protein of rhodococcus equi: implications for early detection of r. equi disease in foals identification of continuous bcell epitopes on the protein moiety of the 58-kilodalton cell wall mannoprotein of candida albicans belonging to a family of immunodominant fungal antigens a 193-amino acid fragment of the sars coronavirus s protein efficiently binds angiotension converting enzyme 2 false-positive results in a recombinant severe acute respiratory syndrome-associated coronavirus (sars-cov) nucleocapsid enzyme-linked immunosorbent assay due to hcov-oc43and hcov-229e rectified by western blotting with recombinant sars-cov spike polypeptide identification of b-cell epitope of dengue virus type 1 and its application on diagnosis of patients prediction of the secondary structure and b cell epitopes for the m protein of sars coronavirus prediction of the b cell epitope for the s protein of sars coronavirus a dna vaccine induces sars coronavirus neutralization and protective immunity in mice changes in expression of j11d on murine b cells during activation and generation of memory identification of immunodominant epitopes on the membrane protein of the severe acute respiratory syndrome-associated coronavirus we are grateful to guangdong cdc for providing patient serum samples and standard strains of human coronaviruses hcov-229e and hcov-oc43.supported by a grant from the national natural science foundation (30340013); guangdong province key problems in science and technology project (532014202028). key: cord-308835-999kewdw authors: leibowitz, julian l.; wilhelmsen, kirk c.; bond, clifford w. title: the virus-specific intracellular rna species of two murine coronaviruses: mhv-a59 and mhv-jhm date: 1981-10-15 journal: virology doi: 10.1016/0042-6822(81)90250-6 sha: doc_id: 308835 cord_uid: 999kewdw abstract seven virus-specific, polyadenylated rna species have been identified in mouse cells infected with the murine coronaviruses mhv-a59 (a59v) or mhv-jhm (jhmv). mhv-infected 17cl·1 cells were labeled with [32p]orthophosphate in the presence of actinomycin d and the cytoplasmic rna was extracted and analyzed by agarose gel electrophoresis. these rna species range in size from 6.3 × 105 to 6.1 × 106 daltons. the a59v and jhmv-specific rnas have identical molecular weights and comigrate in agarose gels. the largest intracellular rna species is identical to rna isolated from purified virions, as determined by agarose gel electrophoresis and oligonucleotide fingerprint studies of ribonuclease t1 digests. oligonucleotide fingerprints of the six subgenomic rnas show that the sequences they contain are present in virion rna, confirming their virus-specific nature. the fingerprinting studies also demonstrate that the six subgenomic rna species make up a nested set. the sequences present in each rna species are also present in all larger rna species. these larger rnas also contain additional sequences consistent with their greater size. the subgenomic rnas fulfull many of the criteria for mrnas. possible mechanisms for generating these rnas are discussed. coronaviruses are widespread in nature and have been associated with several diseases in infected hosts (robb and bond, 1979a) . coronaviruses are defined as pleomorphic enveloped particles about 100 nm in diameter which have characteristic bulbous projections on their surface, bud through the endoplasmic reticulum rather than the plasma membrane, and contain rna (tyrell et al., 1978) . several studies of coronavirus genomic rna have been reported (lomneczi, 197'7; lomneczi and kennedy, 1977; yogo et al., 1977; lai and stohlman, 1978; macnaughton and madge, 1978; schochetman et al., 1977; tannock and hierholzer, 1978; guy and brian, 1979; wege et al., 1978) . these studies have indicated that the coronavirus genome is a large (5.4-8.1 x lo6 daltons) singlestranded rna which is polyadenylated and is infectious. these properties identify the coronaviruses as positive-stranded rna viruses. in contrast to the data obtained on coronavirus virion rna, few data have been published on intracellular coronavirusspecific rna. mishra and ryan (1973) reported that porcine kidney cells infected with transmissible gastroenteritis virus (tgev) contained actinomycin d-resis-tent rna species which sedimented between 18 and 28 s. robb and bond (1979b) have studied murine hepatitis virus (mhv)-infected cells and found that deproteinized rna from virus-specific polysomes sedimented between 10 and 28 s. rna in this size range was recently demonstrated to code for two mhv structural proteins (siddell et al., 1980) . stern and kennedy (1980a,b) have identified six virus-specific rna species in cells infected with avian infectious bronchitis virus (ibv). we have been studying the virus-specific rna species synthesized in cells infected with two strains of mhv:mhv-a59 (a59v) and mhv-jhm (jhmv). jhmv is highly neurotropic and produces an encephalomyelitis with demyelination in its natural host, the mouse (bailey et al., 1949) . a59v is weakly neurotropic (robb et al., 1979) . we have been investigating the replication of these viruses in the hope of eventually understanding the molecular mechanisms by which jhmv produces demyelination. we report here the identification of seven major mhv-specific intracellular rna species. these rnas have been characterized by agarose gel electrophoresis, poly(u) sepharose affinity chromatography, and oligonucleotide fingerprinting. materials and methods materials. [3h]uridine was obtained from new england nuclear and ["piorthophosphate (carrier free) from new england nuclear, amersham, or icn. phenol was obtained from mallinckrodt and redistilled prior to use. actinomycin d and ribonuclease t1 (sankyo) were purchased from calbiochem and hexadecyltrimethylammonium bromide (ctab) and poly(u) sepharose from sigma. glyoxal was purchased from fisher chemicals as a 40% solution and deionized by the method of mcmaster and carmichael (1977) before use. methyl mercuric hydroxide was purchased from alpha chemicals, low melting point agarose from bethesda bioresearch laboratories, standard low m, agarose from bio-rad, linear polyacrylamide from british drug house, and proteinase k from beckman chemicals. acrylamide, xylene cyan01 ff, and bromphenol blue were obtained from eastman chemical. hydroxylapatite (ha; dna grade) was obtained from bio-rad and prepared as described by stern and kennedy (1980a) . cells. the origin and growth of the murine cell line 17cl. 1 has been previously described (sturman and takemoto, 1972; bond et al., 1979) . hela cells were obtained from dr. john holland and grown in 32-0~. prescription bottles in dulbecco's modified eagle's medium with 10% calf serum. viirms. the origin and growth of a59v and jhmv virus stocks have been described (robb and bond, 1979b) . for experiments 17cl. 1 cells were removed from the glass substrate with 0.1% twice crystallized trypsin in puck's saline a supplemented with 0.01 m tris (ph 8.1) and 0.1% egta [ethyleneglycol-his@-amino ethyl ether)-n,llrtetraacetic acid], resuspended in dulbecco's modified eagle's medium containing 10% fetal bovine serum (dmelo), centrifuged at 500g for 2 min and resuspended in medium containing 2% fetal bovine serum (dmez). sufficient virus was added to give a multiplicity of infection (m.o.i.) between 0.1 and 0.15 plaque forming units/cell. virus adsorption was at 37" for 30 min. following adsorption the cells were centrifuged at 500 g for 2 min, resuspended in dme prewarmed to 37", and plated into 35-mm (1.5 x lo6 cells/dish), 60-mm (5 x lo6 cells/ dish), loo-mm (1.5 x lo'cells/dish), or 150mm (4-5 x 10' cells/dish) plastic culture dishes and further incubated at 37". for most experiments utilizing [32p]orthophosphate as label the cells were resuspended and plated in phosphate-free dme-2. the strain of mengovirus used was that described by plagemann and swim (1966 extraction of intracellular rna. cytoplasmic extracts of infected or mock-infected 17cl. 1 cells were prepared using np-40 (borun et al., 1967) . monolayers were rinsed once with cold phosphatebuffered saline (pbs), scraped into cold reticulocyte standard buffer (rsb, 0.01 m tris, ph 7.4,o.ol m nacl, 0.0015 m mgcl.& with a rubber policeman and transferred to a tube containing sufficient np-40 to give a final concentration of 1% . the cell suspension was vortexed gently, incubated on ice for 5 min, vortexed again, and the nuclei removed by centrifugation at 1500 g for 2 min. the cytoplasmic extract was adjusted to 1% sds, 0.4 m nacl, 0.01 m edta, and 1.0 mg/ml proteinase k and incubated at 50" for 5 min and at room temperature for an additional 25 min. the rna was then extracted with phenol chloroform (robb and bond, 1979b) and precipitated with 3 volumes of ethanol at -20' in the presence of 50 pg of yeast carrier trna. preparation of mhvvirion rna. 17cl. 1 cells were infected with a59v or jhmv in phosphate-free dme2 as described above. following virus adsorption the cells were plated in loo-or 150-mm culture dishes and incubated at 33". at 4 hr postinfection (hpi) [32p]orthophosphate was added to a concentration of 100-1000 &i/ ml. at 16-18 hpi the cell-associated virus was released by two cycles of freeze thawing and the resulting lysate clarified by centrifugation at 10,000 g for 30 min at 4'. virus was concentrated by centrifugation for 60 min at 35,000 rpm in the sw40 rotor through a 0.5-ml pad of 15% (w/w) potassium tartrate in mse buffer (0.01 m morpholinopropane sulfonic acid, 0.15 mnacl, 0.001 m edta, ph 6.8). the virus pellets were resuspended by sonication in 0.5 ml of mse buffer, layered onto a 12 ml gradient of 5-25% (w/w) potassium tartrate in mse buffer, and centrifuged at 35,000 rpm for 45 min in the sw40 rotor. the gradient was fractionated and the virion peak located by counting aliquots of each fraction. for the preparation of highly purified virus this material was diluted with mse, layered on a 9 ml lo-40% (w/ w) potassium tartrate gradient, and centrifuged in the sw40 rotor at 37,090 rpm for 4 hr. the gradient was fractionated, aliquots were counted, and the peak of radiolabeled virus collected. this material had a buoyant density between 1.19 and 1.17 g/cm3. the virus was diluted with mse buffer, pelleted at 45,000 rpm for 30 min in the sw50.1 rotor, and resuspended by sonication in 1 ml of mse buffer. virion rna was extracted in a similar manner to intracellular rna and precipitated with ethanol after the addition of 50 rg of carrier trna. virion rna for fingerprinting was prepared by a slightly different procedure. virus was concentrated and banded on a 5-25% potassium tartrate gradient and this partially purified virus was diluted with mse buffer, pelleted in the sw50.1 rotor, and the rna was extracted and precipitated with ethanol as described above. the rna was collected by centrifugation, dried under a stream of nitrogen, and dissolved in 0.1 ml of sds buffer (0.01 m tris, 0.01 m nacl, 0.001 m edta, 0.1% sds, ph 7.4). it was then overlaid on a 5-ml lo-30% (w/w) sucrose in sds buffer gradient and centrifuged at 46,000 rpm for 107 min at 20" in the sw50.1 rotor. the gradient was fractionated and the peak of 48 s virion rna (robb and bond, 1979b ) located by counting aliquots of each fraction. these fractions were pooled, adjusted to 0.4 m nacl and precipitated with ethanol in the presence of 100 rg of trna carrier. this material was used for fingerprinting studies and was homogeneous upon analytical electrophoresis. isolation of poly(a)-containing rna. intracellular rna was extracted and precipitated with ethanol as described. the polyadenylated rna species were isolated by affinity chromatography over poly(u) sepharose as described by wilt (1977) . agarose gel electrophoresis 1. analytical electrophoresis. agarose gel electrophoresis following glyoxal denaturation was essentially as previously described (mcmaster and carmichael, 1977) . nucleic acids were recovered from ethanol by centrifugation, dried under a stream of nitrogen and dissolved in glyoxal buffer. rna or dna was reacted with glyoxal at 50" for 5 min followed by an additional 55 min at room temperature. samples were electrophoresed at 100 v for 4 hr in horizontal slab gels containing 0.7, 0.8, or 1.0% agarose, 0.01 m phosphate buffer, ph 7.0, 0.002 m edta. when appropriate, the gels were strained with acridine orange and the positions of rrna were marked with stainless-steel wire clips inserted into the gel. the gels were fixed in ethanol, dried under vacuum, and exposed to kodak xr film at room temperature. to quantitate the individual rna species present in cytoplasmic extracts two methods were used. autoradiographs were prepared with two exposure times to ensure linearity of the film and scanned with a densitometer (e. c. corporation). the area under the peaks was quantitated by cutting out and weighing tracings of the scans. alternatively, the regions of the gel corresponding to the bands present in the autoradiographs were excised and the amount of rna present in the band was quantitated by liquid scintillation counting. these methods gave similar results. molar ratios of rna species were calculated by dividing the counts per minute or weight of paper in each peak by its molecular weights x lop6 and normalizing all values such that the major rna species, rna7 (see results), was set at 100. 2. preparative electrophwesis. labeled intracellular rna was extracted from 1.2 x lo*-1.6 x 10' cells, the poly(a)-containing rna species selected by affinity chromatography, and then electrophoresed in gels containing 1% low melting point agarose. two gel systems were used. in initial experiments the phosphate buffer system used for analytical agarose electrophoresis was employed without prior denaturation of the rna with glyoxal and dimethyl sufoxide (mcmaster and carmichael, 1977) . electrophoresis was for 4 hr at 100 v. the above conditions did not reproducibly allow complete resolution of all virus-specific rna species. later experiments employed methyl mercury de-naturation of the rna and electrophoresis in agarose gels containing 5 mm methyl mercuric hydroxide (bailey and davidson, 1976) . electrophoresis was at 100 v for 6 hr in a horizontal 1% low melting point agarose gel. following electrophoresis, the majority of the methyl mercury was removed from the gel by soaking in two changes of 750 ml sterile 0.5 m ammonium acetate (bailey and davidson, 1976) . for both gel systems rna species were located by autoradiography of the wet gels wrapped in saran. agarose strips corresponding to the bands seen in the autoradiographs were cut from the gel with a flamed scalpel and placed in tightly capped screw top tubes. rna was recovered from gels by melting the agarose at 70" for 5 min. five milliliters of a slurry of ha, prewarmed to 37", was added to each tube. samples containing methyl mercury had 2-mercaptoethanol added to a concentration of 1%. the mixture was cooled to 37", ha was collected by centrifugation, and washed twice with tris acetate buffer (10 mm tris, 20 mm sodium acetate, 5 mm edta, ph 7.3) in a 37" warm room to remove the liquified agarose. for samples which had been treated with methyl mercury, the tris acetate buffer was supplemented with 1% 2-mercaptoethanol. the ha was resuspended in 5 ml of tris acetate buffer and transferred to ethanolwashed disposable columns (quick-sep, &s-q obtained from isolab) placed in heat sterilized 16 x 125-mm culture tubes. the culture tubes containing the columns were centrifuged at 1000 q for 10 min at room temperature to remove the tris acetate buffer. the rna was eluted from ha columns by two washes of 1.0 ml of 0.4 m sodium phosphate, 1 mm edta (ph 7.0) which were similarly forced from the columns by centrifugation at 1ooog for 10 min. the phosphate eluates were pooled and the rna precipitated with ctab as described by stern and kennedy (1980a) . the ctab precipitates were dissolved in 0.2 ml of 50 mm tris, ph 7.4, containing 1 m nacl and 1 mm edta and the rna precipitated with 1.0 ml of ethanol. ribonuclease tl $nger;orinting. rna was recovered from ethanol by centrifugation, dissolved in 0.1 ml of sterile water, transferred to a sterile 1.5-ml eppendorf centrifuge tube, and the water was evaporated with a stream of nitrogen. the rna was digested with 10 ~1 of ribonuclease t1 (1 mg/ml) in 10 mm tris, ph 7.6, at 37" for 30 min. ten microliters of a solution containing 5 m urea, 50% sucrose, 0.1% bromphenol blue, and 0.1% xylene cyan01 ff was added to the digestion products. the rnase t1-resistent oligonucleoties were separated by two-dimensional polyacrylamide gel electrophoresis as described by stern and kennedy (1980a) with the following modification. after electrophoresis in the first dimension the gel strips were washed for 25 min with two changes of 100 mm tris borate buffer containing 2.5 mm edta, ph 8.3, prior to pouring the second dimension gel (lee et al., 1979) . following electrophoresis the gels were wrapped with polyethylene sheets and exposed at -70" to kodak xr film in the presence of cronex lightning plus (du-pont) intensifying screens (laskey and mills, 1977) . exposures ranged from 2 days to 3 weeks. preparation of molecular weight markers. mengovirus was purified from infected 17cl. 1 cells labeled with [32p]orthophosphate from 4 to 16 hpi. the culture fluids were clarified by centrifugation at 10,000 g for 30 min. sodium dodecyl sulfate was added to the clarified fluids to a concentration of 0.1% and the virus pelleted through a 1.5-ml 30% suclose pad by centrifugation at 25,000 rpm in the sw 27.1 rotor for 3 hr at 20". the virus was resuspended in 1 ml of 0.01 m tris buffer, ph 7.4, layered onto a 16-ml cesium chloride gradient (1.2-1.4 g/cm3) and centrifuged overnight in the sw27.1 rotor at 24,000 rpm. the gradient was fractionated, aliquots were counted and the peak of radioactive virus at a density of 1.34 g/cm3 was pooled. this material was diluted with 0.01 m tris, ph 7.4, and pelleted in the sw50.1 rotor at 45,000 rpm for 1 hr. the pelleted virus was resuspended in 0.01 m tris buffer, the rna deproteinized with sds and proteinase k, extracted with phenol chloroform, and precipitated with ethanol. the rna was collected by centrifugation and prepared for analytical electrophoresis. vsv rna was prepared from vsvinfected bhk 21 cells labeled with [32p]orthophosphate from 2 to 16 hpi. culture fluids were clarified by centrifugation at 10,000 g for 30 min. vsv was pelleted from the clarified supernate by centrifuging in the sw40 rotor at 37,000 rpm for 1 hr, the virus pellet was taken up in 0.01 m tris buffer, ph 7.4, the rna deproteinized with sds and proteinase k, and precipitated with ethanol. this vsv rna preparation was used as a molecular weight marker without further purification although it was contaminated with significant amounts of 28 s and 18 s rna. hela cells were infected with ad2 and labeled with [32p]orthophosphate from 16 to 40 hpi. infected cells were scraped from the substrate, pelleted by centrifugation, and resuspended in 1 ml of 0.01 m tris, ph 8.1. the cells were disrupted by sonication and the labeled virus was purified as previously described (horwitz, 1971) . the purified virus was diluted with 0.01 m tris, ph 8.1, and pelleted by centrifugation at 37,000 rpm for 1 hr. the virus was resuspended in 1 ml of 0.01 m tris buffer (ph 7.4), extracted with phenol (petterson and sambrook, 1973) , and the dna precipitated with ethanol. the dna was recovered from ethanol by centrifugation and denatured with glyoxal prior to electrophoresis. ribosomal rna was purified from cytoplasmic extracts of 17cl. 1 or bhk-21 cells which were labeled for 24 hr with [32p]orthophosphate. sds was added to cytoplasmic extracts to a final concentration of 1% and the 18 and 28 s rrna were purified by centrifugation through 5-30% (w/w) sucrose gradients (containing 0.01 m tris, 0.01 m nacl, 0.001 m edta, 0.1% sds, ph 7.4) at 37,000 rpm for 4 hr in the sw40 rotor at 20". the 28 and 18 s peaks were located by counting aliquots of the gradients, treated with proteinase k and extracted with phenol chloroform. the rna was precipitated with ethanol, collected by centrifugation, and denatured with glyoxal prior to electrophoresis. escherichia coli rrna was purchased from miles biochemicals and denatured 1'7cl. 1 cells were infected with a59v, jhmv, or mock-infected, resuspended in dme2, seeded in replicate 35-mm dishes, and incubated at 3'7". at 1 hr intervals postinfection actinomycin d was added to 5 pg/ml to triplicate dishes. the dishes were incubated for 15 min and then labeled for 1 hr with 5 &i/ml of ['hjuridine. at the end of the labeling period the dishes were removed from the incubator, washed twice with cold phosphate-buffered saline (pbs), scraped from the dishes into cold pbs, and frozen at -20". the samples were thawed 24 hr later and the amount of trichloroacetic acid precipitable radioactivity determined. the results are expressed as the mean of triplicate samples. the amount of radioactivity incorporated into mock infected dishes (about 2000 cpm) has been substracted from the values shown. with glyoxal prior to electrophoresis. the 16 and 23 s rrnas were located following electrophoresis by staining with acridine orange and their locations marked with wire clips inserted in the gel. uridine incorporation robb and bond (1979b) have shown that mhv-specific rna synthesis is resistant to actinomycin d. we have taken advantage of this to determine the kinetics of mhv-specific rna synthesis by assaying the incorporation of rh]uridine (in the presence of actinomycin d) into acid-precipitable material. 17cl. 1 cells were in-fected with jhmv, a59v, or mock infected and at hourly intervals replicate dishes were incubated for 15 min with actinomycin d and then labeled for 1 hr in the presence of the drug. the results of this experiment are shown in fig. 1 . after an initial lag phase of 3 hr in a59v-infected cells and 5 hr in jhmv-infected cells, actinomycin d-resistant uridine incorporation increases exponentially until 7 hpi, when it plateaus. these results are consistent with the one step growth curves of these two viruses (bond et al., 1979) . in view of the above results, we routinely labeled cells from 4 to 8 hpi in subsequent experiments. identification of mhv-specific rna species a59v, jhmv, and mock-infected cells were labeled with [32p]orthophosphate from 4 to 8 hpi in the presence of actinomycin d. the cytoplasmic rna was extracted, denatured with glyoxal, and analyzed by electrophoresis on an agarose gel (fig. 2) . seven rna species, designated l-7 in decreasing order of size, were reproducibly present in jhmv-and a59vinfected cells. jhmv-infected cells synthesize less rna5 than a59v-infected cells (table 1) . although the band representing jhmv-specific rna5 is barely visible in fig. 2 (lane j), this rna species could be seen with a longer exposure of the autoradiograph, was reproducibly present in infected cell extracts, and can be better seen in fig. 6 . in addition, three minor rna species were often observed, although not in every experiment. the largest of these rnas migrated between rnas 3 and 4 just faster than 28 s rrna. two minor rnas migrating faster than rna7 were also sometimes observed. these rnas were not studied extensively due to difficulty in preparing sufficient material and their inconsistent appearance. no discrete bands were identified in mock-infected extracts. the largest mhv-specific rna, rna 1, coelectrophoreses with virion rna and represents the intracellular form of the genome. in contrast to the size differences we have observed between a59v. and jhmv-specific proteins (bond et al., 1979) , a59v-and jhmv-specific rnas comigrate in agarose gels. the amount of virus-specific rna synthesized in a59v-infected cells is, however, about 30% greater than that synthesized in jhmvinfected cells as determined by the incorporation of rsh]uridine or [=plorthophosphate into acid-insoluble cytoplasmic material in the presence of actinomycin d. the molecular weights of mhv-specific intracellular rna the molecular weights of mhv-specific rna species l-7 were determined by agarose gel electrophoresis with appropriate molecular weight markers. glyoxal denaturation allows the use of dna as well as rna size markers (mcmaster and carmichael, 1977) . a plot of the log,, molecular weight against electrophoretic mobility is shown in fig. 3 . a linear relationship was obtained over a molecular weight range of 5.5 x 105-1.15 x 107. the molecular weights obtained for the seven major mhv specific rnas are shown in table 1 . cytoplasmic rna was extracted from infected cells labeled from 4 to 8 hpi and analyzed by agarose gel electrophoresis. the relative molar amounts of the individual mhv-specific rnas were quantitated as described under materials and methods. the results are shown in table 1 . a59v-and jhmv-infected cells synthesize virus-specific rna species in similar ratios with one exception. the relative amount of rna5 synthesized in jhmvinfected cells is about one-tenth of that synthesized in cells infected with a59v. this was a consistent finding in multiple experiments. to determine if there is temporal regulation of mhv-specific rna synthesis, replicate cultures of a59v, jhmv, or mock-infected cells were pulse labeled for 1 hr at hourly intervals and the intracellular rna was extracted and analyzed by gel electrophoresis. the results for a59vinfected cells are shown in fig. 4 . virusspecific rna synthesis was barely detectable in infected cells labeled from 3 to 4 hpi. rnas 4,5,6, and 7 were the only spe-ties present in detectable quantities at this time. at later times all a59v-specific rna species were synthesized coordinately with respect to time. virus-specific rna synthesis was maximal in this experiment at 5-6 hr and then declined. the results of a similar experiment with jhmv are shown in fig. 5 . virus-specific rna synthesis was first detected at 4-5 hpi. at this time all rna species were present in detectable amounts although only rnas 6 and '7 are visible in the photograph. at later times all of the jhmvspecific rna species were easily detectable and were synthesized coordinately. rna extracted from a59v, jhmv, and mock-infected cells was fractionated by chromatography over poly(u) sepharose and then analyzed by gel electrophoresis (fig. 6) . the seven major mhv-specific rnas were present in the material which bound to the poly(u) sepharose (pool 2). in addition, the two minor mhv-specific rna species smaller than rna 7 were also present in this pool. these rnas are therefore presumably polyadenylated. that this is the case was confirmed by ribonuclease t1 fingerprinting (see below). the rna which did not bind to poly(u) sepharose (pool 1) consists of a heterogeneous mixture of rna species. discrete bands which coelectrophoresed with mhvspecific rnas were present superimposed on a smear of heterogeneous material. the sum of the molecular weights of the six subgenomic rna species exceeds the molecular weight of the genome by approximately 50%. to investigate this observation further, the seven major mhvspecific rnas were purified and compared to each other and to virion rna using the technique of ribonuclease t1 fingerprinting. the individual rna species and virion rna were purified from cells labeled with [32p]orthophosphate as described under materials and methods. the purified rnas were digested with ribonuclease tr and the resulting oligonucleotides separated by two-dimensional gel electrophoresis. the results are shown in figs. 7 and 8. poly(a) tracts are seen as streaks in the upper left hand corner of the fingerprints of a59v and jhmv virion rnas. this confirms the findings of others (yogo et al., 1977; lai and stohlman, 1978; wege et al., 1978) that the mhv genome is polyadenylated. the seven major a59v-and jhmv-specific intracellular rnas are also polyadenylated. a comparison of the fingerprints of the seven a59v-specific intracellular rnas with each other and with a59v virion rna reveals several things. the fingerprint of the largest intracellular rna, species 1, is almost identical to that of virion rna. there are, however, several oligonucleotides which are underrepresented in the fingerprint of rna1 as comfig. 4 . the kinetics of a59v-specific rna synthesis. 1'7cl.l cells were infected with a59v or mockinfected, resuspended in dme2, and seeded in replicate 60-mm dishes and incubated at 37". at 1-hr intervals individual dishes were removed from the incubator, media removed, washed twice with phosphate-free dmel, and then incubated for 15 min in phosphate-free dme2 containing 5 pg/ml actinomycin d. the cells were then labeled for 1 hr with 250 &i/ml [aap]orthophosphate and the cytoplasmic nucleic acids were then extracted and precipitated with ethanol. the rna was collected by centrifugation, glyoxalated, and electrophoresed on a 1% agarose gel. the times of labeling are indicated in the figure. the lane containing rna from mock-infected cells is labeled m. fig. 7 , panel l), as well as an oligonucleotide which is overrepresented in the fingerprints of rna1 (closed triangle) and rnas 6 and 7 (see below). all the oligonucleotides contained in rna species 2 are present in rna species 1 and are a subset of these oligonucleotides. similarly, the fingerprint of rna species 3 is a subset of the fingerprint of rna species 2. an examination of the fingerprint of rna species 4 reveals a new spot which is not present in any of the other a59v-specific rnas (arrow in fig. 7, panel 4) . the remaining oligonucleotides observed in rna species 4 are a subset of the larger rna species. the fingerprint of rna species 5 also contains a spot which is not present in any other rna species (arrow in fig. 7 , panel 5). the remaining oligonucleotides in rna species 5 are contained in the fingerprints of the larger rna species. the fingerprint of rna species 6 is a subset of that of rna species 5. similarly, all the oligonucleotides of rna species 7 are contained in rna species 6. however, one oligonucleotide (closed triangle, panels 6 and 7) is overrepresented in rnas 6 and 7 when compared to virion rna and rnas 2, 3, 4, and 5. this oligonucleotide has the same cytoplasmic extracts were prepared and the nucleic acids extracted and precipitated with ethanol. the nucleic acids were dissolved in a buffer containing 1 mm tris, 360 mm nacl,l mm edta, 0.5% sds, ph 7.4, chromatographed over poly(u) sepharose as described by wilt (1977) , and fractionated into two pools. pool 1 was not retained by the column; pool 2 contains the rna which bound to the poly(u) sepharose. both pools were precipitated with ethanol after the addition of carrier trna, collected by centrifugation, glyoxalated, and analyzed by electrophoresis on a 0.8% agarose gel. the positions of rrnas are marked by wire clips. electrophoretic mobility as the oligonucleotide which is overrepresented in rna1 and presumably is identical with it. an analysis of the data obtained with jhmv ( fig. 8 ) reveals similar results. rna1 is essentially identical to genomic rna isolated from purified virions with the exception of an oligonucleotide which is underrepresented in rna1 (open triangle). the six subgenomic jhmv-specific rnas form a nested set as described above for the a59v specific rna species, however, as for a59v, one oligonucleotide (closed triangle) is markedly overrepresented in rnas 6 and 7 when compared to rnas 2-5. the fingerprint of rna species 4 contains a spot which is not present in any other jhmv-specific rna (arrow in fig. 8, panel 4) . the reason for the overrepresentation of some oligonucleotides and the underrepresentation of others in these fingerprints is unclear at present. the spots which are underrepresented in rna1 when compared to virion rna disappear in rna 2. these may represent oligonucleotides from the 5' end of the genome which are selectively lost during the poly(u) sepharose selection in the purification of rnal. a more extensive analysis of the structure of mhv rna should clarify this point. a comparison of the fingerprints of a59v and jhmv virion rnas shows only 10 to 20% sequence homology between these two viruses. this is in contrast to data obtained by hybridization analysis fig. 7 . oligonucleotide fingerprints of a596-specific rna. a59v virion (panel v) and intracellular rnas (panels l-7) were purified as described under materials and methods. the purified rnas were digested with rnase t1 and the resulting oligonucleotides separated by two-dimensional electrophoresis and autoradiographs prepared as described under materials and methods. only the portion of the autoradiographs which contains oligonucleotides migrating more slowly than the xylene cyan01 dye marker is shown. the positions of the bromphenol blue dye markers are idicated by asterisks. unique spots are indicated by arrows. which indicates 75% homology between the two genomes (weiss and leibowitz, in press) and the serologic cross reactions obtained with antisera to these viruses (bond et al., 1979) . this discrepancy is not surprising since t1 fingerprinting detects single base changes and will underestimate the degree of homology. the data presented above indicate that mhv-infected cells synthesize at least seven virus-specific rnas. the largest of these rnas, rnal, is nearly identical to virion rna, as judged by oligonucleotide fingerprinting. the oligonucleotides of the six subgenomic rnas, rnas 2-7, are contained in the virion rna. they are therefore of the same polarity as the virion rna. oligonucleotide fingerprints of these fig. 8 . oligonucleotide fingerprints of jhmv-specific rna. jhmv virion (panel v) and intracellular rnas (panels 1-7) were purified as described under materials and methods. the purified rnas were digested with rnase ti, the oligonucleotides separated by two-dimensional electrophoresis, and autoradiographs prepared. the positions of the bromphenol dye marker are shown by asterisks. unique spots are indicated by arrows. rnas show them to make up a nested set in which any rna contains the sequences present in any other smaller rna plus additional sequences consistent with its larger size. similar data have been obtained with another coronavirus, ibv (stern and kennedy, 1980a) . a "northern blot" analysis using representative and short 3' specific cdna probes indicates that the six subgenomic rnas share sequences at their 3' ends (weiss and leibowitz, in press ). furthermore, stern and kennedy (1980b) have established a 5' to 3' oligonucleotide spot order for the ibv genome. their data show that the ibv subgenomic rnas form a nested set with common 3' ends. taken together, these data suggest that the mhv-specific subgenomic rnas map from the 3' end of the genome in a similar manner to ibv. a possible model of this arrangement is shown in fig. 9 . it is unlikely that any of the six subgenomic rnas are defective interfering rnas. the stock virus used in these experiments was 3 passages (a59v) or 4 passages (jhmv) removed from cloning by limiting dilution (robb and bond, 1979b) . the low m.o.i. used to grow virus stocks (10m4 pfu/cell) and to initiate infections in these experiments (0.1-0.15 pfu/cell) selects against the generation of defective interfering particles. furthermore, after 10 serial undiluted passages of a59v and jhmv in 17cl. 1 cells, the rna gel pattern observed is unchanged from early passage virus (leibowitz, unpublished data) . the six subgenomic rnas are almost certainly mrnas. they are polyadenylated, as expected of mrnas, and robb and bond (1979) have shown that multiple mhv-specific rna species spanning the size range of the subgenomic rnas 2-7 are present on polysomes of infected cells. furthermore, siddell et al. (1980) have recently demonstrated that polyadenylated rna isolated from jhmv-infected cells can be translated in vitro. using sucrose gradients they were able to partially resolve 1'7 and 19 s jhmv-specific rna species. their in vitro translation studies suggest that the 17 s rna codes for the 60,000-dalton virion nucleocapsid protein (sturman, 1977; wege et al., 1979) , while the 19 s rna codes for the 23,000-dalton virion glycoprotein (sturman, 1977; wege et al., 1979) . the relatively large amounts of these two rnas and their sedimentation properties suggest that they correspond to rnas 6 and 7. this has recently been confirmed by the in vitro translation of gel purified rna (leibowitz and weiss, in press ; van der zeijst, in press). van der zeijst has also demonstrated that rna3 codes for the 150,000-dalton protein described by bond et al. (1979) . this protein is the intracellular counterpart to the peplomer protein described by sturman (1977) and sturman and holmes (1977) . of the five nonstructural proteins described by bond et al. (1979) , the 57,000-and 54,000-dalton proteins have been shown to have similar tryptic maps to the nucleocapsid protein (leibowitz, unpublished data) and are presumably translated from the same mrna. this leaves the three other nonstructural proteins described by bond et al. (1979) and possibly a fourth nonstructural protein which has been described by siddell et al. (1979) to be assigned to rnas 2, 4, and 5. the in vitro translation of all the mhv-specific rna species and appropriate characterization of the resulting products will be required for completion of these coding assignments. nomic 26 s rna of alphaviruses is generated in this manner (brzeski and kennedy, 1978) . the second mechanism, again, involves the synthesis of a full length negative strand copy of the genome. this is then transcribed to a full length positive strand rna. the subgenomic rnas are then derived by nucleolytic cleavage of this full length positive strand rna. in the third model, the subgenomic rnas are each transcribed from subgenomic negative strand templates. these templates could originate by nucleolytic cleavage of genome length negative strand rna or by internal termination of transcription. we have been unable to demonstrate any rnase-resistant, and therefore presumably double-stranded, rnas in infected cells. present data are not sufficient to distinguish between these three models or a combination of them. further experiments using ultraviolet mapping of transcription initiation sites and the use of positive strand probes to identify negative strand templates may elucidate the mechanism of transcription of coronavirus-specific rna. synthesis of alphavirus-specified rna bovine coronavirus genome intermediates in the synthesis of type 2 adenovirus deoxyribonucleic acid rna of mouse hepatitis virus enhanced autoradiographic detection of %p and '%i using intensifying screens and hypersensitized film sequence studies of poliovirus rna. iv. nucleotide sequence complexities of poliovirus type 1, type 2, and two type 1 defective interfering particles rnasl and fingerprint of the poliovirus type 3 genome murine coronavirus rna biological properties of avian coronavirus genome of infectious bronchitis virus the genome of human coronavirus strain 2293 analysis of single-and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange ribonucleic acid synthesis in porcine cell cultures infected with transmissible gastroenteritis virus amount of viral dna in the genome of cells transformed by adenovirus type 2 replication of mengovirus. i. effect on synthesis of macromolecules by host cell coronaviridae. in "comprehensive virology pathogenic murine coronaviruses. i. characterization of biological behavior in vitro and virus specific intracellular rna of strongly neurotropic jhmv and weakly neurotropic a59v viruses pathogenic murine coronaviruses. iii. biological and biochemical characterization of temperature sensitive mutants of jhmv 197'7). presence of infectious polyadenylated rna in the coronavirus avian bronchitis virus coronavirus jhm: cell-free synthesis of structural protein ~60 coronavirus multiplication strategy. i. identification and characterization of virus-specified rna coronavirus multiplication strategy. ii. mapping the avian infectious bronchitis virus intracellular rna species to the genome characterization of a coronavirus. i. structural proteins: effects of preparative conditions on the migration of protein in polyacrylamide gels enhanced growth of a murine coronavirus in transformed mouse cells presence of genomic polyadenylate and absence of detectable virion transeriptase in human coronavirus oc-43 coronaviridae isolation and characterization of the virus rna and the subgenomic mrnas of mouse hepatitis virus mhv a59 genomic rna of the murine coronavirus jhm structural polypeptides of the murine coronavirus jhm comparison of the rnas of murine and human coronaviruses the dynamics of maternal poly (a)-containing mrna in fertilized sea urchin eggs polyadenylate in the virion rna of mouse hepatitis virus the authors would like to thank dr. ian kennedy for teaching us the technique of oligonucleotide fingerprinting and the method for recovering rna from agarose gels. we are grateful to david stern and dr. kennedy for making their results available to us prior to publication. the encouragement of dr. peter lampert and dr. michael oxman are gratefully acknowledged. we would like to thank jim de vries and eric paavola for excellent technical assistance. finally, the question arises about how the subgenomic rnas are synthesized. three mechanisms are possible. in the first mechanism, a full length negative strand copy is made from the positive strand genomic rna. each subgenomic rna species is then separately transcribed from this negative strand template. this model would involve internal initiation of transcription. key: cord-325423-d212h4bp authors: carrion, ricardo; ro, youngtae; hoosien, kareema; ticer, anysha; brasky, kathy; de la garza, melissa; mansfield, keith; patterson, jean l. title: a small nonhuman primate model for filovirus-induced disease date: 2011-11-01 journal: virology doi: 10.1016/j.virol.2011.08.022 sha: doc_id: 325423 cord_uid: d212h4bp ebolavirus and marburgvirus are members of the filovirus family and induce a fatal hemorrhagic disease in humans and nonhuman primates with 90% case fatality. to develop a small nonhuman primate model for filovirus disease, common marmosets (callithrix jacchus) were intramuscularly inoculated with wild type marburgvirus musoke or ebolavirus zaire. the infection resulted in a systemic fatal disease with clinical and morphological features closely resembling human infection. animals experienced weight loss, fever, high virus titers in tissue, thrombocytopenia, neutrophilia, high liver transaminases and phosphatases and disseminated intravascular coagulation. evidence of a severe disseminated viral infection characterized principally by multifocal to coalescing hepatic necrosis was seen in ebov animals. marv-infected animals displayed only moderate fibrin deposition in the spleen. lymphoid necrosis and lymphocytic depletion observed in spleen. these findings provide support for the use of the common marmoset as a small nonhuman primate model for filovirus induced hemorrhagic fever. ebolavirus (eboz) and marburgvirus (marv), members of the filoviridae family, are causative agents of hemorrhagic fever. filovirusinduced disease begins with flu-like symptoms and progresses rapidly to the final stages of viral hemorrhagic fever infection, which are characterized by fever, hemorrhage, and hypotensive shock (groseth et al., 2007; zampieri et al., 2007) . infection is fatal in up to 90% as in the case of zaire ebolavirus or marburg angola. the increased frequency of filovirus outbreaks in central and western africa and the potential use of such agents as biological weapons underscore the need to understand pathogenesis of these viruses and to develop effective intervention strategies (groseth et al., 2007; peterson et al., 2004a,b) . nonhuman primates (nhps) are the preferred animal model for human filovirus infection because infection with eboz and marv isolated from humans results in fatal hemorrhagic disease. numerous species of nhps have been used, including baboons, african green monkey, rhesus and cynomolgus macaques, to study filovirus pathogenesis because the pathology of infected nhps is similar to that seen in humans (bente et al., 2009; geisbert et al., 2003b) . the sporadic nature of disease outbreaks and the ethical issues associated with conducting a human vaccine trial make such a study difficult to execute. early-stage development of vaccines has historically occurred in mouse and guinea pig models of filovirus disease, but these models use adapted viruses obtained through sequential passage in the rodent species because the wild-type virus does not cause uniform lethality (bray et al., 1999; connolly et al., 1999) . the disease observed in rodents does not reproduce the extensive disseminated intravascular coagulation seen in human filovirus disease. besides the requirement for an adapted virus, there are reports that the rodent models are not necessarily predictive of efficacy in nhps (feldmann et al., 2003; geisbert et al., 2002; jahrling et al., 1996) . nhps are relevant models to study infectious disease as their immune system is similar to humans and they are good predictors of efficacy in vaccine development and other intervention strategies. licensure of a filovirus vaccine will require testing in a nhp species. the macaque model is the most frequently used model in filovirus efficacy studies involving vaccines or therapies. given the drawbacks of rodent models of filovirus disease, there is a critical need for development of a small animal model that develops disease consistent with human infection using wild-type virus. the common marmoset (callithrix jacchus) is an anthropoid primate weighing between 320 and 450 g that is used extensively in biomedical research and provides an attractive alternative to other nhps (mansfield, 2003) . this small-bodied primate is especially valuable in maximum containment research, where space is generally at a premium. additionally, reagents are available to characterize immunological response to vaccines. marmosets have been used to study a number of viral diseases including hemorrhagic fever viruses such as lassa and junin viruses, as well as eastern equine encephalitis virus, sars-cov, and hepatitis gb virus b, as well as other human syndromes (adams et al., 2008; avila et al., 1985; bright et al., 2004; carrion et al., 2007a; greenough et al., 2005; jacob et al., 2004; lukashevich et al., 2008; mansfield, 2003; weatherford et al., 2009; weissenbacher et al., 1982) . marmoset models of lassa hemorrhagic fever and argentine hemorrhagic fever have been used to validate countermeasures against the hfvs (avila et al., 1987; carrion et al., 2007b; samoilovich et al., 1984; weissenbacher et al., 1986a,b) . here we report that a single intramuscular injection of a common marmoset with as little as 10 plaque forming units (pfu) of either marv or ebov resulted in fatal hemorrhagic disease. the experimental filovirus infections induced a systemic disease with histological features similar to human infection, most notably hepatocellular necrosis and extensive fibrin deposition. the availability of a marmoset model for filovirus induced disease is especially attractive since it provides a small animal model for preclinical trials that is susceptible to viruses isolated from humans without adaptation. all animals appeared normal until 3 days post-infection (dpi) when weight loss and anorexia was observed. animals infected with 1000 pfu ebov were also febrile (n103°f) at this time point. animals infected with 10 pfu of ebov developed a fever 4 dpi. similarly, marv infected animals developed a fever at days 5 and 6 for the high dose and low dose, respectively. after the onset of fever, the behavior of animals changed: they became depressed, had reduced stool production, and weight loss (5-7% from baseline). just before death, febrile response resolved and some animals became hypothermic. marmosets infected with ebola virus succumbed to disease at 4 to 5 days dpi while marv infected animals succumbed to disease at six to eight dpi. complete blood counts and biochemical analyses (table 1) were performed on blood collected from marmosets. beginning at 4 dpi, blood contained elevated levels of serum alanine aminotransferases (alt), a marker of hepatocellular necrosis, which continued to increase until the animal was euthanized. ggt (gamma-glutamyltransferase) and alp (alkaline phosphatase), measured to evaluate excretory liver functions, were elevated n30-fold above baseline. further evidence of hepatic involvement was noted in marv animals as a significant increase in total bilirubin (tbil) observed just prior to death (n3 mg/dl). hematological data showed a reduction in numbers of platelet in all animals over the course of infection with development of leukocytosis ( fig. 1) . all animals experienced an increase in neutrophils and concomitant decrease in lymphocytes beginning at 2 dpi (fig. 1 ). the virulence of filoviruses in humans is related to viremia, suggesting that rate of virus replication is an important factor of pathogenesis (towner et al., 2004) . at necropsy, tissue was collected from marmosets for determination of viral load by qrt-pcr. virus was detected by 3 dpi, resulting in viremia greater than 5 to 7 log 10 genome equivalents (ge) per milliliter on the day of necropsy. all tissues contained amounts of viral rna comparable with levels of viremia (table 2) . at necropsy, differences in peak viral rna level in the tissues of animals infected with low (10 pfu) and high (1000 pfu) doses of ebov was observed. viral load in blood from marv infected animals ranged from 2.88 log 10 at day 2 to 8.15 log 10 at day 7. lesions seen in both ebov marmosets and marv marmosets were similar. hepatomegaly was seen in all animals and was often accompanied by multifocal pigmentation and overall discoloration of the liver. splenomegaly was also a common finding at necropsy. most animals had lung abnormalities in most lobes, primarily hemorrhage and congestion. hemorrhage was frequently noted at the site of venipuncture. other findings included hemorrhage in bladder and adrenals as well as light pigmentation present in lymph nodes. microscopic examination of tissue sections stained with hematoxylin and eosin revealed a number of pathologic lesions (tables 3 and 4) . renal lesions were observed in all ebov-infected animals ( fig. 2a) , and consisted of fibrin microthrombi lodged within capillary beds of the glomerular tufts. thrombi were apparent diffusely and often associated with expansion of bowman's space and accumulation of proteinaceous fluid. polymorphonuclear cells were infrequently observed in glomerular tufts and glomerular cells were necrotic. in conjunction, hypoxic nephrosis was evident in the proximal convoluted tubules of most animals and was likely secondary to microthrombosis of the glomerular tufts. marv-infected animals were dissimilar: renal morphology was within normal limits (fig. 3a ). lungs from all animals revealed fibrin thrombi present within large-to medium-sized pulmonary arteries (fig. 2b ). thrombi often contained increased numbers of neutrophils and pyknotic debris. perivascular lymphatics adjacent to these vessels were often dilated and contained protein. lungs were diffusely congested and multiple foci of fibrin microthrombi were evident in many small arterioles, extending into capillary beds and associated with congestion and hemorrhage. occasionally extravasated erythrocytes were mixed with evidence of proteinaceous exudates in alveolar spaces. in contrast, pulmonary changes were minimal to mild in marvinfected marmosets. one animal (29015) receiving 10 pfu and one receiving 1000 pfu developed mild edema surrounding small pulmonary arteries. in addition, three animals had evidence of a mild focal interstitial pneumonitis characterized by neutrophilic infiltration, septal necrosis and variable alveolar edema. it is unlikely that the pulmonary findings were of clinical significance. hepatic lesions in ebov-infected marmosets were observed in all animals but differed dependent on the dose and day of death (fig. 2c ). in animals inoculated with 1000 pfu of ebov, hepatic lesions were mild and consisted of multifocal infiltrates of polymorphonuclear cells within hepatic sinusoids associated with evidence of early necrosis of sinusoidal cells. viral inclusions were not observed. in animals inoculated with the 10 pfu and surviving to 5 dpi, hepatic disease was more severe and characterized by multifocal to coalescing hepatocellular coagulative necrosis infiltrated by small to moderate numbers of neutrophils. hepatocytes within and adjacent to these regions of necrosis often contained large amphophilic intracytoplasmic inclusions (fig. 2d ). inclusions had an asymmetric perinuclear distribution. these more severe changes were accompanied by intracellular biliary stasis. the findings suggest initial targeting of resident sinusoidal cells (kupffer and monocytes) with subsequent extension to adjacent hepatocytes. marv-infected animals all displayed multifocal to coalescing hepatocellular necrosis. necrotic foci varied from individual hepatocytes to small aggregates of cells up to 300 μm in diameter. necrotic hepatocytes had rounded and deeply eosinophilic homogeneous to granular appearing cytoplasm with pyknotic or karyorrhectic nuclei (fig. 3b ). occasionally, hepatocytes in the early phases of degeneration had well defined eosinophilic bodies present within the cytoplasm (eosinophilic degeneration) but the amphophilic inclusions were infrequently observed (fig. 3c) . dispersed within and adjacent to these regions of necrosis were increased numbers of neutrophils found within sinusoids and mixed with the necrotic debris. in four animals there was evidence of mild to moderate hepatocellular dissociation. the degree of hepatocellular necrosis was similar regardless of inoculum. spleens of all ebov-infected animals were depleted of lymphocytes and contained deposits of fibrin (fig. 2e) . extensive fibrin deposits were observed within the red pulp. these changes were accompanied by necrosis of cells within the cords of billroth in severely affected animals. lymphocyte depletion was severe and appears to be focused on periarteriolar sheaths (t cell regions) and to a lesser extent follicular (b cell) regions. in most residual areas of white pulp, pyknotic and karyorrhectic nuclear debris was evident and these regions were infiltrated by polymorphonuclear cells at multiple foci. in marmosets given 1000 pfu of virus, involvement of b cell regions was significantly less. in contrast, all animals receiving 1000 pfu marv inoculum had evidence of marked lymphocyte depletion and lymphoid necrosis in the spleen (figs. 3d and e). periarteriolar sheaths and follicular b cell areas were reduced in size and contained pyknotic and karyorrhectic debris. the changes were present to a lesser degree in the 10 pfu group, suggesting that viral dose may have been responsible for the observed differences. fibrin deposition was observed in the medullary cords and correlated with the degree of lymphoid necrosis and lymphocytic depletion. in two ebov animals, mild to moderate lymphocytic hyperplasia was observed in cortical parafollicular regions, accompanied by increased numbers of tingible body macrophages. in these nodes, scattered areas of hemorrhage and necrosis were occasionally apparent. in contrast, in the two remaining animals, there was evidence of extensive lymphocytic necrosis that was focused in medullary and parafollicular regions, while follicles were spared. these changes were accompanied by hemorrhage, neutrophilic infiltration and extensive fibrin deposition (fig. 2f) . changes in lymph node morphology were apparent in only 2 of 6 marv-infected animals, and consisted of mild to moderate lymphoid necrosis and lymphocytic depletion. in the remaining animals, there was an absence of secondary follicles with germinal centers but lymphocyte numbers appeared normal (fig. 3f) . fibrin microthrombi were observed in the adrenal glands of some ebov-infected animals and were most frequently observed in the subcapsular region in the zona glomerulosa. thrombi were accompanied by congestion and hemorrhage. multifocal dissociation and necrosis of cortical adrenocytes was evident in a subset of animals and was occasionally accompanied by infiltration by a small number of polymorphonuclear cells. no viral inclusions were evident. fibrin was not observed in marv animals. within adrenal glands there were multiple scattered areas of necrosis of individual and small aggregates of adrenocytes accompanied by the presence of erythrocytes, lymphocytes and mononuclear cells. lesions were observed inconsistently in other organs and were often related to the effects of disseminated intravascular coagulation and terminal hypoxia. these included evidence of myocardial hypoxic necrosis, and microhemorrhages in the brain, heart, urinary bladder and pancreatic islets. two animals infected with 1000 pfu of marv had evidence of a mild focal fibrosis, which dissected along muscle fascicles and was associated with scant histiocytic and lymphocytic infiltrates. findings in other organs were unremarkable. we report for the first time that the common marmoset is susceptible to experimental infection with viruses from the family filoviridae. the intramuscular inoculation of as little as 10 pfu of either ebov or marv induced pathological features similar to those observed in human disease. most notably, animals experienced thrombocytopenia, neutrophilia and disseminated intravascular coagulation. furthermore, the small nonhuman primate experiences a disease syndrome comparable to what has been reported in other nonhuman primate models currently used to study filovirus disease. cynomolgus macaques are frequently used in preclinical testing of filovirus vaccine and therapeutic strategies and are considered the "gold standard" animal model (bente et al., 2009) . experimental ebov infection of macaques by intramuscular injection of 1000 pfu of virus results in a rapid fatal disease. beginning at 4 dpi, macaques experienced anorexia coinciding with the onset of fever. shortly after these initial findings, petechial rash was seen with varying degrees of recumbency, culminating in prostration and death at 7 days post-challenge (geisbert et al., 2003d) . we observed a similar, although shortened (death 4-5 dpi), course of disease in marmosets infected with ebov. previous work has shown that intramuscular inoculation of macaques with marv results in an analogous course of disease; however, overall disease progression was delayed with death occurring between days 8 and 13 (bente et al., 2009; geisbert et al., 2007; jaax et al., 1996) . consistent with the macaque model, experimental inoculation of marmosets with marv also results in delayed onset of disease, with death occurring 3 to 4 days later than that seen with marmosets infected with ebov. however, the course of marburg disease in marmosets was more rapid than that seen in macaques, with death occurring at 6 to 8 dpi. although not statistically significant, it is interesting to note that marv infected marmosets had an average time to death 1 day shorter than those receiving a high dose of virus. human filovirus infection is marked by neutrophilia, lymphopenia and thrombocytopenia. these findings are observed in the macaque model of filovirus disease and are used as surrogate markers of disease progression for in vivo studies (geisbert et al., 2003b; simpson, 1969; simpson et al., 1968) . marmosets infected with either of the filoviruses also display these hematological abnormalities. beginning at 2 dpi, overall platelet counts decreased while neutrophil numbers increased, with a concomitant decrease in lymphocyte numbers (fig. 1) . autopsy findings from fatal human cases reveal hepatomegaly with necrotic foci observed throughout liver. likewise, experimentally infected macaques developed numerous hepatic lesions (geisbert et al., 2003b (geisbert et al., ,c,d, 2008 jaax et al., 1996) . the marmoset showed biochemical signs of liver involvement early in infection: markers of liver function (alt, alp, ggt) reached levels in excess of 30-fold above baseline at necropsy. consistent with high-levels of liver enzymes, gross examination of the liver revealed hepatomegaly with pale foci throughout all lobes. microscopic examination of sections from the liver revealed necrosis with mild to moderate inflammation. hepatic changes did not appear to be virus dose-dependent, although further animal or morphometric studies would be needed to confirm this. evidence of mild to moderate hepatocellular fatty change was present. similar findings have been documented in the macaque model of filovirus infection and most importantly, the findings reported here are comparable to those reported in fatal human cases (geisbert et al., 2003b (geisbert et al., ,c, 2008 jaax et al., 1996) . one difference between the macaque model of filovirus infection and marmosets is that marmosets do not develop a petechial rash. in this respect, they appear to be more similar to the african green monkey model of filovirus infection (bente et al., 2009; davis et al., 1997) . we speculate that the rapid disease progression preempts its development. nonetheless, clear signs of coagulation abnormalities are pronounced, as animals experience thrombocytopenia, hemorrhage and bleeding at sites of venipuncture. fatal human cases are characterized by hemorrhage and bleeding at site of venipuncture and other coagulation abnormalities. the coagulopathy observed in humans at times exists in the absence of rash: only 50% of patients infected with ebov develop a maculopapular rash (bwaka et al., 1999) . further evidence that the marmoset mimics human disease is that microscopic examination of tissue from ebov-infected animals reveals widespread fibrin deposition that is a hallmark of coagulation abnormalities (geisbert et al., 2003c,d; jaax et al., 1996) . ebov infection of the marmoset caused a severe disseminated viral infection characterized principally by microthrombosis in multiple organs (disseminated intravasculature coagulation). to a lesser degree, marvinfected animals displayed moderate fibrin deposition in the spleen. a similar observation has been reported for rhesus macaques infected with marburgvirus angola where deposition of polymerized fibrin was less pronounced than in ebov infected macaques (geisbert et al., 2007) . these findings are similar to that seen in human infection and the macaque (geisbert et al., 2003a,b,c; jaax et al., 1996) . signs of coagulopathy characteristic of primate infections are observed variably in rodent models (bente et al., 2009; bray et al., 1999; connolly et al., 1999) . high levels of viremia in human filovirus infections are associated with high mortality. a 2-log difference in titers is sufficient to predict clinical outcomes (towner et al., 2004) . inoculation of macaques with either ebov or marv results in high viral loads in both blood and tissue (geisbert et al., 2003d ). in the current study, all marmosets had high virus loads (n10 6 ) in blood and tissue regardless of dose or agent. consistent with human cases, virus was widely disseminated, with the spleen and liver frequently having levels 1-log higher than other tissue. in animals given 1000 pfu ebov, viral load was 2-logs less than those in animals receiving 10 pfu of virus. this difference could be a result of a rapid onset of morbidity affecting viral load. virus load in animals infected with marv was similar and likely the result of relatively longer time to death compared to ebov animals. it is worthwhile noting that recently it has been shown that filovirus replication results in a surplus of genomes with varying degrees of packaging efficiency and infectious particles. in the in vitro studies, the ratio of rna to focus forming units was 100-fold more in ebov versus marv (weidmann et al., 2011) . taking this into account, the difference in virus load between marv and ebov infected marmosets may be correspondingly different. differences in histopathology were observed between animals inoculated with marv and ebov. ebov infection resulted in widespread intravascular coagulation apparent in multiple organs including the spleen, adrenal gland, kidney and lung ( fig. 2 and table 3 ). there was evidence of extensive lymphocytic necrosis focused in medullary and parafollicular regions, while follicles were spared. these changes were accompanied by hemorrhage, neutrophilic infiltration and extensive fibrin deposition (fig. 2f) . findings suggest initial activation within t lymphocyte areas followed by subsequent and rapid lymphocytic depletion, most likely through apoptotic mechanisms. comparing changes to changes observed in the spleen suggests alterations in the lymph node lag behind those observed in the spleen. marv animals inoculated with 1000 pfu had moderate fibrin deposition in the spleen, widespread intravascular coagulation was not observed, suggesting a significant difference in pathogenesis between the two agents ( fig. 3 and table 4 ). the observation that hepatic lesions are less severe in animals receiving high dose of virus versus a lower dose is difficult to explain with a small sample size. the virus preparation used to infect the monkeys was prepared at a low moi in order to minimize the effect of di particles. it is known in filovirus infection, hepatic lesions early in infection are less severe than those at terminal endpoints (geisbert et al., 2003a, b,c,d) . perhaps the relatively quick time to death in those animals infected with high dose of virus preempted the development of severe liver pathology. our study has shown that intramuscular inoculation of marmosets with ebov or marv causes hemorrhagic fever reminiscent of human infection. since nonhuman primates are more predictive of filovirus therapeutic efficacy than rodents, the small-sized marmoset is a viable alternative small animal model. the marmoset size is comparable to that of a guinea pig, making it attractive for testing therapeutic reagents that are only available in small quantities. in addition, the neotropical monkey can serve to alleviate the shortage of macaques available for research (patterson & carrion, 2005; satkoski et al., 2008) . that the common marmoset is susceptible to wild-type, non-adapted virus is especially attractive to those wishing to evaluate countermeasures in a small animal model with viruses derived from fatal human cases. biosafety eboz and marv are risk group 4, category a biothreat agents. all experiments with this virus were performed within a biosafety level 4 (bsl4) facility by personnel in a biosafety suit at the texas biomedical research institute (san antonio, tx). both the institutional animal care and use committee and the institutional biohazards committee at texas biomed approved experimental protocols. virus strains ebov (kikwit) and marv musoke were provided by dr. peter jahrling (usamriid, fort detrick, maryland) . stocks were used to infect tissue culture flasks containing vero e6 cells at 90% confluency. the flasks were infected at an moi of 0.01 then incubated until cytopathic effect greater than 80% was observed then harvested and stored in aliquots in an ultralow temperature freezer. infectivity of virus stocks was determined by plaque titration (moe et al., 1981) . ten adult marmosets ranging in weight from 331 g to 425 g were obtained from the southwest national primate research center in san antonio, texas. one week before the start of the study, animals were transferred to the bsl4 facility at texas biomed and housed individually in a climate controlled caging. at day 0, animals were injected intramuscularly (0.5 ml) with either ebov (10 pfu, n =2; 1000 pfu, n =2) or marv (10 pfu, n = 2; 1000 pfu, n = 4) diluted in pbs. animals were sedated and blood was collected at days 0, 2, 5 and 7 for analysis. animals were euthanized when moribund using approved methods. all procedures were approved by the texas biomed institutional animal care and use committee and were performed in accordance with applicable federal guidelines. biochemical analysis of plasma samples was performed using mammalian liver enzyme profile rotor on vet scan analyzer (abaxis, inc., union city, ca). complete blood counts were performed using a vet hmt machine (abaxis, inc., union city, ca). tissue was collected using sterilized scalpels and forceps. to minimize contamination of tissue with blood, tissue samples were harvested after blood collection. aseptically collected tissues were fixed in phosphatebuffered 4% paraformaldehyde (ph 7.2) and embedded in paraffin for histological examination. paraffin-embedded tissues were cut in 5-μm sections, deparaffinized and stained with hematoxylin and eosin. total rna isolation and qrt-pcr viral load in tissues were measured by quantitative rt-pcr (qrt-pcr). tissue was homogenized with trizol reagent (invitrogen) using the tissuelyser (qiagen inc., valencia, ca) homogenization system and rna extracted per the manufacturer's recommendations. real-time qrt-pcr was performed using an abi prism 7700 sequence detection system (applied biosystems, foster city, ca) and rna ultrasense onestep real-time qrt-pcr system (invitrogen). the ebov qrt-pcr assays were performed as previously described (sun et al., 2009) . marv qrt-pcr was performed as described above using a primer/probe set directed at a region within the marv glycoprotein (magp-f: 5′ggccttcaggg-caggtgta 3′, magp-r : 5′ cctgtgcatgagggttttga 3′, mmgp-probe: 5′ fam-ccttgctgttagatcctcctaccaa-tamra 3′). common marmosets (callithrix jacchus) as a nonhuman primate model to assess the 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ebola virus we thank jerritt nunneley, michele reynolds, robert geiger, hilary staples, and juan zapata for technical assistance and helpful discussions. we thank bernadette guerra and robert lanford of the texas biomed molecular core laboratory for assistance with qrt-pcr assays. we thank sandra rios, maria messenger and april hopstetter for assistance in preparing this manuscript and anthony griffiths for critical review of this manuscript.financial support: this work was supported by the national institutes of health (nih) for regional centers of excellence for biodefense and emerging infectious diseases (grants u54 ai057156 and u54 ai57168), an nih laboratory construction grant (c06 rr12087), and a grant from the southwest foundation forum. we acknowledge the new england primate center (grant p51 rr00168-45) for histopathology support and the southwest national primate research center base grant (p51 rr013986). key: cord-305564-dj3vj4tk authors: dediego, marta l.; pewe, lecia; alvarez, enrique; rejas, maria teresa; perlman, stanley; enjuanes, luis title: pathogenicity of severe acute respiratory coronavirus deletion mutants in hace-2 transgenic mice date: 2008-07-01 journal: virology doi: 10.1016/j.virol.2008.03.005 sha: doc_id: 305564 cord_uid: dj3vj4tk recombinant severe acute respiratory virus (sars-cov) variants lacking the group specific genes 6, 7a, 7b, 8a, 8b and 9b (rsars-cov-δ[6-9b]), the structural gene e (rsars-cov-δe), and a combination of both sets of genes (rsars-cov-δ[e,6-9b]) have been generated. all these viruses were rescued in monkey (vero e6) cells and were also infectious for human (huh-7, huh7.5.1 and caco-2) cell lines and for transgenic (tg) mice expressing the sars-cov receptor human angiotensin converting enzyme-2 (hace-2), indicating that none of these proteins is essential for the viral cycle. furthermore, in vero e6 cells, all the viruses showed the formation of particles with the same morphology as the wt virus, indicating that these proteins do not have a high impact in the final morphology of the virions. nevertheless, in the absence of e protein, release of virus particles efficacy was reduced. viruses lacking e protein grew about 100-fold lower than the wt virus in lungs of tg infected mice but did not grow in the brains of the same animals, in contrast to the rsars-cov-δ[6-9b] virus, which grew almost as well as the wt in both tissues. viruses lacking e protein were highly attenuated in the highly sensitive hace-2 tg mice, in contrast to the minimal rsars-cov-δ[6-9b] and wt viruses. these data indicate that e gene might be a virulence factor influencing replication level, tissue tropism and pathogenicity of sars-cov, suggesting that δe attenuated viruses are promising vaccine candidates. the etiologic agent causing severe acute respiratory syndrome (sars) is a novel coronavirus (cov) named sars-cov fouchier et al., 2003; ksiazek et al., 2003; kuiken et al., 2003; marra et al., 2003; peiris et al., 2003; rota et al., 2003) . the disease, which caused an average mortality of approximately 10% and for which no defined therapy is available, was reported for the first time in guandong province, china, at the end of 2002, and rapidly spread to 32 countries. after july 2003, only four community acquired cases were reported in china, although there have been three instances of laboratory-acquired infections described (http://www.who.int/csr/ sars/en/). initial investigations indicated that sars-cov spread to humans from infected wild animals in wet markets of southern china, such as himalayan palm civets (paguna larvatta) and chinese ferret badgers (melogale moschatta) . nevertheless, the recognition of sars-like covs in bats suggests that these species are most likely the natural reservoir of sars-cov li et al., 2005) . therefore, the reemergence of the virus remains a possibility and the engineering of attenuated viruses as research tools and vaccine candidates is of high interest. sars-cov is an enveloped virus of the coronaviridae family, and has a single-stranded, positive sense 29.7 kb rna genome (gorbalenya et al., 2004; snijder et al., 2003) . human coronaviruses have been divided into different groups (enjuanes et al., 2008b) . group 1 includes the human coronavirus 229e (hcov-229e), generally associated with the common cold, and hcov-nl63, which causes more severe lower respiratory diseases (fouchier et al., 2004; kaiser et al., 2005; van der hoek et al., 2004) . group 2 human covs include hcov-oc43, which has been associated with common colds, the recently described hcov-hku1, which was identified in adults with pneumonia , and sars-cov. among human covs, sars-cov causes the most severe disease (weiss and navas-martin, 2005) . coronaviruses replicate in the cell cytoplasm and encode a nested set of mrna molecules of different sizes. viral genome expression begins with the translation of two large polyproteins, pp1a and pp1ab, including the viral replicase genes . these genes are involved in genome replication and transcription of subgenomic mrnas (sg mrnas), encoding structural proteins such as the spike (s), envelope (e), membrane (m), and nucleocapsid (n), and a set of groupspecific proteins, whose sequence and number differs among the different species of coronavirus (enjuanes et al., 2008b) . in the case of sars-cov, open reading frames (orfs) 3a, 6, 7a, and 7b encode additional structural proteins (huang et al., 2006 ito et al., 2005; schaecher et al., 2007; shen et al., 2005) . genes 3a, 3b, 6, 7a, 7b, 8a, 8b and 9b of sars-cov are not found in other covs and their functions in the viral cycle are not well understood. some of these genes are implicated in the pathogenesis of the virus. genes 6 and 3b inhibit interferon function kopecky-bromberg et al., 2007) . in addition, it has been shown that sars-cov gene 6 accelerates murine coronavirus infections (tangudu et al., 2007) and enhances virulence of an attenuated murine hepatitis virus (mhv) (pewe et al., 2005) . similarly, it has been shown that the group specific genes of other covs contribute to the pathogenesis of the virus in vivo, but are not essential for viral replication (de haan et al., 2002; ortego et al., 2003) . sars-cov proteins 3a, 3b, e, m, 7a, and 8a induce apoptosis via several mechanisms khan et al., 2006; lai et al., 2006; law et al., 2005; schaecher et al., 2007; tan et al., 2007; yang et al., 2005) . furthermore, induction of t-cell apoptosis by e protein may contribute to the lymphopenia that is observed in sars patients . coronavirus proteins m and e are key factors in virus assembly and budding (corse and machamer, 2000 de haan et al., 1998; fischer et al., 1998; kuo and masters, 2003) . in addition, we have shown that deletion of e protein leads to a loss of virulence in hamsters (dediego et al., 2007) . furthermore, sars-cov proteins 7a and 9b appear to be associated with intracellular vesicle structures, suggesting a possible role in morphogenesis and replication (meier et al., 2006; nelson et al., 2005) . there is evidence that sars-cov group-specific genes can be deleted individually with very limited or no impact on in vitro and in vivo growth in a murine model (sims et al., 2008; yount et al., 2005) . some of these genes may have redundant functions, as it has been shown that genes 3b, 6, n, nsp1, and nsp3 may all act as interferon antagonists (devaraj et al., 2007; frieman et al., 2007; kopecky-bromberg et al., 2007; wathelet et al., 2007) . these results prompted us to study the role of e gene in the context of full-length genome or using a virus lacking six accessory genes (6, 7a, 7b, 8a, 8b, and 9b). sars-cov infects and replicates in mice, ferrets, hamsters, cats, and several species of non human primates (cynomolgus and rhesus macaques, african green monkeys, and marmosets) (subbarao and roberts, 2006) . nevertheless, an ideal animal model that completely reproduces human clinical disease and pathological findings is still missing. to overcome these limitations, several approaches have been taken. in one of them, a mouse adapted sars-cov was selected and shown to cause an infection that reproduced many aspects of severe sars . interestingly, in other approaches, tg mice expressing the host receptor for sars-cov, hace-2, have been developed (mccray et al., 2007; tseng et al., 2007) . these mice are very susceptible to sars-cov and are especially useful for pathogenesis studies. in this paper, the pathogenicity of a collection of sars-cov deletion mutants, including those with deletion of the group-specific proteins 6, 7a, 7b, 8a, 8b and 9b, the envelope protein e, or a combination of both, has been evaluated in a tg mouse in which the expression of hace-2 was targeted to epithelial cells. the data presented herein show that genes e, 6, 7a, 7b, 8a, 8b, and 9b are not essential for in vitro and in vivo replication even when all are simultaneously deleted. interestingly, viruses lacking gene e are attenuated and do not grow in the central nervous system of hace2 tg mice, whereas virus lacking genes 6 to 9b, which is not significantly attenuated, grows in the brains of infected mice as well as wt virus. together, the data indicate that gene e behaves as a virulence factor that contributes to pathogenicity by favoring sars-cov replication within specific tissues. to study the contribution of the group specific genes 6, 7a, 7b, 8a, 8b and 9b, and of the structural gene e to viral pathogenesis in hace-2 tg mice, viruses with these genes deleted were constructed using an infectious cdna clone assembled as a bac (almazan et al., 2006) . to abolish expression of genes 6 to 8b, a deletion comprising nucleotides 27065 to 27977, covering completely orfs 6, 7a, 7b, 8a, and part of orf 8b was introduced (fig. 1a) . to abolish expression of gene 9b, point mutations that abrogated the initiator atg codon and two in phase downstream atgs, and one introducing a stop codon were engineered (fig. 1a) . it was not possible to delete 9b gene, as it totally overlaps with gene n, an essential gene for the virus replication (almazan et al., 2004) . expression of e gene was abolished as previously described (dediego et al., 2007) . infectious viruses were rescued in vero e6 cells transfected with plasmids pbac-sars-cov-δ[6-9b], pbac-sars-cov-δe, pbac-sars-cov-δ[e,6-9b], or pbac-sars-cov fl as a control, indicating that genes e, 6, 7a, 7b, 8a, 8b and 9b are not essential for virus viability. nevertheless, smaller plaques were detected after infection with rsars-cov-δe or rsars-cov-δ[e,6-9b] viruses but not with rsars-cov-δ[6-9b] virus, suggesting that e gene is responsible for this plaque phenotype (fig. 1b) . the viruses were cloned by three rounds of plaque purification and amplified twice to obtain working viral stocks. the viruses were sequenced to confirm that all point mutations and deletions were maintained. to analyze whether the deleted genes affected viral replication, growth kinetics of the defective viruses were determined by infecting different cell lines at a moi of 0.05. deletion of genes 6 to 9b did not reduce viral titers in monkey vero e6 cells, whereas a reduction of 20fold was observed for viruses with e gene deleted (rsars-cov-δe and rsars-cov-δ[e,6-9b]) ( fig. 2a) . cytopathic effect was detected at 24 h post-infection. maximal virus titers were detected at 24-48 h postinfection. in human caco-2, huh7.5.1, and huh-7 cells, titers decreased between 5-10 and 100-1000-fold, for rsars-cov-δ[6-9b] and rsars-cov-δe or rsars-cov-δ[e,6-9b], respectively (figs. 2b and c, and data not shown). in human cells, no cytopathic effect was observed, and maximal titers were reached at 48-72 h post-infection. although titers of rsars-cov-δe or rsars-cov-δ[e,6-9b] viruses in caco-2 cells are limited, an increase in virus titers of at least 5-fold was observed at 24-48 h post-infection compared to 0 h post-infection, indicating that the viruses replicate in this cell line. the similarity of the growth kinetics exhibited by the δe and the δ[e,6-9b] viruses indicated that none of the accessory genes 6, 7a, 7b, 8a, 8b, and 9b complemented any function of e gene required for replication in tissue culture. the effect of deleting e, 6, 7a, 7b, 8a, 8b and 9b genes on viral morphogenesis was examined using electron microscopy. the number of intracellular mature virions present in the cellular cytoplasm was lower in cells infected with viruses lacking e gene than in cells infected with either rsars-cov or rsars-cov-δ[6-9b] viruses, which is consistent with the lower titers reached by δe viruses. similarly, the number of virions in the endoplasmic reticulum-golgi intermediate compartment (ergic) regions where nucleocapsid invagination occurs (ng et al., 2003) , and in intracellular vesicles, where the virus accumulates before budding, was lower in cells infected with δe viruses ( fig. 3c and d) but not with rsars-cov or rsars-cov-δ[6-9b] ( fig. 3a and b ). together, these data suggest that the structural e protein influences the efficacy of virus morphogenesis, whereas the other accessory structural proteins 6, 7a, 7b, play a minor role in this process. extracellular virion morphology observed in infected cells ultrathin sections by electron microscopy (fig. 4a) showed the presence of virions with standard spherical morphology in all cases, indicating that the e, 6, 7a, 7b, 8a, 8b and 9b proteins were not essential for final virion morphology. although the morphology of the δe viruses was basically normal, a significant fraction of ellipsoid particles was observed in preparations of rsars-cov-δ[e,6-9b] virus, suggesting that the e protein in conjunction with at least one of the other six deleted proteins influences stability or final virion structure. in the two δe mutants, a higher accumulation of virions within the budding process was observed in relation to the viruses with e protein (fig. 4b ). 62.5% of surface-associated δe viruses, as compared to only 16.6% of e-containing viruses were captured in the process of budding, strongly suggesting that e protein plays a major role in this process. negative staining of concentrated virus preparations revealed virions with spherical morphology in all of the mutants, corroborating that none of these proteins were essential to form viral particles with normal morphology. nevertheless, disrupted particles were more frequently seen in negatively stained δe virion preparations than in those of rsars-cov-δ[6-9b] virions (data not shown), suggesting that these deletion mutants are more sensitive to mechanical shearing forces. the pathogenicity of the defective viruses was evaluated in the tg mice expressing hace2, which are highly susceptible to sars-cov (mccray et al., 2007) . weight loss and mortality of animals intranasally infected with 12,000 pfu were daily evaluated (fig. 5 ). mice infected with rsars-cov or rsars-cov-δ[6-9b] viruses rapidly lost weight, in marked contrast to mice infected with rsars-cov-δe or rsars-cov-δ[e,6-9b] (fig. 5a) , suggesting that δe viruses were attenuated in these mice. furthermore, all animals infected with rsars-cov or rsars-cov-δ[6-9b] died by 5 and 6 days post-infection, respectively, whereas those infected with viruses lacking the e gene survived (fig. 5b) , indicating that the δe viruses were fully attenuated even in this highly sensitive animal model. to further analyze rsars-cov-δ[6-9b] virulence in hace-2 tg mice, a range of virus doses was used to inoculate mice (figs. 5c and d) and compared to those infected with wt rsars-cov. all mice lost weight and died even at relatively low doses (800 pfu) of wt or rsars-cov-δ[6-9b] viruses. only some mice inoculated with a very low dosage (240 pfu) of either rsars-cov-δ[6-9b] or rsars-cov, survived. the survival and weight profiles were very similar for mice infected with wild type or rsars-cov-δ[6-9b] viruses suggesting that rsars-cov-δ[6-9b] was not significantly attenuated in hace-2 tg mice. virus titers in lung (fig. 6a ) and brain (fig. 6b ) of animals infected with 12,000 pfu of the indicated virus mutants were evaluated at 2 and 4 days post-infection. titers were very high and nearly identical in rsars-cov and rsars-cov-δ[6-9b]-infected tissues, suggesting that genes 6 to 9b have little effect on viral replication in these animals. in contrast, titers in lungs of mice infected with the δe viruses were about 100-fold lower, indicating that this gene is important for efficient in vivo virus replication. interestingly, no infectious virus was detected in the brains of rsars-cov-δe and rsars-cov-δ[e,6-9b]infected mice (fig. 6b) , even when the virus was intracranially inoculated (data not shown). in contrast, rsars-cov-δ[6-9b] virus was detected at high titers in the brains of infected hace2 tg mice suggesting that the e protein is important for virus replication and dissemination within this tissue. infected lungs and brains (cerebrum) were further analyzed for histological changes and for viral antigen. at day 4 p.i., viral antigen was present in high amounts in the alveoli of mice infected with rsars-cov or rsars-cov-δ[6-9b], whereas it was detected in low amounts in the alveoli of rsars-cov-δe or rsars-cov-δ[e, 6-9b]infected mice (fig. 7) . cellular infiltrates were detected in peribronchial regions and in the parenchyma mostly in mice infected with viruses expressing the e protein (fig. 7) . in contrast, viral antigen was detected throughout the central nervous system (cns) only in mice infected with rsars-cov or rsars-cov-δ[6-9b], with prominent infection of the cerebrum, thalamus and brainstem, but not the cerebellum. in the olfactory bulb, only the mitral layer of the olfactory bulb was infected (data not shown). the extent of labeling was nearly the same in mice infected with rsars-cov or rsars-cov-δ[6-9b]. consistent with the titer data, no viral antigen was detected in the brains of mice infected with either δe virus. our results show that neither the e protein nor several sars-cov accessory proteins (6a, 7a, 7b, 8a, 8b, and 9b) are required for virus replication in vitro or in hace2 tg mice. growth kinetics assays in monkey and human cell lines, studies of morphogenesis by electron microscopy, and analyses in the tg mice all suggest that sars-cov e gene is a virulence factor. interestingly, rsars-cov-δ[6-9b] virus, in which six genes were deleted, grew as well as the wt virus in monkey vero e6 cells whereas mutants in which e gene was deleted (rsars-cov-δe, and rsars-cov-δ[e,6-9b]) showed a 20-fold titer reduction. in human cells, titers of rsars-cov lacking either 6-9b or e genes were reduced up to 10-or 1000-fold, respectively, suggesting that gene e is relevant for viral replication as previously described (dediego et al., 2007) , and that genes 6 to 9b have a significant, although very limited influence on viral replication at least in some cell systems. the reduced growth of the wt and the three deletion mutants in human as opposed to vero e6 cells, could be due to the absence of type 1 interferon expression in the latter (emeny and morgan, 1979) . the hace-2 tg mice is a suitable animal model to evaluate the virulence of the defective viruses, as it has been shown that all the animals died even when infected with low doses of wt sars-cov. using highly susceptible hace-2 tg mice, we found, surprisingly, that virus titers of wt virus and rsars-cov-δ[6-9b], were similar in both the lung and brain. furthermore, weight loss and survival curves were also similar, indicating that rsars-cov-δ[6-9b] is not significantly attenuated in these mice. the virulence of the δ[6-9b] deletion mutant came as a surprise as in the coronaviridae, the functions of the group specific orfs are generally associated with counteracting host defenses. in fact, recombinant mhv, feline infectious peritonitis virus (fipv) and transmissible gastroenteritis virus (tgev) lacking one or more of these group specific orfs efficiently replicate in cell culture but are attenuated in vivo (de haan et al., 2002; haijema et al., 2004; ortego et al., 2003) . on the other hand, the virulence of rsars-cov-δ[6-9b] virus is in agreement with previous results showing that recombinant sars-covs individually lacking each of the group specific genes 3a, 3b, 6, 7a, and 7b were not significantly impaired in replication in vitro or in non-transgenic balb/c mice (sims et al., 2008; yount et al., 2005) . similarly, in the mhv system, it has been shown that deletion of orf 4 in mhv does not affect virulence (ontiveros et al., 2001) . titers of e protein deletion mutants (rsars-cov-δe and rsars-cov-δ[e,6-9b]) in the lungs of hace2 tg mice were about 100-fold lower than in mice infected with wt virus or virus lacking genes 6 to 9b. the observed reduction in virus titers probably is only partially responsible for the attenuated phenotype of the δe viruses, as we showed that viruses lacking e gene did not spread to the central nervous system, even when the virus was intracranially inoculated (data not shown). these data suggest that gene e is important for virus tissue tropism. changes in virus tissue tropism frequently are an important cause of virus attenuation as shown for other coronaviruses such as tgev, in which loss of virulence is associated with loss of enteric tropism (sanchez et al., 1999) . similarly, live poliovirus vaccines are attenuated because of their reduced neurotropism with a concomitant increase in enteric growth (sutter et al., 2004) . it is likely that the infection of the central nervous system is a major factor contributing to the fatal outcome observed for sars-cov-infected tg mice (mccray et al., 2007; tseng et al., 2007) . while the brain is not considered a major target for the virus in humans, there are reports showing the presence of the virus in this tissue (ding et al., 2004; gu et al., 2005; xu et al., 2005) . further, some sars survivors have neurological and psychological sequelae that are not well understood and could result from infection of the central nervous system (lee et al., 2004; xu et al., 2005) . the molecular mechanism involved in the attenuation and changes in tropism of e protein deletion mutants is under further investigation and might suggest new biological functions for the e protein. sars-cov lacking e gene is attenuated in the highly sensitive hace-2 tg mice model and in hamsters (dediego et al., 2007; enjuanes et al., 2008a) suggesting that sars-cov mutants defective in e gene may also be attenuated in other species including humans. accordingly, the induction of protection conferred by these viruses has been studied in hamsters showing complete protection. therefore, these deletion mutants could be considered promising vaccine candidates. nevertheless, more detailed experiments in this and other animal models, including non-human primates, are needed in order to further evaluate the safety and efficacy of these attenuated viruses as vaccine candidates. another application of these genetically attenuated viruses of practical interest is their use as the starting material for the generation of chemically inactivated vaccines. thus, in the event of incomplete chemical inactivation of the vaccine virus, the remaining infectious virus will be highly attenuated, reducing the likelihood of untoward consequences qin et al., 2006; qu et al., 2005; spruth et al., 2006; zhou et al., 2005) . african green monkey kidney-derived vero e6 cells, human liverderived huh-7 cells, human colon carcinoma-derived caco-2 cells, and the huh7.5.1 clone derived from huh-7 cells (gillim-ross et al., 2004; hattermann et al., 2005; mossel et al., 2005; zhong et al., 2005) were kindly provided by e. snijder (university of leiden, the netherlands), r. bartenschlager (university of heidelberg, germany), the european collection of cell cultures and f. v. chisari (scripps research institute, la jolla, california), respectively. in all cases, cells were grown in dulbecco's modified eagle's medium (dmem, gibco, grand island, ny) supplemented with 25 mm hepes and 10% fetal bovine serum (fbs) (biowhittaker, verviers, belgium). virus titrations were performed in vero e6 cells following standard procedures using closed flasks or plates sealed in plastic bags. for plaque assays, cells were fixed with 10% formaldehyde and stained with crystal violet three days post-infection. all work with infectious viruses and infected animals was performed in biosafety level (bsl) 3 facilities by personnel wearing positive-pressure air purifying respirators (3 m hepa airmate, saint paul, mn). hace2 tg mice were generated as previously described and were used after backcrossing 6-9 generations to c57bl/6 mice (mccray et al., 2007) . no differences were observed in disease or histological outcome when mice that were backcrossed to different extents were compared. all animal experiments were approved by the university of iowa animal use and care committee. the pbac-sars-cov-δ[6-9b] plasmid encoding a rsars-cov lacking accessory genes 6, 7a, 7b, 8a, 8b and 9b was constructed using a previously described full-length infectious cdna clone coding for sars-cov, urbani strain in a bacterial artificial chromosome (bac) (plasmid pbac-sars-cov fl ) (almazan et al., 2006) . the pbac-sars-cov-δe plasmid encoding a rsars-cov lacking the gene e was constructed from plasmid pbac-sars-cov fl as described (dediego et al., 2007) . the pbac-sars-cov-δ[e,6-9b] plasmid encoding a rsars-cov lacking the e, 6, 7a, 7b, 8a, 8b and 9b genes was constructed from plasmid pbac-sars-cov-δe. to generate plasmids pbac-sars-cov-δ[6-9b] and pbac-sars-cov-δ[e,6-9b], deletion of genes 6, 7a, 7b, 8a and 8b was introduced by overlap extension pcr using as templates the plasmids pbac-sars-cov fl and pbac-sars-cov-δe, respectively. the final pcr products were digested with the enzymes bamhi and nhei and cloned in the intermediate plasmid psl1190+bamhi/sacii sars-cov to generate the plasmids psl1190+ bamhi/sacii 7, 8] and psl1190+bamhi/sacii sars-cov-δ [e,6,7,8] . the plasmid psl1190+bamhi/sacii sars-cov contains a fragment corresponding to nucleotides 26045 to 30091 of the sars-cov infectious cdna clone (almazan et al., 2006) engineered into plasmid psl1190 (pharmacia) using the unique restriction sites bamhi and sacii. to abrogate expression of gene 9b, overlap pcr extension was performed using as template the infectious cdna clone (almazan et al., 2006) . the final pcr product was digested with the enzymes kpni and nhei and cloned into plasmids psl1190+bamhi/sacii sars-cov-δ[6,7,8] and psl1190+bamhi/sacii sars-cov-δ [e,6,7,8] , to assemble plasmids psl1190+bamhi/sacii sars-cov-δ[6-9b] and psl1190+ bamhi/sacii sars-cov-δ[e,6-9b]. finally, fragment bamhi/sacii of these plasmids was exchanged with that of plasmid pbac-sars-cov fl to generate plasmids pbac-sars-cov-δ[6-9b] and pbac-sars-cov-δ[e,6-9b]. details on the cloning can be obtained from the authors upon request. transfection and recovery of infectious viruses from the cdna clones bhk cells grown to 90% confluence in 12.5 cm 2 flasks were transfected with 6 μg of the plasmids pbac-sars-cov-δ[6-9b], pbac-sars-cov-δe, and pbac-sars-cov-δ[e,6-9b], or the plasmid pbac-sars-cov fl as a control, using 18 μg of lipofectamine 2000 (invitrogen) according to manufacturer's instructions. recombinant viruses were cloned as described (dediego et al., 2007) . rsars-cov-δ[6-9b], rsars-cov-δe, and rsars-cov-δ[e,6-9b] growth kinetics subconfluent monolayers (90% confluency) of vero e6, huh-7, huh-7.5.1 and caco-2 cells were inoculated at a multiplicity of infection (moi) of 0.05 with the viruses rsars-cov, rsars-cov-δ[6-9b], fig. 7 . histopathology and immunohistochemistry. hace2 tg mice were intranasally inoculated with 12,000 pfu of the indicated viruses and sacrificed at day 4 p.i. zinc formalin-fixed lungs (left) and brain (cerebrum) (right) were analyzed for viral antigen as described in materials and methods. original magnification was 10×. rsars-cov-δe, and rsars-cov-δ[e,6-9b]. after an absorption period of 1 h, inoculum medium was removed and fresh medium added. then, culture supernatants were collected at different times postinfection and virus titers were determined as described above. total rna from vero e6-infected cells was purified using the qiagen rneasy kit according to the manufacturer's instructions and used for reverse transcription (rt)-pcr analysis. the regions comprising gene e and genes 6 to 9b were analyzed. rt reactions were performed using murine leukemia virus reverse transcriptase (ambion) and the reverse primers sars-e231-rs (5′-ttagaccagaagatcaggaactcc-3-), complementary to nt 208 to 231 of e gene, to analyze gene e deletion and mutations, and sars-28397-rs (5′-gggtagctcttcggtagtagcc-3′), complementary to nucleotides 28376 to 28397 of the sars-cov genome, to analyze deletion of genes 6 to 8b and 9b mutations. the cdnas were amplified by pcr using sense primers sars-25211-vs (5′-ggatgactctgagccagttctcaaggg-3′), complementary to nucleotides 25212 to 25238 of sars-cov genome and sars-27024-vs (5′-cgccggtagcaacgacaatattgc-3′), complementary to nucleotides 27025 to 27048, and the reverse primers indicated above. rt-pcr products were visualized by electrophoresis in 0.8% agarose gels and sequenced using the primers used for the rt-pcr reactions. for conventional electron microscopy, vero e6 cell monolayers were infected with rsars-cov, rsars-cov-δ[6-9b], rsars-cov-δe, and rsars-cov-δ[e,6-9b] at a moi of 0.5. cells were fixed in situ 20 h post-infection with 2% glutaraldehyde in phosphate na/k buffer (ph 7.4) for 1 h at room temperature. cells were removed and transferred to eppendorf tubes. after centrifugation, cells were washed three times in phosphate na/k buffer (ph 7.4) and processed for embedding in epoxy, taab 812 resin (taab laboratories, berkshire, england) according to standard procedures (dediego et al., 2007) . ultrathin sections of the samples were stained with saturated uranyl acetate and lead citrate and examined at 80 kv in a jeol jem-1010 (tokyo, japan) electron microscope. for negative staining electron-microscopy, supernatants of vero e6 cells infected for 20 h were fixed with 10% formaldehyde, concentrated using a beckman airfuge, negatively stained and examined by electron microscopy as described (dediego et al., 2007) . mice were lightly anesthetized with isoflurane and inoculated intranasally with the indicated doses of virus in 30 μl of dmem. infected mice were examined and weighted daily. in parallel experiments, to obtain tissues for virus titrations, animals were sacrificed at 2 and 4 days post-infection and lungs and brains were aseptically removed into phosphate buffered saline (pbs). tissues were homogenized using a manual homogenizer, and titrated in vero e6 cells as described above. virus titers are expressed as pfu/g tissue with a lower limit of detection of 420 pfu/g. brains (cerebrum) and lungs were removed from mice at 4 days p.i., fixed in zinc formalin and processed as described previously (mccray et al., 2007) . for routine histology, sections were stained with hematoxylin and eosin. to detect virus antigen, cells were pretreated with 3% hydrogen peroxide and stained with biotinylated mouse anti-nucleocapsid mab (kindly provided by dr. john nicholls, university of hong kong), followed by streptavidin-hrp (jackson immunoresearch, west grove, pa). slides were developed with dia-minobenzidine and counterstained with nuclear fast red (lungs) or hematoxylin (brains). the nucleoprotein is required for efficient coronavirus genome replication construction of a sars-cov infectious cdna clone and a replicon to study coronavirus rna synthesis open reading frame 8a of the human severe acute respiratory syndrome coronavirus not only promotes viral replication but also induces 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coronavirus vaccine in rhesus monkeys we thank p. pérez-breña and ana falcón from the human health department (instituto de salud carlos iii key: cord-302486-z36hcvrx authors: cobo, fernando; talavera, paloma; concha, ángel title: diagnostic approaches for viruses and prions in stem cell banks date: 2006-03-30 journal: virology doi: 10.1016/j.virol.2005.11.026 sha: doc_id: 302486 cord_uid: z36hcvrx some stem cell lines may contain an endogenous virus or can be contaminated with exogenous viruses (even of animal origin) and may secrete viral particles or express viral antigens on their surface. moreover, certain biotechnological products (e.g. bovine fetal serum, murine feeder cells) may contain prion particles. viral and prion contamination of cell cultures and “feeder” cells, which is a common risk in all biotechnological products derived from the cell lines, is the most challenging and potentially serious outcome to address, due to the difficulty involved in virus and prion detection and the potential to cause serious disease in recipients of these cell products. stem cell banks should introduce adequate quality assurance programs like the microbiological control program and can provide researchers with valuable support in the standardization and safety of procedures and protocols used for the viral and prion testing and in validation programs to assure the quality and safety of the cells. the new treatments of cell therapy based on the transplant of cells of human origin still have problems, such as the difficulties of culture and differentiation of the cells, the possibility of chromosomal alterations and potential for tumorigenicity. of particular importance is the possibility of transmission of infectious diseases to the recipients of these cell products. any microbial contamination of the donor's biological products or introduced during manufacturing process can potentially present a serious hazard to recipients even if it is not an overt pathogen. the most common potential forms of contamination (e.g. bacteria (include mycoplasma), yeast, fungi) can be readily assessed on a routine basis (european pharmacopeia, 2004a pharmacopeia, , 2004b cobo et al., 2005) . however, viral contamination of cell cultures and feeder cells, which is a common risk in all biotechnological products derived from the cell lines, is the most challenging and potentially serious outcome to address, due to the difficulty involved in virus detection and the potential to cause serious disease in recipients of these cell products (cobo et al., 2005) . to ensure the provision of safe and reliable cells and tissues for these applications, it is necessary to regulate the procurement, processing, testing, preservation, storage and distribution of all cells that will apply in the human body (directive 2004/ 23/ce). stem cell banks must assure the quality and safety of these cells, and these aims are particularly important in the avoidance of transmissible diseases like viral and prion infections that are difficult to diagnose. these establishments should introduce adequate quality assurance programs like the microbiological control program. in this respect, accredited stem cell banks can provide researchers with valuable support in the standardization and safety of procedures and protocols used for viral and prion testing and in validation programs to assure the quality and safety of the cells. this review will discuss the methodology that should be used in the stem cell banks in order to assure the quality of cell and biotechnological products and avoid the transmission of infections, in particular those that involve virus and prion particles. some lines or cell cultures may contain an endogenous virus or can be contaminated with exogenous viruses, and may secrete viral particles or express viral antigens on their surface. moreover, certain biotechnological products (e.g. bovine fetal serum) may contain prionic particles. the primary sources of potential viral and prionic contamination come from infected animal tissue used to prepare biological reagents and media, biological products from donors (e.g. bone marrow, preembryos) and contamination during laboratory manipulation. in addition, infected laboratory workers may cause contamination of stem cell lines during culture manipulation. the requirement for ''feeder'' cells of animal origin (e.g. murine cells) to maintain undifferentiated growth in human embryonic stem cells (hescs) cultures provides intimate contact between the potential therapeutic cells and the feeder cells which are an ideal setting to transmit infectious microorganisms or bioactive molecules in the final therapeutic product. there is evidence that certain mouse viruses, like hantaan virus, lymphocytic choriomeningitis virus (lcmv) and reovirus-3 have all been detected in mouse colonies (kraft and meyer, 1990 ) and these viruses have notably caused serious infection, and even fatalities in laboratory workers (lloyd and jones, 1986; mahy et al., 1991) and may also be transmitted in cell lines and reagents (nicklas et al., 1993) in the event of using feeder cells of human origin (draper et al., 2004; genbacev et al., 2005) , there are numerous viruses and other infectious agents that are susceptible to being transmitted to the recipient because all human cells have the potential to transmit infectious diseases. the screening should apply to testing for human feeder cells for clinical use, the same as the present regulations that require the screening of cell and tissue products from donors for a spectrum of viruses which cause serious human infectious diseases (uk msbt, 2000; aatb, 2002) . virological screening should firstly be carried out for hiv-1/2, hepatitis b and hepatitis c; moreover, recently, the products for transfusion containing cellular material have been tested for the cell associated organisms such as hcmv and htlv-i/ii (consensus document, 1999) , although tests for hepatitis a and hepatitis e should also be recommended in such products. other potentially viral agents that could be contaminants of cells from healthy individuals include human herpesviruses (hhv-6, hhv-7, hhv-8, epstein -barr virus, herpes simplex virus), parvovirus b19, ttv virus and human polyomaviruses (jc and bk virus), and these viruses remain latent and detectable in humans from early childhood. the risk of transmission of these microorganisms is considered extremely low, so tests for them are not currently required for transplantation. however, the oncogenic risk of viral contamination must also be considered because some of these agents have been involved in human cancers (takeuchi et al., 1996; garbuglia et al., 2003) . some microbial agents have marked variation in their geographical distribution, producing infectious epidemics in different areas like the lymphocytic choriomeningitis virus in usa (wnv update, 2004) and the very recent outbreak of severe acute respiratory syndrome (sars) virus in humans in south east asia. furthermore, two new retroviruses (htlv-3 and htlv-4) have been recently identified among african bush-meat hunters (wolfe et al., 2005) and recent cases of transplant transmitted disease due to rabies virus in usa and germany (srinivasan et al., 2005) have been reported, which clearly shows the potential emergence of new serious pathogens or the re-emergence of known pathogens. obviously, any new entity that arises should be considered as a contamination risk factor, and specific tests may be required, and these may be developed for surveillance initiatives, as in the case of sars, for which detection methods are being developed for the causative coronavirus agent (juang et al., 2004) . the use of bovine fetal serum in stem cell cultures requires an urgent need for a risk assessment for transmissible spongiform encephalopathies (tses) by means of a sensitive and specific test in all products derived from ruminants (u.s. food and drugs administration, 1999; directive 2004/c 24/ 03). cell cultures represent a good medium to promote and to allow a persistent replication of prp sc and to maintain the infectivity, even for heterologous cell culture models (solassol et al., 2003) . human beings can be exposed to secondary infections of tse using medical procedures or by administering biological products derived from humans including blood (llewelyn et al., 2004; peden et al., 2004) . thus, preventive measures should be taken into account with respect to blood products, cell or tissue grafts in order to avoid prion transmission in the recipients. in vitro assay for viral detection: viral culture viruses are obligate intracellular parasites that require living cells in order to replicate; since viruses require cellular machinery for replication, living systems must be used. the viral culture is an amplification method that increases the amount of the pathogen, facilitating detection and characterization. this method is unique among detection methods in that it provides an isolate of viable virus that can be further characterized and stored for future studies. another important feature is that culture methods allow the detection of many different viruses, including some not suspected at the time the culture is established; even viruses not previously known can be discovered. a cell lysate or other specimens (e.g. biological products, specimens from donors) should be introduced into the cell culture capable of detecting wide ranges of viruses. the instructions for obtaining and transporting such specimens have been previously reported (storch, 2001) . usually, a minimum of three cell lines that include a human diploid cell line (mrc-5), a monkey kidney cell line (vero) and a cell type of the same species and tissue type used for production (schiff, 2005) is recommended. additional cells may be required depending on the cell source, passage history and raw materials used. after inoculation, cultures are incubated at 35 to 37 -c for either 14 or 28 days and inspected periodically (e.g. daily) and observed for the cytopathic effect of several viruses, including the recently reported method of analysis of hiv virus cytopathicity by using hi-cd4 jurkat t cells cultures (speirs et al., 2005) . in certain circumstances, the observation period can be greater than 28 days for the identification of many viruses (e.g. human cytomegalovirus). virus detection by testing the inoculated cell culture for hemadsorption and/or hemagglutination (ayala et al., 2004) should be necessary for viruses with only minimal visible cytopathic effect. however, viral culture also has significant disadvantages as a diagnostic method, including the need for specialized facilities and expertise, expense and relatively prolonged time to detection. the european medicines evaluation agency (emea, 1997) and the u.s. food and drugs administration (u.s. food and drugs administration, 1998) recommend the use of studies in animals as a screening method for a wide range of viruses. these studies consist of inoculation in mice, pigs and embryonated chicken eggs of the cell lysate or biological products that can cause clinical changes in animals. species-specific viruses that are potentially present in rodent cells or stem cells with murine feeders are tested by the antibody production test like the mouse, hamster and rat test [map (16 murine viruses), hap (5 viruses) and rap (9 viruses)]. these tests are carried out by inoculating into virusfree animals and then after a specific time, examining the serum antibody levels. however, these tests can take a number of weeks, and therefore, can prove difficult to implement in the scheduling for release testing of time critical products. viral infections can occasionally be diagnosed using electron microscopy for the direct visualization of viral particles in specimens. with the introduction of negative staining in the late 1950s (brenner and horne, 1959) and the wider availability of electron microscopes, electron microscopy became essential in characterizing many new isolates detected in cell cultures and clinical samples (plummer et al., 1985; biel and gelderblom, 1999) . pattern recognition (e.g. information of size, particle morphology) leads to rapid identification of infectious agents. the ''open procedure'' of electron microscopic testing allows an unbiased, rapid detection of viruses and other agents if sufficiently high particle concentrations exist. due to this capability, electron microscopic testing must be a frontline method applied to cell cultures of cultivable agents. a specimen can be ready for examination and an experienced virologist or technologist can identify, by electron microscopy, a viral pathogen morphologically within a few minutes of arrival in the electron microscopy laboratory. other advantages of electron microscopy include the lack of requirement for viral viability, the fact that many different kinds of viral particles can potentially be seen, and allow the differential diagnosis of many agents contained in the specimen. disadvantages include the cost and complexity of maintaining an electron microscope (although this fact can be corrected by working with other institutions with centralized services), the need for a skilled operator and relative lack of sensitivity related to the fact that a relatively high concentration of viral particles (10 5 to 10 6 per milliliter) is required for visualization (miller, 1995) . with respect to these questions, there is a paper in which these relevant points are discussed (biel and madeley, 2001) . there are two types of electron microscopy methods: direct or immunoelectron microscopy. with direct methods, negative staining is normally used which requires little special equipment, in contrast to thin sectioning techniques. the specimens may be used directly or the virus particles may be concentrated before negative staining. several methods are available for concentration, including differential centrifugation, ammonium persulfate precipitation and the agar diffusion method. immunoelectron microscopy is a means of increasing the sensitivity and specificity of electron microscopy and is particularly useful if the number of virus particles present is small. details for efficient sample collection, preparation and particle enrichment have been published previously (gelderblom and hazelton, 2000; hazelton and gelderblom, 2003) . the detection of viral antigens directly in clinical specimens or cell cultures has become an essential component of the methodological repertoire of diagnostic virology. these methods can provide diagnostic information within a few hours of the receipt of the specimen. the lack of requirement for virus viability is another important advantage over viral culture, especially when specimen transport time is prolonged or otherwise suboptimal. antigen detection methods can be applied when the following conditions are met: (i) viral antigen is expressed and is present in an accessible specimen, (ii) an appropriate antibody is available, (iii) antigenic variability does not preclude recognition by immunologic reagents of different strains of the target virus and (iv) the antigen being detected is sufficiently stable so that it does not degrade during transport and processing of the specimen. methods used for viral antigen detection include fluorescent antibody staining (fas), immunoperoxidase staining (is) and enzyme immunoassay (eia). fas is widely used for detection of cell associated viral antigens. in the direct format, a fluorescent label, usually fluorescein isothiocyanate (fitc), is conjugated directly to the antibody that recognizes the viral antigen. in the indirect format, the antiviral antibody is unlabeled and is detected by a second antibody that recognizes immunoglobulins from the animal species of origin of the antiviral antibody. the second antibody carries the fluorescent label. after staining, the specimen is viewed with epiilumination using ultraviolet light of the wavelength needed to excite the fluorescent label. the direct method is simpler to use but requires conjugation of each antiviral antibody with the fluorescent label. the indirect method is slightly more sensitive and more versatile because only the antiimunoglobulin antibody has to be conjugated with the fluorescent label. the fas method is widely used for the detection of hsv and hcmv viruses. is is similar in principle to fas except that horseradish peroxidase is used in place of a fluorescent label. the advantage of this method is that the staining can be viewed by light microscopy, thus obviating the need for a fluorescent microscope. the disadvantages of the ip method are that it is more cumbersome that fas, and endogenous peroxidases in some specimens can produce background staining. eia is a widely used method that can be applied to the detection of antigens regardless of whether they are cell associated. since intact cells in the specimen are not required, specimen integrity is less important than for fas and is. advantages of eia include applicability to diverse specimens and potential for automation. viruses for which antigen eias have been widely used are hsv, hbv and hiv. the measurement of antiviral antibodies was one of the first methods used for the specific diagnosis of viral infections. the role of serology in the viral diagnosis of the cell cultures may be to determine the immune status of donors of biological materials. it is important to use sensitive assays, such as eia, immunofluorescence, western blot assays, etc. however, research has shown that the detection of antibodies exclusively runs the risk of samples for tests being taken during an antibody-negative window period of these infections, where an individual has been exposed to viral infection and indeed can be viremic (hitzler and runkel, 2001) . this finding has led to the introduction of nucleic acid amplification techniques, such as polymerase chain reaction (pcr), in which the presence of a virus can be observed by means of the amplification of sequences in the viral genome. the addition of nucleic acid test methods to screening of tissue donors, and to the testing of derived cell lines, should reduce the risk of these infections among recipients of stem cell lines (zou et al., 2004) . diagnostic virology is being revolutionized by the application of nucleic acid detection techniques (tang et al., 1997) . nucleic acid-based diagnostic tests detect only the specific virus to which the diagnostic reagent is directed. these methods detect specific nucleic acid sequences and can be applied to the detection of virtually any virus. depending on the target sequence, the assays can be specific for a single virus species or for a group of related viruses. nucleic acid amplification assays are particularly attractive for viruses that are difficult or impossible to culture, viruses that grow slowly in culture and viruses for which antigen detection cannot be applied because of antigenic diversity or because the level of viral antigen is too low to permit successful detection. pcr is the prototype of target amplification assays invented by kary mullis in 1983 (mullis and faloona, 1987; mullis, 1990) . pcr is based on the ability of dna polymerase to copy a strand of dna by elongation of complementary strands initiated from a pair of closely spaced chemically synthesized oligonucleotide primers and includes repeated cycles of amplifying selected nucleic acid sequences (mullis and faloona, 1987; mullis, 1990) . after pcr amplification, the pcr product (or amplicon) is detected by gel electrophoresis or one of several probe-hybridization techniques, such as southern blot. numerous modifications of the standard pcr procedure have been developed since its inception (erlich et al., 1991; wagar, 1996) . some of these modifications effectively expand the diagnostic capabilities of pcr and have increased its utility in the microbiology laboratory. rt-pcr was developed to amplify rna targets. in this process, rna targets are first converted to complementary dna (cdna) by reverse transcriptase (rt), and then amplified by pcr. rt-pcr has played an important role in diagnosing rna containing virus infections (young et al., 1993) . several new pcr assays have been developed in which the synthesis of the pcr product is detected in real-time (realtime quantitative pcr) (gibson et al., 1996; heid et al., 1996) . real-time quantitative pcr is a homogeneous method that includes both amplification and analysis with no need for slab gels, radioactivity or sample manipulation. reaction products are detected with a fluorescence detection system consisting of a light-emitting diode that delivers excitation light to each reaction tube and an optical unit with three detection channels to record emitted light. the fluorescence of dna dyes or probes is monitored each cycle during pcr. the simplest system for detection of real-time pcr products uses the dnabinding dye sybr green, which fluoresces when its binds to double-stranded dna. these methods have several important advantages over conventional pcr. since the accumulation of pcr product is monitored in the reaction tube, no separate detection method, such as gel electrophoresis, is required, thus shortening the effective assay markedly. furthermore, the possibility of contamination by amplicons is decreased because the systems are closed, with no handling of the reaction contents after completion of pcr. the use of multiple fluorescent dyes with different emission wavelengths makes it possible to perform multiplex reactions with simultaneous amplification of more than one product. moreover, other molecular methods have been described and introduced for the viral diagnosis, like nucleic acid probes (denniston et al., 1986) , branched dna signal amplification (urdea et al., 1991) , nested pcr (erlich et al., 1991) and multiplex pcr (dineva et al., 2005) , etc. real-time multiplex pcr can analyze multiple viruses simultaneously within a single reaction. the main advantages of multiplexing over single-target analysis are the ability to provide internal controls, lower reagent costs and preservation of precious samples. multiplexing can be particularly important when there is a need to analyze several viruses from the samples. there are several assays that use a real-time multiplex rt-pcr technology for diagnosis of hepatitis b virus, hepatitis c virus and hiv-1 virus (candotti et al., 2004) . there are other authors who have described several assays using this procedures for retroviruses (vet et al., 1999) and herpesviruses (o'neill et al., 2003) . the advantages of molecular methods, especially the pcr technique, are their extremely high sensitivity (they may detect down to one viral genome per sample volume), they are easy to set up and have a fast turnaround time. however, the main inconvenience is that for each virus or group of virus one pcr is necessary, so if the amount of viruses to carry out is large, these techniques are the same unviable for the laboratory. retroviruses are one of the main contaminants of the cell cultures. for these viruses, reverse transcriptase assays, electron microscopy techniques and infectivity assays must be included. a variety of infectivity assays are available for rodent cell lines or stem cell lines with murine feeders. there are two retrovirus infectivity assays for the ecotropic and xenotropic viruses: xc plaque assay using indicator cells (xc) to form syncytia (plaques) for detection of ecotropic viruses (lenz and haseltine, 1983) and mink s + l à assay for the detection of xenotropic viruses (li et al., 1999) . however, these tests are not suitable to detect and quantify the levels of the ecotropic recombinant virus, thus a serological focus assay, based on specific antimurine leukemia virus (mulv) viral envelope antibodies is required to detect ecotropic recombinant virus (deo et al., 1994) . moreover, for low levels of murine retroviruses, amplification may be achieved using cocultivation of cells with a susceptible cell line such as mus dunni cells. the reverse transcriptase assay is an enzymatic technique to detect the presence of extracellular retrovirus particles. this assay is based on the ability of reverse transcriptase associated with retroviruses to synthesize radiolabeled nucleotides into complementary dna (cdna) copied from synthetic templates. due to the fact that a variety of enzymes are capable of incorporating labeled deoxynucleotide into an acid-insoluble material, this assay is susceptible to false positives. a useful test for eliminating false positives is to compare the results from both a dna template and a ribonucleic acid (rna) template because cellular dna polymerase and viral reverse transcriptase have different template preferences. furthermore, the pcr-based reverse transcriptase assay is more sensitive than the standard enzymatic reverse transcriptase assay. electron microscopy (see below) is used to visualize both the ultrastructural morphology of the cell substrate and the presence of virus and virus-like particles. the development of microarrays has been fueled by the application of robotic technology to routine molecular biology, rather than by any fundamental breakthrough. southern and northern blotting techniques for the detection of specific dna and mrna species provided the technological basis for microarray hybridization. the construction of arrays involves the spotting of specific dna sequences on a glass slide or small silicon chip by photolithographic combinatorial chemistry methods similar to those used to make electronic chips. membrane-based arrays may be in the format of line probe blots. the results of hybridization between the bound probe and labeled sequences in the sample applied and tested are revealed by scanning or imaging the array surface. confocal microscopy is used to scan the chip, detecting fluorescent signals that reveal hybridization at precise locations on the chip. use of probes representing all possible nucleotide sequence variations within a target sequence allows rapid determination of nucleotide sequence (pease et al., 1994) . high density arrays, which may have thousands of individual probes per cm 2 , are referred to as microarrays. these microarrays on silicon surfaces are therefore known as ''dna chips''. as many dna sequences can be present on a slide, it is possible for microarray analysis to test for multiple pathogens (including viruses) simultaneously. the first application in diagnostic virology has been for rapid sequencing to detect hiv mutations associated with resistance to antiretroviral drugs (kozal et al., 1996) . other roles in virology are in diagnosis, to recognize the causative agent of an illness; for molecular typing (e.g. patient management, epidemiological reasons, purposes related to vaccine use) and in research, to investigate the interactions between the virus and the host cells (clewley, 2004) . the rna expression of human cmv in cell culture in the presence or absence of cycloheximide or ganciclovir was analyzed with an array of oligonucleotides representing human cmv orfs (chambers et al., 1999) . other groups have developed microarrays that detect simultaneously and discriminate several viruses like orthopoxvirus species (laassri et al., 2003) , respiratory viruses (coiras et al., 2005) , herpesviruses, enteroviruses and flaviviruses (korimbocus et al., 2005 ) and hepatitis c virus (xu et al., 2005) . also, wilson et al. (2002) developed a multi-pathogen identification microarray system for the identification of 18 pathogenic prokaryotes, eukaryotes and viruses. the simplicity of the microarray protocols, together with their use of a large number of species-specific oligoprobes and their ability to analyze multiple samples in a short time, offers clear advantages. however, such techniques will need to be carefully validated for sensitivity and specificity before being applied in the safety testing of human tissue products and stem cell lines for therapy. table 1 shows a comparison of diagnostic methods for viruses. proteomics also holds a key position in the new functional genomics biology and is a term for large scale analysis of proteins. proteomics encompasses different methods to identify all the proteins present in a cell or tissue. protein arrays are being prepared with antigens or antibodies bound to a solid phase (analogous to oligonucleotide or amplicon probes) and used to capture specific antibodies or antigens (emili and cagney, 2000; walter et al., 2000) . essentially, these are immunoassays in a microarray format (schweitzer and kingsmore, 2002) . a protein array has been described for torch screening and toxoplasma gondii, rubella virus, cmv and hsv-1 and 2 antigens were printed on glass slides. the slides were first incubated with serum samples and subsequently with fluorescently labeled secondary antibodies. human igg and igm bound to the printed antigens were detected by confocal scanning microscopy. good concordance was obtained between the microarray results and those of elisas (mezzasoma et al., 2002) . a microarray of oligosaccharides on nitrocellulose has been developed to capture carbohydrate-recognizing proteins, and it might be possible to develop this for viral diagnosis (fukui et al., 2002) . biosensors have been defined as small devices which use biological reactions to detect target analytes (wang, 2000) . there are two ways of doing this; bioaffinity arrays involve the target analyte binding to a ligand immobilized on a solid phase (e.g. an oligonucleotide) and, instead of there being a necessity for detection of hybridization of the probe and target by colorimetric or radioactive means, the hybridization is detected by electronic means. biocatalytic arrays involve an immobilized enzyme being used to recognize the substrate of the enzyme, which is, in this case, the target of the array. a signal is generated when the enzyme catalyzes a specific reaction because of the presence of the target in the analyte applied to the array. the reaction may be recognized either by colorimetric ally or via an electronic transducer at the surface of the array. with more technological advances, protein/antibody arrays are likely to impact initially on infectious disease research with profiling sera, body fluids to discover diagnostic markers of particular infections. proteomic techniques also will offer the potential for discovering markers for diagnostic tests of viral infections in vitro (e.g. stem cell cultures). an interesting potential for infected cell cultures may be the discovery of biological markers of prion infection, mainly by comparing control versus infected cultures. this type of experiment was first performed at a genetic level. with the development of sophisticated proteomic approaches, several groups are looking for differentially expressed proteins that could be used as diagnostic markers or at least could give some clue as to the physiopathological event leading to prion propagation. having developed cell lines highly susceptible to prion infection (bosque and prusiner, 2000; nishida et al., 2000) , another potential of cell culture consists of the detection of infectivity in various biological samples. the main sources of transmission of prion proteins to culture cells are the bovine fetal serum, the feeder cells from murine animals and the biological products from donors with tse to establish stem cell lines. in stem cell banks, there is an urgent need for tests for the agents of tses such as the creutzfeldt-jakob disease (u.s. food and drugs administration, 1999) . at the moment, there are several types of tests that can be used for this question. the cell prion protein (prp c ) is essential for pathogenesis and transmission of prion diseases (prusiner, 1989) . during the course of prion disease, a largely protease resistant aggregated form of prp, designated prp sc , accumulates mainly in the brain, and may be the main or only constituent of the prion (mckinley et al., 1991) . the detection of this protein can be carried out by means of antibodies of the monoclonal igg1 subtype, anti-prp 6h4 (enari et al., 2001; parizek et al., 2001) ; this antibody recognizes the sequence dyedryyre in the prion protein (human prp: amino acids 144 -152). this sequence is conserved in most known mammalian prp sequences (human, cattle, sheep, rabbit, mink and a variety of primates). 6h4 can be used for western blotting and elisa (prionics, 2002) . there are other monoclonal antibodies for prion protein such as 34c9 that recognizes the sequence lihfg in the bovine prion protein and a polyclonal antibody r029 that also recognizes bovine prp. recently, sanquin reagents (sanquin, 2005) has developed a new monoclonal antiprion antibody for use in research applications (clone 1e4). in contrast to many other antibodies used for detection of prion protein, 1e4 has a broad species reactivity. detection of prion protein has been demonstrated with western blot for humans, cattle, sheep, deer, mice and hamsters. most of the currently available tse tests are based on the fact that prp c , normal prion protein, is digested by proteinase k, whereas prp sc , tse specific prion, is relatively resistant to degradation by proteases. the special feature of 1e4 is that its epitope is almost hidden on non-digested bovine prp sc , but after proteinase k digestion, the epitope becomes available resulting in a significant increased detection. after digestion with protease k, 1e4 binds to prp sc with high affinity, whereas it has a low affinity for non-digested prp sc . the 1e4 antibody has been tested in a broad variety of methods, such as western blot, ria, elisa, eliblot, facs and immunohistochemistry. cyclic amplification of protein misfolding saborio et al. (2001) have developed a procedure, conceptually similar to polymerase chain reaction cycling, involving cyclic amplification of protein misfolding prp sc . this method could allow a rapid conversion of excess prp c into a protease resistant prp sc -like form. in this method, aggregates formed when prp sc is incubated with prp c are disrupted by sonication to generate multiple smaller units for the continued formation of new prp sc . after cycling amplification, more than 97% of the protease resistant prp present in the sample correspond to newly converted protein. this method could be applied to diagnose the presence of undetectable prion infectious agent in tissues and biological fluids. in this respect, recently, castilla et al. (2005) have been the first to detect the resistant-protein prp sc biochemically in hamster blood by means of the cyclic amplification of protein misfolding. this procedure enables detection of prions in blood with 89% sensitivity and 100% specificity. the high level of sensitivity and specificity indicated that this assay offers promise for the design of a sensitive biochemical test for blood diagnosis of transmissible spongiform encephalopathies. the implementation of a similar blood-detection procedure for humans and culture-detection method for stem cell lines undoubtedly contributes to minimizing the risk of infection with agents causing transmissible spongiform encephalopathies. the diagnosis of infections produced by tses can be carried out by means of the detection of prionic protein (prp sc ) by different methods (see below). although the specificity of these diagnostic methods is nearly 100% (ironside, 1996; lee et al., 2000; wadsworth et al., 2001) , the sensitivity is still inadequate to assure the value of a negative result. an alternative to these methods would consist of the use of transgenic mice; these would be vaccinated with tissues or fluids that have unknown infectivity of the cell lines which have used bovine serum to be obtained. stem cell banks arise from the necessity to guarantee the existence of an appropriate source of cell lines in a standardized way for their use in research or human therapies through clinical trials. moreover, these establishments should assure the safety of biological products for use in cell therapy. the major risks associated with the use of biological products in regenerative medicine are related to cell contamination. these include both serious human pathogens but also human and animal viruses and prions capable of multiplying and producing transmissible infectious diseases. selecting and testing of stem cell lines and biotechnological products (e.g. bovine serum, tripsine, culture media) is one part of a strategy for establishing a viral safety program (cobo et al., 2006) . while the technology to avoid the animal products in the culture manufacturing is being developed, the safety of the human and/or animal products used in the cell cultures with respect to viral and prion contamination could be obtained for application of an exhaustive program of viral screening by means the combination of techniques above-mentioned (tables 2 and 3 ). the presence of viruses (including non-pathogenic types) or prions in a human therapeutic product would most likely render it unacceptable for clinical use. at the moment, the cell banks must have a panel of tests to detect serious pathogens like endogenous viruses, exogenous viruses and prions. this panel of tests should necessarily include reverse transcriptase detection as a general test for retroviruses, electron microscopy that can detect different kinds of viral particles and characterize many unknown isolates present in cell cultures and molecular techniques like pcr (conventional or real-time) and rt-pcr tests to include all the viruses that we know pose a risk to the product. for prion detection, these banks must have a procedure based on the western blot or elisa technology for the detection of antibodies (e.g. 6h4, 1e4) that can be present in the cell cultures. some of these tests may also need to be applied to culture reagents of animal origin, and any testing performed by the manufacturer should be carefully evaluated before accepting the reagents for use. with respect to the use of ''feeder cells'' of animal origin (e.g. murine) for embryonic stem cell culture, there is a need to evaluate several viruses that are capable of infecting humans or primates and that potentially can cause serious infections in laboratory workers. for these viruses, the antibodies production tests (e.g. map, rap, hap) are the most convenient procedures in addition to the evaluation by means of electron microscopy. several tests could be included in the screening of other biological products for a wide range of viruses: the in vitro cell culture assay and the study in animals (emea, 1997). these are long standing techniques that rely on the ability of many viruses to cause cytopathic changes in cell culture or clinical changes in animals. however, there is a difficulty for implementation in most of the laboratories due to the need for specialized facilities and expertise, relatively prolonged time to detection (e.g. can prove difficult to implement the testing of time critical products) and the high cost. in the immediate future, the technologies based on the use of hybridization chips, using microarrays of immobilized oligonucleotides or antigens/antibodies for viruses, and the method of cyclic amplification of protein misfolding prp sc for prions, can provide a rapid and useful methodology of the identification of contaminants. in our opinion, there is a need to design and to implement this methodology in stem cell cultures, so new research will be needed. finally, it is not possible to assert the absolute absence of viral contaminants because of both the impossibility of covering all potential viral contaminants and the absence of adequate levels of sensitivity and specificity of diagnostic techniques in several cases. so, stem cell banks could potentially permit transmission of some of these viral contaminants to many patients receiving future stem cell therapies where the balance of risk and benefit may be different. table 3 virus tests recommended in the characterization of cell lines (modified from dellepiane et al., 2000 and emea, 1997) test master bank work bank cell at the limit electron microscopy + à + reverse transcriptase a + à + in vitro cell inoculation + à + in vivo animal inoculation + à + antibody production tests b + à à infectivity c + à + other virus specific tests d + à + a not necessary if positive by retrovirus infectivity test. b e.g., map, rap, hap-usually applicable for rodent cell lines. c for retroviruses and other endogenous viruses. d tests for cell lines derived from human, non-human primate or other cell lines as appropriate. american association of tissue banks. standards for tissue banking, 10th edr american association of tissue banks development of an antigen for the diagnosis of kilham rat parvovirus by hemagglutination inhibition test diagnostic electron microscopy is still a timely and rewarding method diagnostic virology-the need for electron microscopy: a discussion paper cultured cell sublines highly susceptible to prion infection negative staining method for high resolution electron microscopy of viruses detection of prions in blood multiplex realtime quantitative rt-pcr assay for hepatitis b virus, hepatitis c virus, and human immunodeficiency virus type 1 dna microarrays of the complex human cytomegalovirus genome: profiling kinetic class with drug sensitivity of viral gene expression a role for arrays in clinical virology: fact or fiction? microbiological control in stem cell banks: approaches to standardisation a. general safety guidances in stem cell banks oligonucleotide array for simultaneous detection of respiratory viruses using a reverse line blot hybridization assay selection criteria in the organ donors respect to transmission of infections new challenges in assuring vaccine quality cloned fragment of the hepatitis delta virus rna genome: sequence and diagnostic application detection and characterization of murine ecotropic recombinant virus in myeloma and hybridoma cells simultaneous visual detection of multiple viral amplicons by dipstick assay directive 2004/c 24/03. note for guidance on minimising the risk of transmitting animal spongiform encephalopathy agents via human and veterinary medicinal products (emea 410/01 rev 2 culture and characterization of human embryonic stem cells note for guidance on quality of biotechnological products: viral safety evaluation of biotechnology products derived from cell lines of human or animal origin (cpmp/ich/295/95) large-scale functional analysis using peptide or protein arrays scrapie prion protein accumulation by scrapie-infected neuroblastoma cells abrogated by exposure to a prion protein antibody recent advances in the polymerase chain reaction european pharmacopeia section 2.6.1 (sterility). maisonneuve sa european pharmacopeia section 2.6.7 (mycoplasma). maisonneuve sa oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate -proteins interactions detection of tt virus in lymph node biopsies of b-cell lymphoma and hodgkin's disease, and its association with ebv infection specimen collection for electron microscopy serum-free derivation of human embryonic stem cell lines of human placental fibroblast feeders a novel method for real time quantitative rt-pcr electron microscopy for rapid diagnosis of infectious agents in emergent situations real time quantitative pcr screening of blood donations by hepatitis c virus polymerase chain reaction (hcv-pcr) improves safety of blood products by window period reduction review: creutzfeldt -jakob disease coupling multiplex rt-pcr to a gene chip assay for sensitive and semiquantitative detection of severe acute respiratory syndrome coronavirus dna probe array for the simultaneous identification of herpesviruses, enteroviruses and flaviviruses extensive polymorphisms observed in hiv-1 clade b protease gene using high-density oligonucleotide arrays seromonitoring in small laboratory animal colonies. a five year survey: 1984 -1988 detection and discrimination of orthopoxviruses using microarrays of immobilized oligonucleotides monitoring plasma processing steps with a sensitive western blot assay for the detection of the prion protein localization of the leukemogenic determinants of sl3-3, an ecotropic, xc-positive murine leukaemia virus of akr mouse origin pg-4 cell plaque assay for xenotropic murine leukaemia virus possible 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infectious diseases protein arrays for gene expression and molecular interaction screening from dna biosensors to gene chips west nile virus screening of blood donations and transfusion associated transmission-united states emergence of unique primate t-lymphotropic viruses among central african bushmeat hunters changes of ecm and cam gene expression profile in the cirrhotic liver after hcv infection: analysis by cdna expression array detection of hepatitis c virus by a combined reverse transcription-polymerase chain reaction assay probability of viremia with hbv, hcv, and htlv among tissue donors in the united states ms. angela barnie for the translation and english correction of the manuscript. key: cord-320590-irybhp4j authors: wang, zhitao; sun, bangyao; gao, qi; ma, yong; liang, yumeng; chen, zhijie; wu, hanguang; cui, lu; shao, yuhao; wei, ping; li, hai; liu, shengwang title: host src controls gallid alpha herpesvirus 1 intercellular spread in a cellular fatty acid metabolism-dependent manner date: 2019-08-13 journal: virology doi: 10.1016/j.virol.2019.08.011 sha: doc_id: 320590 cord_uid: irybhp4j viral spread is considered a promising target for antiviral therapeutics, but the associated mechanisms remain unclear for gallid alpha herpesvirus 1 (iltv). we previously identified proto-oncogene tyrosine-protein kinase src (src) as a crucial host determinant of iltv infection. the present study revealed accelerated spread of iltv upon src inhibition. this phenomenon was independent of either viral replication or the proliferation of infected cells and could not be compromised by neutralizing antibody. neither extracellular vesicles nor the direct cytosol-to-cytosol connections between adjacent cells contributed to the enhanced spread of iltv upon src inhibition. further genome-wide transcriptional profile analyses in combination with functional validation identified fatty acid metabolism as an essential molecular event during modulation of the intercellular spread and subsequent cytopathic effect of iltv by src. overall, these data suggest that src controls the cell-to-cell spread of iltv in a cellular fatty acid metabolism-dependent manner, which determines the virus's cytopathic effect. the spread of infectious viruses between host cells is important for viral replication, virulence, and pathogenesis (flint et al., 2009) . two routes exist for viral spread: cell-free spread, which involves infection of new cells through extracellular virions or vesicles containing infectious materials released by infected cells, and cell-to-cell spread, which involves viral particle transmission between cells via cell-to-cell contacts or direct connections (mothes et al., 2010; sattentau, 2008) . cell-free spread is well established and important for distant viral dissemination, such as transmission between hosts and tissue-to-tissue spread within the host body through blood, lymph and cerebrospinal fluid. during distant spread, viruses must be released from host cells. however, once released into the extracellular environment, viruses are exposed to several barriers that block their subsequent cell-free spread, including biophysical barriers such as mucous membranes, cell intrinsic barriers associated with host immunosurveillance, and kinetic barriers due to the processes of random fluid-phase diffusion, attachment of a virus to the host cell membrane and the penetration of viruses bound to host cells. to achieve success in distant dissemination, all of these barriers must be overcome by viruses. considering these disadvantages of cellfree spread, many viruses have evolved to exploit cell-to-cell spread as an alternative strategy to overcome the above barriers. cell-to-cell spread has been proven to be important for infection with many clinically important viruses, such as herpesviruses, poxviruses, measles virus, hepatitis c virus, human immunodeficiency virus type 1, and rabies virus (christian et al., 1971; ritzenthaler, 2011; sattentau, 2008 sattentau, , 2011 wheeler, 1960) . for alphaherpesviruses, the main route of spread in tissues is cell-tocell spread during primary infections in mucocutaneous tissue, establishment of latent infection in sensory neurons, and reactivation from latency, during which the virus spreads intercellularly between the mucocutaneous tissue and sensory neurons (abaitua et al., 2013; arvin et al., 2007) . the cell-to-cell spread inhibitory activity of infectionelicited neutralizing antibodies has been found to be extremely limited in herpes simplex virus 1 (hsv-1)-and hsv-2-infected subjects https://doi.org/10.1016/j.virol.2019.08.011 received 2 may 2019; received in revised form 10 july 2019; accepted 12 august 2019 (criscuolo et al., 2019) . avian gallid alpha herpesvirus 1 (iltv) belongs to the family herpesviridae and the subfamily alphaherpesvirinae, which continues to cause great economic losses in the poultry industry (ou and giambrone, 2012) despite successful protection by vaccination (coppo et al., 2013) . similar to other alphaherpesviruses, iltv establishes latency in the nervous system after acute infection of the upper respiratory tract and cannot be cleared from the host by any currently available therapeutic treatment; therefore, reactivation of iltv from latent infection frequently occurs once the host is under stress or host immunity is compromised (hughes et al., 1989 (hughes et al., , 1991 ou and giambrone, 2012) . thus, elucidation of the mechanisms responsible for cell-to-cell spread can facilitate the development of novel therapeutic strategies to control virus reactivation and the design of effective vaccines to combat iltv. proto-oncogene tyrosine-protein kinase src (src) was originally isolated from a chicken sarcoma and was thought to contribute to oncogenesis (vogt, 1971) . src has been shown to be an important factor for a wide range of biological processes, such as cell proliferation, adhesion, division, and death, angiogenesis, and organization of the cytoskeleton. src has also been reported to be involved in infections with a wide range of viruses, including polyoma virus, dengue, hiv-1, coronaviruses, influenza virus, and herpesvirus (bolen et al., 1984; burkard et al., 2015; cheshenko et al., 2005; gaur et al., 2012; krishnan et al., 2006; kumar et al., 2016; mccarthy et al., 2016) , suggesting that src may be a new potential target for antiviral therapeutic agents and may have potential for repurposing existing fda-approved src inhibitors. however, no clinical applications of antiviral src-targeting agents have been reported, which may be at least partially due to the complicated roles of src in its regulation of the multiple biological processes in which it is involved. the biological effects of src are complex and sometimes controversial. for example, some studies suggest that src is essential in pdgf-induced dna synthesis (barone and courtneidge, 1995; twamley-stein et al., 1993) ; however, other studies of pdgf-induced dna synthesis indicate that src is not absolutely required and even plays the opposite role (demali and kazlauskas, 1998; rosenkranz et al., 2000) . in addition, src is considered both a survival factor and a death factor depending on specific cellular conditions (fan et al., 2007 (fan et al., , 2013 (fan et al., , 2014 iqbal hossain et al., 2015) . our previous study identified src as the key host determinant of iltv infection (li et al., 2015) . we found that activation of host src upon iltv infection raises the threshold of host cells for iltv infection-induced cell death, thus maintaining the survival of infected cells and ensuring a high level of viral replication. however, therapies targeting src not only repress viral replication but also exacerbate the pathological effects of iltv and even increase the likelihood of death of the host. because the underlying molecular mechanisms have not been sufficiently investigated, the development of src-based therapeutics is limited. viral replication requires host cellular metabolism. while similarities exist, virus-specific metabolic requirements by different viruses have been identified. many viruses have been shown to induce fatty acid synthesis in host cells, which provides a stable carbon source and sufficient energy for viral replication, viral spread and even the prolonged survival of infected cells (rodríguez-sánchez and munger, 2019; sanchez and lagunoff, 2015; sanchez et al., 2017) . although the mechanism remains unclear for iltv, the utilization of host fatty acid metabolism has been found to be important for both latent and lytic infections with human gamma herpesvirus, kaposi's sarcoma-associated herpesvirus (kshv), and the survival of infected cells (delgado et al., 2012; sanchez et al., 2017) . thus, a better understanding of the involvement of fatty acid metabolism in viral infection may lead to the development of novel therapeutic approaches targeting specific cellular metabolic processes. in the present study, we observed accelerated cellto-cell spread of iltv with src inhibition in a cellular fatty acid metabolism-dependent manner, which determines the cytopathic effect (cpe) of iltv. given the importance of cell-to-cell spread during iltv infection as evidenced by the efficient protection against iltv infection provided to a host by cell-mediated immunity rather than humoural immunity (coppo et al., 2013) , the mechanism that we revealed may be valuable for the future development of more rational anti-iltv vaccination. the virulent iltv-ljs09 strain (genbank accession no. jx458822) is stored at the harbin veterinary research institute of caas. this strain can be propagated in a chemically immortalized leghorn male hepatoma (lmh) cell line with clear cpes observed zhao et al., 2013) . iltv-egfp was constructed by replacing the us9 gene of iltv-ljs09 with the egfp coding sequence as described previously (li et al., 2018) . lmh cells were maintained in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal bovine serum (fbs), 100 units/ml penicillin, 100 μg/ml streptomycin and 2 mm l-glutamine. cell cultures were incubated at 37°c in 5% co 2 . the src inhibitors pp1 (2 μm) and pp2 (20 μm) were purchased from sellckchem.com (selleck chemicals llc, houston, tx). the 5-tetradecyloxy-2-furoic acid (tofa, 60 μm), an allosteric inhibitor of acetyl co-a carboxylase, and the c75 (8 μm), a synthetic fatty-acid synthase (fasn) inhibitor, were purchased from sigma aldrich (sigma aldrich, st. louis, mo). given that all inhibitors we used were dissolved in dimethyl sulfoxide (dmso), cells treated with dmso at the same volumes were used as chemical control. dil and calcein am were purchased from beyotime biotechnology (beyotime biotech, shanghai, china). a short-interfering rna (sirna) pool that specifically recognizes different sequences of the src mrna and a control sirna with no specific target site in chickens were used (li et al., 2015) . transfection of sirna was conducted using an n-ter nanoparticle sirna transfection system (sigma aldrich) according to the manufacturer's instructions. lmh cells were infected with iltv at a multiplicity of infection (moi) of 0.1. the indicated moi was obtained according to the number of cells to be infected and the estimated number of infectious particles, based on plaque-forming units detected in lmh cells. levels of virus replication were determined using plaque assays and iltv-specific qpcr assays as previously described (li et al., 2018) . to determine the total level of viral replication, both cell-associated viruses and the viruses released into supernatant were collected for virus quantification. cells were lysed via three rounds of freezing-thawing. we conducted facs analyses using a bd facscan and cellquest software version 4.0.2 (bd, mountain view, ca). the activation of src was assayed using an antibody against phosphorylated src at y416 (emd millipore, billerica, ma, usa), followed by a secondary goat antimouse antibody conjugated to apc (jackson laboratory, bar harbor, me). mouse igg was used as an isotype control (abcam trading company, ltd., shanghai, china). the proportion of iltv-infected cells was determined via detection of egfp-positive cells or by using a rabbit polyclonal antibody against iltv glycoprotein i (li et al., 2015) , followed by a secondary goat anti-rabbit antibody conjugated to fitc (jackson laboratory). the background was determined by normal rabbit control serum from non-immunized rabbits. cell death was assayed by propidium iodide (pi) staining of living cells. for immunofluorescent examination, a fluorescent signal was detected with an evos fl fluorescence microscope (amg, bothell, wa). all cell nuclei were highlighted with hoechst 33342 (sigma aldrich). iltv-egfp-infected cells and uninfected cells were co-cultured at a ratio of 1:50 in the absence or presence of src inhibitors, pp1 and pp2, in 35-mm dishes. dishes were placed on a heated chamber at 37°c in an environmentally controlled mini-incubator maintained at 5% co 2 . images were captured on an ultraview vox confocal imaging system (perkinelmer, waltham, ma) in a nikon eclipse ti stand (nikon instruments, melville, ny) with a 20 × lens under the control of volocity software (v3.3.0, perkinelmer). extracellular vesicle (including exosomes and microvesicles) preparation and examination were performed according to previous description (jung and mun, 2018) . lmh cells were cultured to near confluency in a 75 cm dish, and cells were mock infected or infected at a moi of 1 in serum-free dmem at 4°c for 1 h. then cells were washed once with pbs and cultured in extracellular vesicle-free full dmem medium in the absence or in the present of pp1 or pp2. medium was harvested after 24 hpi and extracellular vesicles were purified by differential centrifugation at 4°c (300×g for 10 min, 2000×g for 20 min, 10000×g for 40 min using a beckman ja-25.15 fixed angle rotor, then ultracentrifuged 100000×g for 90 min using a beckman sw-41 swinging bucket rotor). the extracellular vesicle pellets generated were resuspended in serum-free dmem, fixed and dried. then samples were coated with gold/palladium alloy by sputter coating and examined under a hitachi h-7650 transmission electron microscope (hitachi high technologies, shanghai, china), and images were taken using an amt ccd camera (advanced microscopy techniques, woburn, ma). western blotting was performed under reduced denaturing conditions according to previously described procedures (li et al., 2014) . briefly, cells were washed with ice-cold pbs, and soluble proteins were extracted with cell lysis buffer (100 mm tris-hcl ph = 8, 150 mm nacl, 1% np-40, phosphatase and protease inhibitor cocktail tablets (abcam) according to the manufacturer's protocol). the protein concentration was determined using the bio-rad bradford assay (bio-rad, hercules, ca) and bsa standards (sigma aldrich). an equal amount of protein was separated by sds-page. antibodies against src (emd millipore) and actin (sigma aldrich) were used. genome-wide gene expression profiling of lmh cells was performed using rna deep sequencing by annoroad gene technology co., ltd, (beijing, china). library construction was performed following illumina (san diego, ca) manufacturer instructions. samples were sequenced on an illumina hiseq 2500 instrument. rna was isolated from lmh cells using rneasy plus mini kit (qiagen, hilden, germany) according to the manufacturer's protocol. rt-qpcr was performed using the sybr primescript™ kit (takara bio inc, tokyo, japan) as described previously (cong et al., 2013) . primer sequences are presented in table 1 . data is calculated with 2 −δδct method and results were presented as log 2 fold change. rna sequencing data was analyzed with a web-based tool, galaxy (blankenberg et al., 2010) . pathway analysis was performed with david (gene-enrichment analysis using ease score, a modified fisher exact p-value, as threshold) . rna sequencing raw data was uploaded to ncbi with accession number gse98629. the spss software package (spss for windows version 13.0, spss inc., chicago, il, usa) was used for all statistical analyses. data obtained from several experiments are reported as the mean ± standard deviation (sd). the significance of differences between two groups was determined with student's t-test. one-way or two-way analysis of variance with the bonferroni correction was employed for multigroup comparisons. for all analyses, a probability (p) value of < 0.05 was considered statistically significant. an iltv-lsj09 strain expressing enhanced green fluorescent protein (egfp), which exhibits no significant difference from the wild type in either viral replication or the cpes of infection as described previously (li et al., 2018) , was employed to monitor viral infection. with this egfp strain, we investigated the effect of src repression on the transmission of iltv in leghorn male hepatoma (lmh) cells with a multiplicity of infection (moi) of 0.1. we first performed src depletion using pooled sirnas targeting different sequences of src. src depletion was confirmed by western blotting (fig. 1a) . upon iltv infection, an egfp signal was observed in approximately 10% of cells at 24 h post infection table 1 list of rt-qpcr primers. wang, et al. virology 537 (2019) 1-13 (hpi), which met the expected proportion at an moi of 0.1 and was increased significantly by src depletion as proven by the detection of egfp-positive cells via fluorescence-activated cell sorting (facs) (fig. 1b) . a similar conclusion was obtained using an antibody specifically targeting the gi protein of iltv as assayed by facs (fig. 1c) , suggesting that the proportion of egfp-positive cells can be used to reflect iltv spread in our model. consistent with these findings, identical results were obtained for cells with repression of src activity by one of two widely used src inhibitors, pp1 or pp2, both of which repressed src transcription and src phosphorylation at y416 in lmh cells ( fig. 1d and e) as assayed by facs ( fig. 1f and g). the above findings demonstrate induced spread of iltv with repression of src activity in lmh cells. to address whether viral replication contributed to the accelerated iltv dissemination by src inhibition in lmh cells, we measured the total viral titre, including both cell-associated viruses and the viruses released into supernatant, using iltv-specific qpcr ( fig. 2a) and plague formation assays (fig. 2b) . consistent with our previous findings that src is important for iltv to maintain a high level of viral production (li et al., 2015) , the final production of viruses was significantly reduced by both src inhibitors as assayed at 5 days post infection (dpi) for cells treated with pp1 or pp2 and at 6 dpi for cells treated with dmso when all infected cells were dead (corresponding the depletion of src in lmh cells was performed with a pool of two sirnas targeting different sequences of src, and the protein levels of src were assessed by immunoblotting. actin was used as a loading control. (b and c) the percentage of infected cells at 24 h post infection upon depletion of src was quantified by facs via detecting egfp-positive cells (b) or using a polyclonal antibody against glycoprotein i of iltv produced in rabbits followed by an apc-conjugated anti-rabbit second antibody (c). the background was determined by normal rabbit serum. (d and e) src inhibition by pp1 or pp2 was assayed by rt-qpcr (d) and facs using an antibody specifically recognizing phosphorylated src at y-416 followed by an apc-conjugated anti-rabbit secondary antibody (e). the background was determined by a normal rabbit igg control. (f and g) the percentage of infected cells at 24 h post infection upon inhibition of src was quantified by facs via detecting egfp-positive cells (f) or using a polyclonal antibody against glycoprotein i of iltv produced in rabbits followed by an apc-conjugated anti-rabbit second antibody (g). the background was determined by normal rabbit serum. the results in b, c, d, f and g are presented as the mean ± sd, n = 3. asterisks indicate statistical difference (p < 0.05). z. wang, et al. virology 537 (2019) 1-13 time points are indicated by "end" in fig. 2a and b) . however, neither method revealed any difference in viral titres between cells in the absence or presence of src inhibitors within three dpi, and during this period, the viral genome copies continued to increase in all infected groups. the existence of virions was even undetectable at 1 dpi (fig. 2b) . the similar levels of viral replication within 3 dpi demonstrated that the accelerated viral spread by src inhibition that we observed at 24 hpi is independent of viral replication. next, a detailed investigation of iltv dissemination was carried out by monitoring the spread of egfp using time-lapse microscopy. iltv-egfp-infected cells (used as donor cells) and uninfected cells (used as target cells) were co-cultured at a ratio of 1:50 in the absence or presence of a src inhibitor. as shown by time-lapse microscopy, egfp signals appeared between 3 and 6 hpi in all iltv-infected groups. the transmission of the egfp signal first occurred in cells treated with the src inhibitor after 12 hpi but was rarely observed in cells in the absence of src inhibition throughout the observation period (fig. 3a) . interestingly, the egfp signal mainly spread from infected cells to adjacent uninfected cells rather than uninfected cells without direct contact with infected cells. therefore, we hypothesized that the distant transmission by extracellular iltv particles or extracellular vesicles did not contribute to the enhanced iltv spread that we observed. to address this hypothesis, a rabbit polyclonal antibody against iltv glycoprotein i produced in our lab (li et al., 2015) was utilized as a neutralizing antibody to block virions released into culture medium by infected cells. the neutralization of extracellular virions by the antibody was verified by the observation of egfp-positive cells (fig. 3b) . however, the promotion of iltv spread by src inhibition was not compromised by the addition of this neutralizing antibody as assayed by facs (fig. 3c) . this hypothesis was further confirmed by the fact that neither the amount nor the infectivity of extracellular vesicles (including both exosomes and microvesicles) secreted into culture medium was affected by src inhibition in the presence of the neutralizing antibody (fig. 3d) . taken together, the above findings suggest that src inhibition promotes iltv spread mainly in a cell-to-cell manner. in addition to viral replication, the initial infection and the proliferation of infected cells may have also contributed to the enhanced viral spread by src inhibition in our experimental system. given that the initial infection was the same for all groups in our studies, we next addressed whether the accelerated iltv transmission by src inhibition was due to the proliferation of infected cells. to answer this question, iltv-egfp infected cells (used as donor cells) and uninfected cells labelled with dil (used as target cells) were co-cultured at a ratio of 1:50 in the absence or presence of a src inhibitor upon the addition of neutralizing antibodies, as indicated in fig. 3e (left panel) . src inhibition by either inhibitor significantly increased the proportion of cells that were positive for both egfp and dil and the proportion of cells that were positive for egfp but negative for dil (fig. 3e, right panel) as assayed by facs, suggesting that both the proliferation of infected cells and the cell-to-cell spread of viruses contributed to the enhanced spread of iltv by src inhibition in our system. src inhibition most likely promoted iltv spread via the latter mechanism since the proportion of egfp/dil double-positive cells was approximately nine-times that of cells positive for egfp only. taken together, the above findings suggest that src inhibition promotes iltv transmission mainly in a cell-to-cell manner. inhibition of src promotes cell-to-cell spread of iltv independently of direct cytosol-to-cytosol connection. alphaherpesviruses can transmit intercellularly from infected cells to neighbouring cells by either passing through cytosol-to-cytosol connections directly or crossing the contacts between cells (kadiu and gendelman, 2011; lucas, 2006) . to determine the pathway by which src inhibition promotes cell-to-cell spread of iltv, the dissemination of calcein am, a dye that is soluble in cytoplasm and can freely pass through cytosol-to-cytosol connections, was assayed. iltv-infected or uninfected cells stained with calcein am were used as donors, and cells labelled with dil were used as targets. given that the proportion of cells positive for both calcein and dil detected by facs was too low to be analysed at ratio of 1:50 (lower than 1% in all groups), donors and targets were co-cultured at a ratio of 1:4 in the presence of a src inhibitor or dmso as indicated (fig. 4a) . as assayed by facs, direct cytosol-to-cytosol connections occurred naturally between uninfected cells and were significantly promoted by iltv infection (fig. 4b) . however, these connections were not affected by src activity. together with the low frequency at a ratio of 1:50, this finding excluded the possibility of the involvement of direct cytosol-to-cytosol connections in the enhanced intercellular spread of iltv by src inhibition. inhibition of src promotes iltv cell-to-cell spread in a cellular fatty acid metabolism-dependent manner. to elucidate the molecular mechanism underlying the accelerated cell-to-cell spread of iltv by src inhibition, a genome-wide transcriptional profile analysis was conducted. rna isolated from mock cells or iltv-infected cells with distinct src activities was subjected to rna sequencing. bioinformatics analyses identified 3599 genes differentially expressed among groups based on the following criteria: (i) a pvalue < 0.01, (ii) a q-value < 0.001, and (iii) a fold change > 1.5 (table s1 ). hierarchical clustering analysis using these differentially expressed genes demonstrated efficient clustering of biological replicates for each group of cells (fig. 5a ). among these groups, the genome-wide transcription profiles of uninfected cells and the genomewide transcription profiles of infected cells were clustered separately. a total of 721 up-regulated genes and 1028 down-regulated genes were identified upon iltv infection in the absence of src inhibition (fig. 5b) . for validation purposes, the transcription levels of 15 randomly selected genes were examined using an rt-qpcr assay. the rt-qpcr results corresponded to those from the rna-seq assays (fig. 5c) . among the 1749 genes significantly altered by iltv infection in the absence of src inhibition, the up-regulation of 309 genes and the downregulation of 497 genes were halted by pp1 treatment, and the up(a and b) the replication of iltv in lmh cells was determined by iltv-specific qpcr (a) and plaque assays (b), respectively. the results are presented as the mean ± sd, n = 3. asterisks indicate statistical difference (p < 0.05). z. wang, et al. virology 537 (2019) 1-13 regulation of 341 genes and the down-regulation of 449 genes were attenuated by pp2 treatment (fig. 5b) . accordingly, 234 up-regulated genes and 331 down-regulated genes were commonly regulated by pp1 and pp2 treatments (fig. 6a , left panel) and named 'genes uniquely upregulated/down-regulated by iltv infection'. among the genes unaffected by iltv infection, 167 genes were up-regulated and 270 genes were down-regulated by pp1 treatment, and 301 genes were up-regulated and 459 genes were down-regulated by pp2 treatment upon iltv infection (fig. 5b) , with 83 up-regulated genes and 128 down-regulated genes in common (fig. 6a , left panel), which were named 'genes uniquely altered by src inhibitors'. considering the difference in transcriptional profiles between cells treated with pp1 or pp2, to avoid offtarget effects of the inhibitors, only these gene subsets commonly altered by pp1 and pp2 treatments in iltv-infected cells were included for further analyses. pathway analysis using david functional annotation with a p-value < 0.05 revealed pathways involved in fatty acid metabolic processes that were significantly enriched by 'genes uniquely down-regulated by iltv infection' (fig. 6a , right panel, table s2 ) but did not uncover any pathway enriched by either 'genes uniquely altered by src inhibitors' or 'genes uniquely up-regulated by iltv infection'. further david functional annotation clustering analysis (enrichment score > 1.5) using 'genes uniquely down-regulated by iltv infection' identified fatty acid metabolism-related biological processes as the cluster of the biological processes with the highest enrichment score (fig. 6b) . to verify the role of fatty acid metabolism in the accelerated intercellular spread of iltv by src inhibition, two widely used chemicals that specifically inhibit fatty acid synthesis, namely, the allosteric inhibitor of acetyl co-a carboxylase tofa and the synthetic fatty-acid synthase inhibitor c75, were employed with or without the addition of palmitic acid (pal), as shown in fig. 7a . both inhibitors did not affect the replication of viral dna but significantly repressed virion production (p < 0.05) as assayed by iltv-specific qpcr (fig. 7b ) and plague formation assays (fig. 7c) , suggesting that fatty acid metabolism is not required for viral genome replication but is essential for virion production. considering the potential influence of the effects of tofa and c75 on iltv replication, neutralizing antibodies were added while investigating the role of fatty acid metabolism in the promotion of viral spread and cell death by src repression via facs (fig. 7d and e) . both tofa and c75 compromised the promoting effect of src inhibition on viral spread (fig. d) . the inhibitory effects of tofa and c75 on virion production and viral cell-to-cell spread in lmh cells upon src inhibition were rescued by the addition of pal (fig. c and d) . the enhanced death of infected cells by src inhibition was also reduced by tofa and c75 (fig. e) . no significant effect on cell death was observed in cells treated with only pp1, pp2, tofa, or c75 (fig. e) , suggesting that these chemicals are not cytotoxic to lmh cells. thus, our data demonstrate that fatty acid metabolism is essential for the regulation of the cell-to-cell spread and cytopathogenicity of iltv by src. cell-to-cell spread of intracellular pathogens is one of the mechanisms that emerge during the co-evolution of a virus and its host, which is important not only for the pathogenesis of viral infection but also anti-microbial therapies. currently, no antiviral drug targeting the latent infection of alphaherpesvirus is available, which can lead to outbreaks when the host is under stress or the host's immune system is compromised. during both the establishment of latent infection in sensory neurons and reactivation from latency, the virus spreads intercellularly between the mucocutaneous tissue and sensory neurons (arvin et al., 2007; abaitua et al., 2013) . thus, elucidation of the underlying mechanisms modulating the cell-to-cell spread of alphaherpesviruses may provide promising targets for the development of novel therapeutic strategies and for refining the rational design of effective vaccines to combat alphaherpesviruses. despite extensive research efforts, the precise intercellular dissemination mechanisms remain largely unclear. iltv shares common characteristics with other alphaherpesviruses, including its resistance to host humoural immune responses (coppo et al., 2013) , indicating the importance of intercellular virus spread during iltv infection. our previous studies identified src as a key host determinant of iltv infection. in the present study, we found that src acts as the key orchestrator between iltv and its host during infection through its control of viral cell-to-cell transmission and subsequent cell death in a cellular fatty acid metabolismdependent manner. our findings not only provide new insights regarding the mechanisms by which host src regulates iltv infection but may also help further reveal the mechanism of the cell-to-cell spread of other alphaherpesviruses. numerous efforts have been made to elucidate the mechanisms involved in the intercellular spread of viruses. the simplest and most widely used model of cell-to-cell spread is plaque formation in cell cultures with or without the addition of a neutralizing antibody, which reflects the cumulative processes of the host-virus interaction, including viral binding, penetration and replication, reorganization of cell-to-cell junctions, viral intercellular transport, and restriction mechanisms used by the host to resist infection, such as antiviral immune responses and factors (dingwell et al., 1994; johnson et al., 2001; mettenleiter et al., 2009 ). such models have been frequently used to investigate the processes of cell-to-cell spread and the essential viral and host factors for these processes (farnsworth and johnson, 2006; krummenacher et al., 2003; sourvinos and everett, 2002) . recent studies utilizing advanced techniques and equipment to visualize viral infections in living cells, such as time-lapse microscopy, have revealed some novel features of viral cell-to-cell spread that had previously been unexpected and unrecognized. for instance, many viruses have been observed to spread z. wang, et al. virology 537 (2019) 1-13 faster than the limits imposed by their replication kinetics. for poxviruses, through the repulsion of superinfecting virions by infected cells mediated by viral proteins a33, a36, a56, k2, and b5, the speed of viral spread is four-times faster than that expected according to their replication kinetics (doceul et al., 2010 (doceul et al., , 2012 . rapid viral cell-to-cell spread has also been observed for hiv and herpesviruses, and paracrine-dependent migration of uninfected cells to the site of infection (abaitua et al., 2013; igakura et al., 2003; jolly and sattentau, 2004) as well as intercellular spread via nanotubes (eugenin et al., 2009) were thought to contribute to this phenomenon. cytoplasmic connections can promote the spread of viruses such as hiv-1 and herpesviruses by transporting viral genomes, viral capsids or vesicles containing viral particles between adjacent cells (digel et al., 2006; fan et al., 2014; lucas, 2006) . the present study suggests that despite being enhanced by iltv infection, direct cytosol-to-cytosol connections do not contribute to the promoted intercellular spread of iltv by src inhibition z. wang, et al. virology 537 (2019) 1-13 ( fig. 4) . in addition, herpesviruses can travel from infected cells to neighbouring cells via budding at the basolateral intercellular junction or by crossing the tight junctions between cells (cocchi et al., 2000; dingwell et al., 1994; york and johnson, 1993) . whether these mechanisms are responsible for the accelerated spread of iltv that we observed remains unclear. further studies utilizing transmission electron microscopy are needed to investigate the exact mechanism of the enhanced cell-to-cell spread of iltv by src inhibition. our previous and present studies suggest that src plays a very complex role during iltv infection. on one hand, src activation raises the threshold of infected host cells for infection-induced host cell death, which prolongs the duration of iltv replication in each infected cell and allows the infiltrated virus to achieve optimal replication. meanwhile, along with src activation, iltv infection also tends to accelerate viral cell-to-cell spread. unexpectedly, according to the findings of the present study, this process is restricted by the activation of host src triggered by the iltv infection itself. in sum, src activation by iltv not only ensures the ultimate replication of viruses in infected cells but also limits viral dissemination. why does iltv activate host src to achieve these opposite functions? as we showed in previous z. wang, et al. virology 537 (2019) 1-13 studies, src inhibition not only represses iltv replication but also reduces the threshold of infected cells for iltv-triggered cell death and pathologic damage (li et al., 2015) . considering the rapid establishment of latent infection during the initial infection with iltv, the opposite effects of src activation by iltv infection most likely reflect a strategy that evolves during the co-evolution of iltv and its host, which establishes a balance for the pathogen-host interaction. host metabolic requirements for maximal viral production have been examined in several human herpesviruses, including human cytomegalovirus (hcmv), herpes simplex virus 1 (hsv-1), and kshv (delgado et al., 2012; munger et al., 2006 munger et al., , 2008 sanchez et al., 2017; vastag et al., 2011) , and the results suggest that inhibition of specific cellular metabolic pathways can both eliminate latently infected cells and block lytic replication, thus providing opportunities for the development of novel therapeutic approaches for herpesviruses. although similarities exist, significant differences in the utilization of host metabolic resources by these herpesviruses have been reported. for example, hcmv increased the levels of the tricarboxylic acid (tca) metabolites fuelling fatty acid synthesis, while hsv-1 increased the level of flux to the tca cycle to drive pyrimidine synthesis. the metabolic z. wang, et al. virology 537 (2019) 1-13 signature of iltv has not yet been addressed. interestingly, our data demonstrate that src inhibition promotes intercellular iltv spread in a host cellular fatty acid metabolism-dependent manner (fig. 7) . src is a myristoylated protein and can be found both within and outside of lipid rafts, as well as within endosomes, before translocating to the plasma membrane (seong et al., 2009) . src can be activated by exogenous saturated fatty acids, such as palmitate and myristic acid, or the overexpression of cluster of differentiation 36 (cd36, also named fatty acid translocase), which is known to facilitate long-chain fatty acid uptake through the myristoylation of src, leading to activation of its downstream signalling, such as jnk signalling (holzer et al., 2011; huang et al., 2017; kim et al., 2017 kim et al., , 2019 park et al., 2016) . nonetheless, given the increased fatty acid oxidation observed in mice with knockout of fyn, another src kinase family member (bastie et al., 2007) , src activation by iltv infection may limit viral intercellular spread by balancing host fatty acid metabolism, which can be negated by src inhibition, resulting in accelerated viral spread and an exacerbated cpe of infection as observed in our model. this hypothesis is partially supported by our findings that inhibition of src rescues the reduced transcription of genes involved in fatty acid metabolism (fig. 6 , table s2 ), and that the promoting effects of src inhibition on both intercellular viral spread and the cytopathogenicity of iltv can be significantly attenuated by modulation of host fatty acid synthesis (fig. 7) . to exclude the potential off-target effect of src inhibitor, two inhibitors were employed in our studies, and conclusion was made only when similar results were obtained using both inhibitors. the generation of lmh cell line and chicken embryo with src knockout background using crispr/cas9 system will be greatly helpful to further investigations of src-mediated mechanisms in iltv infection by excluding any cell type or chemical specific effect. considering the heterogeneous environment and the difficulty associated with controlling and monitoring the cell-to-cell spread of iltv in vivo, in vitro studies were carried out to investigate the effect of src on iltv spread. although primary cells isolated from chicken embryos can better mimic the in vivo process of iltv infection, the instability of the biological characteristics of different isolates and the short lifespan of primary cells during in vitro culture limit their use in cellular and molecular mechanistic studies. lmh is the only cell line susceptible to iltv infection in vitro. our previous studies found that this cell line infected with iltv can sufficiently mimic iltv in ovo infection, and all srcmediated mechanisms that we identified in lmh cells were validated in ovo, suggesting that lmh cells infected with iltv are reliable in vitro models for studying iltv infection (li et al., 2015 (li et al., , 2018 . in addition, we also analysed the only publicly available, genome-wide gene expression data of primary lung cells isolated from chicken embryos infected with a different virulent iltv strain (lee et al., 2010) , which again revealed that host src is the central regulator of the molecular network induced by iltv infection in host cells (li et al., 2015) . all these results support the notion that the mechanism that we identified in lmh cells is universal in normal cells and tissues rather than a cell line-specific event. thus, the lmh cell line was selected for the mechanistic study of src regulation of iltv infection in the present research. further investigations using primary cultures of tissue samples or cells isolated from the mucosal epithelium of tracheal and conjunctival specimens infected with multiple iltv strains are needed to determine the universality of the regulatory mechanism of src on iltv spread that we revealed in the present study. iltv causes an economically important chicken disease. despite the extensive administration of live attenuated vaccines since the midtwentieth century and the administration of recombinant vaccines in recent years, infectious laryngotracheitis outbreaks due to iltv occur annually worldwide, and there is no cure for it. similar to other alphaherpesviruses, iltv establishes latency in the trigeminal ganglia after acute infection of the upper respiratory tract, and reactivation of viruses from latent infection occurs frequently once host immunity is compromised. no drug or efficient treatment targeting iltv latent infection is currently available. we previously identified src as a key host determinate of iltv infection. however, therapies targeting src not only repress viral replication but also exacerbate the pathological effects of iltv. the underlying mechanisms of the side effects remain unknown. our present study suggests that host src controls the cell-tocell spread of iltv in a cellular fatty acid metabolism-dependent manner, which determines the cpe of iltv. given the importance of cell-to-cell spread during iltv infection, our findings not only provide new insights regarding the mechanisms of iltv infection but may also be valuable for the future development of novel therapeutic strategies and for refining the rational design of effective vaccines. hl, sl and pw designed experiments and wrote the manuscript; zw, bs, qg, ym, yl, zc, hw, lc, and ys performed the experiments; zw, bs, qg, hl sl and pw analyzed the data. all authors reviewed the results and approved the final version of the manuscript. the authors declare that they have no conflicts of interest with the contents of this article. polarized cell migration during cell-to-cell transmission of herpes simplex virus in human skin keratinocytes human herpesviruses: biology, therapy, and immunoprophylaxis integrative metabolic regulation of peripheral tissue fatty acid 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plasma membrane by fret imaging visualization of parental hsv-1 genomes and replication compartments in association with nd10 in live infected cells the src family tyrosine kinases are required for platelet-derived growth factor-mediated signal transduction in nih 3t3 cells divergent effects of human cytomegalovirus and herpes simplex virus-1 on cellular metabolism spontaneous segregation of nontransforming viruses from cloned sarcoma viruses further studies on the effect of neutralizing antibody upon the course of herpes simplex infections in tissue culture direct contact with herpes simplex virus-infected cells results in inhibition of lymphokine-activated killer cells because of cell-to-cell spread of virus detection of infectious laryngotracheitis virus by real-time pcr in naturally and experimentally infected chickens supplementary data to this article can be found online at https:// doi.org/10.1016/j.virol.2019.08.011. key: cord-300470-vgd1ol2z authors: conradie, andelé m.; stassen, liesel; huismans, henk; potgieter, christiaan a.; theron, jacques title: establishment of different plasmid only-based reverse genetics systems for the recovery of african horse sickness virus date: 2016-09-19 journal: virology doi: 10.1016/j.virol.2016.07.010 sha: doc_id: 300470 cord_uid: vgd1ol2z in an effort to simplify and expand the utility of african horse sickness virus (ahsv) reverse genetics, different plasmid-based reverse genetics systems were developed. plasmids containing cdnas corresponding to each of the full-length double-stranded rna genome segments of ahsv-4 under control of a t7 rna polymerase promoter were co-transfected in cells expressing t7 rna polymerase, and infectious ahsv-4 was recovered. this reverse genetics system was improved by reducing the required plasmids from 10 to five and resulted in enhanced virus recovery. subsequently, a t7 rna polymerase expression cassette was incorporated into one of the ahsv-4 rescue plasmids. this modified 5-plasmid set enabled virus recovery in bsr or l929 cells, thus offering the possibility to generate ahsv-4 in any cell line. moreover, mutant and cross-serotype reassortant viruses were recovered. these plasmid dna-based reverse genetics systems thus offer new possibilities for investigating ahsv biology and development of designer ahsv vaccine strains. african horse sickness (ahs), of which african horse sickness virus (ahsv) is the causative agent, is one of the most lethal systemic diseases of horses with a mortality rate in naïve animals up to 95% (stassen et al., 2014) . the virus is transmitted primarily by certain species of biting midges in the genus culicoides, which become infected after taking a blood meal from an infected viraemic host (mellor et al., 2000; venter et al., 2009) . although ahs is endemic in sub-saharan africa, outbreaks with devastating effects have occurred in north africa, the middle east, the arabian peninsula and southern europe (howell, 1960; rafyi, 1961; diaz montilla and panos marti, 1967; lubroth, 1988; rodriguez et al., 1992; portas et al., 1999) . different factors such as climate change (purse et al., 2008; tabachnick, 2010) and increased international trade and movement of animals (maclachlan and guthrie, 2010; martinez-lopez et al., 2011) have raised concerns that ahsv may be re-introduced in european countries. due to the economic impact of disease outbreaks, severity of disease in horses, and its capacity for sudden and rapid expansion, ahs is listed by the world organization for animal health (oie) as a notifiable equine disease. ahsv is a member of the genus orbivirus within the family reoviridae. like other members of this genus, ahsv is non-enveloped and has a complex capsid structure (manole et al., 2012) . the virion is comprised of two concentric protein shells surrounding the genome of 10 linear segments of double-stranded (ds) rna, which are designated from segment 1 (s1) to s10 in decreasing order of size (bremer et al., 1990) . the outer capsid comprises two proteins, vp2 and vp5, which together form a continuous layer that covers the inner capsid or core. the core particle is composed of two major (vp3 and vp7) and three minor (vp1, vp4 and vp6) proteins, in addition to the dsrna genome (manole et al., 2012) . the outer capsid proteins are removed during cell entry and transcriptionally active core particles are released into the cytosol where virus replication occurs (forzan et al., 2007) . within the core particle, each of the dsrna genome segments are repeatedly transcribed by the core-associated enzymes vp1 (rna-dependant rna polymerase), vp4 (capping-enzyme) and vp6 (helicase), resulting in extrusion of newly synthesized capped viral singlestranded rna (ssrna). the extruded positive-sense transcripts have the dual function of serving as templates for the synthesis of contents lists available at sciencedirect journal homepage: www.elsevier.com/locate/yviro viral proteins and for negative-sense rna synthesis during dsrna replication (mertens and diprose, 2004; patel and roy, 2014) . in addition to the structural proteins, other ahsv-encoded proteins (ns1, ns2, ns3 and ns4) are also produced in infected cells where they are thought to be involved in virus replication, morphogenesis and release of progeny virus particles (uitenweerde et al., 1995; van staden et al., 1995; maree and huismans, 1997; zwart et al., 2015) . although the veterinary (guthrie, 2007; clift and penrith, 2010) and vaccine-related aspects of ahsv have received some attention (scanlen et al., 2002; guthrie et al., 2009; alberca et al., 2014) , ahsv replication and the role of individual viral proteins in this process, as well as ahsv-host cell interactions remain largely uncharacterized. a reverse genetics system to engineer viable virus containing targeted sequence modifications has become an essential tool for the molecular dissection of viral gene products, studies regarding viral replication and pathogenesis, and for the development of vaccines. the development of a reverse genetics system for the reoviridae family has lagged behind that of other rna virus families due to technical complexities associated with the manipulation of multi-segmented dsrna genomes (komoto and taniguchi, 2013; trask et al., 2013) . nevertheless, in recent years, reverse genetics systems have been developed for rotavirus (genus rotavirus), reoviruses (genus orthoreovirus), as well as ahsv, bluetongue virus (btv) and epizootic haemorrhagic disease virus (ehdv) (genus orbivirus) (komoto et al., 2006; kobayashi et al., 2007; boyce et al., 2008; kaname et al., 2013; vermaak et al., 2015; yang et al., 2015) . for rotavirus, single gene replacement systems that require helper virus, selection, or both have been developed (komoto et al., 2006; trask et al., 2010; troupin et al., 2010) . in contrast, helper virus-independent reverse genetics systems have been established for reoviruses and orbiviruses. ahsv, btv and ehdv can be recovered by the transfection of in vitro-transcribed and capped rnas into permissive cell lines (boyce et al., 2008; vermaak et al., 2015) or into cells that had previously been transfected with helper expression plasmids that synthesize the core proteins and the two non-structural proteins ns1 and ns2 (kaname et al., 2013; matsuo and roy, 2013; yang et al., 2015) . an entirely plasmid only-based reverse genetics system has been developed for mammalian orthoreovirus (kobayashi et al., 2007) and, more recently, for btv (pretorius et al., 2015) and a fusogenic bat-borne orthoreovirus (kawagishi et al., 2016) . the development of reverse genetics has revolutionized the study of reoviruses and btv (roy, 2013) by providing a powerful tool for investigating virus replication and disease. here, we report the development of a plasmid-only reverse genetics system for ahsv-4 that allows for recovery of the virus from 10 cdna clones representing the viral genome. we subsequently improved the basic ahsv-4 reverse genetics system by consolidating the viral genome segment cdnas into five plasmids, which permits more efficient virus recovery compared to the 10plasmid system. finally, we increased the flexibility of the 5-plasmid reverse genetics system by including a t7 rna polymerase expression cassette onto the genetic backbone of the reverse genetics plasmid, thus liberating the rescue of viable virus from its dependence on t7 rna polymerase-expressing cell lines. the performance of the reverse genetics systems were validated by recovery of mutant and directed cross-serotype reassortant viruses. the enhancements reported here represent a significant advance for the application of reverse genetics to the study of ahsv. previous work from our laboratory established a reverse genetics system for the prototype orbivirus, btv, using a bacteriophage t7 rna polymerase system, whereby infectious virus could be generated reproducibly entirely from cdna clones (pretorius et al., 2015) . in this study, we generated two reverse genetics plasmids, pjad1 and pjad2, as described under materials and methods. both the pjad1 and pjad2 plasmids contain an identical transcription cassette, which was obtained from the prg15 reverse genetics plasmid, and is flanked by newly introduced t7te transcription terminators to prevent transcription into or out of the cloned dna. moreover, with a view to reducing the number of plasmids required for ahsv-4 recovery, unique restriction enzyme sites were also incorporated into the reverse genetics plasmids. in the case of pjad1, unique stui restriction sites flanking the transcription cassette was incorporated, as well as a unique pmei restriction site adjacent to the downstream t7te terminator. in the case of pjad2, unique pmei restriction sites that flank the t7te terminator sequences were incorporated (fig. 1 ). to generate ahsv-4 from cloned cdna, plasmids encoding each viral genome segment were engineered by incorporating cdna copies of genome segments s1 through s5 into pjad1 and cdna copies of genome segments s6 through s10 into pjad2. each plasmid thus contains one full-length genome segment cdna placed under control of the bacteriophage t7 rna polymerase promoter and appended with the hepatitis delta virus (hdv) ribozyme at the 3′ terminus. after transcription with t7 rna polymerase, these plasmids are anticipated to generate 10 full-length positive-sense rnas, each containing native 5′-and 3′-ends ( fig. 2a) . the 10 ahsv-4 constructs were co-transfected into bsr-t7 cells, which constitutively express t7 rna polymerase. the cells were harvested 5 days post-transfection, lysed and plaque assays were performed on bsr cells. plaques were recovered from cells transfected with the 10 ahsv-4 cdna plasmids, indicating the presence of replicating virus. in contrast, no plaques were observed in control, mock-transfected cells (fig. 2b ). to confirm virus recovery, individual plaques were picked, amplified in bsr cells and genomic dsrna purified from infected cells was subsequently analysed on a non-denaturing polyacrylamide gel. the electropherotype of plasmid-derived ahsv-4 was indistinguishable from that of wild-type ahsv-4 derived from cell infection (fig. 2c) . to exclude the possibility that recovered ahsv-4 represents contamination by wild-type ahsv-4, a silent mutation (a to g at nucleotide 1010) resulting in the introduction of a unique psti site was introduced into the s5 genome segment by site-directed mutagenesis of the pjad-s5 rescue plasmid. since this change has not been observed in any reported ahsv s5 genome sequences, it thus serves as a signature for virus derived from plasmid-based rescue. the full-length s5 genome segment (1.748 kb) was amplified using rt-pcr of viral dsrna extracted from bsr cells infected with either plasmid-derived ahsv-4 or wild-type ahsv-4. the s5 genome segment rt-pcr product derived from wild-type ahsv-4 was not digested with psti, whereas the s5 product from plasmid-derived ahsv-4 produced the expected dna fragments of 1012 and 736 bp upon digestion. the nucleotide sequence of the purified amplicons were also determined, the results of which confirmed the presence of the unique psti restriction enzyme site in the s5 genome segment from plasmid-derived ahsv-4 (fig. 2d) . to determine whether the plasmid-derived ahsv-4 and wild-type ahsv-4 have similar replication kinetics, bsr cells were infected with the respective viruses at a moi of 0.1 pfu/cell and virus titres were determined at different times post-infection. there was essentially no difference in growth kinetics, and the titres of the plasmid-derived ahsv-4 and wild-type ahsv-4 were virtually identical at all the time points tested (fig. 2e ). taken together, the data indicate that ahsv-4 can be recovered entirely from plasmid cdna and that the plasmid-derived virus retained the properties of wild-type ahsv-4. 2.3. generation of a simplified reverse genetics system using five plasmids previous reports have indicated that a reduction in the number of plasmids required to rescue infectious virus may improve the efficiency of plasmid-based reverse genetics systems (neumann et al., 2005; zhang et al., 2011; kobayashi et al., 2010) . thus, to reduce the number of plasmids required to rescue ahsv-4 by plasmid-based rescue, the ahsv-4 s6 through s10 genome segment transcription cassettes flanked by t7te transcriptional terminators were recovered from the respective recombinant pjad2 plasmids by digestion with pmei and blunt-end cloned into the unique pmei site of the recombinant pjad1 plasmids (fig. 1) . the derived reverse genetics plasmids were designated pjad-s1-s8, pjad-s2-s6, pjad-s3-s7, pjad-s4-s10 and pjad-s5-s9 (fig. 3a) . as expected, infectious ahsv-4 was recovered following plaque assays on bsr cell monolayers of the transfection cell lysate and subsequent characterization of the plaque-purified virus confirmed their plasmid origin ( fig. 3b-d) . reassortant viruses based on the targeted exchange of specific genes may facilitate not only the functional mapping of genes, such as virulence factors, but also allow for the generation of vaccine strains of particular serotypes. consequently, we attempted to modify ahsv-4 by exchange of the s2 and s6 genome segments of ahsv-4 with those of ahsv-1. the s2 and s6 genome segments encode for the outer capsid proteins vp2 and vp5, respectively. to this end, a dual reverse genetics plasmid was constructed, designated pjad(a1)-s2-s6, which contained transcription cassettes of the s2 and s6 genome segments from ahsv-1. bsr-t7 cells were co-transfected with 5 plasmids in which the wild-type pjad-s2-s6 construct was substituted with the pjad (a1)-s2-s6 construct. reassortant viruses from randomly selected plaques were amplified in bsr cells, dsrna extracted and the genotype of the recovered viruses was determined by electrophoresis of the dsrna on a non-denaturing polyacrylamide gel. the electrophoresis pattern of a representative reassortant virus, a4 ahsv-1 vp2/vp5 , clearly shows co-migration of the s2 genome segment dsrna with that of ahsv-1, but it was not possible to distinguish the origin of the s6 genome segment (fig. 4a ). however, since genome segment s6 of ahsv-4, but not the s6 genome segment of ahsv-1, has a distinctive bamhi restriction site, the s6 genome segments of ahsv-4, ahsv-1 and the reassortant virus a4 ahsv-1 vp2/vp5 were amplified by rt-pcr and digested with bamhi. when analysed on an agarose gel, it was confirmed that the s6 genome segment of ahsv-4 was cleaved but not the s6 genome segments of ahsv-1 and the reassortant virus (fig. 4b ). the identity of the s2 and s6 genome segments of the reassortant virus as originating from ahsv-1 was furthermore confirmed by nucleotide sequencing of cdna copies of the respective genome segments (data not shown). these results thus demonstrate the ability of the reverse genetics system to potentially generate reassortant viruses with any desired genetic combination between different ahsv strains or serotypes. 2.4. generation of a modified 5-plasmid reverse genetics system that encodes both the ahsv-4 genome and t7 rna polymerase in both of the above reverse genetics systems, recovery of infectious ahsv-4 from plasmid dna relies on the expression of t7 rna polymerase within cells transfected with the ahsv-4 cdna plasmids. despite the success of both these approaches, they exclude the use of cells other than bsr-t7 cells for plasmid-based . mocktransfected cells were included as a control (well 1). (c) electropherotypes of wild-type (wt) and plasmid-derived ahsv-4 (lanes 1 and 2). bsr cells were infected with wildtype ahsv-4 or rescued ahsv-4. at 72 h post-infection the viral dsrna was extracted and electrophoresed in a non-denaturing polyacrylamide gel, followed by staining with ethidium bromide to visualize viral genome segments (s1-s10). (d) digestion of the s5 genome segment rt-pcr products of wild-type ahsv-4 (wt) and plasmid-derived ahsv-4 (lanes 1 and 2) with psti to confirm the presence of a novel mutation introduced into the s5 genome segment of plasmid-derived viruses. size markers (m) are indicated in base pairs. the nucleotide sequence of the rt-pcr products was also determined and compared. shown is the sequence chromatogram demonstrating the a-g substitution at nucleotide 1010 that creates a novel psti restriction site in the s5 genome segment of recombinant ahsv-4. (e) growth kinetics of wild-type (wt) ahsv-4 and plasmid-derived ahsv-4 (rahsv-4) in bsr cells. cells were infected with virus at a moi of 0.1 pfu/ml and incubated for the intervals shown. virus titres in cell lysates were determined by plaque assays on bsr cells. the results are presented as the mean virus titres of three independent experiments and error bars indicate the standard deviation. rescue. to increase the flexibility of ahsv-4 plasmid-based reverse genetics, we subsequently modified the 5-plasmid reverse genetics system by cloning a t7 rna polymerase expression cassette, in which expression of the enzyme is under control of the constitutive cytomegalovirus (cmv) promoter, into the genetic backbone of the pjad-s2-s6 reverse genetics vector. the derived plasmid was designated pjad-s2-s6-t7pol and thus serves as a source of t7 rna polymerase. expression of the t7 rna polymerase was confirmed by co-transfection of bsr cells with the pjad-s2-s6-t7pol plasmid and a pjet-mkalama1 reporter plasmid, in which expression of the mkalama1 fluorescent protein is under transcriptional control of a t7 rna polymerase promoter. examination of the transfected cells at 16 h post-transfection by confocal microscopy indicated that, in contrast to mock-transfected cells, cells transfected with both plasmids fluoresced blue and thus confirmed expression of the plasmid-encoded t7 rna polymerase ( supplementary fig. s1 ). to determine whether the modified 5-plasmid system allows recovery of ahsv-4, monolayers of bsr and l929 cells were cotransfected with the 5 plasmids encoding the ahsv-4 genome and t7 rna polymerase (fig. 5a) . the cells were harvested at 3 days post-transfection, lysed and plaque assays were performed in bsr . improved plasmid-based reverse genetics system for ahsv-4. (a) two genome segment transcription cassettes encoding ahsv-4 cdna flanked by the t7 rna polymerase promoter and hdv ribozyme sequences were combined into a single plasmid, creating 5 plasmids encoding the viral genome. the procedure for ahsv-4 recovery is near identical to that for the 10-plasmid system, except that bsr-t7 cells are co-transfected with the 5 plasmids. (b) plaque assays of transfection cell lysates performed 3 days post-transfection (well 2). mock-transfected cells were included as a control (well 1). (c) electropherotypes of wild-type (wt) and plasmid-derived ahsv-or l929 cells. in contrast to mock-transfected cells, plaques were recovered from the different cells transfected with the modified 5-plasmid set ( fig. 5b ). at different time points post-transfection, viral titres in cell culture supernatants were also determined by plaque assays. the results showed that the titres of virus recovered following transfection of bsr and l929 cells with the modified 5-plasmid set increased over time, albeit that the titres of virus recovered in l929 cells were consistently lower than that in bsr cells (fig. 5c ). to confirm that ahsv-4 was indeed recovered following plasmid transfection, the recovered viruses were propagated in bsr and l929 cells, viral dsrnas were extracted and analysed by non-denaturing polyacrylamide gel electrophoresis. the profiles of viral dsrnas from wild-type ahsv-4 as well as ahsv-4 recovered with the modified 5-plasmid system using bsr and l929 cells, were identical (fig. 5d ). furthermore, both of the plasmid-derived ahsv-4 retained the psti restriction site in their s5 genome segments as a rescue-specific marker, thus confirming that the recovered ahsv-4 originated from plasmid dna (data not shown). these results provide evidence that the modified 5-plasmid reverse genetics system can be used to recover ahsv-4 in cells that have not been engineered to express t7 rna polymerase. to compare the efficiencies of ahsv-4 recovery between the 10-plasmid, 5-plasmid and modified 5-plasmid reverse genetics systems, monolayers of bsr-t7 or bsr cells were co-transfected with the appropriate plasmid sets as indicated above. the virus titres in the culture supernatant were determined at different time points post-transfection by plaque assay on bsr cells. for each of the reverse genetics systems, the viral titre in the supernatant of transfected cells was below the limit of detection at 12 h post-transfection. however, the titre of recovered virus increased over time and maximal yields of virus were recovered at 5 days post-transfection, but significant yields were detected at 3 days post-transfection. the mean titres at days 3 and 5 posttransfection were approximately 51 and 2 â 10 2 pfu/ml, respectively, when bsr-t7 cells were transfected with the 10-plasmid system, whereas the virus yield using the 5-plasmid system determined at the same time points was approximately 4 â 10 2 and 3 â 10 3 pfu/ml, respectively. virus yield was also high in the bsr cells co-transfected with the modified 5-plasmid system, with approximately 0.9 â 10 2 and 5 â 10 2 pfu/ml estimated at days 3 and 5 post-transfection, respectively (fig. 6) . from this data we conclude that ahsv-4 can be recovered using each of the developed reverse genetics systems and that reduction in plasmid number substantially improves rescue efficiency. amongst the orbiviruses, bluetongue virus (btv) has been studied the most extensively and thus serves as a model system for the distantly related ahsv. despite similarities in their overall morphology, coding strategy and virus replication cycle, ahsv is, however, significantly different from btv at both the genetic and structural levels and in the proteins it encodes (kaname et al., 2013) . moreover, ahsv causes disease in horses and its pathogenesis is different from that caused by btv infections in sheep and cattle (coetzer and guthrie, 2004; maclachlan et al., 2009) . to address this imbalance, attempts have been made to study the structure-function relationships of individual ahsv proteins through a strategy of mutagenesis and re-expression of the proteins in heterologous hosts (uitenweerde et al., 1995; maree and huismans, 1997; van niekerk et al., 2001; de waal and huismans, 2005; stassen et al., 2011; bekker et al., 2014) . however, a definitive role for many of these proteins in the context of a replicating virus remains unresolved. consequently, the purpose of this study was to establish a reverse genetics system for the recovery of recombinant ahsv based entirely on cloned copies of the viral genome and, once established, to introduce improvements to the system that will expand the utility of reverse genetics for studies on ahsv biology. during the development of reverse genetics systems for different rna viruses, instability of cloned cdna sequences in escherichia coli have been reported for coronaviruses (casais et . the non-denaturing polyacrylamide gel was stained with ethidium bromide to visualize the viral genome segments (s1-s10). davidson, 2008; pu et al., 2011) and influenza virus (zhou et al., 2011) . likewise, we occasionally found that cdna copies of selected ahsv-4 genome segments, especially s3, were prone to deletions and rearrangements when cloned into the previously constructed prg15 reverse genetics plasmid and cultured in e. coli. although the reasons for this phenomenon was not investigated, such instability of cloned dna sequences may be caused by different factors, including long repeat sequences, secondary and tertiary structures, the presence of active e. coli-like promoter sequences within the insert and the generation of toxic proteins (godiska et al., 2010; pu et al., 2011) . based on previous reports indicating that the stability of inserts in circular vectors can be increased by the use of transcriptional terminators (godiska, 2005; zhou et al., 2011) , we thus incorporated bidirectional t7te terminators flanking the t7 rna polymerase transcription cassettes present in the pjad reverse genetics vectors. the successful cloning of cdna copies of the ahsv-4 genome segments into the pjad vectors and their stable maintenance using standard protocols suggest that the backbone of these vectors may provide enhanced stability for unstable ahsv-4 virus genome segments. the initial ahsv-4 reverse genetics system developed here consists of 10 plasmids, each containing a full-length cdna copy of single ahsv-4 genome segments flanked by t7 rna polymerase promoter and hdv ribozyme sequences. these plasmids are presumed to generate 10 transcripts corresponding to native positivesense rnas that serve as templates for translation and dsrna replication. the ahsv-4 replication cycle is initiated by transfection of the 10 plasmids encoding the viral genome into cells expressing t7 rna polymerase. using this system, we demonstrated production of ahsv-4 from the cloned cdnas and showed that this 10-plasmid reverse genetics system permits the selective introduction of desired mutations into the viral genome without the need for a helper virus and selection system. the growth kinetics and genomic electrophoretic profiles were indistinguishable between plasmid-derived ahsv-4 and wild-type ahsv-4, indicating that the replication characteristics of the ahsv-4 generated from cloned cdna reflect that of the wild-type ahsv-4. it is interesting to note that contrary to current rna-based reverse genetics systems that all depend on the use of capped synthetic transcripts (boyce et al., 2008; kaname et al., 2013; vermaak et al., 2015; yang et al., 2015) , the rnas transcribed in situ from the recombinant plasmids will not contain a 5′ cap. these uncapped transcripts can nevertheless functionally substitute for viral transcripts at all stages of the ahsv replication cycle, as evidenced by the recovery of viable ahsv-4. these results are in agreement with those reported for reoviruses (kobayashi et al., 2007; kawagishi et al., 2016) and btv (pretorius et al., 2015) . it is plausible that the sustained synthesis of rna transcripts in situ results in sufficient levels of expression to allow the assembly of core particles, which themselves are transcriptionally active (vermaak et al., 2015) , and thus lead to an amplification of gene transcription. a second aim of this study was to improve the performance of the developed ahsv-4 reverse genetics system by increasing the efficiency of virus recovery. to accomplish this aim, we reduced the number of plasmids required to deliver the entire ahsv-4 genome into bsr-t7 cells from 10 to 5 by combining two genome segment transcription cassettes into a single plasmid. compared to the 10-plasmid reverse genetics system, this 5-plasmid strategy enhanced the efficiency of virus recovery by reducing the time to virus isolation and supporting increased total yields. the efficiency of virus recovery may have been enhanced by different mechanisms. firstly, there are fewer plasmids that need to be transfected into each cell, and thus there is a higher probability that a full set of viral rnas will accumulate in a cell for initiation of replication. secondly, it is possible that only a small fraction of transfected plasmids avoid degradation and remain intact for transcription. by decreasing the number of plasmids the probability that a single cell will contain all of the transcription cassettes in a sufficient number is enhanced. thirdly, constriction of transcription and translation of individual rnas to the same intracellular microenvironment may greatly facilitate protein-protein and protein-rna interactions required for virus recovery (neumann et al., 2005; kobayashi et al., 2010) . the 5-plasmid ahsv-4 reverse genetics system was sufficiently robust to allow the recovery of a directed cross-serotype reassortant virus. technical complexities associated with the construction and manipulation of the dual reverse genetics plasmids may make their use less appealing. in this regard, the 10-plasmid reverse genetics system remains a useful alternative for demanding cloning tasks. for example, we generated the a4 ahsv-1 vp2/vp5 reassortant virus by combining four ahsv-4 dual plasmids and a dual plasmid containing cdna copies of the s2 and s6 genome segments from ahsv-1. however, the same reassortant virus was also recovered successfully by combining the four ahsv-4 dual vectors with two single reverse genetics plasmids, which contained cdna copies of the s5 and s6 genome segments, respectively. the efficiency of virus recovery was slightly lower compared to the use of 5 dual plasmids and is likely due to a lower transfection efficiency associated with the requirement for an additional plasmid for transection (data not shown). such an approach may be useful to generate so-called "serotyped" vaccine strains in which the immunogenic outer capsid proteins are exchanged with those of other virus serotypes (van gennip et al., 2012; feenstra et al., 2014 feenstra et al., , 2015 van de water et al., 2015) . thus, the single reverse genetics plasmids remain important adjuncts to the 5-plasmid reverse genetics system by enhancing flexibility with gains in efficiency. although both the 10-and 5-plasmid reverse genetics systems are sufficient for generating recombinant ahsv-4, it would be an advantage if the dependence of virus recovery on the use of bsr-t7 cells could be overcome. to this end, we have cloned a t7 rna polymerase expression cassette onto the genetic backbone of the pjad-s2-s6 dual reverse vector and subsequently demonstrated that ahsv-4 could be recovered in bsr and l929 cells following transfection of the cells with the modified 5-plasmid set. in these experiments, the titre of ahsv-4 recovered in l929 cells was lower than that of virus recovered in bsr cells. this difference in virus titres may be ascribed to the ability of l929 cells to produce interferons (takano-maruyama et al., 2006) , whereas bsr and bsr-t7 cells lack an interferon response (rieder et al., 2011) . these findings nevertheless provide evidence that this alternative 5-plasmid reverse genetics system can be employed using cell lines that have not been engineered to express t7 rna polymerase, and thus provides for a streamlined reverse genetics system for recovery of ahsv-4 in different cell lines. fig. 6 . comparison of virus recovery using the 10-plasmid, 5-plasmid and modified 5-plasmid ahsv-4 reverse genetics systems. bsr-t7 cells were co-transfected with the 10-plasmid set or 5-plasmid set, and bsr cells were co-transfected with the modified 5-plasmid set. the titres of virus released into the supernatant at different times post-transfection were determined by plaque assays on bsr cells. the results are presented as the mean titres of three independent experiments and error bars indicate the standard deviation. in conclusion, we have established a basic reverse genetics system for ahsv-4 and made improvements to the system that increase the flexibility and efficiency of reverse genetics, and also allows for the recovery of ahsv-4 in cells that have not been engineered to express t7 rna polymerase. these entirely plasmidbased reverse genetics systems opens new possibilities for investigating virus infection at the molecular level, including virus protein function, virus replication, host-virus-vector interactions and pathogenesis. moreover, the ability to recover mutant and reassortant viruses indicates that these reverse genetics systems can be exploited to generate further recombinant viruses that could be potential live-attenuated vaccines. bsr cells (a clone of baby hamster kidney-21 cells) were cultured in eagle's minimum essential medium (emem) supplemented with earle's balanced salt solution (ebss), 2 mm l-glutamine, 5% (v/v) foetal bovine serum (fbs), 1% (v/v) non-essential amino acids (neaa), and antibiotics (10,000 u/ml of penicillin, 10,000 mg/ml of streptomycin, 25 mg/ml of amphotericin b) (hyclone). l929 (mouse fibroblast) cells were cultured in dulbecco's modified eagle's medium (dmem) supplemented with 5% (v/v) fbs and antibiotics (100 u/ml of penicillin, 100 mg/ml of streptomycin) (gibco). bsr-t7 cells, which stably express bacteriophage t7 rna polymerase (buchholz et al., 1999) , were maintained in the same manner as bsr cells, except for the addition of 1 mg/ml of geneticin (invitrogen) with every second passage of the bsr-t7 cells. all mammalian cell lines were grown at 37°c with 5% co 2 . ahsv serotypes 1 (ahsv-1) and 4 (ahsv-4) were used in this study. the origin of ahsv-4 has been described previously (van de water et al., 2015) and is a live-attenuated vaccine strain (ahsv-4lp) that was obtained by attenuation of virulent ahsv hs32/62 through passage in suckling mice and bhk-21 cells, followed by selection of large plaques on vero cells (erasmus, 1972) . for cell table 1 oligonucleotides used in this study. oligonucleotide sequence (5′-3′) use and properties ipcr f cacaaagccggcgcttacagacaagctgtg construction of pjad1 and pjad2; t7te terminator sequences are underlined, stui restriction sites are indicated in italics and pmei restriction sites are indicated in bold cgaaggtttaaacggctcaccttcgggtgggcctttctgcgcaaaaaacccctcaagacccgtttag invf ggccggcatggtcccagc linearization of pjad1 and pjad2 for cloning invr tatagtgagtcgtatta s1f gttatttgagcgatggtcatcaccg ahsv-4 genome segment-specific primers s1r gtaagtgttttgagctgtgggg s2f gttaattcactatggcgtccgag s2r gtatgtgtattcacatggagcaac s3f gtttattttctgctgtgaactccag s3r gtaagtgtaattctgcccgc s4f gttatttaggatggaaccttacg s4r gtaaggttattaggattcccc s5f gttaaagaacctaggcggtttgg s5r gtaagtttgtgaaccaggggg s6f gttatttttccagaagccatgg s6r gtaagtgtttttcccgcccac s7f gttaaattcggttaggatggacg s7r gtaagtgtattcggtattgacgtattac s8f gttaaaaatccgttcgtcatcatgg s8r gtatgttgaaatccgcggttac s9f gttaaataagttgtctcatgtcttcggc s9r gtaagttttaagttgcctgcaag s10f gttaattatcccttgtcatgaatctagc s10r gtaagttgttatcccaccccctag the pjad1 and pjad2 reverse genetics vectors share the same puc19 genetic backbone, which lacks the lacz' region inclusive of the multiple cloning site, and was prepared by an inverse pcr using puc19 as template (genbank accession no. m77789.2) together with primers ipcr-f and ipcr-r (table 1) . to construct pjad1, a transcription cassette comprising a t7 rna polymerase promoter, followed by two inverted bsmbi restriction enzyme sites, a hepatitis delta virus (hdv) ribozyme sequence and a t7 rna polymerase terminator sequence, was pcr-amplified from plasmid prg15 (pretorius et al., 2015) using primers cas1f and cas1r (table 1 ). the amplicon was phosphorylated using t4 polynucleotide kinase (thermo scientific) and then blunt-end ligated to the modified puc19 genetic backbone. a similar strategy was used to construct pjad2, except that the transcription cassette was pcr-amplified from plasmid prg15 with primers cas2f and cas2r (table 1 ). the nucleotide sequence of the cloned insert dnas was verified by automated sequencing procedures using the abi prism bigdye terminator cycle sequencing ready reaction kit v3.1 (perkin-elmer applied biosystems). the pjad1 and pjad2 reverse genetics vectors thus harbour identical transcription cassettes flanked by bidirectional t7te terminators (reynolds et al., 1992; reynolds and chamberlin, 1992) and contain unique restriction enzyme sites (stui and/or pmei) to facilitate cloning procedures (fig. 1) . total rna was extracted from bsr cells infected with ahsv-4 using the nucleospin rna ii kit (macherey-nagel) and ssrna was removed by precipitation with 2 m licl and centrifugation at 17,000g for 30 min at 4°c. first-strand cdna was synthesized from 3 mg of dsrna using the revertaid h minus first strand cdna synthesis kit (thermo scientific), and cdna copies of the fulllength ahsv-4 genome segments were amplified by pcr using genome segment-specific primer pairs (table 1) and high-fidelity velocity dna polymerase (bioline). the purified amplicons were phosphorylated and then blunt end-ligated to the respective pjad vectors that had been linearized by means of an inverse pcr using primers invf and invr (table 1) . these back-to-back primers were designed to flank the poorly digestible bsmbi cloning site present in the pjad vectors and thus resulted in the amplification of the vector dna excepting the cloning site between the two primers. cdna copies of ahsv-4 genome segments s1 through s5 were ligated to the pjad1 vector, and cdna copies of genome segments s6 through s10 were ligated to the pjad2 vector to generate pjad-s1 through pjad-s10. the sizes of the recombinant plasmids are provided in supplementary table s1 . plasmid pjad-s5, encoding the entire ahsv-4 s5 genome segment, has a psti restriction site as a genetic marker that was created by introducing a single nucleotide change at position 1010 in the s5 genome segment using the quickchange site-directed mutagenesis kit (stratagene) and primers s5psti-f and s5psti-r (table 1) recombinant ahsv-4 generation from five plasmids was facilitated by combining two genome segment transcription cassettes flanked by t7te terminators onto one plasmid. to this end, recombinant plasmids pjad-s6 through pjad-s10 were digested with pmei, and the excised dna fragments were blunt-end cloned into the pmei site of pjad-s1 through pjad-5. the derived reverse genetics plasmids were named pjad-s1-s8, pjad-s2-s6, pjad-s3-s7, pjad-s4-s10 and pjad-s5-s9 (supplementary table s1 ). the integrity of the recombinant plasmids was verified by nucleotide sequencing of the genome segment transcription cassettes present in each of the recombinant constructs. to enable the recovery of recombinant ahsv-4 from plasmid dna in cell lines that have not been engineered to express t7 rna polymerase (genbank accession no. am946981), an expression cassette encoding for t7 rna polymerase was incorporated into the genetic backbone of the pjad-s2-s6 rescue plasmid. the coding region of the t7 rna polymerase gene was pcr-amplified with phusion high-fidelity dna polymerase (thermo scientific) using chromosomal dna from e. coli bl21(de3) as template and primers t7bam-f and t7bam-r (table 1) , which both harbour a bamhi site. the purified amplicon was digested with bamhi and cloned into the bamhi site of the pcdna3.1( þ) mammalian expression vector (invitrogen) to generate pcdna-t7pol. the recombinant plasmid dna was characterized by nucleotide sequencing to confirm that the insert dna was cloned under transcriptional control of the cytomegalovirus (cmv) immediate-early promoter. subsequently, pcdna-t7pol was used as template together with primers cmv-f and cmv-r (table 1) , both of which harbour an ecorv site, to pcr amplify the t7 rna polymerase expression cassette. this cassette comprised the t7 rna polymerase coding region flanked by the upstream cmv promoter sequence and the downstream bovine growth hormone poly (a) sequence. the amplicon was digested with ecorv and bluntend cloned into the sspi site of plasmid pjad-s2-s6 to yield pjad-s2-s6-t7pol. the integrity of the cloned insert was verified by nucleotide sequencing. using similar approaches to those described above, full-length cdnas of ahsv-1 genome segments s2 and s6 were amplified by rt-pcr using viral dsrna extracted from virus-infected cells as template. segment-specific cdnas were amplified by using primers 2.1f and 2.1r for s2, and primers 6.1f and 6.1r for s6 (table 1). the amplified cdna for the ahsv-1 s2 and s6 genome segments were cloned into pjad1 and pjad2, respectively, and the derived recombinant plasmids were designated pjad(a1)-s2 and pjad(a1)-s6. the integrity of the cloned cdna in each of the plasmids was confirmed by nucleotide sequencing. the s6 genome segment transcription cassette flanked by t7te terminators was recovered from pjad(a1)-s6 by digestion with pmei and cloned into pmei-digested pjad(a1)-s2 to generate plasmid pjad(a1)-s2-s6. plasmid dna used in cell transfections was purified using the genopure plasmid maxi kit (roche life science) according to the manufacturer's instructions. monolayers of bsr-t7 cells at 70% confluency in 6-well tissue culture plates were co-transfected with plasmids representing the cloned ahsv-4 genome using transit-lt1 transfection reagent (mirus). briefly, 7.5 ml of the transfection reagent was added to 250 ml of optimem i reduced serum medium (life technologies) and incubated at room temperature for 30 min. for co-transfection of the cells with the 10 recombinant pjad plasmids, 250 ng of each plasmid was mixed in an eppendorf tube and the plasmid dna mixture was then added to the diluted transfection reagent. following incubation at room temperature for 1 h, the transfection mixture was added to the bsr-t7 cells and optimem i reduced serum medium was added to a final volume of 3 ml. at 16 h post-incubation, the medium was replaced with complete emem. an identical protocol to that above was followed to recover recombinant ahsv-4 from bsr-t7 cells transfected with the 5-plasmid set, except that 500 ng of each plasmid was used. the same protocol was followed to generate a reassortant virus, except that the pjad-s2-s6 plasmid was substituted with the pjad (a1)-s2-s6 plasmid prior to transfection of the bsr-t7 cell monolayer. recombinant ahsv-4 was likewise recovered in monolayers of bsr and l929 cells using the modified 5-plasmid reverse genetics system, except that bsr or l929 cells were cotransfected with 500 ng of each dual (pjad-s1-s8, pjad-s3-s7, pjad-s4-s10 and pjad-s5-s9) plasmid and 1 mg of the pjad-s2-s6-t7pol plasmid, and the volume of the transfection reagent used was adjusted to 9 ml. following 1 to 5 days of incubation, cells and medium were harvested, lysed and used in a plaque assay on bsr cells (oellermann, 1970) . viral dsrnas were extracted from infected cells, electrophoresed on non-denaturing 10% polyacrylamide gels and visualized by ethidium bromide staining. to discriminate between wildtype and plasmid-derived ahsv-4, full-length cdna products of genome segment s5 from recovered viruses were digested with psti and the digestion products were resolved by electrophoresis on a 1% agarose gel. as a final confirmation, the nucleotide sequence of the amplified s5 genome segments was also determined. to characterize reassortant viruses, cdna was synthesized from the viral dsrna and pcr amplification was performed using s2 and s6 genome segment-specific primer pairs for ahsv-1. the purified genome segment s6 pcr product was analysed by agarose gel electrophoresis following digestion with bamhi, and the nucleotide sequences of the s2 and s6 pcr products were also determined. confluent bsr cell monolayers in 6-well tissue culture plates were infected with either wild-type ahsv-4 or plasmid-derived ahsv-4 at a moi of 0.1 pfu/cell. virus adsorptions were performed at 37°c for 1 h, after which the medium with unbound virus was removed. the monolayers were washed once with serum-free emem, 2 ml of complete emem was added and incubation was continued. at different time intervals post-infection the cells and medium were harvested. the samples were passed through a 22g needle and subjected to low-speed centrifugation to pellet cell debris. the virus titres were subsequently determined by serial dilution and plaque assays on bsr cells. monolayers of bsr-t7 cells were co-transfected with the 10plasmid or 5-plasmid reverse genetics constructs and bsr cells were co-transfected with the modified 5-plamsid set, as described above. aliquots (1 ml) of the supernatant of transfected cells were collected and replaced with 1 ml fresh medium at 12 h, 3 days and 5 days post-transfection. virus titres in the culture supernatants were determined by plaque assays on bsr cells. to confirm expression of the plasmid-encoded t7 rna polymerase, bsr cell monolayers, grown on sterile glass coverslips in 6-well tissue culture plates, were co-transfected with pjad-s2-s6-t7pol (2 mg) and the reporter plasmid pjet-mkalama1 (800 ng) sing transit-lt1 transfection reagent (mirus). in the reporter plasmid, the coding region of mkalama1 is under transcriptional control of a t7 rna polymerase promoter. at 16 h post-transfection, the glass coverslips were removed and mounted onto glass slides, which were viewed with a zeiss lsm s10 meta confocal microscope. the images were captured with a zeiss axiovert series 5 digital camera and analysed with zeiss lsm image browser 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transcribed in vitro from a cdna copy of the human coronavirus genome cloned in vaccinia virus comparative analysis of reoviridae reverse genetics methods dual selection mechanisms drive efficient single-gene reverse genetics for rotavirus rearranged genomic rna segments offer a new approach to the reverse genetics of rotaviruses the multimeric nonstructural ns2 proteins of bluetongue virus, african horsesickness virus, and epizootic haemorrhagic disease virus differ in their single-stranded rnabinding ability vp2 exchange and ns3/ns3a deletion in african horse sickness virus (ahsv) in development of disabled infectious single animal vaccine candidates for ahsv bluetongue viruses based on modified-live vaccine serotype 6 with exchanged outer shell proteins confer full protection in sheep against virulent btv8 expression of non-structural protein ns3 of african horsesickness virus (ahsv): evidence for a cytotoxic effect of ns3 in insect cells, and characterization of the gene products in ahsv-infected vero cells the oral susceptibility of south african field populations of culicoides to african horse sickness virus directed genetic modification of african horse sickness virus by reverse genetics reverse genetics and the study of dengue virus development of a reverse genetics system for epizootic haemorrhagic disease virus and evaluation of novel strains containing duplicative gene rearrangements strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model a one-plasmid system to generate influenza virus in cultured chicken cells for potential use in influenza vaccine reverse genetics plasmid for cloning unstable influenza a virus gene segments characterising non-structural protein ns4 of african horse sickness virus supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2016.07.010. key: cord-326688-a1djgqpa authors: dubois-dalcq, monique e.; doller, elizabeth w.; haspel, martin v.; holmes, kathryn v. title: cell tropism and expression of mouse hepatitis viruses (mhv) in mouse spinal cord cultures date: 1982-06-30 journal: virology doi: 10.1016/0042-6822(82)90092-7 sha: doc_id: 326688 cord_uid: a1djgqpa abstract mouse hepatitis viruses (mhv) are coronaviruses which cause various infections in mice affecting lung, intestine, liver, and other organs as well as the central nervous system. the replication of three different mhv strains was studied in mouse dissociated spinal cord cultures containing differentiated neurons and nonneuronal cells (nn) (including astrocytes). cell tropism and maturation of each virus strain was analyzed by immunolabeling methods using antisera to the virion or to purified membrane glycoproteins (e1 and e2) and by electron microscopy (em). wt-jhm, which causes acute encephalitis in mice, produces acute cytopathic changes in both neurons and nn cells. in neurons, virions mature in smooth er cisternae closely associated to the golgi apparatus. as judged by em, fewer virions are produced by neurons than nn cells and neurons do not fuse or stain for e2 as do nn cells. nn cells contain large inclusions made of nucleocapsid strands. a temperature-sensitive mutant of jhm, ts8-jhm, which causes demyelination in mice, infects nn cells but not neurons. infected nn cells synthesize e1 and e2, and contain large inclusions but few mature virions, even at permissive temperatures. these inclusions appear granular and rarely contain nucleocapsid strands in contrast to wt-jhm infection. nn cells infected with this mutant also display numerous membrane whorls. the hepatotropic strain a59 lacks tropism for neurons and primarily infects nn cells, thus resembling ts8-jhm. infected nn cells become loaded with intracytoplasmic virions which are secreted from the cells. e1 can only be detected in the perinuclear area of these cells while e2 rapidly spreads throughout the cytoplasm. the cytoplasm of a59 infected nn cells frequently contains large tubular structures often in the lumen of the rer. in conclusion, in primary cns cultures consisting of neurons and nn cells: (1) wt-jhm replicates in both neurons and nn cells but has different effects on these cells; (2) ts8-jhm exhibits no productive infection of neurons, and in nn cells appears to be defective in assembly and to stimulate membrane synthesis; (3) a59 also shows tropism restricted to nn cells which produce many viruses and display differential distribution of the two virion glycoproteins. thus, in the absence of the immune system, the mhv strains assayed exhibit differences in viral tropism, cytopathic changes, and viral assembly in cns cells, and these differences may account for the different disease patterns. (3) a59 also shows tropism restricted to nn cells which produce many viruses and display differential distribution of the two virion glycoproteins. thus, in the absence of the immune system, the mhv strains assayed exhibit differences in viral tropism, cytopathic changes, and viral assembly in cns cells, and these differences (knobler et al., 1981b) or a demyelinating disease, depending upon the age of the animal at the time of inoculation and the virus passage level (haspel et al., 19'78; herndon et al., 1975; lampert et al., 1973; stohlman and weiner, 1981; weiner, 1973) . in contrast, a chemically induced temperature-sensitive mutant of wt-jhm, ts8-jhm, rarely induces fatal encephalomyelitis in the same mouse strains but regularly leads to demyelination (haspel et al., 1978) . in viva electron microscopic and immunocytochemical studies have shown that neurons do not appear to support replication of ts&jhm, while oligodendrocytes do (knobler et al., 1981a) . finally, a hepatotropic mhv strain, a59 (sturman and takemoto, 1972) , can also produce chronic cns disease in cbh mice (k. v. holmes and p. 0. leinikki, unpublished observations.) a59 is presently the best characterized coronavirus strain: it has three virion-associated polypeptides which have been isolated and purified holmes et al., 1981 sturman and holmes, 1977; sturman et al., 1980) . the membrane glycoprotein el appears to be involved in virus assembly, while the peplomeric glycoprotein ez is required for infectivity and virus-induced cell fusion. monospecific antibodies have been raised against these proteins as well as to detergent-disrupted virions (sturman et al., 1980) . we have compared the tropism and maturation of two jhm virus strains with that of the a59 strain in cns cells, using immunolabeling and electron microscopy. our results suggest that specific properties of each virus strain and host factors present in differentiated nerve cells act in concert to control the features and outcome of mhv cns infection at the target cell level. cells. cultures of dissociated cns cells were obtained from spinal cord dissected from 12-to 13-day-old c57b1/6 mouse embryos as described before (faulkner et al., 1979; hooghe-peters et al., 1979) . c57b1/6 mice are permissive for the jhm strains. cells were grown on 12-mm glass coverslips or 35-mm petri dishes coated with collagen and supplemented with medium as described (faulkner et al., 1979; hooghe-peters et al., 1979) . fibroblast growth was inhibited by a 2-day antimitotic treatment with a mixture of fudr and uridine after 1 week in vitro. cultures were used after 2 to 3 weeks of in vitro maturation when neurons and astrocytes had acquired their mature stage (figs. la and b). three cell types were identified in these cultures. the neurons represent 5 to 10% of the cells; they are either isolated or in clusters and show a characteristic morphology with long processes as seen by phase microscopy ( fig. la) (faulkner et al., 1979; hooghe-peters et al., 1979) . the nonneuronal (nn) cells are comprised of at least two cell types as judged on the basis of positive or negative immunoreactivity for glial fibrillary acidic protein (gfap), a specific protein of astrocytes (bignami et al., 1980) . gfap+ cells represent 50 to 70% of the nn cells; these astrocytes have a pleiomorphic shape, contain radiating fibrils, and form groups under the neurons. gfap-cells probably represent primary fibroblastic cells of cns origin (meningeal or perivascular). oligodendrocytes were not detected in these cultures. vimlses. the a59 virus, a hepatotropic strain of mouse hepatitis virus, was used as described before (sturman and takemoto, 1972; sturman and holmes, 1977; sturman et al., 1980) . the origin and propagation of the wt and ts8 strains of jhm have been published (haspel et al., 1978) . the multiplicity of infection of 10 plaqueforming units (pfu)/cell was used with a59, whereas a multiplicity of infection of 0.3 to 1 pfu/cell was used with both jhm strains (the jhm stock viruses had much lower titers). viral adsorption was carried out for 1 hr at 37" as previously described (knobler et al., 1981a) . cultures were incubated at 37", although in a few experiments paired cultures were also incubated at 34". the cultures were observed daily with a zeiss inverted phase scope. viral infectivity of supernatant fluids was determined by plaque assay in l2 or 17cl 1 cells as described (sturman and takemoto, 1972) . antiserum and immunolubeling. four different rabbit antisera were used. rabbit antiserum against gfap was kindly provided by dr. rebecca pruss; the rabbit had been injected with a suspension of human spinal cord. when reacted with cord proteins separated by agar gel electrophoresis, immune reactivity of this serum was found to be restricted to gfap. preparation and characterization of monospecific polyclonal rabbit antisera to the isolated el and ez glycoproteins of a59 virion envelope has been previously described (sturman et al., 1980) . rabbit antisera directed against gradient-puri-fied np40-disrupted a59 virions were also prepared. this hyperimmune serum immunoprecipitated the three major structural polypeptides of both a59 and jhm from radiolabeled infected cells. for fluorescent antibody (fa) staining, dissociated cns cells grown on glass coverslips were fixed in a mixture of 5% acetic acid and 95% ethyl alcohol at -20" (for gfap staining) or in acetone (for el or ez staining) before being incubated for 1 hr in various dilutions of antisera. in some instances, living cells were incubated with antiviral sera at 4' or room temperature in order to detect only antigens on the cell surface. the second reagent used to detect bound antibodies was either protein afluorescein (pharmacia, piscataway, n. j.) or goat anti-rabbit igg coupled to fluorescein. this conjugate was absorbed with liver powder or 17cl 1 cells. for immunoperoxidase labeling, staining was performed as described before (knobler et al., 1981a) . briefly, the cells were fixed in 4% formaldehyde with 0.3 to 0.5% glutaraldehyde for 20 min, frozen and thawed in a sucrose-glycerol mixture in order to render the cell membrane permeable to antibody molecules. the cells were then allowed to react with antibody to the virion or to glycoprotein el or ez. protein a-peroxidase was used as a second layer and cells were further incubated in the peroxide-diaminobenzidine substrate. immediate observations by light microscopy were done on a zeiss photomicroscope iii using a water immersion phase contrast (40x) objective for immunoperoxidase or an oil immersion (40x) objective for fluorescence. when double labeling with antisera to both gfap and viral antigens was performed, glass coverslip cultures of cns cells were fixed with acid-alcohol at -20". after washing with serum containing medium, the coverslips were incubated successively in: (1) rabbit antiserum to gfap, (faulkner et al., 1979) , glass coverslips covered with cells were used for scanning electron microscopy @em), whereas 35mm petri dishes were used for transmission electron microscopy (tem) studies. cells were usually fixed with karnovsky fixative consisting of a mixture of 1.25% glutaraldehyde and 1% formaldehyde in 0.06 m cacodylate buffer at ph 7.2. for sem studies, cells were then postfixed in 1% osmium tetroxide in 0.1 m cacodylate buffer for 2 hr, and dehydrated in methanol, critical point dried in coz in a balzer apparatus, coated under high vacuum with gold-palladium alloy, and observed in an etec autoscan at 20 kv and a tilt angle of 45'. for tem studies, monolayers in petri dishes that had been labeled by the immunoperoxidase method (as described above) or fixed in situ with the karnovsky fixative, were postfixed with osmium tetroxyde 1% in cacodylate buffer for 30 min, stained with uranyl acetate at ph 5, progressively dehydrated in ethanol, and embedded in epon 812. the technique for circling and drilling out immunolabeled cells or syncytia from the embedded monolayer has been published in detail (knobler et al., 1981a) . sections were cut with a diamond knife and examined in philips 201 or philips 400-t electron microscopes. thin sections were usually counterstained with uranyl acetate and lead salts, and examined at 80 kv. proteins between 16 and 18 hr. titers of jhm was examined successively in inreleased virus were always lower than ti-fected neurons and nn cells. infected neuters of cns cells infected with a59 strain rons showed diffuse immunofluorescent (table 1) pi, the organization of the cytoplasm of infected neurons was not altered since they had an extensive rough er system and a normal distribution of other organelles. budding and free virions with electron-lucent centers were detected in cisternae of smooth membranes closely associated with the golgi apparatus (figs. 2a, b) . virus was also found occasionally in small vacuoles within dendrites (fig. 2~ ) as seen in infected mouse spinal cord in tivo (knobler et al., 1981a) . at 48 hr pi, the cytoplasm of infected neurons showed disorganization of rer membranes and vacuolization. scattered budding virions were seen in association with intracytoplasmic membranes as described before (knobler et al., 1981a) . fewer cytoplasmic vacuoles containing virions were detected in thin sections of infected neurons than in giant cells formed by nn cells (illustrated in figs. 3 and 4) . similarly, clusters of extracellular virions were rarely found around infected neurons, whereas they were numerous around nn cells (fig. 4~ ). this correlates with previous in viva observations on infected neurons of the spinal cord (knobler et al., 1981a) . infected nn cells contained both el and ez glycoproteins at 16 hr pi. these proteins were predominantly localized in the perinuclear area of giant cells (fig. 3, these fused nn cells revealed nucleocapsid loosely folded nucleocapsid strands apstrands accumulating in large inclusions proximately 8 nm in diameter were seen and numerous virions budding in vacu-in close apposition to the er membrane oles (fig. 3) . before budding occurred, (figs. 4a and b) . during budding, the nu-cleocapsid was closely apposed to the virion membrane of the bud which had acquired definite spikes (fig. 4b) . virions were budding from rer as well as smooth er. using antiserum against purified a59 virions, mhv antigens were detected in all membranes directly associated with virion .formation and in the virion surface (data not shown). numerous free virions were released by giant cells and formed clusters firmly attached to infected cell surface and heavily labeled after immunoperoxidase staining (fig. 4~) . free virions in the extracellular space resembled those seen in vacuoles and were 80 to 100 nm in diameter (fig. 4c, inset) . neurons showed no cytopathic changes or viral antigens after inoculation with this strain. however, scattered foci of nn cells slowly developed fusion over 3 to 5 days and contained viral antigen. both gfap+ and gfap-cells were infected as demonstrated by double labeling for viral-and cell-specific antigens. the glycoproteins, ez and el, were detected in the perinuclear area of giant cells at 24 hr and stayed in these regions throughout infec: tion. slow progression of infection led to disintegration of the monolayer after 5 to 7 days. nn cells infected with ts8-jhm released at least 10 times less virus than cells infected with wt-jhm (table l) , even at the permissive temperature (34') (data not shown). by em, inclusions present in nn cells infected with ts&jhm were markedly different from those observed in wt-jhm infection of nn cells. wt-jhm inclusions contained nucleocapsid strands 8 nm in diameter as well as crescent-shaped corelike structures (37-40 nm in diameter), usually with an electronlucent center (fig. 5a) . ts8-jhm inclusions contained numerous dense granules (20 to 30 nm in diameter). strands of nucleocapsid could not be clearly identified in these inclusions (fig. 5b ). in addition, rows of dense granules (18 nm in diameter) were seen aligned along cytoplasmic tubules of ts&jhm-in-fected nn cells (fig. 5~) . the possibility that these granules represent ribosomes is not excluded. nn cells infected with ts8-jhm showed less intracellular and extracellular virions than in wt-jhm infection (fig. 4~) . nn cells infected with ts&jhm displayed numerous membrane whorls sometimes organized in myelin-like figures (fig. 6) . however, our cultures do not normally contain oligodendrocytes or show myelination. these membrane whorls surrounded cytoplasmic compartments containing er, dense granules, and, occasionally, budding virions (fig. 6) . some whorls were made of 40 pairs of membranes with an interspace of 12 to 20 nm. immunoperoxidase labeling of viral antigens did not reveal staining of membrane whorls. as with ts8-jhm, no infected neurons were found in cns cultures inoculated with a59 and the infection appeared restricted to nn cells. mild fusion of nn cells was observed and infection lasted for 5 to 10 days. infected gfap+ cells progressively retracted their fibrillar processes. a59-infected cultures produced 10 times more infectious virus than those infected with wt-jhm (table 1 ) even though wt-jhm replicated in both neurons and nn cells. infected astrocytes were wrapped around healthy, apparently uninfected neurons as seen by em (fig. 7) . these astrocytes were very rich in intermediate filaments and contained numerous viral particles in their er system (fig. 7) . the inset of fig. 7 shows a virion in a vacuole close to the cell surface. the glycoprotein ez was detected as early as 8 hr pi and localized throughout the cytoplasm of infected nn cells, while el, first detected at 16 hr, remained predominately perinuclear (figs. 8a and b) . such differential localization of these proteins has been observed in infected 17cl 1 fibroblasts (e. w. doller et al., submitted for publication). interestingly, virus maturation in perinuclear cisternae was frequently noted by em (fig. 8~) the lumen of the rer (fig. 8d) . these tubular structures had a higher density than cellular microtubules. a59-infected nn cells also showed some membrane whorls but these were smaller than those observed in ts&jhm infection of nn cells (fig. 7) . discussion the present study describes how three different mhv strains interact in vitro with cultured neurons and nn cells, including astrocytes, isolated from mouse spinal cords. previous studies have demonstrated that persistent infection with the jhm strain could be obtained in cloned cell lines of glial and neuronal origin (lucas et al., 1977 (lucas et al., ,1978 stohlman and weiner, 1978) . here, we have used primary cultures of differentiated nerve cells, an in vitro system that seems closer to the in viva situation. our observations are summarized in table 2 . wt-jhm is the only strain able to replicate in mature neurons as well as nn cells and this correlates well with in viva observations (knobler et al., 1981a) . wt-jhm never produces fusion of neurons as do other viruses , but rather induces neuronal rounding and lysis with little virus release. simultaneously, extensive fusion and virus production occur in the nearby nn cells. thus wt-jhm has very different effects in these two cns cell types. in addition, the peplomeric glycoprotein ez was not detected in infected neurons but was observed in nn cells. thus, jhm virions produced in neurons may either lack ez or have an altered e2 no longer reacting with antisera used. virions lacking peplomers and e2 are formed in a59-infected 17cl 1 cells treated with tunicamycin (holmes et al., 1981 . thus it is possible for buds lacking ez to form but such buds are known to be noninfectious (holmes et al., 1981 . the possible differences in e2 glycoprotein synthesis between neurons and nn cells infected with wt-jhm should be further studied provided that purified neuron cultures could be obtained (knobler et al., 1982) . the other two mhv strains, ts8-jhm and a59, infect only nn cells. similarly, in weanling mice, ts&jhm does not appear to replicate in neurons (knobler et al., 1981a) . one possible explanation for this restricted tropism is that ts8-jhm and a59 may lack some structural aspects of the surface glycoprotein e2 necessary for its interaction with virus receptors on neurons. alternately, the virus may have infected the neurons but intracellular factors of an unknown nature may have blocked its replication. in a study of restricted tropism of vsv ts mutants, it was suggested that "secondary" virulence genes may control viral replication in cells through adequate or inadequate interactions of virus specific polymerase complexes with some specific host cell factor (preble et al., 1980) . and a59 is closely similar to that observed in fibroblast cell lines and liver cell cultures (david-ferreira and manaker, 1965 ) but more stages are captured in primary cns cells. tem analysis of these stages reveals that a close interaction between a folded nucleocapsid strand and a virus-modified er membrane is occurring during the budding process, as is the case for other enveloped rna viruses. there is a close association of the virus maturation sites with the golgi system. virus contained in vacuoles is "secreted" since these vacuoles eventually fuse with the plasma membrane . this release process is also observed with other enveloped viruses undergoing intracellular maturation such as herpes simplex (rodriguez and dubois-dalcq, 1978) . a59 infected cns cultures produce more virus than wt-jhm infected ones, even though a59 infects only nn cells in a slow, progressive way. in addition, differential but not in wt-jhm-infected nn cells. in a59 infections, the membrane glycoprotein el appears to remain in the golgi area while the peplomeric glycoprotein e2 migrates rapidly to the plasma membrane ( figs. 8a and b) . in contrast, the e2 synthesized in nn cells by the jhm strains did not disperse rapidly but remained near the cell center (fig. 3, inset) . possibly the signals for migration of the e2 glycoprotein are different for the jhm and a59 strains of mhv. these differences may account, at least in part, for the different yields of virus from cells infected with wt-jhm or a59. ts8-jhm-infected nn cells showed viral inclusions different from those observed in wt-jhm infection. these alterations suggest a defect in the ribonucleoprotein, resulting in defective assembly of t&3-jhm occurring even at permissive temperatures. such an alteration was not observed in fibroblastoid cell lines but only in cns cells (dubois-dalcq et al., unpublished observations) . thus the host cell appears to influence expression and maturation of this virus. in addition, the cytopathic changes produced by ts&jhm in nn cells were slowly progressive and resulted in the accumulation of abnormal whorls of cytoplasmic membranes. such accumulation was not observed in fibroblastoid cell lines. in vivo, oligodendrocytes infected with jhm strains appear to lay down more myelin lamellae (powell and lampert, 1975) . thus membrane proliferation induced by mhv infection may be a specific event occurring in differentiated glial cells. analysis of rna and protein synthesis of ts8-jhm might elucidate the molecular basis of the ability of this mutant to produce chronic demyelination in vivo. there is no evidence yet that the host immune response plays a critical role in the induction of virus persistence in vivo since immunosuppression does not alter the course of the disease (weiner, 1973) . rather, our in vitro study demonstrates differences in viral tropism and cytopathic changes between neurons and nn cells which may be correlated with differences in disease patterns in vivo. our study also points to the advantages of using cultures of specialized nerve cells to elucidate virus-nerve cell interactions. advances in cellular neurobiology an electron microscope study of the development of mouse hepatitis virus in tissue culture cells antibody-induced modulation of rhabdovirus infection of neurons in vitro defective interfering particles modulate vsv infection of dissociated neuron cultures 19'78). temperature-sensitive mutants of mouse hepatitis virus produce a high incidence of demyelination mouse hepatitis virus-induced recurrent demyelination evolution of a coronavirus during persistent infection in vitro analysis of the functions of coronavirus glycoproteins by differential inhibition of synthesis with tunicamycin tunicamycin resistant glycosylation of a coronavirus glycoprotein: demonstration of a novel type of viral glycoprotein. %o.!ogv 11 visna virus induced fusion of nerve cells in vitro host and virus factors associated with cns cellular tropism leading to encephalomyelitis or demyelination induced by the jhm strain of mouse hepatitis virus selective localization of wild type and mutant mouse hepatitis virus (jhm strain) antigens in cns tissue by fluorescence, light and electron microscopy mouse hepatitis virus type 4 (jhm strain) induced fatal central nervous system disease. i. genetic control and the murine neuron as the susceptible site of disease mechanism of demyelination in jhm virus encephalomyelitis. electron microscopic studies in vivo and in vitro models of demyelinating disease: tropism of the jhm strain of murine hepatitis virus for cells of glial origin in vivo and in vitro models of demyelinating diseases. ii. persistence and host-regulated thermo-sensitivity in cells of neural derivation infected with mouse hepatitis and measles viruses oligodendrocytes and their myelin-plasma membrane connections in jhm mouse hepatitis virus encephalomyelitis neurovirulence mutant of vesicular stomatitis virus with an altered target cell tropism in vivo chronic measles virus infection of mouse nerve cells in vitro intramembrane damages occurring during maturation of herpes simplex virus type i: freeze-fracture study stability of neurotropic mouse hepatitis virus (jhm strain) during chronic infection of neuroblastoma cells chronic central nervous system demyelination in mice after jhm virus infection enhanced growth of a murine coronavirus in transformed mouse cells characterization of a coronavirus. ii. glycoproteins of the viral envelope: tryptic peptide analysis isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid pathogenesis of demyelination induced by a mouse hepatitis virus (jhm virus) we wish to thank ray rusten, key: cord-301293-jqy7lcbk authors: gupta, vandana; tabiin, tani m.; sun, kai; chandrasekaran, ananth; anwar, azlinda; yang, kun; chikhlikar, priya; salmon, jerome; brusic, vladimir; marques, ernesto t.a.; kellathur, srinivasan n.; august, thomas j. title: sars coronavirus nucleocapsid immunodominant t-cell epitope cluster is common to both exogenous recombinant and endogenous dna-encoded immunogens date: 2006-03-30 journal: virology doi: 10.1016/j.virol.2005.11.042 sha: doc_id: 301293 cord_uid: jqy7lcbk correspondence between the t-cell epitope responses of vaccine immunogens and those of pathogen antigens is critical to vaccine efficacy. in the present study, we analyzed the spectrum of immune responses of mice to three different forms of the sars coronavirus nucleocapsid (n): (1) exogenous recombinant protein (n-gst) with freund's adjuvant; (2) dna encoding unmodified n as an endogenous cytoplasmic protein (pn); and (3) dna encoding n as a lamp-1 chimera targeted to the lysosomal mhc ii compartment (p-lamp-n). lysosomal trafficking of the lamp/n chimera in transfected cells was documented by both confocal and immunoelectron microscopy. the responses of the immunized mice differed markedly. the strongest t-cell ifn-γ and ctl responses were to the lamp-n chimera followed by the pn immunogen. in contrast, n-gst elicited strong t cell il-4 but minimal ifn-γ responses and a much greater antibody response. despite these differences, however, the immunodominant t-cell elispot responses to each of the three immunogens were elicited by the same n peptides, with the greatest responses being generated by a cluster of five overlapping peptides, n(76–114), each of which contained nonameric h2(d) binding domains with high binding scores for both class i and, except for n(76–93), class ii alleles. these results demonstrate that processing and presentation of n, whether exogenously or endogenously derived, resulted in common immunodominant epitopes, supporting the usefulness of modified antigen delivery and trafficking forms and, in particular, lamp chimeras as vaccine candidates. nevertheless, the profiles of t-cell responses were distinctly different. the pronounced th-2 and humoral response to n protein plus adjuvant are in contrast to the balanced ifn-γ and il-4 responses and strong memory ctl responses to the lamp-n chimera. sars coronavirus nucleocapsid immunodominant t-cell epitope cluster is common to both exogenous recombinant and endogenous dna-encoded immunogens vaccines designed to prevent viral infection must promote the adaptive expansion of t cells that are associated with band t-cell long-term memory of epitopes identical to or crossreactive with those of the natural pathogen (welsh et al., 2004; crotty and ahmed, 2004) . the processing and presentation of antigen epitopes of vaccine immunogens must therefore mimic that of pathogens. the conventional understanding has been that epitopes presented by mhc class i are derived from endogenous cellular proteins that have been cleaved by proteasomal and post-proteasomal peptidases (villadangos et al., 1999; yewdell and bennink, 2001; kloetzel 2004) and that the epitope loading of mhc class ii molecules generally involves exogenous proteins that are taken into antigenpresenting cells (apcs) by specialized endocytic receptors and processed by proteases of the endosomal/lysosomal compartments (moreno et al., 1991; bryant and ploegh, 2004) . it has been widely reported that various antigen-processing pathways may affect immune epitope formation either positively or negatively and may influence the repertoire of t-cell epitope-specific immune responses either quantitatively or qualitatively (pamer and cresswell, 1998; kessler et al., 2002; lautwein et al., 2004; kloetzel, 2004) . such specificity in antigen processing would suggest that the form and delivery of an immunogen can be critical to the generation of biologically effective antigen epitopes and thus to vaccine efficacy. there is, however, abundant evidence for the presentation of exogenous antigens by mhc class i and of endogenous proteins by mhc class ii through a variety of alternative antigen trafficking and processing mechanisms, including specialized processing pathways (reimann and schirmbeck, 1999; norbury et al., 2001; bonifaz et al., 2002; imai et al., 2004) , cross priming and presentation (brode and macary, 2004; tewari et al., 2005) , alternative endolysosomal pathways (brooks et al., 1991; lich et al., 2000; chen and jondal, 2004) , vesicular translocation (reis e sousa and germain, 1995; ackerman and cresswell, 2004) , and phagocytic or autophagic mechanisms for antigen delivery to both the mhc class i and ii compartments (houde et al., 2003; guermonprez et al., 2003; nimmerjahn et al., 2003; rocha and tanchot, 2004; paludan et al., 2005) . nevertheless, little is known about the consequences of these alternative antigen trafficking and processing pathways for the production of pathogen-specific antigen epitopes. thus, an examination of t-cell epitopes is a critical early step in the development of a new generation of vaccines, including dna vaccines, chimeras with other protein sequences, or epitopebased antigens, all of which involve antigens as non-viral structures that may access processing pathways that differ from those followed by the native pathogens. these issues regarding the pathways used for antigen delivery and processing are particularly relevant to mhc class ii epitopes. presentation of antigen epitopes and activation of cd4 t cells by mhc class ii molecules are critical steps in generating memory b cell and cd8 + t-cell phenotypes (wang and livingstone, 2003; , and failure to elicit immune memory is one of the recognized problems of conventional genetic vaccines (maecker et al., 1998) . this defect may reflect an inadequate level of antigen expression or restricted access to the mhc ii processing and presentation compartments by dna-encoded endogenous antigens that lack a dedicated trafficking pathway (martins et al., 1995; maecker et al., 1998; oehen et al., 2000; rush et al., 2002) . in response to this problem, we have characterized dna vaccine constructs synthesized in the form of chimeras that link the antigen sequences to the trafficking/targeting signals of the lysosome-associated membrane protein-1 (lamp-1) (chen et al., 1985) . lamp molecules are known to traffic to lysosomes and to specialized multilaminar vesicular compartments of immature apcs, termed miic, where mhc ii antigen processing and the formation of antigenic peptide -mhc ii complexes occur (kleijmeer et al., 1997; geuze 1998; murk et al., 2004) . studies of several antigens (including hiv-1 env and gag, hpv e7, cytomegalovirus pp65, carcinoembryonic antigen, dengue prem/e and yellow fever env, and the catalytic subunit of the telomerase reverse transcriptase (htert)) expressed from viral vectors and naked dna or rna have shown that lamp-targeted antigens generated greater antigenspecific lymphoproliferative activity, antibody titers, and cytotoxic t-lymphocyte activities than do dna constructs encoding wild-type antigens (rowell et al., 1995; wu et al., 1995; ruff et al., 1997; nair et al., 1998; raviprakash et al., 2001; bonini et al., 2001; su et al., 2002; lu et al., 2003; marques et al., 2003; chikhlikar et al., 2004; barros de arruda et al., 2004; anwar et al., 2005; su et al., 2002) . most recently, in a clinical study, patients who received dendritic cells transfected with rna encoding an htert/lamp chimera developed higher frequencies of htert-specific t-cell responses than did subjects receiving dendritic cells transfected with the unmodified htert template (su et al., 2005) . despite this abundance of evidence of increased immune responses to lamp chimeras of antigen proteins, the biological consequences of the enhanced immune responses in relation to protective immunity have not been defined, and there remains a critical question of the identity of t-cell epitopes of lamp-targeted antigens as compared to those of natural pathogens. lamp trafficking is not a normal mechanism for antigen access to the mhc class ii compartment of antigen-presenting cells, and the epitope specificity of antigen processing and presentation in vivo in comparison to other antigen delivery systems remains to be demonstrated. in the present study, we have used the severe acute respiratory syndrome coronavirus (sars cov) nucleocapsid (n) as a model antigen to address this issue of immune response and t-cell epitope presentation as a function of antigen formulation. the n protein of 422 amino acids is abundantly expressed and highly immunogenic (lau et al., 2004; huang et al., 2004a) . the primary function of this structural protein in virus assembly is to bind to sars cov rna during the formation of the ribonucleoprotein complex (huang et al., 2004b) . previous studies have shown that both the n protein (lin et al., 2003; zhao et al., 2005) and dna encoded n (kim et al., 2004; zhu et al., 2004; yang et al., 2004) elicit antibody and cytotoxic t-cell responses in mice. in this analysis of the effect of immunogen formulation and delivery on immune responses in vivo, we have utilized three forms of n delivery: (1) recombinant n-gst protein combined with freund's adjuvant as a classical exogenous antigen under conditions of profound activation of the innate immune response; (2) n encoded in a dna plasmid as an unmodified endogenous cytoplasmic/nuclear antigen; and (3) a dna construct with n inserted into the luminal domain of the entire lamp-1 molecule as an unnatural endogenous antigen targeted to the luminal compartment of endosomal/lysosomal vesicles (guarnieri et al., 1993) . validation of n expression by p-n and p-lamp-n dna constructs was carried out by analyzing cells transfected in vitro. cdna encoding the n protein was prepared by rt-pcr with n-specific primers and sars rna purified from a singapore clinical isolate (genbank accession no. ay307165). the corresponding 1269-bp n sequence was inserted into the p43 vector (kessler et al., 1996) to form the wild-type p-n dna expression vector and into the p43-hlamp-1 construct in a position 5v-proximal to the lamp transmembrane domain to make the p-hlamp-n vector (fig. 1a) . western blot analysis of n in cell lysates and culture supernatants of transfected cos-7 cells showed the presence of the¨42 kda n protein at comparable levels in cell lysates and culture supernatants of p-n and p-hlamp-n-transfected cos-7 cells and of various forms of the higher molecular weight lamp-n chimera in the cell lysate and culture supernatant of p-hlamp-n-transfected cells (fig. 1b) . the n-like protein in the cell lysate of p-hlamp-n-transfected cells is attributed to the cleavage of n from the protease-resistant lamp at some stage in the cellular trafficking of the molecule. intact or near-intact hiv-1 gag protein was also observed with cells transfected with the corresponding lamp/gag dna vaccine construct . several forms of the larger lamp-n molecule were also present in both the cell lysate and culture supernatants of the transfected cells. the intact lamp-n is found at about 150 kda, and the other bands are attributed to various degradation products. however, we are cautious about the presumed cell trafficking pathway for the newly synthesized n, which lacks an endoplasmic reticulum translocation (signal) sequence, is to the cell cytoplasm. in contrast, the lamp-n chimera is expected to traffic to endosomal/ lysosomal vesicles because of the lamp vesicular trafficking sequences. we used confocal microscopy to validate these trafficking destinations for the immunoreactive unmodified n antigens and n of the lamp-n chimera expressed in transfected mouse lb 27.4 b cells and human melanoma mel-juso cells, comparing their cellular distribution to that of the endogenously expressed lamp-1 and mhc class ii proteins (fig. 2) . the colocalization of endogenous lamp and mhc class ii proteins in vesicular compartments of apcs has been extensively documented (kleijmeer et al., 1996; geuze, 1998; murk et al., 2004) . trafficking of the lamp-n chimera protein to the lamp and mhc ii compartments of the transfected cells was validated by the colocalization of the lamp-n chimera (labeled by anti-n antibody) with endogenous lamp-1 and mhc class ii in both mouse b ( figs greater definition of the localization of n in transfected cells was obtained by analyzing of immunolabeled ultrathin cryosections of mouse lb 27.4 b cells. the endocytic compartments of these b cells are morphologically similar to those of other reported mouse b-cell lines (6h5.dm and a20.a b ), with well-defined multivesicular and multilaminar miics and end organelles (geuze, 1998) . double immunogold labeling of the transfected b cells confirmed that n of the lamp-n chimera was present with both endogenous lamp and mhc class ii molecules in the miic multilaminar vesicles (figs. 3a, b). in contrast, unmodified n was predominantly localized in the cytoplasm or in vacuolated structures with no evident colocalization with lamp or mhc class ii (figs. 3c-e). remarkably, lower power magnification showed that some of the cytoplasmic vesicles contained large amounts of n. these vesicles may correspond to the strongly labeled vesicular structures shown by immunofluorescent microscopy, and the presence of n in these structures suggests a possible route for the secretion of the protein (fig. 3e ). n antigen in three forms, representing differences in antigen delivery, adjuvants, and peptide processing pathways, were administered according to the following protocols: (1) n as an exogenous n-gst protein was administered three times, on day one and 3 and 6 weeks later, initially with cfa and then with ifa. (2) n as an unmodified endogenous, dna-encoded, cytoplasmic protein, and (3) n as an artificial chimera encoded in the luminal domain of lamp and targeted to cellular lysosomal and mhc ii compartments were administered five times, on day 1, and 3, 6, 9, and 14 weeks later, without added adjuvant but subject to the molecular adjuvant effects of the dna cpg motifs and the targeting of the lamp-n chimera to the mhc ii compartment. dna encoding p-hlamp was used as the negative control. t-cell responses were assayed by ifn-g elispot and elisa assays using spleen cells collected 8 days after the final dna immunization. for these assays, the t cells were stimulated with n-his protein as a positive control or with a library of overlapping n peptides, 15 to 20 amino acids in length. the results were confirmed in three experiments and in multiple assays with three independent sets of peptides from different sources. the ifn-g responses were also assayed with an elisa protocol, with similar results (data not shown). the strongest ifn-g responders were cells from mice immunized with p-hlamp-n, with comparable but weaker responses by the p-n-immunized mice, and minimal responses by cells of the n-gst-immunized mice (table 1; fig. 4 ). the major grouping of ifn-g responses was elicited by peptides of amino acid sequence n 76 -114 , encompassing 5 of the overlapping peptides. more recently, these results have also been confirmed in a new experimental protocol currently being conducted that also show that all of the peptides that encompass n 76 -114 elicit both cd4-and cd8-specific elispot responses (data not shown). other dominant responses were to peptides n 130 -149 and n 241 -258 and the overlapping peptides n 352 -366 and n 353 -370 . all significant epitope-specific responses of the pn or n-gst-immunized mice were shared by the p-hlamp-nimmunized mice. the weak ifn-g responses of the n-gst protein immunized mice were observed only with peptides that elicited the strongest responses with splenocytes of the dnaplasmid-immunized mice (figs. 4a -c). this comparatively weak ifn-g response of n-gst-immunized mice was in marked contrast to the strong il-4 responses as shown below and to the exceptional strong antibody responses. the n-specific antibody titers to n-gst immunization on day 42 (after the first boost) was 1:25,000 and at day 63 (after the second boost) was 1:67,500, as compared to titers increasing to a maximum of 1:3200 on days 84 and 105 in response to immunization with the p-n and p-hlamp-n dna constructs. memory immune responses (total igg antibody titers, ctl activity, and elispot ifn-g and il-4 responses) were examined by use of a separate experimental protocol with groups of mice treated with the same three n antigen preparations but receiving only three inoculations over 6 weeks. after an interval of 36 weeks, the memory immune response was activated by a single injection at week 42 of the p-n construct as a surrogate of virus infection. antibody and tcell elispot and ctl responses were measured immediately before p-n injection at 42 weeks and three times at week intervals thereafter (figs. 5 and 6). the t-cell assays were conducted with the selected peptides that had elicited immunodominant responses in pilot experiments for and ctl, and n 353 -370 . the residual t-cell ifn-g and il-4 responses of mice sacrificed on day 1 before p-n memory activation were very low and were significant only in mice initially immunized with p-hlamp-n (fig. 5) . following the recall p-n injection, increased ifn-g and il-4 responses were observed on day 8, reaching an apparent maximum on days 15 to 22. the strongest memory t-cell responses in all assays were those of mice initially immunized with p-hlamp-n, with strong ifn-g and il-4 responses to both peptides. n-gst plus freund's adjuvant elicited strong il-4 responses but minimal ifn-g responses, as seen for the primary response. the responses of mice initially immunized with p-n were uniformly lower than those seen for the lamp chimera and the il-4 responses to n-gst. however, the ifn-g responses were greater for p-n than for n-gst. memory ctl responses were strongest in p-hlamp-n-immunized mice, with a much weaker response to p-n dna immunization and little if any after immunization with n-gst with adjuvant (fig. 6) . memory antibody responses followed the pattern of the primary response, with a much stronger igg titer of about 1:25,000 on days 15 and 22 following activation of the n-gst-immunized mice as compared to the responses of about 1:7000 by the p-hlamp-n-immunized mice. there was relatively little response (1:1000) by mice initially immunized with p-n. peptide epitope activation of t cells relies on receptor (tcr) recognition of complexes of antigen peptide sequences bound to the nine amino acids of the binding clefts of mhc class i and ii molecules (benacerraf, 1978) . it is postulated that the kinetic stability of the mhc-peptide complexes is a key parameter that dictates immunodominance (lazarski et al., 2005) . in the further analysis of the t-cell epitope specificity of n, the predicted h2 d class i (h2-k d , -l d , -d d ) and class ii (i-e d , i-a d ) binding motifs of the 60 sars n overlapping peptides were examined by a murine immunoinformatics system based on quantitative matrices, pred balb/c (zhang et al., 2005) . each of the 414 nine-amino-acid sequences of n was analyzed for binding to the core sequence of the 5 h2 d alleles. all of the nine ifn-g elispot-positive peptides scored for a high probability of binding to class i and/or class ii alleles (table 2) , and, in ongoing experiments, all of a new set of peptides encompassing n 76 -114 elicit both cd4-and cd8specific responses (data not shown). in the present study, we have demonstrated that the dominant t-cell immune responses of mice immunized with the sars cov n protein plus adjuvant or with dna encoding two different cellular trafficking forms of n are directed to the same peptide epitopes of all three immunogens. this finding points to the existence of versatile antigen-processing mechanisms that are not defined by the initial site of delivery of the protein, whether exogenous or endogenous, or the state of the innate responses to the immunogens. the data are consistent with multiple in vivo routes for antigen access to proteolytic processing compartments that produce the same or comparable mhc i and ii peptide epitopes. this versatility would not all elispot responses were to a group of 9 dominant peptides, shown by peptide number, amino acid region in n, sequence, and number of ifn-g-positive cells of mice immunized with the three immunogens. a cluster (n 76 -114 ) of 5 peptides of 15 to 20 aa, overlapping by 10 to 16, contains 3 of the strongest dominant epitopes. preclude quantitative variation in epitope presentation as a result of differences in the levels of antigen, the degree of processing, or access to cellular compartments for antigen presentation. for example, in this study, the greater response to the lamp-n chimera could be related to the quantitatively greater delivery of n to the mhc class ii compartment of apcs. there can also be major differences in the repertoire or functionality of t cells responding to any given epitope, such as the differences in the ifn-g and il-4 responses of mice immunized with dna or with n-gst plus cfa. others have reported similar findings that the same ctl-defined epitope peptide is generated by an exogenous protein and by plasmid dna-encoded antigens (schirmbeck et al., 1998) and that the efficiency of mhc class i cross-presentation of peptides derived from exogenous antigens is comparable to that for presentation by the classical mhc class ii pathway (storni and bachmann, 2004) . additionally, ria et al. (2004) have compared the response to hen egg-white lysozyme (hel) and its reduced and carboxymethylated form (rcm-hel) and have found that rcm-hel induces an in vivo t-cell response that is focused on the same immunodominant determinant that characterizes the response to native hel but that the rcm-hel response is skewed toward the th1 pathway. no difference in the efficiency of processing was observed between hel and rcm-hel. we conclude that the makeup of the t-cell epitopes of an antigen is primarily determined by the amino acid composition of the protein in the context of the peptide binding sites of mhc molecules, and not by the mechanism of delivery of the protein. these findings support the usefulness of novel immunogens, such as antigen chimeras and dna vaccines encoding antigens with modified cell trafficking signals, as vaccine candidates, despite their differences from normal pathogen proteins in terms of antigen structure, delivery, and processing mechanisms. the differences in the results we obtained for the three antigen preparations were largely related to the character and magnitude of the immune responses. mice immunized with exogenous n protein plus freund's adjuvant responded with a greatly enhanced antibody response and a high ratio of t-cell il-4 to ifn-g responses to the antigen peptides. in contrast, the same peptides elicited a balanced ifn-g and il-4 response and lower antibody responses in mice immunized with dna vaccines. freund's adjuvant has been associated with the preferential induction of t-cell th2 responses (shibaki and katz, 2002; yip et al., 1999; billiau and matthys, 2001) , and different t-cell cytokine responses to the same epitope (varga et al., 2000) have been attributed to variable expression patterns of single cells and populations that reflect transient rather than heritable differences in the expression profile (kelso and groves, 1997; kelso et al., 2002) . another difference between the vaccines was the comparatively poor memory response to the n-gst protein and p-n dna construct as compared to the p-hlamp-n chimera construct. the limited efficacy of the conventional p-n dna construct is consistent with many previous studies of our and other laboratories (martins et al., 1995; maecker et al., 1998; oehen et al., 2000; rush et al., 2002; marques et al., 2003; barros de arruda et al., 2004) . although it is likely that there are multiple factors involved in memory t-cell differentiation (masopust et al., 2004) , we attribute the enhanced t-cell and memory responses to p-lamp-n, at least in part, to the increased efficiency of trafficking of the lamp-n chimera to the mhc ii compartments of apcs. the localization of the dominant t-cell responses to a cluster of overlapping peptides epitopes within n 76 -114 is similar to findings from other ongoing studies of hiv-1 gag and yellow fever virus envelope proteins where the major t-cell responses fig. 4 . primary t-cell ifn-g elispot responses of (a) p-hlamp-n, (b) p-n, and (c) n-gst-immunized mice. as described in materials and methods, mice were immunized five times with the dna immunogens and three times with n-gst plus adjuvant, and the mice were sacrificed for elispot assay of antigenspecific splenocyte ifn-g secretion 8 days after the final immunization. the splenocytes were stimulated with recombinant n-his protein and with n peptides from panels of overlapping peptides spanning the entire molecule, with mice immunized by hlamp plasmid as the negative control. all significant t-cell peptide-specific ifn-g responses elicited by each immunogen were stimulated by the same peptides, with the strongest responses to p-hlamp-n and the weakest to n-gst. also involve clustered epitopes (unpublished observations). regions of overlapping t-cell epitopes are also reported in studies of hiv-1 proteins (shankar et al., 1996; surman et al., 2001; brown et al., 2003; berzofsky et al., 1991) , the outer membrane protein of chlamydia trachomatis (kim and demars, 2001) , and in predictions of hla t-cell epitopes (srinivasan et al., 2004; zhang et al., 2005) . clustered t-cell epitopes appear to be a common, perhaps ubiquitous occurrence and, as such, may facilitate the development of epitope-based vaccines if a single or few hot spots elicit all of the required tcell functions. the basis for such hot spots is unknown but is likely to be related in some measure to the binding affinities of the peptide sequences to mhc molecules as well as to other functional parameters that affect mhc -peptide binding to tcell receptors during t-cell activation (chen et al., 2005) . analysis of the nonameric mhc binding motifs of sars n by a newly developed pred balb/c computational system for predicting the probability of binding of each nine-amino-acid sequence of an antigen to the h2 d class i (h2-k d , -l d , -d d ) and class ii (i-e d , i-a d ) binding motifs (zhang et al., 2005) showed that each of the sars n peptides that elicited elispot-positive ifn-g t-cell responses contained highprobability binding motifs for mhc class i and/or ii alleles. there may also be protein structural effects in the selection of epitope hot spots. the three-dimensional structure of the n amino-terminal domain, n 45 -181 , of a truncated model lacking n 1 -44 and n 182 -422 has been determined by nmr spectroscopy (huang et al., 2004b) . two of the elispot-positive sequences, n 76 -114 and n 130 -149 , were present on the exposed surface of this structure. the n 76 -114 t-cell hot spot sequence also contains the n 76 -101 sequence reported to comprise a b cell epitope (huang et al., 2004a ). an association between tand b-cell epitopes has also been found for the hiv envelope; these are seen to be present in exposed loops or strands when mapped onto the crystal structure of the protein (brown et al., 2003; del porto et al., 2002; shirai et al., 1999) . mouse b lymphoma and monkey kidney cell lines (lb27.4 and cos-7) were obtained from atcc (rockville, md). human melanoma cell line (mel-juso) was obtained from german collection of microorganisms and cell cultures (braunschweig germany). insect cell lines sf9 and high5 were obtained from invitrogen. all the cell lines were maintained according to the supplier's protocols. the sars cov nucleocapsid (n) cdna was prepared from a singapore clinical isolate and purified total rna. the 1269 bp n gene sequence was obtained by pcr with primers 5v-cggctagcatgtctgataatggaccccaatc-3v and 5v-gcggtaccttatgcctgagttgaatcagcag-3v, having nhei and kpni sites. the pcr product was cloned into the p43 expression vector containing aav-itr sequences flanking the expression elements and a cmv promoter (kessler et al., 1996) to make the wild-type plasmid p-n. this n-sequence (without the stop codon) was amplified, and xholi and ecori sites were introduced by using primers 5v-cgctcgagatgtctga-taatggaccccaatc-3v and 5v-cggaattctgcct-gagttgaatcagcagaa-3v. the p-hlamp-n chimera vector was constructed by substitution of the hiv-gag sequence into the p-hlamp-gag vector . due to the presence of internal nhei or xhoi sites in the n sequence, the p-n and p-hlamp-n plasmids were constructed by sequential cloning of the two fragments of the n gene. sars-cov n was amplified from the rt-pcr sequence using forward primer 5v-acgtcagaattcatgtctga-taatggaccccaa with an ecori site and reverse primer 5v-agtttagcggccgctgcctgagttgaatcagcag with a noti site. the cdna sequence was cloned into the ecori and noti sites of the pgex6p-1 (invitrogen, a kind gift from dr. roland of the institute of molecular and cell biology, singapore) and pfastbac ht-a (invitrogen, carlsbad, ca) vectors. all clones were verified by sequencing. the n-specific h2 d binding motifs were analyzed by the pred balb/c system. each of the ifn-g elispot peptides contained multiple nine amino acids binding motifs that were within the top 5% of all binding scores to the five h2 d alleles. all of the ifn-g elispot peptides except n 241 -258 scored were predicted to contain class i binding motifs. similarly, class ii binding motifs were predicted in all except n 76 -93 and n 130 -149 . expression of the n protein from the vaccine plasmids p-n and p-lamp-n in the cos-7 cell line was analyzed by western blot analysis. cells were transfected with the sars plasmids using polyfect transfection reagent (qiagen gmbh). cell culture and polyacrylamide gel electrophoresis were carried out as described . the n protein was detected by incubation with polyclonal serum from mice immunized with n-gst protein followed by horseradishperoxidase-conjugated goat anti-mouse igg monoclonal antibody (pierce biotechnology). binding of secondary antibody was visualized with the supersignal west pico chemiluminescent kit or dab (pierce biotechnology). recombinant n protein was generated by use of two dna plasmid expression systems, n-gst in e. coli for use as an antigen for mouse immunization and n-his in a baculovirus system as an antigen for elisa and elispot assays. recombinant n protein was expressed as a gst fusion in top10 e. coli cells (invitrogen) after iptg induction. the protein was purified by affinity chromatography with glutathione s -sepharose beads (amersham pharmacia biotech ab), and the preparation was validated by western blot analysis using mouse monoclonal anti-gst (santa cruz biotechnology) and rabbit polyclonal anti-n (imgenex) antibodies. the bac to bac baculovirus expression system version c (invitrogen) was used for n protein expression in insect cells. pfastbacht-a-n was constructed by cloning the ecori-and noti-digested sars cov n fragment into the pfastbacht-a vector. n protein was purified from lysates of infected high5 cells using ni-nta agarose beads (invitrogen), and the protein preparation was verified by western blot analysis with mouse monoclonal penta-his-antibody (qiagen, gmbh) and anti-n (imgenex) antibodies. purification of the culture products of each expression system yielded fractions enriched for the 73-kda n-gst and 55-kda n-his proteins. the cellular localization of the expressed viral proteins in transfected mouse b lymphoma cells was studied by confocal and immunoelectron microscopy and confirmed by confocal microscopy of transfected human mel-juso cells. cells were transfected with the sars cov plasmids (p-hlamp-n and p-n) using polyfect (qiagen, gmbh). the expression of the recombinant viral proteins was visualized by the use of rabbit polyclonal anti-n (imgenex) or mouse monoclonal anti-human lamp1 antibody h4a3 (obtained from supernatant of hybridoma cultures prepared in our laboratory). localization of the transgenic proteins in mhc-class-ii-containing compartments and lysosomes of b cells was determined by the use of anti-mouse mhc class ii antibody m5/114.15.2 (bd biosciences) and anti-mouse lamp1 (mlamp1) id4b antibody (prepared in our laboratory). localization of the viral proteins in the mhc class ii-containing compartments of mel-juso cells was analyzed with anti-hla-dr, dp, and dq antibodies (bd pharmingen) and rabbit anti-n antibody (img-549; imgenex). three immunization protocols were carried out: (1) three groups (p-hlamp control, p-n, and p-hlamp-n immunogens) of 5 female (6 to 8 weeks old) balb/c mice per group were immunized subcutaneously at the base of the tail with 50 ag of specified endotoxin-free dna plasmid diluted in pbs on days 1, 22, 43, 64, and 100. blood samples were collected by phlebotomy from the tail vein on days 0, 21, 42, 63, 84, 99, and 108, and the animals were sacrificed on day 108, 8 days after the last immunization. (2) another group was immunized intraperitoneally with 10 ag of n-gst protein emulsified with cfa followed by two booster immunizations 3 weeks apart with n-gst protein emulsified with ifa. blood samples were collected as outlined above on days 0, 21, and 42, and the animals were sacrificed on day 50, 8 days after the last immunization. protocols 1 and 2 were carried out twice with five mice per group each time. (3) an additional protocol was carried out for analysis of memory responses: four groups of 16 mice each (p-hlamp control, p-n, p-hlamp-n, and n-gst immunogens) were immunized as outlined above, but with only three injections over 6 weeks. at week 42, after an interval of 36 weeks, the memory immune response was activated by a single injection of the p-n construct with p-hlamp as the negative control. three mice of each group were sacrificed 1 day before the final p-n injection and at weekly intervals for 3 weeks thereafter. these experiments were performed as approved by the johns hopkins university animal care and use committee under protocol number mo04m178. anti-sars cov n igg antibodies in the serum from each mouse were assayed by elisa for seroconversion at a dilution of 1:300, and the total igg titers of 3-fold serial dilutions, starting from 1:100, were determined by standard quantitative elisa. in brief, elisa plates (96-well, maxisorb f96; nunc inc.) were coated overnight at 4 -c with 1 ag/ml recombinant n-his protein in pbs followed by blocking with elisa buffer (pbs containing 2.5% milk and 0.05% tween 20). after washing, appropriately diluted mouse serum was added and incubated for 2 h at room temperature. total igg was detected with the mouse extravidin alkaline phosphatase staining kit (sigma). the response of antigen-specific t cells from immunized mice was measured by the use of ifn-g and il-4 elispot sets (bd pharmingen) according to the manufacturer's protocol. splenocytes were stimulated with 5 ag/ml of recombinant n-his (baculovirus-produced) or 10 ag/ml of the overlapping sars cov synthetic peptides from three sources (nih aids research and reference reagent program, rockville, md; synpep corp, dublin, ca; the johns hopkins biochemistry core facility). negative control stimulation produced less than five spots per well in >90% of experiments. the average number of spots in the negative control wells was 1.67 t 1.87. each experiment was repeated at least twice on different groups of mice from two immunizations. cytotoxic t-lymphocyte assay ctl assays were performed using the cytotox 96 nonradioactive cytotoxicity assay kit (promega corporation, usa). the pooled splenocytes (from two mice in the n-gst group and three mice in each of the other groups) obtained at each time point (days 1, 8, 15, and 22) after memory recall with pn were stimulated in vitro with each of the two peptides, n 80 -99 and n 353 -370 , at a final concentration of 10 ag/ml for 4 days. sp2/o target cells were pulsed overnight separately with 10 ag/ ml of each of the two peptides, washed two times, resuspended in rpmi supplemented with 2% fcs, and seeded onto a 96well u-bottom plate (1 â10 4 cells/well in 50 al of culture medium) with an equal volume of pooled, in-vitro-stimulated splenocytes from recalled mice (effector cells) at different effector/target ratios, 20:1, 40:1, and 80:1. all incubations were done in quadruplicate. the negative control contained an equal concentration of unrelated peptide, and controls for effector cell spontaneous release, target spontaneous release, and target maximum release were included in the assays. all the values were computed as the mean t standard deviations of the four replicate assays after subtracting the average of the values obtained for the negative controls. the use of overlapping peptides for antiviral cd8 and cd4 t-cell responses has been described (maecker et al., 2001; draenert et al., 2003) . balb/c mouse h2 d t-cell binding motifs of the 60 overlapping n peptides were predicted by use of the pred balb/c class i (h2-k d , -l d , -d d ) and class ii (i-e d , -a d ) models (zhang et al., 2005) . the pred balb/c system scores the binding index of all immunogen nine-amino-acid sequences to h2 d molecules, based on quantitative matrices. cellular mechanisms governing crosspresentation of exogenous antigens west nile premembrane-envelope genetic vaccine encoded as a chimera containing the transmembrane and cytoplasmic domains of a lysosome associated membrane protein: increased cellular concentration of the transgene product, targeting to the mhc ii compartment and enhanced neutralizing antibody response dna vaccine encoding human immunodeficiency virus-1 gag, targeted to the major histocompatibility complex ii compartment by lysosomal-associated membrane protein, elicits enhanced long-term memory response a hypothesis to relate the specificity of t lymphocytes and the activity of i region-specific ir genes in macrophages and b lymphocytes construction of peptides encompassing multideterminant clusters of human immunodeficiency virus envelope to induce in vitro t cell responses in mice and humans of multiple mhc types modes of action of freund's adjuvants in experimental models of autoimmune diseases efficient targeting of protein antigen to the dendritic cell receptor dec-205 in the steady state leads to antigen presentation on major histocompatibility complex class i products and peripheral cd8 + t cell tolerance targeting antigen in mature dendritic cells for simultaneous stimulation of cd4 + and cd8 + t cells cross-presentation: dendritic cells and macrophages bite off more than they can chew class ii-restricted presentation of an endogenously derived immunodominant t-cell determinant of hen egg lysozyme clustering of th cell epitopes on exposed regions of hiv envelope despite defects in antibody activity class ii mhc peptide loading by the professionals endolysosomal processing of exogenous antigen into major histocompatibility complex class i-binding peptides identification of two lysosomal membrane glycoproteins structural and kinetic basis for heightened immunogenicity of t cell vaccines inverted terminal repeat sequences of adeno-associated virus enhance the antibody and cd8 (+) responses to a hiv-1 p55gag/lamp dna vaccine chimera immunological memory in humans high prevalence of hypervariable region 1-specific and-cross-reactive cd4 (+) t cells in hcv-infected individuals responsive to ifn-alpha treatment comparison of overlapping peptide sets for detection of antiviral cd8 and cd4 t cell responses the role of endosomes and lysosomes in mhc class ii functioning the motif tyr -x -x-hydrophobic residue mediates lysosomal membrane targeting of lysosome-associated membrane protein 1 er-phagosome fusion defines an mhc class i crosspresentation compartment in dendritic cells phagosomes are competent organelles for antigen cross-presentation evaluation of antibody responses against sars coronaviral nucleocapsid or spike proteins by immunoblotting or elisa structure of the n-terminal rna-binding domain of the sars cov nucleocapsid protein exogenous antigens are processed through the endoplasmic reticulum-associated degradation (erad) in cross-presentation by dendritic cells a single peripheral cd8 + t cell can give rise to progeny expressing type 1 and/or type 2 cytokine genes and can retain its multipotentiality through many cell divisions the genes for perforin, granzymes a -c and ifn-gamma are differentially expressed in single cd8 (+) t cells during primary activation gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein mhc class i antigen processing regulated by cytosolic proteolysis-short cuts that alter peptide generation epitope clusters in the major outer membrane protein of chlamydia trachomatis generation and characterization of dna vaccines targeting the nucleocapsid protein of severe acute respiratory syndrome coronavirus characterization of mhc class ii compartments by immunoelectron microscopy major histocompatibility complex class ii compartments in human and mouse b lymphoblasts represent conventional endocytic compartments generation of major histocompatibility complex class i antigens: functional interplay between proteasomes and tppii detection of severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein in sars patients by enzyme-linked immunosorbent assay human b lymphoblastoid cells contain distinct patterns of cathepsin activity in endocytic compartments and regulate mhc class ii transport in a cathepsin s-independent manner the kinetic stability of mhc class ii: peptide complexes is a key parameter that dictates immunodominance cytoplasmic processing is a prerequisite for presentation of an endogenous antigen by major histocompatibility complex class ii proteins identification of an epitope of sars-coronavirus nucleocapsid protein dengue 2 prem-e/lamp chimera targeted to the mhc class ii compartment elicits long-lasting neutralizing antibodies cytotoxic t cell responses to dna vaccination: dependence on antigen presentation via class ii mhc use of overlapping peptide mixtures as antigens for cytokine flow cytometry hiv-1 p55gag encoded in the lysosome-associated membrane protein-1 as a dna plasmid vaccine chimera is highly expressed, traffics to the major histocompatibility class ii compartment, and elicits enhanced immune responses dna vaccination against persistent viral infection the role of programming in memory t-cell development processing of an endogenous protein can generate mhc class iirestricted t cell determinants distinct from those derived from exogenous antigen 3-d structure of multilaminar lysosomes in antigen presenting cells reveals trapping of mhc ii on the internal membranes induction of primary carcinoembryonic antigen (cea)-specific cytotoxic t lymphocytes in vitro using human dendritic cells transfected with rna major histocompatibility complex class ii-restricted presentation of a cytosolic antigen by autophagy multiple antigen-specific processing pathways for activating naive cd8 + t cells in vivo antiviral protection after dna vaccination is short lived and not enhanced by cpg dna endogenous mhc class ii processing of a viral nuclear antigen after autophagy mechanisms of mhc class i-restricted antigen processing synergistic neutralizing antibody response to a dengue virus type 2 dna vaccine by incorporation of lysosome-associated membrane protein sequences and use of plasmid expressing gm-csf alternative pathways for processing exogenous and endogenous antigens that can generate peptides for mhc class i-restricted presentation major histocompatibility complex class i presentation of peptides derived from soluble exogenous antigen by a subset of cells engaged in phagocytosis selection of similar naive t cell repertoires but induction of distinct t cell responses by native and modified antigen towards a cellular definition of cd8 + t-cell memory: the role of cd4 + t-cell help in cd8 + t-cell responses lysosome-associated membrane protein-1-mediated targeting of the hiv-1 envelope protein to an endosomal/lysosomal compartment enhances its presentation to mhc class ii-restricted t cells the enhanced immune response to the hiv gp160/lamp chimeric gene product targeted to the lysosome membrane protein trafficking pathway efficient priming of cd4 + and cd8 + t cells by dna vaccination depends on appropriate targeting of sufficient levels of immunologically relevant antigen to appropriate processing pathways similar as well as distinct mhc class i-binding peptides are generated by exogenous and endogenous processing of hepatitis b virus surface antigen three regions of hiv-1 gp160 contain clusters of immunodominant ctl epitopes induction of skewed th1/th2 t-cell differentiation via subcutaneous immunization with freund's adjuvant t cell recognition of hypervariable region-1 from hepatitis c virus envelope protein with multiple class ii mhc molecules in mice and humans: preferential help for induction of antibodies to the hypervariable region prediction of class i t-cell epitopes: evidence of presence of immunological hot spots inside antigens loading of mhc class i and ii presentation pathways by exogenous antigens: a quantitative in vivo comparison enhanced induction of telomerase-specific cd4 (+) t cells using dendritic cells transfected with rna encoding a chimeric gene product cd4 + t cells are required for the maintenance, not programming, of memory cd8 + t cells after acute infection localization of cd4 + t cell epitope hotspots to exposed strands of hiv envelope glycoprotein suggests structural influences on antigen processing a cytosolic pathway for mhc class ii-restricted antigen processing that is proteasome and tap dependent the attachment (g) glycoprotein of respiratory syncytial virus contains a single immunodominant epitope that elicits both th1 and th2 cd4 + t cell responses proteases involved in mhc class ii antigen presentation cutting edge: cd4 + t cell help can be essential for primary cd8 + t cell responses in vivo immunological memory to viral infections engineering an intracellular pathway for major histocompatibility complex class ii presentation of antigens a dna vaccine induces sars coronavirus neutralization and protective immunity in mice cut and trim: generating mhc class i peptide ligands adjuvant-guided type-1 and type-2 immunity: infectious/noninfectious dichotomy defines the class of response pred balb/c : a system for prediction of peptide binding to the h2d molecules, a haplotype of the balb/c mouse immune responses against sars-coronavirus nucleocapsid protein induced by dna vaccine induction of sars-nucleoprotein-specific immune response by use of dna vaccine this research has been funded in part by the national institute of allergy and infectious diseases, national institutes of health, department of health and human services, contract no. hhsn266200400085c, and by contracts from the agency for science, technology and research, singapore to the division of biomedical sciences, johns hopkins in singapore, and institute for infocomm research, singapore. we thank a/p ng mah lee and a/p charanjit kaur (national university of singapore) for critical analysis of the iem images and j.l. koh (institute for infocomm research) for computational analysis of the balb/c proteome. the support of the nih aids research and reference reagent program in providing the sars n peptides is gratefully acknowledged. key: cord-309469-2naxn580 authors: an, hongliu; cai, zhichao; yang, yuying; wang, zhaoxiong; liu, ding xiang; fang, shouguo title: identification and formation mechanism of a novel noncoding rna produced by avian infectious bronchitis virus date: 2019-01-05 journal: virology doi: 10.1016/j.virol.2018.12.019 sha: doc_id: 309469 cord_uid: 2naxn580 viral noncoding (nc) rnas have been shown to play important roles in viral life cycle. many viruses employ different mechanism to produce ncrnas. here, we report that coronavirus infectious bronchitis virus (ibv) produces a novel ncrna in virus-infected cells. this ncrna consists of 563 nucleotides excluding a poly(a) tail, is mainly derived from the 3′-untranslated region of ibv genome, and contains a 63-nt-long of terminal leader sequence derived from the 5′ end of the viral genome. using mutagenesis and reverse genetics, we reveal that this ncrna is a subgenomic rna generated by discontinuous transcription mechanism. viruses employ different mechanisms to produce a number of noncoding (nc) rnas excluding microrna. these ncrnas mainly include: (1) viral ncrnas transcribed by rna polymerase (pol) iii. for example, two virus-associated (va) rnas encoded by human adenovirus (reich et al., 1966; steitz et al., 2010) , eber1 and eber2 encoded by epstein-barr virus (skalsky and cullen, 2015) , mirna precursors encoded by murine γ-herpesviral trna-pre-mirna chimeras (bogerd et al., 2010; bowden et al., 1997; diebel et al., 2010) and retrovirus (kincaid et al., 2012) , and intragenic viral small ncrna encoded by human bocavirus 1 (wang et al., 2017) ; (2) viral ncrnas transcribed by rna polymerase ii. for example, polyadenylated nuclear (pan) rna encoded by kaposi's sarcoma-associated herpesvirus (sun et al., 1996; zhong and ganem, 1997) , the~2-kb latency-associated transcript (lat) expressed by herpes simplex virus (bloom, 2004 ), a con-served~5-kb intron expressed by human cytomegalovirus (hcmv) (kulesza and shenk, 2004) , and a 7.2-kb rna expressed by mouse cmv (kulesza and shenk, 2006) , u-rich rnas (hsurs) produced by herpesvirus saimiri (hvs), (albrecht and fleckenstein, 1992; ensser and fleckenstein, 2005) ; (3) subgenomic ncrnas from single-stranded rna viruses by incomplete degradation of genomic rna by the cellular 5-3′ exonuclease xrn1. for example, subgenomic rna (sfrna) produced by flaviviruses, such as dengue virus (denv), west nile virus (wnv), yellow fever virus (yfv), and zika virus [reviewed in (bidet and garcia-blanco, 2014; roby et al., 2014; pijlman et al., 2008; akiyama et al., 2016) , and subgenomic ncrna generated by some plant viruses such as barley yellow dwarf virus and red clover necrotic mosaic virus using similar mechanism (miller et al., 2016a) . viral ncrnas play important roles in viral life cycle. adenovirus va rnas are characterized for their role in counteracting the host antiviral defense through inhibition of protein kinase r (pkr) (mathews and shenk, 1991; wilson et al., 2014) . eber2 regulates the expression of a subset of ebv latent genes throung the interaction of eber2 and a cellular transcription factor paired box protein 5 (pax5) (arvey et al., 2012; lee et al., 2015) . degradation of mir-27 by mediated hsur 1 promotes activation and presumably proliferation of hvs-infected host t cells guo et al., 2014) . kshv pan rna is essential for virion production (borah et al., 2011) .β-herpesvirus hcmvencoded β2.7 prevents mitochondria-induced apoptosis, enabling steady atp production for viral processes and persistent infection (campbell et al., 2008; stern-ginossar et al., 2012) . sfrna produced by flaviviruses is required for cytopathicity and pathogenicity (pijlman et al., 2008) . it has been demonstrated to 1) interfere with cellular rna decay pathways by inhibiting xrn1 (moon et al., 2012) , 2) dampen the antiviral activity of type i interferon (schuessler et al., 2012) and 3) inhibit the rnai pathway in both the vertebrate and arthropod hosts, most likely by serving as a decoy substrate for dicer (schnettler et al., 2012) . inhibition of the host interferon response appears to be, at least in some flaviviruses, achieved by binding and inactivating cellular https://doi.org/10.1016/j.virol.2018.12.019 received 28 september 2018; received in revised form 19 december 2018; accepted 26 december 2018 regulators of translation of interferon-upregulated mrnas (bidet et al., 2014) . ncrnas encoded by some plant rna viruses can inhibit host translation and overwhelm host's rna interference system to favor virus infection (miller et al., 2016b) . avian infectious bronchitis virus (ibv) belongs to the genus gammacoronavirus within the order nidovirales. ibv is an enveloped positive-sense, single-stranded rna virus causing the acute highly contagious poultry disease infectious bronchitis (cavanagh, 2005) . like other coronaviruses, ibv can produce sgrnas via a discontinuous transcription mechanism to encode its structural proteins and specific accessory proteins (masters, 2006; sawicki et al., 2007) . briefly, ibv produces six mrna species in the infected cells, including its genomic mrna1, sgrna2 encoding spike (s) protein, sgrna3 encoding 3a, 3b and envelope (e) protein, sgrna4 encoding membrane (m) protein, sgrna5 encoding 5a and 5b, and sgrna6 encoding nucleocapsid (n) protein. recently, a low-abundance sgrna located between the sgrna4 and 5 has been identified (bentley et al., 2013) . in this study, we identify firstly an ncrna in the ibv-infected cells. moreover, we prove that this ncrna is derived mainly from 3′ utr of viral genome and is generated by discontinuous transcription process. vero and chicken embryo fibroblast df1 cells were maintained in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal bovine serum (fbs), penicillin (100 units/ml) and streptomycin (100 units/ml) (invitrogen). a recombinant ibv (ribv) (fang et al., 2007) generated from an infectious clone reference genome (ibv beaudette p65, genbank accession number dq001339.1) was used as the wild-type control. all ibv mutants were propagated in vero cells in fbs-free dmem. virus stocks were made through three repeated freezethaw cycles and kept at −80°c in 0.5-1 ml aliquots until use. constructs containing mutation or deletion were produced by using a quikchange site-directed mutagenesis kit (stratagene). the fulllength cdna was assembled as previously described (fang et al., 2007) by replacing the corresponding fragment with the mutant fragment. the mutations were verified by automated nucleotide sequencing. fulllength transcripts were generated in vitro using the mmessage mmachine t7 kit (ambion, austin, tx) according to the manufacturer's instructions, and electroporated into vero cells with one pulse at 450 v and 50 µf with a bio-rad gene pulser ii electroporator. the transfected vero cells were cultured overnight in 1% fbs-containing dmem and further cultured in dmem without fbs. the transfected cells were monitored daily for formation of cytopathic effect (cpe). recovered viruses were plaque purified and passaged on vero cells. vero cells in 6-well plates were infected with a dilution series of viruses for 1 h, washed twice with medium. cells were overlaid with 0.4% agar in fbs-free dmem, incubated at 37 ℃ for 3-4 days, fixed with 10% formaldehyde, and stained with 0.2% crystal violet. the number of plaques was counted and the virus titer was calculated as plaque-forming unit (pfu) per ml. ten-day-old embryonated, pathogen-free chicken eggs were inoculated with ibv as described previously (shen et al., 2009) . the allantoic fluid and different organs were harvested after the embryos were chilled at 4°c overnight. total rna was extracted from the homogenized tissues and used for rt-pcr analysis. total rna was isolated from ibv-infected cells using trizol reagent® (invitrogen) according to the manufacturer's instructions. the concentration of the total rna extracted was quantified using a nanodrop™ 1000 spectrophotometer (nanodrop technologies, inc., thermo fisher scientific, usa). reverse transcription (rt) was performed with oligo(dt)18 or specific primer using reverse transcriptase (promega) according to the manufacturer's instructions. for the detection of viral positive-stranded subgenomic (sg)rna, oligo(dt)18 was used for cdna synthesis; for negative-stranded sgrna, ibv-5′end-f (5′-1 acttaagatagatattaatatata) was used. ibv-5′end-f and ibv-3′end-r (5′-27608 tgctctaactctatactagc) were used for pcr. ibv-infected cells at different time point post-infection were washed with pbs and lysed with 2 ×sds loading buffer containing 100 mm dithiothreitol (dtt), boiled at 100°c for 5 min, and clarified. the proteins were separated by sds-page and transferred to a polyvinylidene difluoride (pvdf) membrane (stratagene). the membrane was blocked overnight at 4°c or for 2 h at room temperature in blocking buffer (5% fat-free milk powder in phosphate-buffered saline (pbs) buffer containing 0.1% tween 20 (pbst)) and then was incubated with diluted primary antibodies in blocking buffer for 2 h at room temperature. after the membrane was washed three times with pbst, it was incubated with 1:2000 diluted anti-mouse or anti-rabbit igg antibodies conjugated with horseradish peroxidase (dako) in blocking buffer for 1 h at room temperature. after the membrane was washed three times with pbst, the polypeptides were detected with a chemiluminescence detection kit (ecl kit; amersham biosciences) according to the manufacturer's instructions. the films were exposed and developed. quantitative real-time pcr (qpcr) was used to validate gene expression changes in infected cells. total rna (2 µg) was reversedly transcribed to cdna, and the resulting cdna was subjected to qpcr using power sybr green pcr master mix (applied biosystems). amplification and data collection were performed as manufacturer's instruction (applied biosystems 7500 real-time pcr system). the relative gene expression levels were measured using gapdh as an internal reference, and normalized to gene expression in mock-infected cells (relative expression = 1). all experiments were performed in triplicate. in the virus-infected cells, ibv produces six mrna species, including the genome-length mrna1 and five subgenomic mrnas via a discontinuous transcription mechanism. this mechanism is mediated by transcription-regulating sequences (trss) in the 3′ end of the leader (trs-l) and the preceding each mrna body (trs-b). trss comprise a conserved core sequence (cs) [cu(u/g)aacaa] in ibv. each mrna possesses a leader sequence of 64 nucleotides derived from the 5′-end of the genome. in general, six mrnas are readily detected by northern blotting in the infected cells. when probing the viral positive-or negative-stranded mrna6 by rt-pcr, an unexpected pcr product was concurrently amplified in the ibv-infected vero cells as well as in chicken embryos (fig. 1) . it is smaller than mrna6. subsequently, this product was cloned and sequenced. sequence analysis indicated that it consists of 563 nucleotides excluding a poly(a) tail, is mainly derived from the 3′-utr (from nucleotides 27104-27608) of ibv genome, and contains a 63-nt-long of terminal leader sequence derived from the 5′ end of the viral genome (fig. 2) . the results showed the generation of a novel sgrna in the ibvinfected cells. this sgrna may be overlooked in previous studies because of its smaller size and lower level of transcription. due to lack of start codon aug, this sgrna is designated as a noncoding rna (ncrna). insertion of an orf encoding egfp (carrying its own start codon) between 27149 and 27150 nt of ibv 3′-utr allowed the recovery of recombinant virus. rt-pcr analysis showed the presence of the egfp-containing sgrna and fluorescence confirmed egfp expression in virus-infected cells (fig. 3) , implying that the ncrna is an mrna. however, this virus was unstable and a deletion of 377 nucleotide acids (from 81 to 457 nt) of egep sequences was detected in passage 3 in vero cells (data not shown). further sequence comparison revealed that a sequence motif (uaaca), located in the junction between the 5′ end leader sequence and the 3′-utr of ncrna, is shared by 5′-terminus of 3′-utr and the core sequence (cuuaacaa) within ibv trs (fig. 2) . these results prompt us to speculate that this ncrna, like the other viral sgrna, may be generated by a discontinuous transcription mechanism via template switch mediated by a noncanonical core sequence (uaaca). to confirm this hypothesis, we analyzed the effect of several mutations on ncrna generation by mutagenesis and reverse genetics. as shown in fig. 4 , compared to wild-type ribv, single mutation u27104a and a27108u had no or minor effect on ncrna synthesis; a27105u resulted in a significant reduction in the ncrna generation, while mutation c27107g, a27106u and deletion of five nucleotides (δ27104-08) completely abolished the synthesis of both positive-stranded and negative-stranded ncrna (fig. 4) , suggesting that at least three fig. 1 . detection of a novel sgrna in virus-infected vero cells and chicken embryo by rt-pcr. total rna was extracted from the ribv-infected vero cells and chicken embryo. cdna was synthesized by reverse transcription using oligo (dt)18 as a primer. pcr was performed using primers ibv-5′end-f and ibv-3′end-r. the amplicons were analyzed on 1% agarose gel electrophoresis. fig. 2 . sequence of the junction between ibv 3′-utr and the leader at 5′-end of viral genome, indicating the formation of a novel sgrna mediated by 27104 uaaca 27108 . vero cells were infected with ribv at an moi of 1 pfu/cell. total rna extracted from the infected cells was used for reverse transcription using primer oligo(dt)18. pcr was carried out using primers ibv-5′end-f and ibv-3′end-r. pcr product was cloned and sequenced. nucleotides (a27105/a27106/c27107) are involved in the effective ncrna generation. the results confirmed previous report that ibv can synthesize sgrna via template switch mediated by a noncanonical core sequence (bentley et al., 2013) notably, the mutant virus carrying four mutations (a27100u/a27111u/g27113c/g27114c) was also unable to produce ncrna (fig. 4) , suggesting these nucleotides are required for ncrna production. because the sequence motif (a27100/a27111/ g27113/g27114) located downstream of the truncated cs (uaaca) also exists downstream of the cs (cuuaacaa) in the leader trs (table 1 , marked in bold), our result prove that the sequences downstream of the cs exert a stronger influence on coronavirus sgrna synthesis (sola et al., 2005) . taken together, the results confirm that the ncrna generation involves a discontinuous transcription process in which the 5′ leader sequence and 3′-utr are fused through the transcription-regulating sequences in the 3′ end of the leader and in the 5′ end of the 3′-utr. the mutant virus c27107g was selected for further experiments because it is not capable of producing ncrna and carries only one nucleotide change, compared to ribv. plaque assay on vero cells showed both viruses did not display major differences in plaque morphology and in virus growth properties (fig. 5a) . moreover, real-time rt-pcr and western blot were performed to analyze the expression of s and n genes at different time points post-infection, respectively. similarly, no significant differences were detected at both rna level and protein level (fig. 5b, c) . the results suggest that ncrna has little effects on viral replication and viral cytopathicity in vero cells. in this report, we have identified an ncrna in ibv-infected cells and revealed that this ncrna is generated via discontinuous transcription mechanism by reverse genetics and mutagenesis for the first time. although the ncrna has no or little effect on viral replication and pathogenesis in vero cells, roles in ibv pathogenesis in chicken and virus-host interplay are unknown, needing to further study. coronaviruses employ a discontinuous transcription mechanism to synthesize subgenomic mrnas through template switch taking place in the transcription-regulating sequences in the 3′ end of the leader (trs-l) and in the intergenic region preceding each mrna body (trs-b) during negative rna synthesis (baric and yount, 2000; zúñiga et al., 2004; masters, 2006; sawicki et al., 2007) . the five sgrnas (mrnas 2-6) of ibv, which are readily detected by northern blotting, possess the canonical core sequence (5′-cu(u/g)aacaa-3′) ( table 1 ). it has been reported that coronaviruses, such as severe acute respiratory syndrome coronavirus, mouse hepatitis virus and ibv, can use noncanonical cs to synthesize sgrna via discontinuous transcription mechanism (zhang and liu, 2000; hussain et al., 2005; bentley et al., 2013) . in this report, we have identified the existence of a novel sgrna derived mainly from the 3′ utr of ibv in the ibv-infected cells (fig. 1) . the synthesis of this sgrna is mediated by a truncated cs ( 27104 uaaca 27108 ) identical to nucleotides of 3-7 of ibv cs. among which at least three nucleotides (a27105/a27106/c27107) are involved in the effective sgrna generation (figs. 2 and 4) , providing more evidence for the use of noncanonical transcriptional signals in synthesis of coronavirus sgrnas. in addition, we verified that the sequence motif (a27100/a27111/g27113/g27114) located downstream of the truncated cs (uaaca) is necessary for ncrna generation (fig. 4) , reinforcing the importance of nucleotides immediately flanking cs in coronavirus sgrna synthesis (sola et al., 2005) . interestingly, when blast search in genbank, we find that the sequence motif (uaaca) is conserved at 5′ end of 3′ utr of ibv strain beaudette and its derivants, arkansas dp1, and turkey coronavirus but not for strain m41, h120, h52, a2, and several field isolates in china. therefore, whether these viruses can produce ncrna and how ncrna affects the viral pathogenecity remain to be determined. fig. 5 . effect of ncrna on plaque morphology and viral replication. a. plaque assay. vero cells in 6-well plates were infected with a dilution series of ribv and mutant virus ibv-c27107g for 1 h, respectively. after washing twice with medium, cells were overlaid with 0.4% agar in fbs-free dmem, incubated at 37 ℃ for 3-4 days, fixed with 10% formaldehyde, and stained with 0.2% crystal violet. b. quantitative analysis of sgrna synthesis of n and s. total rna (2 μg) extracted from the vero cells infected with ribv and ibv-c27107g at an moi of 0.5 pfu/cell at 8 and 20 h post-infection was used for reverse transcription using primer oligo(dt)18 respectively. amplification and data collection were performed as manufacturer's instruction (applied biosystems 7500 real-time pcr system). the relative gene expression levels were measured using gapdh as an internal reference, and normalized to gene expression in mock-infected cells (relative expression = 1). all experiments were performed in triplicate. c. western blotting. vero cells were infected with ribv and ibv-c27107g at an moi of 0.5 pfu/cell at the indicated time point. cell lysates from the indicated time point were prepared for western blotting using antibody against s or n protein. zika virus produces noncoding rnas using a multi-pseudoknot structure that confounds a cellular exonuclease nucleotide sequence of hsur 6 and hsur 7, two small rnas of herpesvirus saimiri an atlas of the epstein-barr virus transcriptome and epigenome reveals host-virus regulatory interactions subgenomic negative-strand rna function during mouse hepatitis virus infection identification of a noncanonically transcribed subgenomic mrna of infectious bronchitis virus and other gammacoronaviruses g3bp1 g3bp2 and caprin1 are required for translation of interferon stimulated mrnas and are targeted by a dengue virus non-coding rna hsv lat and neuronal survival a mammalian herpesvirus uses noncanonical expression and processing mechanisms to generate viral micrornas a viral nuclear noncoding rna binds re-localized poly(a) binding protein and is required for late kshv gene expression murine gammaherpesvirus 68 encodes trna-like sequences which are expressed during latency aedes aegypti uses rna interference in defense against sindbis virus infection coronaviruses in poultry and other birds down-regulation of a host microrna by a herpesvirus saimiri noncoding rna mature and functional viral mirnas transcribed from novel rna polymerase iii promoters t-cell transformation and oncogenesis by gamma2-herpesviruses an arginine-to-proline mutation in a domain with undefined functions within the helicase protein (nsp13) is lethal to the coronavirus infectious bronchitis virus in cultured cells identification of novel subgenomic rnas and noncanonical transcription initiation signals of severe acute respiratory syndrome coronavirus rna virus microrna that mimics a b-cell oncomir human cytomegalovirus 5-kilobase immediate-early rna is a stable intron murine cytomegalovirus encodes a stable intron that facilitates persistent replication in the mouse an ebv noncoding rna base pairs with nascent rna to enable pax5 transcription factor binding to viral dna the molecular biology of coronaviruses adenovirus virus-associated rna and translation control noncoding rnas of plant viruses and viroids: sponges of host translation and rna interference machinery noncoding rnas of plant viruses and viroids: sponges of host translation and rna interference machinery a noncoding rna produced by arthropod-borne flaviviruses inhibits the cellular exoribonuclease xrn1 and alters host mrna stability a highly structured, nuclease-resistant, noncoding rna produced by flaviviruses is required for pathogenicity rna of low molecular weight in kb cells infected with adenovirus type 2 a contemporary view of coronavirus transcription noncoding flavivirus rna displays rna interference suppressor activity in insect and mammalian cells west nile virus noncoding subgenomic rna contributes to viral evasion of the type i interferon-mediated antiviral response towards construction of viral vectors based on avian coronavirus infectious bronchitis virus for gene delivery and vaccine development ebv noncoding rnas role of nucleotides immediately flanking the transcription-regulating sequence core in coronavirus subgenomic mrna synthesis noncoding rnps of viral origin decoding human cytomegalovirus polyadenylylated nuclear rna encoded by kaposi sarcoma-associated herpesvirus parvovirus expresses a small noncoding rna that plays an essential role in virus replication dissection of the adenoviral va rnai central domain structure reveals minimum requirements for rna mediated inhibition of pkr identification of a noncanonical signal for transcription of a novel subgenomic mrna of mouse hepatitis virus: implication for the mechanism of coronavirus rna transcription characterization of ribonucleoprotein complexes containing an abundant polyadenylated nuclear rna encoded by kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) sequence motifs involved in the regulation of discontinuous coronavirus subgenomic rna synthesis this work was financially supported by grants from the national natural science foundation of china (no. 31572490) and the department of science and technology, hubei provincial people's government, china (no. 2013bhe020) key: cord-311255-zaa8i9vh authors: kim, youngnam; lee, changhee title: porcine epidemic diarrhea virus induces caspase-independent apoptosis through activation of mitochondrial apoptosis-inducing factor date: 2014-07-31 journal: virology doi: 10.1016/j.virol.2014.04.040 sha: doc_id: 311255 cord_uid: zaa8i9vh abstract the present study sought to investigate whether porcine epidemic diarrhea virus (pedv) induces apoptosis and to elucidate the mechanisms associated with apoptotic cell death after pedv infection. pedv-infected cells showed evidence of apoptosis in vitro and in vivo. however, experimental data indicated that the caspase cascade is not involved in pedv-induced apoptotic cell death. interestingly, mitochondrial apoptosis-inducing factor (aif) was found to translocate to the nucleus during pedv infection, and aif relocalization was completely abrogated by the presence of cyclosporin a (csa), an inhibitor of cyclophilin d (cypd) that is an essential component of the mitochondrial permeabilization transition pore (mptp) complex. csa treatment resulted in significant inhibition of pedv-triggered apoptosis and suppressed pedv replication. furthermore, direct inhibition of aif strongly impaired pedv infection and virus-induced apoptosis. altogether, our results indicate that a caspase-independent mitochondrial aif-mediated pathway plays a central role in pedv-induced apoptosis to facilitate viral replication and pathogenesis. porcine epidemic diarrhea (ped) is a devastating disease of swine that was initially recognized in the united kingdom in 1971 and that has then spread to swine-producing european countries (oldham, 1972; pensaert et al., 1981) . since the 1990s, the disease has become rare in europe and is more often associated with postweaning diarrhea in adult pigs (saif et al., 2012) . ped epidemics were first reported in asia in 1982 and since then, ped has continued to threaten swine health causing substantial financial losses in the asian swine industry (chen et al., 2008; kweon et al., 1993; li et al., 2012; puranaveja et al., 2009; takahashi et al., 1983) . in 2013, ped outbreaks suddenly appeared in the united states and have swept the national pork industry with high mortality in newborn piglets, posing significant economic and public health concerns (mole, 2013; stevenson et al., 2013) . in 1978, the etiological agent of this disease was identified as a coronavirus, ped virus (pedv), which belongs to the genus alphacoronavirus within the family coronaviridae of the order nidovirales (pensaert and de bouck, 1978; saif et al., 2012) . pedv is a large, enveloped virus possessing a single-stranded positivesense rna genome of approximately 28 kb with a 5 0 cap and a 3 0 polyadenylated tail (pensaert and de bouck, 1978; saif et al., 2012) . the pedv genome is composed of a 5 0 untranslated region (utr), at least 7 open reading frames (orf1a, orf1b, and orf2 through 6), and a 3 0 utr (kocherhans et al., 2001) . the two large orf1a and 1b cover the 5 0 two-thirds of the genome and encode the nonstructural replicase genes. the remaining orfs in the 3 0 terminal region code for four major structural proteins, the 150-220 kda glycosylated spike (s) protein, 20-30 kda membrane (m) protein, 7 kda envelop (e) protein, and 58 kda nucleocapsid (n) protein (duarte et al., 1994; saif et al., 2012) . pedv is a pathogenic enterocyte-tropic coronavirus of swine, and its infection is marked by acute enteritis with mortality approaching 100% in suckling piglets up to one week of age (debouck and pensaert, 1980; saif et al., 2012) . the virus is transmitted via the fecal-oral route and the virus replicates in the cytoplasm of villous epithelial cells throughout the small intestine. pedv infection results in severe villous atrophy, leading to a reduction in the villous height:crypt depth ratio from the normal 7:1 to 3:1. this interrupts digestion and absorption of nutrients and electrolytes, thereby causing malabsorptive watery diarrhea followed by serious and fatal dehydration in piglets (saif et al., 2012) . in addition, pedv can be propagated in vero (african green monkey kidney) cells in vitro, where the cytopathic effects contents lists available at sciencedirect journal homepage: www.elsevier.com/locate/yviro (cpes) consist of vacuolation and formation of syncytia with up to 100 nuclei (hofmann and wyler, 1988) . however, the mechanisms inducing cell death in pedv-infected target cells, both in vitro and in vivo, remain poorly understood. apoptosis is a tightly regulated mechanism of cell death that is triggered by various extracellular (extrinsic) or intracellular (intrinsic) stimuli and modulated by anti-and pro-apoptotic cellular factors (galluzzi et al., 2012) . cells undergoing apoptosis are accompanied by characteristic morphological changes, including rounding-up of the cell, chromatin condensation, nuclear fragmentation, and plasma membrane blebbing (kroemer et al., 2009) . apoptosis is considered to be a host innate defense mechanism that disrupts viral replication by eliminating virus-infected cells. therefore, viruses have developed strategies to avoid apoptosis, which, in turn, prevent premature cell death and thus maximize progeny viral production (thomson, 2001) . however, many viruses have the ability to actively induce apoptosis as a response to viral replication, thereby facilitating the release and dissemination of viral progeny to neighboring cells. this pro-apoptotic event is one of the cytolytic properties of viral infections causing cpe in vitro or/and plays a pathogenic role contributing to cell damage, tissue injury, and disease severity in vivo dediego et al., 2011; eleouet et al., 1998; favreau et al., 2012; lan et al., 2013; lee and kleiboeker, 2007; st-louis and archambault, 2007; sarmento et al., 2006; suzuki et al., 2008) . at present, it is unknown whether pedv triggers apoptotic cell death, and if so, whether virusinduced apoptosis aids or worsens viral replication and pathogenicity. therefore, in this study, we aimed to determine if pedv induces apoptosis following infection in vitro and in vivo and to define the specific pathways involved in apoptotic death of virus-infected cells. apoptotic cell death occurred after in vitro and in vivo infections of pedv, suggesting that pedv-induced apoptosis may play an important role in cytotoxicity and pathogenesis. treatment with a pancaspase inhibitor failed to suppress pedv-induced apoptosis and pedv infection. however, pedv replication and virus-triggered apoptosis were significantly impaired by inhibitors of cyclophilin d (cypd) and apoptosis-inducing factor (aif). these data indicate that pedv promotes aif-mediated and caspase-independent apoptosis, which is essential for the replication of pedv. overall, our study will improve the understanding of the correlation between pedvinduced apoptosis and its pathogenic mechanisms and show the potential of anti-apoptotic therapeutic strategies for combating pedv infection. to investigate whether apoptotic cell death was triggered by pedv, virus-infected cells were first examined for the presence of dna fragmentation, which is a hallmark of cells undergoing apoptosis. vero cells were infected with pedv, and cpe was monitored after infection. light microscopy of the pedv-infected vero cells indicated that typical cpes characterized by vacuolation and syncytia formation were visible starting at 24 hpi and became prominent by 48 hpi (fig. 1a , upper panels). to detect intracellular fragmented dna, cellular dna from mock-or virus-infected cells was harvested and subjected to a dna laddering assay. consistent with the cpe observation results, intracellular dna fragmentation appeared at 24 hpi, and the dna ladder pattern was apparent at 48 hpi (fig. 1a, lower panel) . this characteristic of apoptosis was further confirmed using a tunel assay. as shown in fig. 1b , compared to mock-infected cells, tunel-labeled cells were only observed within virus-infected cells. the process of pedv-induced apoptosis was then assessed using annexin v/pi flow cytometry. virus-or mock-infected cells were stained with annexin v and pi and then examined using facs flow cytometry to quantitatively determine the percentage of viable, apoptotic, and dead cells. pedv infection produced a high level of apoptosis (annexin v positive/pi negative) appearing at 6 hpi, and the percentage of early apoptotic cells increased with infection time, reaching a maximum of 87.4% at 48 hpi (fig. 1c ). in addition, we sought to determine whether pedv induces apoptosis in the natural host. to accomplish this, a tunel assay was performed to detect and quantify apoptosis in small intestine sections prepared from piglets experimentally inoculated with pedv. immunofluorescence using n protein-specific mab revealed that abundant viral antigens were primarily present in enterocytes over the entire villi in all areas of the small intestine at 3 dpi and increasingly dominant in the interior of the villi at 5 dpi; the majority of virus-infected cells were tunel-positive (fig. 2) . in contrast, no tunel-positive enterocytes were observed in the tissue specimens prepared from a non-infected animal (fig. s1 ). taken together, these data demonstrate that pedv infection induces apoptosis in vitro and in vivo. caspases, a family of aspartate-specific cysteine proteases, play critical roles in the execution phase of apoptosis of the cell leading to morphological and nuclear changes . in order to elucidate the mechanism and the type of apoptotic cell death, we initially assessed whether the broad-spectrum pancaspase inhibitor, z-vad-fmk, blocks pedv-induced apoptosis. based on the results of the mtt assay, none of the doses of z-vad-fmk tested in the present study resulted in any change in cell viability (data not shown). vero cells were treated with z-vad-fmk at concentrations of 100 μm followed by viral infection. at the indicated time points post-infection, virus-infected and z-vad-fmk-treated cells were analyzed based on facs quantification of annexin v binding. notably, treatment with z-vad-fmk was incapable of protecting cells from pedv-induced apoptosis (fig. 3a) . the percentage of early apoptotic cells in the presence of z-vad-fmk was almost similar to that in vehicle (dmso)-treated cells (second panel) and pedv-infected cells during the course of infection (compared to fig. 1c ). these results indicated that caspases are not associated with pedv-triggered apoptosis. for further confirmation, we tried to determine the activity of caspase-3 upon pedv infection by western blot analysis. caspase-3 is the main executioner caspase activated by the both extrinsic and intrinsic apoptotic pathways causing the morphological features of apoptosis. as shown in fig. 3b , no detectable level of cleaved caspase-3 was observed in pedv-infected cells up to 48 hpi (first panel), whereas the n protein was first detected at 6 hpi, and its production greatly increased thereafter, indicating pedv replication (third panel). on prolonged exposure of the western blot, however, it was apparent that a small amount of activated caspase-3 appears only at 48 hpi (second panel), which seems to be a basal level normally present at late time of infection. we next investigated if the replication of pedv is also affected by the same caspase inhibitor. vero cells were pretreated with z-vad-fmk at concentrations of 10-100 μm or with dmso as a vehicle control for 1 h prior to infection. z-vad-fmk was present during the entire period of infection. viral production was measured by monitoring cpe and confirmed by immunofluorescence at 48 hpi (fig. 3c ). apparent cpe and n-specific staining were equally evident in cells treated with z-vad-fmk when compared to vehicle-treated control cells. furthermore, pedv replication, as quantified by the growth kinetics results, was not reduced by z-vad-fmk treatment, indicating that activation of caspases is irrelevant to pedv replication (fig. 3d ). altogether, our data reveal that caspase cascades are not activated by pedv and their chemical inhibition diminished neither pedv-induced apoptotic cell death nor viral replication, suggesting that caspase activation is dispensable for the process. since caspases appear to be non-essential factors in pedvtriggered apoptosis, we investigated an alternative caspaseindependent apoptotic pathway mediated mainly by aif in relation to apoptotic cell death induced by pedv. aif is a flavoprotein embedded in the outer surface of the mitochondrial inner membrane. following apoptotic stimuli, aif is proteolytically cleaved and truncated aif is released from the mitochondria and translocates to the nucleus, where it causes high-molecular weight dna fragmentation and chromatin condensation independently of caspases (cregan et al., 2002; daugas et al., 2000; galluzzi et al., 2012; joza et al., 2001; lu et al., 2013; yuste et al., 2005) . mitochondria plays a central role in the intrinsic apoptotic pathway by releasing proapoptotic factors, such as aif and cytochrome c (cytc), through the mitochondrial permeability transition pore (mptp), which is permeabilized in response to intracellular stress (galluzzi et al., 2012) . in order to study the potential role of mitochondrial aif in the caspase-independent pathway of pedvinduced apoptosis, thus, we first aimed to determine the subcellular localization of aif in pedv-infected cells using immunofluorescence confocal microscopy. although aif continued to localize broadly in the mitochondria after pedv infection shown by its colocalization with a mitochondrial-specific marker, the majority of cells infected with pedv also displayed a condensed aif staining pattern within the nucleus, and its intensity was for the dna fragmentation assay, dna was extracted from mock-or pedv-infected cells and nucleosomal dna fragmentation of the cells was then analyzed by agarose gel electrophoresis (bottom panel). as a positive control, cells were treated with staurosporine for 24 h to induce apoptosis. lane m represents a 1-kb ladder as a dna molecular-weight size marker. (b) tunel labeling of pedv-infected cells. mock-infected control and pedv-infected cells fixed at 48 hpi were labeled with tunel (green) and sequentially stained with an anti-pedv-n antibody (red). the cells were then counterstained with dapi and photomicrographs of tunel labeling and n protein staining in virus-infected cells was observed using a confocal microscope at 400 â magnification. in a merged image, all tunel-positive cells were localized within the nuclei of corresponding pedv-infected cells. (c) cell death analysis by flow cytometry with dual annexin v-pi cell labeling. pedv-infected cells collected from different time periods were dually labeled with annexin v and pi and analyzed by facs. the lower left quadrants represent intact cells (annexin v negative/pi negative); the lower right quadrants represent early apoptotic cells (annexin v positive/pi negative); the upper right quadrants indicate late apoptotic and/or necrotic cells (annexin v positive/pi negative); the upper left quadrants indicate necrotic cells (annexin v negative/pi positive). the figure is representative of three independent experiments. the graph on the right represents the percentage of each quadrant and the non-significant percentages of annexin v-positive and pi-negative cells were excluded. tissue specimens were collected from the duodenum, jejunum, and ileum of infected animals at the indicated days postinoculation. the formalin-fixed and paraffin-embedded tissue sections were deparaffinized and sequentially labeled with tunel (green) followed by an anti-pedv-n antibody (red). the sections were then counterstained with dapi and examined using a confocal microscope at 200 â magnification. yellow regions indicate merged images, where cells are positive for both tunel and pedv infection. significantly elevated at 48 hpi ( fig. 4a , arrows). this observation was confirmed by western blot analysis of mitochondrial and nuclear extracts. as shown in fig. 4b , an uncleaved 62 kda aif was detected in both extracts during the course of infection, whereas a cleaved form of aif was observed only in nuclear extracts of pedvinfected cells. we then analyzed the translocation of another proapoptotic protein, cytc, which is released to the cytoplasm by mitochondrial outer membrane permeabilization to execute caspase proteolytic cascade-dependent intrinsic apoptosis. in contrast to aif, however, cytc was sustainedly located in the mitochondria of pedv-infected cells, as demonstrated by the colocalization of cytc with mitotracker and the detection of cytc in mitochondrial fractions (fig. 4c and d) . this cytc retention in the mitochondria during pedv infection was concurrent with the caspase-independent apoptotic cell death induced by pedv as described above. consequently, our findings demonstrate that pedv infection specifically triggers mitochondrial-nuclear translocation of aif but not the release of mitochondrial cytc. cyclophilin d (cypd) is localized to the mitochondrial membrane matrix and is a main component in the process of mptp opening (baines et al., 2005; schinzel et al., 2005) . therefore, we aimed to determine whether inhibition of cypd could prevent apoptotic cell death induced by pedv. cells were treated with csa, a chemical inhibitor of cypd, to inhibit mptp formation upon pedv infection. the dna laddering assay indicated that the presence of csa in cells infected with pedv completely abolished intracellular dna fragmentation (fig. 5a, lane 4) , while cells infected with pedv alone or combined with z-vad-fmk clearly displayed a dna laddering pattern (fig. 5a, lanes 2 and 3) . upon csa treatment, pedv-induced apoptosis was then quantitatively evaluated by annexin v/pi flow cytometry. as shown in fig. 5b , treatment with csa notably decreased the percentage of early apoptotic cells induced by pedv (second panel), ranging from 19.1% to 29.9% during the course of pedv infection (compared to fig. 3a ). these results demonstrate that inhibition of cypd efficiently suppresses apoptotic cell death induced by pedv, indicating that pedv infection triggers the mitochondrial apoptotic pathway. to examine the effect of csa on pedv replication, cells were pretreated with csa at concentrations of 1-10 μm, and csa was present during the entire period of infection. pedv replication was determined by monitoring virus-specific cpe and verified by immunofluorescence at 48 hpi (fig. 6a) . in contrast to vehicletreated control cells, csa had a strong inhibitory effect on pedv propagation by dramatically decreasing virus-induced cpe and viral gene expression even at the lowest concentration used. the number of cells expressing viral antigen, as quantified by n protein staining results, was also reduced during csa treatment, resulting in almost complete inhibition in response to 10 μm (fig. 6b ). to further assess the antiviral activity of csa against pedv replication, viral yield was determined during treatment with csa. upon infection, viral supernatants were collected at 48 hpi, and viral titers were measured. as fig. 6c shows, the presence of csa significantly reduced the release of viral progeny in a dosedependent manner. the peak viral titer was determined to be 10 6.68 pfu/ml in the vehicle-treated control, whereas the addition of 10 μm csa declined the titer of pedv to 10 1.69 pfu/ml (an almost 5-log reduction compared to the control). the growth kinetics study further demonstrated that the overall process of pedv replication was significantly delayed when cells were treated with csa (fig. 6d ). in addition, we investigated whether viral protein translation and viral rna synthesis were affected by csa. the expression level of the pedv n protein in the presence or absence of csa was evaluated at 48 hpi by western blot analysis. densitometric analysis of the western blot revealed that intracellular expression of the viral protein was entirely prevented by csa at a concentration of 10 μm (fig. 7a) . similarly, levels of both genomic rna and sg mrna were greatly diminished in a dosedependent manner and their synthesis was fully abolished at the highest csa concentration (fig. 7b ). taken together, our data strongly indicate that csa proficiently suppresses the replication of pedv, which is correlated with inhibition of pedv-induced apoptosis by csa. the present study shows that mitochondrial aif is translocated to the nucleus following pedv infection. because cypd is involved in alteration of mitochondrial membrane permeability, followed by release of proapoptotic factors, it is conceivable that inhibition of cypd would block aif nuclear translocation induced by pedv, leading to impaired pedv-induced apoptosis and viral replication. we therefore determined whether csa specifically affects the nuclear translocation of aif mediated by pedv infection. immunofluorescence confocal microscopy results demonstrated that aif nuclear translocation in pedv-infected cells was distinctly hampered following cypd inhibition by csa, exhibiting a cytoplasmic distribution that colocalized with the mitochondrial marker during the entire course of pedv infection (fig. 8a) . mitochondrial retention of aif in the presence of csa was confirmed by a cell fractionation assay. as expected, the level of truncated aif in the nuclear fractions was barely detectable in the presence of csa, demonstrating that inhibition of cypd failed to cause the relocalization of aif from the mitochondria to the nucleus (fig. 8b) . altogether, these results suggest that upon pedv infection, cypddependent mptp directly modulates apoptotic cell death by relocating aif to the nucleus, resulting in successful viral replication. considering that aif is a key proapoptotic protein released from the mitochondria in response to pedv infection to trigger apoptosis, we sought to determine whether chemical inhibition of aif could control pedv-induced apoptosis and viral replication. to accomplish this, we first used an aif inhibitor, n-phmi, that prevents aif-induced dna fragmentation (susin et al., 1996; wang et al., 2007) . treatment with n-phmi resulted in substantial reduction of the percentage of apoptotic cells during pedv infection (fig. 9) . addition of n-phmi retained an average cell viability of over 80% during the entire time of pedv infection, demonstrating that inhibition of aif robustly represses apoptosis induced by pedv. to further confirm the importance of aif in apoptosis triggered by pedv infection, we examined whether this drug alters the nuclear relocalization of aif following pedv infection by confocal microscopy analysis. as depicted in fig. 10a , n-phmi treatment completely abrogated aif nuclear translocation induced by pedv, leading to mitochondrial accumulation of aif. western blot analysis provided further support by showing the absence of truncated aif in the nuclear fractions of pedv-infected cells in the presence of n-phmi (fig. 10b) . these results strongly indicated that nuclear targeting of aif is required for pedv-induced apoptotic cell death. finally, to determine whether aif function is necessary for pedv replication, vero cells were treated with n-phmi for 1 h prior to infection, and the drug was allowed to remain during infection and subsequent incubation. at 48 hpi, the level of pedv replication was measured indirectly, as viral antigen production, by quantifying cells expressing the n protein through ifa (fig. 11a) . treatment of cells with 5 μm n-phmi resulted in a 490% decrease in pedv production compared to the untreated vehicle control. to further examine the inhibitory effect of n-phmi on pedv infection, virus production was measured using viral supernatants propagated for 48 h under each condition. as shown in fig. 11b , inhibition of aif by n-phmi markedly decreased the titer of pedv in a dosedependent manner, and the lowest viral titer was determined to be 10 2.61 pfu/ml at the concentration of 5 μm (an almost 4-log reduction compared to the control). furthermore, the replication process of pedv severely interfered with the presence of n-phmi (fig. 11c) . taken together, our data show that aif is an essential host determinant in a caspase-independent apoptosis process activated by pedv, which is indispensable for pedv replication. apoptosis is a tightly controlled multistep process of cell death that occurs in response to a wide range of stimuli, including viral infections. viruses possess various mechanisms to inhibit apoptosis that allow them to evade the innate immune defenses, which restrict viral infection by eliminating infected cells through the interactions at different stages of the apoptotic pathway. however, some viruses induce apoptosis to facilitate the release and dissemination of viral progeny for further invasion, which are important biological parts in viral pathogenesis and disease processes that promote cell death and tissue injury. although various viruses are known to modulate apoptosis as a critical armament to complete their replication cycle, it remains so far even undetermined whether pedv induces apoptosis. in the present study, we characterized pedv-induced cytotoxicity and demonstrated that apoptotic cell death is triggered in pedv-infected cells in vitro, displaying biochemical features of apoptosis including oligonucleosomal dna fragmentation and ps exposure. this finding strongly suggests that the cytopathology of pedv infection, represented by vacuolation and syncytia formation in vitro, is associated with the apoptotic process. the capability of pedv to induce apoptosis was also verified by an in vivo study using the small intestinal tissues of piglets experimentally inoculated with virulent pedv. the results showed that the great part of the villus enterocytes in all segments of the small intestine infected with pedv experienced apoptotic cell death at 3 dpi, and thereafter, cells undergoing apoptosis were more evident inside the small intestine (fig. 2) . since a specific hallmark of pedv infection in neonatal piglets is the severe destruction of the villi in the small intestine causing lethal diarrhea and dehydration, pedv-induced massive apoptosis in vivo appears to be one of the viral pathogenic mechanisms to destroy the target enterocytes leading to villus atrophy or vacuolation. the morphological characteristics of apoptosis are typically caused by the sequential activation of caspases, which are normally present in mammalian cells as inactive precursors . upstream initiator caspases are first activated by both extrinsic and intrinsic apoptotic pathways and subsequently, are responsible for processing and activation of downstream corresponding effector caspases to mediate apoptosis galluzzi et al., 2012) . to understand the molecular mechanisms underlying apoptosis caused by pedv, we focused on the potential involvement of caspases in apoptotic cell death upon pedv infection. in the present study, caspase-3, the primary effector caspase, was not proteolytically cleaved to trigger the execution phase of apoptosis during the course of pedv infection, although its inactive precursor was continuously present in cells infected with pedv. however, our western blot data, after long exposure, showed only slight levels of caspase-3 activation at 48 hpi (fig. 3b, second panel) . this observation ruled out the possibility that the absence of active caspase-3 was caused by problems with the antibody used or by technical issues. likewise, chemical suppression of caspase activation was found to be absolutely incapable of abrogating pedv-induced apoptosis and viral replication. these results strongly indicate that the failure of the pan-caspase inhibitor to protect cell survival is likely a consequence of caspase-independent cell death, which is indispensable for the replication of pedv. our data here reveal that caspases are irrespective of both pedv-induced apoptosis and viral replication. thus, we sought to investigate other pro-apoptotic factors that could be involved in pedv-induced apoptosis, independent of caspases. in response to various intracellular stress conditions, both pro-and antiapoptotic signals converge toward a mitochondrion-centered control mechanism to trigger intrinsic apoptosis. under this circumstance, mitochondrial outer membrane permeabilization (momp) can begin directly at the outer mitochondrial membrane due to the pore-forming activity of the pro-apoptotic bax subfamily that translocate from the cytoplasm to the mitochondria or can result from the mitochondrial permeability transition at the inner mitochondria membrane caused by the opening of the mptp (brenner and grimm, 2006; galluzzi et al., 2012; tait and green, 2013) . this phenomenon permits the release of several proteins normally retained within the mitochondrial intermembrane space to the cytosol (galluzzi et al., 2012) . among these, cytosolic cytc participates with other pro-apoptotic factors in the formation of the apoptosome, which triggers the caspase-dependent proteolytic cascade (galluzzi et al., 2012; li et al., 1997) , whereas aif, a ubiquitously expressed flavoprotein, critically functions in caspase-independent apoptosis by relocating to the nucleus and promoting large-scale dna fragmentation and cell death (galluzzi et al., 2012; joza et al., 2001; susin et al., 1999) . we demonstrate here that the translocation of mitochondrial aif to the nucleus was actively manifested during pedv infection. conversely, displacement of cytc from the mitochondria to the cytoplasm was completely absent in pedv-infected cells. this result further supports the hypothesis that pedv triggers apoptosis in a caspase-independent manner. the association of pedv-induced apoptosis with aif translocation to the cytosol, without the release of cytc followed by caspase activation, indicates that aif is the sole pro-apoptotic factor released via altered mitochondrial membrane permeability and primarily acts directly to execute apoptotic cell death in pedv infection. the unique relocation of aif, but not cytc, leads us to speculate that momp may be regulated by the opening process following the blot was also reacted with anti-β-actin antibody to verify equal protein loading. pedv n protein expression was quantitatively analyzed by densitometry in terms of the relative density value to the β-actin gene, and csa-treated sample results were compared to dmso-control results. (b) total cellular rna was extracted at 48 hpi, and strand-specific viral genomic rnas (black bars) and sg mrnas (white bars) of pedv were amplified by quantitative real-time rt-pcr. viral positive-sense genomic rna and sg mrna levels were normalized to monkey gapdh mrna, and relative quantities (rq) of mrna accumulation were evaluated. csa-treated sample results were compared with dmso-treated results. values are representative of the mean from three independent experiments and error bars denote standard deviations. *, p ¼0.001 to 0.05; †, po 0.001. mptp formation during pedv infection, in which cypd functions as a major inducing factor to constitute the mptp complex in the mitochondrial membrane. the data clearly show that inhibition of cypd by csa blocked apoptosis induced by pedv and was accompanied by 70-80% cell survival after pedv infection, comparable to mock-infected cells. furthermore, csa could exert the antiviral activity via potent inhibition of multiple steps of the pedv life cycle, including viral translation, rna synthesis, and spread of viral progeny. notably, chemical suppression of cypd impaired aif nuclear translocation, leading to absolute mitochondrial retention of aif. thus, cypd-mediated mptp modulation plays a pivotal role in caspase-independent apoptotic cell death by controlling aif translocation upon pedv infection. numerous putative cellular proteins are known to bind with cypd to promote mptp formation. bax is one such factor, which has been shown to be displaced toward the mitochondria and to interact subsequently with cypd to facilitate the formation of pores in the mitochondrial membrane in response to apoptotic stimuli, including viral infection (favreau et al., 2012; kumarswamy and chandna, 2009 ). although it is unclear whether bax is directly involved in pedv-induced apoptosis, this mechanism is less plausible because pedv infection was irrelevant to mitochondrial cytc release, which is mainly mediated by mitochondrial translocation of bax. similarly, direct inhibition of aif with a chemical inhibitor completely confined aif protein in the mitochondria without nuclear localization, and robustly diminished pedvinduced apoptotic cell death and pedv replication. the inhibitory activity of n-phmi on pedv infection was less efficient than that of csa, although treatment with n-phmi also exhibited significant suppression of pedv replication in its own way. this difference can be attributed to the wide-spectrum antiviral effect of csa via multiple simultaneous mechanisms; csa is known to inhibit the replication of several rna viruses, including nidoviruses, by affecting the function of various members of the cellular cyclophilin protein family (de wilde et al., 2013; kambara et al., 2011; nakagawa et al., 2004; pfefferle et al., 2011; qing et al., 2009; ) . moreover, it was recently demonstrated that csa fully blocks arterivirus replication by suppressing viral rna synthesis (wilde et al., 2013) . we also showed here that treatment of cells with csa dramatically reduces synthesis of both genomic rna and sg mrna pedv-specific cpes were observed daily and were photographed at 48 hpi using an inverted microscope at a magnification of 200 â (first panels). at 48 hpi, virus-infected cells were subjected to ifa with anti-pedv n antibody (second panels) followed by dapi counterstaining (third panels) and examined using a fluorescent microscope at 200 â magnification. viral production in the presence of n-phmi was assessed exactly as described in the legend to fig. 6b. (b) progeny virus release in the presence of n-phmi. vero cells were pretreated with dmso or n-phmi for 1 h and were mock-or pedv-infected (moi of 0.1). at 48 hpi, the virus supernatants were collected and viral titers were measured. (c) growth kinetics of pedv upon n-phmi treatment. at the indicated time points post-infection, culture supernatants were harvested and viral titers were determined. results are expressed as the mean values from three independent experiments and error bars represent standard deviations. †, p o 0.001. (fig. 7b) . therefore, csa appears to synergistically elicit its antiviral activity on the replication of pedv through inhibition of apoptotic cell death and interference with viral rna synthesis. in summary, our findings described here reveal that pedv induces apoptotic cell death, which represents a cytolytic mechanism causing cpes in vitro and contributes to pedv pathogenicity by damaging the villous enterocytes of the small intestine, leading to malabsorptive diarrhea. however, pedv-induced apoptosis was neither inhibited by z-vad-fmk nor accompanied by the activation of caspase cascades and the concomitant release of cytc from the mitochondria, strongly indicating that this cell death event by pedv is caspase-dispensable. on the other hand, we demonstrated the essential roles of cypd and aif in pedv-induced apoptosis; in response to pedv infection, momp occurs through opening of the mptp dependent on cypd, which collapses the mitochondrial potential, resulting in the characteristic release of aif to the nucleus and thereby mediating large-scale dna fragmentation. furthermore, inhibition of either cypd or aif robustly abrogated the life cycle of pedv at subcytotoxic doses. to our knowledge, this is the first report of cypd-involved, aif-indispensable apoptosis, independent of both caspase activation and cytc release following viral infection. although mitochondrial cypd and aif necessarily contribute to pedv-induced apoptosis, the upstream intracellular regulatory signal(s) and the viral protein(s) that are associated with this mitochondrion-centered apoptotic pathway mediated by pedv infection are still unclear and accordingly, identification of such cellular and viral factors would be the next issue to be addressed in future studies. in conclusion, the results presented here indicate that pedv induces apoptotic cell death via a caspaseindependent, aif-mediated pathway that plays a critical role in pedv replication and pathogenesis and suggest that the antiapoptotic approach may be one of appropriate strategies for the development of an anti-pedv compound to combat ped. vero cells were cultured in alpha minimum essential medium (α-mem; invitrogen) with 10% fetal bovine serum (fbs; invitrogen) and antibiotic-antimycotic solutions (100 â ; invitrogen). the cells were maintained at 37 1c in a humidified 5% co 2 incubator. pedv strain sm98-1 was kindly provided by the korean animal and plant quarantine agency and propagated in vero cells as described previously (hofmann and wyler, 1988) . staurosporine was purchased from cayman chemical company and used at a concentration of 1 μg/ml to induce apoptosis. z-vad-fmk (r&d systems), cyclosporin a (csa; cell signaling technologies), and nphenylmaleimide (n-phmi; sigma-aldrich) were dissolved in dimethyl sulfoxide (dmso) to yield a 20 mm, 10 mm, or 1 m stock, respectively. the pedv n protein-specific monoclonal antibody (mab) was a kind gift from sang-geon yeo (kyungpook national university, daegu, korea). antibodies specific for aif, cytc, sp1, and β-actin and horseradish peroxidase (hrp)-conjugated secondary antibodies were obtained from santa cruz biotechnology. the caspase-3, voltage-dependent anion channel (vdac), and αtubulin antibodies were purchased from sigma-aldrich. the cytotoxic effects of reagents on vero cells were analyzed using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) assay (sigma-aldrich) to detect cell viability. briefly, vero cells were grown at 1 â 10 4 cells/well in a 96-well tissue culture plate with z-vad-fmk, csa or n-phmi treatment for 24 h. after 1 day of incubation, 50 μl of mtt solution (1.1 mg/ml) was added to each well, and the samples were incubated for an additional 4 h. the supernatant was then removed from each well, after which 150 μl of dmso was added to dissolve the colored formazan crystals produced by the mtt. the absorbance of the solution was measured at 540 nm using an enzyme-linked immunosorbent assay plate reader. all mtt assays were performed in triplicate. vero cells were grown at 3.5 â 10 5 cells/well in 6-well tissue culture plates for 1 day and then mock infected or infected with pedv at a multiplicity of infection (moi) of 0.1. in addition, cells were pretreated with z-vad-fmk or csa for 1 h followed by pedv infection. at the indicated times, cells were harvested, washed with pbs, and then incubated in a cell lysis buffer (10 mm tris, ph 7.5, 1 mm edta, and 0.2% triton x-100) containing 500 μg/ml protease k for 24 h at 55 1c. the dna was then extracted twice with phenol/chloroform, precipitated with isopropanol, and resuspended in distilled water. next, the purified dna was incubated with 20 μg/ml ribonuclease a for 1 h at 37 1c, electrophoresed on a 1.2% agarose gel containing midori green advanced dna stain (nippon genetics), and photographed. vero cells were grown in 6-well tissue culture plates for 1 day and mock infected or infected with pedv at an moi of 0.1. to examine the effect of each inhibitor on pedv-induced apoptosis, cells were treated with z-vad-fmk, csa, or n-phmi and then infected with pedv. the virus-inoculated cells were further propagated in the presence of z-vad-fmk (100 μm), csa (5 μm), n-phmi (5 μm) or dmso (vehicle control). phosphatidylserine exposure was determined by measuring annexin v binding at the indicated times using an alexa fluor 488 annexin v/dead cell apoptosis kit (invitrogen), according to the manufacturer's protocol. in brief, cells were harvested, washed with cold pbs, and suspended in 100 μl 1 â annexin-binding buffer. the cells were then incubated with alexa fluor 488-conjugated annexin v and propidium iodide (pi) at room temperature (rt) for 15 min in the dark. following the incubation period, 400 μl of annexin-binding buffer was added to each sample, and the samples were mixed gently and kept on ice. the fluorescent signals of annexin v and pi were detected at channels fl-1 and fl-2, respectively, and analyzed using a fluorescence-activated cell sorter (facs) aria iii flow cytometer (bd biosciences). cells negative for pi uptake and positive for annexin v were considered apoptotic. vero cells were grown on microscope coverslips placed in 6well tissue culture plates and mock infected or infected with pedv at a moi of 0.1. the virus-infected cells were fixed at 48 h postinfection (hpi) with 4% paraformaldehyde for 25 min at 4 1c and permeabilized with 0.2% triton x-100 in pbs at rt for 5 min. for tissue labeling, two 4 day-old piglets were orally inoculated with 1 ml of small intestine homogenate containing 10 5 tcid 50 of a pedv field isolate as described previously (oh et al., in press) , and tissue specimens were collected from the duodenum, jejunum, and ileum of piglets at 3 and 5 days post-inoculation (dpi) at necropsy. each small intestinal tissue (o3 mm thick) was fixed with 10% formalin for 24 h at rt and embedded in paraffin according to standard laboratory procedures. the formalin-fixed, paraffin-embedded tissues were cut to slices 5-8 μm thick on a microtome, floated on a 40 1c water bath containing distilled water, and transferred onto glass slides. the tissues were then deparaffinized in xylene for 5 min and washed in decreasing concentrations of ethanol (100%, 95%, 85%, 70%, and 50%), for 3 min each. the sections were fixed with 4% paraformaldehyde for 15 min, permeabilized with 20 μg/ml proteinase k solution at rt for 10 min, and fixed again with 4% paraformaldehyde for 5 min. a terminal deoxynucleotidyl transferase-catalyzed deoxyuridine phosphate-nick end labeling (tunel) assay was performed using a deadend fluorometric tunel system kit (promega) according to the manufacturer's instructions with some modifications. briefly, pedv-infected vero cells and the deparaffinized intestinal tissue sections were rinsed twice with pbs, and the tunel reaction mixture was added, incubated for 60 min at 37 1c, immersed in 2 â ssc buffer for 15 min, and washed 3 times in pbs. tunellabeled cells were subjected to an immunofluorescence assay using n-specific mab and alexa fluor 594-conjugated goat antimouse antibody as described below. the samples were mounted on microscope glass slides in mounting buffer (60% glycerol and 0.1% sodium azide in pbs) and analyzed under a confocal laser scanning microscope (carl zeiss) using an excitation wavelength in the range of 450-500 nm and an emission wavelength in the range of 515-565 nm. vero cells grown on microscope coverslips placed in 6-well tissue culture plates were pretreated with z-vad-fmk, csa or n-phmi for 1 h and mock infected or infected with pedv at a moi of 0.1. the virus-infected cells were subsequently grown in the presence of inhibitors until 48 hpi, fixed with 4% paraformaldehyde for 10 min at rt and permeabilized with 0.2% triton x-100 in pbs at rt for 10 min. the cells were blocked with 1% bovine serum albumin (bsa) in pbs for 30 min at rt and then incubated with nspecific mab for 2 h. after being washed five times in pbs, the cells were incubated for 1 h at rt with a goat anti-mouse secondary antibody conjugated to alexa fluor 488 (invitrogen), followed by counterstaining with 4 0 ,6-diamidino-2-phenylindole (dapi; sigma-aldrich). the coverslips were mounted on microscope glass slides in mounting buffer and cell staining was visualized using a fluorescent leica dm il led microscope (leica). for study of colocalization, mitotracker red cmxros (200 nm; invitrogen) was added to viable vero cells and left for 45 min at 37 1c prior to fixation. the cells were then stained with aif-or cytc-specific antibody as described above, and cell staining was analyzed using a confocal laser scanning microscope (carl zeiss). vero cells were grown in 6-well tissue culture plates for 1 day and were mock infected or infected with pedv at an moi of 0.1. at the indicated times, cells were harvested in 50 μl of lysis buffer (0.5% tritonx-100, 60 mm β-glycerophosphate, 15 mm ρ-nitro phenyl phosphate, 25 mm mops, 15 mm, mgcl 2 , 80 mm nacl, 15 mm egta [ph 7.4], 1 mm sodium orthovanadate, 1 μg/ml e64, 2 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 mm pmsf) and sonicated on ice 5 times for 1 s each. homogenates were lysed for 30 min on ice, and clarified by centrifugation at 15,800 â â â g (eppendorf centrifuge 5415r) for 30 min at 4 1c. for cell fractionation, inhibitor-treated, pedv-infected vero cells were fractionated using a nuclear/cytosol or mitochondria/cytosol fractionation kit (biovision) according to the manufacturer's manuals. the total protein concentrations in the supernatants were determined using a bca protein assay (pierce). equal amounts of total protein were separated on a nupage 4-12% gradient bis-tris gel (invitrogen) under reducing conditions and electrotransferred onto immunobilon-p (millipore). the membranes were subsequently blocked with 3% powdered skim milk (bd biosciences) in tbs (10 mm tris-hcl [ph 8.0], 150 mm nacl) with 0.05% tween-20 (tbst) at 4 1c for 2 h and incubated at 4 1c overnight with the primary antibodies. the blots were then incubated with corresponding secondary hrp-labeled antibodies at a dilution of 1:5000 for 2 h at 4 1c. proteins were visualized using enhanced chemiluminescence (ecl) reagents (ge healthcare) according to the manufacturer 0 s instructions. to quantify viral protein production, band densities of pedv n proteins were quantitatively analyzed using a computer densitometer with the wright cell imaging facility (wcif) version of the imagej software package (http://www.uhnresearch.ca/facilities/wcif/imagej/) based on the density value relative to the β-actin protein. vero cells were pedv infected and treated with z-vad-fmk, csa, n-phmi or dmso as described above. the culture supernatant was collected at different time points (6, 12, 24, 36, and 48 hpi) and stored at -80 1c. the pedv titer was determined by a plaque assay using vero cells and quantified as plaque-forming units (pfu) per ml. vero cells were pedv inoculated with treatment of csa for 1 h at 37 1c. the virus inoculum was subsequently removed and the infected cells were maintained in fresh medium containing csa for 48 h. total rna was extracted from lysates of the infected cells at 48 hpi using trizol reagent (invitrogen) and treated with dnase i (takara) according to the manufacturer 0 s protocols. the concentrations of the extracted rna were measured using a nanovue spectrophotometer (ge healthcare). quantitative real-time rt-pcr was conducted using a thermal cycler dice real time system (takara) with gene-specific primer sets described previously (kim and lee, 2013) . the rna levels of viral genes were normalized to that of mrna for the glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene, and relative quantities (rq) of mrna accumulation were evaluated using the 2 à δδct method. to detect alteration of genomic rna and subgenomic (sg) mrna levels in the presence of csa during pedv infection, the results obtained using csa-treated samples were compared with vehicle-treated results. all statistical analyses were performed using student 0 s t-test, and p-values of less than 0.05 were considered statistically significant. loss of cyclophilin d reveals a critical role for mitochondrial permeability transition in cell death the permeability transition pore complex in cancer cell death molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in china fas-mediated apoptotic signaling in the mouse brain following reovirus infection apoptosis in animal models of virus-induced disease apoptosis-inducing factor is involved in the regulation of caspase-independent neuronal cell death apoptosis-inducing factor (aif): a ubiquitous mitochondrial oxidoreductase involved in apoptosis experimental infection of pigs with a new porcine enteric coronavirus, cv 777 severe acute respiratory syndrome 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mitochondrial apoptosis-inducing factor bcl-2 inhibits the mitochondrial release of an apoptogenic protease equine coronavirus induces apoptosis in cultured cells mitochondrial regulation of cell death. cold spring harbor perspect an outbreak of swine diarrhea of a newtype associated with coronavirus-like particles in japan viruses and apoptosis actinomycin d enhances trail-induced caspase-dependent and -independent apoptosis in sh-sy5y neuroblastoma cells cysteine protease inhibition prevents mitochondrial apoptosis-inducing factor (aif) release supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2014.04.040. key: cord-311774-fhdvcvi0 authors: o'donnell, vivian; pacheco, juan m.; larocco, michael; burrage, tom; jackson, william; rodriguez, luis l.; borca, manuel v.; baxt, barry title: foot-and-mouth disease virus utilizes an autophagic pathway during viral replication date: 2011-02-05 journal: virology doi: 10.1016/j.virol.2010.10.042 sha: doc_id: 311774 cord_uid: fhdvcvi0 foot-and-mouth disease virus (fmdv) is the type species of the aphthovirus genus within the picornaviridae family. infection of cells with positive-strand rna viruses results in a rearrangement of intracellular membranes into viral replication complexes. the origin of these membranes remains unknown; however induction of the cellular process of autophagy is beneficial for the replication of poliovirus, suggesting that it might be advantageous for other picornaviruses. by using confocal microscopy we showed in fmdv-infected cells co-localization of non-structural viral proteins 2b, 2c and 3a with lc3 (an autophagosome marker) and viral structural protein vp1 with atg5 (autophagy-related protein), and lc3 with lamp-1. importantly, treatment of fmdv-infected cell with autophagy inducer rapamycin, increased viral yield, and inhibition of autophagosomal pathway by 3-methyladenine or small-interfering rnas, decreased viral replication. altogether, these studies strongly suggest that autophagy may play an important role during the replication of fmdv. foot-and-mouth disease (fmd) is a highly contagious viral disease of cloven-hoofed animals caused by foot-and-mouth disease virus (fmdv), the type species of the aphthovirus genus within the picornaviridae family (grubman and baxt, 2004) . the positivestranded rna virus contains a genome of approximately 8400 nucleotides (grubman and baxt, 2004) . infection with positive-strand rna viruses results in a number of changes in the cell, including the rearrangement of intracellular membranes into vesicular structures where viral genome replication takes place (armer et al., 2008; bienz, et al., 1973; bienz et al., 1987; egger et al., 2002; monaghan et al., 2004; pedersen et al., 1999; schwartz et al., 2004) . poliovirus, the most extensively studied picornavirus, induces a large number of membranous vesicles, most of them surrounded by double lipid bilayers (dales et al., 1965; jackson et al., 2005; suhy et al., 2000) . characterization of these membranes modified during poliovirus infection demonstrated that they contained cellular markers from the endoplasmic reticulum (er) as well as membranes from other origins, including lysosomes and the golgi (cho et al., 1994; datta and dasgupta, 1994; schlegel et al., 1996; suhy et al., 2000) . other studies have suggested that several features displayed by the vesicles induced during poliovirus infection, such as double-membrane morphology, vesicular content, and apparent er origin, are all consistent with an autophagic origin (schlegel et al., 1996; suhy et al., 2000; taylor and kirkegaard, 2007) . autophagy is a cell-regulated pathway designed to degrade and recycle long-lived proteins and cellular components, an important aspect of organelle turnover and management of starvation (levine and klionsky, 2004) . autophagy was first characterized morphologically in mammalian cells; however the molecular components of autophagy were first described in yeast (wang and klionsky, 2003) . a number of signals can induce autophagy in mammalian cells including starvation, differentiation, development, specific drugs, and infection with intracellular pathogens. during autophagy a double-membrane vesicle, the autophagosome, forms in the cytoplasm sequestering cytoplasmic components or small organelles. the formation and maturation of the autophagosome requires the modification of two ubiquitin-like molecules, microtubule-associated protein light-chain kinase 3 (lc3) and atg12 (autophagy-related protein) (espert et al., 2007) . autophagosomes fuse with lysosomes to generate singlemembrane vesicles named autophagolysosomes, in which the cargo is degraded. initial studies provided evidence that the source of the membranes forming this structure was both the golgi and the er. however, recent studies suggest that the source of these membranes is primarily from the er (dunn, 1990; espert et al., 2007; juhasz and neufeld, 2006) . more recently, hailey et al. (2010) suggested that the mitochondrial outer membrane contributes to their biogenesis during starvation. the autophagic pathway is also used to degrade intracellular pathogens including bacteria and viruses and may be a component of the innate and adaptive immune responses (deretic, 2010 , kirkegaard et al., 2004 , lin et al., 2010 sumpter and levine, 2010) . interestingly, recent studies have shown that certain intracellular bacteria and a number of positive-strand rna viruses, including picornaviruses, induce the formation of autophagic membranes and inhibit either their final maturation or their ability to fuse with lysosomes (egger et al., 2002; jackson et al., 2005; lee et al., 2008; fig. 1 . analysis of redistribution of autophagy marker gfp-lc3 and fmdv proteins in mcf-10a cells. a. mcf-10a cells were transfected with a plasmid that expresses a gfp-lc3 (green) fusion protein as indicated. forty-eight hours after transfection, cells were infected with fmdv o 1 campos or mock infected and processed for immunofluorescence staining as described in materials and methods. fmdv non-structural proteins were detected with specific mabs and visualized with alexa fluor 594 (red). b. mcf-10a cells were infected with fmdv o 1 campos and processed for immunofluorescence staining as describe in materials and methods. fmdv viral capsid protein vp1, was detected with a mab and visualized with alexa fluor 594 (red); atg5 was detected with a rabbit antibody and visualized with alexa fluor 488 (green). pedersen et al., 1999; schlegel et al., 1996; suhy et al., 2000) . furthermore the effect of specific autophagy inducers and inhibitors on virus yield supports the idea that autophagosome structures may provide a scaffold for rna replication by rna viruses (dreux et al., 2009; jackson et al., 2005; taylor and kirkegaard, 2007; wong et al., 2008) . these studies also suggested that these double-membrane vesicles, late in infection, may provide a pathway for the non-lytic release of virus taylor and kirkegaard, 2007) , which would be essential in the establishment of persistent infections, such as that established by fmdv (alexandersen et al., 2002; salt, 1993) . in this study, we analyzed the role of autophagy during fmdv replication by using confocal microscopy to monitor the co-localization of specific autophagosome-associated proteins and viral proteins in infected cells. in addition, we explored the effect of inducing or inhibiting the autophagy pathway on fmdv replication. our results indicate that fmdv non-structural proteins 2b, 2c and 3a, co-localize with the autophagosome marker lc3. additionally, co-localization of the viral capsid protein vp1 with atg5 (autophagy-related marker) and lc3 with lamp-1 in fmdv-infected cells was observed. furthermore, chemical stimulation or inhibition of the autophagy process directly correlated with increase or decrease in virus production, respectively. finally, knockdown of lc3 and atg12, two proteins required to initiate the process of autophagy, led to decreased virus replication. altogether, these results strongly suggest that autophagy plays an important role in the replication of fmdv. to study if fmdv replication complexes contain constituents of autophagosomes, we used confocal microscopy to monitor the localization of both lc3, a specific marker of autophagosomes, and viral non-structural proteins that have been characterized as critical components of picornavirus replication complex (bienz et al., 1987; cho et al., 1994; jackson et al., 2005; moffat et al., 2005; suhy et al., 2000) . for most of our experiments we utilized mcf-10a epithelial cells (o'donnell et al., 2005) , unless otherwise indicated. to visualize autophagosomes we expressed lc3 as a fusion product with green fluorescent protein (gfp). in uninfected cells, gfp-lc3 was detected as small discrete foci distributed throughout the cell cytoplasm (fig. 1a) . upon infection, gfp-lc3 co-localized with fmdv nonstructural proteins 2b, 2c, and 3a, by 5 hours post-infection (hpi), adopting a perinuclear pattern (fig. 1a) . by 5 hpi we also show colocalization of the viral capsid protein vp1 with atg5, an autophagyrelated protein that is essential for autophagosome formation (espert et al., 2007; matsushita et al., 2007) (fig. 1b) . to examine the role of autophagy in a system applicable to fmdv pathogenesis studies, we used primary cell cultures derived from bovine pharynx, a site where fmdv primary replication occurs (arzt et al., 2010; pacheco et al., 2010a) . these cultures, known to be negative for fmdv by virus isolation, rt-pcr and immunostaining (data not shown), were infected with fmdv type o 1 campos at a multiplicity of infection (moi) of 10. after infection, distribution of viral nonstructural protein 3a was compared to the distribution of lc3 by using double-label immunofluorescence and confocal microscopy. fig. 2 shows a clear co-localization of lc3 and fmdv 3a proteins, with both of the proteins showing a small punctuated distribution pattern. it has been demonstrated that both lc3 and lysosomal membrane protein 1 (lamp-1) are components of the mature autophagosome membrane (espert et al., 2007; kirkegaard and jackson, 2005) . in order to confirm that autophagy is induced during fmdv replication, we examined the distribution of these two markers in uninfected and infected cells, as well as in the presence of rapamycin, a well-known autophagy inducer. lc3 and lamp-1 do not appear to co-localize in uninfected untreated cells. conversely, both in fmdv-infected and in rapamycin-treated cells, lc3 and lamp-1 proteins clearly co-localize with a perinuclear distribution (fig. 3) , confirming that autophagy occurs in cells infected with fmdv. well characterized autophagy modulators were used to assess the role of autophagy during fmdv replication in infected cells. fmd viral fig. 2 . analysis of redistribution of autophagy marker lc3 and non-structural fmdv protein 3a in bovine pharynx epithelial primary cultures. primary cultures of bovine pharynx were infected with fmdv o 1 campos or mock infected and processed for immunofluorescence staining as described in materials and methods. fmdv non-structural protein 3a was detected with a mab and visualized with alexa fluor 594 (red). lc3 was detected with a rabbit antibody and visualized with alexa fluor 488 (green). yield was analyzed in the presence of the autophagy inducer rapamycin or the autophagy inhibitor 3-methyladenine (3-ma). mcf-10a cell cultures were pre-treated either with 50 nm of rapamycin or 20 mm of 3-ma, 2 h prior to infection with fmdv o 1 campos (moi of 1). assessment of virus yield was performed at 5 hpi. yield of virus increased approximately 2 logs when cells were pre-treated with rapamycin ( fig. 4a ). similar results were obtained when cells were infected in the presence of rapamycin and examined by using immunofluorescence and confocal microscopy. at 5 hpi, it was evident that twice the number of rapamycin-treated cells was fmdv-positive when compared to untreated infected cells (data not shown). conversely, when infected cells were pre-treated with 3-ma, fig. 3 . detection of gfp-lc3 and lamp-1 in fmdv-infected cells and in the presence of an autophagy inducer, rapamycin. mcf-10a cells were transfected with a plasmid that expresses a gfp-lc3 (green) fusion protein as indicated. forty-eight hours after transfection, cells were mock infected or infected with fmdv o 1 campos, or treated with rapamycin. cells were processed for immunofluorescence staining as described in materials and methods. lamp-1 was detected with a mab and visualized with alexa fluor 594 (red). an autophagy inhibitor, virus yield decreased by nearly 2 logs (fig. 4a ). moreover, we evaluated the effect of these two compounds in bovine pharynx epithelial primary cultures infected with fmdv o 1 campos. we found that, as observed in mcf-10a cells, when autophagy is induced by rapamycin, viral yield increases up to 1 log. when the fmdv-infected bovine pharynx epithelial cells were treated with 3-ma, viral yield was markedly reduced, more than 2 logs (fig. 4b) . these results suggest that the induction of autophagy during fmdv infection promotes virus replication. sirna silencing of the autophagy proteins atg12 and lc3 results in a reduction of fmdv replication to confirm the role of autophagy during virus replication, we analyzed the effect of knockdown on transcription levels of autophagy-related proteins lc3 and atg12. both proteins have been demonstrated to be critical in the autophagy process (jounai et al., 2007; kirkegaard et al., 2004; kirkegaard and jackson, 2005; wang and klionsky, 2003) . when cells were transfected with sirnas targeting lc3 prior to infection with fmdv o 1 campos, an approximately 3.8fold and 4.5-fold inhibition in extra-and intracellular viral yield, respectively, was observed (fig. 5a) . furthermore, an even stronger inhibitory effect on viral replication (greater than 3 logs) was observed when cells were transfected with sirna targeting atg12 (fig. 5b ). these results reinforce the hypothesis that autophagy is actively involved in the process of virus replication in cells infected with fmdv. upon induction of autophagy, a membrane cisterna (fold of membrane) known as the isolation membrane (sometimes referred as the phagophore in mammalian cells) appears and curves around part of the cytoplasm. sealing of the edges of the isolation membrane results in a unique double-membrane vesicle, the autophagosome (juhasz and neufeld, 2006) . ultrastructural analysis studies of cells infected with picornaviruses have revealed the presence of virions and other cytoplasmic material within the lumen of doublemembrane vesicles (kirkegaard et al., 2004; miller and krijnse-locker, 2008; schlegel et al., 1996) . to determine whether formation of double-membrane vesicles is linked to autophagy during fmdv infection, electron microscopy was performed on mock and fmdvinfected cells. quantitative analysis demonstrated an increase in the quantity of the single-and double-membrane vesicles in the fmdvinfected cells (single membrane 160.2 ± 108.2, double membrane 8.1 ± 4.1, and size 0.15 ± 0.04 μm) compared to uninfected cells (single membrane 1.2 ± 3.03, double membrane 0.28 ± 0.89, and size 0.46 ± 0.15 μm) (fig. 6a) . examination by immunoelectron microscopy (iem) also showed an increased expression of autophagy-related protein lamp-1 during virus replication in virus-infected cells compared to uninfected cells (fig. 6b) . interestingly, iem also showed a close association of non-structural viral protein 2b with the membranes associated with fmdv infection within the infected cells (fig. 6b) . therefore, fmdv-infected cells showed evidence of an autophagy-related process, which is absent in mock-infected cells, associated with the expression of non-structural viral proteins. picornaviruses, as with other positive-strand rna viruses, generally induce the rearrangement of intracellular membranes to allow the assembly of virus replication complexes (bienz et al., 1987; mackenzie, 2005) . co-localization of viral rna replication complexes with singleor double-membrane vesicles in infected cells has been described several times for various positive-stranded rna viruses (bienz et al., 1987; suhy, et al. 2000; wong et al, 2008) . the double-membrane vesicles induced during poliovirus infection shared several features with cellular structures termed autophagosomes, double-membrane vesicles that ultimately mature to degrade their cytoplasmic contents (dales et al., 1965; schlegel et al., 1996) . induction of autophagy has been shown to be beneficial for the replication of several viruses including poliovirus, rhinovirus, equine arterivirus, dengue virus, coxsackievirus, hepatitis c virus, and severe acute respiratory syndrome, although the detailed mechanisms remain elusive (egger et al., 2002; espert et al., 2007; jackson et al., 2005; lee et al., 2008; pedersen et al., 1999; wileman, 2006; wong et al., 2008) . based on these previous findings, it was hypothesized that autophagy may play a role in fmd viral replication. the findings in this report demonstrate that fmdv triggers the autophagy machinery, and this enhances viral replication. this observation is supported by the redistribution of autophagic hallmarks, notably lc3 and lamp-1, along with virus antigens in fmdv-infected cells, the effect of autophagy modulating compounds on viral yield, and the inhibition of intra-and extracellular virus replication by knockdown of autophagy-related genes. to understand how autophagy plays a role during fmdv replication, we analyzed the localization of autophagosome markers with viral proteins after infection. we monitored the localization of lc3 with fmd non-structural viral proteins by confocal microscopy, demonstrating that lc3 co-localizes with viral non-structural proteins 2b, 2c and 3a. we also evaluated autophagy in a primary cell culture derived from bovine pharynx, a relevant bovine tissue as a primary site of virus replication (arzt et al., 2010; burrows et al., 1981; pacheco et al., 2010a) . studies performed using bovine pharynx epithelial cell cultures showed a clear co-localization of lc3 and 3a proteins by 5 hpi. for poliovirus the expression of 2bc and 3a in combination, but not separately, induces the formation of double-membrane vesicles that display biochemical markers and fractionate similarly to the vesicles induced during poliovirus infection suhy et al., 2000) . taylor and kirkegaard (2007) showed that the expression of the individual poliovirus protein 2bc induces lipid modification of lc3. conjugation of lc3 to phosphatidylethanolamine (pe) is one of the early steps of autophagosome formation and, it is thought, of vesicle formation during poliovirus infection. in agreement with those results observed for poliovirus infection, within 4 h of fmdv infection, lc3 and lamp-1, two components of the mature autophagosome membrane, co-localized in both fmdv-infected cells and in rapamycin-treated cells, whereas in uninfected cells no co-localization was seen. additionally, co-localization of viral capsid protein vp1 with the autophagy-related protein atg5, was observed (fig. 1b) . co-localization of autophagosome markers, atg5 and lc3, with viral replication complexes suggested that at least some rna replication complexes are present on or near autophagosome-like vesicle. co-localization of autophagosome markers and viral proteins could have two explanations. first, the virus could succumb to autophagy as a destructive innate immune response. this has been shown to occur in several viruses and bacteria, including hsv-1, sindbis virus, gas, and tb (lin et al., 2010; xu and eissa, 2010) . some pathogens, however, subvert the cellular autophagy pathway to promote their own replication, including enterovirus 71 and anaplasma phagocytophilum (huang et al., 2009; niu et al., 2008) . wong et al. (2008) have shown that inhibition of signaling pathways or autophagy genes critical for autophagosome formation reduces coxsackievirus b3 (cvb3) viral protein expression and the viral progeny titer. conversely, increasing the cellular number of autophagosomes prior to viral infection by cellular starvation, rapamycin treatment, or lysosomal inhibition promotes viral replication, suggesting that the host double-membrane autophagosome is likely utilized by cvb3 to facilitate its own replication (wong et al., 2008) . poliovirus also subverts the autophagy pathway, inducing structures resembling cellular autophagosomes on which viral rna replication complexes are assembled. treatment with either rapamycin or tamoxifen, both inducers of autophagy, increased poliovirus yield 3-5-fold . therefore, autophagosome formation increases poliovirus load suggesting that the autophagic machinery serves a valuable role in viral replication . accordingly, in our study, viral loads increased when mcf-10a cells as well as the bovine pharynx epithelial primary cells were infected with fmdv in the presence of the autophagy inducer rapamycin (figs. 4a-b). the increase of viral yield was manifest to a greater extent in the extracellular viral yield as opposed to intracellular virus in mcf-10a cells (data not shown). our results clearly indicate that fmdvinduced autophagic machinery is favorable for fmdv replication. in contrast, when autophagy is blocked in both cell types by 3-ma, viral loads are reduced (figs. 4a-b) . interestingly, when autophagy is inhibited by 3-ma, fmdv replication still continues at a reduced level, suggesting that the autophagy machinery may play a promoting role in the replication of fmdv rather than being a critical factor. based on these results, it is therefore likely that the autophagic machinery does not have a destructive role during fmdv infection and instead contributes to the formation of viral replication complexes. to further examine the role of autophagy, we tested the effect of lc3 and atg12 knockdown on fmdv replication. by using sirna to reduce the amount of intracellular autophagy proteins lc3 and atg12, an inhibitory effect on viral replication was detected, which was more significant in extracellular than in intracellular viral yields, indicating that the expression of autophagy-related proteins is important and necessary for the virus. jackson et al. (2005) also reported a larger effect on extracellular than intracellular virus yield when the abundance of autophagy proteins atg12 and lc3 was reduced by sirna. these authors suggest that the reduction in autophagosome machinery decreased cell lysis early in infection, or that reduced abundance of autophagosomal machinery decreased non-lytic viral escape, contributing to the decrease in extracellular virions. we show here a similar effect on fmdv, indicating that inhibition of autophagy has a stronger effect on extracellular than intracellular viral yields. em analyses of cells infected with poliovirus showed the presence of large numbers of membranous vesicles with diameters of 200-400 nm, which due to the 'cytoplasmic matrix' present in the lumen of the vesicles, were postulated to develop by a mechanism comparable to that of the formation of "autolytic vesicles" (dales et al., 1965; schlegel et al., 1996) . as mentioned above, positive-strand rna viruses, such as poliovirus, equine arterivirus and murine hepatitis virus, all require membrane surfaces on which to assemble their rna replication complexes (mackenzie, 2005) . for instance, in poliovirusinfected cells the membranes that are induced during infection resemble autophagosomes due to the double-membrane-bound morphology that is present even early during the infection, their specific labeling with anti-lamp-1 antibodies, and their low buoyant densities suhy et al., 2000) . these facts obtained using other rna viruses support our observations regarding membranous vesicles that resemble autophagosomes and their role during fmdv replication. mcf-10a cells infected with fmdv demonstrated an increase of both single-and double-membrane vesicles, as shown in fig. 6 , which are associated with the autophagy-related protein lamp-1 as well as fmdv non-structural protein 2b. it has been reported that 2bc expression leads to the accumulation of large single-membrane vesicles (cho et al., 1994; suhy et al., 2000) with buoyant densities similar to those of the vesicles induced during poliovirus infection (suhy et al., 2000) . further investigation of the ultrastructure and biochemistry of these vesicles formed during autophagy and the expression of individual viral proteins may reveal additional steps to the lipid sequestration, cytosolic wrapping, and maturation of these unique intracellular compartments. a hypothesis for the presence of autophagosome-like membranes in poliovirus-infected cells is that these membranes are induced as a component of the innate immune response . autophagy is increasingly appreciated as a functional pathway in the innate immune response to several viruses, intracellular bacteria, and parasites. the specific mechanism by which the autophagosome is utilized during replication in host cells has not yet been elucidated, however it is likely that the autophagosome provides a physical scaffold where the virus complex may reside as has been suggested for poliovirus and coxsackievirus taylor and kirkegaard, 2007; wong et al., 2008) . this report constitutes the first demonstration that autophagy appears to be an important part of the fmdv replication cycle, although the mechanism by which fmdv infection induces the activation of autophagic machinery remains to be determined. the correlation between the autophagy pathway and increase of viral yield supports this observation. it could be speculated that autophagy may be a possible mechanism fmdv uses to maintain a persistent infection. a number of studies have shown the importance of the pharyngeal area tissues during persistent fmdv infection (burrows et al., 1971 (burrows et al., , 1981 prato murphy et al., 1994; zhang and kitching, 2001) . the evidence of autophagic machinery activation in fmdv-infected bovine pharynx epithelial primary cultures, a relevant tissue during persistent infection in the natural host, may indicate that under certain circumstances, autophagy enables the non-lytic release of the virion from persistently infected cells. human mammary gland epithelial cells (mcf-10a) were obtained from atcc (catalogue no. crl-10317) and maintained in a mixture of dulbecco's minimal essential medium (dmem, invitrogen, ca) and f12 ham media (1:1, invitrogen) containing 5% heat-inactivated fetal bovine serum (hi-fbs, hyclone), 20 ng/ml epidermal growth factor (sigma, st.lois, mo), 100 ng/ml cholera toxin (sigma), 10 μg/ml insulin (sigma), and 500 ng/ml hydrocortisone (sigma). fmdv type o 1 strain campos (o 1 campos) was derived from the vesicular fluid of an experimentally infected steer. the virus was grown in baby hamster kidney-21 (bhk-21) cells and the titer determined by plaque assay on (bhk) cells using standard techniques (pacheco et al., 2010b) . plasmid designed to express gfp-lc3 fusion protein was produced and described elsewhere . bovine pharynx epithelial primary cultures were produced by using a modification of preparing keratinocyte cultures and described elsewhere (barlow and pye, 1989; o'donnell et al., 2001) . monoclonal antibody (mab) 12fb, directed against the fmdv type o 1 structural protein vp1, has been previously described (stave et al., 1986) . monoclonal antibodies to the non-structural fmdv proteins 2b, 2c and 3a, were obtained from dr. e. brocchi, istituto zooprofilattico sperimentale della lombardia e dell emilia-romagna, brescia, italy. rabbit antibodies to lc3 (anaspec) and atg5 (santa cruz) were used as autophagy markers. anti-lamp-1 monoclonal antibody (bd biosciences) was used to detect lamp-1, a marker of late endosomes and lysosomes. an autophagy inducer, rapamycin (cell signaling technology, beverly, ma), prepared as a 100 μm stock solution in methanol, was used at a 50 nm concentration diluted in mem media, and the inhibitor 3-methyladenine (3-ma, sigma, st louis), was used at a 20 mm concentration diluted in mem medium. sub-confluent monolayers of mcf-10a cells or bovine pharynx epithelial primary cultures grown on 12 mm glass coverslips in 24well tissue culture dishes were infected with fmdv o 1 campos at an moi of 10 pfu/cell, for 1 h at 4°c in mem containing 0.5% hi-fbs and 25 mm hepes, ph 7.4. after the 1 h adsorption period the supernatant was removed and the cells rinsed with ice-cold 2-morpholinoethanesulfonic acid (mes) buffered saline (25 mm mes, ph 5.5, 145 mm nacl) to remove unadsorbed virus. the cells were washed once with media before fresh media was added, then incubated at 37°c. to express gfp-lc3 protein, monolayers of mcf-10a cells were transfected with 1 μg of plasmid dna by using fugene (roche, mannheim, germany), following the manufacturer's recommendations. at 24 h post-transfection the cells were infected as described above, fixed with 4% paraformaldehyde (ems, hatfield, pa) at the appropriate times and analyzed by confocal microscopy. to study whether rapamycin (50 nm) or 3-ma (20 mm) affected viral replication, cells were pre-incubated with each compound for 2 h at 37°c, then subsequently infected with fmdv o 1 campos (moi 1). immunofluorescence and confocal microscopy using the antibodies listed above were performed as previously described (o'donnell et al., 2005) . in order to get a representative image for each experiment, we analyzed 5 to 10 independent fields examining 15-20 cells/field at a low magnification (40×) and an additional observation at higher magnification (63×) where variable numbers of fields with 2-3 cells/ field were examined. of note, each of the experiments shown in figs. 1, 2, 3 and 6 was run at least 3-4 independent times. viral replication in the presence of compounds mcf-10a cells or bovine pharynx epithelial primary cultures were incubated with the compounds at the concentrations listed above for 2 h at 37°c prior to infection. the cells were then infected with fmdv o 1 campos at an moi of 1 in the presence of either rapamycin or 3-ma. at the end of the adsorption period the supernatant was removed, followed by a single rinse with ice-cold mes buffered saline to inactivate unadsorbed virus. the cells were washed once with media, before fresh media with or without the rapamycin or 3-ma was added. one set of cultures was immediately frozen at − 70°c, and the other set of plates was incubated for an additional 4 h at 37°c, and then removed to −70°c. the plates were then thawed and cell debris was removed by centrifugation and viral titer was determined by plaque assay on bhk-21 cell monolayers. sirna smartpools consisting of four rna duplexes targeting the gene of interest and a control sirna (sirna glo) were purchased from dharmacon (lafayette, co). for lc3, both lc3a and lc3b were targeted, bringing the total number of transfected duplexes to eight. mcf-10a cells were grown to densities of 1.0 × 10 5 cells per well in 24-well tissue culture dishes in 1 ml of media without antibiotics, and transfected using lipofectamine 2000 (invitrogen) as previously described . forty-eight hours after transfection, the cells were infected with fmdv o 1 campos at an moi of 1 for 1 h at 37°c. after adsorption, the inoculum was removed, and the cells were rinsed with ice-cold mes to remove residual virus particles, rinsed with mem containing 1% fbs and 25 mm hepes, ph 7.4, then incubated at 37°c. at appropriate times post-infection, the cells were frozen at −70°c, and the thawed lysates were used to determine titers by plaque assay on bhk-21 cell monolayers. sub-confluent monolayers of mcf-10a cells grown on tissue culture dishes (mattek, ashland, ma) were infected with fmdv o 1 campos, at an moi of 10 pfu/cell for 1 h at 4°c in mem containing 0.5% fbs and 25 mm hepes, ph 7.4. at the end of the adsorption period the supernatant was removed and the cells were rinsed once with ice-cold mes to remove unadsorbed virus. the cells were then washed once with media followed by addition of fresh media, and incubated at 37°c for 4 h. at this time, the media was removed and the cells were fixed with a solution containing 2.5% glutaraldehyde, 100 mm nacl, and 2 mm calcium chloride, in hepes buffer, ph 7.4 for 1 h at room temperature (rt). the cells were rinsed twice with a solution containing 100 mm nacl, 2 mm calcium chloride, and 3.5% sucrose in hepes buffer, ph 7.4 and post-fixed with 1% osmium tetroxide and 1.5% potassium ferricyanide. the cells were stained en bloc with 2% aqueous uranyl acetate overnight, dehydrated with ethanol and infiltrated with embed 812 resin (ems). to view sequential organization of the replication complexes and other membrane vesicles, ribbons of 70 angstrom sections were picked up on slot grids and grid stained with methanolic uranyl acetate and lead citrate. the sections were examined with a hitachi t-7600 electron microscope operated at 80 kv. digital images were captured with an advanced microscopy techniques camera (danvers, ma). for in situ iem analysis, fmdv-infected and mockinfected mcf-10 cell monolayers were fixed in the mattek dishes using a protocol described elsewhere and ultra-small gold and other reagents from aurion (wageningen, netherlands) were used. briefly, the cell monolayers were fixed using a solution containing 1% lysine, 4% paraformaldehyde (electron microscopy grade 16%) (ems), 1% sodium meta-periodate and 0.1% glutaraldehyde in hepes buffer, ph 7.4 for 1 h at rt. primary antibodies were incubated overnight at 4°c. the cell monolayers were rinsed extensively in binding buffer and incubated with an anti-mouse conjugated to ultra-small gold (dil 1/300, aurion, ems) for 1 h at rt. the cell monolayers were rinsed 5 times with binding buffer, 2 times with dulbecco's phosphate buffered saline (pbs) and fixed with 2% glutaraldehyde in pbs. after extensive washing with milliq water (millipore), the ultra-small gold was enlarged to approximately 10 nm using gold en-hance (aurion) for 20 min at rt. the cell monolayers were incubated with a solution of 0.5% osmium tetroxide for 30 min at 4°c on wet ice, rinsed with water, dehydrated with ethanol and embedded in spurr's resin per manufacturer's instructions. aspects of the persistence of foot-andmouth disease virus in animals -the carrier problem foot-and-mouth disease virus, but not bovine enterovirus, targets the host cell cytoskeleton via the nonstructural protein 3cpro the early pathogenesis of foot-and-mouth disease in cattle after aerosol inoculation: identification of the nasopharynx as the primary site of infection keratinocyte culture virus replication, cytopathology, and lysosomal enzyme response of mitotic and interphase hep-2 cells infected with poliovirus association of polioviral proteins of the p2 genomic region with the viral replication complex and virus-induced membrane synthesis as visualized by electron microscopic immunocytochemistry and autoradiography the growth and persistence of foot-and-mouth disease virus in the bovine mammary gland the pathogenesis of natural and simulated foot-and-mouth disease infection in cattle membrane rearrangement and vesicle induction by recombinant poliovirus 2c and 2bc in human cells electron microscopy study of the formation of poliovirus expression and subcellular localization of poliovirus vpgprecursor protein 3ab in eukaryotic cells: evidence for glycosylation in vitro autophagy in infection the autophagy machinery is required to initiate hepatitis c virus replication studies on the mechanism of autophagy: formation of the autophagic vacuole expression of hepatitis c virus proteins induced distinct membrane alterations including a candidate viral replication complex involvement of autophagy in viral infections: antiviral function and subversion by viruses foot-and-mouth disease mitochondria supply membranes for autophagosome biogenesis during starvation enterovirus 71-induced autophagy detected in vitro and in vivo promotes viral replication subversion of cellular autophagosomal machinery by rna viruses the atg5-atg12 conjugate associates with innate antiviral immune responses autophagy: a forty-year search for a missing membrane source topology of double-membraned vesicles and the opportunity for non-lytic release of cytoplasm cellular autophagy: surrender, avoidance and subversion of microorganisms autophagic machinery activated by dengue virus enhances virus replication development by self-digestion: molecular mechanisms and biological functions of autophagy viral interactions with macroautophagy: a double-edged sword wrapping things up about virus rna replication structure of atg5.atg16, a complex essential for autophagy modification of intracellular membrane structures for virus replication effects of foot-and-mouth disease virus nonstructural proteins on the structure and function of the early secretory pathway: 2bc but not 3a blocks endoplasmic reticulum-to-golgi transport the ultrastructural of the developing replication site in foot-and-mouth disease virus-infected bhk-38 cells subversion of cellular autophagy by anaplasma phagocytophilum subcellular distribution of foot-and-mouth disease virus 3a protein in cells infected with viruses encoding wild-type and bovine-attenuated forms of 3a analysis of foot-and-mouth disease virus internalization events in cultured cells early events in the pathogenesis of foot and mouth disease virus in cattle after controlled aerosol exposure domain disruptions of individual 3b proteins of foot-and-mouth disease virus do not alter growth in cell culture or virulence in cattle open reading frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulumderived double-membrane vesicles which carry the viral replication complex analysis of sites of foot and mouth disease virus persistence in carrier cattle via the polymerase chain reaction the carrier state in foot-and-mouth disease -an immunological review cellular origin and ultrastructure of membranes induced during poliovirus infection alternate, virus-induced membrane rearrangements support positive-strand rna virus genome replication analysis of fmdv type o 1 brugge neutralization epitopes using monoclonal antibodies remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagylike origin for virus-induced vesicles autophagy and innate immunity: triggering, targeting and tuning modification of cellular autophagy protein lc3 by poliovirus the molecular mechanism of autophagy aggresomes and autophagy generate sites for virus replication autophagosome supports coxsackievirus b3 replication in host cells autophagy in innate and adaptive immunity the localization of persistent foot and mouth disease virus in epithelial cells of the soft palate and pharynx this work was supported by the us department of agriculture, agricultural research service, through current research information system project (cris) no. 1940-32000-052-00d and a specific cooperative agreement no. 58-1940-2-245, with the department of pathobiology and veterinary sciences, university of connecticut, storrs, ct, usa. we thank ms. melanie v. prarat for editing the manuscript. key: cord-289045-vft163v0 authors: thackray, larissa b.; turner, brian c.; holmes, kathryn v. title: substitutions of conserved amino acids in the receptor-binding domain of the spike glycoprotein affect utilization of murine ceacam1a by the murine coronavirus mhv-a59 date: 2005-03-30 journal: virology doi: 10.1016/j.virol.2005.01.016 sha: doc_id: 289045 cord_uid: vft163v0 the host range of the murine coronavirus (mhv) is limited to susceptible mice and murine cell lines by interactions of the spike glycoprotein (s) with its receptor, mceacam1a. we identified five residues in s (s33, l79, t82, y162 and k183) that are conserved in the receptor-binding domain of mhv strains, but not in related coronaviruses. we used targeted rna recombination to generate isogenic viruses that differ from mhv-a59 by amino acid substitutions in s. viruses with s33r and k183r substitutions had wild type growth, while l79a/t82a viruses formed small plaques. viruses with s33g, l79m/t82m or k183g substitutions could only be recovered from cells that over-expressed a mutant mceacam1a. viruses with y162h or y162q substitutions were never recovered, while y162a viruses formed minute plaques. however, viruses with y162f substitutions had wild type growth, suggesting that y162 may comprise part of a hydrophobic domain that contacts the mhv-binding site of mceacam1a. viruses of the coronaviridae family form three distinct antigenic and phylogenetic groups (lai and holmes, 2001) . coronaviruses in group ii infect mice, rats, cattle or humans, as well as several other host species. only one cellular glycoprotein has been identified as a receptor for a group ii coronavirus. the murine coronavirus [murine hepatitis virus (mhv)] utilizes as receptors murine carcinoembryonic antigen cell adhesion molecule 1a (mceacam1a) and related murine glycoproteins in the cea family of glycoproteins in the immunoglobulin (ig) superfamily (dveksler et al., 1993a (dveksler et al., , 1993b yokomori and lai, 1992) . the envelope of most group ii coronaviruses contains a hemagglutinin esterase (he) glycoprotein that binds to 9-oacetylated neuraminic acid, but the a59 strain of mhv (mhv-a59) does not express he (yokomori et al., 1991) . thus, mhv-a59 infection of susceptible mice and murine cell lines depends solely on interactions of the viral spike glycoprotein (s) with mceacam1a and related murine glycoproteins (hemmila et al., 2004) . although the variety of hosts infected by coronaviruses illustrates that these viruses can emerge in a new host species, the mechanisms required for the introduction of a coronavirus into a new host are not well understood. the interaction of the viral spike glycoprotein with a specific cellular glycoprotein receptor is a major determinant of coronavirus host range. cell lines from host species that are normally resistant to mhv, porcine coronavirus [transmissible gastroenteritis virus (tgev)] or human coronavirus strain 229e (hcov-229e) are rendered susceptible to infection by transfection with cdna encoding the specific coronavirus receptors mceacam1a, porcine aminopeptidase n (papn) (delmas et al., 1992; dveksler et al., 1991; yeager et al., 1992) . the 180-kda s of mhv is a type i viral fusion protein that mediates both receptor binding and membrane fusion activities (bosch et al., 2003; gallagher and buchmeier, 2001) . s of mhv-a59 is post-translationally cleaved by a cellular protease into 90 kda s1 and s2 proteins that remain non-covalently associated on the viral envelope. variations in the amino acid (aa) sequence of s1 of mhv strains are associated with differences in tissue tropism and pathogenesis (leparc-goffart et al., 1998; phillips et al., 2001) . the membrane-anchored s2 contains a coiled-coil domain that is likely associated with membrane fusion and is more highly conserved among mhv strains than s1 (bosch et al., 2003) . mhv and the blocking anti-mceacam1a monoclonal antibody, mab-cc1, bind to the amino (n)-terminal ig-like domain of mceacam1a (dveksler et al., 1993a (dveksler et al., , 1993b . binding of the n-terminal domain of soluble mceacam1a to s1 on mhv virions at 37 8c induces conformational changes in s1 and s2 proteins, and neutralizes virus (gallagher, 1997; lewicki and gallagher, 2002; matsuyama and taguchi, 2002; miura et al., 2004; zelus et al., 1998) . incubation of purified mhv-a59 virions with soluble mceacam1a at neutral ph, or without receptor at ph 8.0, triggers major conformational changes in s2 . these conformational changes may expose a hydrophobic domain in s that allows virus to bind liposomes and presumably initiates fusion of the viral envelope with host cell membranes, as well as cell-to-cell fusion. mutational analyses of determinants in mcea-cam1a that alter mhv binding and entry implicated residues in the predicted ccv loop and the cv h sheet in the n-terminal domain of mceacam1a (rao et al., 1997; wessner et al., 1998) . the predicted ccv loop was also shown by mutational analyses to be critical for recognition of human ceacam1 by bacterial pathogens such as neisseria, as well as for homophilic cell adhesion (bos et al., 1999; virji et al., 1999; watt et al., 2001) . the crystal structure of mceacam1a [1, 4] showed that the ccv loop has a convoluted conformation unlike other ig superfamily glycoproteins (tan et al., 2002) . the side chain of an isoleucine at residue 41 (i41) projects upwards away from the membrane and was predicted to play an important role in mceacam1a recognition by mhv s proteins. the structural characterization of s is limited to fusion cores of s2 proteins of mhv and severe acute respiratory syndrome coronavirus (sars-cov) (bosch et al., 2003; liu et al., 2004; tripet et al., 2004; xu et al., 2004) . domains of s that are responsible for receptor binding have been identified for several coronaviruses. aa 417-547 of s of hcov-229e comprise a minimal receptor binding domain (rbd) for hapn expressed on cell membranes breslin et al., 2003) , while aa 1-330 of s of mhv (s330) comprise the minimal domain for binding to soluble mceacam1a in vitro and initiating infection via anchored mceacam1a (kubo et al., 1994; tsai et al., 2003) . aa 318-510 of s of sars-cov comprise a minimal domain for binding to angiotensin converting enzyme 2 (ace2) in vitro (babcock et al., 2004; li et al., 2003; wong et al., 2004; xiao et al., 2003) . recently, we demonstrated that the n-terminal region of s1 containing 21 aa substitutions and a 7-aa insert derived from mhv/bhk, a virus variant generated during persistent mhv-a59 infection of murine cells, is sufficient to extend the host range of mhv-a59 in vitro (sawicki et al., 1995; schickli et al., 1997 schickli et al., , 2004 thackray and holmes, 2004) . residues critical for receptor utilization and host specificity have not been identified for most coronaviruses, although several residues that play a role in the interactions of the mhv s protein with mceacam1a or of the sars-cov s protein with ace2 have been identified (saeki et al., 1997; suzuki and taguchi, 1996; wong et al., 2004) . with the emergence of sars-cov in humans and the search for effective drugbased interventions for coronavirus infection, the need to identify residues critical for the interactions of coronaviruses with their cellular receptors is greater than ever. we compared the n-terminal 330 aa of s proteins of seven mhv strains with the n-termini of s proteins of the extended host range variant mhv/bhk, as well as related group ii coronaviruses of rats, cattle and humans. we identified five residues that are highly conserved in s330 of all mhv strains and mhv/bhk, but are not found in s330 of rat, bovine or human coronaviruses of group ii. to examine the roles of these residues in the receptor specificity of s, we used targeted rna recombination (trr) to generate isogenic viruses that differ from mhv-a59 at one or two aa in the rbd of s. we showed that y162 is critical for the recovery of mhv-a59 from cells expressing mceacam1a. we also found that certain aa substitutions at s33, l79, t82 or k183 can inhibit the recovery of mhv-a59 from murine cells. to identify residues that are involved in the receptor specificity of the s glycoprotein of mhv, we compared the n-terminal 330 aa of s proteins of seven mhv strains with the n-termini of s proteins of related coronaviruses of rats, cattle and humans. we identified four residues, s33, t82, y162 and k183, that are conserved in s330 of all mhv strains, but not in the corresponding domains of rat, bovine and human coronaviruses in group ii (fig. 1a) . another residue, l79, is conserved in s330 of all mhv strains, except mhv 2, but not in s330 of rat, bovine or human group ii coronaviruses. these five residues are also conserved in the extended host range variant mhv/bhk that infects a wide range of non-murine cell lines while maintaining the ability to infect murine cells (schickli et al., 2004; thackray and holmes, 2004) . to examine the roles of these residues in the receptor specificity of s, we used trr to introduce single aa substitutions at s33, y162 or k183 and double aa substitutions at l79 and t82 into the genome of mhv-a59. the aa substitutions were chosen to reflect residues found in rat, bovine or human group ii coronaviruses, a conservative y to f substitution, or neutral alanine substitutions (fig. 1a) . although aa substitutions at t62 or l65 in s330 of mhv-jhm were previously associated with reduced mceacam1a binding (saeki et al., 1997; suzuki and taguchi, 1996) , we observed that t62 and l65 were conserved in s330 of all mhv strains, as well as s330 of related rat, bovine and human coronaviruses (fig. 1a) . to examine the roles of these residues in the receptor specificity of s in the context of infectious isogenic viruses, we used targeted rna recombination (trr) to introduce aa substitutions at t62 or l65 into the genome of mhv-a59. the aa substitutions were chosen to reflect substitutions made previously, s and h for t62 and l65, respectively, (saeki et al., 1997; suzuki and taguchi, 1996) or neutral alanine substitutions. donor rnas, transcribed in vitro from pmh54 constructs, were transfected into feline (fcwf) cells that had been inoculated with the chimeric helper virus fmhv (kuo et al., 2000) . rna recombination occurs between fmhv, which contains a chimeric s gene with the ectodomain of feline infectious peritonitis virus and the rest of the mhv-a59 genome, and the pmh54 donor rna containing the 3vmost 7.4 kb of the mhv-a59 genome (kuo et al., 2000) . the infected and transfected fcwf cells were immediately overlaid onto monolayers of murine 17 cl 1 cells to select for isogenic recombinant viruses that had gained the ability to infect murine cells. for each mutant pmh54 construct, three recombinant viruses (a, b and c) were independently recovered and plaque-purified to control for adventitious mutations that might arise during trr. in addition, in every experiment, pmh54 rna encoding wild-type mhv-a59 s protein was used to reconstitute wild-type mhv-a59 virus (sa59) in triplicate. extensive cytopathic effects (cpe) were seen at 48 h in 17 cl 1 cell monolayers overlaid with fmhv-inoculated fcwf cells transfected with pmh54 rna or pmh54 rnas encoding s33r, t62s, t62a, y162f or k183r substitutions. in contrast, 17 cl 1 cell monolayers overlaid with fmhv-inoculated fcwf cells transfected with pmh54 rnas encoding l65h, l65a or l79a/t82a substitutions exhibited less cpe at 48-72 h, while 17 cl 1 cell monolayers overlaid with fmhv-inoculated fcwf cells mock-transfected or transfected with pmh54 rnas encoding s33g, l79m/t82m, y162h, y162q, y162a or k183g substitutions exhibited no detectable cpe even after 72 h. most of the recombinant viruses formed uniform plaques on 17 cl 1 monolayers, although recombinant viruses derived from the l65h or l65a constructs formed clear and opaque plaques (data not shown). several mutant viruses were never recovered from 17 cl 1 cell monolayers (table 1) , either because the mutant s proteins interacted inefficiently with mceacam1a or because the mutant s proteins were inefficiently incorporated into mhv virions. trr using fmhv-inoculated fcwf cells transfected with pmh54 rnas encoding s33g, l79m/t82m, y162h, y162q, y162a or k183g substitutions was repeated several times, but none of these mutant viruses could be recovered from 17 cl 1 cell monolayers. in order to isolate these crippled viruses with mutant s proteins, we used hamster (bhk) cells stably expressing mceacam1a [1, 4] containing an i41r substitution [bhk + mceacam1a(i41r)]. these cells expressed 10-fold more receptor than either 17 cl 1 cells or bhk cells stably expressing wild type mceacam1a[1,4], as measured by flow cytometry using anti-mceacam1a mab cc1 (data not shown). bhk + mceacam1a(i41r) cell monolayers overlaid with fmhv-inoculated fcwf cells transfected with pmh54 or pmh54 rnas encoding s33r, l79m/t82m or k183g substitutions exhibited extensive cpe at 48 h, while no cpe was detected even at 72 h in cells transfected with pmh54 rnas encoding y162h, y162q or y162a substitutions. all three recombinant viruses derived from each of the sa59, s33g, l79m/t82m or k183g constructs formed uniform plaques on bhk + mceacam1a(i41r) cell monolayers, as well as on 17 cl 1 cells (data not shown). we do not know whether the recovery of the s33g, l79m/ t82m or k183r viruses from bhk + mceacam1a(i41r) cells was facilitated by the high level of receptor expression in these cells or the i41r substitution in the n-terminal domain of mceacam1a. however, the recovery phenotypes and plaque morphologies shared by the three replicates of each mutant virus suggest that the engineered mutations in s330 were responsible for the observed phenotypes of these viruses. we further characterized the s33g, l79m/t82m and k183g viruses that were subsequently plaque-purified and propagated in 17 cl 1 cells. viruses derived from the y162h, y162q and y162a constructs were not recovered from either 17 cl 1 or bhk + mceacam1a(i41r) cell monolayers (table 1) . trr using fmhv-inoculated fcwf cells transfected with pmh54 rnas encoding y162h, y162q or y162a substitutions was repeated several times, but these mutant viruses were not recovered from bhk + mceaca-m1a(i41r) cell monolayers. to examine the infectivity and spread of these crippled viruses with mutant s proteins immediately after trr, we used a pmh54-egfp plasmid that contains the enhanced green fluorescent protein (egfp) gene in place of gene 4 (das sarma et al., 2002) . gene 4 is not essential for replication of mhv-a59, since mhv-a59 egfp has wild type growth in vitro and in vivo. donor rnas, transcribed in vitro from mutant pmh54-egfp constructs, were transfected into fcwf cells that had been inoculated with the chimeric helper virus fmhv (kuo et al., 2000) . the infected and transfected fcwf cells were immediately overlaid in duplicate onto monolayers of 17 cl 1 or bhk + mceacam1a(i41r) cells. for each mutant pmh54-egfp construct, two recombinant viruses (a and b) were independently recovered. in addition, wild-type pmh54-egfp rna was used to reconstitute wild type mhv-a59 egfp virus (sa59-egfp) in duplicate. nearly all of the 17 cl 1 or bhk + mceacam1a(i41r) cells overlaid with fmhv-inoculated fcwf cells transfected with pmh54-egfp or pmh54-egfp encoding a y162f substitution exhibited extensive cpe and strong egfp expression at 48 h (fig. 2) . in contrast, less than 0.01% of the 17 cl 1 or bhk + mceacam1a(i41r) cells overlaid with fmhv-inoculated fcwf cells transfected with pmh54-egfp encoding y162h, y162q or y162a substitutions exhibited cpe and egfp expression at 48 h. although y162a-egfp viruses induced several large syncytia in bhk + mceacam1a(i41r) cells, the number of cells infected by y162a-egfp viruses decreased between 24 and 48 h (fig. 2) . these results suggested that h, q or a substitutions for y162 that reflect residues found at corresponding residues in s330 of rat, bovine or human coronaviruses in group ii altered the interaction of mhv-a59 virions with mceacam1a. the detection of the mutant egfp viruses in 17 cl 1 cells prompted us to examine the plaque forming ability of the y162h, y162q and y162a-egfp viruses on 17 cl 1 cell monolayers. both replicates of the sa59, y162a and y162f-egfp viruses formed plaques that exhibited egfp expression in 17 cl 1 cells monolayers (data not shown). both of the y162f-egfp viruses formed large, 4-5 mm plaques on 17 cl 1 cell monolayers like mhv-a59 (fig. 3) . however, both of the y162a-egfp viruses formed minute, 0.5 mm plaques that were difficult to visualize with neutral red staining, but were readily apparent using immunoperoxidase labeling of viral nucleocapsid (n) protein in 17 cl 1 cell monolayers (fig. 3) . the yields of the y162a-egfp viruses from 17 cl 1 cells were 10,000-fold lower than those of the sa59 and y162f-egfp viruses. consequently, the egfp viruses were not studied further. to identify mutant viruses that were free of adventitious mutations in the s gene, we sequenced the s genes of plaque-purified recombinant viruses derived from mutant pmh54 constructs. point mutations were found in the s genes of the sa59 a, s33g a and b, l65h a and b, l65a a and b and l79a/t82a a viruses ( table 2 ). the s genes of the sa59 b, s33r a, s33g c, t62s a, t62a a, l65h c, l65a c, l79m/t82m a, l79a/ t82a b and k183g a viruses were free of adventitious mutations, while the s genes of the y162f a and k183r a viruses had only non-coding adventitious mutations ( table 2 ). the lack of adventitious mutations in the s genes of the s33g c, l79m/t82m a, b, and c, and k183g a, b, and c viruses suggests that second-site mutations in the s gene were not responsible for the observed ability of these viruses to form plaques on 17 cl 1 cells after their recovery from bhk + mceaca-m1a(i41r) cell monolayers. most of the plaque-purified recombinant viruses released between 2 ã� 10 6 and 2 ã� 10 7 pfu/ml into the tissue culture medium by 48 h p.i., except for all three replicates of the l79a/t82a virus that released only 1 ã� 10 3 pfu/ml, as determined by plaque assay on 17 cl 1 cell monolayers. to determine whether aa substitutions in the rbd of s altered the growth of mhv-a59 in murine cells, we examined the plaque morphologies of the plaque-purified recombinant viruses on 17 cl 1 cell monolayers. when examined using neutral red stained cells, the sa59, s33r, l79m/t82m, y162f and k183r viruses formed clear plaques like mhv-a59, whereas the s33g, t62s, t62a and k183g viruses formed turbid plaques (fig. 4) . the plaque-purified l65h and l65a viruses formed opaque plaques that were difficult to visualize with neutral red staining, but were readily apparent using immunoperoxidase labeling of viral n protein in 17 cl 1 cell monolayers. most of the recombinant viruses formed large, 4-5 mm plaques at 72 h like mhv-a59, whereas the three l79a/t82a viruses formed small, 2.9 mm plaques (fig. 4) . the plaque morphologies shared by the three replicates of each mutant virus indicate that the engineered mutations in s330 were responsible for the observed phenotypes of these viruses. differences in plaque morphology could be caused by alterations in receptor binding or membrane fusion activities of mutant s proteins on virions. to investigate whether aa substitutions in the rbd of s altered the susceptibility of mhv-a59 virions to neutralization by soluble mcea-cam1a, we incubated the sa59 b, s33r a, s33g c, t62s a, t62a a, l65h c, l65a c, l79m/t82m a, y162f a, k183r a and k183g a viruses at neutral ph and 37 8c with 70 nm of purified, anchorless mceacam1a [1, 4] . this concentration of mceacam1a[1,4] was 100-fold higher than the nd 50 for mhv-a59, but did not neutralize the extended host range variant mhv/bhk (schickli et al., 1997; zelus et al., 1998) . like mhv-a59, all of the recombinant viruses were neutralized by mceacam1a [1, 4] at neutral ph and 37 8c (fig. 5) .the ability of soluble mceacam1a to neutralize the l79a/t82a b virus was not measured due to the low titer of this virus. the neutralization of the mutant viruses by soluble mceacam1a [1, 4] at neutral ph and 37 8c suggested that the soluble receptor bound to mutant s1 proteins on the recombinant virions and induced a conformational change in s2 like that observed for mhv-a59 (gallagher, 1997; matsuyama and taguchi, 2002; zelus et al., 1998 zelus et al., , 2003 . to further characterize the mceacam1a utilization of the mutant viruses, we measured the yields of the sa59 b, s33r a, s33g c, t62s a, t62a a, l65h c, l65a c, l79m/t82m a, l79a/t82a b, y162f a, k183r a and k183g a viruses from 17 cl 1 cells treated with anti-mceacam1a mab-cc1. mab-cc1 binds to the nterminal domain of mceacam1a using an epitope that overlaps, but is not identical to, the virus-binding site (dveksler et al., 1993a (dveksler et al., , 1993b wessner et al., 1998) . like mhv-a59, none of the recombinant viruses infected 17 cl 1 cells in the presence of mab-cc1 (fig. 6) . thus, aa substitutions at s33, t62, l65, l79/t82, or k183 in s did not prevent binding of mhv-a59 virions to soluble or anchored mceacam1a. since some of the aa substitutions in the rbd of s were chosen to reflect residues found in s330 of related rat, bovine or human coronaviruses in group ii (fig. 1a) , we examined the ability of the sa59 b, s33r a, s33g c, t62s a, t62a a, l65h c, l65a c, l79m/t82m a, l79a/ t82a b, y162f a, k183r a and k183g a viruses to infect non-murine cells lines that are normally resistant to mhv-a59 infection. as determined by immunofluorescence labeling of viral n protein and plaque formation, none of the mutant viruses infected rat (rie), human (hrt-18g) or hamster (bhk) cells. rie cells were susceptible to infection by rat coronavirus (rcov), hrt-18g cells were susceptible to infection by bovine coronavirus and human coronavirus strain oc43, while all three cell lines were susceptible to infection by the extended host range variant mhv/bhk (schickli et al., 1997) . in addition, viruses derived from the y162h, y162q and y162a constructs did not infect rie, hrt-18 or bhk cell lines. thus, aa substitutions at s33, t62, l65, l79/t82, y162 or k183 did not extend the host range of mhv-a59. we used comparative analysis of s proteins from coronaviruses in group ii to identify residues in the receptor binding domain, s330, that may be important for the mceacam1a specificity of mhv virions. to study the biological significance of aa substitutions in the rbd of s, we used targeted rna recombination (trr) to generate isogenic recombinant viruses that differ from mhv-a59 at one or two residues in s330. aa substitutions were selected by identification of five residues, s33, l79, t82, y162 and k183, that are highly conserved in s330 of seven mhv strains and the extended host range variant mhv/bhk, but not in s330 of related group ii coronaviruses of rats, cattle and humans. since mhv-a59 does not bind or infect rat, bovine or human cell lines and rcov does not bind to or utilize mceacam1a (compton et al., 1992; gagneten et al., 1996) , we reasoned that these residues might be important for the interactions of mhv-a59 with mceacam1a. in this study, we found that y162 in the rbd of s is critical for the recovery of mhv-a59 from cells expressing mceacam1a. fcwf cells inoculated with fmhv and transfected with pmh54-egfp or pmh54-egfp rnas encoding y162h, y162q, y162a or y162f substitutions showed limited egfp expression when plated alone (data not shown). however, fmhv-inoculated fcwf cells transfected with pmh54-egfp or pmh54-egfp rnas encoding y162h, y162q, y162a or y162f substitutions that were overlaid onto 17 cl 1 or bhk + mceacam1a(i41r) cell monolayers showed increased cpe and egfp expression (fig. 2) . these data strongly suggested that the majority of cpe and egfp expression observed was due to infection of 17 cl 1 or bhk + mceacam1a cells. recombinant viruses with y162h and y162q substitutions inefficiently infected 17 cl 1 and bhk + mceacam1a(i41r) cell monolayers (fig. 2) and these viruses were not recovered from either 17 cl 1 or bhk + mceacam1a(i41r) cells (table 1) . although viruses with y162a substitutions were recovered from 17 cl 1 and bhk + mceacam1a(i41r) cells, the y162a viruses formed minute plaques on 17 cl 1 cell monolayers (fig. 3) and had markedly lower yields than mhv-a59. in contrast, f substitutions for y162 were well tolerated by the s proteins of mhv-a59, since the y162f viruses infected both 17 cl 1 and mceacam1a(i41r) cells as well as mhv-a59 (figs. 2-4 and 6) . these data suggest that the phenyl ring of y162 may form an essential hydrophobic contact with the mhv-binding domain of mceacam1a, and that this hydrophobic contact is disrupted in s proteins with h, q or a substitutions at y162. alternatively, it is possible that h, q or a substitutions at y162 may alter the local conformation or stability of s proteins, since a s310g substitution in the s of mhv-jhm is associated with decreased s1-s2 stability (ontiveros et al., 2003) and a q294l substitution in the s of infectious bronchitis virus has been shown to inhibit maturation and incorporation of s proteins into virions (shen et al., 2004) . additional studies will be needed to determine whether y162 participates directly in mcea-cam1a binding, triggers the fusion activity of s and/or maintains the conformation of the rbd of the mhv s protein. in this study, we found that viruses with s33r, l79a/ t82a or k183r substitutions in the rbd of s were readily recovered from 17 cl 1 cells. the s33r and k183r viruses had wild-type growth in 17 cl 1 cells, while the l79a/t82a viruses formed small plaques on murine cell monolayers (fig. 4) and had reduced viral yields compared to mhv-a59. viruses with s33g, l79m/t82m or k183g substitutions in s330 were never recovered from 17 cl 1 cells. however, these viruses could initially be recovered from bhk + mceacam1a(i41r) cells that express 10-fold more receptor than 17 cl 1 cells or bhk cells stably expressing wild type mceacam1a. the s33r, s33g, l79m/t82m, k183r and k183g viruses were neutralized like mhv-a59 by soluble mceacam1a at neutral ph and 37 8c (fig. 5 ). in addition, just like mhv-a59, the s33r, s33g, l79m/t82m, l79a/t82a, k183r and k183g viruses bound mcea-cam1a using an epitope that overlaps that of the blocking anti-mceacam1a mab-cc1 (fig. 6) . the recovery, growth and plaque phenotypes shared by each independently generated replicate of each mutant virus strongly suggests that the engineered mutations in s330, rather than adventitious mutations outside of the s gene, were responsible for the observed phenotypes of these viruses. the failure of the s33g, l79m/t82m and k183g viruses to be recovered from 17 cl 1 cells may have been due to impaired interactions between mutant s proteins and mceacam1a. during trr, feline fcwf cells infected with fmhv and transfected with mutant mhv-a59 rnas may generate virions whose envelopes contain both chimeric fmhv s proteins and mutant mhv-a59 s proteins due to phenotypic mixing. the phenotypically heterogeneous s33g, l79m/t82m and k183g virions may have impaired interactions with mceacam1a. however, phenotypically homogenous s33g, l79m/t82m and k183g virions, generated in bhk + mceacam1a cells, may regain the ability to interact with mceacam1a on 17 cl 1 cells. alternatively, the high level of receptor expression on bhk + mceacam1a(i41r) cells may facilitate the recovery of these crippled viruses by increasing the number of receptors available on cell membranes. in a similar manner, increased expression of its cellular receptor partially restores the infectivity of a friend murine leukemia virus with aa substitutions in its receptor binding pocket (davey et al., 1999) . in addition, the i41r substitution in the nterminal domain of mceacam1a may facilitate the recovery of crippled viruses with mutant s proteins. several residues in s330 that may be important for binding to mceacam1a have previously been identified. a single t62s substitution or a triple t212s, y214s, y216s substitution in soluble s330 of mhv-jhm reduced binding to soluble mceacam1a in vitro (suzuki and taguchi, 1996) . soluble receptor resistant (srr) mutants of mhv-jhm with l65h substitutions in s330 also had reduced binding to soluble mceacam1a in vitro (saeki et al., 1997) . however, these srr mutants had wild-type interactions with anchored mceacam1a expressed in hamster cells (matsuyama and taguchi, 2000) . in this study, we found that isogenic recombinant mhv-a59 viruses with t62s, t62a, l65h or l65a substitutions had wild-type growth in 17 cl 1 cells and were neutralized, like mhv-a59, by soluble mceacam1a at neutral ph and 37 8c (figs. 5 and 6) . the t62s and t62a viruses formed turbid plaques on 17 cl 1 cell monolayers, while the l65h and l65a viruses formed opaque plaques (fig. 4) . thus, although t62 and l65 are not essential for mceacam1a utilization by mhv-a59, these residues may nevertheless influence spike-receptor interactions (rao and gallagher, 1998) . we noted that t62 and l65 are conserved in s330 of murine, rat, bovine and human coronaviruses in group ii (fig. 1a) . since mhv-a59 does not bind rat, bovine or human tissues or infect rat, bovine or human cell lines (compton et al., 1992) , t62 and l65, individually, most likely do not determine the receptor specificity of mhv. in this study, we demonstrated that single aa substitutions at s33, t62, l65, y162 or k183 and double aa substitutions at l79 and t82 in the rbd of s of mhv-a59 did not allow the virus to interact with alternative receptors on murine or non-murine cell lines. like wild type mhv-a59, infection of murine cells by viruses with substitutions at s33, t62, l65, l79/t82, y162 or k183 was blocked by anti-mceacam1a mab-cc1 (fig. 6) . none of the viruses with substitutions at s33, t62, l65, l79/t82, y162 or k183 infected rat, hamster or human cell lines. perhaps multiple aa substitutions are needed to change the host range of mhv-a59. we recently showed that 21 aa substitutions and a 7-aa insert in the n-terminal region of s1 of mhv-a59 are sufficient to extend the viral host range (schickli et al., 2004; thackray and holmes, 2004) . isogenic recombinant viruses that differ from mhv-a59 by the 21 aa substitutions and the 7-aa insert in s not only utilize mceacam1a, but also as yet unidentified receptors on murine and non-murine cells . alternatively, residues in the s proteins of rat, bovine and human coronaviruses in group ii critical for receptor binding may not be found in s330. an aspartic acid at residue 454 in the rbd of the distantly related sars-cov s protein is essential for binding to ace2 in vitro (wong et al., 2004) . the mceacam1a-binding site in the mhv s glycoprotein has previously been proposed to be conformationdependent based on the inhibition of mhv infection by conformation-dependent anti-s mabs (suzuki and taguchi, 1996) . we postulate that y162 may comprise part of a hydrophobic pocket or patch in the rbd of s that interacts with hydrophobic residues in the mhv-binding site of mceacam1a. hydrophobic residues such as y, f and w are known to play important roles in spike-receptor interactions of other enveloped viruses such as herpes simplex virus, human immunodeficiency virus and murine retroviruses (carfi et al., 2001; connolly et al., 2003; kwong et al., 1998; wu et al., 1996; zhang et al., 2003) . residues s33, t62, l65, l79, t82 and k183 are most likely located peripheral to this hydrophobic binding site, since binding hot spots in spike-receptor interactions are often surrounded by energetically less critical residues, predominantly charged in nature (bogan and thorn, 1998; wang, 2002) . residues that extend the host range of mhv-a59 may also be located peripheral to the hydrophobic binding site. during persistent mhv infection in murine cells, the important mceacam1a binding residues in s are retained (baric et al., 1999; schickli et al., 1997) , while additional aa substitutions may be selected that enhance the affinity or avidity of s for mceacam1a and allow mhv to utilize as yet unidentified alternative receptors on murine and nonmurine cells. a mouse monoclonal antibody (mab) to the mhv nucleocapsid protein (n) (anti-n mab) was provided by julian leibowitz (department of pathology and laboratory medicine, texas a and m university, college station, tx). mouse anti-mceacam1a mab-cc1 blocks binding of mhv to mceacam1a and infection of murine cells that express mceacam1a, such as 17 cl 1 cells (dveksler et al., 1993a (dveksler et al., , 1993b williams et al., 1990) . a mouse mab directed against the h subunit of cholera toxin (mab-ctrl) was used as an isotype matched control for anti-n mab and mab-cc1. the 17 cl 1 cell line of spontaneously transformed balb/c 3t3 fibroblasts, rat intestinal epithelial (rie) cells, baby hamster kidney bhk-21 (bhk) cells, felis catus whole fetus (fcwf) cells, felis catus lung epithelial (ak-d) cells and african green monkey kidney (vero 76) cells were propagated as previously described (schickli et al., 1997; thackray and holmes, 2004) . human rectal tumor clone g cells (hrt-18g) were kindly provided by johannes storz (department of veterinary microbiology and parasitology, louisiana state university school of veterinary medicine, baton rouge, lo) and propagated in dulbecco's modified eagle medium (dmem; gibco, invitrogen corporation, grand island, ny) supplemented with 5% heat-inactivated fetal bovine serum (fbs; hyclone laboratories, inc., logan, ut), 2% antibiotic-antimycotic (psf; gibco) and 1.5 g/l sodium bicarbonate. to generate bhk cells stably transfected with a murine ceacam1a cdna encoding an i41r substitution [bhk + mceacam1a(i41r)], site-directed mutagenesis of mcea-cam1a [1, 4] in pci-neo (invitrogen) was performed with the mutagenic forward primer 5vctacggctagagac-aaagaaattg, and reverse primer 5vcaatttctttg-tctctagccgtag. bhk cells were transfected with cdna encoding the mceacam1a(i41r) or wild type mcea-cam1a construct using lipofectamine 2000 (invitrogen), as specified by the manufacturer, and selected using 500 ag/ml of geneticin (gibco). stably transfected bhk cells were sorted twice on a cytomation moflo cell sorter (ft. collins, co) for high levels of mceacam1a expression using anti-ceacam1a mab-cc1 followed by phycoerythrin (pe)conjugated goat anti-mouse igg (jackson immunoresearch laboratories, inc., west grove, pa). mhv-a59 and fmhv were propagated in 17 cl 1 and fcwf cells, respectively, as previously described (kuo et al., 2000; schickli et al., 1997) . virus titers were measured by plaque assay on 17 cl 1, fcwf, ak-d, rie or bhk cells as previously described (gagneten et al., 1995; kuo et al., 2000) . virus-inoculated hrt-18g cells were incubated under 0.8% seakem agarose (biowhittaker molecular applications, rockland, me) and mem (gibco) with 8% fbs and 2% psf. virus inoculation of cells grown on coverslips and detection of newly synthesized viral nucleocapsid (n) protein by immunofluorescence were performed as previously described . virus-inoculated cell monolayers were incubated under agar, and plaques were visualized by neutral red staining or immunolabeling of viral n protein in cell monolayers. cell monolayers were washed with isotonic phosphate buffered saline and fixed in methanol/acetic acid at ã�20 8c for 10 min. expression of n protein was detected using anti-n mab followed by biotinylated anti-mouse igg and avidin dh/biotinylated horseradish peroxidase (hrp) h complexes (vectastain elite abc kit; vector laboratories inc., burlingame, ca). avidin-hrp complexes were visualized by deposition of 3,3v-diaminobenzidine (dab; vector laboratories inc., burlingame, ca). s330 sequences of mhv strains and the corresponding domains of other group ii coronaviruses were obtained using the following genbank accession numbers: mhv-a59 (ay497328), mhv-jhm (x04797), mhv-1 (d83333), mhv-2 (d83334), mhv-3 (d83335), mhv-s (d83337), mhv-u (d83336), mhv/bhk (ay497331), rcov (aaf97738), bcov strain l9 (p25191) and hcov-oc43 (z21849). deduced aa sequences were aligned and five residues that were highly conserved in s330 of all mhv strains, but not in s330 of other group ii coronaviruses were identified (fig. 1a) . the s constructs used in this paper were assembled in pbc sk+ (stratagene, la jolla, ca) (fig. 1b) and used to replace the s gene of pmh54 (kuo et al., 2000) or pmh54-egfp (das sarma et al., 2002) . the transcription vector pmh54-egfp, containing the 3v-most 7.4 kb of the mhv-a59 genome and the gene for enhanced green fluorescent protein (egfp) in place of gene 4, was kindly provided by susan weiss (department of microbiology, university of pennsylvania school of medicine, philadelphia, pa). s gene sequences in this paper were numbered according to genbank accession number ay497328 (schickli et al., 2004) . site-directed mutagenesis was used to introduce point mutations into a cdna encoding the s protein, the s/pbc sk+ construct containing an avrii site at nt 37 . mutant s constructs were amplified with cloned pfu dna polymerase (pfu; stratagene, la jolla, ca) using various primer pairs (table 3 ) essentially as previously described (wentworth and holmes, 2001) . mutant s constructs were screened by restriction enzyme digestion and/or sequence analysis. all 14 s constructs were subcloned into the s gene of pmh54 using avrii and a draiii site at nt 1271. y162h, q, a and f mutant s constructs were also subcloned into pmh54-egfp. the 5v ends of each construct were sequenced by the university of colorado cancer center dna sequencing and analysis core facility using abi prism kits (applied biosystems, foster city, ca) as previously described (wentworth and holmes, 2001) . six primers: sstart, g133d-, a59.3, a59.4, a59.30 and a59.22 (schickli et al., 2004; thackray and holmes, 2004) were used to generate overlapping sequences for each construct. mutations were introduced into the genome of mhv-a59 by targeted rna recombination (trr) as previously described . briefly, fcwf cells were inoculated with the chimeric helper virus fmhv, transfected with pmh54 or pmh54-egfp derived donor rnas containing engineered mutations in the s gene, and immediately overlaid onto 17 cl 1 or bhk + mceaca-m1a(i41r) cell monolayers in individual 25-cm 2 flasks. after 48 h, culture media were collected, clarified by centrifugation, and flash frozen. independently recovered replicates were plaque-purified and propagated in 17 cl 1 cells. total cellular rna from 17 cl 1 cells inoculated with each of the recombinant viruses was extracted and reverse transcribed as previously described . the cdna was amplified with primers sstart and a59.6 or a59.7 and 4a(igs) as previously described . amplification products were sequenced by the university of colorado cancer center dna sequencing and analysis core facility as described above. eleven primers: 572, a59.3, a59.30, a59.c7, s(xbai)+, a59.20, s(noti)+, a59.19, a59.10, a59.11, and bz25 [table 3 and (schickli et al., 2004; thackray and holmes, 2004) ] generated overlapping sequences for each s gene. virions were incubated with purified, anchorless mcea-cam1a[1,4] essentially as described previously (zelus et al., 1998) . briefly, 30 al of virus (5000 pfu) were incubated with 180 al of soluble mceacam1a diluted in trisbuffered saline with 5% glycerol (tbs-g) and 0.1 mg/ml bovine serum albumin fraction v (bsa) or tbs-g with 0.1 mg/ml bsa alone. after incubation for 1 h at 37 8c, virus survival was determined by plaque assay on 17 cl 1 cell monolayers. percent virus neutralization was calculated as: 100 ã� [(number of plaques from virus incubated with mceacam1a/number of plaques from virus incubated with buffer alone) ã� 100]. virus inoculation of murine cells in the presence of 0.05 mg/ml anti-ceacam1a mab-cc1 or isotype matched mab-ctrl was performed as previously described (schickli et al., 1997; thackray and holmes, 2004) . amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor persistent infection promotes cross-species transmissibility of mouse hepatitis virus anatomy of hot spots in protein interfaces identification of a receptor-binding domain of the spike glycoprotein of human coronavirus hcov-229e homologue scanning mutagenesis reveals cd66 receptor residues required for neisserial opa protein binding the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex human coronavirus 229e: receptor binding domain and neutralization by soluble receptor at 37 degrees c herpes simplex virus glycoprotein d bound to the human receptor hvea coronavirus species specificity-murine coronavirus binds to a mouse-specific epitope on its carcinoembryonic antigen-related receptor glycoprotein structure-based mutagenesis of herpes simplex virus glycoprotein d defines three critical regions at the gd-hvea/ hvem binding interface enhanced green fluorescent protein expression may be used to monitor murine coronavirus spread in vitro and in the mouse central nervous system identification of a receptorbinding pocket on the envelope protein of friend murine leukemia virus aminopeptidase-n is a major receptor for the enteropathogenic coronavirus tgev cloning of the mouse hepatitis-virus (mhv) receptor-expression in human and hamster-cell lines confers susceptibility to mhv several members of the mouse carcinoembryonic antigen-related glycoprotein family are functional receptors for the coronavirus mouse hepatitis virus-a59 mouse hepatitis-virus strain-a59 and blocking antireceptor monoclonal-antibody bind to the n-terminal domain of cellular receptor interaction of mouse hepatitis-virus (mhv) spike glycoprotein with receptor glycoprotein mhvr is required for infection with an mhv strain that expresses the hemagglutinin-esterase glycoprotein attachment glycoproteins and receptor specificity of rat coronaviruses a role for naturally occurring variation of the murine coronavirus spike protein in stabilizing association with the cellular receptor coronavirus spike proteins in viral entry and pathogenesis ceacam1a(ã�/ã�) mice are completely resistant to infection by murine coronavirus mouse hepatitis virus a59 localization of neutralizing epitopes and the receptor-binding site within the amino-terminal 330 amino-acids of the murine coronavirus spike protein retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier structure of an hiv gp120 envelope glycoprotein in complex with the cd4 receptor and a neutralizing human antibody coronaviridae and their replication targeted recombination within the spike gene of murine coronavirus mouse hepatitis virus-a59: q159 is a determinant of hepatotropism quaternary structure of coronavirus spikes in complex with carcinoembryonic antigen-related cell adhesion molecule cellular receptors angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus interaction between heptad repeat 1 and 2 regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors impaired entry of soluble receptorresistant mutants of mouse hepatitis virus into cells expressing mhvr2 receptor receptor-induced conformational changes of murine coronavirus spike protein n-terminal domain of the murine coronavirus receptor ceacam1 is responsible for fusogenic activation and conformational changes of the spike protein enhanced virulence mediated by the murine coronavirus, mouse hepatitis virus strain jhm, is associated with a glycine at residue 310 of the spike glycoprotein multiple regions of the murine coronavirus spike glycoprotein influence neurovirulence intracellular complexes of viral spike and cellular receptor accumulate during cytopathic murine coronavirus infections identification of a contiguous 6-residue determinant in the mhv receptor that controls the level of virion binding to cells identification of spike protein residues of murine coronavirus responsible for receptor-binding activity by use of soluble receptor-resistant mutants persistent infection of cultured-cells with mouse hepatitis-virus (mhv) results from the epigenetic expression of the mhv receptor the murine coronavirus mouse hepatitis virus strain a59 from persistently infected murine cells exhibits an extended host range the n-terminal region of the murine coronavirus spike glycoprotein is associated with the extended host range of viruses from persistently infected murine cells a single amino acid mutation in the spike protein of coronavirus infectious bronchitis virus hampers its maturation and incorporation into virions at the nonpermissive temperature analysis of receptor-binding site of murine coronavirus spike protein crystal structure of murine sceacam1a[1,4]: a coronavirus receptor in the cea family amino acid substitutions and an insertion in the spike glycoprotein extend the host range of the murine coronavirus mhv-a59 structural characterization of the sars-coronavirus spike s fusion protein core the n-terminal domain of the murine coronavirus spike glycoprotein determines the ceacam1 receptor specificity of the virus strain critical determinants of host receptor targeting by neisseria meningitides and neisseria gonorrhoeae: identification of opa adhesiotopes on the n-domain of cd66 molecules protein recognition by cell surface receptors: physiological receptors versus virus interactions homophilic adhesion of human ceacam1 involves n-terminal domain interactions: structural analysis of the binding site molecular determinants of species specificity in the coronavirus receptor aminopeptidase n (cd13): influence of n-linked glycosylation mutational analysis of the virus and monoclonal antibody binding sites in mhvr, the cellular receptor of the murine coronavirus mouse hepatitis virus strain a59 purification of the 110-kilodalton glycoprotein receptor for mouse hepatitis-virus (mhv)-a59 from mouse-liver and identification of a nonfunctional, homologous protein in mhv-resistant sjl/j mice a 193-amino acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme 2 kinetic and structural analysis of mutant cd4 receptors that are defective in hiv gp120 binding the sars-cov s glycoprotein: expression and functional characterization structural basis for coronavirus-mediated membrane fusion-crystal structure of mouse hepatitis virus spike protein fusion core human aminopeptidase-n is a receptor for human coronavirus-229e mouse hepatitis-virus utilizes 2 carcinoembryonic antigens as alternative receptors heterogeneity of geneexpression of the hemagglutinin-esterase (he) protein of murine coronaviruses purified, soluble recombinant mouse hepatitis virus receptor, bgp1(b), and bgp2 murine coronavirus receptors differ in mouse hepatitis virus binding and neutralizing activities conformational changes in the spike glycoprotein of murine coronavirus are induced at 37 degrees c either by soluble murine ceacam1 receptors or by ph 8 identification of the receptor binding domain of the mouse mammary tumor virus envelope protein the authors are grateful for technical assistance from jason bartsch, jacinta cooper, sonia tusell and mark young. we also thank mk smith and sonia tusell for many helpful discussions.this work was supported by nih grant r01-ai-25231. sequencing of dna samples at the university of colorado cancer dna sequencing and analysis core facility and facs analysis at the university of colorado cancer center flow cytometry core were supported by a nih/nci cancer core support grant (p30 ca046934). key: cord-316460-ibprgdh4 authors: rodríguez-grille, javier; busch, lisa k.; martínez-costas, josé; benavente, javier title: avian reovirus-triggered apoptosis enhances both virus spread and the processing of the viral nonstructural muns protein date: 2014-06-17 journal: virology doi: 10.1016/j.virol.2014.04.039 sha: doc_id: 316460 cord_uid: ibprgdh4 avian reovirus non-structural protein muns is partially cleaved in infected chicken embryo fibroblast cells to produce a 55-kda carboxyterminal protein, termed munsc, and a 17-kda aminoterminal polypeptide, designated munsn. in this study we demonstrate that muns processing is catalyzed by a caspase 3-like protease activated during the course of avian reovirus infection. the cleavage site was mapped by site directed mutagenesis between residues asp-154 and ala-155 of the muns sequence. although muns and munsc, but not munsn, are able to form inclusions when expressed individually in transfected cells, only muns is able to recruit specific arv proteins to these structures. furthermore, munsc associates with arv factories more weakly than muns, sigmans and lambdaa. finally, the inhibition of caspase activity in arv-infected cells does not diminish arv gene expression and replication, but drastically reduces muns processing and the release and dissemination of progeny viral particles. avian reoviruses (arvs) are important pathogens that cause great economic losses in the poultry industry. infection by these viruses has been associated with a variety of disease conditions, including viral arthritis, chronic respiratory diseases, and malabsorption syndrome (jones, 2000; van der heide, 2000) . arvs are members of the orthoreovirus genus, one of the 12 genera of the reoviridae family. they are non-enveloped fusogenic viruses whose particles contain 10 double-stranded rna genome segments enclosed within two concentric protein shells (benavente and martinez-costas, 2007) . viral genomic segments can be separated electrophoretically into three different size classes, named l (large; three segments), m (medium; three segments) and s (small; four segments). the arv genome expresses at least 8 structural and four non-structural proteins (bodelon et al., 2001) , but the protein repertoire of arv is increased to at least 12 structural proteins and six non-structural proteins by posttranslational cleavage of some viral proteins (busch et al., 2011; ji et al., 2010; varela et al., 1996) . genome replication and assembly of arvs takes place in distinctive cytoplasmic globular inclusions called viral factories where viral components concentrate to increase the efficiency of these processes (benavente and martinez-costas, 2006; touris-otero et al., 2004a) . the framework of the factories is thought to be formed by the non-structural muns protein, because it is the only viral protein that accumulates in factory-like inclusions when expressed individually in transfected cells (touris-otero et al., 2004b) . furthermore, the fact that viral core protein lambdaa and the non-structural protein sigmans redistribute to inclusions when individually co-expressed with muns in transfected cells suggests that muns is able to recruit these proteins to viral factories of infected cells (touris-otero et al., 2004a) . similar to many other viruses, arvs induce apoptotic cell death of infected cells, and the activation of the intracellular apoptotic program takes place during an early stage of the arv life cycle (labrada et al., 2002) . apoptosis is triggered by arv in the absence of viral gene expression, but is no longer induced when intracellular viral uncoating is blocked, suggesting both that apoptosis does not depend on viral protein synthesis and that it is triggered from within the infected cell by viral products generated after intraendosomal uncoating of parental reovirions (labrada et al., 2002) . a previous report revealed that the m3 gene of arv expresses three muns isoforms in infected cells and that the two smaller isoforms originate by a specific post-translational cleavage near the amino terminus of muns. this cleavage produces a 55-kda carboxyterminal protein, termed munsc, and a complementary 17-kda aminoterminal polypeptide, designated munsn. cleavage of muns occurs with $30% efficiency, so precursor muns and its cleavage products are all present in arv-infected cells (busch et al., 2011) . in this study we show that muns processing is indirectly promoted by arv infection through activation of an effector caspase that cleaves muns between amino acid residues 154 and 155. we have previously shown that arv muns is cleaved in arvinfected cells to produce the aminoterminal peptide munsn and the carboxyterminal protein munsc (busch, et al., 2011) . to assess whether muns cleavage is promoted by avian reovirus proteins and/or by changes induced in the host during infection, we compared muns-to-munsc conversion in arv-infected cells with that in transfected cells and in insect cells infected with a munsexpressing recombinant baculovirus (fig. 1a ). for this, extracts from cef monolayers either mock-infected (lane 1) or infected with arv s1133 (lane 2), from cef monolayers transfected with either empty pcineo plasmid (lane 3) or pcineo-muns plasmid (lane 4), and from sf9 cells infected with either wild-type baculovirus (lane 5) or recombinant baculovirus acnpv-s1133-muns (lane 6) were subjected to immunoprecipitation (upper panel) and immunoblot (lower panel) analyses. the results revealed that while a significant amount of munsc was generated in arv-infected cells (its position is marked with an asterisk at the left of lane 2), this protein was hardly detected in transfected cells (lane 4) or in insect cells infected with a muns-expressing recombinant baculovirus (lane 6). these data strongly suggest that intracellular processing of muns is promoted by arv infection. two additional experiments were subsequently performed to confirm this suggestion. in the first one, a plasmid expressing gfp fused to the amino terminus of muns (gfp-muns) was lipofected into cef monolayers and 10 h later the monolayers were mockinfected or infected with arv s1133 for 16 h. extracts from these cells were analyzed by western blotting using antibodies specific for both muns and gfp. the results shown in fig. 1b reveal that while cleavage products derived from gfp-muns are not detected in mock-infected cells (lanes 1 and 3) , a $ 45-kda polypeptide that is recognized by antibodies against both gfp and muns is present in arv-infected cells (lanes 2 and 4). its electrophoretic mobility and its capacity to interact with the two antibodies suggest that this polypeptide is gfp-munsn. in the second confirmation experiment ( fig. 1c) , 35 s-labeled in-vitro-generated muns (lane 2) was incubated with extracts obtained from either mock-infected cells (lane 3) or arv-infected cells (lane 4), and the resulting samples, as well as radiolabeled extracts from arv-infected cef that had been immunoprecipitated against muns (lane 1), were analyzed by sds-page and autoradiography. the results revealed that muns-to-munsc conversion took place in the sample incubated an immunoblot analysis of nonradiolabeled samples is shown in the lower panel. (b) cef monolayers are transfected with gfp-muns for 10 h and then mock-infected (lanes 1 and 3) or infected with 10 pfu/cell of arv s1133 for 16 h (lanes 2 and 4). the cells are then lysed in ripa buffer and analyzed by western blot with polyclonal antibodies against both gfp (lanes 1 and 2) and muns (lanes 3 and 4). (c) 35 s-radiolabeled in-vitro-synthesized muns (lane 2) is incubated for 2 h at 37 1c with extracts from mock-infected cells (lane 3) or from arv-infected cells (lane 4). these samples, as well as an immunoprecipitated extract of arv-infected cef (lane 1), are analyzed by 10% sds-page and autoradiography. positions of protein markers are indicated on the left, and those of nonstructural arv proteins on the right. the position of the munsc band is marked with an asterisk. (d) western blot analysis of extracts from cef monolayers either mock-infected (lane 1) or infected for 16 h with 10 pfu/cell of the arv isolates 1733 (lane 2), 2408 (lane 3), and s1133 (lane 4). in lane 5 the analysis is performed with an extract of vero cells infected for 24 h with 50 pfu of arv s1133, and in lane 6 with an extract of df-1 cells infected for 16 h with 10 pfu of arv s1133. the membranes in lanes 1-4 and 6 are exposed for 5 s and the one in lane 5 for 30 s. with extracts from arv-infected cells (lane 4), but not in the one incubated with extracts from mock-infected cells (lane 3). taken together, these results indicate that muns cleavage is promoted by arv infection. the muns protein expressed by three different arv isolates is processed to a similar extent in infected cef cells (fig. 1d , lanes 2-4), indicating that muns processing is not a unique property of the arv s1133 isolate, but a more general property of arvs. to assess whether muns cleavage is influenced by cell-type specific factors we examined muns-to-munsc conversion in cells other than cef. the results revealed that partial cleavage of arv muns also took place in arv-infected monkey vero cells (fig. 1d , lane 5) and in human hela cells (fig. 5a, lane 2) . surprisingly, the load of munsc is highly reduced following infection with arv s1133 of the cefderived avian cell line df-1 (fig. 1d , lane 6), suggesting that muns processing is controlled by cell factors. the immortal df-1 cell line was established spontaneously from line 0 endogenous-virus negative embryos and had been widely used for the propagation of various avian viruses (bacon et al., 2000) . a genome-wide transcription profile of df-1 cells and a comparison of their global gene expression with that of primary cef revealed that df-1 cells are characterized by enhanced molecular mechanisms for cell cycle progression and proliferation, suppressing cell death pathways, downregulation of caspase 3, altered cellular morphogenesis, and accelerated capacity for molecule transport (kong et al., 2011) . the observation that reduced caspase activity of df1 cells is accompanied by diminished muns processing when arv infects these cells raised the interesting possibility that caspase activation and muns cleavage are interlinked events. this hypothesis is reinforced by our finding that muns is not processed when expressed in baculovirus-infected cells (fig. 1a) , probably because baculoviruses express antiapoptotic factors that prevent caspase activation (clem, 2007; clem et al., 1991) , although this awaits experimental confirmation. to explore whether there was a connection between caspase activity and muns processing we first investigated the effect that two broad-spectrum caspase inhibitors, z-vad-fmk and q-vd-oph, exert on these processes when added to arv-infected cells at the onset of infection. the effect of these inhibitors on the apoptotic state of infected cells was monitored by two different approaches. in the first one, we used immunofluorescence analysis of histone h2ax phosphorylation to determine the percentage of cells containing damaged dna (cook et al., 2009; yuan et al., 2010) . for this, the cells were fixed, stained with a monoclonal antibody against phosphorylated h2ax and counterstained with dapi. polyclonal antiserum against the nonstructural muns protein was also used to visualize arv-infected cells ( fig. 2a) . quantification of the stained cells revealed that, similar to staurosporine treatment (lane 5), arv infection caused a 10 fold increase in the percentage of cells containing phosphorylated h2ax (compare lanes 1 and 4). however, this increase was highly attenuated when the infected cells were incubated in the presence of either of the two caspase inhibitors (lanes 2 and 3). in the second approach, we used the luminiscent caspase-glo 3/7 assay kit (promega) for measuring caspase-3/7 activities. the cells were lysed in caspase-glo 3/7 substrate and protease activity was measured as relative light units (rlu). the results shown at the bottom of fig. 2b indicate that infection of cef cells with arv induces a 10 fold increase in caspase activity, but this increase is significantly reduced when the infected cells are incubated in the presence of either of the two pancaspase inhibitors. once demonstrated that the two inhibitors were highly efficient in preventing arv-induced caspase activation, we next examined their capacity to prevent muns processing in arvinfected cells. both immunoblotting and immunoprecipitation analyses of extracts from arv-infected cef revealed that munsto-munsc conversion was dramatically reduced when the cells were incubated in the presence of either of the two inhibitors ( fig. 2c , compare lane 1 with lanes 2 and 3). in subsequent experiments we exclusively used the inhibitor q-vd-oph because it blocks apoptosis at very low nontoxic concentrations, and also because it inhibits caspase activity, but not cathepsin activity (caserta et al., 2003; kuželová et al., 2011) . the results shown in fig. 2d reveal that the ability of q-vd-oph to inhibit both apoptosis and muns processing in arv-infected cef is dosedependent, and further show that the load of munsc is drastically reduced at a q-vd-oph concentration as low as 5 μm. a time course of muns processing and caspase activation performed in arv-infected cells in the presence or absence of q-vd-oph ( fig. 2e ) revealed that even though the presence of muns was first detected at 3 hpi, munsc was not detected until 6 hpi, coinciding with a large increase in caspase 3/7 activity. furthermore, both muns processing and an increase in caspase activity were not observed when q-vd-oph was present during the infection (fig. 2e , þ q). taken together, these results strongly suggest that there is a correlation between arv-induced apoptosis and muns processing. to confirm this suggestion additional experiments were performed. we first observed that the capacity of extracts of arvinfected cells to cleave in-vitro-synthesized muns was largely abolished when the infected cells were incubated in the presence of q-vd-oph (fig. 3a , compare lanes 1 and 2). in addition, the reduced ability of arv to induce muns processing in df-1 cells was significantly enhanced when the cells were incubated during the last 6 h of infection with the pro-apoptotic agents actinomycin d and staurosporine (fig. 3b , compare lane 1 with lanes 2 and 4), but the ability of these compounds to enhance muns processing was blocked in the presence of the apoptotic inhibitor q-vd-oph ( fig. 3b , lanes 3 and 5). finally, these pro-apoptotic agents were also effective in promoting muns processing in transfected cells (fig. 3c ). our findings that caspase inhibitors prevent muns processing in arv-infected cells and that apoptosis enhancers promote muns cleavage in both transfected cells and arv-infected df1 cells indicate that muns cleavage is catalyzed by an effector caspase activated during arv infection. in a first attempt to map the muns cleavage site we compared the electrophoretic mobility of munsc with that of several muns amino-terminal truncations whose translation initiates at different internal methionine codons (fig. 4a ). plasmids expressing these truncations (lanes 3-5) and full-length muns (lane 2) were transfected into cef and the transfected cells, as well as arvinfected cef (lane 1), were incubated with [ 35 s]amino acids at 24 h after transfection or 16 hpi. the cells were lysed with ripa buffer, the extracts were immunoprecipitated with muns-specific antiserum and the resulting samples were analyzed by sds-page and autoradiography. the results reveal that munsc migrates slightly faster than the muns truncation 140-635, suggesting that the cleavage site should be located several residues downstream of met-140 (fig. 4a) . a similar conclusion has been recently reached when comparing the electrophoretic mobility of munsc with that of in-vitro-translated muns truncations (busch et al., 2011) . a survey of the deduced amino acid sequence downstream of met-140 for an appropriate caspase cleavage motif revealed the presence of the sequence 151 dspd↓a 155 (fig. 4b, underlined) , which is a canonical cleavage motif for caspases 3 and 7 (dxxd↓y; "x" indicates any amino acid; "y" indicates g, a, t, s or n) (thornberry et al., 1997; timmer and salvesen, 2007) . to verify that this putative caspase recognition sequence is a bona fide cleavage site we first examined whether the electrophoretic mobility of muns, munsc and munsn matched with that of transiently expressed polypeptides comprising muns residues 1-635, the resulting extracts were immunoprecipitated with polyclonal anti-muns serum and the imunoprecipitated proteins were resolved on an electrophoresis gel system (8% tricine-sds-page gel) specifically designed to resolve small peptides (schägger, 2006) . the results reveal that the muns, munsc and munsn proteins present in arv-infected cells (fig. 4c , lane 1) have the same electrophoretic mobility as that of the munsderived recombinant polypeptides comprising residues 1-635, 155-635 and 1-154, respectively (fig. 4c, lanes 3-5) . it should be however noted that partial conversion of muns to munsc took place in transfected cef cells when the cell monolayers were kept in serum-free medium for more than 5 h during lipofection (fig. 4c, lane 3) . this is probably due to the fact that apoptosis is induced when the cells are incubated for long periods of time in serum-free medium (not shown). to confirm that muns is cleaved after asp-154 we next mutated this residue to ala and investigated abrogation of cleavage. to rule out the possibility that a function of the protein other than proteolysis could be modified by the mutation, we examined the capacity of the d154a mutant to form inclusions and to recruit the viral proteins lambdaa and sigmans to these structures. the immunofluorescent images shown in fig. 4d reveal that the single site mutant d154a maintains the ability of wild-type muns to form inclusions (lane 1) and to recruit the two arv proteins to these structures (compare top and bottom rows of lanes 2 and 3). these observations suggest that the d-to-a mutation does not significantly alter the spatial conformation and activity of the nonstructural viral protein. we next compared the capacity of staurosporine to promote processing of both wild-type muns and its d154a mutant in transfected cells. the results revealed that staurosporine promoted the cleavage of wild-type muns, but not of the mutant d154a, in transfected cells (fig. 4e) . similarly, extracts of arv-infected cef promoted the cleavage of wild-type muns, but not of the d154a mutant (fig. 4f ). taken together, these observations indicate that arv muns is cleaved by caspases between muns residues asp-154 and ala-155. the results presented so far indicate both that muns cleavage is catalyzed by a caspase and that caspase 3 and/or caspase 7 are activated in arv infected cells. our observation that muns cleavage takes place in arv-infected cells of both avian and mammalian origin indicates that muns is a substrate for avian and mammalian caspases. to assess the role of caspase 3 in muns processing we compared muns-to munsc conversion in arv-infected hela and mcf-7 human cells. hela is a caspase 3-competent cell line, whereas mcf-7 is a breast cancer cell line that does not express a functional caspase 3 due to a 125 bp deletion in exon 3 of the caspase 3 gene (jänicke, 2009) . the results of the immunoblot analysis shown in fig. 5a reveal that muns-to-munsc conversion takes place in arv-infected hela cells (lane 2) and that this conversion is prevented by the pancaspase inhibitor q-vd-oph (lane 3). however, muns-to-munsc conversion did not occur in arv-infected mcf-7 cells (lane 5), even when these cells were treated for the last 6 h of infection with pro-apoptotic staurosporine (lane 6). this result suggests that caspase 3 or a protease activated by caspase 3 is directly involved in the cleavage of muns. we next examined whether a recombinant muns protein purified from baculovirus-infected insect cells is a substrate for active caspases 3 and 7 of human origin available commercially. the samples obtained when muns was incubated with these caspases, either in the presence or absence of q-vd-oph, as well as an extract of arv-infected cef, were subjected to western blot analysis with muns-specific antiserum. the results shown in fig. 5b reveal that partial muns-to-munsc conversion occur in the samples incubated with caspase 3, and that this conversion is prevented by the pancaspase inhibitor q-vd-oph. although the result shown in lane 5 of fig. 5b appears to suggest that muns is not a substrate for caspase 7, a faint band with the same electrophoretic mobility as munsc, which disappears in the presence of q-vd-oph, is detected in some caspase 7 incubations after long exposures of the membrane (not shown). we first analyzed the capacity of three muns isoforms to form inclusions and to recruit other viral proteins to these structures. the pictures shown in fig. 6a reveal that both muns and munsc, but not munsn, collect into globular inclusions when expressed individually in transfected cells. furthermore, all muns truncations containing the intercoil region (residues 477-542) including munsc, but not ha-tagged munsn, collected into inclusions when co-expressed with muns (brandariz-nuñez et al., 2010a). however, these results should be taken with caution, since the attached tag could modify the properties of the proteins. on the other hand, the immunofluorescent images presented in fig. 6b show that muns, but not munsc, is able to recruit lambdaa and sigmans to inclusions (compare lanes 2 and 3). this result suggests that the portion of muns corresponding to munsn contains sequences required for the interaction with each of the two arv proteins. we next examined the strength with which muns and munsc associate with arv factories. for this we compared the capacity of triton-x-100-containing buffer (tx) to extract muns and munsc from arv-infected cells. this buffer has been previously used to discriminate between soluble and cytoskeleton-associated mammalian reovirus proteins, as well as soluble and inclusionassociated arv proteins (mora et al., 1987; touris-otero et al., 2004a) . in the first approach, mock-infected (fig. 6c, lanes 1 and 3) and arv-infected cef (fig. 6c, lanes 2 and 4) were lysed with the triton-x-100-containing buffer and the extracts resolved into tx-soluble (lanes 1 and 2) and tx-insoluble (lanes 3 and 4) fractions. the resulting samples were subjected to western blot analysis with muns-specific antiserum. the results revealed that while similar amounts of muns were present in the tx-soluble and -insoluble fractions, most munsc was extracted by the tx buffer. in the second approach, arv-infected cells were pulsed with [ 35 s]amino acids for 10 min, then chased in nonradioactive medium for different time periods, and finally fractionated into tx-soluble and -insoluble fractions. the samples were immunoprecipitated with muns-specific antiserum and analyzed by 8% tricine-sds-page and autoradiography (fig. 6d) . the results revealed that newly-synthesized muns and sigmans distributed between the two fractions, with a slightly higher proportion in the tx-insoluble fraction. in contrast, most munsc and almost all munsn were extracted by the tx buffer at all chasing times. the reliability of the extracting procedure was evidenced by the finding that the structural protein lambdaa coimmunoprecipitated with muns exclusively in the tx-insoluble fraction at all chasing times (lanes 6-8). these findings indicate that muns, sigmans and lambdaa associate with arv factories more tightly than munsc, despite that munsc, but not lambdaa or sigmans, collects into inclusions when expressed individually in transfected cells. it is of interest to determine the role that muns processing plays on arv replication. in the absence of an established reverse genetics system for arv that would allow us to generate a recombinant virus that expresses an uncleavable muns protein, like d154a, we examined the effect of q-vd-oph on arv replication in cef cells, since this compound had been shown to block both apoptosis and muns processing. the western blot shown in fig. 7a reveals that although the presence of the inhibitor from the onset of the infection completely blocks muns processing, it does not significantly alter the intracellular concentration of the arv nonstructural protein sigmans. this result indicates that apoptosis and muns processing do not have significant effects on arv gene expression and on stages of the virus life cycle prior to gene expression. furthermore, the inhibitor did not significantly change the production of infective arv particles, as determined by a virus plaque assay (fig. 7b, total virus) , indicating that both apoptosis and muns processing are not required for the production of infective progeny in cultured cells. we next investigated the effect of q-vd-oph on arv release by titrating the infectious viral particles released into the incubation medium. the results showed that virus release was drastically reduced when the infected cells were incubated in the presence of q-vd-oph (fig. 7b, released virus) , suggesting that arv-induced apoptosis enhances dissemination of progeny reovirions from infected cells. this suggestion was further supported by our finding that the presence of q-vd-oph during plaque assay of an arv stock, where the agar overlay limits virus dissemination to neighboring cells, caused a significant reduction in the size of the viral plaques (fig. 7c) , suggesting that the inhibitor reduces cellto-cell viral spread over the different rounds of replication, release and re-infection that occur during plaque formation. activation of programmed cell death is one of the first protective defences setup by the host cell to restrict viral amplification and spread. when triggered early in infection apoptosis may limit both the time and the cellular machinery available for virus replication. therefore, it is not surprising that many viruses try to overcome the apoptotic threat by using a battery of different strategies and by expressing a variety of antiapoptotic products aimed to block or delay the induction of apoptosis (galluzzi et al., 2010) . however, other viruses induce apoptosis actively at late stage of infection to facilitate virus release and spread with limited induction of inflamatory and immune host responses (teodoro and branton, 1997) . the results shown in fig. 2e confirm previous findings from our laboratory that arv infection triggers apoptosis in cultured cells at an early stage of the viral life cycle (labrada et al., 2002) , yet the results of this study demonstrate that early apoptosis triggering does not have adverse effects on either arv gene expression or infectious progeny production. these apparently conflicting results can be reconciled considering that arv has a short replication time (benavente and martinez-costas, 2006) , which would allow the virus to proceed its life cycle successfully until completion and to reach a satisfactory intracellular production of progeny viral particles before the infected cell dies or is severely damaged by the execution of apoptosis. a growing number of viruses have been documented to take advantage of apoptosis induction and caspase activation to promote their own replication. for instance, viruses as diverse as mammalian reovirus, human astrovirus, measles virus, porcine circovirus 2, african swine fever virus, bovine herpesvirus 1, coronavirus, influenza virus and the moscow strain of ectromelia virus, all have been reported to usurp apoptosis for facilitating the release of progeny viral particles from infected cells (best, 2008; galluzzi et al., 2010) . the results of the present study suggest that arv should be included in that list, since this virus activates rather than suppress caspases and this activation is necessary for efficient virus release and spread. on the other hand, many apoptosisinducing viruses express proteins that are processed by caspases to generate cleavage products with novel properties, ranging from apoptosis downregulation to enhancement or attenuation of viral replication (richard and tulasne, 2012) . still, the functional consequences of many protein caspase cleavages remain elusive so far, yet the conservation of caspase targets in many viral proteins and the stability of their processed products suggest that caspase cleavage of viral polypeptides can constitute an evolutionary advantage that benefits virus replication. in this study we have provided compelling evidence that caspases activated during arvinduced apoptosis catalyze the cleavage of arv nonstructural muns protein to produce the 17-kda n-terminal peptide munsn and the 55-kda c-terminal protein munsc. in the literature to date, there are several examples of other non-structural viral proteins that are caspase targets, like the ns1 protein of aleutian mink disease parvovirus, the ns5a protein of hepatitis c virus, the ns1 0 protein of japanese encephalitis virus, and the eia protein of adenovirus 12 (best et al., 2003; grand et al., 2002; satoh et al., 2000; sun et al., 2012) . we also observed that a substantial amount of full-length muns remains uncleaved both in infected cells and after 6 h of incubation with recombinant caspase 3, implying that caspase-catalyzed muns cleavage is a regulated process, as has been reported for several cellular and viral proteins (best et al., 2003; chaudhry et al., 2012; cheng et al., 2010; mashima et al., 1999; sun et al., 2012) . incomplete processing of a precursor viral protein could be a strategy to reduce the activity of the intact protein or it could be used to increase the protein repertoire encoded by size-limited viral genomes, thus generating cleaved viral products with novel properties/activities. that avian and mammalian reoviruses use different mechanisms to express their munsc isoforms (busch et al., 2011) , and that both the muns precursor and its truncated forms coexist in avian/ mammalian reovirus-infected cells suggest that there should be distinct functional roles for both the precursor and its truncated proteins. it should be noted however that avian and mammalian reovirus munsc proteins display different properties. thus, while mammalian reovirus munsc still maintains its precursor's ability to associate with the major core protein lambda1 (miller et al., 2010) , this ability is not shared by its arv counterpart (fig. 6b) , because sequences of arv muns required for binding to lambdaa are lacking in munsc. it has been reported that the release of astrovirus from infected cells depends on both the processing of the capsid precursor polypeptide vp90 and the activities of executioner caspases (banos-lara and mendez, 2010) . accordingly, the possibility exists that muns processing is required for enhanced release and dissemination of arv, although it is unlikely, since apoptosis has also been reported to promote the release of mammalian reovirus from infected cells without enhancing the production of its munsc protein (marcato et al., 2007) . in the absence of an established reverse genetics system for avian reoviruses, the consequences of muns cleavage on viral replication and spread remain unclear. despite the facts that arv munsc is able to form inclusions when expressed in isolation and that it collects into inclusions when co-expressed with muns (brandariz-nuñez et al., 2010b) , munsc associates with factories of infected cells more weakly than full-length muns, sigmans or lambdaa, as evidenced by extraction with tx buffer (fig. 6c and d) . this observation suggests that arv factories are complex structures formed by a diverse array of protein-protein and protein-rna interactions and that these entities are much more complex than muns-derived inclusions. this is supported by our observation that the inclusions formed by expressing muns versions in baculovirus-infected insect cells can be easily purified (brandariz-nuñez et al., 2010a) , whereas we are unable to purify the factories formed in arvinfected cells. the results shown in fig. 5 demonstrate that muns is a substrate for mammalian caspase 3, yet the absence of both commercial recombinant caspases of avian origin and caspase 3-deficient avian cells does not allow us to identify the avian caspase that catalyzes arv muns cleavage in infected avian cells. nevertheless, this cleavage is probably catalyzed by an avian caspase 3-like protease, since caspase sequences and targets are highly conserved in different species (sakamaki and satou, 2009) , and since we found both that caspase 3/7 is active in arv-infected avian cells and that muns-to-munsc processing is promoted by the caspase 3 activator staurosporine (chae et al., 2000) . the presence of a caspase 3/7 consensus sequence ( 151 dspd↓a 155 ) within the muns cleavage region allowed us to map the muns cleavage site between residues asp-154 and ala-155, by using sitedirected mutagenesis. this finding suggests that this region of muns is placed in a flexible and exposed loop accessible to caspases. interestingly, the caspase 3/7 consensus sequence found in arv s1133 muns is not fully conserved among avian reoviruses isolated from chickens, since it is only present in 8 isolates, and in one quail isolate, but not in other 9 chicken isolates. thus, in the muns protein from 4 chicken isolates asp-151 is replaced by his, while in the protein from another 5 isolates asp-151 and ser-152 are replaced by his and glu, respectively (fig. 8 ). if cleavage of these "nonconsensus" arv muns proteins still occurs between asp-154 and ala-155, it would imply that accessibility is more important than the presence of specific amino acids at positions 3 and 4 of the caspase consensus sequence, which is in agreement with previous reports indicating that caspases do not have a strict requirement for positions 2-4 of the consensus cleavage site (timmer and salvesen, 2007; thornberry et al., 1997) . curiously, the muns protein encoded by both duck and goose reoviruses does not contain a caspase 3/7 consensus sequence spanning residues 151-155, because asp-154 is replaced by a gly residue. however, these muns proteins contain two putative caspase targets, one spanning residues 142-146 ( 142 gtmda 146 ), and the other comprising residues [156] [157] [158] [159] [160] . surprisingly, the former caspase target is also present in the muns protein from four chicken isolates (fig. 8) . it should be of interest to check whether duck/goose reoviruses induce apoptosis, whether the muns protein of these viruses is cleaved in infected cells and whether its cleavage takes place at one or both of these caspase consensus sequences. nevertheless, the presence of specific residues in the muns 140-177 region could serve to assign a poultry reovirus as an avian/quail or a goose/duck reovirus. thus, the muns from reoviruses of avian/quail origin has leucine in position 144, alanine or valine in 150, aspartic acid in 154, cysteine in 156 and valine in 159, whereas the muns from reoviruses of duck/ goose origin has methionine, glycine, glycine and aspartic acid in those positions. primary cultures of cefs were prepared from 9-to 10-day-old chicken embryos and grown in monolayers in medium 199 supplemented with 10% tryptose phosphate broth and 5% calf serum. avian df-1, human hela and mcf-7, and monkey vero cell lines, all were grown in monolayers in dulbecco's modified eagle's medium (dmem; invitrogen) supplemented with 10% fetal bovine serum (fbs). mcf-7 cells were a kind gift from maría dolores blanco (universidad complutense de madrid). sf9 cells were grown in suspension culture in serum-free sf-900 ii media at 27 1c. strains s1133, 1733 and 2408 of avian reovirus were grown in semiconfluent monolayers of primary cefs. conditions for growing and titrating these viruses have been described previously (grande and benavente, 2000) . the recombinant baculovirus acnpv-s1133-muns was grown in sf9 cells as previously described (brandariz-nuñez et al., 2010b) . rabbit polyclonal sera against arv s1133 reovirions and cores, as well as arv s1133 proteins muns and sigmans were raised in our laboratory (touris-otero et al., 2004b) . monoclonal antigamma histone h2a.x (phospho s139) antibody was purchased from abcam plc. rabbit anti-actin polyclonal antibody was purchased from santa cruz biotechnology. peroxidase-conjugated goat anti-rabbit antibody was purchased from sigma. pancaspase inhibitors z-vad-fmk and q-vd-oph were from calbiochem. caspases 3 and 7 were from enzo life science (cat. # c-3: alx-201-059; c-7: alx-201-061-u025). all other reagents were purchased from sigma. semiconfluent monolayers of cells were infected with 10 pfu/ cell of arvs for the times indicated for each experiment. in all experiments, except for some of fig. 1 and those of fig. 5a , cef cell monolayers were infected with the arv s1133. for metabolic radiolabeling, the cultured medium was removed and the cells were incubated for the indicated times in methionine/cysteinefree medium containing 100 μci/ml of [ 35 s]methionine-cysteine. for immunoprecipitation and western blot analyses the cells were lysed in ripa buffer (50 mm tris-hcl ph 7.5, 150 mm nacl, 1% np40, 0.5% doc, 0.1% sds, supplemented with "complete protease inhibitor cocktail" from roche diagnostics), and muns-derived proteins were detected with a muns-specific antiserum (touris-otero et al., 2004b) . to isolate tx-soluble and -insoluble fractions, 2 â 10 6 cells were lysed in 200 μl of tx buffer (10 mm pipes ph 6.8, 3 mm mgcl 2 , 100 mm kcl, 300 mm sucrose, 1% triton x-100, supplemented with "complete protease inhibitor cocktail" from roche diagnostics) and the soluble fraction was removed. the plates were washed again with tx buffer and the insoluble fraction that remained attached to the plates was directly solubilized in 200 μl of ripa buffer (touris-otero et al., 2004a) . for pulse-chase analysis, mock-infected and reovirus-infected cells were incubated for 2 h at 12 h p.i. in medium lacking methionine and cysteine, and then incubated for 10 min in the same medium supplemented with 500 μci/ml [ 35 s]amino acids. the cells were chased for the indicated times in non-radioactive medium supplemented with an excess of nonradiolabelled methionine and cysteine. infection of sf9 insect cells with the recombinant baculovirus acnpv-s1133-muns and purification of recombinant muns have already been described (brandariz-nuñez et al., 2010b) . the construction of recombinant plasmids expressing fulllength arv s1133 muns, gfp-muns, and the aminoterminal truncations comprising muns residues 84-635, 127-635 and 140-635 has already been described (brandariz-nuñez et al., 2010b) . plasmid pcineo-muns was used as template to generate constructs expressing the following proteins. the d154a muns point mutant was generated with the quikchange site-directed mutagenesis kit (stratagene), according to the manufacturer's specifications. the forward primer was: 5 0 -ccaccgctgattcc-cccgctgcctgcgtcccagtcacc-3 0 ; and the reverse primer was: 5 0 -ggtgactgggacgcaggcagcgggggaatcagcggtgg-3 0 . to generate the construct expressing muns(1-154), the forward primer was: 5 0 -gcggaattcatcatggcgtcaaccaagtgg-3 0 ; and the reverse primer was: 5 0 -gcgtctagattaatcgggggaatcagcggtgg-3 0 . to generate the construct expressing muns (155-635), the forward primer was: 5 0 -gcggaattcatcatggcctgcgtcccagtc-3 0 ; and the reverse primer was: 5 0 -gcgtctagatcacagatcatccac-caattcttc-3 0 . the correctness of the recombinant plasmids was confirmed by nucleotide sequencing. transfection of preconfluent cell monolayers was done using lipofectamine plus reagent (invitrogen) following the manufacturer's instructions, with 1 μg of dna per well of a 12-well dish. transfected cells were incubated at 37 1c for 24 h, unless otherwise stated. cell monolayers grown on coverslips were infected or transfected as indicated in the figure legends. at the indicated times, the monolayers were washed twice with pbs and fixed in paraformaldehyde for 15 min at 4 1c. fixed cells were washed twice with pbs, incubated for 10 min in permeabilizing buffer (0.1% triton x-100 in pbs), incubated 20 min with blocking buffer (2% bsa in pbs), and then incubated for 1 h at room temperature with primary antibodies diluted in blocking buffer. the cells were washed three more times with pbs and then incubated with secondary antibodies and dapi (4,6-diamidino-2-phenylindole) for 1 h at room temperature. coverslips were then washed six times with pbs and mounted on glass slides. images were obtained with an olympus dp-71 digital camera mounted on an olympus bx51 fluorescence microscope. images were processed with adobe photoshop (adobe systems). the recombinant plasmids used as templates were linearized with noti, purified by extraction with phenol/chloroform, precipitated with ethanol and resuspended in sterile water at a final concentration of 1 mg/ml. in vitro transcription from the t7 promoter was performed by using a ribomax large scale rna production system (promega). in vitro translation was carried out by using a rabbit reticulocyte lysate system (nuclease-treated; promega) following the manufacturer's instructions, for 90 min at 30 1c in the presence of 50 mg/ml rna and 0.4 mci/ml [ 35 s] methionine (hartmann analytic ksm-01). the intracellular activity of caspases 3/7 was determined with the caspase-glo s 3/7 assay kit from promega, following the manufacturer's instructions. in vitro caspase cleavage assays were performed by mixing 1 μg of muns purified from baculovirusinfected insect cells, as described previously (brandariz-nuñez et al., 2010a) , with 1 unit of recombinant caspase in 10 μl of caspase buffer (20 mm hepes ph 7.4; 100 mm nacl; 0.05% np-40). in some samples 10 μm of the pancaspase inhibitor q-vd-oph was also included. mixtures were incubated for 4 h a 37 1c, mixed with 5 μl of 3x laemmli electrophoresis sample buffer and boiled for 5 min at 100 1c. cleavage of muns was analyzed by western blotting. to evaluate the capacity of extracts of arv-infected cells to promote muns cleavage, extracts from mock-infected and infected cells were prepared as follows. samples of 2 â 10 7 cef cells, mockinfected or infected with 0.1 pfu/ml of arv s1133, in the presence or absence of 10 μm q-vd-oph, were washed twice with pbs at 24 hpi and overlayed with 1 ml of caspase buffer. the cells were then collected by scrapping and lysed by one cycle of freezing and thawing. a typical protease cleavage assay was performed by mixing 5 μl of cell extract with 5 μl of reticulocyte lysate containing 35 s-radiolabeled muns or its d154a mutant. samples were incubated for 2 h at 37 1c, then supplemented with 5 μl of 3x laemmli sample buffer and boiled for 5 min at 100 1c. processing of muns was monitored by sds-page and autoradiography. competitividad (bfu2010-22228) and from the xunta de galicia (cn 2012/018). a review of the development of chicken lines to resolve genes determining resistance to diseases role of individual caspases induced by astrovirus on the processing of its structural protein and its release from the cell by a non-lytic mechanism early steps in avian reovirus morphogenesis avian reovirus: structure and biology viral subversion of apoptotic enzymes: escape from death row caspase cleavage of the non-structural protein ns1 mediates replication of aleutian mink disease parvovirus the avian reovirus genome segment s1 is a functionally tricistronic gene that expresses one structural and two nonstructural proteins in infected cells a versatile molecular tagging method for targeting proteins to avian reovirus muns inclusions. use in protein immobilization and purification avian reovirus muns protein forms homo-oligomeric inclusions in a microtubule-independent fashion, which involves specific regions of its c terminal domain avian and mammalian reoviruses use different molecular mechanisms to synthesize their μns isoforms q-vd-oph, a broad spectrum caspase inhibitor with potent antiapoptotic properties molecular mechanism of staurosporine-induced apoptosis in osteoblasts prostate apoptosis response 4 (par-4), a novel substrate of caspase 3 during apoptosis activation the capsid proteins of aleutian mink disease virus activate caspases and are specifically cleaved during infection baculoviruses and apoptosis: a diversity of genes and responses prevention of apoptosis by a baculovirus gene during infection of insect cells tyrosine dephosphorylation of h2ax modulates apoptosis and survival decisions viral strategies for the evasion of immunogenic cell death caspase-mediated cleavage of adenovirus early region 1a proteins optimal conditions for the growth, purification and storage of the avian reovirus s1133 mcf-7 breast carcinoma cells do not express caspase-3 intracellular cleavage of sa protein of avian reovirus avian reovirus infections genome-wide differential gene expression in immortalized df-1 chicken embryo fibroblast cell line dose-dependenteffects of the caspase inhibitor q-vd-oph on different apoptosis-related processes avian reoviruses cause apoptosis in cultured cells: viral uncoating, but not viral gene expression, is required for apoptosis induction ras transformation mediates reovirus oncolysis by enhancing virus uncoating, particle infectivity, and apoptosis-dependent release caspase-mediated cleavage of cytoskeletal actine plays a positive role in the process of morphological apoptosis localization of mammalian orthoreovirus proteins to cytoplasmic factory-like structures via nonoverlapping regions of μns association of avian reovirus proteins with the structural matrix of infected cells caspase cleavage of viral proteins, another way for viruses to make the best of apoptosis caspases: evolutionary aspects of their functions in vertebrates cleavage of hepatitis c virus nonstructural protein 5a by a caspase-like protease(s) in mammalian cells tricine-sds-page japanese encephalitis virus ns1 0 protein depends on pseudoknot secondary structure and is cleaved by caspase during virus infection and cell apoptosis regulation of apoptosis by viral gene products a combinatorial approach defines specificities of members of the caspase family and granzyme b. functional relationships established for key mediators of apoptosis caspase substrates avian reovirus morphogenesis occurs within viral factories and begins with the selective recruitment of sns and λa to μns inclusions avian reovirus nonstructural protein μns forms viroplasm-like inclusions and recruits protein sns to these structures the history of avian reovirus intracellular posttranslational modifications of s1133 avian reovirus proteins focus on histone variant h2ax: to be or not to be we would like to thank rebeca menaya for technical assistance and other members of our laboratory for helpful discussions on the manuscript. we also thank laboratorios msd españa (salamanca, spain) for providing pathogen-free embryonated eggs. this work was funded by grants from the ministerio de economia key: cord-303238-us3dybue authors: kanjanahaluethai, amornrat; chen, zhongbin; jukneliene, dalia; baker, susan c. title: membrane topology of murine coronavirus replicase nonstructural protein 3 date: 2007-05-01 journal: virology doi: 10.1016/j.virol.2006.12.009 sha: doc_id: 303238 cord_uid: us3dybue mouse hepatitis virus (mhv) is a member of the family coronaviridae. these positive strand rna viruses encode a replicase polyprotein that is processed into 16 nonstructural proteins (nsps). the nsps assemble with membranes to generate double membrane vesicles, which are the sites of viral rna synthesis. mhv nsp3 contains multiple domains including two papain-like protease domains, plp1 and plp2, and a predicted transmembrane (tm) domain. in this study, we determined the membrane topology of nsp3-tm and showed that tm-mediated tethering of plp2 is important for processing at cleavage site 3. biochemical analysis revealed that nsp3 is an integral membrane protein that is inserted into endoplasmic reticulum (er) membranes co-translationally and glycosylated at asparagine-2357. proteinase k digestion experiments indicate that the tm domain of nsp3 has 4 membrane-spanning helices. we show that nsp3-tm is sufficient to mediate er membrane association of a cytosolic protein. this study is the first detailed analysis of the topology and function of the coronavirus nsp3 tm domain. proteolytic processing of a replicase polyprotein and the generation of a membrane-associated replication complex are common themes in studies analyzing the replication of positive strand rna viruses. for the majority of coronaviruses, the proteolytic processing of the viral replicase polyprotein is mediated by three distinct viral proteinases to generate 16 replicase products [reviewed in (ziebuhr, 2005) ]. both coronavirus and the related arterivirus replicase products have been shown to assemble with cellular membranes to generate double-membrane vesicles that are the sites of viral rna synthesis (goldsmith et al., 2004; gosert et al., 2002; pedersen et al., 1999; snijder et al., 2006) . the goal of our research is to characterize the coronavirus replicase proteolytic processing cascade and identify factors that mediate membrane-association of the replication complex. our model system is the replication of mhv, one of the prototype coronaviruses. mhv is a ∼31.5-kilobase (kb) positivestrand rnavirus that replicates in the cytoplasm of infected cells. the 5′-most 22 kb of the mhv genomic rna contains two large open reading frames (orfs), termed orf1a and orf1b (lee et al., 1991) . during translation of the genomic rna, the two orfs are joined via a ribosomal frameshifting mechanism to produce a polyprotein of ∼800 kilodaltons (kda) in size (brierley et al., 1987; lee et al., 1991) . this polyprotein is the viral rnadependent rna polymerase, termed the replicase. the mhv replicase is processed by three distinct proteases contained within orf1a of the replicase polyprotein. two of the proteases are papain-like cysteine proteases, termed plp1 and plp2. the third protease domain is distantly related to the picornavirus family of 3c-like proteases, and is termed 3clpro. these three proteases process the replicase polyprotein to produce intermediates and products that function in the replication of genomic rna and the synthesis of a nested set of subgenomic mrnas [reviewed in (brian and baric, 2005; sawicki and sawicki, 2005) ]. studies have shown that the replicase products alone are sufficient to mediate viral rna synthesis , and to generate the double-membrane structures that serve as the sites for viral rna synthesis (pedersen et al., 1999) . however, the role of proteolytic processing in regulating the assembly and function of the mhv replication complex is not yet understood. therefore, we wanted to identify regions of the replicase polyprotein that may direct or regulate proteolytic processing and/or membrane association. the focus of this study was to identify the regions of mhv nsp3 that direct membrane association and plp2 activity. previously, we showed the mhv plp2 can act in trans to process cleavage site 3 (cs3) at the nsp3/nsp4 junction (kanjanahaluethai and baker, 2000; kanjanahaluethai et al., 2003) . other coronaviruses, such as the human coronavirus 229e, have been shown to utilize plp2 (also termed pl2pro) to process sites both upstream and downstream of the catalytic domain (ziebuhr et al., 2001) . the papain-like protease (plpro) encoded by the coronavirus that causes severe acute respiratory syndrome (sars-cov) processes three sites in the replicase polyprotein (harcourt et al., 2004) , and has recently been shown to have de-ubiquitinating activity (barretto et al., 2005; lindner et al., 2005) . the crystal structure of this enzyme has been resolved and is currently being targeted for anti-viral drug development (ratia et al., 2006) . a better understanding of the coronavirus papain-like proteases may facilitate anti-viral drug development for sars and also other recently identified human coronavirus infections caused by nl63 (van der hoek et al., 2004) and hku1 (woo et al., 2005) , which can cause pneumonia and respiratory tract infections in children and the elderly. the aims of this study were to determine the topology of mhv nsp3 and to identify the regions in nsp3 required for plp2 activity. we used a trans-cleavage assay to determine if an expressed plp2 domain was sufficient for the recognition and processing of a substrate containing cs3. we found that constructs containing plp2 and the downstream putative tm domain were able to efficiently process the substrate. bioinformatic analysis of the nsp3-tm domain indicated the presence of putative membrane-spanning helices and consensus sites for n-linked glycosylation. site-directed mutagenesis of the asparagine residues and analysis of endoglycosidase h (endo h) sensitivity revealed that asparagine-2357 in nsp3-tm is glycosylated. to investigate the topology of nsp3-tm, we tested for sensitivity to digestion with proteinase k and found that at least two lumenal domains are protected, consistent with 4 membranespanning regions in nsp3-tm. we showed that nsp3-tm alone is sufficient to confer membrane association to a normally cytosolic protein, enhanced green fluorescent protein (egfp), and that a predicted multi-spanning tm domain is conserved in nsp3 of all coronaviruses. thus, the nsp3-tm domain is important for membrane association of the replicase and tethering the plp2 domain for viral polyprotein processing activity. the mhv nsp3-tm domain is important for plp2 processing at cleavage site 3 analysis of sars-cov plpro activity showed that the nsp3 hydrophobic domain downstream of plpro is essential for processing at cs3 (harcourt et al., 2004) . to determine if a similar domain is important for mhv plp2 activity, we generated a series of plp2 c-terminal deletion constructs and tested each construct for expression and processing activity. plp2 expression constructs were generated by polymerase chain reaction (pcr) amplification and cloning of the amplified region into pcdna3.1/v5-his, as described in materials and methods. to determine if each clone was expressing a protein of the expected size, we analyzed the products after t7-mediated expression and immunoprecipitation (fuerst et al., 1986; kanjanahaluethai and baker, 2000) . we detected the expected series of truncated plp2 proteins that ranged in size from ∼96 kda to ∼ 50 kda (fig. 1b, lanes 1-6) . to determine if these plp2 products were sufficient to mediate processing of cs3, we tested each construct in the trans-cleavage assay by co-transfection with the substrate (fig. 1c, lanes 7-12) . we found that only two of the plp2 expression products, pplp2-2485 and pplp2-2390, were able to efficiently process the substrate and produce the 44-kda cleavage product, nsp4 (fig. 1c, lanes 7 and 8). these two constructs encompass all or a major part of the predicted nsp3-tm domain indicating that membrane tethering of plp2 is important for recognition and processing at cs3. thus, both sars-cov plpro (harcourt et al., 2004) and mhv plp2 require the downstream tm domain for recognition and processing at the nsp3/nsp4 junction. bioinformatic analysis of nsp3-tm predicts a series of putative membrane-spanning sequences previously, we showed that mhv nsp3 is indeed an integral membrane protein, but the role of the tm in mediating this membrane association was not investigated (gosert et al., 2002) . initial bioinformatic analysis indicated two transmembrane helices in nsp3 (ziebuhr et al., 2001) . to extend these studies of membrane association of coronavirus replicase products, we analyzed the amino acid sequence of mhv-jhm nsp3 (from glycine-833 to glycine-2840) for probability of transmembrane helices using the five different programs designed to search for putative membrane-spanning sequences: phobius, tmhmm, hmmtop, sosui and tmpred (fig. 2) . interestingly, each program generated a unique prediction for the topology of nsp3 (fig. 2c ). the number of predicted membrane-spanning domains varied from three (phobius) to seven (tmpred). however, since both the n-and c-termini of nsp3 are cleaved in the cytosol, the number of membrane-spanning helices must be either two [as previously predicted (ziebuhr et al., 2001) ], four or six. to better understand the topology of the nsp3-tm domain, we performed membrane-association, fractionation and proteinase k protection experiments. to determine if the nsp3-tm is indeed required for membrane association, we performed in vitro transcription and translation of the plp2 expression constructs in the absence or presence of canine microsomal membrane (cmm) and assayed for membrane association. the newly translated proteins were metabolically radiolabeled with [ 35 s]-translabel, subjected to centrifugation to separate the membrane-associated pelleted fraction from the soluble fraction. protein products of both fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page), and visualized by autoradiography (fig. 3) . the percentage of total proteins detected in soluble and pelleted fractions was quantitated by phosphoimaging analysis. in the absence of cmm, the translated protein products were detected predominantly in the soluble fraction (fig. 3a) . in contrast, when cmms were added to the mixture, protein products that included all or part of nsp3-tm (plp2-2485, -2390 and -2258) were detected predominantly in the pelleted fraction, consistent with membrane association (fig. 3b ). to determine if the membrane association occurred co-translationally or post-translationally, we added the cmms after termination of translation, and assessed membrane association. previous studies with hepatitis c virus revealed post-translational insertion of the ns5a replicase protein (brass et al., 2002) . we found that the translation product of plp2-2485 is inserted co-translationally, with no detectable post-translational insertion into membranes (fig. 3c ). these results indicate that mhv nsp3 membrane insertion likely occurs via the normal, signal recognition particle (srp)-dependent er translocation mechanism, and not via a tail-anchoring or post-translational mechanism. to distinguish between membrane association versus integral membrane insertion, membrane extraction experiments were performed. protein products expressed from pplp2-2485, pplp2-2390 and pplp2-2258 constructs were translated in vitro in the presence of cmm and the pelleted fraction was subsequently subjected to differential extraction methods as indicated in fig. 4 . as expected, treatment with nte buffer alone had no effect and the proteins pelleted with membranes, whereas treatment with detergent triton x-100 disrupted the membranes, and the proteins were detected in the soluble fraction (fig. 4a) . treatments with urea, nacl or sodium carbonate, ph 11.5 have been shown to disrupt the association between peripheral, membrane-associated proteins, but such treatments do not disrupt integral membrane proteins (bordier, 1981) . we tested for disruption of plp2-tm proteins and found that majority of the proteins remained in the pelleted membrane fraction, consistent with integral membrane proteins (fig. 4b ). we noted that the protein product expressed from pplp2-2258, which contains only one or two predicted membrane spanning helices, was dissociated by treatment with high ph, but that the protein products expressed from constructs that extended further into the tm were not disrupted from the membranes by these treatments, consistent with the characteristics of integral membrane proteins. bioinformatic analysis also revealed two consensus sequences for n-linked glycosylation [the consensus being nx(t/s), where x is any amino acid (helenius and aebi, 2001) ] in nsp3-tm. if one or more of the predicted sequences was indeed a transmembrane helix, then the sites for n-linked glycosylation could be lumenal, and subject to modification. to determine if either of the putative sites was modified by glycosylation, we the amino acid numbering is according to ncbi accession number nc006852. conserved domains previously identified by comparative sequence analysis (ziebuhr et al., 2001) and the recently described adp-ribose-1ʺ-phosphatase (adrp) domain (saikatendu et al., 2005) are indicated. two putative sites for n-linked glycosylation were predicted at asparagine residues 2281 and 2357. (b) the diagram shows the results of an analysis of the nsp3 for predicted transmembrane helices using tmhmm program. the predicted tm domain designates the boundaries of the putative membrane-spanning regions a, b, c, d, and e. (c) sequence of the plp2-tm (amino acid residues 1610 to 2485) were analyzed for the prediction of transmembrane domains using phobius, tmhmm, hmmtop, sosui and tmpred programs. subjected the proteins expressed from wild-type pplp2-cen (kanjanahaluethai and baker, 2000) and two asparagine-toalanine substitution mutants to endo h treatment (fig. 5) . expressed proteins were radiolabeled with [ 35 s]-translabel, lysates were prepared, and subjected to immunoprecipitation with anti-v5 antibody as described in materials and methods. the plp2-cen protein was either untreated, or treated with endo h for 16 h, then analyzed for mobility by electrophoresis through a 5% polyacrylamide gel. we found that the untreated wild-type protein migrated at approximately 110 kda (lane 1). however, after digestion with endo h, the protein migrated at 108 kda (lane 2), consistent with the loss of one n-linked modification. the plp2-cen-n2357a protein with an asparagine-to-alanine substitution migrated more quickly than the wild type or n2281a protein, indicating that n2357 is the site modified by n-linked glycosylation (compare lanes 3-5). furthermore, proteins generated by endo h treatment of the pplp2-cen-n2357a and pplp2-cen-n2281a migrated more quickly than wild type plp2-cen (lanes 6-8). these results indicate that nsp3 is glycosylated at asparagine-2357. overall, these experiments demonstrate that nsp3-tm does have transmembrane and lumen sequences that can tether the plp2 domain to intracellular membranes. to further investigate the topology of nsp3-tm, we tested for sensitivity to proteinase k digestion. cytosolic domains are sensitive to proteinase k, whereas transmembrane and lumenal domains are protected from digestion with proteinase k. analysis of plp2-2485 revealed two major fragments of 28 kda and 10 kda protected from proteinase k digestion (fig. 6, lane 2) . we noted the predicted size of the tm domain from k2227-y2469 is 28.0 kda, and the predicted size of the k2227-i2308 (a and b helices) is 9.6 kda. analysis of plp2-2390 revealed two protected fragments of 19 kda and 10 kda, respectively (fig. 6, lane 4) . the predicted size of the k2227-s2390 fragment is 18.8 kda. by combining the results of the integral membrane assays (figs. 3 and 4) , the glycosylation assay (fig. 5) , and the proteinase k sensitivity assay (fig. 6a ), we were able to generate a topology model for nsp3-tm that is consistent with all the data (figs. 6b and c). our results indicate that nsp3-tm has four membrane-spanning sequences and two lumenal domains (fig. 6b ) and that the 10 kda fragment likely represents the proteinase k resistant fragment generated by the first two membrane-spanning sequences. our model is most similar to the predicted models generated by the tmhmm and sosui programs with the exception that the fourth predicted membrane-spanning sequence (which is the only domain not consistent in these two predictions) is not a membrane-spanning sequence, but instead remains lumenal, and the final membranespanning domain is in the reverse orientation. our results, and the results of others (hugle et al., 2001; miller et al., 2006) , demonstrate the importance of experimental validation of bioinformatics predictions of membrane-spanning sequences. for example, in the case of dengue virus type 2 nonstructural protein 4b, miller and co-workers found that two computerpredicted transmembrane helices were in fact lumenal and one was glycosylated (miller et al., 2006) . in addition, we note the value of using multiple programs to better estimate the complexity of the bioinformatic prediction. the differences in the predicted models could then be tested experimentally. here, we provide an initial model of nsp3-tm that should be refined by further experimentation. similar studies should also be performed to determine the topology of nsp4 and nsp6, the other coronavirus replicase products with multiple predicted transmembrane helices. finally, to determine if the tm domain of mhv nsp3 was sufficient to confer membrane association to a cytosolic protein, we appended nsp3-tm to egfp and determined the localization of the fusion protein using confocal microscopy (fig. 7) . egfp normally is distributed throughout the cell (fig. 7a) . we found that appending the mhv nsp3-tm sequence to egfp fig. 4 . membrane extraction experiments of the plp2 expressed protein products treated with triton x-100, urea, nacl or sodium carbonate solution ph 11.5. in vitro transcription and translation reactions of pplp2-2485, pplp2-2390 or pplp2-2258 were performed in the presence of cmm. subsequently, reaction mixtures were centrifuged to sediment cmm containing associated plp2 protein. the supernatant were removed and the pellets were resuspended in nte buffer or 0.5% triton x-100 (a), 4 m urea, 1 m nacl or 100 mm sodium carbonate solution ph 11.5 (b) and incubated for 20 min at 4°c. subsequently, membrane sedimentation analyses were performed as described under materials and methods. soluble (s) and pellet (p) fractions were applied in equivalent amounts and subjected to 12% sds-page analysis. quantitation was performed by phosphoimaging and values expressed in % were given at the bottom and depicted as bars. was sufficient to tether it to membranes, as shown by the intense, perinuclear localized signal (fig. 7b ). to determine if the nsp3-tm domain is retained in the er membranes or is transported through the medial golgi, we radiolabeled the efgp-nsp3tm protein in transfected cells, immunoprecipitated the protein and subjected the immunoprecipitated products to endo h. we found that efgp-nsp3tm is sensitive to treatment with endo h, indicating that the protein is retained in the er and does not pass through the medial golgi. thus, the nsp3-tm domain is sufficient to confer membrane-localization and retention in the er. these studies are in agreement with our previous findings showing that the tm domain of sars-cov nsp3 (previously termed the hd) confers membrane association of egfp (harcourt et al., 2004) . in summary, using biochemical fractionation and proteinase k protection assays, we show that nsp3-tm likely has four membrane-spanning domains, and that lumenal residue asparagine-2357 is modified by glycosylation. furthermore, we found the region nsp3-tm domain is required for efficient mhv plp2 process activity at cleavage site 3 in the polyprotein. why does mhv plp2 require membrane association for proteolytic processing of the plp2 cleavage site? one possible explanation is that membrane-tethering brings plp2 into close proximity with a membrane-associated substrate. it is also possible that the nsp3-tm membrane tether is important for anchoring the replicase complex to intracellular membranes. bioinformatic analysis of the nsp3 of 10 other coronaviruses revealed that the membrane-spanning features of nsp3-tm are conserved in all viruses (fig. 8 , analysis using the tmhmm program is shown as an example), even though the amino acid identity is relatively low (18-32% identity within nsp3). therefore, the tm domain is likely to be important for both plp2 activity and assembly of the replication complex. further studies will be required to determine the precise topology of the tm domain in other coronaviruses, and if the lumenal sequences in nsp3-tm play any role in interacting with host factors during viral replication. hela cells expressing the mhv receptor, hela-mhvr cells (gallagher, 1996) were used for all transfection experiments. the cells were grown in dulbecco's modified eagle's medium supplemented with 10% fetal bovine serum (invitrogen, carlsbad, ca), 0.5% penicillin/streptomycin, 2% glutamine, and 5 mm sodium n-2-hydroxyethylpiperazine-n′-2ethanesulfonic acid, ph 7.4. recombinant plasmid dna constructs expressing the plp2 coding region were generated using specific primers (listed in table 1 ) to amplify the designated region from the parental plasmid pplp2-cen (kanjanahaluethai and baker, 2000) . the region of interest was generated by pcr amplification using la-taq polymerase according to the manufacturer's instructions (clontech, palo alto, ca). the amplified region was then digested with restriction enzymes bamhi and xhoi and ligated into the corresponding sites in the pcdna3.1/v5-his expression vector (stratagene, la jolla, ca) using t4 ligase (new england biolabs). the ligated dna product was transformed into xl-1 blue competent cells according to the manufacturer's instructions (stratagene), except that the bacteria were grown at 25°c. hela-mhvr cells were infected with a recombinant vaccinia virus expressing the bacteriophage t7 polymerase (vtf7-3) at a multiplicity of infection of 10. then, infected cells were co-transfected with recombinant plasmid dnas encoding the mhv-jhm indicated protease domain and the substrate using lipofectamine according to manufacturer's instruction as previously described (fuerst et al., 1986; kanjanahaluethai and baker, 2000) . newly synthesized proteins were metabolically labeled with 50 μci/ml [ 35 s]-translabel (icn, costa mesa, ca) from 5.5 to 10.5 h post-infection (hpi). to harvest the cells, radioactive labeled cells were washed with phosphate buffered saline (pbs), and cell lysates were prepared by scraping the cells in lysis buffer a [4% sds, 3% dtt, 40% glycerol and 0.065 m tris, ph 6.8 (schiller et al., 1998) ]. the lysates were either used directly for immunoprecipitation assays or stored at − 70°c for future studies. radiolabeled cell lysate was diluted in 1.0 ml ripa buffer [0.5% triton x-100, 0.1% sds, 300 mm nacl, 4 mm edta, and 50 mm tris-hcl, ph 7.4 (schiller et al., 1998) ] and subjected to immunoprecipitation with anti-v5 monoclonal antibody (invitrogen) and protein-a sepharose beads (amersham biosciences, piscataway, nj). the immunoprecipitated products were eluted with 2× laemmli sample buffer, incubated at 30°c for 30 min, and analyzed by electrophoresis on a 5.0-12.5% gradient polyacrylamide gel containing 0.1% sds. following electrophoresis, the gel was fixed in 25% methanol-10% acetic acid, enhanced with amplify (amersham biosciences) for 60 min, dried, and exposed to kodak x-ray film at − 70°c. site-directed mutagenesis of putative glycosylation sites in mhv-jhm nsp3-tm domain plasmid dna pplp2-cen which encompasses mhv-jhm gene 1 amino acid residues 1525-2485 (kanjanahaluethai and baker, 2000) was subjected to site-directed mutagenesis at positions 7055 and 7056 for pplp2-n2281a and positions 7283 and 7284 for pplp2-n2357a using synthetic oligonucleotides with mismatches encoding specific nucleotides changes as shown in table 1 . mutagenesis was performed according to the manufacturer's instructions (quickchange site-directed mutagenesis; stratagene), and as previously described (kanjanahaluethai and baker, 2000) . mutations were confirmed by dna sequence analysis. the tnt t7-coupled reticulocyte lysate system (promega, madison, wi) was used according to the manufacturer's instructions. the recombinant plasmid dna encoding the designated plp2 region was linearized by digestion with pmei. in vitro transcription and translation was performed for 90 min at 30°c in the presence of 0.8 μci of [ 35 s]-translabel per ml in a volume of 25 μl. where indicated, 1.0 μl of cmm (promega) was added prior to the incubation. for analysis of membrane association, the products of in vitro transcription and translation were centrifuged at 14,000 rpm for 10 min. the supernatant was removed, the pellet that may contain aggregated or membrane-associated protein was suspended in 2× laemmli sample buffer, heated at 95°c for 5 min, and both fractions were analyzed by sds-page and subjected to autoradiography. protection of translation products by microsomal membranes was examined by digestion with proteinase k (hugle et al., 2001) . following translation, reaction mixtures (after incubation with rnase a) were adjusted to 0.5 mg/ml of proteinase k (roche, indianapolis, in) and incubated for 30 min on ice. proteinase k digestion was terminated by addition of phenylmethylsulfonyl fluoride to 1 mg/ml and incubation was continued for 5 min on ice. a portion of each reaction mixture (generally 4 μl) was mixed with 40 μl of 2× laemmli sample buffer, heated at 95°c for 5 min, analyzed by 15% sds-page and subjected to autoradiography. for endo h treatment, lysates from vtf7.3-infected and pplp2-tm transfected cells were prepared and subjected to immunoprecipitation as described above. protein-a sepharoseantibody-antigen complexes were washed once in ripa buffer and endo h treatment was performed as suggested by the manufacturer (roche). briefly, the complexes were resuspended in 20 μl of 50 mm sodium phosphate buffer, ph 6.0, and incubated in the presence or absence of a final concentration of 1 unit/μl of endo h for 16 h at 37°c. following the incubation, 25 μl of 2× laemmli sample buffer was added to each sample, mixed, and incubated for 30 min at 37°c. the sepharose beads were pelleted by a brief, high-speed spin in a microfuge, and the supernatant loaded directly for analysis by 12% sds-page and subjected to autoradiography. for membrane extraction experiments, the pellets from 15 μl in vitro transcription-translation reactions performed in the presence of cmm were resuspended in 40 μl nte buffer, 0.5% triton x-100, 4 m urea, 1 m nacl, or 100 mm sodium carbonate (ph 11.5) and incubated for 20 min at 4°c (hugle et al., 2001) . subsequently, supernatant and pellet fractions were separated by centrifugation at 14,000 rpm for 10 min, and analyzed by sds-page and autoradiography. quantitation was performed by using phosphoimaging analysis. the putative transmembrane domain 1 region of mhv-jhm nsp3 (nt 6896 to 7648) was pcr amplified from the pplp2-cen (kanjanahaluethai and baker, 2000) with primers b410 and b411 (table 1) , cloned into the mammalian expression vector for egfp, pegfp-c1 (bd biosciences), and designated pegfp-nsp3tm. the plasmid dna encoding egfp or egfp-nsp3tm was transfected into hela-mhvr cells in 8well chamber culture slides with lipofectamine according to manufacturer's instructions, for 48 h. expression of egfp and egfp-nsp3tm fusion protein products was detected by confocal microscopy (zeiss lsm 510 lazer-scanning confocal microscope). egfp-nsp3tm was amplified by primers zcp1 and zcp2 (table 1) using pegfp-nsp3tm as template, and then cloned into bamhi and xbai sites of pcdna3.1/v5-hisb (invitrogen) to generate the construct of pcdna3.1-egfp-nsp3tm. the plasmid dna was expressed via the vaccinia virus-t7 expression system, proteins radiolabeled with 35 stranslabel, cell lysates were subjected to immunoprecipitation with anti-v5 antibody, and products were either incubated with endo h or buffer alone, and analyzed by electrophoresis on 10% sds-page. the papain-like protease of severe acute respiratory syndrome coronavirus has 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authors: sungsuwan, suttipun; jongkaewwattana, anan; jaru-ampornpan, peera title: nucleocapsid proteins from other swine enteric coronaviruses differentially modulate pedv replication date: 2020-01-15 journal: virology doi: 10.1016/j.virol.2019.11.007 sha: doc_id: 316134 cord_uid: lkd2mj27 porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev) and porcine deltacoronavirus (pdcov) share tropism for swine intestinal epithelial cells. whether mixing of viral components during co-infection alters pathogenic outcomes or viral replication is not known. in this study, we investigated how different coronavirus nucleocapsid (cov n) proteins interact and affect pedv replication. we found that pdcov n and tgev n can competitively interact with pedv n. however, the presence of pdcov or tgev n led to very different outcomes on pedv replication. while pdcov n significantly suppresses pedv replication, overexpression of tgev n, like that of pedv n, increases production of pedv rna and virions. despite partial interchangeability in nucleocapsid oligomerization and viral rna synthesis, endogenous pedv n cannot be replaced in the production of infectious pedv particles. results from this study give insights into functional compatibilities and evolutionary relationship between cov viral proteins during viral co-infection and co-evolution. porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev) and porcine deltacoronavirus (pdcov) belong to the family coronaviridae (enjuanes, 2000) . pedv and tgev have been classified into the alphacoronavirus genus, whereas pdcov belongs to the deltacoronavirus genus (jung et al., 2016a; jung and saif, 2015a) . they share similar genome architectures, with a 25-30 kb positivesense, single-stranded rna genome. the 5′ two-thirds of the viral genome encodes non-structural proteins from open reading frames (orf) 1a and 1b necessary for viral genome replication. the rest of the genome encodes a number of unique accessory proteins such as pedv orf3, tgev 3a/3b/7, pdcov ns6/ns7, and four common structural proteins, namely the spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins (kocherhans et al., 2001; lee and lee, 2014; penzes et al., 2001) . these enteric swine coronaviruses (covs) infect epithelial cells lining the small intestine and cause villous atrophy, resulting in malabsorption and severe diarrhea (jung et al., 2016a ). an outbreak of these viruses, especially pedv, can lead to up-to-100% mortality in neonatal piglets, prompting huge economic losses in the swine production industry worldwide. unless they are examined by laboratory-level diagnosis, these covs produce almost indistinguishable pathogenesis. co-infection of enteric pathogens are common. tgev and pdcov have been found to co-circulate with pedv in the field (song et al., 2015; wang et al., 2016) . in pdcov-positive samples, the rate of pedv co-infection as detected by rt-pcr varies from 33% to 50% (jung et al., 2016a; jung and saif, 2015a) . although tgev infection has become rarer nowadays, it has still been detectable in samples in china, and often together with pedv and/or pdcov (dong et al., 2015; wang et al., 2013) . despite substantial epidemiological evidence of co-infection, the effects of these events on disease outcomes have not yet been formally described. since these enteric swine covs share cell tropism, co-infection of these viruses can theoretically cause mixing of viral components in the same cellular compartments, possibly leading to direct or indirect effects on viral replication kinetics or pathogenic outcomes. to the best of our knowledge, there are currently no reports on studies at molecular or cellular levels on how viral components from different cov species interact with or affect other viruses. investigation of possible molecular interactions between components of pedv, pdcov and tgev and their influence on replication of each virus would provide a crucial insight into comprehensive understanding of these covs. of all viral proteins, we have chosen to start with the n protein, as it is among the most abundant and ubiquitous structural proteins in infected cells. the cov n protein is functionally conserved across the family coronaviridae (chang et al., 2009; cong et al., 2017) , with its primary function being to form a scaffold for packaging viral genomic https://doi.org/10. 1016/j.virol.2019.11.007 received 20 september 2019; received in revised form 4 november 2019; accepted 5 november 2019 rna (grna) into the internal core of virions (de haan and rottier, 2005) . besides scaffolding, other functions of the cov n protein (primarily based on studies of common representatives of the family like severe acute respiratory syndrome-cov (sars-cov) or mouse hepatitis virus (mhv)) include acting as rna chaperones (zuniga et al., 2007 (zuniga et al., , 2010 , promoting viral genome transcription or replication (hurst et al., 2010 (hurst et al., , 2013 masters et al., 1994; zuniga et al., 2007 zuniga et al., , 2010 , facilitating viral assembly (de haan and rottier, 2005; kuo et al., 2016) , suppressing antiviral rna-interference activity from their hosts (cui et al., 2015) , and suppressing host immunity (ding et al., 2014 (ding et al., , 2017 xu et al., 2013; zhang et al., 2018) . based on sequence alignment and limited structural data from some representative covs, all cov n proteins are predicted to consist of three structural domains: the n-terminal domain (ntd), linker region (lkr) and c-terminal domain (ctd) (chang et al., 2014; mcbride et al., 2014) . ntd binds rna through electrostatic interaction with its charged amino acids as well as interaction between conserved aromatic residues in the proteins and nucleotide bases in the rna (chang et al., 2014; huang et al., 2004; tan et al., 2006) . lkr is a disordered domain between ntd and ctd. studies of sars-cov n indicate roles for lkr in rna binding, virion assembly and self-association (chang et al., 2009 (chang et al., , 2013 he et al., 2004a) . although it is also reported to have rna binding capacity, ctd is a more hydrophobic domain mainly responsible for self-association to form stable dimers and subsequent oligomers of cov n (chen et al., 2007; yu et al., 2006) . studies of n proteins from sars-cov and mhv revealed that n dimerization followed by multimerization is a common process in the formation of viral ribonucleoprotein that initiates viral genome packaging in the virion assembly process among covs (cong et al., 2017; fan et al., 2005; ma et al., 2010; yu et al., 2006) . examples from other viruses suggest that cross-association between viral nucleocapsid proteins could determine different outcomes during co-infection. for instance, mixed infection between two types of plant tospoviruses results in interspecies interaction between their n proteins and more severe symptoms compared to single infections, suggesting synergy between these viruses (bag et al., 2012; tripathi et al., 2015) . on the other hand, intertypic interference between types a and b influenza viruses could be partially explained by the inhibitory effect of type b influenza virus nucleoprotein exerted on its type a counterpart (aoki et al., 1984; jaru-ampornpan et al., 2014) . given structural similarities, we expect that cov n proteins would likely cross-interact with each other during a co-infection event. whether this cross-interaction has beneficial or detrimental effects on viral replication has never been explored. investigation of the interaction between cov n proteins could lead to insights into virus evolution, anti-cov drug development and vaccine design (chang et al., 2016; lo et al., 2013) . human embryonic kidney (hek) 293t cells, wild-type veroe6 cells and veroe6-based cell lines stably expressing cov n were maintained in optimem supplemented with 10% fetal bovine serum (fbs) and antibiotics at 37°c with 5% co 2 . the virus pedv-avct12-mcherry (pedv-mcherry), its infectious clone (psmart-bac-mcherry-pedv avct12 [ppedv-mcherry]) and pcaggs-pedv n-myc, a plasmid for c-terminally myc-tagged pedv n expression, have been described previously (jaru-ampornpan et al., 2017; jengarn et al., 2015) . tgev and pdcov n encoding genes were codon-optimized for high expression in mammalian cells and to avoid expression of pdcov ns7 embedded in the pdcov n gene ) (genbank: aag30228.1, afd29191.1, respectively). pcr products of the corresponding n genes with indicated tags at the c-termini were ligated into pcaggs vectors to give plasmids for expression of tagged cov n (pcaggs-tgev n-flag and pcaggs-pdcov n-ha). pcaggs-ha-pedv n for n-terminally ha-tagged pedv n expression was similarly constructed based on the same pedv n dna sequence in pcaggs-pedv n-myc. pgex-4t3-pedv n was constructed by in-frame insertion of the pedv n gene sequence, codon-optimized for bacterial expression based on the sequence from avct12 strain (accession number lc053455), into the pgex-4t3 vector (amersham) following the coding sequence of n-terminal gst. all plasmids were verified by dna sequencing. cells were lysed with lysis buffer (25 mm tris-hcl ph 7.4, 150 mm nacl, 1 mm edta, 1% np-40 and 5% glycerol, supplemented with a protease inhibitor cocktail (halt™ protease inhibitor cocktail, thermo scientific)). proteins in the cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page), and then transferred onto nitrocellulose membranes (bio-rad laboratories). the membrane was blocked in 5% skim milk prior to incubation with indicated primary antibodies. horseradish peroxidase (hrp)-conjugated goat anti-mouse igg (biolegend) or (hrp)-conjugated donkey antirabbit igg (biolegend) was used as secondary antibodies. primary antibodies used in this study include mouse-anti-myc (thermo scientific), rabbit-anti-flag (cell science technology), rabbit-anti-ha (cell science technology), mouse-anti-s1 (a kind gift from dr. qigai he, huazhong agricultural university), and mouse-anti-pedv n (sd 6-29, medgene labs). hek293t cells were co-transfected with pcaggs-pedv n-myc (or the empty pcaggs vector for negative controls) and either pcaggs-ha-pedv n, pcaggs-tgev n-flag or pcaggs-pdcov n-ha using fugene hd (promega) according to the manufacturer's instructions. at 48 h post-transfection (hpt), cell lysates were prepared as described previously and pre-adsorbed to control agarose beads (thermo scientific) for 1 h at 4°c to reduce non-specific binding. then, the treated lysates were incubated with anti-myc-conjugated agarose beads (thermo scientific) at 4°c overnight. the mixtures were washed three times with lysis buffer supplemented with 250 mm nacl. ip complexes were eluted by boiling the bead mixtures in 2× non-reducing sample buffer (thermo scientific) for 5 min and supplemented with 2 mm dtt before sds-page and western blot analysis. gst and gst-pedv n were expressed in bl21 (de3*) cells. after 3h protein induction by 0.5 mm isopropyl ß-d-1-thiogalactopyranoside, bacteria were lysed by sonication in 1× phosphate-buffered saline (pbs). the lysates were clarified and stored in 100-μl aliquots at -80°c until use. hek293t cells were transfected with plasmids as indicated and were lysed at 48 hpt with lysis buffer. the bacterial lysate containing the bait protein (gst/gst-pedv n) and the hek293t cell lysate containing the prey protein (ha-pedv n, tgev n-flag, pdcov n-ha, or combinations thereof) were incubated with pre-equilibrated gsh ff resin (amersham) overnight and washed twice in high-salt gst buffer and four times in low-salt gst buffer (20 mm tris-hcl ph 8.0, 20% glycerol, 1 mm edta, 0.1% np-40 and 1 m/100 mm nacl) (nguyen and goodrich, 2006) . the bound proteins were released by boiling the resin in 2× sds-page loading buffer and analyzed by coomassie staining (for bait) and western blotting with indicated antibodies (for prey). figures were representatives of three independent experiments. for fig. 2 , quantification of eluted protein bands in each gst pulldown reaction was performed using the biorad image lab software 6.0.1 and expressed as the ratios of pulled-down pedv n (western blot with anti-ha) to eluted gst-pedv n (coomassie stain). because of variability between independent experiments, these ratios were normalized to the conditions without extra cov n protein performed side-by-side in each experiment before statistical comparison. for the rnasea experiment, the combined lysates were treated with 10 μg of rnasea (thermo scientific) or pbs buffer for 30 min at 4°c to avoid protein precipitation at high temperature. to verify the absence of rna after the treatment, total rnas were purified from parts of treated lysates by rna purification kit (thermo scientific) and were used at equal volumes as templates for rt-pcr. the rt-pcr products were visualized by ethidium bromide staining. veroe6 cell lines expressing pedv n-myc, tgev n-flag and pdcov n-ha were constructed using the same protocol as veroe6-pedv n cells as described previously (liwnaree et al., 2019) . briefly, lentiviruses carrying each cov n gene were generated by co-transfection of psin-csgw-ubem carrying the inserted cov n gene with a packaging plasmid encoding gag, pol, rev and tat (pcmv-δr8.91) and a plasmid expressing lentiviral vsv envelope glycoprotein (pmd2.g) into hek293t cells. at 48 hpt, supernatants containing the lentiviruses were harvested and filtered through a 0.45-μm filter. the filtered supernatants were then used to transduce veroe6 cells. a single clone of the transduced cells expressing the corresponding n protein was selected based on similar levels of the transgene protein expression. uniform expression of exogenous n proteins in the engineered veroe6 cell lines was verified by immunofluorescence analysis. briefly, cells were grown in an eight-well slide. at 24 h, cells were fixed with 80% cold acetone for 10 min. after washing twice with pbs, cells were blocked with 10% fbs, 1% bovine serum albumin (bsa) in pbs for 1 h at room temperature. cells were then incubated with mouse anti-pedv n antibodies (sd 6-29) or anti-pdcov n (sd55-197, medgene labs) or anti-tgev n (211.56, median diagnostics, republic of korea). after washing twice with pbs, cells were incubated with goat antimouse igg alkaline phosphatase antibodies (abcam). fluorescence was developed by adding alkaline phosphatase substrate (immpact™ vector® red). fluorescence images were taken under a fluorescence microscope (olympus). the n-deficient pedv infectious clone (ppedv-mcherry-δn) was constructed using a strategy described previously (jengarn et al., 2015; wanitchang et al., 2019) . a frameshift mutation was used to silence n expression by mutating the original start codon in the n orf, atggct, to atatgt in an intermediate plasmid, ptz-gh, containing a pedv genome fragment (designated 'gh'; fig. 6a ) with the 5′ end of the n gene as described previously (jengarn et al., 2015) . the site-directed mutagenesis was performed in the ptz-gh cloning vector. the mutated fragment was amplified by primers that facilitated subsequent in-fusion ligation (in-fusion hd, clontech) into a pre-digested psmart-bac plasmid containing the rest of the pedv genome (jengarn et al., 2015) to yield psmart-bac-pedv-mcherry-δn (designated ppedv-mcherry-δn). hek293t cells were transfected with 2 μg of the indicated infectious clone (or co-transfected with the indicated amount of a cov n expressing plasmid) using fugene hd (promega) according to the manufacturer's instructions. at 72 hpt, cell lysates were prepared for western blot analysis as described previously, and supernatants were transferred for adsorption on veroe6 cells for 1 h at 37°c. for viral rescue of pedv-mcherry-δn, veroe6 cells stably expressing pedv n was further transfected with 2 μg of pcaggs-pedv n for 24 h before virus adsorption to ensure sufficient expression of pedv n in the infected cells. the inocula were then removed, and the cells were washed once with pbs and supplied with 2 ml fresh optimem with 0.1% tryple (thermo scientific). at indicated time points, infected cells were imaged under a fluorescence microscope (olympus), and the supernatants were collected for subsequent experiments or viral titer analysis by tcid 50 assay. veroe6-based cell lines (5×10 5 cells/ml) were plated in a six-well plate. at 24 h, the cells were washed once with pbs and treated with 1 ml of virus at the indicated multiplicity of infection (moi). at 1 h post-adsorption, the inoculum was removed, and the cells were washed once with pbs and supplied with 2 ml of fresh optimem containing 0.1% tryple. extent of pedv infection was monitored by mcherry fluorescence under a fluorescence microscope. cell lysates or supernatants were harvested at the indicated time points for further analysis. for viral infection in the transient cov n expression experiment, veroe6 cells were transfected with 1 or 2 μg of pcaggs-pedv n-myc, pcaggs-pdcov n-ha, pcaggs-tgev n-flag, or the empty pcaggs vector and were incubated for 24 h to allow for protein expression. cells were then infected with pedv-mcherry (moi = 0.0001) as described above. monolayers of veroe6 cells in 96-well plates were washed once with pbs. one hundred microliters of 10-fold serially diluted virus in optimem with 0.1% tryple was added to the cells (8 wells per each dilution). at 72 h post-infection (hpi), the infected cells were scored by mcherry expression under a fluorescence microscope. tcid 50 titers were calculated using the reed-muench method (reed and muench, 1938) . for analysis of viral rna synthesis, total rna was extracted from veroe6 cell lines expressing various n infected with pedv-mcherry (moi = 0.0001) at indicated time points using the rna extraction kit (thermo scientific). dnasei (fermentas) was used to treat the rna (15 min at 37°c) before inactivation with edta (10 min at 65°c). onestep rt-qpcr was performed with the luna universal one-step rt-qpcr mix (new england biolabs) as described previously (liwnaree et al., 2019) . relative quantities of rna accumulation were evaluated using the 2 -δδct method normalized against viral rna levels from infected veroe6 cells at 24 hpi. values are reported as averages ± sem from three independent experiments. during co-infection, viruses can influence the course of infection of other viruses via direct physical interactions between viral components. to explore the possibility that swine cov n proteins might affect replication of other co-infecting covs, we first investigated cross-species interaction between these n proteins. specifically, we asked if n proteins from tgev or pdcov can interact with pedv n and possibly affect pedv replication. we first assessed interspecies protein-protein interaction by co-immunoprecipitation. hek293t cells were co-transfected with pcaggs-pedv n-myc and pcaggs expressing ha-pedv n, other cov n proteins bind competitively to pedv n. gst pulldown by gst-pedv n was performed with hek293t cell lysates containing ha-pedv n and varying amounts (0-2 μg transfected plasmids) of pdcov n-ha or tgev n-flag. the input and eluates were separated by sds-page. coomassie staining was used to detect gst-pedv n, and western blotting was used to detect haand flag-tagged proteins. the figure is representative of three independent experiments. quantification was performed using the biorad image lab software 6.0.1 as described in materials and methods. the pulldown ratios from the conditions without pdcov or tgev n proteins in each independent experiment were set to one. *p < 0.05, **p < 0.01 compared to the conditions without pdcov or tgev n proteins. fig. 3 . effect of rna on homo-and hetero-oligomerization of pedv n. gst pulldown by gst-pedv n was performed with hek293t cell lysates containing ha-pedv n, pdcov n-ha or tgev n-flag. the lysate mixtures were treated with 10 μg rnasea (+) or buffer (-) for 30 min at 4°c prior to incubation with gsh ff resin. the input and eluates were analyzed with sds-page (coomassie staining for gst-pedv n) and western blotting for the other n proteins. rna was extracted from a portion of the lysate mixtures, treated with dnasei, and subjected to rt-pcr with primers specific to the nucleocapsid genes studied in each experiment. expected rt-pcr products were about 1200 bpand were analyzed by agarose gel electrophoresis. pdcov n-ha or tgev n-flag. at 48 hpt, lysates were prepared and incubated with agarose beads coupled to anti-myc antibodies prior to elution and analysis by western blotting. as expected, ha-tagged pedv n could be precipitated with myc-tagged pedv n but not with empty beads, indicating specificity of the homo-oligomeric interaction (fig. 1a) . interestingly, both n proteins from related covs also displayed specific interaction with pedv n, both co-eluting with myctagged pedv n (fig. 1a) . nevertheless, we observed slight non-specific binding in the case of pdcov n-ha despite stringent washing conditions. therefore, we utilized a gst pulldown assay as an alternative and independent verification of these results. for gst pulldown, the bait proteins, gst and gst-pedv n, were expressed in bacterial bl21 (de3*) cells, and the prey proteins, ha-pedv n, pdcov n-ha and tgev n-flag, were expressed in hek293t cells. bacterial and hek293t cell lysates were incubated with gsh ff resin overnight to allow for bait-prey interaction. after removing nonspecific binding by multiple rounds of washing, the bound proteins were released and analyzed by sds-page. bait and prey proteins were detected by coomassie staining and western blotting with indicated antibodies, respectively. ha-pedv n could only be observed when pulled down with gst-pedv n, while no ha-pedv n was observed when pulled down with gst, demonstrating the specificity of the assay (fig. 1b) . next, we performed the gst pulldown experiment with pdcov n and tgev n as prey proteins. similarly, we found that pdcov n-ha and tgev n-flag only co-eluted with gst-pedv n, indicating that n proteins from both tgev and pdcov bound to pedv n specifically (fig. 1b) . both co-immunoprecipitation and gst pulldown results indicated that interspecies hetero-oligomers of cov n proteins are possible, suggesting conservation of the oligomerization motifs and function of nucleocapsid proteins among swine covs. these results encouraged us to further probe the factors affecting the nature of these interactions. the cross-association capability of pdcov n or tgev n could possibly interrupt homo-oligomerization between pedv n and lead to competitive binding between pedv n and other cov n proteins. using the gst pulldown assay, we further probed the interplay between these cov n proteins to see how pedv n homo-oligomers would be affected in the presence of other cov n proteins. hek293t cells were cotransfected with pcaggs-ha-pedv n and varying amounts of pcaggs-pdcov n-ha or pcaggs-tgev n-flag. at 48 hpt, lysates were prepared and incubated with gst-pedv n-containing bacterial lysate and gsh ff resin overnight. gst pulldown revealed that, upon increasing amounts of co-eluting pdcov or tgev n, co-eluting hatagged pedv n decreased reciprocally (fig. 2) . this indicates, to a certain level, a competition between homo-oligomerization of pedv n and hetero-oligomerization with nucleocapsid proteins from other covs, and suggests that these cov n proteins utilize the same motifs or binding interfaces in oligomeric complex formation or harness common factors such as rna that can mediate oligomeric interactions. one of the primary functions of cov n proteins is to bind and organize viral rna genomes for viral assembly (mcbride et al., 2014) . however, there have been conflicting reports about the necessity of viral rna in promoting or assisting cov n self-oligomerization. some reports observed rnasea susceptibility of the oligomeric complex (narayanan et al., 2003; verheije et al., 2010) , while others detected intact higher cov n oligomers despite the absence of rna (cong et al., 2017; jayaram et al., 2006; ma et al., 2010) . to investigate whether the presence of rna influences interspecies association between these swine enteric cov n proteins, gst pulldown experiments were performed with or without rnasea treatment. first, however, we tested whether or not rna affected pedv n homo-oligomerization in this assay setup. hek293t cell lysate containing ha-tagged pedv n was mixed with gst-pedv n bacterial lysate and split into two equal parts. the lysate mixtures were either treated with rnasea or pbs buffer for 30 min at 4°c. the absence of rna after the treatment was assured by performing rt-pcr reactions with primers specific to the pedv n gene (fig. 3) . the lysate mixtures were then incubated with gsh ff resin overnight to compare the amount of ha-pedv n pulled down by gst-pedv n in the presence or absence of rna. our results across three independent experiments consistently showed a slight but noticeable increase in gst-pedv n in the elution fraction after rnasea treatment (fig. 3) . moreover, the amount of ha-pedv n pulled down in the absence of rna seemed to be greater than that observed in the presence of rna, suggesting that rna interferes with the ability of pedv n to bind to each other. we also consistently observed a slight decrease in the level of tagged pedv n in the input fraction after the lysates were treated with rnasea, implying decreased overall stability of pedv n in the absence of rna. similar decreases in protein levels were not observed with tgev or pdcov n proteins (see below), suggesting specificity of the phenomenon. interestingly, when the experiments were performed on lysates containing pdcov n-ha or tgev n-flag, a completely opposite trend was observed for hetero-oligomerization. while increased gst-pedv n was still similarly observed in the eluted fraction in the absence of rna, both pdcov n and tgev n were pulled down by gst-pedv n much less efficiently in the absence of rna (fig. 3) . it should be noted that the total amount of the tagged cov n proteins did not decrease, arguing against the possibility that tgev or pdcov n proteins were more prone to degradation without the bound rna (fig. 3, 'input' lanes) . these results suggest that the presence of rna immensely helps strengthen hetero-oligomerization between pedv n and other cov n proteins but renders homo-oligomerization between pedv n weakened. although not an absolute requirement for interspecies complex formation, rna could act as a bridge for n proteins from different species to form the complex, implying the heterologous protein-protein interactions are significantly weaker or less specific than the homologous interaction. even though the levels of rna purified from rnasea-treated lysates were below the detection limit of spectrophotometer (data not shown) and could not produce visible rt-pcr products, we could not rule out the possibility of leftover small rna fragments mediating heterologous cov n protein binding, giving rise to much fainter tgev n or pdcov n bands that co-eluted with gst-pedv n. in summary, the results in this section demonstrate conserved association capability among different cov n proteins but suggest some differentiating features, especially the influence of rna on each complex, between homo-and hetero-oligomers involving pedv n. hetero-oligomer formation as demonstrated in the previous section formed a basis for our investigation into n-mediated virus-virus interaction. as it has been shown for other viruses, it is not unreasonable to assume that the specific and competitive binding between n proteins of pedv and other covs could interrupt regular functions of pedv n during viral growth. to investigate how this protein-protein interaction might affect pedv replication, we transiently transfected veroe6 cells with varying amounts of the pcaggs plasmid expressing n proteins from either pdcov or tgev for 24 h before infection with pedv-mcherry (moi = 0.0001) and followed the course of viral replication for each condition. the levels of cov n expression were verified by western blotting against tags attached to each n protein (fig. 4a) . cell culture supernatants were collected at 24, 48 and 72 hpi to determine virus titers by tcid 50 assay (fig. 4b-d) . the results showed that veroe6 cells transiently transfected with either pedv n or tgev n showed a dose-dependent increase, up to an order of magnitude, in pedv-mcherry titers compared to veroe6 cells transfected with a blank vector ( fig. 4b and d) . interestingly, veroe6 cells transiently transfected with pdcov n showed suppression on pedv-mcherry growth, especially at high levels of pdcov n expression (fig. 4c ). due to relatively low transfection efficiencies in veroe6 cells and the uncontrollable proportion of exogenous n expression and pedv infection occurring in the same cells, the actual effects of these cov n proteins on pedv growth might be even more pronounced than observed from the transfection experiments. to test this, we constructed veroe6-based cell lines stably expressing n from pedv, pdcov and tgev by lentivirus transduction. veroe6 clones expressing relatively equal amounts of n proteins from either pedv, pdcov and tgev were selected based on western blot analysis (fig. 5a) . uniform expression of exogenous n proteins in these engineered veroe6 cells was also assessed by immunofluorescence to eliminate the concern from the transient transfection experiment (fig. 5a) . equal numbers of cells were plated to confluence in 24-well plates and infected with pedv-mcherry (moi = 0.0001). spread of pedv infection and syncytium formation was monitored daily by fluorescence microscopy. supernatants were collected at 24, 36 and 48 hpi to monitor pedv growth by tcid 50 assay. veroe6 cells expressing n from pedv and tgev clearly accelerated pedv syncytia formation and spread. by 36 hpi, most cells displayed signs of profuse infection; by 48 hpi, infected cells were dead and detached compared to infection in wild-type veroe6 cells (fig. 5b) . interestingly, pdcov n stably expressed in veroe6 cells significantly suppressed pedv spread; even at 48 hpi, no extensive syncytia had formed (fig. 5b) . quantification of supernatant pedv-mcherry titers by tcid 50 assay also reflected the extent of syncytium formation. at 36 hpi, veroe6 cells expressing tgev n or pedv n yielded about a hundred times more infectious pedv virus particles than veroe6 cells, while those expressing pdcov n resulted in an infectious titer about ten times lower. by 48 hpi, pedv replication in wild-type veroe6 cells had started to catch up and closed the gap to about one order of magnitude. however, veroe6-pdcov n cells still showed significantly slower pedv replication kinetics (fig. 5c ). as expected, we noted that the suppressive effect from veroe6-pdcov n cells was stronger than that observed in transiently transfected veroe6 cells. this could be because the stable cell line homogeneously expresses pdcov n which can directly exert an effect on the infecting virus, while only a fraction of infected cells was transfected (resulting in the effect being diluted out by cells that were infected but not transfected). previously, we observed a significant enhancement of pedv rna synthesis and increased viral titers in veroe6-pedv n cells (liwnaree et al., 2019) . to explore if similar mechanisms were employed by tgev n and pdcov n on pedv replication, we then probed viral rna production inside these veroe6-based cell lines using rt-qpcr. total rna was extracted from cells infected with pedv-mcherry at 24, 36 and 48 hpi. specific primers were used to quantify viral genomic rna (orf1a gene; grna) and subgenomic rna (5′utr-s gene; sgrna) production. the levels of each rna species were normalized to the levels of gapdh mrna from the same conditions and were expressed as relative to rna levels at 24 hpi from infected veroe6 cells. as previously observed, veroe6-pedv n cells drastically increased sgrna and grna production at 36 and 48 hpi (fig. 5d ). on the other hand, veroe6-pdcov n cells displayed stunted pedv rna transcription and replication; even at 48 hpi, the levels of sgrna and grna were still lower than those observed in veroe6 cells at 24 hpi (fig. 5d ). veroe6-tgev n cells moderately enhanced pedv rna synthesis compared to wild-type veroe6 cells but did not reach the same level as veroe6-pedv n cells (fig. 5b ). according to the results, the presence of extraneous n proteins posed greater effects on grna replication than sgrna transcription. these data corroborated with visual observation and quantitative measurement of infectious viral particle production. together, they strengthened the findings in the transient expression experiments and suggest opposing directions of influence on pedv replication mediated by n proteins derived from related covs. based on their ability to cross-associate and affect pedv rna synthesis and replication, we speculated that n proteins from other covs, especially tgev, may have interchangeable functions with pedv n in pedv replication. in an attempt to answer which roles and functions of pedv n could be replaced by other cov n proteins, we constructed an n-deficient pedv infectious clone, ppedv-mcherry-δn, and determined whether supplying cov n proteins in trans could replace the missing pedv n protein from the pedv's viral genome. since the nucleotide fragment of the n gene in the viral genome may play other important roles in viral genome packaging or transcription, we employed a frameshift mutation strategy to silence n expression from the infectious clone while minimally disturbing the nucleotide sequence of the n gene. the original start codon in the n orf, atggct, was mutated to atatgt, thereby shifting the start codon by two nucleotides, resulting in a +2 frameshift during translation (fig. 6a) . the mutated sequence theoretically produced a short unrelated peptide of 21 amino acids with early termination. we intentionally kept the mcherry gene in the construct as a visual indicator for sub-genomic mrna transcription and protein expression in the virus rescue process. to verify the lack of n expression during pedv reverse genetics rescue, we transfected the n-deficient infectious clone into hek293t cells. western blot analysis confirmed that n expression was completely diminished in cells transfected with the n-deficient infectious clone compared to the wild-type ppedv-mcherry (fig. 6b) . with the n-deficient infectious clone in hand, we next investigated pedv reverse genetics rescue in the presence of other cov n proteins. s. sungsuwan, et al. virology 540 (2020) 45-56 hek293t cells were co-transfected with ppedv-mcherry-δn and a pcaggs plasmid expressing cov n protein. expression of mcherry from the infectious clone was used as a visual proxy for production of viral rna and proteins in hek293t cells during the reverse genetics step. no detectable fluorescence was observed from co-transfection with the empty pcaggs vector, confirming the role of pedv n protein during at least the first round of viral rna transcription and viral protein translation (fig. 7a ). co-transfection with pcaggs-pedv n-myc yielded a substantial fraction of red cells, while slightly lower amount of mcherry protein expression was observed from cells cotransfected with pcaggs-tgev n-flag. co-transfection with pcaggs-pdcov n-ha resulted in very low, yet detectable, numbers of mcherrypositive cells (fig. 7a ). cell lysates were prepared at 72 hpt and probed for s expression using anti-s antibody to corroborate visual inspection. as a negative control, cells transfected with ppedv-mcherry-δn and pcaggs vector displayed no detectable trace of s (fig. 7b) . cells co-transfected with pcaggs expressing cov n proteins showed varying levels of s expression, with pedv n-myc giving the highest s expression and pdcov n-ha giving the lowest (fig. 7b) . tgev n-flag expression resulted in noticeably lower s expression compared to pedv n-myc but still higher than that of pdcov n-ha (fig. 7b) . the levels of spike protein expression from the n-deficient clone in each condition correlated with the level of mcherry expression as observed by fluorescence microscopy (fig. 7a and b) . these results suggested that cov n proteins from alphacoronaviruses can aid viral rna and protein production from infectious pedv clones more efficiently than that from deltacoronavirus. to determine if pedv rescue successfully produced viable infectious particles with help from various cov n proteins in trans, hek293t cell supernatants were collected at 72 hpt to inoculate veroe6-pedv n cells pre-transfected with pcaggs-pedv n. we found that additional transfection could ensure sufficient pedv n expression in infected cells and facilitated pedv replication better than untreated veroe6-pedv n cells (data not shown). limited syncytium formation was observed only in veroe6-pedv n cells inoculated with the supernatant derived from hek293t cells co-transfected with pcaggs-pedv n-myc (fig. 7c) . however, the infecting viruses could not continuously propagate to produce extensive syncytia as routinely observed with the rescue of ppedv-mcherry. this is probably due to insufficient pedv n protein levels as supplied by the stable cell line compared to virally encoded pedv n. remarkably, supernatants from hek293t cells co-transfected with a plasmid encoding tgev n or pdcov n did not contain infectious progeny from virus rescue, displaying no second-round pedv-mcherry replication in veroe6-pedv n cells as determined by mcherry expression or syncytia formation (fig. 7c) . these results suggest that, despite some conserved function of n proteins among porcine enteric covs in self-oligomerization and assistance during viral rna synthesis or protein expression, n is not fully interchangeable among covs for generating pedv infectious virus progeny. these results imply that the suppressive or enhancing effects on pedv replication in cells expressing pdcov n or tgev n may reflect their effects on viral rna replication and/or viral protein expression, rather than their abilities to supply complementary n proteins during rnp core formation or virion assembly. although the swine industry has focused on pedv alone as one of its largest threats, few have attempted to understand the implications of swine enteric cov co-infection despite multiple epidemiological studies. as the true impact of co-infection on disease outcomes still awaits further investigation, these viruses could predictably exert influence on each other, considerably shaping the course of their infection or evolution. molecular interactions underlying these possible viral interferences also remain to be identified and characterized. in this work, we tried to understand how different enteric swine cov n proteins, one of the most abundant structural proteins, interact and affect functions of pedv n and replication kinetics of pedv. first, we provided evidence of cross-interaction between cov n proteins despite low sequence similarity among them. amino acid sequence analyses on pedv, tgev and pdcov n proteins revealed that the proteins can be divided roughly into two structural domains (ntd fig. 6 . construction of the n-deficient pedv infectious clone, ppedv-mcherry-δn. (a) schematic representation of a frameshift mutation strategy to silence n expression from the ppedv-mcherry-derived infectious clone to yield ppedv-mcherry-δn. cmv, cytomegalovirus immediateearly promoter; hdv, hepatitis delta virus ribozyme self-cleavage site; bgh, bovine growth hormone termination; t, transcription terminator. (b) western blot analysis of lysates prepared from hek293t cells transfected with ppedv-mcherry or ppedv-mcherry-δn (2 μg each) at 72 hpt. and ctd) with relatively high conservation and three intrinsically disordered regions (idrs) with relatively low conservation located in the middle and at the two termini (fig. 8) . this domain analysis is consistent with previous crystallographic studies on sars-cov and mhv n, which also suggested conserved general structural organization of cov n (chang et al., 2009; cong et al., 2017; ma et al., 2010; yu et al., 2006) . moreover, immunological data documenting cross-reactivity of antibodies against n proteins from tgev or pdcov with pedv n further implied shared topological similarity between these cov n proteins (gimenez-lirola et al., 2017; lin et al., 2015; ma et al., 2016) . nevertheless, precise molecular mechanisms mediating interspecies oligomerization of cov n proteins remain to be determined. structural and biochemical studies of sars-cov n strongly suggest a role of ctd as a major mediator for cov n dimerization. moreover, ctds of n proteins from sars-cov and porcine reproductive and respiratory syndrome virus (prrsv), despite diverse sequences, could adopt well-conserved structures, implying similar approaches to oligomerization, at least at a gross structure level (yu et al., 2006) . however, since cov n ctds formed intricate molecular interaction networks during dimerization (chang et al., 2014) , it is unlikely that cov n proteins from different species could replace all these specific molecular interactions. consistent with our results that hetero-oligomer formation is highly sensitive to the presence of rna, a more likely possibility is that rna-protein interactions play a more dominant role in interspecies cov n interactions than protein-protein interactions. next, we asked if n proteins from different covs can replace pedv n function during viral rna and protein synthesis. during the rescue of the n-deficient pedv infectious clone, we showed that supplying n protein from any cov in trans can initiate first-round sgrna transcription and protein expression. most likely, this function stems from the shared intrinsic ability of cov n to bind viral rna and act as an rna chaperone that initiates rna replication and/or transcription processes (zuniga et al., 2007 (zuniga et al., , 2010 . these results support previous notions that cov n proteins play important roles during cov rescue. for instance, rescue of tgev infectious rna required at least co-transfection of mrna encoding the n protein (yount et al., 2000) . schelle et al. demonstrated that an n-deficient hcov229e rna vector replicon carrying the gfp gene can produce sgrna and express gfp from the vector when co-transfected with n from pedv, a closely related virus, but not from mhv, a more distantly-related one (schelle et al., 2005) . we noted fig. 7 . other cov n proteins can partially function to replace pedv n. for pedv rescue experiments, hek293t cells were co-transfected with ppedv-mcherry-δn and 1 μg of pcaggs plasmid expressing n from pedv, pdcov or tgev. at 72 hpt, (a) cells were imaged under fluorescence microscopy to monitor mcherry expression from ppedv-mcherry-δn, and (b) cell lysates were prepared for western blot analysis with an anti-s antibody. supernatants from (a) were adsorbed onto veroe6-pedv n cells, which were additionally transfected with 2 μg pcaggs-pedv n to ensure sufficient n expression. at 72 hpi, (c) cells were imaged under fluorescence microscopy to monitor mcherry expression and syncytium formation as a sign for successful pedv infection into veroe6-pedv n cells (scale bar, 100 μm). a similar preference in our experiments. between tgev n and pdcov n, the former is more effective in replacing the indigenous pedv n than the latter. since both pedv and tgev belong to the alphacoronavirus genus, while pdcov is classified as a deltacoronavirus, tgev n would be expected to be functionally and structurally closer to pedv n, and hence able to form replication/transcription complexes and participate in pedv replication with higher compatibility than pdcov n. although both tgev n and pdcov n can oligomerize and perform some functions during g/sgrna transcription, they alone cannot totally replace all pedv n's functions. without native pedv n, infectious pedv virions could not be detected in the presence of tgev or pdcov n, implying that other functions are not interchangeable. these critical functions might involve viral core assembly as cov n participates in many steps during viral core or virion assembly (mcbride et al., 2014) . for instance, several reports showed that cov n is involved, directly or indirectly, with recognition of specific packaging signals for genome packaging (hsieh et al., 2005; hsin et al., 2018; kuo et al., 2014) . since rna packaging signals among covs are distinct in terms of length and sequence, even closely related cov n might not be able to recognize pedv packaging signals. similar compatibility issues have been raised in the case of viral interference between different types of influenza viruses (baker et al., 2014) . another critical step that could be disrupted by heterologous cov n is viral assembly through interactions between n and m proteins. it is widely accepted that cov n and m proteins interact to form the viral core, but different covs utilize different regions of n in binding to their own m proteins. for instance, while mhv utilizes the very c-terminal end of n to bind m, sars-cov n uses the middle disordered linker for the interaction (he et al., 2004b; narayanan et al., 2000) . in addition, although both tgev and mhv n proteins used their c-termini to bind m, the n binding sites on tgev m and mhv m are very distinct (hurst et al., 2005; kuo and masters, 2002) , pointing to high specificity for each virus. it could be argued that our use of c-terminally tagged cov n proteins might have contributed to failure in rescuing infectious pedv from ppedv-mcherry-δn, possibly due to interference with n-m protein interaction. nevertheless, our results showed that addition of a myc tag at the c-terminus of pedv n did not completely ruin protein-protein interaction required for viral assembly as pedv n-myc remained capable of producing infectious virions. moreover, using untagged pedv n and tgev n during reverse genetics rescue of ppedv-mcherry-δn gave similar results to those presented in fig. 7 (data not shown) , suggesting that the inability of tgev n or pdcov n to rescue infectious pedv lacking endogenous nucleocapsid protein stemmed largely from incompatibility of interspecies protein interaction rather than the interference from epitope tags. in all, it is conceivable that not all of n functions could be replaced by the protein originating from other viral species, even though n proteins from pedv, tgev and pdcov can cross-assemble, form highorder oligomers and aid in rna synthesis processes. notably, we observed completely opposite effects of tgev and pdcov n proteins on pedv replication. we noticed that, in the presence of rna, homologous binding between pedv n itself is weakened and cross binding between pedv n and the other n proteins is strengthened. during co-infection, where viral grna is ubiquitous, the rna could act as a tethering bridge to stabilize the formation of heterologous oligomers, which may not be a productive core structure for pedv virion assembly and may interrupt the optimal processes in viral fig. 8 . analysis of cov n sequences reveals low sequence similarity but conserved structural organization. multiple amino acid sequence alignment was performed with t-coffee (notredame et al., 2000) . the color scheme denotes consistency between different alignment methods ranging from bad (low agreement; blue and green) to good (high agreement; pink). bold and normal lettering denote ordered and disordered regions as predicted by the prdos program (ishida and kinoshita, 2007) . (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) replication that prefer the homo-oligomer of n. such interruption could explain the suppressive effect of pdcov n. nevertheless, the existence of interspecies cov n oligomers might not be the only factor dictating the effect of extraneous cov n proteins on pedv replication. tgev n also forms rna-dependent interspecies oligomers with pedv n and could have resulted in similar suppression. instead, we observed a small enhancement effect on pedv replication in the presence of tgev n. this could be the net outcome of other oligomerization-independent roles of tgev n that indirectly enhance pedv replication. a study by zhao et al. showed that, when compared to single-virus infection, coinfection of tgev and pedv could synergistically enhance rupture to tight and adherens junctions of the intestinal epithelial cells ipec-j2 by suppressing expression of proteins that help form the junction (zhao et al., 2014) . such altered barrier integrity is possibly caused by cov n and facilitate viral replication. another possible role of pedv or tgev n that could lead to enhancement of pedv replication is to act as a viral suppressor of rna silencing (cui et al., 2015) . using mhv n as a model, they found that cov n can bind and suppress host antiviral rnai, leading to increased viral protein expression and subsequent viral replication. more remarkably, they found that n proteins of alphacoronaviruses, including tgev and pedv, also have this ability. indeed, among different cov n proteins, they found that tgev n was among the most active and mers-cov n was the least active (cui et al., 2015) . therefore, n from tgev may also employ this mechanism in promoting pedv replication, while some other cov n proteins might not be capable of this function. these are interesting hypotheses currently under investigation, which could reveal more complexity of co-infection between these viruses. in summary, this work shows how other swine enteric cov n proteins can substitute for endogenous pedv n in some functions but not in others, demonstrating both conservation and specificity among related viruses in the coronaviridae family. furthermore, co-expression experiments revealed interesting phenomena in which different cov n proteins exert vastly different effects on pedv replication. these results raise even more questions. for example, in the context of co-infection, in which all other viral components are present, what would be the net outcomes on replication kinetics of each virus? are there effects from other viral components? considering the opposing effects on pedv replication of n proteins from tgev, an older virus emerging prior to pedv, and pdcov, a newly emerged one, is it possible that nucleocapsid proteins could act as one of the factors in permitting or limiting emergence of enteric swine covs? if so, what is the effect of pedv n on newer covs such as swine acute diarrhea syndrome coronavirus (sads-cov), which has been detected predominantly in farms encountering pedv epidemics (zhou et al., 2018) , and vice versa? further investigation into these questions would give valuable insights for us to better understand co-infection events and their implications for the emergence of novel enteric swine viruses. all authors of this manuscript have no conflict of interest to declare. mechanism of interference between influenza a/wsn and b/kanagawa viruses complementation between two tospoviruses facilitates the systemic movement of a plant virus silencing suppressor in an otherwise restrictive host influenza a and b virus intertypic reassortment through compatible viral packaging signals transient oligomerization of the sars-cov n protein-implication for virus ribonucleoprotein packaging the sars coronavirus nucleocapsid protein-forms and functions multiple nucleic acid binding sites and intrinsic disorder of severe 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and up-regulates interleukin-8 expression strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model crystal structure of the severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein dimerization domain reveals evolutionary linkage between corona-and arteriviridae type iii interferon restriction by porcine epidemic diarrhea virus and the role of viral protein nsp 1 in irf1 signaling transmissible gastroenteritis virus and porcine epidemic diarrhoea virus infection induces dramatic changes in the tight junctions and microfilaments of polarized ipec-j2 cells coronavirus nucleocapsid protein facilitates template switching and is required for efficient transcription coronavirus nucleocapsid protein is an rna chaperone the authors would like to thank dr. qigai he for anti-pedv s antibody, and benjamas liwnaree for technical assistance. we thank the members of virology and cell technology research team, especially dr. samaporn teeravechyan, for critical comments and language help on the manuscript. this work was supported by biotec's fellow grant [grant number p-15-51261]. key: cord-351197-xv6ymc4l authors: cibulski, samuel; alves de lima, diane; fernandes dos santos, helton; teixeira, thais fumaco; tochetto, caroline; mayer, fabiana quoos; roehe, paulo michel title: a plate of viruses: viral metagenomics of supermarket chicken, pork and beef from brazil date: 2020-09-28 journal: virology doi: 10.1016/j.virol.2020.09.005 sha: doc_id: 351197 cord_uid: xv6ymc4l a viral metagenomics study was conducted in beef, pork, and chicken sold in supermarkets from southern brazil. from chicken, six distinct gyroviruses (gyv) were detected, including gyv3 and gyv6, which for the first time were detected in samples from avian species, plus a novel smacovirus species and two highly divergent circular rep-encoding ssdna (cress-dna) viruses. from pork, genomes of numerous anelloviruses, porcine parvovirus 5 (ppv5) and 6 (ppv6), two new genomoviruses and two new cress-dna viruses were found. finally, two new cress-dna genomes were recovered from beef. although none of these viruses have history of transmission to humans, the findings reported here reveal that such agents are inevitably consumed in diets that include these types of meat. meat and meat products are the source of numerous bacteria (e.g. salmonella 33 enterica), protozoa (e.g. toxoplasma gondii) and worms (e.g. trichinella spiralis) that 34 may cause significant illness in humans. therefore, it has been rising the overall 35 concern of viral transmission by food products, especially the ones causes by meat and 36 meat-derived. meats have been pointed in charge, once is well-known that food-source 2009; morse et al., 2012) . today, emerging viruses are a major concern, since 44 devastating, occasionally life-threatening epidemics -e.g., covid-19 and pandemic 45 influenza -are caused by viruses which originally spilled over from animals to humans. 46 in 2014, an interesting study identified a series of viral genome sequences in 47 approximately 531,000 high-quality reads were generated by metagenomic 131 sequencing. after assembly, a total of 2,370 contigs were generated; 50.3% were viral 132 contigs. from these, 0.75% were assigned to eukaryotic viruses and 99.25% to 133 bacteriophages. however, despite the relatively small number of contigs associated with 134 viral genomes previously known to infect eukarya, ~20% of the reads were mapped 135 back to these contigs, indicating a high sequence coverage. a detailed taxonomic 136 classification, including the numbers of reads for each eukarya-related viral contig 137 recovered is this study, is provided in supplementary gives them the ability to persist and spread in the environment. chicken meat has been 149 reported to carry numerous gyroviruses, including the highly prevalent cav, the 150 worldwide distributed, and recognized pathogen of chicken. over the last decade, 151 twelve new putative gyrovirus species were described, most of these recovered from this fragment shares low nt and aa identity with previously reported gyv3 isolates 163 (~72% of nt and ~75% at amino acid level), and probably represents another genome 164 variant, since the degree of vp1 identity between other gyv3 is greater than 70%. 165 another interesting remark is that, to date, gyv3 has only been identified in feces from 166 mammalian hosts (human and cat). therefore, it seems very likely that gyv3 may, in sequences described in this study are marked in red. 206 the last two genomes recovered were cress-dna viruses that could not be 207 allocated to any previously described viral family. cress 53f (5,532) exhibited four 208 orfs encoding rep, cap and two hypothetical proteins (with homologues in other 209 cress-dna viruses) ( figure 3a ). the n-terminus of cap presented a r-rich region, 210 whereas the rep showed motifs implicated in rcr (supplementary table 2 approximately 465,000 high-quality reads were produced by metagenomic 241 sequencing of pork sample. after assembly, a total of 2,922 contigs were generated; 242 55% of these were representative of viral sequences. from these, 1.4% were assigned to 243 eukaryotic viruses, whilst the remaining sequences were representatives of 244 bacteriophages. a detailed taxonomic classification, including the numbers of 245 j o u r n a l p r e -p r o o f sequenced reads of each eukarya-related viral contig recovered in this study, is 246 provided in supplementary table 1 . 247 in the same way that gyroviruses were found in large amounts in chicken meat, 248 anelloviridae contigs (torque teno sus gascuel, 2003). sequences described in this study are marked in red. 328 j o u r n a l p r e -p r o o f approximately 523,000 high-quality reads were obtained at metagenomic 330 sequencing. after assembly, a total of 1,739 contigs were generated; 51.6% were viral 331 contigs. from these, 0.5% were assigned to eukaryotic viruses, whereas remaining 332 contigs corresponded to bacteriophage sequences. a detailed taxonomic classification, 333 including numbers of sequenced reads of each eukarya-related viral contig recovered in 334 this study, is provided in supplementary table 1 . meat is among the most perishable foods and a favorable environment for the 393 replication of microorganisms because of its high concentrations of nutrients and high 394 water activity (odeyemi et al., 2020) . during slaughterhouse processing, meats may be 395 contaminated by the skin, intestinal content, personnel, and processing equipment. the 396 microbial load varies from 10 2 and 10 4 cfu/cm 2 (russo et al., 2006 fastqc: a quality control tool for high throughput sequence data 440 the virome of an endangered stingless bee suffering from annual mortality trimal: a tool for 443 automated alignment trimming in large-scale phylogenetic analyses novel astroviruses in 446 insectivorous bats viral metagenomics in brazilian pekin ducks identifies two gyrovirus, including a 450 new species, and the potentially pathogenic duck circovirus virus detection and 453 identification using random multiplex (rt)-pcr with 3′-locked random primers liver virome of healthy pigs reveals diverse small ssdna viral genomes new polyomavirus species identified in nutria, myocastor coypus 464 polyomavirus 1 identification of diverse circular single-stranded dna viruses in adult 467 dragonflies and damselflies (insecta: odonata) of arizona and oklahoma, usa. 468 primeiros cantos spoilage microbiota 472 associated to the storage of raw meat in different conditions direct sequencing of human gut virome 475 fractions obtained by flow cytometry divergent gyroviruses in the feces of tunisian 479 children helicases: amino acid sequence comparisons 481 and structure-function relationships a new superfamily of putative ntp-484 binding domains encoded by genomes of small dna and rna viruses 487 new algorithms and methods to estimate maximum-likelihood phylogenies: 488 assessing the performance of phyml 3.0 a simple, fast, and accurate algorithm to estimate 491 large phylogenies by maximum likelihood conserved sequence motifs in the initiator proteins 494 for rolling circle dna replication encoded by diverse replicons from eubacteria, 495 eucaryotes and archaebacteria a 498 broadly reactive one-step real-time rt-pcr assay for rapid and sensitive detection 499 of hepatitis e virus mafft multiple sequence alignment software 502 version 7: improvements in performance and usability multiple origins of 505 prokaryotic and eukaryotic single-stranded dna viruses from bacterial and 506 archaeal plasmids pervasive chimerism in the 508 replication-associated proteins of uncultured single-stranded dna viruses. 509 viruses global organization and proposed megataxonomy of the 512 genomoviridae: a new 514 family of widespread single-stranded dna viruses cressdnaviricota: a virus phylum unifying 7 families of rep-encoding 519 viruses with single-stranded, circular dna genomes sms: smart model selection in 522 phyml faecal 525 eukaryote viral 526 community, including novel circular ssdna viruses the 530 intestinal virome of malabsorption syndrome-affected and unaffected broilers 531 through shotgun metagenomics detection 535 of alphacoronavirus in velvety free-tailed bats (molossus molossus) and brazilian 536 free-tailed bats (tadarida brasiliensis) from urban area of southern brazil the 539 role of swine in the generation of novel influenza viruses 542 recombination in eukaryotic single stranded dna viruses torque teno virus (ttv) is highly prevalent in the european wild boar 546 (sus scrofa) detection patterns of 548 porcine parvovirus (ppv) and novel porcine parvoviruses 2 through 6 (ppv2-549 ppv6) in polish swine farms metagenomics and future perspectives in 551 virus discovery prediction and prevention 554 of the next pandemic zoonosis understanding 557 spoilage microbial community and spoilage mechanisms in foods of animal origin marine phage genomics: the tip of the iceberg a third gyrovirus species in human 564 faeces high frequency and extensive genetic heterogeneity 567 of ttsuv1 and ttsuvk2a in pcv2-infected and non-infected domestic pigs and 568 wild boars from uruguay complete nucleotide and amino acid 571 sequences and genetic organization of porcine kobuvirus, a member of a new 572 species in the genus kobuvirus, family picornaviridae prevalence of the novel torque teno sus virus species k2b from pigs in 576 the united states and lack of association with post-weaning multisystemic 577 wasting syndrome or mulberry heart disease a field guide to eukaryotic circular single-580 stranded dna viruses: insights gained from metagenomics behaviour of brochothrix 583 thermosphacta in presence of other meat spoilage microbial groups. food 584 microbiol the general 586 composition of the faecal virome of pigs depends on age, but not on feeding with a 587 probiotic bacterium the fecal 589 virome of pigs on a high-density farm consensus statement: virus 596 taxonomy in the age of metagenomics hepatitis 599 e virus and related viruses in wild, domestic and zoo animals: a review torque teno sus virus 1 (ttsuv1) 603 and 2 (ttsuv2) viral loads in serum of postweaning multisystemic wasting 604 syndrome (pmws)-affected and healthy pigs in brazil torque teno sus virus 608 (ttsuv) in tissues of pigs and its relation with the occurrence of postweaning 609 multisystemic wasting syndrome viral dna genomes in sera of 614 farrowing sows with or without stillbirths smacoviridae: a new family of animal-associated 617 single-stranded dna viruses sequence-based taxonomic framework for the 620 classification of uncultured single-stranded dna viruses of the family 621 animals as reservoir for human 623 norovirus resistance of porcine 625 circovirus and chicken anemia virus to virus inactivation procedures used for blood 626 products viral metagenomics analysis 628 demonstrates the diversity of viral flora in piglet diarrhoeic faeces in china what is for dinner? 631 viral metagenomics of us store bought beef, pork, and chicken 634 identification of a chicken anemia virus variant-related gyrovirus in stray mfold web server for nucleic acid folding and hybridization 637 prediction key: cord-317333-unrd76bo authors: danesh, ali; cameron, cheryl m.; león, alberto j.; ran, longsi; xu, luoling; fang, yuan; kelvin, alyson a.; rowe, thomas; chen, honglin; guan, yi; jonsson, colleen b.; cameron, mark j.; kelvin, david j. title: early gene expression events in ferrets in response to sars coronavirus infection versus direct interferon-alpha2b stimulation date: 2011-01-05 journal: virology doi: 10.1016/j.virol.2010.10.002 sha: doc_id: 317333 cord_uid: unrd76bo type i interferons (ifns) are essential to the clearance of viral diseases, however, a clear distinction between genes upregulated by direct virus–cell interactions and genes upregulated by secondary ifn production has not been made. here, we investigated differential gene regulation in ferrets upon subcutaneous administration of ifn-α2b and during sars-cov infection. in vivo experiments revealed that ifn-α2b causes stat1 phosphorylation and upregulation of abundant ifn response genes (irgs), chemokine receptors, and other genes that participate in phagocytosis and leukocyte transendothelial migration. during infection with sars-cov not only a variety of irgs were upregulated, but also a significantly broader range of genes involved in cell migration and inflammation. this work allowed dissection of several molecular signatures present during sars-cov which are part of a robust ifn antiviral response. these signatures can be useful markers to evaluate the status of ifn responses during a viral infection and specific features of different viruses. viral respiratory infections are a major worldwide cause of morbidity and mortality (kolling et al., 2001; thompson et al., 2003) . emerging viral threats, such as the severe acute respiratory syndrome coronavirus (sars-cov), avian influenza h5n1 and pandemic influenza h1n1 virus are well poised to cause epidemics or pandemics that could be socially and economically disastrous (dushoff et al., 2006; weiss and mcmichael, 2004; dawood et al., 2009) . for decades, ferrets have been used for the investigation of influenza infection since they are susceptible to influenza viruses (hull and loosli, 1951) . more recently, ferrets have also been shown to be a good model of human sars-cov infection (martina et al., 2003) . we have previously characterized ferret cytokine and chemokine genes as well as have developed immunological assays for evaluating the ferret immune system following sars and influenza infection danesh et al., 2008; ochi et al., 2008) . ligation of the interferon (ifn) alpha receptors 1 and 2 (ifnar1 and ifnar2) with ifn-α induces ifn signaling pathways and promotes ifn gene induction. formation of the signal transducer and activator of transcriptions 1 and 2 (stat1-stat2) heterodimer occurs following the phosphorylation of janus kinase 1 (jak1) and tyrosine kinase 2 (tyk2) that are associated with ifnar2 and ifnar1, respectively (marijanovic et al., 2007) . these two kinases phosphorylate stat1 and stat2, which together form a complex with interferon regulatory factor 9 (irf9) (takaoka and yanai, 2006) . the interferon stimulatory factor 3 complex (isgf3) binds to interferon-stimulated response element (isre) and activates transcription of ifn-α inducible genes, including 2'-5' oligoadenylate synthase 1 (oas1), myxovirus resistance 1 (mx1), interferon stimulated gene 15 (isg15) and many other ifn-response genes (irgs) (uddin and platanias, 2004) . ifn-α stimulation ultimately promotes a cellular antiviral state which is hallmarked by the upregulation of irgs (chevaliez and pawlotsky, 2007) . although ifn signaling gene upregulation during viral infection has been the subject of previous reports, there is little information regarding the host immune responses directly induced by viruses versus those that are upregulated due to secondary ifn stimulation (chelbi-alix and wietzerbin, 2007; haagmans et al., 2004; loutfy et al., 2003; cameron et al., 2007) . here we used our previously described ferret model (chu et al., 2008) to identify genes that were regulated by sars-cov infection compared to ifn-α2b stimulation in the ferret model to elucidate immune responses during viral infection. we examined the phosphorylation status of signaling molecules in ifn-α2b-stimulated peripheral blood mononuclear cells (pbmcs) . we also analyzed the in vivo gene expression profiles of ferret pbmcs and lung necropsies following ifn-α2b injection during the time course. evaluation of gene expression patterns in pbmcs and lung necropsies of sars-cov-infected ferrets led us to the identification of 7 upregulated irgs that also were upregulated in response to ifn-α2b injection. our findings in ferrets suggested ifn-α2b injection and sars-cov infection led to similar as well as unique gene expression signatures in a global point of view. increased knowledge of the interaction of these gene expression signatures may improve our understanding of the immune system of ferrets as a preferred animal model of severe respiratory viral illnesses. we first investigated the phosphorylation of the ifn-α receptor downstream signaling molecule stat1 to determine the signaling potential of ifn-α2b in ferrets. the phosphorylation status of stat1 was evaluated using phosphorylated amino acid specific monoclonal antibody for flow cytometry analysis that cross-reacted with the phosphorylated ferret protein. pbmcs demonstrated a significant stat1 phosphorylation response 15 min post-stimulation with ifn-α2b in vitro compared to the control stimulated with pbs alone ( supplementary fig. 1 ). since stat1 phosphorylation was observed in vitro, we then determined whether ifn-α2b could activate stat1 in vivo. four ferrets were subcutaneously injected with 1 μg/kg ifn-α2b and peripheral blood samples were taken at 0, 3 and 6 h post-stimulation for flow cytometry examination. by 3 h, samples extracted from all ferrets demonstrated significant stat1 phosphorylation in the pbmcs. control ferrets injected with pbs did not demonstrate marked stat1 phosphorylation at any time point. the stat1 average mean fluorescent intensity (mfi) of the ifn-α2b-injected group was significantly increased compared to the average of its control group (p b 0.01) (fig. 1a) . these results indicated that stat1 was also inducible by ifn-α2b in vivo. ifn signaling is critical to successful antiviral responses during infection (haller et al., 2007) . therefore, we next investigated the phosphorylation status of stat1 following sars-cov infection. we infected ferrets with sars-cov or pbs control and measured the phosphorylation of stat1 by flow cytometry. three ferrets infected with sars-cov demonstrated significant stat1 phosphorylation in pbmcs post-infection with a maximum peak at day 3 (p b 0.01). control ferrets mock-infected with pbs did not demonstrate significant stat1 phosphorylation at any time point (fig. 1b) . since stat1 was phosphorylated following sars-cov infection and ifn-α2b injection, we investigated select irg expression by qrt-pcr following in vitro stimulation of ferret peripheral whole blood with ifn-α2b. in vitro stimulation with ifn-α2b led to significant upregulation of stat1 and several irgs such as mx1, oas1, isg15, isg20, irf7 and interferon-induced protein 35 (ifi35). as expected, activation of ifn-α receptor signaling therefore initiated transcriptional activation of interferon response genes ( supplementary fig. 2 ). we then assessed genome-wide gene expression following in vivo ifn-α2b administration in ferrets. ferrets were subcutaneously injected with pbs (control) or ifn-α2b and blood samples were drawn for rna isolation 1 and 2 days after injection. without a commercially available ferret microarray, the rna was then used for microarray analysis on the affymetrix genechip canine genome 2.0 array (see materials and methods), because ferret genes show a high degree of homology with canine genes as we have previously established rowe et al., 2010; fang et al., table 1 ). the peripheral blood gene expression data from ifn-α2b-injected group were normalized to the control group. the t-test analyses showed the highest number of significant changes occurred at day 2, with a total of 2717 upregulated and 1230 downregulated genes in peripheral blood of the ifn-α2b-injected ferrets (table 1) . a threshold of at least 1.5 fold-change and a p value for the t-tests of less than 0.05 were chosen. the peak upregulation of a cluster of irgs, including mx1, oas1, oas2, isg15, ifi44 and ubiquitin specific protein 18 (usp18), occurred at day 1, while peak upregulation of irgs such as jak1, jak2, protein inhibitor of activated stat1 (pias1) irf1, interferon-γ receptor 1 (ifngr1), and eukaryotic translation initiation factor 2-α kinase 2 (eif2ak2) occurred at day 2 post-injection ( fig. 2a and supplementary table 2) . after assessing the large scale gene expression profile following ferret in vivo ifn-α2b stimulation, we validated the expression of selected irgs by qrt-pcr according to the availability of the ferret specific primers. we found stat1 and irgs such as mx1, oas1 and isg15 were significantly upregulated in ferrets injected with ifn-α2b compared to the controls (fig. 2b ). the lack of ferret sequences for other irgs prevented us from confirming the upregulation of these genes. to determine if stat1 phosphorylation was correlated with irg activation in our ferret animal model of sars-cov infection (chu et al., 2008) , we went on to analyze host gene expression following sars-cov infection. the gene expression data at day 2 post-infection with sars-cov was normalized to the mock control dataset. unfortunately, blood samples from day 1 post-infection did not meet minimal rna quality for microarray analysis, preventing us from performing a timecourse study on the peripheral blood. there were 138 upregulated and 414 downregulated genes (p b 0.05, n1.5 fold change) as ascertained by t-test analysis at day 2 post-infection (table 1) . irgs, including stat1, mx1, oas1, oas2, isg15, ifi44, suppressor of cytokine signaling 1 (socs1), radical s-adenosyl methionine domain containing 2 (rsad2), usp18 and oas ligand (oasl) were significantly upregulated ( fig. 2c and supplementary table 2 ). the upregulation of stat1, mx1, oas1 and isg15 were validated with qrt-pcr (fig. 2d ). these gene expression and stat1 phosphorylation findings suggested that robust ifn responses were activated following sars-cov infection 2 days post-infection. interferon canonical pathway analysis confirmed the similarities between the expression patterns of irgs at day 2. stat1, mx1, oas1, usp18, rsad2, isg15 and ifi44 were upregulated in the peripheral blood of ifn-α2b-injected and sars-cov-infected ferrets. in contrast, oasl, oas2 and socs1 were upregulated during sars-cov infection alone (fig. 3a) . since sars-cov infection causes severe lung pathology we went on to compare and contrast the genes upregulated by ifn-α2b stimulation and sars-cov infection in the lungs of ferrets. microarray analysis was performed on lung necropsies of ifn-α2b-injected ferrets compared to controls. the peak gene expression occurred at day 2 with a total of 512 upregulated and 550 downregulated (p b 0.05) genes (table 1) . interestingly, the strongest upregulation of several irgs, such as stat1, mx1, oas1, oas2, isg15, ifi44, ifi44 ligand (ifi44l) and eif2ak2, occurred on day 1 ( fig. 2a and supplementary table 1 ). there was a marked increase in the total number of regulated genes from lung necropsies of sars-covinfected ferrets compared to lungs from ifn-α2b-stimulated ferrets ( table 1 ). the sars-cov infected ferrets had a peak in gene expression at day 1 with 4222 upregulated versus 1248 downregulated genes (p b 0.05). both the number of upregulated irgs and the expression levels peaked at day 1, including stat1, mx1, oas1, oas2, isg15, irf1, interferon-induced protein with tetratricopeptide repeats 1( ifit1), ifi35, ifi44, ifi44l, proteasome subunit multifunctional beta 8 (psmb8), eif2ak2 and ifnrg1. jak1 was the only irg that was downregulated at day 1 (fig. 2c ). the upregulation of stat1, mx1, oas1 and isg15 was validated with qrt-pcr on lung necropsies of ferrets injected with ifn-α2b or infected with sars-cov (fig. 2d ). the comparison of microarray results between the lung tissue of ifn-α2b and sars-cov ferrets at day 1 revealed commonalities in the expression patterns of most irgs. stat1, mx1, oas1, oas2, isg15, ifi44, ifi44l and eif2ak2 were among the overlapping genes (fig. 3b ). to further model the pathways involved in the host response to sars-cov and the direct effects of ifn-α2b administration, functional analysis of the regulated genes was performed using ingenuity pathway analysis software. for each experimental group, genes showing changes in their expression levels were mapped into highlevel gene ontology categories: cellular process, metabolic process, intracellular signaling cascade, cell cycle and immune response ( table 1 ). the number of genes present in each functional category is representative of the level of biological activity in each experimental group with respect to the controls. analysis of the ifn signaling canonical pathway showed the upregulation of stat1, mx1, oas1, oas2, isg15 and ifi44 in lung necropsies of ifn-α2b injected and sars-cov infected ferrets (fig. 4) . functional classification of upregulated genes showed that ifn-α2b induces increased expression of phagocytosis-related genes, such as fc fragment of igg, high affinity ia, receptor cd64 (fcgr1a) and dynamin 1-like (dnm1l), leukocyte transendothelial migration genes, such as integrins beta 1 and 2 (itgb1 and itgb2), and upregulation of chemokine receptors, chemokine c-c motif receptors 3, 7 and 9 (ccr3, ccr7, ccr9) and chemokine c-x-c motif receptor 4 (cxcr4) (supplementary table 3 ). these results suggest that ifn-α2b is able to activate specific functions of the leukocyte responses in blood samples after exposure. the lungs of ferrets infected with sars-cov showed broader immune responses than ifn-α2b-injected ferrets, as demonstrated by the higher number of regulated genes in several functional categories related to the activation of the immune responses, including: complement and coagulation, cell adhesion molecules and leukocyte activation (fig. 5 ). type i ifns are a critical component of the innate immune response during viral infections. the function of many downstream genes has been studied in-depth, however, it is likely that the presence of the virus and subsequent tlr-mediated signaling are required to deploy full irg-mediated antiviral activity (bosinger et al., 2009) . in this study we investigated the gene signatures induced following subcutaneous administration of ifn-α2b in ferrets. we also analyzed the signaling pathways during an infection with sars-cov, and by means of comparative analysis we profiled ifn gene responses in the context of a respiratory infection. we used an experimental model of infection with sars-cov in ferrets, which causes mild symptoms without mortality. the pathological features of this model were previously published (chu et al., 2008) and a summary of the clinical information can be found in the supplementary information (supplementary table 4 ). we assessed the capacity of subcutaneous administration of ifn-α2b to activate antiviral responses in ferrets. the activation levels of several intracellular signaling proteins were studied by using phospho-specific antibodies and subsequent facs analysis. stat1 plays a key role downstream of ifn signaling while stat3 and stat5 are thought to be involved at a lesser extent, and/or weak participation of stat4, mitogen activated protein kinase 38 (p38) and extracellular signal-regulated kinase (erk) (li et al., 2007) . in vitro incubation of ferret pbmcs with ifn-α2b led to strong phosphorylation of stat1, weak phosphorylation of stat3 and stat5 and no phosphorylation of stat4, p38 and erk. furthermore, the activation of the stat1 signaling pathway in vitro was confirmed at the mrna level with the presence of many downstream irgs, including mx1, oas1, oas2, isg15, and ifi44. the in vivo effects of ifn-α2b were also investigated. stat1 showed increased phosphorylation levels in the peripheral blood at early hours post-injection, while stat3 and stat5 remained unchanged. moreover, we did not observe mrna gene expression of interleukin 8 (il-8) and suppressor of cytokine signaling 3 (socs3) at the mrna level (data not shown), suggesting that stat3 (gharavi et al., 2007) and stat5 (barclay et al., 2007) , respectively, do not participate in vivo in response to ifn-α2b. the global numbers of regulated genes found in the microarray results constitute good estimators of the intensity of the host response at different time-points. in vivo effects of ifn-α2b can be observed 24 h after the injection and their peak is reached 48 h post-injection. irgs are markedly increased in both blood and lung tissue, however the responses in the blood show greater breadth and magnitude as compared with the responses observed in lung tissue (supplementary table 2 ). this suggests that the administration protocol of ifn-α2b used in this study is only capable of inducing a limited activation in lung tissue. therefore, alternative protocols including direct administration of ifn-α2b into the respiratory tract or subcutaneous administration at higher doses should be explored in order to achieve stronger antiviral responses at the infection sites. gene expression during sars-cov infection, on the other hand, shows the presence of strong antiviral and inflammatory responses in the lungs 24 h postinfection, fading on day 2 post-infection in both blood and lung tissue. as expected, ifn-α2b stimulates the increased expression of a variety of irgs that play a central role in the clearance of viral infections, including mx1, oas1, oas2 and isg15. they exert their effects through different mechanisms of action, such as direct targeting of viral entry, inhibition of protein synthesis or degradation of viral rna. mx1 is a dynamin-like large guanosine triphosphatase (gtpase), which has antiviral activity against a wide range of rna viruses. the antiviral activity of mx1 is effective at the early stages of the viral cycle in the nucleus or cytoplasm (haller et al., 2007) . oas is an adenylate synthetase, which uses adenosine triphosphate to synthesize 2',5'-oligoadenylates. the latter activates latent rnase l that is involved in the degradation of viral rna (bonnevie-nielsen et al., 2005) . isg15 is a ubiquitin-like enzyme that covalently conjugates to a large number of cellular proteins; however this does not usually lead to protein degradation. in the case of hiv-1, isg15 inhibits the release of virions (okumura et al., 2006) . upregulation of similar sets of irgs by sars-cov and ifn-α2b were observed, including 7 irgs (stat1, isg15, mx1, oas1, oas2, ifi44 and ifi44l) in the peripheral blood and lung tissue of both groups. in contrast, several irgs, including ifi35, ifit1 and psmb8, were only upregulated in the lungs during sars-cov infection. these results suggest that the expression of certain irgs lie beyond the direct control of ifn-α2b, and additional signals such as activation of tlr-signaling by viral components are probably required to assemble a fully functional antiviral response. although the induction of irgs by ifn-α2b is the hallmark feature of ifn responses, a full understanding of the biological effects of antiviral ifns requires a comprehensive study of the additional functional responses triggered by ifn-α2b. in the blood of ferrets injected with ifn-α2b, the upregulation of genes that participate in glycolysis-gluconeogenesis (e.g. acyl-coa synthetases and lactate dehydrogenases) (supplementary table 3 ) are indicators of higher levels of metabolic activity. moreover, ifn also induces the expression of genes related with apoptosis (e.g. caspases and tnfsf10) and cell cycle (e.g. cyclins and smad family members). it is unclear whether ifn-α2b alone is capable of inducing apoptosis and/or cell replication in vivo, however, the upregulation of these genes may indicate that pbmcs are now more responsive to signals capable of triggering cell cycle events. upregulation of chemokine receptors, such as ccr3, ccr7, ccr9 and cxcr4 may indicate that ifn-α2b can increase the responsiveness of pbmcs to locally produced chemokines. likewise, increased levels of genes that are involved in leukocyte transendothelial migration and fc-gamma receptor-mediated phagocytosis (supplementary table 3 ) suggest that ifn-α2b enhances leukocyte responses (corssmit et al., 1997) . interestingly, a number of genes that are part to the wnt signaling pathway were found to be upregulated (supplementary table 3 ). this indicates that in vivo administration of ifn-α2b also has effects over lymphocyte maturation and differentiation (staal et al., 2008) . the lungs of ferrets infected with sars-cov show the upregulation of a broader variety of genes, as compared with ifn-α2b administration, and depicts a more complex biological environment dominated by the antiviral responses, leukocyte infiltration and other inflammatory responses (fig. 5b) . a number of chemokine ligands, such as chemokine c-c motif ligand 2 (ccl2), ccl4, ccl14, ccl19 and ccl25, and cell adhesion molecules, such as activated leukocyte cell adhesion molecule (alcam) and intercellular adhesion molecule 1 (icam1) are upregulated during sars-cov infection, but these were not induced by the administration of ifn-α2b ( fig. 5a and supplementary table 3) . sars-cov also induced the upregulation of genes of the complement system such as complement component 3 (c3) and complement factor b (cfb) (fig. 5b) . taken together, these results depict how irgs and other arms of the innate immune responses are capable of resolving the respiratory infection caused by sars-cov infection. previously, gene regulation has been investigated using microarray analysis with the intent on revealing molecular pathways imperative to h1n1 infection (shapira et al., 2009 ). here we investigated gene regulation of sars-cov infected and ifn-α2b injected ferrets. microarray analysis was conducted on rna from lungs and blood on day 1 and day 2. the number of upregulated genes was quantified and compared to the number of downregulated genes for each sample type. the number of downregulated genes was greater than upregulated genes in the day 1 ifn-α2b lungs and in the day 2 sars-cov infected blood samples. to expose the molecular signature of this finding we then broke down the genes from each group into their respective functional pathways: cellular process, metabolic process, intracellular signaling cascade, cell cycle, and immune response. interestingly, we found that for every functional pathway the day 1 ifn-α2b-injected lungs had more downregulated genes than upregulated genes (except the immune response) where blood samples from the same animals had the opposite trend of more upregulated genes. furthermore, the sars-cov-infected animals had the opposite trend where day 1 lungs had more upregulated genes and in the blood of day 2 there were more downregulated genes. these findings may be indicative of the activity of the stimulant ifn-α2b compared to sars-cov. moreover, the difference in the number of genes regulated shows that ifn-α2b and sars-cov have different spatial stimulation which may be an important finding when determining the therapeutic efficacy of ifn-α2b. it is possible that the increase in gene expression in the blood samples following ifn-α2b injection is indicative of activation of systemic immunity where the sars-cov infection had an increase of lung gene expression signifying possible local inflammation. type i ifns play a critical role during antiviral responses, however their functions in vivo have not yet been fully resolved. additional research is required to define the optimal irgs profile that is present during successfully cleared viral infections. moreover, fine tuning of the irgs responses may achieve more prolonged and wider protection by therapeutic agents such as attenuated vaccines against respiratory viruses. male, 1 kg, 6-month-old ferrets (mustela putorius furo) were purchased from marshall farms inc. (oak park, il) and housed at the toronto general research institute animal facility (toronto, canada) or at southern research institute (birmingham, al, usa). ferrets were quarantined and monitored 5 days prior to tissue and blood collection. the ferrets' diet was based on a low fat/high protein regimen as recommended by marshal farms. animal protocols were approved and monitored by the animal care committee of the university health network or of the southern research institute. in vitro blood stimulation with ifn-α2b whole blood was drawn from 4 ferrets and diluted ¼ with cell culture media (invitrogen, ca). two ml of diluted blood from 4 animals was stimulated with 0.001 μg/ml ifn-α2b (pegylated ifn-α2b, schering-plough, pointe-claire, canada) in separate wells and incubated at 37°c (5% co 2 ) for 1 and 3 h. pbs was added to 3 control wells. the cultured blood was then harvested and injected to paxgene tubes. rna was purified according to manufacturer's protocol (invitrogen, ca). one ml of blood stimulated with ifn-α2b and or pbs was also added to 10 ml lyse/fix buffer (bd biosciences, usa) for evaluation of phosphorylation status of signaling molecules at 0, 15, 30, 45, 60, 75 and 90 min, using phosflow antibodies (bd biosciences, usa) . subcutaneous injections of 1 ml of pbs (control) or 1 μg/ml of ifn-α2b were performed on the back of each ferret. two ml of blood was collected directly into paxgene tubes. one gram of the lung necropsy was added to trizol® reagent (invitrogen ca). collected blood and lung tissues were used for rna isolation according to the manufacturer's protocol and used for microarray, and quantitative real-time pcr analysis. one ml blood was added to lyse/fix buffer (bd biosciences, san jose, ca) for analysis of the signaling molecule phosphorylation status. ferrets were infected with sars-cov in the animal biohazard safety level 3 (absl3) facility at southern research institute (birmingham, al, usa), in accordance with the approved protocols. three male ferrets, weighing approximately 800-1000 g, were infected intranasally with 10 3 tcid 50 sars-cov tor2 strain (isolated from a patient in toronto and sequenced at cdc, vancouver, bc) in 1 ml pbs. an additional 3 animals were mock-infected with 1 ml pbs. animals were anesthetized and blood and lung necropsies were collected for rna purification. infection of ferrets with the above mentioned dose results in weight loss, decreased activity, temperature increase and histology lesions with no mortality during the disease course (chu et al., 2008) . a summary of natural history of ferrets infected with sars-cov has been provided as supplementary table 4 . one ml of in vitro-stimulated blood with ifn-α2b or pbs and/or 1 ml of blood drawn from the ifn-α2b or pbs injected ferrets and/or 1 ml blood from infected ferrets with sars-cov or mock controls (in vivo) was added to 10 ml lyse/fix buffer (bd biosciences, usa) and incubated in a 37°c water bath for 10 min. tubes were then centrifuged at 300g for 5 min and the cells were washed twice with cold pbs. one ml perm iii (bd biosciences, usa) was added to each tube and the tube was incubated on ice for 20 min to permeabilize cells for intracellular staining. cells were washed with perm/wash (bd biosciences, usa) and 10 6 cells were added to each tube for flow cytometry. twenty microliters of phosphorylated (p)-stat1, p-stat3, p-stat4, p-stat5, p-p38 and p-erk antibodies conjugated with alexa-fluor 488 was added to separate tubes (bd biosciences, usa). matched isotype control was added to one tube as a negative control. tubes were incubated at room temperature in the dark for 30 min. cells were washed with cold perm wash (bd biosciences, usa) and fixed with 2% paraformaldehyde in pbs. twenty thousand events were acquired with a bd facscalibur (bd biosciences, usa) and data were analyzed, using flowjo software (tree star inc., usa). cloning and sequencing was performed as described previously . briefly, purified rna was reverse transcribed to cdna using invitrogen rt-kits (invitrogen, carlsbad, ca). genespecific degenerate primers were designed based on multiple gene sequence alignment analysis of several species using clustalw (1.83) and then used to clone the cdnas for each gene. standard pcrs were performed and specific bands were gel-purified (qiagen, mississauga, canada) and cloned into the pcr 2.1-topo vector (invitrogen, carlsbad, usa). sequences of positive clones were confirmed using an abi 3730xl dna analyzer (applied biosystems, foster city, ca). the identification of genes was performed using basic local alignment search tool (blast) analyses against national centre for biotechnology information (ncbi) database. the following components were added to the reaction mixture plus cdna to a total volume of 10 μl in distilled water: 0.2 μl cdna, 250 nmol forward gene-specific primer, 250 nmol reverse genespecific primer and 5 μl cyber green (applied biosystems, foster city, ca). for every experiment, each reaction was performed in triplicate. an abi 7900 sequence detection system (applied biosystems, foster city, ca) was used for amplification. initial denaturation was 15 min at 95°c, followed by 40 cycles of amplification. each cycle consisted of a denaturation step (15 seconds at 95°c) and an annealing/extension step (1 min at 60°c). expression levels were normalized to β-actin and data were analyzed by sds 2.1 software (applied biosystems, foster city, ca). briefly, 0.5 μg of total rna was isolated using paxgene whole blood purification kits or trizol® reagent. oligonucleotide microarray analysis was performed using affymetrix two-cycle crna synthesis and ivt kits according to the manufacturer's protocols (affymetrix, santa clara, ca). crna samples (20 μg) were labelled and hybridized to affymetrix genechip canine genome 2.0 arrays to monitor the gene expression of over 18,000 canis familiaris mrna/est-based transcripts and over 20,000 non-redundant predicted genes. as described earlier, canine arrays were used following the observation of high levels of homology between canine and ferret nucleotide sequences (average of 89% identity) rowe et al., 2010) . supplementary table 1 demonstrates the amino acid identity of genes in this study compared to available orthologues of human and mouse. the arrays were scanned using an affymetrix gcs3000 7g system according to standard affymetrix protocols. probe-level analysis was performed using probe logarithmic error intensity estimate (plier). the raw intensity values for each individual target on the affymetrix chips were pre-processed with variance stabilization, log 2 -transformation and were then normalized against the time zero datasets with arrayassist v 5.5.1 (stratagene, usa). student's t-tests or edge time course differential expression analysis (storey et al., 2005) were performed with benjamini-hochberg false discovery rate (fdr) correction. genes with a significant difference were selected for agglomerative hierarchical clustering with pearson distance metrics and average linkage distance measurements between clusters using genelinker platinum v 4.6.1 (improved outcomes software, kingston, canada). ingenuity pathway analysis 5.0 software (ingenuity systems inc., redwood city, ca) was used to annotate and organize the gene expression data into networks and pathways. pathways and functional categories were considered as over-represented when fisher's exact test p value was ≤0.05. datasets are publicly available at the ncbi's gene expression omnibus (http:// www.ncbi.nlm.nih.gov/geo) accession number gse22581. t tests or edge analyses were used for statistical analysis considering a biological filter of 1.5 fold change compared to controls and a p value of ≤0.05 as significant. supplementary materials related to this article can be found online at doi:10.1016/j.virol.2010.10.002. regulation of suppressor of cytokine signaling 3 (soc3) by growth hormone in pro-b cells variation in antiviral 2', 5'-oligoadenylate synthetase (2'5'as) enzyme activity is controlled by a single-nucleotide polymorphism at a splice-acceptor site in the oas1 gene global genomic analysis reveals rapid control of a robust innate response in siv-infected sooty mangabeys interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome gene expression analysis of host innate immune responses during lethal h5n1 infection in ferrets interferon, a growing cytokine family: 50 years of interferon research interferon-based therapy of hepatitis c the sars-cov ferret model in an infectionchallenge study effects of interferon-alpha (ifn-alpha) administration on leucocytes in healthy humans cloning, expression and characterization of ferret cxcl10 emergence of a novel swine-origin influenza a (h1n1) virus in humans mortality due to influenza in the united states-an annualized regression approach using multiplecause mortality data molecular characterization of in vivo adjuvant activity in influenza-vaccinated ferrets role of the jak/ stat pathway in the regulation of interleukin-8 transcription by oxidized phospholipids in vitro and in atherosclerosis in vivo pegylated interferon-alpha protects type 1 pneumocytes against sars coronavirus infection in macaques interferon-induced mx proteins in antiviral host defense adrenocorticotrophic hormone (acth) in the treatment of experimental air-borne influenza virus type a infection in the ferret leucocyte response and anti-inflammatory cytokines in community acquired pneumonia the interferon signaling network and transcription factor c/ebp-beta interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study comparable potency of ifnalpha2 and ifnbeta on immediate jak/stat activation but differential down-regulation of ifnar2 virology: sars virus infection of cats and ferrets cloning, expression and immunoassay detection of ferret ifn-gamma innate antiviral response targets hiv-1 release by the induction of ubiquitin-like protein isg15 modeling host responses in ferrets during a/california/07/2009 influenza infection a physical and regulatory map of host-influenza interactions reveals pathways in h1n1 infection wnt signalling in the immune system: wnt is spreading its wings significance analysis of time course microarray experiments interferon signalling network in innate defence mortality associated with influenza and respiratory syncytial virus in the united states mechanisms of type-i interferon signal transduction social and environmental risk factors in the emergence of infectious diseases we are indebted to nikki kelvin for her editing and critical review of this manuscript. we also would like to thank lixia guo and zujiang li for their assistance in cloning of the ferret genes. key: cord-305143-mqd4ioj4 authors: zmasek, christian m.; knipe, david m.; pellett, philip e.; scheuermann, richard h. title: classification of human herpesviridae proteins using domain-architecture aware inference of orthologs (daio) date: 2019-01-06 journal: virology doi: 10.1016/j.virol.2019.01.005 sha: doc_id: 305143 cord_uid: mqd4ioj4 we developed a computational approach called domain-architecture aware inference of orthologs (daio) for the analysis of protein orthology by combining phylogenetic and protein domain-architecture information. using daio, we performed a systematic study of the proteomes of all human herpesviridae species to define strict ortholog groups (sogs). in addition to assessing the taxonomic distribution for each protein based on sequence similarity, we performed a protein domain-architecture analysis for every protein family and computationally inferred gene duplication events. while many herpesvirus proteins have evolved without any detectable gene duplications or domain rearrangements, numerous herpesvirus protein families do exhibit complex evolutionary histories. some proteins acquired additional domains (e.g., dna polymerase), whereas others show a combination of domain acquisition and gene duplication (e.g., betaherpesvirus us22 family), with possible functional implications. this novel classification system of sogs for human herpesviridae proteins is available through the virus pathogen resource (vipr, www.viprbrc.org). herpesviruses comprise a large and diverse order (herpesvirales) of double stranded dna viruses that infect humans and a wide range of other hosts (pellet and roizman, 2007; virus taxonomy: the classification and nomenclature of viruses the online 10th report of the ictv, 2017). human diseases caused by herpesviruses range from vesicular rashes to cancer. the order herpesvirales is subdivided into three families, including the herpesviridae, which is further subdivided into three subfamilies, the alpha-, beta-, and gammaherpesvirinae. within subfamilies, groups of related herpesvirus species are classified into genera. the nine species of human herpesviruses are distributed across the three subfamilies and several genera (table 1) ; these viruses are the main focus of this work. prior studies found that the beta-and gammaherpesvirinae are more closely related to each other than to alphaherpesvirinae (montague and hutchison, 2000) . in contrast to some other human viruses, the human herpesviruses have a long evolutionary history, with evidence suggesting that the primordial herpesvirus diverged into the alpha-, beta, and gammaherpesvirinae approximately 180 million to 220 million years ago (mcgeoch et al., 1995) . coupled with their genome complexity and the availability of numerous complete genome sequences, this deep evolutionary history makes herpesviruses a tractable and informative model to study virus genome evolution at the levels of gene duplication and protein domain rearrangement. nehrt et al., 2011; rogozin et al., 2014) , due to its importance for computational sequence functional analysis (eisen, 1998; zmasek and eddy, 2002) and the significance of gene duplications for biological evolution (zhang, 2003) . orthologs (or groups/clusters of orthologs) have often been inferred by indirect methods based on (reciprocal) pairwise highest similarities [e.g. (remm et al., 2001; tatusov et al., 1997) ]. in this work, we used explicit phylogenetic inference combined with comparison to a trusted species tree for orthology inference, as this approach is likely to yield more accurate results eddy, 2002, 2001) . many eukaryotic proteins, and by extension, proteins of eukaryotic viruses, are composed of multiple domains, components that can each have their own evolutionary history and functional implications. the architecture of a protein is a product of the ordered arrangement of its several domains and their overall tertiary structure. evolutionarily, individual domains can combine with other partner domains, enabling formation of a vast number of domain combinations, even within the same species (moore et al., 2008) . assembling multiple domains into a single protein creates a distinct entity that can be more than the sum of its constituent parts. the emergence of proteins with novel combinations of duplicated and then diverged domains is considered to be a major mechanism for rapid evolution of new functionality in eukaryotic genomes (itoh et al., 2007; peisajovich et al., 2010) . it is especially important in the evolution of pathways, where novel linkages between existing domains may result in the rearrangement of pathways and their behaviors in the cell (peisajovich et al., 2010) . the modular structure of eukaryotic proteins provides a mechanism that enables evolutionarilyrapid differentiation and emergence of a multitude of novel protein functions from an initially limited array of functional domains. proteins can gain (or lose) new domains via genome rearrangements, creating (or removing) domain combinations, in addition to modification of domains themselves by small-scale mutations (patthy, 2003; ye and godzik, 2004) . here we present a systematic classification of proteins catalogued in the ncbi refseq entries for each of the nine human herpesviruses plus selected comparisons with homologs from non-human herpesviruses based on phylogenetic inferencing and domain architecture analysis using domain-architecture aware inference of orthologs (daio). this analysis resulted in the classification of proteins into "strict ortholog groups" (sogs), in which all proteins are orthologous to each other (related by speciation events) and exhibit the same domain architecture. the sog classification also enabled the development of an informative name convention for each sog that includes information about the protein's function (if known) and a suffix indicating the taxonomic distribution of the protein. for example, an "abg" suffix would indicate that proteins of this group are found in some (but not all) human alphaherpesvirinae species (lowercase "a"), and all human beta-and gammaherpesvirinae species (uppercase "b" and "g"). such suffixes allow for the quick understanding of presumed conserved protein function and minimal common genome across the herpesviridae family. the sog classification results have been made publicly available through the virus pathogen resource (vipr) (pickett et al., 2012) at https://www.viprbrc.org. for this analysis, we developed a rational, phylogeny-and domain architecture-aware classification approach for human herpesvirus proteins, the domain-architecture aware inference of orthologs (daio) method, which produces strict ortholog groups (sogs) of proteins. before we present genome-wide findings, we show results for a few instructive sog examples, including protein groups that have evolved in a "simple" manner, recapitulating the herpesviridae evolutionary tree without gene duplications or domain rearrangements, and protein groups in which domain rearrangements (domain gains) and/or gene duplications have occurred. table 2 lists the 23 sogs common to all nine human herpesviruses. for every sog, a suggested name is provided, composed of a protein names and a suffix indicating the taxonomic distribution (a, b, g: present in all human members of the alpha-, beta-, gammaherpesvirinae, respectively; a, b, g: present in some but not all human members of the alpha-, beta-, gammaherpesvirinae, respectively). gene names/symbols (a forward slash is either part of the accepted gene name or is used to separate multiple gene names) and pfam domain architecture names are also included. the table is organized into three sections. the first section lists protein families that have apparently evolved without gene duplication or domain rearrangements [e.g., uracil dna glycosylase and the capsid scaffolding protein protease (cspp)]; the second section lists proteins that have evolved with domain rearrangements and or duplications [e.g., glycoprotein b (gb), dna polymerase, and multifunctional regulator of expression proteins (mre)], and the third section lists proteins that share some function (and even genome region) but have been formed from distantly or unrelated domains (e.g., gl, gn, and dna polymerase processivity factor). uracil dna glycosylases catalyze the first step -removal of the rna base uracil from dna -in base excision repair, the mechanism by which damaged bases in dna are removed and replaced (krusong et al., 2006) . uracil dna glycosylases are found in eukaryotes, bacteria, and archaea, as well as in herpesviruses and poxviruses (chen et al., 2002) . our phylogenomic analysis shows that for all nine human herpesviruses, uracil dna glycosylase is well conserved and contains one pfam domain, udg (uracil dna glycosylase superfamily). in addition, the gene tree for human herpesvirus uracil dna glycosylases ( fig. 1b ) precisely recapitulates the herpesvirus species tree (fig. 1a) ; therefore, this protein family can be inferred to have evolved from a single common ancestor and without any gene duplications or domain rearrangements (see table 2 for virus-specific gene names). capsid scaffolding protein proteases are essential for herpesvirus capsid assembly and maturation, and have an essential serine protease activity (liu and roizman, 1993) . these proteins contain one pfam domain, peptidase_s21. in contrast to uracil dna glycosylases, currently available data indicate that protease-scaffolding proteins with a peptidase_s21 domain are unique to herpesvirales. like uracil dna glycosylases, cspp evolved without domain architecture rearrangements or gene duplications (fig. 1c , table 2 ). other examples of herpesviridae genes that have evolved without any domain architecture rearrangements or gene duplications are listed in the first section of table 2 . herpesvirus virions have an envelope that consists of an outer lipid bilayer studded with 12 or more surface glycoproteins (originally defined in hsv). after virion glycoprotein engagement with cell surface receptors, the envelope fuses with the plasma membrane -a process which, for herpes simplex virus 1 (hsv-1), requires four of its 12 envelope glycoproteins, namely glycoproteins gb, gd, gh, and gl (cai et al., 1988; forrester et al., 1992; ligas and johnson, 1988; roop et al., 1993; spear and longnecker, 2003) . in contrast, for other herpesviruses, only glycoproteins gb, gh, and gl have been reported to be required for membrane fusion (alhajri et al., 2017) . gb and gh are highly conserved across all nine human herpesviruses (table 2) . a protein annotated as gl is also present in all nine human herpesviruses, yet its occurrences in members of the alpha-, beta-and gammaherpesvirinae are homologous within, but not between subfamilies. gls from different subfamilies contain unrelated protein domains (pfam: herpes_ul1, cytomega_gl, and phage_glycop_gl). gl is discussed in more detail below. detailed phylogenetic analysis of the human herpesvirus gb family ( fig. 2a) , including proteins from selected non-human members of the herpesviridae, shows a picture of a protein that has evolved without gene duplications (or, at the very least, duplicated genes have not been retained) and with nearly completely conserved domain architectures. the one exception to this is that human cytomegalovirus (hcmv) glycoprotein b (gb) has a short region of about 40 amino acids near its nterminus that comes in two forms that differ by approximately 50% at the amino acid level. this sequence variant was identified in hcmv strains isolated from chinese patients (shiu et al., 1994) and is identified in pfam as "hcmvantigenic_n domain". in our global hmmscan analysis (applying the same threshold of e = 10 −6 for every pfam domain) evalue support for presence of this domain in some strains is strong (e < 10 -22 ) and matching over the entire pfam model while other hcmv strains do not exhibit significant sequence similarity with this domain. it has been suggested that this domain polymorphism may be implicated in hcmv-induced immunopathogenesis, as well as in strainspecific behaviors, such as tissue-tropism and the ability to establish persistent or latent infections (pignatelli et al., 2004) . in our new systemic naming approach (see below) we term the sog of the protein with hcmvantigenic_n domain "glycoprotein b_ abg.b", whereas all other proteins fall into the "glycoprotein b_ abg.abg" sog. all members of the herpesviridae encode six conserved proteins that play essential roles at the replication fork during viral dna replication: a single-strand dna binding protein (major dna binding protein), a dna polymerase composed of two independently coded subunits (the catalytic dna polymerase subunit and a dna polymerase processivity factor encoded by three distantly related genes in members of the alpha-, beta-, and gammaherpesvirinae, see below), and a three subunit helicase/primase complex (dna replication helicase, dna helicase primase complex associated protein, and dna primase) (pellet and roizman, 2007) . our analysis shows that the catalytic dna polymerase subunits of all members of the herpesviridae contain two domains: an n-terminal dna polymerase family b exonuclease domain, and a c-terminal polymerase domain from dna polymerase family b (fig. 2b ). cellular family b dna polymerases are the main polymerases involved with nuclear dna replication and repair in eukaryotes and prokaryotes, and include dna polymerases ii and b, and polymerases α, δ, and ε (garciadiaz and bebenek, 2007) . family b dna polymerases are also found in other dsdna viruses, such as the insect ascoviridae, and members of the iridoviridae (e.g., fish lymphocystis disease virus) and phycoviridae (e.g., chlorella virus) (villarreal and defilippis, 2000) . in addition to these two large and ubiquitous domains, simplexvirus (which include human simplex virus 1 and 2) and mardivirus also possess a small c-terminal domain, called the dna polymerase catalytic subunit pol (dnapoly-mera_pol) domain in pfam (zuccola et al., 2000) , and are longer by about 45 aa on average than dna polymerase proteins from other herpesviridae. according to currently available genomic data, dnapo-lymera_pol is found in members of the simplexvirus genus of the alphaherpesvirinae. while varicella-zoster virus (human herpesvirus 3) and other members of the varicellovirus genus of the alphaherpesvirinae possesses dna polymerases that also tend to be longer, similarity of these protein regions to the dnapolymera_pol domain is low, using the current pfam model for dnapolymera_pol (pfam version 31.0). the function of this third domain is to mediate interaction between dna polymerase and its cognate processivity factor (bridges et al., 2000; loregian et al., 2000) based on the observation that a peptide corresponding to the 27 c-terminal amino acids of hsv-1 dna polymerase has been shown to inhibit viral replication by disrupting the interaction between dna polymerase and ul42 (digard et al., 1995; loregian et al., 1999) . in this context, it is interesting to note that the dna polymerase processivity factors are only distantly-related across the alpha-, beta-, and gammaherpesvirinae (see below). it is therefore conceivable that the interactions of beta-, and gammaherpesvirinae dna polymerase processivity factors with their corresponding dna polymerases (which lack a dnapolymera_pol domain) is different in nature than for alphaherpesvirinae. as for varicellovirus it is unclear whether they possess a functional dnapolymera_pol domain, and a definitive answer will require similar biochemical assays as have been performed for hsv-1. phylogenetic analysis of human herpesvirus dna polymerase proteins, plus related proteins from selected mammalian herpesviruses, shows that, similar to the glycoprotein b family, dna polymerases of the herpesviride evolved without gene duplication. nonetheless, in contrast to gb, dna polymerases acquired a new domain early in alphaherpesvirinae evolution. this domain might have been lost again, or underwent significant mutations, during varicellovirus evolution. the presence of the longer domain in varicelloviruses suggests that the longer domain emerged prior to the varicellovirus/simplexvirus split. multifunctional regulator of expression (mre; also known as immediate-early protein ie63, infected cell protein 27, icp27, and α27) is a protein with homologs in all human herpesviruses (for gene names see table 2 ). multifunctional regulator of expression is a regulatory protein that plays a role in the prevention of apoptosis during hsv1 infection (aubert and blaho, 1999) . multifunctional regulator of expression interacts directly with a number of proteins in performing its many roles. in particular, multifunctional regulator of expression protein contributes to host shut-off by inhibiting pre-mrna splicing by interacting with essential splicing factors, termed sr proteins, and affecting their phosphorylation (sciabica et al., 2003) . furthermore, the mre protein c.m. zmasek et al. virology 529 (2019) [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] has been shown to associate with cellular rna polymerase ii holoenzyme in a dna-and rna-independent manner and to recruit rna polymerase ii to viral transcription/replication sites (dai-ju et al., 2006; zhou and knipe, 2002) . mre also competes with some transport receptors, resulting in the inhibition of host pathways while supporting mrna export factor-mediated transport of hsv-1 mrnas (malik et al., 2012) . all of the multifunctional regulator of expression proteins analyzed here have a single copy of a pfam "herpesvirus transcriptional regulator family" (herpes_ul69) domain that is specific to members of the herpesviridae. in addition to the herpes_ul69 domain, human simplexvirus mre have an additional n-terminal domain, the "herpes viral adaptor-to-host cellular mrna binding domain" (hhv-1_vabd) (tunnicliffe et al., 2011) . besides human simplexvirus, architectures with c-terminal hhv-1_vabd and n-terminal herpes_ul69 domains are also found in chimpanzee herpesviruses (e.g. ncbi reference sequence: yp_009011042 (severini et al., 2013) ), while other non-human simplexviruses lack the hhv-1_vabd domain. using currently available genomic data, we were unable to detect hhv-1_vabd domains outside of the simplexvirus genus. phylogenetic analysis of human herpesvirus mre proteins, including proteins from selected herpesviruses of other mammals, shows that multifunctional regulator of expression proteins evolved without observable gene duplications (since this gene tree recapitulates the herpesvirus species tree). nine groups of human herpesviruses are annotated as performing the same, or very similar function, in the absence of discernable protein sequence similarity (table 2, fig. 3 ). as mentioned above, dna polymerase processivity factor is one of the six proteins that play essential roles at the replication fork during viral dna replication. processivity factors, also called clamp proteins, help to overcome the tendency of dna polymerase to dissociate from the template dna, and thus greatly enhance dna polymerase processivity (weisshart et al., 1999; zhuang and ai, 2010) . in contrast to the protein families discussed so far, dna polymerase processivity factors are only distantly-related across the alpha-, beta-, and gammaherpesvirinae. in the alphaherpesvirinae, the protein is composed of two tandem herpes_ul42 domains; betaherpesvirinae have a single herpe-s_pap domain; gammaherpesvirinae have a single herpes_dnap_acc domain (fig. 3a, b, c) . these three domains are very distant homologs and are members of the dna clamp superfamily (pfam clan cl0060). gl (fig. 3d , e, f) is another example of a protein function performed by different, probably non-homologous domains present in different herpesviridae subfamilies (pfam domains herpes_ul1, glyl_c, cyto-mega_gl, and phage_glycop_gl). interestingly, the open reading frames for these seemingly unrelated proteins are located in analogous conserved genomic contexts, including open reading frame sizes and orientations relative to the surrounding conserved coding regions. the remaining seven groups with these characteristics are: cytoplasmic egress tegument protein, cytoplasmic egress facilitator-1, cytoplasmic egress facilitator-2, encapsidation chaperone protein, glycoprotein n pfam clan herpes_glyco, cl0146), ltp binding protein, and small capsid protein (table 1 and supplementary table 1) . in contrast to the protein families discussed so far, the evolutionary history of human herpesviridae proteins with 7-transmembrane receptor domains is more complex (fig. 4) (spiess et al., 2015) . by comparing this gene tree with a species tree for human herpesviridae (fig. 1a) , we can infer three gene duplication events (marked as red squares in fig. 4) , resulting in four groups of orthologous genes: ul33/u12, us27, u51/orf74, and us28. in our new nomenclature (see below), we call the first group "gprotein coupled receptor homolog ul33/u12_b" because it is found in all four human betaherpesvirinae species (uppercase b suffix). the second group is called "g-protein coupled receptor homolog us27_b" as it is found in some human betaherpesvirinae (lowercase b suffix). the third group is called "g-protein coupled receptor homolog u51/orf74_bg" because it found in some human betaherpesvirinae and in some human gammaherpesvirinae (lowercase "bg" suffix). the fourth group is called "envelope protein us28_b". no orthologous genes were found in the human alphaherpesvirinae. whenever available, we base our names preferably on (mocarski, 2007) or the "recommended name" (under "protein names") from the uniprotkb database (bateman et al., 2017) . for reasons of consistency and objectivity, we used an automated approach to root all trees by mid-point rooting. it is possible, that the true root for the 7-transmembrane domain proteins tree is at the base of the u51-orf74 subtree. in this case there would be only two duplications in the tree, but still the same four ortholog groups: u51/orf74, us28, us27, ul33/u12. functionally, all these proteins appear to be hijacked human proteins that are being used by the virus to modulate the host immune system. in particular, many of them appear to act as chemokine (orphan) receptors (casarosa et al., 2003 (casarosa et al., , 2001 isegawa et al., 1998; murphy, 2001; zhen et al., 2005) (fig. 5) . proteins with us22 domains have the most complex evolutionary history of all herpesviridae proteins, even though among the human herpesviruses, the us22 domain has been found only in c.m. zmasek et al. virology 529 (2019) 29-42 betaherpesviruses (hanson et al., 1999) . us22 domain proteins are also present in gallid herpesvirus 2 (a member of the alphaherpesvirinae), in members of the alloherpesviridae family, in other dsdna viruses (e.g., poxviridae and iridoviridae), and in some animal species. most proteins with us22 domains carry two copies of the domain. us22 is a member of a large group of distantly homologous proteins (the sukh superfamily, pfam clan cl0526), which, for example include bacterial syd proteins. it has been suggested that a function of the us22 family is to act against various anti-viral responses by interacting with specific host proteins (zhang et al., 2011) . here we summarize the results of our phylogenetic analysis of us22 domain proteins of the human bataherpesviruses. unfortunately, the phylogenetic signal across this group of protens is weak, thus some support values are low. two groups of us22 orthologs span all four human betaherpesviruses: cmv tegument protein ul23 is likely to have orthologs in hhv-6a, hhv-6b, hhv-7 (roseolovirus) protein u3 ("tegument protein ul23/protein u3_b"). similarly, cmv tegument protein ul43 is likely to be orthologous to hhv-6a, hhv-6b, hhv-7 (roseolovirus) protein u25 ("tegument protein ul43/protein u25_b"). u3 and u25 are paralogous towards each other, as they are connected by a gene duplication, as are hcmv ul23 and 43. four groups of orthologs specific to roseolovirus are tegument protein dr1, tegument protein dr6, protein u7, and protein u17/u16. in u17/u16 proteins, it is unclear whether they possess a second us22 domain, as the similarity to this domain is weak to the point of insignificance. in contrast, u7 proteins possess at least three us22 domains and an additional c-terminal herpes_u5 domain. proteins u7 are most closely related to cmv ul29, but differ in their domain architecture (lack of herpes_u5 domain). thus cmv ul29 forms its own species-specific group of orthologs. numerous proteins with us22 domains are specific to cmv (and thus all paralogous to each other) given current data: apoptosis inhibitor ul38, early nuclear protein hwlf1, tegument protein ul26, us24, protein ul24, ul29, ul36, us23, us26, protein irs1, and protein trs1. c.m. zmasek et al. virology 529 (2019) 29-42 2.8. the inferred minimal proteomes of the human herpesviruses as described above, we classified viral proteins into "strict ortholog groups," requiring that all proteins exhibit the same domain architecture and are orthologous to each other. we attempted to give an informative name for each of these groups including a suffix that indicates the taxonomic distribution of a protein. for example, an "ag" suffix would indicate that proteins of this group are found in some (but not all) members of human alphaherpesvirus species (lowercase "a"), and members of both human gammaherpesvirus species (uppercase "g"). families which have a (some) domain(s) in common but differ in their domain architectures, are more difficult to rationally name (we found 17 of these cases). an example of such a family is dna polymerase. in such cases, the suffix is split by a period into two parts. the first part indicates overall presence of common domain(s) for all members of this sog, the second part (after the period) relates to specific domain architectures. thus, "dna polymerase_ abg.abg" refers to the simpler dna_pol_b_exo1--dna_pol_b domain architecture present in nearly all alphaherpesvirinae species. "dna polymerase_ abg.a" refers to the dna_pol_b_exo1--dna_pol_b-dnapolymera_pol da that is present in a smaller subset of alphaherpesvirinae species. the rationale behind this approach for labeling members of protein families that have different domain architectures is that it gives users a choice between "traditional" ortholog groups, which do not consider domain architectures (by ignoring the part after the period), and sogs (taking the full name into account). in total, we were able to establish 169 sogs (supplementary table 1 ). of these, 40 (23 +8 +9) functionally similar groups (table 2) are present in all 9 human herpesviridae species and represent the core proteins of human herpesviruses. besides proteins with clearly defined pfam domains, we found 29 protein families for which pfam domains have not been defined. classification of these proteins was based on manual blast searches. an example of such a family is the virion host shutoff protein ul41. another unusual case is the hsv1 ul13 serine threonine protein kinase. all nine human herpesviruses have homologs of this protein, but its associated pfam domain ul97 only matches sequences in betaherpesviruses. extension of the family to alpha-and gammaherpesviruses is thus based on manual blast searches. finally, two protein families could not be classified due to lack of phylogenetic signal: protein b8 of hhv-6a and hhv-6b (associated gene names u92, u93, hn1, hn92d, b8) and protein ul28/ul29/u8 of hhv-6a, hhv-6b, and hhv-7. proteins which are species or strain specific are listed in supplementary table 2. in order to make the results of daio classification available to all herpesvirus researchers for experimental hypothesis testing, we incorporated sog data into the virus pathogen resource (vipr) at https://www.viprbrc.org (pickett et al., 2012) . through vipr, scientists can search, sort, and download sog names (including taxonomic distribution), pfam domain architecture data, and individual protein sequences belonging to selected sogs. fig. 6a shows an example of a search result table, which includes data for some of the protein families discussed above, namely glycoprotein b family members (associated with two distinct sogs: "glycoprotein b_ abg.b" and "glycoprotein b_ abg.abg"), dna polymerase ("dna polymerase_ abg.a" and "dna polymerase_ abg.abg"), and multifunctional regulator of expression ("multifunctional regulator of expression_abg.a" and "multifunctional regulator of expression_abg.abg"). by clicking on the "total # of proteins" table entries, users can view and download the individual protein sequences belonging to a given sog. fig. 6b shows how sog data, including domain architecture information, is part of protein annotations in vipr (simplexvirus "dna polymerase_abg.a" example). as new genome sequence data become available, the sog data in vipr is continuously updated in order to keep current with the ever expanding universe of herpesvirus protein sequences. in addition, sog annotations in vipr will be expanded to include non-human herpesviruses in the future. sog data is also available for pox-and coronaviruses in vipr, and will be applied to other virus families in the future. in this work, we used domain-architecture aware inference of orthologs (daio) to provide a classification for proteins of human herpesviruses, based on domain architecture and phylogenetic history. while the work presented here is limited to human herpesviruses, and thus does not take full advantage of all the sequence data that is currently available, we plan to extend our daio approach to all herpesviruses with a known phylogenetic history. a major contribution of our classification system to herpesvirus biology is that it provides a series of testable hypotheses for further experimental investigations. for example, it informs experimental reconstruction of minimal genome viruses. such synthesized minimal genomes could prove useful for identification of genes responsible for pathogenic and other biological differences between viruses. of particular interest in the field of molecular biology is the relationship between domain architecture and protein function. the detailed analysis of domain architectures presented here suggests studies that investigate the functional effects of removing or swapping domains in viral multidomain protein architectures the fact that simplexvirus dna polymerases contain the extra dnapolymera_pol domain and that this domain architecture is conserved among simplexvirus isolates suggests that it may provide some unique function necessary for efficient replication of simplexviruses. this hypothesis could be explored experimentally. similarly, what would be the consequence of adding a cterminal glyl_c domain to the gl protein of vzv (which contains one herpes_ul1 domain), and so making it similar to the gl protein found in hsv-1 and hsv-2 (which has a herpes_ul1--glyl_c architecture)? interestingly, while it has been noted that domain loss is an important mechanism in eukaryote evolution (probably equally-and possibly even more-important than domain gain) (zmasek and godzik, 2011) ; and references therein), in herpesvirus evolution domain loss seems to play a lesser role, as most of the events we were able to detect are domain gains (according to the parsimony principle). another implication of this work relates to the observation that in some cases proteins that share the same name are composed of either unrelated (e.g. gl) or very distantly related domains (e.g. dna polymerase processivity factor) in different herpesvirus species. this raises the question -are such share named truly justified for proteins composed of unrelated domains? and to what extent has their putative shared function been experimentally validated. our approach is also expected to facilitate the detection and subsequent experimental study of species-(and strain-) specific proteins (listed in supplementary table 2). whereas hsv1 and hsv2 do not have any species specific proteins given current data, vzv has six, and cmv has by far the most with 130 proteins which are not found in any other species. interestingly, many of these 130 proteins are specific to one strain (or isolate) of cmv. unsurprisingly, many of these species-and strain-specific protein do not yet have a pfam domain (and thus were analyzed by manual blast searches in this work). an example of such a protein is the orf45 protein of kshv (zhu and yuan, 2003) . our automated approach provides a starting point for the systematic computational and experimental study of these species-and strain-specific proteins-studies, which eventually will provide answers to such questions as: are these species-and strain-specitic proteins essential under certain conditions? do they result in altered pathology or clinical symptoms? do they function in host interaction? do they possess as of yet undiscovered, but shared protein domains? in summary, we developed a computational approach called domain-architecture aware inference of orthologs (daio) for the classification of viral proteins into groups of orthologous proteins with identical domain architecures (sogs). in addition, we established a nomenclature for sogs that provides the user with information about the biological function and taxonomic distribution for the member proteins of a sog. we applied this classification and nomenclature to the proteomes of all human herpesviridae species and made the results publicly accessible via the vipr database. the acquisition and retention of novel domain architectures suggests that some herpesviridae proteins may have acquired novel functional characteristics, which can now be explored experimentally. we developed a semi automated software pipeline to analyze amino acid sequences for their protein domain based architectures and to infer multiple sequence alignments and phylogenetic trees for the molecular sequences corresponding to these architectures, followed by gene duplication inference. this pipeline contains the following five major steps: (1) sequence retrival; (2) domain architecture anlysis, including the inference of the taxonomic distributions of domain architectureseach of which corresponding to one preliminary sog, and manual naming of domain architecures/preliminary sogs (to be automated in future versions of this pipeline); (3) extraction of molecular sequences corresponding to domain architectures/preliminary sogs; (4) multiple sequence alignment and phylogenetic inference; (5) gene duplication inference, to determine which preliminary sogs contain sequences related by gene duplications and thus need to divided in multiple, final sogs. links to all custom software programs developed for this work are available here: https://sites.google.com/site/cmzmasek/home/ software/forester/daio. in the following the tools and methods used are described in more detail. individual protein sequences were downloaded from the vipr database (pickett et al., 2012) , while entire proteomes were downloaded from uniprotkb (bateman et al., 2017) . multiple sequence alignments were calculated using mafft version 7.313 (with "localpair" and "maxiterate 1000" options) (katoh and standley, 2013; kuraku et al., 2013) . prior to phylogenetic inference, multiple sequence alignment columns with more than 50% gaps were deleted. for comparison we also performed the analyses based on alignments for which we only deleted columns with more than 90% gaps. protein domains were analyzed using hmmscan from hmmer v3.1b2 (eddy, 2011) and the pfam 31.0 database (finn et al., 2016) . c.m. zmasek et al. virology 529 (2019) 29-42 phylogenetic trees were calculated for individual domain architectures (not full-length sequences) except for us22 domain proteins, because us22 domain alignments lack phylogeneticly sufficient signal. distance-based minimal evolution trees were inferred by fastme 2.0 (desper and gascuel, 2002) (with balanced tree swapping and "gme" initial tree options) based on pairwise distances calculated by tree-puzzle 5.2 (schmidt et al., 2002) using the wag substitution model (whelan and goldman, 2001 ), a uniform model of rate heterogeneity, estimation of amino acid frequencies from the dataset, and approximate parameter estimation using a neighbor-joining tree. for maximum likelihood approaches, we employed raxml version 8.2.9 (stamatakis et al., 2005) (using 100 bootstrapped data sets and the wag substitution model). tree and domain composition diagrams were drawn using archaeopteryx [https://sites.google.com/site/cmzmasek/home/ software/forester]. rooting was performed by the midpoint rooting method. unless otherwise noted, pfam domains are displayed ith a e = 10 −6 cutoff. gene duplication inferences were performed using the sdi and rio methods eddy, 2002, 2001) . automated genome wide domain composition analysis was performed using a specialized software tool, surfacing version 2.002 [zmasek cm (2012) , a tool for the functional analysis of domainome/genome evolution [available at https://sites.google.com/site/cmzmasek/home/software/ forester/surfacing]. all conclusions presented in this work are robust relative to the alignment methods, the alignment processing, the phylogeny reconstruction methods, and the parameters used. all sequence, alignment, and phylogeny files are available upon request. the processes for defining and naming strict ortholog groups were formalized into a set of "rules" and then implemented into a semi-automatic domain-centric phyloinformatics pipeline. any unique arrangement of single or multiple pfam domains is considered a domain architecture (da) godzik, 2012, 2011) . most proteins of members of the herpesviridae have das consisting of only a single domain. for example, the udg domain of uracil dna glycosylase is a single domain da, whereas the combination of n-terminal dna_-pol_b_exo1 and c-terminal dna_pol_b (denoted as dna_pol_b_ex-o1--dna_pol_b) of dna polymerases is a da with two domains. in this analysis, we consider a given da "present" in a given herpesviridae species s if the da is present under a set of thresholds in at least one strain of the species s. the rationale for this is that it is possible to miss a da in a genome, due to incomplete or erroneous sequences, erroneous assembly and gene-predication (false negatives), and even recent, actual gene loss. the opposite (false positive), on the other hand, is far less likely. for this work, we used two thresholds: a minimal domain length of 40% of the length set forth in the pfam database (domain fragments are unlikely to be functionally equivalent to full length domains) and a hmmscan e-value cutoff of e = 10 −6 . for every domain architecture, a set of bootstrap resampled phylogenetic trees (gene trees) was calculated by raxml (stamatakis et al., 2005) using protein sequences from one representative for each of the nine human herpesviridae species. for comparison and validation, we also calculated phylogenetic trees that included non-human hosted herpesviridae. for illustrations, gene duplications were inferred by comparing the consensus gene trees to the species tree ( fig. 1) for herpesviridae using the sdi (speciation duplication inference) algorithm (zmasek and eddy, 2001) . to obtain confidence values on orthology assignments (bootstrap support values), we employed the rio approach (resampled inference of orthologs) to compare sets of bootstrap resampled phylogenetic trees with the species tree for herpesviridae (zmasek and eddy, 2002) . in this work, we define a strict ortholog group (sog) as sequences related by speciation events and exhibiting the same domain architecture (based on pfam domains from pfam 31.0, a length threshold of 40%, and e-value cutoff of e = 10 −6 ). based on this approach for defining sogs, we developed the following naming syntax. for protein families such as uracil dna glycosylase, which exhibit the same da in all nine human herpesviridae, and which are related by speciation events only, we base our names on (mocarski and edward, 2007) as the base name and add a case-sensitive suffix that indicates the taxonomic distribution -"abg" in this case, since uracil dna glycosylase appears in each human alpha-, beta-, and gammaherpesvirinae species. therefore, the full name is "uracil dna glycosylase_abg". to indicate presence in some, but not all members of a subfamily, we use lower-case suffixes. "replication origin-binding protein_ ab" implies that members of this sog are present in all human alphaherpesvirinae species ("a"), and in some (but not all) betaherpesvirinae ("b"). while most of the human herpesviridae protein families fall into these basic cases, families which have a (some) domain(s) in common but differ in their da, are more difficult to rationally name. an example of such a family is glycoprotein b described above. because members of this family have different das, namely "glycoprotein_b" and "hcmvantigenic_n-glycoprotein_b", it is composed of two sogs (named "glycoprotein b_ abg.abg" and "glycoprotein b_ abg.b"). in such cases, we split the suffix into two parts, separated by a period. the first part ("abg") indicates overall presence of common domain(s) for all members of this sog, glycoprotein_b in this case. the second part (after the period) relates to entire das. ". abg" of "glycoprotein b_ abg.abg" means that the glycoprotein_b da is present in all human alpha-and gamma-, and some betaherpesvirinae. ".b" of "glycoprotein b_ abg.b" implies that the "hcmvantigenic_n-glycoprotein_b" da is present in some betaherpesvirinae. ovine herpesvirus 2 glycoproteins b, h, and l are sufficient for, and viral glycoprotein ov8 can enhance, cell-cell membrane fusion resolving the ortholog conjecture: orthologs tend to be weakly, but significantly, more similar in function than paralogs the herpes simplex virus type 1 regulatory protein icp27 is required for the prevention of apoptosis in infected human cells uniprot: the universal protein knowledgebase secondary structure and structure-activity relationships of peptides corresponding to the subunit interface of herpes simplex virus dna polymerase role of glycoprotein b of herpes simplex virus type 1 in viral entry and cell fusion constitutive signaling of the human cytomegalovirus-encoded chemokine receptor us28 constitutive signaling of the human cytomegalovirus-encoded receptor ul33 differs from that of its rat cytomegalovirus homolog r33 by promiscuous activation of g proteins of the gq, gi, and gsclasses roles of uracil-dna glycosylase and dutpase in virus replication the ortholog conjecture is untestable by the current gene ontology but is supported by rna sequencing data icp27 interacts with the cterminal domain of rna polymerase ii and facilitates its recruitment to herpes simplex virus 1 transcription sites, where it undergoes proteasomal degradation during infection herpesvirus systematics evolution of the herpesviruses fast and accurate phylogeny minimum-evolution principle specific inhibition of herpes simplex virus dna polymerase by helical peptides corresponding to the subunit interface accelerated profile hmm searches phylogenomics: improving functional predictions for uncharacterized genes by evolutionary analysis the pfam protein families database: towards a more sustainable future distinguishing homologous from analogous proteins construction and properties of a mutant of herpes simplex virus type 1 with glycoprotein h coding sequences deleted multiple functions of dna polymerases transcriptional analysis of the murine cytomegalovirus hindiii-i region: identification of a novel immediate-early gene region human herpesvirus 6 open reading frame u12 encodes a functional beta-chemokine receptor evolutionary history and functional implications of protein domains and their combinations in eukaryotes orthologs and paralogs -we need to get it right mafft multiple sequence alignment software version 7: improvements in performance and usability a comparative study of uracil-dna glycosylases from human and herpes simplex virus type 1 aleaves facilitates on-demand exploration of metazoan gene family trees on mafft sequence alignment server with enhanced interactivity a herpes simplex virus mutant in which glycoprotein d sequences are replaced by beta-galactosidase sequences binds to but is unable to penetrate into cells characterization of the protease and other products of aminoterminus-proximal cleavage of the herpes simplex virus 1 ul26 protein intranuclear delivery of an antiviral peptide mediated by the b subunit of escherichia coli heatlabile enterotoxin the catalytic subunit of herpes simplex virus type 1 dna polymerase contains a nuclear localization signal in the ul42-binding region herpes simplex virus icp27 protein directly interacts with the nuclear pore complex through nup62, inhibiting host nucleocytoplasmic transport pathways molecular phylogeny and evolutionary timescale for the family of mammalian herpesviruses toward a comprehensive phylogeny for mammalian and avian herpesviruses comparative analysis of herpesvirus-common proteins gene content phylogeny of herpesviruses arrangements in the modular evolution of proteins viral exploitation and subversion of the immune system through chemokine mimicry testing the ortholog conjecture with comparative functional genomic data from mammals modular assembly of genes and the evolution of new functions rapid diversification of cell signaling phenotypes by modular domain recombination. science. (80-.) herpesviridae: a brief introduction vipr: an open bioinformatics database and analysis resource for virology research genetic polymorphisms among human cytomegalovirus (hcmv) wild-type strains automatic clustering of orthologs and in-paralogs from pairwise species comparisons gene family level comparative analysis of gene expression n mammals validates the ortholog conjecture a mutant herpes simplex virus type 1 unable to express glycoprotein l cannot enter cells, and its particles lack glycoprotein h tree-puzzle: maximum likelihood phylogenetic analysis using quartets and parallel computing icp27 interacts with srpk1 to mediate hsv splicing inhibition by altering sr protein phosphorylation genome sequence of a chimpanzee herpesvirus and its relation to other primate alphaherpesviruses sequence variation of the amino-terminal antigenic domains of glycoprotein b of human cytomegalovirus strains isolated from chinese patients herpesvirus entry: an update identification and functional comparison of seven-transmembrane g-protein-coupled bilf1 receptors in recently discovered nonhuman primate lymphocryptoviruses raxml-iii: a fast program for maximum likelihood-based inference of large phylogenetic trees a genomic perspective on protein families. science (80-.) structural basis for the recognition of cellular mrna export factor ref by herpes viral proteins hsv-1 icp27 and hvs orf57 a hypothesis for dna viruses as the origin of eukaryotic replication proteins the classification and nomenclature of viruses the online (10th) report of the ictv herpes simplex virus processivity factor ul42 imparts increased dna-binding specificity to the viral dna polymerase and decreased dissociation from primer-template without reducing the elongation rate a general empirical model of protein evolution derived from multiple protein families using a maximum-likelihood approach comparative analysis of protein domain organization a novel immunity system for bacterial nucleic acid degrading toxins and its recruitment in various eukaryotic and dna viral systems evolution by gene duplication: an update the human herpesvirus 6 g protein-coupled receptor homolog u51 positively regulates virus replication and enhances cell-cell fusion in vitro association of herpes simplex virus type 1 icp8 and icp27 proteins with cellular rna polymerase ii holoenzyme the orf45 protein of kaposi's sarcoma-associated herpesvirus is associated with purified virions processivity factor of dna polymerase and its expanding role in normal and translesion dna synthesis rio: analyzing proteomes by automated phylogenomics using resampled inference of orthologs a simple algorithm to infer gene duplication and speciation events on a gene tree this déjà vu feeling-analysis of multidomain protein evolution in eukaryotic genomes strong functional patterns in the evolution of eukaryotic genomes revealed by the reconstruction of ancestral protein domain repertoires the crystal structure of an unusual processivity factor, herpes simplex virus ul42, bound to the c terminus of its cognate polymerase the authors thank sanjay vashee for critical review of the manuscript. we also thank the primary data providers for sharing their data in public archives, including vipr and uniprotkb. this work was funded by the national institute of allergy and infectious diseases (nih/dhhs) under contract no. hhsn272201400028c to rhs. supplementary data associated with this article can be found in the online version at doi:10.1016/j.virol.2019.01.005. key: cord-330847-a84pcc9z authors: edwards, m. c.; petty, i.t.d.; jackson, a. o. title: rna recombination in the genome of barley stripe mosaic virus date: 1992-07-31 journal: virology doi: 10.1016/0042-6822(92)90722-2 sha: doc_id: 330847 cord_uid: a84pcc9z abstract barley stripe mosaic hordeivirus (bsmv) is a positive-strand rna virus requiring three single-stranded rnas (α, β, and γ) for infectivity. a terminal-sequence-dependent cloning strategy was used to clone the entire genome of the cv17 strain. full-length γ cdna clones were obtained when oligonucleotides specific for the 5′-terminal sequence of rna α were used in the cloning procedure, but not when rna γ-specific oligonucleotides were used. sequence analysis of six putative γ cdna clones revealed that nucleotides 1–70 possess 89% homology with the first 70 nucleotides of rna α. this leader region is separated from the γ-specific coding region by an eight-base intervening sequence common to both cv17 rnas α and γ. northern and southern hybridization with oligonucleotide probes specific for either α or γ leader sequences indicated that cv17 γ cdna clones are representative of native cv17 γ rnas. furthermore, bioassays indicated that in vitro transcripts derived from these γ cdna clones were infectious when coinoculated with in vitro transcripts of full-length α and β cdna clones. thus, the evidence suggests that rna γ of bsmv strain cv17 is a recombinant molecule which may have arisen as a result of natural recombination between rnas α and γ. barley stripe mosaic virus (bsmv), a hordeivirus, encodes seven major polypeptides in a segmented genome composed of three (+)-sense, single-stranded (ss) rnas designated (y (3.8 kb), p(3.3 kb), and y (3.2 or 2.8 kb) (i). the length of rna y varies with the presence or absence of a tandem repeat near its 5' terminus. rnas ly and y are able to replicate in protoplasts in the absence of rna fl (z), although the presence of all three rnas is essential for infection of intact plants (3). nucleotide sequence analyses confirm that the cua and ya genes encode the essential viral-derived replicase components (4, 5) . although the sequence of each rna is largely unique, the 3'-terminus of each rna is composed of a highly conserved sequence that can be folded into a trna-like structure and aminoacylated with tyrosine in viva (6). a "bandaid" cloning strategy, which exploits the conservation of the 3' terminal sequence, as well as the unique nature of the 5' ends of the bsmv rnas, has been used previously to obtain full-length cdna clones from which infectious in vitro transcripts can be produced (3, 7). we have now used this strategy to clone the entire genome of bsmv strain cv17. analysis of the resulting cdna clones suggested that cv17 rna y is a naturally occurring chimeric recombinant, composed of a 70nucleotide (nt) a-specific leader sequence preceding a r-specific coding region. further-' to whom reprint requests should be addressed. more, hybridization and bioassay data indicate that these clones are representative of native cv17 y rnas. one of these cv17 y cdna clones has been used previously to investigate the effects of short orfs in the 5' leader of rna y on the systemic movement of bsmv in mcotiana benthamiana (8). the present paper provides evidence for the recombinant nature of cv17 rna y. bsmv strain cv17 was propagated in barley (hordeum vulgare cv. "black hulless," c.i. 666) and purified as previously reported (9). virion rna was extracted using a phenol-sds procedure and was subsequently recovered by ethanol precipitation, followed by 3 a.4 sodium acetate precipitation, and finally by ethanol precipitation again (7). this mixture of rnas (y, p, and y was then used for cdna synthesis and cloning as previously described for the nd1 8 and type strains of bsmv (7). briefly, linearized single-stranded phagemid dna with a t7 promoter sequence at its 5'-end and a short 3'-terminal sequence complementary to the 3'-end of bsmv rna was used as a primer for firststrand cdna synthesis. phagemid-linked full-length cdna was then circularized by annealing to a bandaid oligonucleotide with sequence complementary to both ends of the phagemid:cdna. the use of three different bandaid oligonucleotides, each with 5'-ends complementary to the t7 promoter sequence and with 3'-ends complementary to the 5'-ends of either full-length (y, p, or y cdnas, provided a means of isolating specific fulllength cdna clones in a single cloning operation without the need for prior separation of the (y, 0, and y rnas. full-length clones were initially identified by colony color and gel electrophoresis of ssdna obtained after superinfection of transformed escherichia co/i strain dh5af' with bacteriophage ml 3k07, as described previously (7). the full-length nature of individual clones and the integrity of the t7 promoter/bsmv 5'-end junction were verified by dideoxynucleotide sequencing of ssdna templates (10). full-length cv17 (y and p cdna clones were successfully isolated using the cy and ,f3 bandaid oligonucleotides, respectively. however, all attempts to isolate full-length y clones using the y bandaid oligonucleotide were unsuccessful. instead, the initial size screening of cdna clones obtained with the (y bandaid oligonucleotide resulted in the identification of one with an insert equivalent in size to a 3.2-kb y rna rather than a full-length (y rna (3.8 kb). fourteen additional clones with inserts of this size were identified in subsequent cloning experiments utilizing the (y bandaid oligonucleotide, whereas only one full-length (y clone was isolated. analysis of rsal rflps in these fifteen putative y-specific cv17 cdna clones indicated that all were similar to each other, but that they differed from the bsmv type or nd1 8 strain y cdna clones previously isolated by petty et a/. (3) (fig. 1) . using an m 13 universal primer, six of these putative cv17 y clones were then sequenced in the region of the cdna insert corresponding to the 5'-end of cv17 rna y. a cv17 (y clone was sequenced in a similar manner and also by direct rna sequencing with reverse transcriptase (4) and a primer which bound to nt 195-209 (5'-cgtgcgagca-cagta). the sequences of all six y clones were identical for at least the first 100 nt and the first 70 nt of these cdnas matched the 5'-terminal70 nt of cv17 rna ci in 62 positions (fig. 2) . however, no significant sequence alignment could be made between this region of the putative cv17 y cdna clones and the first 70 nt of the type strain rna y. the 5' terminal 70 nt of the a-specific sequence in the cv17 y cdnas were followed by an eight-base segment common to both cv17 a! and 7 rnas. this sequence was, in turn, followed by a region that is identical to the sequence reported (4) for the type strain rna y for at least an additional 100 nt (fig. 2) . since the cv17 rna y contains a ca. 370-nt tandem repeat near its 5'-terminus similar to that described for the type strain (4) and a variant of the nd1 8 strain (3) the authenticity of the cv17 y cdna clones was not readily verifiable by direct rna sequencing. instead, the authenticity of these clones was assessed by hybridization with oligonucleotide probes specific for cv17 (y and type y leader sequences. northern blots of native rnas of bsmv strains cv17, cv42, and nd1 8 and southern blots of the fifteen cv17 y cdna clones were prepared according to standard protocols (1 i). blots were probed with either an qi leader-specific (5'~aagaatcgattacgattatg) or a y leader-specific (5'-tltagcgttatltggcaagc) 32p-end-labeled oligonucleotide probe complementary to nt 41-60 of the cv17 (y and type y rnas, respectively. results evident in fig. 3 demonstrate that native cv17 y rnas typically possess an (y leader sequence, as do all fifteen of the cv17 y cdna clones. further verification of the authenticity of the cv17 y cdna clones was obtained by infectivity assay. capped rnas were produced by in vitro transcription of four of the cv17 y cdna clones exactly as previously described (3). each was combined with in vitro transcripts of either cv17 or nd1 8 (y and cv17 p cdna clones and inoculated onto 5-to 6-day-old barley seedlings. inoculated seedlings developed symptoms typical of bsmv infection within 4 to 7 days, thus verifying that these cv17 y cdna clones represent full-length and biologically active copies of cv17 rna y. (table 1) our results strongly suggest that rna y of bsmv strain cv17 is a naturally occurring product of recombination between rnas (y and y. although the mechanism of recombination in bsmv is not known, two possible models for rna recombination have been commonly proposed: (i) template switching (copy choice) and (ii) enzymatic cutting/splicing. kirkegaard and baltimore (12) provided convincing evidence for a copy choice mechanism of poliovirus recombination; it appears likely that the recombination reported here proceeded in a similar manner. although template switching may occur in the absence of sequence homology, it should be noted that a short homologous sequence at 10 20 30 40 50 60 70 80 90 100 110 120 130 cv17a cuauguaaguugccuuuggg ugu aaaauuucuugcaugcacauaaucguaa ucgauucuucuugaucucuaaacaacacuuucccguuagc~gcuagcgaugagauuguccgcaaucugaucucccgugagaggu iiiiiiiiiiiiiiiiii i ii iiiii iiii iii1 iiiiiiiiiii lliiiilli iiiiiii iiiiiiiii iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiliiiiiiiiiiiiiiiiiiiiiiii type, guauagcuugagcauuaccgucguguaauugcaacacuuggcuugccaaauaacgcuaaagcguucacgaaacaaacaacaauucggc~gauguugugaagaaauucgccgucaugucagugacuguaguagcagguc 10 20 30 40 50 60 70 80 90 100 110 120 130 140 fig. 2. comparison of the 5'termrnal sequence of barley stripe mosarc vrrus strarn cv17 rna y wrth that of cv17 rna cy and type strain rna y. initiation codons for the cya and ya coding regions are underlrned. vertical bars denote homology between sequences belreved to be evolutionarily related. asterisks denote nucleotide matches that may have ansen by chance between molecules believed to be distinct. the putative crossover point is present in cv17 rnas a and y, but not in rna p. the presence of a tandem repeat in cv17 rna y which is similar to the 372-nt tandem repeat present in the type strain y is also suggestive of a copy choice mechanism. we have no conclusive explanation for the minor differences observed between the cv17 01 and y leader sequences. considering the high degree of sequence conservation among bsmv strains (e.g., the (y leader sequences of the type and cv17 strains differ by only 1 nt), the sequence divergence between the cv17 (y and y leaders may be due to selection for ability to replicate rather than random drift. the normal accumulation of rna y to much higher levels than rna ql in all strains studied thus far, including cv17, is an indication that differences in replication efficiency do exist among these rnas (fig. 3) . alternatively, natural selection pressures on rna n may not apply to rna y, hence allowing genetic drift of the two leader se-quences. as a result of this divergence, only one of the two small overlapping orfs present in the cv17 y leader is present in the cv17 n leader. it has been shown that the small y leader orfs play a significant role in the efficiency of in vitro translation of the cv17 ya gene and can affect systemic movement and pathogenicity of nd1 8kv17 pseudorecombinants in n. benthamiana (8). however, the n origin of these small leader orfs is not significant, since the type strain y leader also contains a small orf with unrelated sequence that has a similar effect on translation and systemic movement (8). thus, phenotypic effects, if any, resulting from recombination of the cv17 rna 01 and y leader regions have not yet been observed. substantial evidence for the occurrence of rna recombination now exists. recombination has been well documented in rna viruses, especially the picornavrruses, coronavrruses, and alphaviruses (12-15). altered pathogenicity as a result of rncorporation of host 1 2 3 4 5 6 7 8 9 1011 12 1314 151617 18 fig. 3. ldentrfrcatron of barley stripe mosaic virus strarn cv17 ly and type strain y-specific leader sequences in 15 putative cv17 y cdna clones and native rnas of strains cv17, cv42, and nd1 8. (a) southern blot of 15 putative bsmv strain cv17 7 cdna clones (lanes 1-l 5), a type strain y cdna clone (lane 16) , and a cv17 u cdna clone (lanes 17-l 8) hybndrzed to an ly leader-specrfrc olrgonucleotrde probe. plasmrd dnas in lanes l-l 7 were lrneanzed with il/l/ul. (b) southern blot identical to a, except that the olrgonucleotide probe used was y leader-specific. (c) northern blot of natrve rnas from bsmv strains cv17, cv42, and nd1 8 hybridized to the same (y leader-specrfic probe as in a. they rnas of cv17 and cv42 are each 3.2 kb in size and comrgrate with rna fi (arrow labeled fly); nd1 8 rna y is 2.8 kb in size (arrow labeled y). (0) northern blot identical to c, except that the y leader-specific olrgonucleotrde probe was used. hybrrdrzatrons were performed in 6x sspe t i % sds at 54" for the n leader-specific probe and at 58" for the y leader-specific probe (t, = t,, -9"). a black hulless barley plants were inoculated with a mixture of in vitro transcripts from cdna clones of both the nd18 and cv17 strains suspended in gkp buffer (50 mm glycine, 30 mm k,hpo, ph 9.2, 1% bentonite, 1% celite). the number of infected plants was determined by visual inspection. no symptoms were observed on control plants mock inoculated with gkp buffer alone proc. nat other examples, such as the deletion of repeated sequences in tobacco mosaic virus mutants, have been reported more recently (19) proc. nat/. acad. sci. usa molecular cloning: a laboratory manual the high degree of nucleotide sequence similarity between tomato ringspot nepovirus rnas 1 and 2 also may be a result of recombination (26). recently, recombination was found to have occurred between turnip crinkle virus genomic rna and satellite rna d retroviruses, viroids, and rna recombination new aspects of positive strand rna viruses acknowledgments 25 this work was supported in part by usda competitive grant 26 key: cord-324321-y96x8x3h authors: cai, yingyun; liu, yin; yu, dongdong; zhang, xuming title: down-regulation of transcription of the proapoptotic gene bnip3 in cultured astrocytes by murine coronavirus infection date: 2003-11-10 journal: virology doi: 10.1016/j.virol.2003.07.007 sha: doc_id: 324321 cord_uid: y96x8x3h murine coronavirus mouse hepatitis virus (mhv) causes encephalitis and demyelination in the central nervous system of susceptible rodents. astrocytes are the major target for mhv persistence. however, the mechanisms by which astrocytes survive mhv infection and permit viral persistence are not known. here we performed dna microarray analysis on differential gene expression in astrocyte dbt cells by mhv infection and found that the mrna of the proapoptotic gene bnip3 was significantly decreased following mhv infection. this finding was further confirmed by quantitative reverse transcription–polymerase chain reaction, western blot analysis, and bnip3-promoter-luciferase reporter system. interestingly, infection with live and ultraviolet light-inactivated viruses equally repressed bnip3 expression, indicating that the down-regulation of bnip3 expression does not require virus replication and is mediated during cell entry. furthermore, treatment of cells with chloroquine, which blocks the acidification of endosomes, significantly inhibited the repression of the bnip3 promoter activity induced by the acidic ph-dependent mhv mutant oblv60, which enters cells via endocytosis, indicating that the down-regulation of bnip3 expression is mediated by fusion between viral envelope and cell membranes during entry. deletion analysis showed that the sequence between nucleotides 262 and 550 of the 588-base-pair bnip3 promoter is necessary and sufficient for driving the bnip3 expression and that it contains signals that are responsible for mhv-induced down-regulation of bnip3 expression in dbt cells. these results may provide insights into the mechanisms by which mhv evades host antiviral defense and promotes cell survival, thereby allowing its persistence in the host astrocytes. viruses can cause different types or patterns of infection in susceptible hosts; host cells in turn respond to viral infection in many different ways. some virus-infected cells undergo rapid destruction, whereas others become immortalized. in addition to immune response, host cells often devise countermeasure mechanisms against virus invasion. one of the common antiviral defense strategies operated by host cells is programmed cell death or apoptosis, for apoptosis would eliminate virus-infected cells, thereby blocking the production of progeny virus and limiting the spread of virus to neighboring uninfected cells. however, apoptosis can also be a viral strategy to invade hosts. many viruses can induce apoptosis in host cells during late stages of the virus life cycle to speed up their release from infected cells and spread to neighboring cells. on the other hand, many viruses have also devised strategies to counter host antiviral defense by inducing an antiapoptotic state in host cells. for examples, human immunodeficiency virus tat protein (zauli et al., 1995) and epstein-barr virus-ebna4 and -lmp-1 proteins (silins and sculley, 1995; henderson et al., 1991) induce the expression of antiapoptotic protein bcl-2, which is the master checkpoint protein of apoptosis through the mitochondrial pathway (hockenbery et al., 1990) . members of the bcl-2 family share up to four conserved regions of homology known as bcl-2 homology (bh) domains, which mediate interactions among themselves (kelekar and thompson, 1998) . a delicate balance of interaction between proapoptotic and antiapoptotic members of the bcl-2 family has been shown to modulate the sensitivity of cells to mitochondrial integrity in response to apoptotic signals . many viral proteins are bcl-2 ho-mologs such as adenovirus e1b 19-kda protein (ad-e1b 19k) (chiou et al., 1994) . bnip3 (bcl-2 and nineteen-kilodakton-protein interacting protein 3) is the proapoptotic member of the bcl-2 family. it was first identified as an ad-e1b 19k and bcl-2 interacting protein (boyd et al., 1994) . bnip3 contains only the bh3 domain (yasuda et al., 1998) . it functions as a proapoptotic protein that both induces apoptosis and increases the sensitivity of cells to apoptosis induced by drugs and granzymes b upon transient expression . moreover, bnip3 can form heterodimers with antiapoptotic bcl-2 family members such as bcl-2 and bcl-xl and promotes apoptosis by sequestering the antiapoptotic members (imazu et al., 1999) . recent evidence from studies in t cells suggests that the bh3-dependent apoptotic pathways can also be turned on by surface receptor via interaction with cd47 (lamy et al., 2003) . it has been reported that the expression of bnip3 gene is induced by hypoxia (bruick, 2000; kubasiak et al., 2002) and a zinc-finger protein, plagl2 (mizutani et al., 2002) , leading to cellular apoptosis. murine coronavirus mouse hepatitis virus (mhv) is an enveloped, positive-strand rna virus. it contains four or five structural proteins, depending on virus strains. the spike protein (s) is the major peplomer protruding from the virion envelope. it is responsible for virion attachment to host cells via interaction with receptors, induction of fusion between viral envelope and cell membranes during infection, and elicitation of neutralizing antibodies and cellular immune responses (lai, 1990) . thus, the s protein is essential for virus infectivity. the hemagglutinin/esterase (he) has sequence homology with the hef protein of influenza c virus (luytjes et al., 1988) . it contains an esterase activity but lacks the receptor-binding activity . it is present on the virion surface only in some mhv strains (such as jhm) but not others (such as a59) la monica et al., 1992) , and it is thus not essential for virus infectivity. the small envelope (e) protein is acetylated (yu et al., 1994) and the membrane (m) protein is glycosylated (niemann et al., 1984) . both proteins are hydrophobic and largely embedded in the virion envelope. they are essential for virion assembly and morphogenesis (vennema et al., 1996; bos et al., 1996) . the nucleocapsid (n) protein is a phosphoprotein that is tightly associated with the viral rna genome to form the nucleocapsid inside the envelope shell (stohlman and lai, 1979) . it has been shown that dephosphorylation of the n protein following viral entry into host cells is a prerequisite step for virus replication (kalicharran and dales, 1995; kalicharran et al., 1996) . during the initiation of virus infection, the s protein binds to a specific receptor on the surface of susceptible cells, which is a murine biliary glycoprotein belonging to the carcinoembryonic antigen (cea) in the immunoglobulin superfamily (williams et al., 1991) . upon viral binding, fusion between viral envelope and plasma membrane is then triggered, allowing the nucleocapsid to release into the cytoplasm. interaction between the s protein and the receptor can also mediate endocytosis (nash and buchmeier, 1997) . in this case, fusion occurs between viral envelope and endosomal membrane at an acidic environment. once in the cytoplasm, the n protein is presumably dephosphorylated and dissociates from the genomic rna, thereby completing the uncoating process. the viral rna-dependent rna polymerase is then translated from the incoming viral genome and viral replication proceeds subsequently. mhv infects rodents, causing hepatitis, nephritis, enteritis, and central nervous system (cns) diseases. infection of rodent cns results in encephalitis and demyelination, the latter of which resembles the multiple sclerosis seen in humans (lampert et al., 1973; herndon et al., 1977) . therefore, mhv has been extensively used as an animal model for studying the pathogenesis of multiple sclerosis and other cns degenerative diseases. in certain mouse strains, demyelination develops during the first week of infection (woyciechowska et al., 1984) and reaches to a peak at approximately 30 days postinfection (p.i.), at which time infectious virus can no longer be isolated but viral rnas are still detectable (fleming et al., 1993; adami et al., 1995) . the predominant cell type in the cns for harboring persistent viruses is the astrocyte (perlman and ries, 1987) . indeed, persistent infection can be readily established in animal cns (adami et al., 1995; rowe et al., 1998) and cultured astrocytes (maeda et al., 1995; chen and baric, 1995) . in contrast to the infection in vivo, infectious virus can be recovered from infected, cultured astrocytes after continuous passage of cells for more than 100 days (chen and baric, 1995) . because astrocyte is one of the antigen-presenting cells in the cns (fierz et al., 1985) , it can produce diverse proinflammatory molecules such as cytokines, chemokines, and nitric oxide upon stimulation (van wagoner and benveniste, 1999; huang et al., 2000; chen and swanson, 2003) . it has been shown that coronaviruses induce the expression of transforming necrosis factor-(tnf) ␣, interleukin-(il) 6, matrix metalloproteinases, and inducible nitric oxide synthase in primary and established astrocytes (joseph et al., 1993; zhou et al., 2001; grzybicki et al., 1997; kyuwa et al., 1994) . thus, it is conceivable that infection of astrocytes with mhv can modulate the development of the cns pathology and alter the course of the disease. in the past, most studies have been focused on how mhv has evolved during persistent infection in the cns and in cultured cells (adami et al., 1995; rowe et al., 1998) . information on coevolvement of the host has been limited to the alteration of receptors by mhv infection (chen and baric, 1996; sawicki et al., 1995) . to understand how mhv infection alters the gene expression of astrocytes, we employed the dna microarray technology to analyze the mrna expression profiles in mhv-infected murine astrocytoma cell line dbt and compared them with those in mock-infected dbt cells. we found that the expression of a substantial number of genes was either increased or decreased upon mhv infection. of these, the expression of the proapoptotic gene bnip3 was significantly decreased. a series of subsequent experiments further confirmed this conclusion. interestingly, the downregulation of bnip3 expression does not require viral replication and is mediated during cell entry. our results may provide insight into the mechanisms by which mhv evades host antiviral defense and promotes cell survival, thereby allowing its persistence in the host astrocyte. to determine altered gene expression of dbt cells by virus infection, dbt cells were infected with mhv strain jhm at an m.o.i. of 2 or mock-infected. at 12 h p.i., intracellular rnas were isolated with the rnaeasy kit (qiagen inc.). the mrna expression profiles were determined using the affymetrix genechip-the murine genome u74av2 array, which contains oligonucleotides representing approximately 8000 known genes and 4000 est of the mouse genome. a ն2-fold difference in mrna amount between virus-infected and mock-infected cells was considered statistically significant. based on this criteria, we identified 125 known genes that were significantly up-regulated by mhv infection and 124 known genes that were significantly down-regulated (data not shown). among those, we found that the mrna of the proapoptotic gene bnip3 was decreased approximately 2.4-fold in mhv-infected dbt cells as compared to that in mock-infected dbt cell. to verify this microarray data, we employed three different approaches. the first approach was to use reverse transcription-polymerase chain reaction (rt-pcr) to directly measure the amounts of the bnip3 mrna expression during the course of virus infection. dbt cells were infected with mhv strain jhm at an m.o.i. of 5. at 4, 8, and 12 h p.i., intracellular rnas were isolated and cdnas were synthesized by rt with a hexomer random primer. cdnas were then amplified by pcr using a primer pair specific to bnip3 (fig. 1a) . we found that cdnas representing the bnip3 gene decreased at 8 and 12 h p.i. in virus-infected cells as compared to those in mock-infected cells after normalizing with ␤-actin gene (data not shown). to further provide quantitative assessment on the extent of bnip3 mrna reduction by mhv infection, we employed a competitive rt-pcr. as depicted in fig. 1a , we generated a competitor dna fragment that contains an internal deletion of about 100 nt but which contains the same two primer binding sites on the target dna. as the result, the amount of the target dna fragment being amplified by pcr would be decreased with an increased amount of competitor dna being added in the pcr reac-tion. the pcr products were analyzed by agarose gel electrophoresis and the intensity of the dna bands following ethidium bromide staining was determined by densitometry. rnas were isolated and were subjected to rt with a random primer. cdnas were amplified by pcr using the primer pair specific to bnip3 as shown in a in the presence of a known amount of the competitor dna. the competitor dna was amplified from a deletion construct and was purified from agarose gel. from lanes 1 to 5 and 6 to 10, a 10-fold serially diluted competitor dna (indicated as copy number) was added to the pcr. the rt-pcr products were analyzed by electrophoresis in 2% agarose gel and visualized by staining with ethedium bromide. images were taken with the digital camera (uvp). the intensity of each band was determined with the software (labworks4). arrows indicate the target pcr products (bnip3) and the competitor pcr product (comp), respectively. an equal amount of the target pcr product to the competitor pcr product is considered the amount of the target template in the original rna sample, which is expressed as percentage of virus-infected samples to mockinfected samples as shown in c. a representative gel is shown in fig. 1b . the amounts of the target dnas and competitor dnas were then plotted, and the value, at which an equal amount of target and competitor dnas was obtained, was considered to be the amount of the target gene. to insure that the pcr products quantitatively reflected on the target gene, we optimized the pcr reaction by reducing the amount of input cdna and the number of pcr cycles so that the amount of the dna in each band was not saturated by ethidium bromide staining. we used this technique to determine the amount of bnip3 mrnas at different time points p.i. as shown in fig. 1c , there was virtually no difference in the amount of bnip3 cdna between virus-infected and mock-infected cells at 4 h p.i., but an approximately 60% reduction was detected in virus-infected cells at 8 h p.i., as compared with that of mock-infected cells. at 12 h p.i., the reduction in bnip3 gene reached to approximately 80% in virus-infected cells. these results were reproducible in at least three independent experiments. these results demonstrate that mhv infection down-regulated bnip3 gene expression at the transcriptional level, thus confirming the results from the dna microarray analysis. the second approach was to determine the expression of the bnip3 protein. we employed western blot in combination with immunoprecipitation to determine the bnip3 protein expression at various time points p.i. as expected, a slight decrease in bnip3 protein was detected in virusinfected cells at 5 and 9 h p.i., as compared to those in mock-infected cells (fig. 2) . the reduction of bnip3 protein in virus-infected cells was more pronounced at 13 h p.i., when normalized with the ␤-actin protein. although the degree of reduction of the bnip3 protein in mhv-infected cells varied among experiments, the pattern of reduction was consistent in several independent experiments. these results indicate that, consistent with the pattern of mrna expression, bnip3 protein expression was inhibited by mhv infection. the third approach was to use a reporter system to determine the promoter activity of the bnip3 gene in re-sponse to mhv infection. the 5ј upstream sequence (promoter) of the bnip3 gene was placed in front of the firefly luciferase gene in the pgl-bnip3 construct ( fig. 3a ) (bruick, 2000) . transient transfection of the pgl-bnip3 dna into dbt cells that were either infected with mhv at dbt cells were infected with mhv jhm at an m.o.i. of 5 or mockinfected. at 1 h postinfection (p.i.), cells were transfected with 500 ng of pgl3-bnip3 plasmid or the promoterless plasmid pgl3. at 18 h p.i., cell lysates were harvested and luciferase activities measured as described under materials and methods. the relative luciferase activity in pgl3-bnip3-transfected cells is expressed as fold increase over the luciferase activity in cells transfected with the promoteless pgl3. data indicate the means of three separate infection/transfection results. error bars denote standand deviations. the data are representative of six independent experiments. (c) same as in b, except that the virus was purified through sucrose-gradient ultracentrifugation. (d) same as in b, except that the transfection was carried out 5 h prior to virus infection and the luciferase activity was determined at 9 and 13 h p.i. o.i. of 5 or mock-infected. at 5, 9, and 13 h p.i., the cells were harvested and lysed. equal amounts of cell lysates were immunoprecipitated with an antibody specific to bnip3 and protein g-sepharose beads. the precipitates were separated by polyacrylamide gel (12%) electrophoresis. proteins were then transfered to nitrocellulose membranes and were detected with the same bnip3 antibody and enhanced chemiluminescence by western blot analysis. an aliquot of an equal amount of cell lysates for each sample was used for analyzing the ␤-actin protein with the ␤-actin-specific antibody in western blot as an internal control. the data are representative of three experiments. m, lysates from mock-infected cells; i, lysates from virus-infected cells. an m.o.i. of 5 or mock-infected as a control resulted in the expression of luciferase activity. consistent with the results from dna microarray, rt-pcr and protein detection described above, the luciferase activity was significantly decreased following mhv infection (fig. 3b ). this result was reproducible in more than 20 independent transfectioninfection experiments. it was also confirmed by using purified virus and uv-irradiated virus (see below). these data thus unequivocally establish that mhv infection downregulated bnip3 gene expression in dbt cells. because the luciferase reporter system is a very sensitive assay that has been well established and widely used for determining promoter activity and because our results also demonstrated its sensitivity and utility, we used this reporter system as a standard method for all subsequent experiments. because our original dna microarray study used rna samples isolated from dbt cells that were cultured or infected with mhv in the presence of 0.5% newborn calf serum, it is possible that the presence of a low level of serum and other unidentified factors secreted by infected-dbt cells in the inoculum might have contributed to the observed down-regulation of bnip3 expression. to rule out this possibility, we used the virus purified through sucrose gradient ultracentrifugation. as shown in fig. 3c , infection of dbt cells with purified virus resulted in a six-fold decrease of luciferase activity as compared to that from mock-infected cells. this result is comparable to that obtained with unpurified virus (fig. 3b) , indicating that the down-regulation of bnip3 gene expression specifically resulted from mhv infection and not from factors present in the viral inoculum. it is also possible that the observed reduction in luciferase activity resulted from inhibition of transfection efficiency by virus infection rather than from direct virus infection because mhv infection could potentially cause alteration of cell membrane permeability. to exclude this possibility, we carried out dna transfection for 5 h to allow complete delivery of the plasmid dna into cells prior to virus infection. we found that a significant reduction of luciferase activity was detected in mhv-infected cells as compared to mock-infected cells (fig. 3d) , although the fold reduction in luciferase activity was less prominent when compared with the results from prior infection (fig. 3c ). these data argue against an indirect effect by dna transfection efficiency. it is known that productive mhv replication results in inhibition of host cell translation hilton et al., 1986) . to establish that the reduction of luciferase activity expressed from the pgl3-luc reporter was not due to mhv-replication-induced inhibition of cell translation, dbt cells were infected with uv-irradiated mhv at an m.o.i. of 5 for 1 h and then transfected with the pgl3-luc reporter dna. mock-infected cells were used as controls. as shown in fig. 4 , luciferase activities were reduced at similar levels in cells infected with live virus and uvinactivated virus as compared to those in mock-infected cells. this result strongly supports the conclusion that the bulk of the reduction of luciferase activity in mhv-infected cells indeed resulted from mhv infection and not from indirect, general inhibition of cellular translation. to establish further the specificity of the down-regulation of bnip3 gene expression, we used vesicular stomatitis virus (vsv) in a parallel experiment, because vsv is also an enveloped rna virus whose infection has a broad host cell range and is mediated by the interaction between the envelope g protein and cell membranes (riedel et al., 1984) . as expected, the luciferase activity was completely inhibited by infection with live vsv because it is known that vsv replication shuts down host cell transcription (fig. 4) . in contrast, little reduction of luciferase activity was detected in dbt cells that were infected with uv-inactivated vsv. nevertheless, the difference in reduction of luciferase activity between uv-inactivated mhv and vsv was significant (fig. 4) . taken together, these results demonstrate that the down-regulation of bnip3 gene expression resulted specifically from mhv infection. because uv-irradiated viruses are no longer capable of replicating themselves, although they are still able to bind cell receptors and to be up-taken by the cells, uv-irradiation of viruses can be used to distinguish the involvement of various steps either before or after virus replication. the data presented in fig. 4 also indicate that the down-regulation of bnip3 gene expression does not require virus replication. this result was reproducible in both neurotropic mhv strains jhm and a59 and in at least six independent experiments with triplicates for each experiment (representative data shown in fig. 4 , further data not shown). these results thus establish that mhv infection induces the downregulation of bnip3 gene expression during early stages of the virus life cycle and requires no virus replication. as the above results pinpointed the involvement of virus entry in regulation of bnip3 gene expression, we were interested in further dissecting the individual steps of the infectious process. we designed experiments to first block downstream processes so that the role of upstream events can be assessed. however, most mhv strains, including jhm and a59, enter cells both via fusion with plasma and endosomal membranes (nash and buchmeier, 1997) . this property poses a technical dilemma for not being able to block fusion by the common lysosomotropic agents. gallagher et al. (1991) isolated several mutants from obl20 cells persistently infected with mhv jhm (mhv-4). one of these mutant isolates, oblv60, strictly depends on acidic ph for entry and infectivity. we therefore used this ph-dependent mutant for this purpose. dbt cells were transfected with the pbnip3-luc reporter plasmid dna for 5 h, treated with 0, 25, and 50 m of chloroquine for 1 h prior to virus infection, and then infected with oblv60 or the wild-type jhm at an m.o.i. of 5 in the presence of chloroquine. at 13 h p.i., cell lysates were isolated and luciferase activity determined. mock-infected and untreated cells were used as controls. as shown in fig. 5 , treatment of cells with an increasing concentration of chloroquine resulted in an increase of luciferase activities by approximately three fold in oblv60 infected cells (fig. 5a) , concomitant with a decrease of virus titer (fig. 5b ). in contrast, neither the luciferase activities nor the virus titers were significantly affected by chloroquine treatment in jhm-infected dbt cells. these results suggest that fusion between viral envelope and endosomal membranes during oblv60 infection provides at least in part the signal(s) that triggers the down-regulation of bnip3 gene expression. by extrapolating the data from the mutant virus, we can envision that fusion between viral envelope and plasma/endosomal membranes during wild-type mhv infection is involved in the down-regulation of bnip3 gene expression. the above results suggest that the promoter region of the bnip3 gene contains both the cis-acting elements that are essential for bnip3 expression and the negative elements that repress bnip3 expression in response to mhv infection. to identify and dissect such cis-acting elements, we made a series of reporter constructs that contain various deletions in the bnip3 promoter. in an initial experiment, we determined the minimal promoter sequences required for bnip3 gene expression. dbt cells were transfected with various deletion reporter constructs. transfection with the promoterless vector was used as a negative (basal level) control. at 18 h posttransfection, cells were lysed and assayed for luciferase activities. as summarized in fig. 6 , when deletions were made from the 5ј-end to nt 262 (construct pr-7) and from nt 91 to 262 (construct pr-6) of the promoter, the luciferase activities retained at 91.5 and 71.1%, respectively, as compared to the full-length promoter, indicating that the upstream half (262 nt) of the promoter is not essential for bnip3 gene expression. in contrast, deletions of nt 262 to 550 (in constructs pr-4 and pr-5) essentially abolished the luciferase activity, indicating that the sequence from nt 262 to 550 of the promoter is the cis-acting element required for driving bnip3 gene expression. further deletion of the downstream sequence (from nt 550 to 591 in pr-10) resulted in approximately 50% reduction of luciferase activity, suggesting that this sequence can influence the transcription efficiency but is not essential for bnip3 gene expression. partial or complete deletions of the 288-nt sequence (from nt 262 to 550) in constructs pr-11, pr-12, pr-14, pr-15, pr-13, and pr-9 essentially abolished the luciferase activity. we thus conclude that the 288-nt sequence (nt 262-550) of the promoter contains the cisacting elements essential for bnip3 gene expression. we next determined the cis-acting elements responsive to mhv infection. cells were transfected with the fulllength and various deletion reporter constructs for 5 h and were then infected with uv-inactivated mhv at an m.o.i. of 5 or mock-infected as controls. as shown in fig. 6 , the luciferase activities were decreased approximately four-to sixfold in mhv-infected cells for various reporter constructs (full-length, pr-7, pr-4, pr-10, pr-15, and pr-6), as compared with those in mock-infected control cells. the reduction of luciferase activities must have resulted from the infection process rather than from general inhibition by mhv replication, because the virus was inactivated by uv irradiation. these results suggest that the negative elements responsive to mhv infection reside within the same region of the promoter that is required for bnip3 expression. we were unable to narrow down further the minimal responsive elements because further deletions within this region in the reporter constructs drastically abrogate the reporter gene expression even in the absence of virus infection (constructs pr-5, pr-11, pr-12, pr-14, pr-13, and pr-9). in this study, we employed dna microarray, rt-pcr, western blot, and a promoter-reporter gene system to explore the potential alteration of cellular gene expression by mhv infection. using such multitude approaches, we discovered that the expression of the cellular proapoptotic gene bnip3 was significantly down-regulated in cultured dbt cells by mhv infection. this finding supports the hypothesis that mhv infection in dbt cells may induce an antithe luciferase activity was expressed as fold increase over that expressed from the promoterless pgl3. the data were the means and standard deviations (sd) of at least three independent experiments. the luciferase activities for the deletion constructs were also expressed as percentage to that derived from mock-infected and full-length plasmid-transfected cells, which was set as 100%. (b) details of the cis-acting elements in the bnip3 promoter region between nt 262 and 550. the sequence was analysed with the computer software signal scan (prestridge, 2000) . the putative consensus sequences for the binding of transcription factors are shown schematically. the numbers in brackets indicate the approximate nt positions in the promoter. apoptotic state through down-regulation of expression of proapoptotic genes. it has been shown that mhv infection induces apoptosis in macrophages and fibroblast cells but not in dbt cells (belyavsky, et al., 1998; an et al., 1999; chen and makino, 2002) . however, overexpression of the single viral e protein in dbt cells by a recombinant vaccinia virus results in apoptosis, which can be blocked by overexpression of an antiapoptotic protein bcl-2, indicating the involvement of the mitochondrial pathway in e proteininduced apoptosis in dbt cells . how mhv infection in dbt cells overcomes the apoptotic effect induced by the e protein expression remained unanswered in that study. it is possible that mhv infection of dbt cells induces an antiapoptotic state during an early stage of the virus life cycle through down-regulation of proapoptotic genes such as bnip3 (this study) or up-regulation of antiapoptotic genes (cai and zhang, unpublished results). such an antiapoptotic response would prevent cells from succumbing to apoptosis induced by the viral apoptotic e protein. consistent with this hypothesis is the observation that when dbt cells were treated with apoptotic inducers, fewer cells underwent apoptosis when infected with mhv than mock-infected cells (cai and zhang, unpublished results). although a direct correlation between down-regulation of bnip3 and inhibition of e protein-induced apoptosis in dbt cells has not been determined, the general proapoptotic effect exerted by bnip3 mizutani et al., 2002; kubasiak et al., 2002; lamy et al., 2003) makes the hypothesis conceivable. moreover, the involvement of the mitochondrial/bcl-2 pathway in e protein-induced apoptosis correlates well with the functional role of bnip3 as a member of the proapoptotic, mitochondrial/bcl-2 family. thus, it is conceivable that down-regulation of bnip3 expression can shift the balance to an antiapoptotic state at the mitochondrial checkpoint. using uv-inactivated virus for infection and chloroquine for blocking the acidification of the endosomes, we demonstrated that the down-regulation of bnip3 gene expression by oblv60 infection was mediated during cell entry and required no virus replication (figs. 4 and 5) . however, treatment of dbt cells with chloroquine did not completely block the down-regulation of bnip3 expression by oblv60 infection (fig. 5) . one possible interpretation is that a part of the signals that trigger the down-regulation of bnip3 expression has already been induced during virus attachment and endocytosis but prior to fusion between viral envelope and endosomal membranes (in the case of oblv60 virus), so that inhibition of fusion by chloroquine cannot completely derepress the bnip3 promoter activity. alternatively, the partial restoration of bnip3 promoter activity may be due to incomplete blockage of fusion by chloroquine. this is possible and even likely because the virus infectivity was reduced by approximately only 1 log10 by chloroquine treatment (at 50 m). although gallagher et al. (1991) reported a reduction of 3 log10 in virus titer when dbt cells were treated with chloroquine at 50 m, they used approximately 3 log10 less virus for infection. in our infection-transfection experiments, we had to use a higher m.o.i. to insure that each transfected cell is infected with a virus. the higher virus infectivity used in this study likely contributes to a lesser effective inhibition by chloroquine. therefore, although it cannot exclude the possibility that virus attachment to cell receptors and the subsequent endocytosis are also involved in signaling bnip3 down-regulation, our results clearly establish that virus-cell fusion during entry triggers the signaling cascade. little is known about the cis-acting elements that regulate bnip3 gene expression. our systematic deletion analyses identified the 288-nt sequence (from nt 262 to 550) of the bnip3 promoter, which is necessary and sufficient for the expression of bnip3 gene. interestingly, this region also contains the two previously identified hypoxia response elements (hre-1 and hre-2) (bruick, 2000) , the tata box, four transcription factor sp1-binding sites, the cac or ccacc consensus sequence, the ccaat box, and the nuclear factor-(nf) 1-binding site (fig. 6b) . it is not known whether and how these transcription factors are involved in the regulation of bnip3 gene expression. it has been reported that the transcription factor hypoxia-inducing factor-1 binds to the hres on the bnip3 promoter and induces the bnip3 expression (bruick, 2000; kubasiak et al., 2002) , whereas the zinc-finger protein plagl2 induces the expression of bnip3 via the same hres, but independent of the hypoxia-inducing factor-1 (mizutani et al., 2002) . our finding that further deletions within this 288-nt region inactivated the promoter suggests that bnip3 transcription in dbt cells likely requires multiple transcription factors acting in concert with these cis-elements. the mechanism by which mhv infection of dbt cells downregulates bnip3 gene expression is currently unknown. we have attempted to identify the negative elements that are responsive to mhv infection. our results showed that the negative, mhv-responsive elements largely overlap with the cis-acting elements essential for bnip3 transcription (fig. 6) . one hypothesis is that mhv infection triggers the induction of one or more transcription factors that act on the 288-nt sequence of the bnip3 promoter to compete with the positive transcription regulators, thereby down-regulating the transcription of bnip3 on infection. in our dna microarray analysis, we found that the expression of a class of early response transcription factors such as c-fos (12-fold increase) and early growth response gene-1 (egr-1) (9-fold increase) was significantly induced by mhv infection (data not shown). preliminary studies employing transient cotransfection of dbt cells with plasmids expressing c-fos or egr-1 gene and the bnip3 promoter reporter plasmid demonstrated that the bnip3 promoter was down-regulated by egr-1 in a dose-dependent manner but not by c-fos, suggesting that the down-regulation of bnip3 gene expression by mhv infection might be mediated through the induction of egr-1 (data not shown). it has been shown that egr-1 and sp1 share the consensus binding sequence and that egr-1 can act as an antagonist of sp1 by binding to the sp1 consensus sequence and represses transcription (huang et al., 1997) . our analysis of the 288-nt bnip3 promoter sequence (from nt 262 to nt 550) identified four sp1-binding sites: two upstream at nt 285 and 320 and two downstream at nt 441 and 496, respectively. coincidently, mhv infection of dbt cells inhibited the luciferase expression in all reporter plasmids that contain the two or four sp1-binding sites (fig. 6) . however, direct evidence for the role of egr-1 in mhv-dependent transcriptional regulation of bnip3 gene remains to be established. a recent study showed that overexpression of egr-1 resulted in down-regulation of bnip3 transcription in prostate cancer cells (virolle et al., 2003) , thus providing supporting evidence for the role of egr-1 in regulating bnip3 gene. the mouse hepatitis virus (mhv) strain jhm ) was used throughout this study. for some experiments, mhv strain a59 (leibowitz et al., 1981) and the mutant oblv60 (gallagher et al., 1991) were also used. oblv60 (kindly provided by michael buchmeier, the scripps research institute, la jolla, california) was originally isolated from the obl20 cells that were persistently infected with mhv jhm (mhv-4). this mutant enters cells via receptor-mediated endocytosis and thus its entry depends on acidic ph. the mouse astrocytoma cell line (dbt) (hirano et al., 1974) was used for virus propagation, plaque assay, and all other experiments involving cell culture throughout this study. dbt cells were cultured in 1x minimum essential medium (mem) containing 10% trypton phosphate broth (tpb) and 7.5% newborn calf serum (ncs) (gibco brl, gaithersburg, md). all mhv strains were propagated in dbt cells in mem either containing 1% ncs or serum-free. for propagation of oblv60 mutant virus, no nahco 3 was added to the medium. a recombinant vesicular stomatitis virus (vsv) expressing the green fluorescence protein (gfp) originally provided by michael whitt (university of tennessee at memphis) was obtained from marie chow (uams). vsv was propagated and titered in hela cells. antibodies specific to mouse bnip3 protein and ␤-actin were purchased from santa cruz biochem and sigma, respectively. dbt cells were infected with mhv at a multiplicity of infection (m.o.i.) of 5 or mock-infected. at various time points postinfection (p.i.), intracellular total rnas were isolated and purified with the rneasy kit according to the manufacturer's instruction (qiagen, inc.). rnas were then treated with rnase-free dnase i (promega). the concen-trations of the rna samples were determined by spectrophotometry (hitatchi, model no. 4500) . for detection of the proapoptotic gene bnip3 mrna, a semiquantitative, competitive rt-pcr was employed. briefly, the rnas were reverse-transcribed into cdnas with maloney murine leukemia virus (mmlv) reverse transcriptase (promega) by using a random hexomer oligonucleotide primer (invitrogen, inc.) in a standard rt reaction as described previously (zhang et al., 1991) . cdnas were then used as templates for pcr amplification with two pairs of primers specific for bnip3 gene and ␤-actin gene as an internal control, respectively. the sense primer for bnip3 of taq dna polymerase, 2 l of the rt products, for 30 cycles with each cycle consisting of denaturation at 95°c for 20 s, annealing at 55°c for 1 min and extension at 72°c for 1 min with a final extension of 10 min. pcr was performed in a thermocycler dna engine (model ptc-200, mj research). for competitive pcr, a plasmid dna containing a 99-nt deletion between the primer sequences (5јbnip3 and 3јbnip3) was constructed (see below) and used as a competitor. for each rna sample, at least 10 pcr reactions were performed with an incremental amount of the competitor dna. pcr products were analyzed by agarose gel electrophoresis, and the gels were stained with ethidium bromide and photographed with a uvp gel document center. the intensity of the dna bands was determined by densitometry, analyzed with manufacture-installed software (uvp), and plotted. the point at which the amount of target pcr products equals to a known amount of competitor dna being added in the pcr reaction is the amount of target cdna of the virus-infected cells, which is expressed as percentage relative to the amounts of target cdna in mock-infected cells (which were set as 100%). mhv strains jhm and a59 and vsv were inactivated by exposing the viruses to uv light for 30 min on ice in a uv crosslinker (fisher scientific). inactivation was confirmed by titration of samples before and after uv-light exposure and by the absence of cytopathic effect after inoculation of dbt cell monolayers with uv-irradiated virus or by the absence of green fluorescence cells (for vsv). dbt cells were grown to monolayer in 60-mm petri dishes and were infected with mhv jhm at an m.o.i. of 5 or mock-infected. at 5, 9, and 13 h p.i., cells were harvested and lysed in 500 l of radioimmunoprecipitation assay (ripa) buffer (50 mm tris-hcl, ph 7.5, 150 mm nacl, 0.5% sodium deoxycholate, 1% np-40) containing protease inhibitor cocktail tablets (roche, mannheim, germany). the cell lysates were passed through a 25-gauge needle several times to sheer the dna and were clarified from cell debris by centrifugation. the protein concentration was measured by using bio-rad protein assay kit (bio-rad, ca). equal amounts of cell lysates (cell number equivalent) were immunoprecipitated with an anti-bnip3 peptide antibody (2 g/ml) (santa cruz, ca) for 1 h at 4°c in a rocking platform (bellco inc.). the antigen-antibody complexes were then precipitated with protein g-sepharose beads (10 l of the 50% slurry) (roche) for 4 h at 4°c in a rocking platform. protein g beads were pelleted down by centrifugation and washed three times with the ripa buffer. twenty microliter of 1x protein sample loading buffer [10 mm tris-hcl, ph 6.8, 100 mm dithiothreitol (dtt), 2% sodium dodecyl sulfate (sds), 0.1% bromphenol blue, 10% glycerol] were added to each sample. samples were boiled for 3 min, chilled on ice, and pelleted by centrifugation before loading onto gels. proteins were separated by sdspolyacrylamide gel (12%) electrophoresis (page). the proteins were then transferred to nitrocellulose membrane (msi, westborough, ma) for 4 h at 100 v in a transfer buffer (25 mm tris, 200 mm glycine, 20% methanol, 0.02% sds). after being blocked with 5% skin milk in tris-buffered saline (tbs) for 1 h at room temperature, the membrane was washed three times in tbs containing 0.5% tween-20 and immunoblotted with the bnip3 antibody (1 g/ml) for 2 h at room temperature, followed by a secondary ab coupled to horseradish peroxidase (1:1000 dilution) (sigma) for 1 h at room temperature. the presence of the bnip3 protein was detected by enhanced chemiluminescence (ecl) using peracid as a substrate (amersham) followed by autoradiography. the luciferase reporter plasmid that contains 588 nt 5ј upstream sequence (the promoter) of bnip3 gene in front of the luciferase orf was kindly provided by richard bruick (university of texas, southwestern medical center in dallas). for construction of a competitor dna for quantitative pcr, the full-length orf of bnip3 gene was cloned. rnas were isolated from dbt cells and cdnas were synthesized by rt using the random hexomer primer described above. the bnip3 gene was amplified by pcr using the sense primer 5јbnip3ecori (5ј-agt gaa ttc acc atg tcg cag agc-3ј, corresponding to the first 15 nt of the bnip3 orf, genbank accession number af041054), which contains an ecori site at the 5ј-end (underlined), and the antisense primer 3јbnip3xhoi (5ј-ctc gag gaa ggt gct agt gga-3, complementary to the last 15 nt of the bnip3 orf), which contains an xhoi site (underlined). the pcr products were directly cloned into pcr2.1 ta cloning vector (invitrogen), resulting in pta-bnip3. the sequence of the clone was confirmed by automatic dna sequencing (ibi, prizm-377) in the departmental core facility. an internal deletion was made in three steps. in the first pcr, a 222-nt fragment of the bnip3 gene was amplified from the full-length clone pta-bnip3 using the primer pair 5јbnip3-rt and 3јbnip3-cl (5ј-ttt ctc gcc aaa gct gtg gc-3ј, complementary to the sequence at nt 230 -249). in the second pcr, a 173-nt fragment of the bnip3 gene was synthesized using the primer pair 5јbnip3-cl (5ј-gc cac agc ttt ggc gag aaa cca gaa aat att ccc ccc aag-3ј, corresponding to sequence at nt 349 -369 with a 20-nt sequence overhang at the 5ј-end that corresponds to nt 230 -249) and 3јbnip3-rt. the pcr products from these two sets of reactions were separated by agarose gel electrophoresis; the corresponding dna fragments were excised from the gels and purified with a gel extraction kit (qiagen, inc.). the two pcr fragments were then mixed and used as templates for a third pcr. the third pcr was carried out with the primer pair 5јbnip3-rt and 3јbnip3-rt. the products from the third pcr were directly cloned into the pcr2.1-ta cloning vector (invitrogen), resulting in generation of pta-bnip3d100. this clone contains the region of bnip3 gene from nt 28 to nt 502 with a deletion between nt 249 and nt 349. this clone was then used as a competitor for the competitive pcr described above. for construction of 5ј-deletion mutants of the bnip3 promoter, plasmid pgl3-bnip3, which contains the fulllength promoter, was doubly digested with hindiii and bstxi, pf1mi, or stui, respectively. the digested plasmids were purified with the gel extraction kit (qiagen) following agarose gel electrophoresis, blunt-ended with t4 dna polymerase, and self religated with t4 dna ligase, resulting in generation of pgl3-bnip3-pr7, pgl3-bnip3-pr4, and pgl3-bnip3-pr5, respectively (fig. 6) . deletion mutants pgl3-bnip3-pr10, pgl3-bnip3-pr11, pgl3-bnip3-pr12, pgl3-bnip3-pr14, pgl3-bnip3-pr15, and pgl3-bnip3-pr6 were made similarly by deleting the internal fragments through double digestion with stui and ncoi, pf1 mi and ncoi, bstxi and ncoi, pf1mi and stui, bstxi and pfl mi, or apai alone (two sites), respectively. for construction of pgl3-bnip3-pr13 and pgl3-bnip3-pr9, the 138-nt pf1mi-stui fragment and the 224-nt apai-apai fragment were blunt-ended and cloned into the smai site of the pgl3-basic vector. the orientation of these fragments in the plasmids were confirmed by dna sequencing. for dna transfection, the lipofectamine reagent was used according to the manufacturer's instruction (gibco-life technologies, inc.). briefly, dbt cells were seeded in a 24-well culture plates at a density of 4 ϫ10 5 cells per well. when the cells reached to approximately 60% confluency, ϸ500 ng of plasmid dnas and 3 l of the lipofectamine reagent were diluted separately in 100 l of serum-free opti medium without antibiotics, mixed, and incubated at room temperature for 30 min before adding to each well of the 24-well plate. dbt cells were washed with pbs and 300 l of opti medium were added to each well of the cell culture. the dna-lipofect amine mixture (200 l) was added to each well and the cell culture plates were incubated at 37°c for various time periods as indicated. for luciferase assay, cells were harvested along with culture medium and lysed by freezing and thawing. one hundred microliters of the cell lysate was assayed for luciferase activity using the luciferase assay system (promega) in a microtiter plate luminometer (dynex technologies, inc.). evolution of mouse hepatitis virus (mhv) during chronic infection: quasispecies nature of the persisting mhv rna induction of apoptosis in murine coronavirus-infected cultured cells and demonstration of e protein as an apoptosis inducer coronavirus mhv-3-induced apoptosis in macrophages the production of recombinant infectious di-particles of a murine coronavirus in the absence of helper virus adenovirus e1b 19 kda and bcl-2 proteins interact with a common set of cellular proteins expression of the gene encoding the proapoptotic nip3 protein is induced by hypoxia murine coronavirus-induced apoptosis in 17cl-1 cells involves a mitochondria-mediated pathway and its downstream caspase-8 activation and bid cleavage the e1b 19k/bcl-2-binding protein nip3 is a dimeric mitochondrial protein that activates apoptosis the e1b 19k/bcl-2-binding protein nip3 is a dimeric mitochondrial protein that activates apoptosis astrocytes and brain injury molecular anatomy of mouse hepatitis virus persistence: coevolution of increased host cell resistance and virus virulence function of a 5ј-end genomic rna mutation that evolves during persistent mouse hepatitis virus infection in vitro functional complementation of the adenovirus e1b 19-kilodalton protein with bcl-2 in the inhibition of apoptosis in infected cells astrocytes as antigen-presenting cells. i. induction of ia antigen expression on astrocytes by t cells via immune interferon and its effect on antigen presentation persistence of viral rna in the central nervous system of mice inoculated with mhv-4 alteration of the ph dependence of coronavirus-induced cell fusion: effect of mutations in the spike glycoprotein nitric oxide synthase type ii expression by different cell types in mhv-jhm encephalitis suggests distinct roles for nitric oxide in acute versus persistent virus infection induction of bcl-2 expression by epstein-barr virus latent membrane protein 1 protects infected b cells from programmed cell death regeneration of oligodendroglia during recovery from demyelinating disease translational control in murine hepatitis virus infection replication and plaque formation of mouse hepatitis virus (mhv-2) in mouse cell line dbt culture bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death chemokines and chemokine receptors in inflammation of the nervous system: manifold roles and exquisite regulation reciprocal modulation between sp1 and egr-1 bcl-2/e1b 19 kda-interacting protein 3-like protein (bnip3l) interacts with bcl-2/bcl-xl and induces apoptosis by altering mitochondrial membrane permeability interleukin-6 induction in vitro in mouse brain endothelial cells and astrocytes by exposure to mouse hepatitis virus (mhv-4, jhm) dephosphorylation of the nucleocapsid protein of inoculum jhmv may be essential for initiating replication regulation of the initiation of coronavirus jhm infection in primary oligodendrocytes and l-2 fibroblasts bcl-2-family proteins: the role of the bh3 domain in apoptosis hypoxia and acidosis activate cardiac myocyte death through the bcl-2 family protein bnip3 modulation of cellular macromolecular synthesis by coronavirus: implication for pathogenesis coronavirus mrna synthesis: identification of novel transcription initiation signals which are differentially regulated by different leader sequences coronaviruses: organization, replication and expression of genome mechanism of demyelination in jhm virus encephalomyelitis: electron microscopic studies cd47 and the bh3-only protein bnip3 in t-cell apoptosis the virus-specific intracellular rna species of two murine coronaviruses: mhv-a59 and mhv-jhm sequence of mouse hepatitis virus a59 mrna 2: indications for rna recombination between coronaviruses and influenza c virus characterization of dbt cell clones derived from cells persistently infected with the jhm strain of mouse hepatitis virus evolution of the 5ј-end of genomic rna of murine coronaviruses during passages in vitro a zinc-finger protein, plagl2, induces the expression of a proapoptotic protein nip3, leading to cellular apoptosis entry of mouse hepatitis virus into cells by endosomal and nonendosomal pathways the carbohydrates of mouse hepatitis virus (mhv) a59: structures of the o-glycosidically linked oligosaccharides of glycoprotein e1 the astrocyte is a target cell in mice persistently infected with mouse hepatitis virus computer software for eukaryotic promoter analysis cell surface expression of fusogenic vesicular stomatitis virus g protein from cloned cdna quasispecies development by high frequency rna recombination during mhv persistence persistent infection of cultured cells with mouse hepatitis virus (mhv) results from the epigenetic expression of the mhv receptor identification of a new transcriptional initiation site and the corresponding functional gene 2b in the murine coronavirus rna genome bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel vdac burkitt's lymphoma cells are resistant to programmed cell death in the presence of the epstein-barr virus latent antigen ebna-4 phosphoproteins of murine hepatitis viruses coronavirus translational regulation: leader affects mrna efficiency interleukin-6 expression and regulation in astrocytes egr1 promotes growth and survival of prostate cancer cells. identification of novel egr1 target genes nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins acute and subacute demyelination induced by mouse hepatitis virus strain a59 in c3h mice adenovirus e1b-19k/bcl-2 interacting protein bnip3 contains a bh3 domain and a mitochondrial targeting sequence biosynthesis, structure, and biological activities of envelope protein gp65 of murine coronavirus heterogeneity of gene expression of the hemagglutinin-esterase (he) protein of murine coronaviruses mouse hepatitis virus gene 5b protein is a new virion envelope protein the human immunodeficiency virus type-1 tat protein upregulates bcl-2 gene expression in jurkat t-cell lines and primary peripheral blood mononuclear cells comparison of the nucleotide and deduced amino acid sequences of the s genes specified by virulent and avirulent strains of bovine coronaviruses regulation of matrix metalloproteinase (mmp) and tissue inhibitor of matrix metalloproteinase (timp) genes during jhmv infection of the central nervous system this work was supported by a public health research grant (ai 47188) from the national institutes of health and in part by the graduate student research fund from the university of arkansas for medical sciences. we thank richard bruick (university of texas southwestern medical center, dallas) and mike buchmeier (the scripps research institute, la jolla, ca) for kindly providing the bnip3 promoter-reporter construct and mhv mutant oblv60, respectively. we also thank marie chow for valuable suggestions during the course of this study. key: cord-304421-xpj6c0vx authors: piñón, josefina d.; teng, henry; weiss, susan r. title: further requirements for cleavage by the murine coronavirus 3c-like proteinase: identification of a cleavage site within orf1b date: 1999-10-25 journal: virology doi: 10.1006/viro.1999.9954 sha: doc_id: 304421 cord_uid: xpj6c0vx abstract the coronavirus mouse hepatitis virus strain a59 (mhv-a59) encodes a 3c-like proteinase (3clpro) that is proposed to be responsible for the majority of the processing events that take place within the replicase polyproteins pp1a and pp1ab. in this study we demonstrate that the q939↓s940 peptide bond, located between the polymerase and zn-finger regions of pp1ab (the pol↓zn site), is processed by the 3clpro, albeit inefficiently. mutagenesis of the pol↓zn site, as well as the previously identified hd1↓3c site in the 1a region of pp1a and pp1ab, demonstrated that the amino acid residues at the p2 and p1 positions of the cleavage site, occupied by l and q, respectively, were important determinants of 3clpro substrate specificity. finally, a direct comparison of the 3clpro-mediated cleavages at the hd1↓3c and pol↓zn sites was made by determining the rate constants using synthetic peptides. the results show that while a larger polypeptide substrate carrying the hd1↓3c site was processed more efficiently than a polypeptide substrate carrying the pol↓zn site, cleavage of the synthetic peptide substrates containing these two cleavage sites occurred at similar efficiencies. this indicates that the overall conformation of a large polyprotein substrate is important in the accessibility of the cleavage site to the proteinase. the family coronaviridae is composed of a group of viruses that cause a variety of diseases in different animal hosts. the murine coronavirus, mouse hepatitis virus (mhv), causes a range of diseases in mouse, including enteritis, hepatitis, encephalitis, and a demyelinating disease (holmes and lai, 1996; houtman and fleming, 1996; lai, 1990) . coronaviruses, along with the arteriviruses, are classified under the newly established order nidovirales based on the similarities in their genome organization and replication strategy (cavanagh, 1997; de vries et al., 1997) . the name nidovirales originates from the latin word nidus, meaning "nest," and refers to the 3ј nested set of subgenomic mrnas that is produced during viral infection (de vries et al., 1997) . as with all positive-strand rna viruses, entry of the viral genome into the cytoplasm is followed by the translation of the positive-strand rna genome, resulting in the expression of viral proteins. the coronavirus genome is organized into seven genes that are separated by stop codons and intergenic sequences (holmes and lai, 1996; lai, 1990) . thus, translation of the viral genome results only in the expression of gene 1 proteins. the rest of the viral genome is expressed through subgenomic mrnas that are transcribed by the viral rna-dependent rna polymerase encoded in gene 1. replication of the viral genome also requires the replicase proteins encoded in gene 1. thus, for both viral replication and subgenomic mrna transcription to take place, the expression of gene 1 products is essential. the replicase gene (gene 1) ( fig. 1) of coronaviruses, spanning 20-22 kb, is organized into two overlapping open reading frames, orf1a and orf1b (bonilla et al., 1994; lee et al., 1991) . the expression of the downstream orf1b is mediated by a ribosomal frameshift event that is aided by the formation of a pseudoknot structure within the overlapping region (bredenbeek et al., 1990; brierley et al., 1987; . thus, two polypeptides, pp1a and pp1ab, are expressed from gene 1, with the translation of pp1ab being only 25-40% as efficient as that of pp1a in in vitro studies (bredenbeek et al., 1990; brierley et al., 1987) . through a series of intricate cotranslational and posttranslational processing events, these polyproteins are converted into a functional complex that in turn is responsible for both genomic rna replication and subgenomic mrna transcription (de vries et al., 1997) . responsible for these processing events are at least two or three viral proteinases encoded within the orf1a region of gene 1 (fig. 1) . two of these proteinase domains, by sequence analysis, share similarities with the cellular proteinase papain. a third proteinase, resembling the poliovirus 3c proteinase, has also been identified (gorbalenya et al., 1989; lee et al., 1991) . the coronavirus 3c-like proteinase (3clpro), flanked on either side by hydrophobic, possibly membrane-spanning regions (hd1 and hd2), is believed to be the prinicipal viral proteinase responsible for the processing events leading to the formation of the viral replicase complex, with as many as 11 potential cleavage sites identified throughout pp1ab (gorbalenya et al., 1989; lee et al., 1991 ) (see fig. 1 ). the presence of the 3clpro is conserved in all coronavirus genomes studied to date (bonilla et al., 1994; boursnell et al., 1987; eleouet et al., 1995; lee et al., 1991) . the 3clpro of mhv-a59 has been identified as a 29-kda protein (p29) both in in vitro study and in mhv-a59infected cells (piñó n et al., 1997) . reported a molecular weight of 27 kda for the same polypeptide.) the catalytic residues of the mhv-a59 3clpro, his3374, and cys3478 have also been identified . treatment of infected cells with e-64d, a known inhibitor of the 3clpro, results in the inhibition of viral rna replication in these cells (kim et al., 1995) , demonstrating the importance of the action of the 3clpro in the events leading to viral replication. van dinten et al. (1999) demonstrated the importance of 3clpro cleavages using an infectious clone of the related arterivirus eav; introduction of mutations into the candidate orf 1b 3clpro cleavage sites had drastic effects on rna synthesis and virus replication. these fndings indicate that this proteinase is a good potential target for antiviral therapy. the cleavage sites of the coronavirus 3clpro ( fig. 1 ) conform to the consensus xq2z (arrow indicates site of cleavage), with x being a hydrophobic residue, usually l, although the amino acids i, m, v, and f are also found in this position (de vries et al., 1997) . at the p1ј position, z is usually a small uncharged residue such as s, a, g, or c (de vries et al., 1997) , with s being the most common residue at this position. data recently obtained for the avian infectious bronchitis virus (ibv), the human coronavirus (hcv-229e), and mhv-a59 demonstrated that many of these predicted cleavage sites are functional. for all three strains, many of the cleavage sites in pp1a have been identified, including a noncanonical lq2n site that had not previously been predicted liu and brown, 1995; liu et al., 1997; ng and liu, 1998; piñó n et al., 1997; tibbles et al., 1996; ziebuhr et al., 1995; ziebuhr and siddell, 1999) . in addition, several of the processing sites in orf1b have also been identified in both ibv and hcv-229e (grötzinger et al., 1996; heusipp et al., 1997a,b; liu et al., 1994 liu et al., , 1998 . however, for mhv, there has been greater difficulty in demonstrating processing by the 3clpro at any of the predicted orf1b cleavage sites. in this study we demonstrate processing by the 3clpro at the putative orf1b the following domains are shown: papain-like proteinase (plp-1 and plp-2), x domain (x), poliovirus 3c-like proteinase (3clpro), hydrophobic domains (hd1 and hd2), growth factor-like domain (gfl), rna-dependent rna polymerase (pol), zinc-finger domain (zn), and helicase (hel). the predicted 3clpro cleavage sites are indicated by the numbers and the cleavage site sequences from the p5 to the p5ј position are listed in table form (arrow indicates site of cleavage) (bonilla et al., 1994; bredenbeek et al., 1990; gorbalenya et al., 1989; lee et al., 1991). cleavage site situated between the putative polymerase (pol) and zinc finger (zn) domains (the pol2zn site) (fig. 2) . furthermore, identification of this site allowed us to make comparisons between the efficiencies of processing at this orf1b site and a previously identified site in orf1a located at the junction between hydrophobic domain 1 (hd1) and the 3clpro (the hd123c site). we have previously demonstrated processing at an orf1a site in mhv-a59, located at the junction between hd1 and the 3clpro (hd123c), by a recombinant mhv-a59 3clpro expressed as a fusion protein with the maltose binding protein (mbp) (piñó n et al., 1997) . the plasmid pet21-nx.3c c3478a, encoding the carboxy-terminal 98 amino acids of hd1 and the inactivated 3clpro, is in vitro transcribed and translated to yield a 40-kda substrate nx.3c (fig. 3a, lane 1) . as previously demonstrated (piñó n et al., 1997), upon addition of the recombinant mbp-3clpro, this substrate is efficiently processed into the 29-kda proteinase (3c) and the 11-kda hd1-derived product (nx) (lane 2). other studies using similar methods have led to the identification of orf1a cleavage sites downstream of the 3clpro, including a previously unidentified, noncanonical lq2n site . however, the demonstration of processing at orf1b sites has proven to be difficult. our work with papain-like proteinase 1 (plp-1) showed that substrate length, and possibly substrate conformation, played an important role in the ability of a substrate to be cleaved efficiently by the proteinase (teng et al., 1999) . we therefore created several substrates of various lengths, encoding different putative cleavage sites in orf1b, in order to investigate processing by the recombinant mbp-3clpro. of these, only the substrate expressed from pet21-pol.zn, encoding mhv-a59 orf1b amino acids l714-q1201, proved to be useful in our investigations. in vitro transcription-translation of the construct pet21-pol.zn resulted in the expression of a 55-kda full-length substrate, pol.zn (predicted molecular weight 56 kda) (fig. 3b , lane 1). upon incubation of this substrate with the recombinant mbp-3clpro enzyme, cleavage between q939 and s940, would give rise to two products, an n-terminal product with a predicted molecular weight (bonilla et al., 1994; bredenbeek et al., 1990; lee et al., 1991) . the positions of the catalytic residues, his3374 and cys3478, are shown. pet 21-ha-hd1.3c encodes mhv-a59 amino acids from s3149 to g3636 directly downstream of the influenza hemagglutinin (ha) tag under the control of the t7 promoter (á). pet21-nx.3c encodes mhv-a59 amino acids from k3258 to q3636. pmal-3c.wt encodes mhv-a59 amino acid sequences from s3334 to g3636 fused to the mal e gene encoding the maltose-binding protein (mbp). the construct pmal-3c is used for the overexpression of the 3clpro in escherichia coli. (c) enlarged map of pol and zn region of orf1b and schematic representation of plasmids derived from this region. the position of the q9392s940 cleavage site at the junction between pol and zn is shown. pet21-pol.zn encodes mhv-a59 orf1b amino acids from l714 to q1201 under the control of the t7 promoter. of 26 kda (pol) and a c-terminal product with a predicted molecular weight of 30 kda (zn). in fig. 3b , lane 2, the addition of the recombinant mbp-3clpro to the pol.zn substrate resulted in the production of a 26-kda protein which presumably corresponds to the n-terminal processing product. mutagenesis of q939 to k (lane 4) or r (lane 6) abolished this processing, suggesting that the cleavage is occurring at the predicted site and that, in view of the substrate specificity demonstrated, the observed processing event is in fact due to the action of the recombinant mbp-3clpro. we were unable to detect the c-terminal 30-kda product by sds-page analysis. one explanation could be that p30 cannot be resolved from p26 in our gel system. (there have been previous reports of viral proteins migrating with electrophoretic mobilities different from that expected. the mhv-a59 3clpro (p29) itself migrates with an electrophoretic mobility faster than its predicted molecular weight of 33 kda (piñó n et al., 1997; lu et al., 1995) ). in addition, the predicted cleavage product p30 has approximately half the methionine content of p26, which may contribute to the difficulty in its detection. the identification of these two cleavage sites, one in orf1a (hd123c) and one in orf1b (pol2zn), allowed us to further define the amino acids required for efficient processing by the 3clpro. for these experiments, we chose to use the pet21-ha.hd1.3c construct, rather than other plasmids encoding 3clpro, because the ha.hd1.3c polypeptide can be efficiently cleaved both in cis and in trans and does not require membranes for its cleavage (piñó n et al., 1997) . several sets of mutations, from the p3 to the p3ј position, were introduced into the construct pet21-ha.hd1.3c by pcr mutagenesis using the mutagenesis primers outlined in table 1 . the effect of these cleavage site mutations on the autocatalytic cis release of the 29-kda 3clpro was assayed by the expression of the radiolabeled, in vitro transcribed, and translated substrate from pet21-nx.3c was incubated with mbp-3clpro (lane 2) or an equal volume of column buffer/20% glycerol (lane 1) and the processed products were separated on a 15% sds-page gel. the arrows on the right of the panel indicate the electrophoretic migration of the 29-kda 3clpro-and the 11-kda hd1-derived cleavage products (nx). (b) trans processing at the pol2zn site. the plasmid pet21-pol.zn was in vitro transcribed and translated. radiolabeled substrate was incubated either with mbp-3clpro or with an equal volume of column buffer/20% glycerol (denoted by plus or minus signs above the lanes, respectively). processed products were analyzed on a 12% sds gel. the electrophoretic migration of p26 is indicated by an arrow on the right of the panel. the molecular weight in kilodaltons of prestained protein markers is indicated on the left of each panel. mutated substrates using in vitro transcription-translation, followed by sds-page analyses of the protein products (fig. 4) . we observed that the l3332 and q3333 residues, at the p2 and p1 positions, respectively, were most sensitive to mutations. any mutation at either one of these positions inhibited the autocatalytic cis processing by the 3clpro (fig. 4 , lanes 6-10). in contrast, mutations at any of the other positions studied were tolerated and the expression of precursor proteins harboring mutations at these sites still resulted in the autocatalytic release of the 29-kda 3clpro. exceptions are the s3334c substitution at position p1ј (lane 12) and the g3335p substitution at position p2ј (lane 14). these mutations also abolish the cis processing by the 3clpro. the effect of the g3335p mutation, however, is expected since the introduction of a p at this site could result in a drastic change in the conformation at the cleavage site. to investigate whether the cleavage sequence requirements for trans cleavage at the hd123c site by the recombinant mbp-3clpro parallel that observed for cis cleavage, the same set of cleavage site mutations were introduced into the construct pet21-ha.hd1.3c c3478a, which also carries an inactivating mutation in the catalytic cysteine residue of the proteinase. the release of the 29-kda 3clpro from this precursor can only be accomplished by incubation with the recombinant mbp-3clpro. figure 5 showed that the effect of these mutations on trans cleavage paralleled the effects on cis cleavage. those mutations centering around the p2 and p1 positions of the cleavage site (l3332 and q3333, respectively) affected trans processing the most. for the pol2zn cleavage site in orf1b, similar mutagenesis studies were conducted in order to determine the cleavage specificity requirements at this site (fig. 6) . mutations from the p3 to the p1ј position were introduced into the plasmid pet21-pol.zn. the mutant substrates were expressed using an in vitro transcriptiontranslation system and then incubated with the recombinant mbp-3clpro. the effect of the mutations on the production of p26 was assayed by sds-page and com-pared to the processing of the wildtype pol.zn substrate (fig. 6, lanes 1 and 2) . the results were similar to that observed with the orf1a hd123c site in that the l938 and q939 residues, at the p2 and p1 positions respectively, were most sensitive to mutations. the l938i mutation (fig. 6 , lanes 5 and 6) resulted in the inhibition of p26 processing, indicating the sensitivity of this site to a conservative change. however, the l938m substitution (fig. 6, lanes 7 and 8) did not abolish processing of p26. mutation of q939 to either k (lanes 9 and 10) or r (lanes 11 and 12) abolished processing at this site. substitution of s940 with an a (lanes 13 and 14) was tolerated, whereas mutating s940 to the bulkier n (lanes 15 and 16) resulted in the inhibition of p26 processing. the results of mutagenesis studies on both the hd123c and pol2zn cleavage sites demonstrate that the p1 and p2 positions at the cleavage site are the primary determinants of cleavage specificity by the 3clpro in both cis and trans processing. we investigated the efficiency of processing at the orf1a site compared to the orf1b site. the substrates, nx.3c and pol.zn, were incubated with a fixed amount of recombinant proteinase for increasing periods of time. we observed that the orf1a substrate, nx.3c, is readily processed within 1 h, with the substrate completely converted into product by 20 h (fig. 7a) . processing of the orf1b substrate, however, is not observed until after 4 h of incubation with the recombinant proteinase. furthermore, cleavage of pol.zn occurs at a much lower level and does not reach completion even after 30 h (fig. 7b ). this inefficiency of processing of the pol2zn site compared to the hd123c site may be explained in two ways. first, although the primary sequences of the two sites reveal no obvious reason why one is processed more efficiently than the other, it is possible that the subtle differences in the sequences of the two sites are enough to make the hd123c site a more efficient substrate than the pol2zn site. alternatively, the difference may not be inherent to the primary sequence of the cleavage sites, but rather to the conformation of the entire substrate as a whole. it is possible that the substrate conformation of nx.3c allows the recombinant proteinase better access to the cleavage site. in the pol.zn substrate, the cleavage site may be more obscured, and in this manner the virus regulates both when and how much of its encoded proteins are produced. to determine whether the difference in processing efficiencies at the hd123c and pol2zn sites can be attributed to the primary sequences of the cleavage sites, we synthesized a 14-mer peptide, representing the p6-p8ј residues of the hd123c cleavage site, and a 15-mer peptide, representing the p6-p9ј amino acids of the pol2zn cleavage site. each peptide was then incubated with the recombinant mbp-3clpro and the resulting cleavage products were separated from each other and from the substrate by reverse-phase chromatography. for the orf1a peptide, reverse-phase chromatography of the reaction at zero time resulted in a single peak, representing the peptide substrate, in the elution profile (fig. 8a) . incubation with mbp-3clpro followed by chromatography resulted in two additional peaks, representing the cleavage products, and a reduction in the fig. 5. hd123c cleavage site mutagenesis: trans processing at the hd123c site. the same cleavage site mutants used in the study of cis processing were introduced into pet21-ha.hd1.3c c3478a, which also harbors a mutation at the catalytic cysteine residue of the proteinase. equivalent counts per minute of radiolabeled substrates expressed from these mutant plasmids by tnt were incubated with mbp-3clpro or an equivalent volume of column buffer/20% glycerol (ϩ/ϫ mbp-3clpro). cleavage products were then separated on a 10% sds-gel. the electrophoretic migration of p29 is indicated by an arrow on the right. molecular weight markers are indicated on the left. substrate peak. microsequencing of the cleavage products confirmed that cleavage had occurred between q3333 and s3334. for the orf1b peptide similar results were observed in that the peptide substrate eluted as a single peak at zero time (fig. 8b) . incubation with mbp-3clpro prior to separation resulted in two new peaks, representing the two cleavage products. a reduction in the substrate peak was also observed. again, the authenticity of cleavage of the synthetic peptide was confirmed by microsequencing of the cleavage products, which showed that cleavage had occurred between q939 and s940. the level of cleavage of the orf1a peptide was not any different from, and in some cases was less than, that of the orf1b peptide. we observed that under identical reaction conditions no more than 30% of the orf1a peptide was cleaved by the mbp-3clpro, whereas with the orf1b peptide the level of cleavage was between 30 and 50%. in order to allow direct comparison between the cleavage efficiencies of the nx23c and pol2zn sites, we determined the k cat and k m values for the reactions with the synthetic peptides described above. results show that cleavage of the orf1a peptide by the recombinant mbp-3clpro yielded a k cat of 0.015 s ϫ1 , and a k m of 2.32 ϯ 0.50 mm. with the orf1b peptide the k cat was slightly slower, with a value of 0.006 s ϫ1 , and the k m was calculated at 0.27 ϯ 0.07 mm. our results therefore show that cleavage (k cat ) of the orf1a peptide occurred at only a slightly higher rate (2.5-fold) than that of the orf1b peptide. the lack of substantial difference between the k cat values is not surprising given that the sequences of the peptides are very similar. interestingly, when the catalytic efficiency (k cat /k m ) was taken into consideration, the orf1b peptide (k cat /k m ϭ 2.1 ϫ 10 ϫ5 m ϫ1 s ϫ1 ) was a slightly better substrate than the orf1a peptide (k cat /k m ϭ 6.4 ϫ 10 ϫ6 m ϫ1 s ϫ1 ). taken together, the peptide cleavage results presented here suggest that the cleavage efficiencies of the two peptides are similar. the action of viral-encoded proteinases is essential to viral replication (dougherty and semler, 1993) . this makes viral-encoded proteinases potentially good targets for antiviral drugs. in the murine coronavirus, two such proteinases are under continued investigation in order to better understand the manner in which these proteinases function. papain-like proteinase 1 (plp-1) fig. 6. pol2zn cleavage site mutagenesis. recombinant mbp-3clpro was used in posttranslational trans cleavage assays with radiolabeled substrates generated from pet21-pol.zn that encoded a wildtype cleavage sequence or harbored mutations around the pol2zn site. substrate volumes containing equivalent counts per minute were incubated with mbp-3clpro (denoted by plus sign above the lanes) or an equivalent volume of column buffer/20% glycerol (denoted by a minus sign above the lanes). the electrophoretic migration of p26 is indicated by the arrow to the right of the panel. molecular weight markers are indicated on the left. has been linked to the processing of several nonstructural proteins encoded in the 5ј end of the viral genome (baker et al., 1989; bonilla et al., 1995; denison et al., 1992; hughes et al., 1995) . these cleavage products, p28 and p65, are not yet linked with any known viral function. however, those viral proteins with presumed functions in viral replication and viral rna transcription, such as the rna-dependent rna polymerase (pol) and the zinc-finger/helicase (zn-hel) proteins, are believed to be processed by the 3c-like proteinase of the virus. the 3clpro is predicted to cleave at, at least, 11 sites in pp1ab. many of the cleavage sites located in the 1a region of pp1ab have been shown to be functional cleavage sites and processing by the 3clpro at these sites has been demonstrated in the coronaviruses ibv (liu et al., 1997; liu and brown, 1995; ng and liu, 1998; tibbles et al., 1996) , hcv-229e (ziebuhr et al., 1995; ziebuhr and siddell, 1999) , and mhv-a59 piñó n et al., 1997) . some of the mature viral products resulting from these cleavages have also been identified in infected cells (liu et al., 1997; lu et al., 1998; ng and liu, 1998; piñó n et al., 1997; ziebuhr and siddell, 1999; denison et al., 1999) . according to computer predictions, further processing at the q9392s940, q15392c1540, q20602s2061, and q24342a2435 sites located in the 1b region of mhv-a59 pp1ab would result in mature viral products of 106, 67, 59, 42, and 33 kda, respectively, corresponding to pol, zn-hel, and the three c-terminal-most cleavage products. in hcv-229e, viral products of 105, 71, and 41 kda, corresponding to the pol, zn-hel, and the second c-terminal-most proteins, have been identified in infected cells and the role of the 3clpro in the processing of these products has been authenticated in vitro (grötzinger et al., 1996; heusipp et al., 1997a,b) . similarly, in the case of ibv, viral proteins of 100, 39, and 35 kda have been identified in infected cells (corresponding to pol and the two c-terminal-most proteins) and cotransfection experiments have implicated the 3clpro in the processing of these viral products (liu et al., 1994 . for mhv-a59, however, demonstration of processing at any mhv cleavage site in the 1b region of pp1ab has lagged behind that of hcv and ibv. here we report the first demonstration of processing at the site between pol and zn in pp1ab by the mhv-a59 3clpro. the cleavage at the pol2zn site is highly inefficient compared to the processing observed at the hd123c site, as evidenced by the time course assays illustrated in fig. 7 . phosphorimager analyses indicate that the pol.zn substrate is cleaved fivefold less efficiently than the nx.3c substrate (data not shown). the differences in these efficiencies, however, could not be explained by the subtle differences in the primary sequences of the cleavage sites alone. in fact, when presented to the enzyme in the context of a 14-or 15-mer peptide substrate, there was no substantial difference in cleavage efficiency between these two peptides, an observation that would seem to contradict the results obtained with the larger polyprotein substrates. recently, ziebuhr and siddell (1999) investigated the efficiency of processing at several hcv-229e 3clpro sites located at the c-terminus of pp1a or the central region of pp1ab. they observed that several viral products were produced less efficiently than others, with reduced cleavage activity at two orf 1a sites, between v-q35462s3547 and l-q38242n3825, compared with cleavage at the sites flanking the 3clpro domain. the corresponding lq2n site in mhv-a59 has also been shown to be less effi-ciently cleaved than the lq2s sites flanking the 3clpro domain . in the case of l-q38242n3825 in hcv-229e, additional peptide cleavage data demonstrated that the properties of the cleavage sequence itself, rather than the overall conformation of the polypeptide and the accessibility of the cleavage site, contribute to the observed inefficiency of processing at this site (ziebuhr and siddell, 1999) . while our results do not contradict theirs, they do demonstrate that, in vivo, the conformation of a larger polypeptide substrate is likely to be as important a determinant of cleavage as is the primary structure and sequence of the cleavage site. our results clearly show that, at least with the case of the pol2zn site of mhv-a59, the observed inefficiency of cleavage was likely due to the overall conformation of the polypeptide, which may directly translate into the accessibility of the cleavage site, rather than the primary sequence of the site. taken together, both sets of results highlight important regulatory mechanisms employed by the virus to coordinate the temporal production and the accumulation of the various replicase proteins. thus, the very slow in vitro processing at the orf 1b cleavage site, compared with that at the orf 1a site, generally correlates with the levels of orf 1a and orf 1b polypeptides found in infected cells. however, there are inherent differences between in vitro cleavage reactions with either recombinant proteins or peptide substrates and in vivo processing; these include the lengths of the substrates, the concentration of the enzyme and substrates, as well as the subcellular localization of replication complexes in vivo (denison et al., 1999; ziebuhr and siddell, 1999) . these differences may all contribute to the extended lengths of time necessary for in vitro cleavages. the substrate specificity of the coronavirus 3clpro has been determined mainly through the identification of functional cleavage sites and a visual inspection of these cleavage site sequences. mutagenesis has been done primarily to verify the authenticity of the cleavage site and most mutagenesis studies have not extended beyond the q residue that is absolutely conserved at the p1 position in all 3clpro cleavage sites identified to date. we have extended our mutagenesis studies to cover the p3 to p3ј positions of the cleavage site sequence. our results demonstrated that the substrate sequence specificity of the 3clpro is primarily influenced by the amino acid residues present at the p2 and p1 positions of the cleavage site. substitution of the q residue at the p1 position with any other amino acid has resulted in substrates that could not be cleaved by the 3clpro, demonstrating the importance of this residue in the substrate. we could not detect any processing in substrates containing mutations at this position (figs. 4-6) . the p2 position of the cleavage site is most often occupied by an l; however, in some cleavage sites identified in hcv-229e, this position is occupied by a v or an i (grötzinger fig. 8 . chromatograms of cleavage products of the synthetic peptides corresponding to orf1a amino acids and orf1b amino acids. a and b show chromatograms of orf1a peptide (1 mm) and orf1b peptide (1 mm), respectively, at 0 h and after 16 h of incubation with mbp-3clpro (17.5 m enzyme) at 30°c. in both panels the chromatograms for t ϭ 0 h are offset to allow comparison between the two time points. the asterisks (*) indicate the cleavage products that were used in peptide sequencing in order to confirm the sites of cleavage. ziebuhr and siddell, 1999) , suggesting that this position is not as strictly conserved as the p1 position and may thus be able to tolerate some mutations. however, our results demonstrated otherwise, in that even a conservative change to i resulted in a reduction of cleavage , to below 20%, as measured by phosphorimager analysis (data not shown). this suggests that perhaps an i at the p2 position of the cleavage site can be functional only when compensated for elsewhere in the substrate sequence. the only change tolerated at this position was a change to m in the p2 position at the pol2zn site (fig. 6) . interestingly, the jhm strain of mhv encodes an m instead of an l at this position (lee et al., 1991) . thus such a mutation resulted in a wildtype jhm pol2zn site and a cleavage efficiency equivalent to wildtype levels. this provides indirect evidence that the pol2zn site in jhm is a functional 3clpro site. although the s found at the p1ј position of both the hd123c and pol2zn sites is not as sensitive to mutations, we observed that some mutations, such as s3334c (in the hd123c site) (fig. 4) and s940n (in the pol2zn site) (fig. 6) , are not tolerated. additionally, both s3334a and s940a substitutions, though tolerated, resulted in a reduction, but not complete inhibition of cleavage , suggesting that the residue at the p1ј position also plays a role in substrate recognition, although to a lesser extent than those at the p1 and p2 positions. aside from the amino acid residues at the p2, p1, and p1ј positions, the amino acid sequences surrounding the scissile q2s(a,g) peptide bonds that are recognized by the 3clpro do not share any other significant primary structure similarity. furthermore, the presence of an lqa tripeptide not cleaved by the proteinase would again suggest the existence of a common conformational determinant shared by all 3clpro susbstrates that is necessary for 3clpro-mediated processing. the pol2zn site is the first functional cleavage site identified in the orf1b region of the mhv-a59 pp1ab. further work remains to be done in order to identify other functional cleavage sites in orf1b. additionally, the mature viral products resulting from these processing events remain to be identified in infected cells. a direct comparison of the cleavage efficiencies of each site will help to elucidate the complex posttranslational processing pattern of the mhv-a59 gene 1 polyprotein, as well as provide insight into the regulatory mechanisms employed by the virus to maintain the production of its proteins under control. the parental plasmids used in this study are illustrated in fig. 2 . the plasmids pet21-nx.3c, pet21-ha.hd1.3c, and pmal-3c.wt have all been described elsewhere (piñó n et al., 1997) . pet21-nx.3c encompasses mhv-a59 nucleotides 9912-10661, encoding the last 98 amino acids of hd1 and the entire 303-aminoacid region encompassing the 3clpro (from k3235 to q3636). pet21-nx.3c c3478a is the same as pet21-nx.3c but carries the inactivating c3478a mutation in the catalytic cysteine residues of the proteinase. the plasmid pet21-ha.hd1.3c encodes mhv-a59 nucleotides 9654-10661, encoding hd1 and the 3clpro from s3149 to q3636, directly behind the influenza hemagglutinin (ha) epitope under the control of the t7 promoter in pet21a. the plasmid pet21-ha.hd1.3c c3478a contains the 3clpro inactivating mutation c3478a in the background of the parental pet21-ha.hd1.3c plasmid. the plasmid pmal-3c.wt encodes mhv-a59 nucleotides 10209-10661, corresponding to the 3clpro region from s3334 to q3636, behind the mal e gene in the pmalc2 vector (new england biolabs). this plasmid encodes the 3clpro domain fused to the coding sequence of the maltose binding protein and is used for overexpression of the recombinant mbp-3clpro enzyme. a region of mhv-a59 gene 1, from nucleotides 15677 to 17140, corresponding to orf1b amino acids l714 to q1201, was pcr amplified from a plasmid encoding the entire orf1b sequence using the primers f1bp 2383-2394 (5ј-ttcgaattccccgggggatcccttatggcatg-caatggacac-3ј) and r1bp 3846-3835 (5ј-cgaattc-ctctagaaagcttgctgaaacgtctcaggcacact-3ј). the resulting pcr fragment was digested with bamhi and hindiii (denoted by the underlined sequences in the primers) and cloned into the corresponding sites of pet21a, resulting in the plasmid pet21-pol.zn. the pet21-ha.hd1.3c cleavage site mutants l3332i, l3332s, q3333k, q3333r, and s3334a were created by two rounds of pcr as described previously hughes et al., 1995) using the fmp and rmp primers listed in table 1 and the primers fij31 (5ј-tg-gcttgtcatgtatggtgc-3ј) and rsp 10661-10644 (5ј-aacatatcctacagaacc-3ј). the resulting mutant fragments were digested with kpni and bamhi and cloned into the same sites in pet21-ha.hd1.3c. all other cleavage site mutants in pet21-ha.hd1.3c were created using the quikchange mutagenesis kit (stratagene) following the manufacturer's protocols. the mutagenic primers used are listed in table 1 . following pcr amplification, the amplified plasmids were digested with the restriction enzyme dpni, which digests methylated and hemimethylated dna, thus destroying the parental plasmid and any hybrids containing one parental strand and one mutated strand. escherichia coli xl1-blue supercompetent cells (stratagene) were then transformed with the mutated plasmids. the presence of the desired mutation was verified by sequencing. the fragments containing the hd1.3c cleavage site mutations were also subcloned into pet21-ha.hd1.3c c3478a using the ndei and bamhi sites in order to create plasmids carrying both the inactivating c3478a mutation and mutations at the cleavage site. these plasmids were used to express substrates used in trans cleavage assays. all pet21-pol.zn cleavage site mutants v937s, l938i, l938m, q939k, q939r, s940a, and s940n were created using the quikchange mutagenesis kit as described above. the primers used in creating these mutants are also listed in table 1 . cell-free expression of plasmid dnas was carried out using the tnt rabbit reticulocyte lysate-coupled transcription-translation system (promega) at 30°c for 2 h, as previously described (piñó n et al., 1997) . the incorporation of [ 35 s]methionine into acid precipitable counts was used as an indicator of protein synthesis. equivalent amounts of acid precipitable counts were directly analyzed by sds-polyacrylamide gel electrophoresis (sds-page) or used in posttranslational proteolytic assays as indicated. radioimmunoprecipitations were carried out as described previously denison et al., 1991) . expression of the recombinant 3clpro from pmal-3c.wt (neb), which expresses 3clpro as an mbp-3cl fusion protein, and the purification of the fusion protein was carried out according to the manufacturer's protocol and as described by herold et al. (1996) . briefly, e. coli tb1 cells transformed with pmal-3c.wt were grown at 37°c in the presence of ampicillin (100 g/ml) until the a 600nm reached 0.8, at which point the cells were induced with isopropylthio-␤-d-galactoside at a final concentration of 0.5 mm for 4 h at 25°. cells were harvested and then resuspended in 10 ml of column buffer [20 mm tris-cl (ph 7.5)], 200 mm nacl, 1 mm edta, 1 mm dtt] per gram of cells. the cell suspension was then lysed by sonication. cell debris was pelleted by centrifugation at 9000g for 30 min. the crude lysates were diluted 1:5 in column buffer and then loaded onto an amylose column (bed volume 6 ml), preequilibrated with column buffer, at a flow rate of 1 ml/min. the column was then washed with 12 column volumes of column buffer and the mbp-3clpro fusion protein was eluted with column buffer containing 10 mm maltose. fractions of 1 ml were collected and those containing the 72-kda recombinant mbp-3clpro were identified by analyzing 10-l aliquots by sds-page. fractions containing the recombinant proteinase were pooled and the concentration of the fusion proteinase was determined using the bradford assay against known concentrations of bovine serum albumin. the recombinant proteinase was stored at ϫ80°c in column buffer supplemented with 20% glycerol. radiolabeled substrates containing the cleavage sequences of hd1.3c or pol.zn (fig. 2) were generated using the tnt rabbit reticulocyte lysate system. lysate volumes containing equivalent counts per minute were incubated with approximately 1-10 g of recombinant proteinase or an equivalent volume of column buffer/20% glycerol at 30°c for 12-16 h or, where applicable, the specified lengths of time. the processed products were analyzed by sds-page followed by autoradiography. phosphorimager analysis was carried out as previously described teng et al., 1999) . recombinant mbp-3clpro enzyme (1.27 mg/ml in elution buffer supplemented with 20% glycerol and 2 mm dtt, 17.5 m enzyme) was incubated with synthetic peptide orf1a (0.02 to 0.6 mm in 10% dmso), with the sequence h 2 n-thr-thr-ser-phe-leu-gln2ser-gly-ile-val-lys-met-val-ser-cooh, corresponding to orf1a amino acids 3328 to 3341 (arrow indicates cleavage site) or orf1b peptide (0.02 to 0.25 mm in 10% dmso), with the sequence h 2 n-arg-ser-ala-val-leu-gln2ser-val-gly-ala-cys-val-val-cys-ser-cooh, corresponding to orf1b amino acids 934 to 948 (arrow indicates cleavage site) in a final reaction volume of 100 l. the reactions were allowed to proceed at 30°c for 60-120 min, at which time the reactions were quenched by addition of trichloroacetic acid (tca) to 1% final concentration. the samples were then chilled on ice, and the denatured protein was precipitated by centrifugation. for zero time point samples, the enzyme was mixed with tca prior to the addition of peptide substrates and the reactions were carried out as described above. separation of cleavage product from substrate was carried out with the ä ktapurifier system (amersham pharmacia biotech) equipped with a sephasil peptide c18 5-m st 4.6/100 reverse-phase column (amersham pharmacia biotech). elution was performed with a linear gradient of 92% eluent a (0.1% aqueous tfa)/8% eluent b (0.1% tfa in 90% acetonitrile/10% water) up to 100% eluent b (seybert et al., 1997) over 26 min (flow rate 0.8 ml/min, detection at 215 nm). with the orf1a peptide the level of cleavage was no more than 30%, whereas with the orf1b peptide the level of cleavage was between 30 and 50%. the data obtained were fitted to the michaelis-menten equation and the k cat and k m values were obtained using kaleidagraph 3.08 (synergy software). identification of a domain required for autoproteolytic cleavage of murine coronavirus gene a polyprotein mouse hepatitis virus strain a59 rna polymerase gene orf 1a: heterogeneity among mhv strains characterization of the leader papain-like proteinase of mhv-a59: identification of a new in vitro cleavage site completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus the primary structure and expression of the second open reading frame of the polymerase gene of the coronavirus mhv-a59; a highly conserved polymerase is expressed by an efficient ribosomal frameshifting mechanism an efficient ribosomal frame-shifting signal in the polymerase encoding region of the coronavirus ibv nidovirales: a new order comprising coronaviridae and arteriviridae identification and characterization of a 65-kda protein processed from the gene 1 polyprotein of the murine coronavirus mhv-a59 the putative helicase of the coronavirus mouse hepatitis virus is processed from the replicase gene polyprotein and localizes in complexes that are active in viral rna synthesis intracellular processing of the n-terminal orf 1a proteins of the coronavirus mhv-a59 requires multiple proteolytic events identification of polypeptides encoded in open reading frame 1b of the putative polymerase gene of the murine coronavirus mouse hepatitis virus a59 the genome organization of the nidovirales: similarities and differences between arteri-, toro-, and coronaviruses expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes complete sequence (20 kb) of the polyprotein-encoding gene 1 of transmissible gastroenteritis virus coronavirus genome: prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis characterization of a 105-kda polypeptide encoded in gene 1 of the human coronavirus hcv 229e nucleotide sequence of the human coronavirus 229e rna polymerase locus an "elaborated" pseudoknot is required for high frequency frameshifting during translation of hcv 229e polymerase mrna characterization of coronavirus rna polymerase gene products identification and subcellular localization of a 41 kda polyprotein 1ab processing product in human coronavirus 229e-infected cells identification of an atpase activity associated with a 71-kilodalton polypeptide encoded in gene 1 of the human coronavirus 229e coronaviridae: the viruses and their replication pathogenesis of mouse hepatitis virus-induced demyelination identification of the murine coronavirus p28 cleavage site coronavirus protein processing and rna synthesis is inhibited by the cysteine proteinase inhibitor e64d coronavirus: organization, replication and expression of genome the complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and rna polymerase a 100-kilodalton polypeptide encoded by open reading frame (orf) 1b of coronavirus infectious bronchitis virus is processed by orf1a products characterization and mutational analysis of an orf1a-encoding proteinase domain responsible for proteolytic processing of the infectious bronchitis virus 1a/1b polyprotein proteolytic mapping of the coronavirus infectious bronchitis virus 1b polyprotein: evidence for the presence of four cleavage sites of the 3c-like proteinase and identification of two novel cleavage products proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein: identification of a 10-kilodalton polypeptide and determination of its cleavage sites identification and characterization of a serine-like proteinase of the murine coronavirus mhv-a59 mouse hepatitis virus 3c-like protease cleaves a 22-kilodalton protein from the open reading frame 1a polyprotein in virus-infected cells and in vitro identification of a 24-kda polypeptide processed from the coronavirus infectious bronchitis virus 1a polyprotein by the 3c-like proteinase and determination of its cleavage sites efficient autoproteolytic processing of the mhv-a59 3c-like proteinase from the flanking hydrophobic domains requires membranes expression and characterization of a recombinant murine coronavirus 3c-like proteinase expression, purification, and activity of recombinant mhv-a59 3clpro expression of murine coronavirus recombinant papain-like proteinase: efficient cleavage is dependent on the lengths of both the substrate and the proteinase polypeptides characterization in vitro of an autocatalytic processing activity associated with the predicted 3c-like proteinase domain of the coronavirus avian infectious bronchitis virus proteolytic processing of the open reading frame 1b-encoded part of the arterivirus replicase is mediated by nsp4 serine protease and is essential for virus replication characterization of a human coronavirus (strain 229e) 3c-like proteinase assay processing of the human coronavirus 229e replicase polyproteins by the virus-encoded 3c-like proteinase: identification of proteolytic products and cleavage sites common to pp1a and pp1ab the authors thank ravi mayreddy for the construction of the orf1bencoding plasmids. peptide sequencing was provided by the protein chemistry laboratory of the school of medicine (university of pennsylvania), supported by core grants of the diabetes and cancer centers (dk-19525 and ca-16520). this work was supported by nih grant ai-17418. key: cord-328046-5us4se5o authors: xu, h. y.; lim, k. p.; shen, s.; liu, d. x. title: further identification and characterization of novel intermediate and mature cleavage products released from the orf 1b region of the avian coronavirus infectious bronchitis virus 1a/1b polyprotein date: 2001-09-30 journal: virology doi: 10.1006/viro.2001.1098 sha: doc_id: 328046 cord_uid: 5us4se5o abstract the coronavirus 3c-like proteinase is one of the viral proteinases responsible for processing of the 1a and 1a/1b polyproteins to multiple mature products. in cells infected with avian coronavirus infectious bronchitis virus (ibv), three proteins of 100, 39, and 35 kda, respectively, were previously identified as mature cleavage products released from the 1b region of the 1a/1b polyprotein by the 3c-like proteinase. in this report, we show the identification of two more cleavage products of 68 and 58 kda released from the same region of the polyprotein. in addition, two stable intermediate cleavage products with molecular masses of 160 and 132 kda, respectively, were identified in ibv-infected cells. the 160-kda protein was shown to be an intermediate cleavage product covering the 100and 68-kda proteins, and the 132-kda protein to be an intermediate cleavage product covering the 58-, 39-, and 35-kda proteins. immunofluorescent staining of ibv-infected cells and cells expressing individual cleavage products showed that the 100-, 68-, and 58-kda proteins were associated with the membranes of the endoplasmic reticulum, and the 39and 35-kda proteins displayed diffuse distribution patterns. coronavirus gene expression involves the expression of six to seven mrna species. in cells infected with the prototype species of the coronaviridae, avian coronavirus infectious bronchitis virus (ibv), six mrna species are detected. these include the genome-length mrna (mrna1) of 27.6 kilobases (kb) and five subgenomic mrna species (mrnas 2-6) with sizes ranging from 2 to 7 kb. the 5ј-terminal unique region of mrna 1 contains two large orfs, 1a and 1b, encoding the 441-kda 1a and 741-kda 1a/1b polyproteins (boursnell et al., 1987) (fig. 1) . the two polyproteins are cleaved by two viral proteinases to produce functional products associated with viral replication (ziebuhr et al., 2000) (fig. 1) . the first proteinase was identified to be included in a 195-kda cleavage product, which contains a papain-like proteinase domain encoded by orf 1a from nucleotides 4242 to 5553 (lim et al., 2000) . this proteinase was shown to be involved in cleavage of the n-terminal region of the 1a and 1a/1b polyproteins at two g-g dipeptide bonds (g 673 -g 674 and g 2265 -g 2266 ) to release two mature products of 87 and 195 kda lim and liu, 1998; lim et al., 2000) . the second proteinase was identified as a 33-kda protein in ibv-infected cells lim et al., 2000; ng and liu, 2000) . this serine proteinase belongs to the picornavirus 3c proteinase group (3c-like proteinase) and was shown to mediate cleavage of the 1a and 1a/1b polyproteins at more than 10 q-s(g, n) dipeptide bonds to release mature cleavage products (liu et al., 1994 (liu et al., , 1997 liu, 1998, 2000) . in addition to the viral proteinases, understanding of the functions of other cleavage products from the 1a and 1a/1b polyproteins is emerging. for example, the 71-kda protein released from the human coronavirus 1a/1b polyprotein was shown to possess atpase and rna duplex-unwinding activities, confirming the previous predication that the protein may be the viral helicase (heusipp et al., 1997a; seybert et al., 2000a,b) . immunofluorescence and biochemical studies demonstrated that several cleavage products are membrane-associated and colocalize with the viral rna replication-transcription machinery (bost et al., 2000; denison et al., 1999; ng and liu, 2000; schiller et al., 1998; shi et al., 1999; sims et al., 2000; ziebuhr and siddell, 1999; , suggesting the involvement of these products in viral rna replication. more recent studies showed that the first cleavage product of mouse hepatitis virus-a59 (mhv-a59) 1a and 1a/1b polyproteins, p28, might play a direct role in viral rna synthesis together with polymerase and helicase (bost et al., 2000; sims et al., 2000) . other cleavage products, such as the mhv 22-kda protein, may segregate into different but tightly associated membrane populations which may serve independent functions during viral replication (bost et al., 2000; sims et al., 2000) . in previous reports, we demonstrated that four previously predicted q-s(g) dipeptide bonds located in the 1b region of the 1a/1b polyprotein are genuine cleavage sites of the 3c-like proteinase (liu et al., 1994 . taken together with the n-terminal cleavage site identified for releasing the 100-kda protein, cleavage at these positions would result in the release of five mature products with molecular masses of approximately 100, 68, 58, 39, and 35 kda, respectively (fig. 1 ). among them, the 100-, 39-, and 35-kda proteins were specifically identified in ibv-infected cells. in this communication, we report the identification of the two previously unidentified products, the 68-and 58-kda proteins, in ibvinfected cells with newly raised region-specific antisera. meanwhile, two relatively stable intermediate cleavage products with molecular masses of approximately 160 and 132 kda were identified. analysis of the expression and processing kinetics showed that both the 160-and 132-kda intermediate cleavage products coexist with their final cleavage products during the viral replication cycle. furthermore, immunofluorescence analysis showed that the 100-, 68-, and 58-kda proteins were associated with the membranes of the endoplasmic reticulum (er), while the 39-and 35-kda proteins displayed diffuse distribution patterns. in our previous reports, four q-s(g) dipeptide bonds, q 891(1b) -s 892(1b) , q 1492(1b) -g 1493(1b) , q 2012(1b) -s 2013(1b) , and q 2350(1b) -s 2351(1b) , located in the orf 1b region and encoded by nucleotides 15129-15134, 16929-16934, 18492-18497, and 19506-19511, respectively , were demonstrated to be the cleavage sites of the 3c-like proteinase (liu et al., 1994 (fig. 1 ). taken together with the q 3928 -s 2929 dipeptide bond (encoded by nucleotides 12310-12315) identified as the n-terminal cleavage site of the 100-kda protein, cleavage at these positions would result in the release of five mature products with molecular masses of approximately 100, 68, 58, 39, and 35 kda. among them, the 100-, 39-, and 35-kda proteins were specifically identified in ibv-infected cells (liu et al., 1994 . to further identify and characterize the cleavage products, four new region-specific antisera, anti-100, anti-68, anti-58, and anti-35, were raised in rabbits against bacterially expressed viral proteins. the ibv proteins used to raise these antisera were encoded by nucleotides 12447-15131, 15536-16787, 16932-18494, and 19509-20414, respectively (fig. 1) . anti-100 was raised to replace v-58, which was raised against the ibv sequence encoded by nucleotides 14492-15520, and was used to identify the 100-kda protein in ibvinfected cells (liu et al., 1994) . the specificities of these antisera were tested by immunoprecipitation assay, showing that they could specifically precipitate their target proteins synthesized in both the in vitro system and intact cells (data not shown). to identify the cleavage products in ibv-infected cells, confluent monolayers of vero cells were infected with ibv at a m.o.i. of approximately 3 pfu per cell. to reduce the background, 5 g/ml of actinomycin d was added to the culture medium at 2 h postinfection and cells were labeled with [ 35 s]methionine and cysteine at 6 h postinfection. cell lysates were prepared from cells harvested at 8 h postinfection and subjected to immunoprecipitation with anti-100, anti-68, anti-58, v17, and anti-35. v17 was raised against the ibv polypeptides encoded by nucleotides 19154-20414 and used to detect the 39-and 35-kda proteins in ibv-infected cells in a previous report . immunoprecipitation with anti-100 resulted in the detection of the 100-kda protein and a protein with an apparent molecular mass of 160 kda (fig. 2a, lane 10) . the 160-kda protein was also detected by anti-68 (fig. 2a, lane 9) . no product corresponding to the 68-kda putative helicase domain-containing protein was detected by anti-68 (fig. 2a, lane 9) . the detection of the 160-kda protein with the two n-terminally specific antisera and the apparent molecular mass of the protein suggest that it may be an intermediate cleavage product containing the 100-kda and the putative 68-kda proteins. immunoprecipitation of the same lysates with anti-58 resulted in the detection of two products with apparent molecular masses of 58 and 132 kda (fig. 2a, lane 8) . the 132-kda protein was also immunoprecipitated by v17 and anti-35 (fig. 2a, lanes 6 and 7) . in addition, v17 also precipitated specifically the 39-and 35-kda proteins (fig. 2a, lane 7) . once again, very weak immunoprecipitation of the 35-kda protein was observed (fig. 2a, lane 7) . the 35-kda protein was also weakly immunoprecipitated by anti-35 (fig. 2a, lane 6 ). the detection of the 132-kda protein by the three c-terminally specific antisera and the apparent molecular mass of the protein suggest that it may be an intermediate cleavage product derived from the c-terminal region of the polyprotein. interestingly, the three c-terminally specific antisera also weakly immunoprecipitated the 160-kda protein (fig. 2a , lanes 6-8). the reason for this result is currently unclear, but it may reflect the interaction among the cleavage products. as immunoprecipitation with anti-68 failed to detect the putative 68-kda protein in ibv-infected vero cells, we then tried to detect the protein by western blot with the same antiserum. for this purpose, cell lysates were prepared from ibv-infected vero cells harvested at 8, 24, and 32 h postinfection, respectively, and subjected to western blotting analysis. as shown in fig. 2b , a polypeptide with an apparent molecular mass of 68 kda was specifically detected in lysates prepared from cells harvested at 24 h postinfection (lane 2), and the expression of the protein was dramatically increased at 32 h postinfection (lane 3). expression and processing kinetics of the orf 1b region of the 1a/1b polyprotein in ibv-infected vero cells as the 160-and 132-kda proteins may represent stable intermediate cleavage products, time-course experiments were carried out to investigate the expression and processing kinetics of the two products in ibv-infected cells. for this purpose, confluent monolayers of vero cells were infected with ibv at a m.o.i. of approximately 3 pfu per cell and were labeled for 2 h with [ 35 s]methionine at 6 h postinfection. the cells were then washed with complete medium and chased with a 10-fold excess of cold methionine until they were harvested at appropriate times. immunoprecipitation of cell lysates with anti-100 showed the detection of both the 100-and 160-kda proteins throughout the time course (fig. 3a) . the 100-kda protein appeared at the beginning of the time course, peaked after chase for 1.5 h, and remained stable at the end of the time course (fig. 3a, lane 2) . the 160-kda protein peaked at the beginning of the time course and remained detectable after chase for 7.5 h (fig. 3a, lanes 1-6) . slight and gradual reduction of the 160-kda protein over time was observed (fig. 3a) . these results demonstrate that the 160-kda protein is a relatively stable intermediate cleavage product coexisting with the 100-kda mature cleavage product during the ibv infection cycle. similarly, immunoprecipitation of cell lysates with anti58 showed the detection of both the 132-and 58-kda proteins (fig. 3b) . the 132-kda protein appeared at the beginning of the time course, peaked after chased for 3 h, and remained detectable at the end of the time course (fig. 3b, lanes 9-12) . the 58-kda protein was first seen after chased for 3 h and gradually increased over time (fig. 3b, lanes 9-12) . interestingly, a product with an apparent molecular mass of 97 kda, representing an intermediate cleavage product containing the 58-and 39-kda proteins , was weakly detected and briefly observed during the time course (fig. 3b , lanes 9 and 10), indicating that it is not a stable intermediate cleavage product. the coexistence of the 132-and 58-kda proteins over time suggests that the 132-kda protein is a stable intermediate cleavage product. further characterization and definition of the coding sequences of products processed from the cterminal 200-kda region of the 1a/1b polyprotein two dicistronic constructs, p3c-cite-ibv20 and p3c-cite-ibv8, were made and expressed to characterize the expression and processing patterns of the c-terminal 200-kda region of the 1a/1b polyprotein. in these two constructs, the region coding for the 3c-like proteinase was placed between the t7 promoter and the internal ribosome entry site (ires) of encephomyolitis virus (emcv), and the ibv sequences from nucleotides 15132-20506 and 15132-18495, respectively, were cloned downstream of ires (fig. 1) . expression of both constructs in cos-7 cells resulted in the detection of the 33-kda 3c-like proteinase, which comigrates on sds-page with the 33-kda protein detected in ibv-infected cells (fig. 4, lanes 2-4) . immunoprecipitation of cell lysates prepared from p3c-cite-ibv20-transfected cells with anti-58 led to the detection of three protein species with apparent molecular masses of 200, 132, and 58 kda, respectively (fig. 4, lane 7) . the 200-kda protein represents the full-length product encoded by the ibv 1b sequence present in this plasmid, and the 132-and 58-kda proteins comigrated with the 132-and 58-kda proteins detected in ibv-infected cells (fig. 4, lanes 6 and 7) . immunoprecipitation of cell lysates prepared from p3c-cite-ibv8-transfected cells with anti-58 led to the detection of the 58-kda protein and a product with an apparent molecular mass of 125 kda (fig. 4, lane 8) . the 125-kda protein may represent the full-length product encoded by the 1b sequence present in this construct. immunoprecipitation of cell lysates prepared from p3c-cite-ibv20-transfected cells with antiserum v17 led to the detection of the 200-, 132-, 39-, and 35-kda proteins (fig. 4, lane 11) . the 132-, 39-, and 35-kda proteins comigrated with the three equivalent fig. 4 . comparative analysis of the 132-and 58-kda proteins expressed and processed in cells transfected with p3c-cite-ibv20, p3c-cite-ibv8, and pibv1b4 and in ibv-infected cells. plasmid dnas were expressed in cos-7 cells using the vaccinia virus-t7 expression system (fuerst et al., 1986) . semiconfluent monolayers of cos-7 cells were infected with a recombinant vaccinia virus (vtf7-3), transfected with plasmid dna using dotap according to the instructions of the manufacturer (roche), and labeled with [ 35 s]methionine and cysteine, and lysates were prepared. cell lysates prepared from mock-infected (lanes 1, 5, and 9) and ibv-infected (lanes 2, 6, and 10) vero cells and transfected cells (lanes 3, 4, 7, 8, 11-13) were immunoprecipitated with anti-3c (lanes 1-4) , anti-58 (lanes 5-8, 12, and 13), and v17 (lanes 9-11). the radiolabeled polypeptides were separated on an sds-12.5% polyacrylamide gel and detected by fluorography. numbers indicate molecular mass in kilodaltons. products detected in ibv-infected cells (fig. 4, lanes 9-11) . as mentioned earlier, the apparent molecular mass and processing pattern of the 132-kda protein suggested that it may be derived from the c-terminal region of the 1a/1b polyprotein covering the 58-, 39-, and 35-kda proteins. to further confirm this possibility, plasmid pibv1b4, which covers nucleotides 16932-20490 and therefore encodes the 132-kda product , was expressed in cos-7 cells. as expected, immunoprecipitation of cell lysates prepared from cells transfected with pibv1b4 with anti-58 resulted in the detection of the full-length 132-kda protein, which comigrated on sds-page with the 132-kda intermediate cleavage detected from cells transfected with p3c-cite-ibv20 (fig. 4, lanes 12 and 13) . to gain clues of the functions of the five cleavage products in the viral replication cycle, indirect immunofluorescence analysis of cells expressing individual cleavage products was carried out and representative confocal microscopy images are present in the proteins were labeled with the fitc-conjugated secondary antibodies. panels b, e, h, k, and n refer to cells stained with r6, a dye for the er. the green images represent fitc-derived green fluorescence, and red images represent rhodamine and texas red-derived red fluorescence. colocalization of viral proteins with the organelle markers is represented by the yellow region within each cell in the merged images (c, f, i, l, and o). panels a-o show cos-7 cells transfected with the empty vector pkt0 and stained with antisera or r6 as indicated. the fluorescence was viewed using a confocal scanning zeiss microscope. nofluorescent pattern of anti-pdi (data not shown). these results suggest that the three proteins may be associated with the er membranes. in cells expressing the 39and 35-kda proteins, a diffuse staining pattern was observed for each protein (figs. 5j and 5m), which does not coalign with the staining pattern of r6 (figs. 5j-5o). this diffuse distribution pattern of the 39-and 35-kda proteins was unexpected, as the majority of cleavage products were shown to be membrane-associated. the same antisera were used to stain cells transfected with the empty vector pkt0 (liu et al., 1994) , showing weak background staining (figs. 5a-5o) . the subcellular localization patterns of the five proteins were then analyzed in ibv-infected cells (fig. 6) . immunofluorescent staining of ibv-infected vero cells with anti-100, anti-68, and anti-58, respectively, showed similar er localization profiles (figs. 6a-6i), and a diffuse staining pattern was observed in cells stained with antiserum v17, which reacts with both the 39-and 35-kda proteins (figs. 6j-6l) . similarly, a diffuse distribution pattern was observed in cells stained with anti-35 (figs. 6m-6o). the same antisera were used to stain mockinfected cells, showing weak background staining (figs. 6a-6o). in our previous reports, we showed the identification of three mature cleavage products of 100, 39, and 35 kda, processed from the 1b region of the 1a/1b polyprotein (liu et al., 1994 . however, we were unable to detect the two other cleavage products of 68 and 58 kda. in this study, we report the identification of the 68-and 58-kda proteins in ibv-infected cells. in addition, two stable intermediate cleavage products of 160 and 132 kda, respectively, were identified, which coexist with the mature cleavage products in virus-infected cells. immunofluorescence analysis showed that the polymerase domain-containing 100-kda protein and the helicase domain-containing 68-kda protein as well as the 58-kda protein may be associated with the er and ic membranes, the viral replication and assembly sites. however, the 39-and 35-kda proteins display diffuse distribution patterns in both ibv-infected cells and cells expressing each of the proteins. it is intriguing that immunoprecipitation and western blot of ibv-infected cells harvested at 8 h postinfection failed to detect the 68-kda protein. the protein was detected by western blot in ibv-infected cells harvested at 24 and 32 h postinfection. as the equivalent human coronavirus 71-kda protein was shown to be an rna helicase (heusipp et al., 1997a; seybert et al., 2000a,b) , this protein is likely the ibv helicase. it is expected that such a functional product directly involved in viral rna replication would be expressed at early stages of the viral replication cycle. in fact, the equivalent 71-kda protein of human coronavirus was first seen in virus-infected cells at 5 h postinfection (heusipp et al., 1997a) . the reason for the failure to detect the 68-kda protein in ibv-infected cells at earlier time point is uncertain, but it might reflect the folding property, as discussed later, of the protein. the dramatic increase in the detection of the 68-kda protein at 24 and 32 h postinfection may partially be due to the secondary infection of cells that remain uninfected during the primary infection. as significantly more cells (over 95% of cells) were infected at 24-32 h postinfection, it is understandable that more protein would be detected. a slight, but gradual increase of the accumulation of the human coronavirus 71-kda protein over a time course of 15 h was also observed (heusipp et al., 1997a) . alternatively, the increase in the detection of the 68-kda protein at 24-32 h postinfection may reflect the genuine accumulation pattern of the protein in virusinfected cells at late stages of the viral replication cycle. if this were the case, it may indicate that the protein might also be involved in processes other than viral rna replication. the identification of two stable intermediate cleavage products of 160 and 132 kda coexisting with the mature cleavage products raised two interesting questions. first, the two products may have certain functions during the viral replication cycle. the 160-kda product is particularly interesting in this aspect, considering the fact that it contains both the polymerase and helicase domains and the 68-kda helicase protein is undetectable at early stages of infection. it is plausible that the 160-kda protein might have both polymerase and helicase functions in the replication of viral rna at early stages of the infection. in fact, some smaller positive-stranded rna viruses encode single proteins containing both the polymerase and helicase domains (buck, 1996) . the second interesting question is why only these two intermediate cleavage products were detectable in ibv-infected cells. as shown in fig. 4 as well as in our previous report , other intermediate cleavage products were also observed when this region was expressed in intact cells. one obvious distinct feature of the cleavage site between these two products is that it is a q-g dipeptide bond, while all the other cleavage sites in this region of the polyprotein are q-s dipeptide bonds (fig. 1) . however, no experimental data indicate that cleavage at the q-g site is more efficient than at the q-s sites. the 68-kda protein migrated on sds-page as a multiprotein species, heterogeneous smear, probably due to the formation of protein aggregates, and deletion analysis showed that a stretch of 30 amino acid residues in the c-terminal region was responsible for the aberrant migration property of the protein (data not shown). it suggests that the 68-kda protein may misfold when expressed in intact cells in the absence of other viral components. in recent years, it was found that the proper folding of certain proteins requires the assistance of molecular chaperones (ellis and van der vies, 1991; gething and sambrook, 1992) . as the 68-kda protein may be a component of the viral rna replication complex, the protein is expected to interact with viral rna and other viral proteins. those viral rna/proteins may act as a chaperone for the correct folding of the 68-kda protein in ibv-infected cells. it would be of interest to define if the region of the 30 amino acid residues that were shown to be responsible for the aberrant migration of the 68-kda protein on sds-page contain either rna binding or protein interacting domains. however, no such domains were found in this or the neighboring regions by computer analysis using relevant programs. the 58-kda protein was recently shown to be able to induce programmed cell death when expressed alone in intact cells . this is the first ibv product that was demonstrated to be a proapoptotic protein. in our previous report, we were unable to identify the 58-kda protein in ibv-infected cells due to the cross-reactivity of the antiserum used with a cellular protein . a newly raised antiserum was used in this study, leading to the successful identification of the protein in virus-infected cells. understanding of the expression, processing, and subcellular distribution pattern of the 58-kda protein would help us to further characterize the proapoptotic property of the protein and to study the functions of the protein in the pathogenesis of ibv-induced infection in chicken, the natural host of ibv. currently, no functions have been assigned to the 39and 35-kda proteins. a counterpart of the ibv 39-kda protein, the 41-kda protein of human coronavirus, was shown to exhibit a punctate, perinuclear distribution pat-tern in virus-infected cells (heusipp et al., 1997b) . interestingly, the 39-and 35-kda proteins display a diffuse distribution pattern in both ibv-infected cells and in cells overexpressing the proteins. as the majority of the cleavage products from the 1a and 1a/1b polyproteins were shown to be associated with cellular membranes at or near the viral replication and assembly sites, the diffuse distribution pattern may exclude the direct involvement of the two proteins in the formation of viral replication complexes. the egg-adapted beaudette strain of ibv (atcc vr-22) was obtained from the american type culture collection (atcc) and was adapted to vero cells as described by alonso-caplen et al. (1984) . briefly, the virus was passaged three times in 11-day-old chicken embryos and then adapted to vero cells (atcc ccl-81) by a series of passages at 24-48 h intervals. the cytopathic effects, including syncytium formation and rounding up of cells, were initially observed after three passages in vero cells. virus stocks were prepared after the 36th passage by infecting monolayers of vero cells at a m.o.i. of approximately 0.1 pfu/cell. the virus was harvested at 24 h postinfection and the titer of the virus preparation was determined by plaque assay on vero cells. vero cells were grown at 37°c in 5% co 2 and maintained in glasgow's modified minimal essential medium (gmem) supplemented with 10% newborn calf serum. confluent monolayers of vero cells were infected with ibv at a m.o.i. of approximately 3 pfu/cell. prior to being labeled, the cells were incubated in methionine-free medium for 30 min. after 4 h of labeling with 25 ci of [ 35 s]methionine, the cells were scraped off the dishes in phosphate-buffered saline (pbs), recovered by centrifugation, and stored at ϫ80°c. open reading frames placed under control of the t7 promoter were expressed transiently in eukaryotic cells as described previously (liu et al., 1994) . briefly, semiconfluent monolayers of vero cells were infected with 10 pfu/cell of a recombinant vaccinia virus (vtf7-3) which expresses the bacteriophage t7 rna polymerase and then transfected with appropriate plasmid dna using the dotap transfection reagent according to the instructions of the manufacturer (roche). after incubation of the cells at 37°c for 4 h, 25 ci/ml of [ 35 s]methionine was added directly to the medium. the radiolabeled cells were harvested at 18 h posttransfection. appropriate primers and template dnas were used in amplification reactions with pfu dna polymerase (stratagene) under standard buffer conditions with 2 mm mgcl 2 . the pcr conditions were 30 cycles of 95°c for 45 s, x°c for 45 s, and 72°c for x min. the annealing temperature (x°c) and the extension time (x min) were adjusted according to the melting temperature of the primers used and the length of the pcr fragments synthesized. plasmid dna-transfected vero cells were lysed with ripa buffer (50 mm tris-hcl, ph 7.5, 150 mm nacl, 1% sodium deoxycholate, 0.1% sds, 1% np-40) and precleared by centrifugation at 12,000 rpm for 5 min at 4°c in a microfuge. immunoprecipitation was carried out as described previously (liu et al., 1994) . sds-polyacrylamide gel electrophoresis (sds-page) of virus polypeptides was carried out using 12.5% polyacrylamide gels (laemmli, 1970) . labeled polypeptides were detected by autoradiography or fluorography of dried gels. cells were grown on four-well chamber slides (iwaki) and infected with ibv or transfected with appropriate plasmid dnas. after washing with pbs, the cells were fixed with 4% paraformaldehyde (in pbs) for 15 min at room temperature and permeabilized with 0.2% triton x-100 (in pbs), followed by incubation with specific antiserum at room temperature for 2 h. antibodies were diluted in fluorescence dilution buffer (pbs with 5% normal goat serum). the cells were then washed with pbs and incubated with anti-rabbit igg conjugated to fluorescein isothiocynate (fitc) (sigma) in the fluorescence dilution buffer at 4°c for 1 h before mounting. confocal microscopy was performed on a zeiss axioplan microscope connected to a bio-rad mrc 1024 laser scanner equipped with an argon laser with appropriate filters. fluorescent images were superimposed to allow fine comparison and colocalization of green (fitc) and red (tritc) signals in a single pixel produces yellow, while separated signals are green or red. plasmid pibv1b4, which contains nucleotides 16932-20490, was previously described . plasmid pibv1b3 contains nucleotides 15132-16931 and codes for the 68-kda protein, pibv1b6 contains nucleotides 16930-18495 and codes for the 58-kda protein, pibv1b9 contains nucleotides 18496-19508 and codes for the 39-kda protein, and pibv1b10 contains nucleotides 19509-20506 and codes for the 35-kda protein. these constructs were made by cloning an ncoi-and bamhi-digested pcr fragment into ncoi-and bamhidigested pkt0 (liu et al., 1994) . the sequences of the two primers used to construct pibv1b3 were 5ј-cgact-tccatggcttgtggcgttј-3 and 5ј-ccaaaggatccta-ttgcagacttg-3ј. the sequences of the two primers used to construct pibv1b6 were 5ј-acaagtcccatggg-tacaggtt-3ј and 5ј-tattggatcctacggagagctg-3ј. the sequences of the two primers used to construct pibv1b9 were 5ј-gtttttcagctcccatggctatcgac-aat-3ј and 5ј-aaccacacgtcggatcctattgaagctg-tg-3ј. the sequence of the two primers used to construct pibv1b10 was 5ј-ccacagcttcccatggcatg-gacgtg-3ј. plasmid pibvpol, which contains nucleotides 12451-15131 and codes for the 100-kda protein with a 37-aminoacid truncation at the n-terminus, was constructed by cloning a bamhi-and xhoi-digested pcr fragment into bamhi-and xhoi-digested pet22b(ϩ) (novagen). the sequences of the primers used were 5ј-gtaataag-gatccagctggtatg-3ј and 5ј-aaggcctcgagttgta-aagtcgtaggagc-3ј. the two dicistronic constructs, p3c-cite-ibv20 and p3c-cite-ibv8, were constructed as follows. the ires sequence was obtained by digestion of pcite-1 (novagen) with ecori, end-repair with klenow, and redigestion with ncoi. this 592-bp fragment was then cloned into pvuii-and ncoi-digested pkt0, giving rise to pkt-cite. the ibv sequence that codes for the 3c-like proteinase was obtained by digestion of pibv3c (liu et al., 1997) with bglii and bamhi and was cloned into bglii-digested pkt-cite, giving rise to p3c-cite. digestion of p3c-cite with ncoi produced a 1510-bp fragment containing both the ibv 3c-like proteinase and the ires sequences. this fragment was then cloned into ncoi-digested pibv1b8 and pibv20, respectively, creating the two dicistronic constructs. plasmids pibv1b8 and pibv20, which cover the ibv sequences from nucleotides 15132 to 18495 and 15132 to 20506, respectively, were constructed by cloning ncoi-and bamhi-digested pcr fragments covering the relevant regions into ncoi-and bamhi-digested pkt0. replication and morphogenesis of avian coronavirus in vero cells and their inhibition by monensin impaired integrinmediated adhesion and signaling in fibroblasts expressing a dominant-negative mutant ptp1b subcellular distribution of normal and mutant vitamin d receptors in living cells four proteins processed from the replicase gene polyprotein of mouse hepatitis virus colocalize in the cell periphery and adjacent to sites of virion assembly completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus comparison of the replication of positive-stranded rna viruses of plants and animals the putative helicase of the coronavirus mouse hepatitis virus is processed from the replicase gene polyprotein and localizes in complexes that are active in viral rna synthesis molecular chaperones eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage t7 rna polymerase protein folding in the cell identification of an atpase activity associated with a 71-kilodalton polypeptide encoded in gene 1 of the human coronavirus 229e identification and subcellular localization of a 41 kda, polyprotein 1ab processing product in human coronavirus 229e-infected cells cleavage of structural proteins during the assembly of the head of bacteriophage t4 characterization of the two overlapping papain-like proteinase domains encoded in gene 1 of the coronavirus infectious bronchitis virus and determination of the c-terminal cleavage site of an 87 kda protein identification of a novel cleavage activity of the first papain-like proteinase domain encoded by orf 1a of the coronavirus avian infectious bronchitis virus and characterization of the cleavage products the missing link in coronavirus assembly: retention of the avian coronavirus infectious bronchitis virus envelope protein in the pre-golgi compartments and physical interaction between the envelope and membrane proteins induction of caspase-dependent apoptosis in cultured cells by the avian coronavirus infectious bronchitis virus characterisation and mutational analysis of an orf 1a-encoding proteinase domain responsible for proteolytic processing of the infectious bronchitis virus 1a/1b polyprotein a 100-kilodalton polypeptide encoded by open reading frame (orf) 1b of the coronavirus infectious bronchitis virus is processed by orf 1a products identification, expression, and processing of an 87-kda polypeptide encoded by orf 1a of the coronavirus infectious bronchitis virus proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein: identification of a 10 kda polypeptide and determination of its cleavage sites proteolytic mapping of the coronavirus infectious bronchitis virus 1b polyprotein: evidence for the presence of four cleavage sites of the 3c-like proteinase and identification of two novel cleavage products identification of a 24 kda polypeptide processed from the coronavirus infectious bronchitis virus 1a polyprotein by the 3c-like proteinase and determination of its cleavage sites further characterization of the coronavirus infectious bronchitis virus 3c-like proteinase and determination of a new cleavage site processing of the coronavirus mhv-jhm polymerase polyprotein: identification of precursors and proteolytic products spanning 400 kilodaltons of orf1a the human coronavirus 229e superfamily 1 helicase has rna and dna duplexunwinding activities with 5ј-3ј polarity biochemical characterization of the equine arteritis virus helicase suggests a close functional relationship between arterivirus and coronavirus helicases colocalization and membrane association of murine hepatitis virus gene 1 products and de novo-synthesized viral rna in infected cells mouse hepatitis virus replicase proteins associate with two distinct populations of intracellular membranes characterization of endoplasmic reticulum by colocalization of b-p and dicarbocyanine dyes localization of mouse hepatitis virus nonstructural proteins and rna synthesis indicates a role for late endosomes in viral replication integral membrane proteins of the nuclear envelope are dispersed throughout the endoplasmic reticulum during mitosis processing of the human coronavirus 229e replicase polyproteins by the virus-encoded 3c-like proteinase: identification of proteolytic products and cleavage sites common to pp1a and pp1ab virus encoded proteinases and proteolytic processing in the nidovirales acknowledgment this work was supported by the national science and technology board of singapore. key: cord-325179-gsf8ad65 authors: jarvis, donald l.; butel, janet s. title: modification of simian virus 40 large tumor antigen by glycosylation date: 1985-03-31 journal: virology doi: 10.1016/0042-6822(85)90250-8 sha: doc_id: 325179 cord_uid: gsf8ad65 abstract the sv40-encoded transforming protein, large tumor antigen (t-ag), is multifunctional. chemical modifications of the t-ag polypeptide may be important for its multifunctional capacity. t-ag is additionally modified by glycosylation. t-ag was metabolically labeled in sv40-infected cells with tritiated galactose or glucosamine, but not with mannose or fucose. the identity of glycosylated t-ag was established by immunoprecipitation with a variety of t-ag-specific antisera, including monoclonal antibodies. incorporation of labeled sugar into t-ag was inhibited in the presence of excess unlabeled sugars, but not in the presence of excess unlabeled amino acids. labeled monosaccharides could be preferentially removed from t-ag with a mixture of glycosidic enzymes. in addition, galactose was removed from purified t-ag by acid hydrolysis and identified as such by thin-layer chromatography. t-ag oligosaccharides were resistant to treatment with endoh, and glycosylation was not inhibited by tunicamycin. together, these data strongly suggest that t-ag is glycosylated. several characteristics, including lack of mannose labeling, endoh resistance, and tunicamycin resistance, suggest that t-ag is not an n-linked glycoprotein. rather, these properties are more consistent with the identification of t-ag as an o-linked glycoprotein. simian virus 40 (sv40) encodes the synthesis of two early proteins, designated the small tumor antigen (t-ag) and the large tumor antigen (t-ag), that exhibit apparent molecular weights (mw) of about 20,900 and 90,000, respectively. both are chemically modified polypeptides, and their entire amino acid sequences are known (fiers et al, 1978; reddy et &, 1978) . it is thought that t-ag mediates most of the events culminating in cellular transformation by sv40. accordingly, a vast array of biochemical and biological functions has been attributed to this polypeptide (reviewed by martin, 1981; tooze, 1981; rigby and lane, 1983) . however, the molecular basis for its multifunctional capacity remains obscure. one possible explanation invokes the known chemical modifications of the t-ag polypeptide. theoretically, those modifi' author to whom reprint requests should be addressed. cations could generate distinct forms of t-ag that might perform different functions in the host cell. the modifications reported for t-ag include phosphorylation (tegtmeyer et al, 1977) , n-terminal acetylation (mellor and smith, 19'78) , poly-adp-ribosylation (goldman et cd, 1981) , fatty acid acylation (klockmann and deppert, 1983) , and glycosylation (schmidt-ullrich et cd, 1977 . however, the evidence suggesting that t-ag possesses the latter modification (i.e., glycosylation) is both limited and indirect. therefore, we have addressed this question by performing detailed biochemical studies. the results of these analyses establish that tag is glycosylated and that its oligosaccharide moiety (or moieties) contains galactose and glucosamine and/or their derivatives. the oligosaccharides probably do not contain mannose or fucose and are resistant to treatment with endo+n-acetylglucosaminidase h (endoh). in addition, glycosylation of t-ag is not inhibited by tunicamycin (tm), suggesting that it is not an n-linked, but rather, is probably an o-linked glycoprotein. glycosylation of viral transformation proteins has been recognized in only a few retrovirus systems and has not previously been conclusively demonstrated with any dna tumor virus. all of the known retroviral transformation glycoproteins are n-glycosylated (dresler et al, 1979; hayman et al, 1983; privalsky et cd, 1983) ; none has been shown to be oglycosylated. only a single viral glycoprotein, the el matrix glycoprotein of certain coronaviruses, is believed to be exclusively 0-glycosylated (holmes et d, 1981; niemann and klenk, 1981) . thus, the demonstration of 0-glycosylation of sv40 tag provides a novel basis for further investigation of t-ag multifunctionality, particularly with respect to the significance of this modification. in addition, tag may be a useful model for the study of o-linked glycoproteins in general, which remain relatively poorly characterized. materials and methods cez.zs and wirus. tc-7 cells (robb and huebner, 1973) were propagated in enriched eagle's minimum essential medium (e-mem; gibco, grand island, n. y.; noonan et al, 1976) . mmfjmt/cl cells (owens and hackett, 1972) , mammary tumor cells that produce mouse mammary tumor virus (mmtv), were propagated in dulbecco's modified minimum essential medium (d-mem, gibco; slagle et d, 1984) . wild-type sv40 was passaged at a low multiplicity of infection (m.o.i.) and plaque assayed in tc-7 cells (noonan and butel, 1978) . vesicular stomatitis virus (vsv, indiana strain) was passaged and titrated as described for sv40, except that it was not subjected to freeze-thawing. c3h mouse mammary tumor virus [(c3h)-mmtv] concentrates were provided by the biological carcinogenesis branch, division of cancer cause and prevention, national cancer institute. antisera normal hamster serum (nhs), hamster ascites fluid containing antibodies against t-ag (haf), normal rabbit serum (nrs), and rabbit antiserum against purified t-ag (racut) or disrupted (c3h)-mmtv (cymmtv) have been previously described (lanford and butel, 1979; slagle et al, 1984) . rabbit antiserum against purified vsv (rbavsv) was generously provided by dr. trudy morrison. t-ag or cellular protein p53-specific monoclonal antibodies used in this study included pab 100, 101, and 122 (gurney et cd, 1980) , pab 204 hoeffler, 1980), and pab 402, 405, 414, 416, 419, 421, 423, and 430 (harlow et al, 1981) . hybridomas were cultured and antibodies were precipitated from culture supernatants as previously described (harlow et cd, 1981; santos and butel, 1984) . iqfeiticm and metabohc labeling. tc-7 monolayers were mock infected with e-mem or infected with sv40 or vsv at an m.0.i. of 50 plaque-forming units (pfu)/cell (jarvis et o& 1984) . for vsv infections, 1.5 pg/ml actinomycin d (sigma chemical co., st. louis, mo.) was included with the inoculum and maintained throughout the infection. at appropriate times postinfection (p.i.), monolayers were washed with tris-buffered saline (tbs) and starved in glucose-free e-mem for 30 min (martineau et cd, 1972) . cells were then labeled with ["s]methionine, d-[l-3h]galactose, ~-[l-~hlglucosamine, d-[2-3h]mannose, or l-[~-~h]fucose (amersham, arlington heights, ill.). labeling times and radioisotope concentrations are specified in the figure legends. mm5mt/cl cells were labeled as previously described (slagle et cd, 1984) . special labeling corditions. in some experiments, sv40-infected cells were labeled with ps]methionine or 3h-monosaccharides in the presence of excess unlabeled amino acids, excess unlabeled sugars, or tm. normal (1x) amino acid concentrations in e-mem were approximately: trp, 0.1 mm; met, 0.3 mm; his, 6.4 mm; gly and phe, 0.5 mm; ser and arg, 0.6 mm; leu, ile, thr, and val, 1.0 mm; lys, 1.4 d; and gln, 3.0 mm. e-mem and glucose-free e-mem containing 5 times (5x), 10 times (10x), or 50 times (50x) the normal concentration of amino acids were prepared. for labeling in the presence of excess amino acids, infected cells were treated with e-mem containing different amino acid concentrations be-ginning at 2 hr p.i. cells were starved from 20.5 to 21 hr p.i. using glucose-free e-mem supplemented with the same amino acid concentrations and were labeled for 3 hr using 25 &i/ml [%s]methionine or 100 rci/ml phlgalactose in the same medium. e-mem normally contained 1 mg/ml glucose. for labeling in the presence of excess unlabeled sugars, e-mem also contained 1 mg/ml galactose for 1x sugar-enriched medium or higher concentrations of both sugars for 5x, 10x, and 50x sugar-enriched medium. infected cells were starved in glucose-free e-mem from 20.5 to 21 hr p.i., then labeled for 3 hr using 25 &i/ml [?s]methionine or 100 &i/ml rh]galactose in e-mem containing different sugar concentrations. finally, for tm experiments, e-mem and glucose-free e-mem containing various concentrations of tm were prepared. treatment was initiated at 15 hr p.i. and maintained throughout the 30-min starvation and 3-hr labeling periods (20.5-24 h p.i.). extmctk, immuvwprecipitation, and gel electrophoresis. labeled monolayers were washed, detergent-extracted in the presence of leupeptin (jarvis et al, 1984) , and extracts were clarified. after incubation with an appropriate antiserum, formalinfixed staphybcoccus aureus strain cowan i (saci) (kessler, 19'75) was added to adsorb immune complexes. if monoclonal antibody was used, a goat anti-mouse igg bridge was included. immunoprecipitates were then washed three times with wash buffer (wb; 50 mm tris, ph 8.0, 100 mm nacl, 1% np-40,1% sodium deoxycholate, and 0.1% sds), disrupted, and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page; soule and butel, 1979) . gels were impregnated with autofluor (national diagnostics, somerville, n. j.), dried, and exposed to x-ray film at -70". treatment of t-ag or mmtv poll/pep tides with gl~cosiduses. sv40-infected tc-7 cells or mmsmt/cl cells were glucose starved, doubly labeled with [%]methionine and phlglucosamine, and extracts were immunoprecipitated with haf or crmmtv and saci. immune complexes were washed three times with wb, then twice with tbs. pellets were resuspended in tbs supplemented with 200 fl leupeptin (sigma) and 1000 kiu trasylol (mobay chemical co., new york, n. y.), mixed, and triplicate samples were removed for determination of pretreatment acid-precipitable 3h and ?s radioactivity. tbs, endoh (miles laboratories, inc., elkhart, ind.), or char&a lampas mixed glycosidases (miles) were added, and mixtures were incubated with shaking at 37" for 15 hr. triplicate samples were then removed for determination of post-treatment acid-precipitable 3h and % radioactivity, and the remainder was processed for sds-page analysis. pre-and posttreatment samples were disrupted, clarified, and spotted onto glass microfiber filters (whatman, clifton, n. j.). filters were dried, then washed once with 10% trichloroacetic acid (tca), twice with 5% tca, and once with 95% ethanol. filters were redried, then assayed for 3h and ?s radioactivity by liquid scintillation spectroscopy. acid hydrolysis of t-ag and analysis of monosaccharides. glucose-starved, sv40infected tc-7 cells were doubly labeled with p5s]methionine and phlgalactose, and t-ag was isolated by immunoprecipitation and sds-page. t-ag was excised, eluted from the gel, concentrated, and desalted on a sephadex g25m column (pharmacia fine chemicals, uppsala, sweden). the sample was then reconcentrated and sds was extracted (henderson et ul, 1979) . carrier galactose was added, and the mixture was suspended in 2.0 n hzs04 and sealed under nitrogen in a glass ampule. after hydrolysis for 4 hr at loo", the reaction mixture was diluted to 0.1 n h2s04 and applied to a dowex 5ow-x8 cation-exchange column. the flowthrough was neutralized, clarified, and lyophilized. this material was then applied to a silica gel g thin-layer plate (fisher scientific co., pittsburg, pa.) and chromatographed with n-propanol:water (71, v/v) in a closed chamber (gal, 1968) . the plate was dried and stained with 30% aqueous ammonium bisulfate for visualization of the carrier and standard mono-saccharides. after measurement of rfvalues, the lanes in which hydrolysates had been chromatographed were fractionated, and fractions were assayed for radioactivity by liquid scintillation spectroscopy. cells with radioactive mcmomcchurides in initial experiments designed to determine if t-ag is glycosylated, we utilized metabolic labeling of sv40-infected cells with [%]methionine or tritiated monosaccharides. a polypeptide of about 88,000 (88k) in apparent mw was detected by immunoprecipitation of ps]methionine-labeled, sv40-infected cell extracts with haf (fig. la, lane 3) . this polypeptide was identified as t-ag because it was not detected in mock-infected cell extracts immunoprecipitated with nhs or haf or in infected cell extracts immunoprecipitated with nhs (lanes 1, 2, and 4). a corn&rating polypeptide, presumably t-ag, was also detected in phlgalactose-(lane 8) and mlucosamine-labeled (lane 16), infected cell extracts immunoprecipitated with haf; it was absent in the corresponding mock-infected or nhs-treated controls (lanes 5-7 and 13-15). labeled t-ag was not recovered from cells pulsed with rh]mannose (lanes 9-12) or rh]fucose (lanes 17-20), although unlabeled t-ag was immunoprecipitated from infected cells with haf as evidenced by coomassie blue staining. incorporation of rh]galactose into t-ag was enhanced when infected cells were pretreated with glucosefree e-mem and the isotope was added in this same medium. this can be seen by comparing lanes 1 and 2 in confluent tc-7 cell monolayers were washed with warm tbs, then mock infected with e-mem (a: lanes l-2, 5-6, 9-10, 13-14, and 17-18) or infected with 50 pfwcell sv40 (a: lanes 3-4, '7-8, 11-12, 15-16, and 19-20; b: lanes 1 and 2). after adsorption for 2 hr at 37", the cells were fed with e-mem and returned to 37". except for the sample in b, lane 1, cells were starved prior to labeling for 30 min in glucose-free e-mem. cells were then labeled from 21 to 24 hr p.i. with 100 &i/ml 4,5, 7, 9.11,13, 15, 17, and 19) or haf (a: lanes 2, 3, 6, 8.10, 12, 14, 16, 18, and 20; b: lanes 1 and, 2). immune complexes were adsorbed using saci, washed thoroughly, then disrupted and analyzed by sds-page on 8% acrylamide gels. numbers to the left of the figure indicate the positions of molecular weight standards, designated by their molecular weights x lo-*. galactose and glucosamine were incorporated into t-ag, suggesting that its putative oligosaccharide moiety(ies) contains those sugars and/or their derivatives. it was possible that the relatively short (3 hr) labeling time used in the initial experiments had not permitted equilibration of intracellular sugar pools with each input labeled monosaccharide, producing false negative results. therefore, sv40infected cells were labeled for 3, 6, or 18 hr using tritiated galactose, glucosamine, or mannose, and samples were processed as before. radioactive t-ag was detected in all of the galactose-labeled samples and in the 18-hr glucosamine-labeled sample, but was not detected in any of the mannose-labeled samples (fig. 2) . these observations support the interpretation that putative t-ag oligosaccharide(s) contain galactose and glucosamine and/or their derivatives but lack mannose. it was not firmly established that the oligosaccharide(s) lack fucose, since that sugar was labeling of sv40 t-ag with various monosaccharides. confluent tc-7 monolayers were infected with sv40 as described in the legend to fig. 1 . cells were labeled without prior glucose starvation from 6 to 24 hr p.i. (18 hr; lanes l-3), 18-24 hr p.i. (6 hr; lanes 4-6), or 21-24 hr p.i. (3 hr; lanes 7-9) with 20 &i/ml djl-%jgalactose (lanes 1, 4, 7), d-cl-%jglucosamine (lanes 2, 5, 8), or d-f% 'hlmannose (lanes 3, 6, 9). samples were then processed and analyzed as described in the legend to fig. 1 , using haf for immunoprecipitation and 8% acrylamide gels for analysis. molecular weight markers are shown on the left. not included in this experiment. however, except for the 18-hr galactose-labeled sample, t-ag was not as intensely labeled in this experiment as in the experiment shown in fig. 1a . thus, shorter pulses with higher concentrations of radioactive sugars generally provided better incorporation than longer pulses with lower concentrations, suggesting that adequate uptake of the input sugars had occurred under those labeling conditions. preliminary experiments using [35s]methionine labeling had established the maximal time of t-ag synthesis under our conditions of infection to be about 20-24 hr pi. (data not shown). this time period was chosen for the experiments described above since a large amount of t-ag would be available for labeling and the detection of monosaccharide-labeled t-ag would be optimized. however, if glycosylated t-ag represents a discrete subpopulation of tag, then its maximal time of synthesis might differ from that of overall t-ag synthesis. therefore, we examined the absolute and relative incorporation of [?s]methionine and [3hlgalactose into tag at various times p.i. the absolute incorporation of both isotopes was greatest at 21-24 hr p.i., suggesting that maximal glycosylation occurred at the same time as maximal overall t-ag synthesis (table 1) . however, the relative incorporation of [3hlgalactose compared to [35s]methionine was highest very early in infection, as shown by 3h:35s ratios (table 1 ). the ratio decreased steadily until about 12 hr p.i., then remained approximately constant until 48 hr p.i. this result might indicate that relatively more t-ag molecules or more sites on t-ag are glycosylated early in infection. although a large degree of variability in the 3h:35s ratios was observed among different experiments, the ratio repeatedly decreased with time after infection. positive ident~catiim of the 88k glvcoprotein as t-ag in the experiments described above, an 88k glycoprotein was identified as t-ag because it was immunoprecipitated from t-ag was immunoprecipitated from clarified cell extracts, gel purified, and eluted from the gel. concentrated eluents were then quantitated for "s and 'h content by double-label liquid scintillation spectroscopy. braw counts per minute (cpm) were determined using two channels on a beckman model ls-250 spectrometer, preadjusted to detect "s or %i. raw cpm were corrected for relative efficiency of counting on those channels as compared to an open channel and, in the case of %i cpm, for a small amount of contamination with "s cpm. correction factors were determined by counting singly labeled samples under conditions identical to those used for the experimental samples. infected cell lysates with haf and was absent in the corresponding mock-infected and nhs controls. however, since haf is a polyclonal antiserum, exhibiting multiple immunologic reactivities, it was necessary to more conclusively identify this 88k polypeptide. several different t-ag-specific and control antisera were used for immunopre-cipitation of galactose-labeled, infected cell extracts (fig. 3) . haf, raat, and all 10 of 10 different monoclonal antibodies against t-ag immunoprecipitated the galactose-labeled 88k polypeptide. this polypeptide was not immunoprecipitated with nhs, nrs, or a control monoclonal antibody directed against human igg. these results identify the 88k glycoprotein as t-ag, since it is extremely unlikely that another protein would possess that many cross-reactive antigenic determinants. mmosaccharicle label an important consideration in the interpretation of metabolic labeling experiments, particularly those involving monosaccharides, is the possibility that randomization of label may occur. the input monosaccharide might become metabolically converted to another form in the cell, such as amino acids, which could then be incorporated into the protein of interest. the following experiments were performed to evaluate this possibility. monoclonal antibody pab 419 recognizes an antigenic determinant on the amino end of t-ag (harlow et al, 1981) . since large t-ag and small t-ag share common amino terminal sequences, that antibody would coimmunoprecipitate both polypeptides from [35s]methionine-or [3hjgalactose-labeled infected cell extracts. both should be labeled with galactose if randomization were occurring. in contrast, small t-ag would not be labeled if randomization of label did not occur and the glycosylation site(s) on large t-ag were located on a portion of the polypeptide distal to those sequences shared with small t-ag. as expected, pab 419 immunoprecipitated both polypeptides from [35s]methionine-labeled infected cell extracts (fig. 4) . coomassie blue staining revealed that both large t-ag -and small t-ag were also immunoprecipitated from galactose-labeled infected cell extracts (data not shown). however, only large tag was detectably labeled with phigalactose. this observation, although infig. 3 . immunoprecipitation of galactose-labeled sv40 tag with various antisera. tc-7 cells were mock infected (-) or sv40 infected (+), glucose starved, and labeled with d-[l-?@&wb3e as described in the legend to fig. 1 . cells were extracted, then clarified extracts were immunoprecipitated with different antisera. immune complexes were adsorbed, washed, disrupted, and analyzed on 8% acrylamide gels. the headings at the top of the figure indicate the antisera used, numbers refer to pab designations for t-ag and p53-specific monoclonal antibodies, cm refers to a control monoclonal antibody directed against human igg. other antisera abbreviations are described under materials and methods. molecular weight markers are shown on the left. direct, suggests that randomization of the input sugar label had not occurred to a significant degree. in addition, it is likely that the glycosylation sites on large t-ag are not located within the region of the polypeptide encoded from nucleotides 5163 to 4917, since that is the region in common with small t-ag. a more direct approach to the potential problem of randomization involved metabolic labeling of t-ag with [?s]methionine or ['hlgalactose in the presence of excess unlabeled amino acids or sugars. the incorporation of ps]methionine should be inhibited by the presence of unlabeled amino acids but unaffected by the presence of unlabeled sugars. conversely, if malactose were incorporated into t-ag in the form of sugar, that incorporation should be unaffected by unlabeled amino acids but inhibited by unlabeled sugars. the results with ["s]methionine labeling were as predicted; 63 and 82% inhibitions were observed when labeling was performed in the presence of 5x and 10x amino acids, respectively (fig. 5a, table 2 ). no inhibition was observed with up to 10x sugars; instead, [35s]methionine incorporation was slightly enhanced with increasing sugar concentration (fig. 5b, table 2 ). at 50x sugars, methionine incorporation was strongly inhibited, for unknown reasons. perhaps uptake of metabolites from the growth medium was inhibited at this sugar concentration. coomassie blue staining revealed little difference in the amount of t-ag synthesized in cells treated with various sugar concentrations (data not shown). for fhkalactose incorporation, excess unlabeled amino acids had little effect, except for a slight inhibition at the 10x level (fig. 5a, table 2 ). coomassie blue staining revealed a slight reduction in the amount of t-ag immunoprecipitated from cells treated with 10x amino acids (data not shown). in addition, cells treated with higher (50x) levels of amino acids were killed. thus, it is likely that the slight inhibition of phjgalactose incorporation observed in the presence of 10x amino acids was due to minor toxicity and reflected a decrease in overall t-ag synthesis. incorporation of phlgalactose into t-ag was significantly inhibited in the presence of unlabeled sugars (fig. 5b) ; 90, 95, 96, and 86% inhibitions were observed for 1x, 5x, 10x, and 50x sugars, respectively (table 2). these results strongly suggest that t-ag was specifically labeled with phjgalactose due to glycosylation events, rather than simply due to randomization of the input sugar label. another possibility not addressed in . after extraction and clarification, pab 419 was used for immunoprecipitation. immune complexes were harvested, washed, disrupted and analyzed on 12% acrylamide gels. t and t on the right-hand side mark the positions of large and small tumor antigens, respectively. molecular weight markers are shown on the left. the randomization control experiments described above is that [3h14galactose could have been metabolically converted to labeled nucleotides. in fact, galactose can be converted to ribose through the phosphogluconate pathway and could then be incorporated into t-ag as poly-adp-ribose or rna oligonucleotides. these products are found in covalent or tight association, respectively, with t-ag (goldman et a& 1981; khandjian et ol, 1982) . however, this possibility was precluded by using galactose tritiated in the number 1 position as the input sugar. that carbon and its hydrogens are lost in the early steps of the phosphogluconate pathway before ribose is produced. thus, nucleotides con-taining ribose generated from d#-~h]galactose would not be radioactive, and labeled t-ag would not be detected as a result of such a conversion. another approach undertaken to substantiate that t-ag is modified by glycosylation involved treatment with a mixture of glycosidic enzymes. t-ag was immunoprecipitated from doubly labeled, 1 and 4) , 5x (lanes 2 and 5), or 10x (lanes 3 and 6) amino acids, as described under materials and methods. cells were labeled with 25 &i/ml ps]methionine (lanes l-3) or 100 &i/ml pi-dgalactose (lanes 4-6), extracted, and tag was immunoprecipitated from clarified extracts with haf. disrupted immunoprecipitates were analyzed on 8% acrylamide gels. (b) glucose-starved, sv40-infected tc-'7 cells were labeled in the presence of ox (lanes 1 and 6), 1x (lanes 2 and 7), 5x (lanes 3 and 8), 10x (lanes 4 and 9), or 50x (lanes 5 and 10) glucose-and galactose-containing e-mem. cells were labeled with 25 &i/ml ps]methionine (lanes l-5) or 100 &i/ml [shlgalactose (lanes 6-lo), then processed and analyzed as described for a. molecular weight markers are shown on the left. "t-ag bands were excised from each of the gel lanes shown in fig. 5 . gel slices were solubilized by the method of mahin and lofberg (1966) infected cell extracts, and the immune complexes were adsorbed to saci, washed, and treated with tbs or various amounts of mixed glycosidases, as described under materials and methods. aliquots of the reaction mixture were removed before and after treatment and assayed for acidprecipitable 3h and y!! radioactivity ( table 3 ). a small decrease in acid-precipitable 3h and 35s radioactivity was observed in the control samples after incubation with tbs for 15 hr at 37". this was probably due to nonspecific degradation or some autoproteolytic activity on the part of tag, as proposed by seif (1982) . incubation in the presence of increasing amounts of mixed glycosidases resulted in a small loss of acid-precipitable as radioactivity, probably reflecting contamination of the enzyme preparation with proteases. however, we observed a much more marked reduction in the recovery of acid-precipitable 3h radioactivity from the same samples; the decrease was greater than 60% relative to the tbs control at the lowest concentration of enzyme. this result suggests that the input 3h label was incorporated into t-ag as a monosaccharide, since it was preferentially removed from t-ag with glycosidic enzymes, and supports the interpretation that t-ag is glycosylated. rewwval and recovery of gahctose from t-ag the most direct evidence for glycosylation would require removal of sugar from "sv40-infected tc-7 cells were glucose starved, then labeled from 21 to 24 hr p.i. with 100 &i/ml each of cj6sjmethionine and n-[l-%ilglucosamine. t-ag was immunoprecipitated with haf and saci, and immunoprecipitates were washed with wb and tbs. saci-bound immune complexes were then resuspended in tbs and treated with 0, 0.5, 1.0, 1.5, or 2.0 mg of mixed giycosidaaes for 15 hr at 37o. aliquots of each sample were removed before and after treatment, tca precipitated, and analyzed for ?s and eh radioactivity. bass and 'h cpm were determined as described in the legend to table 1. purified t-ag, followed by recovery and identification of that sugar. again, t-a@; was doubly labeled, then purified as described under materials and methods and hydrolyzed in 2.0 n hzs04 for 4 hr at 100" in the presence of carrier galactose. this treatment was expected to preferentially release neutral monosaccharides from t-ag with minimal hydrolysis of the polypeptide chain. the hydrolysate was then subjected to cation-exchange chromatography, and the flow-through was neutralized and lyophilized. analysis of the sample before and after cation exchange revealed that most of the ?3labeled material had been removed from the hydrolysate (table 4 ). this was expected, since positively charged %-labeled protein, peptides, and amino acids would bind to the column matrix in the presence of acid. the flow-through was comprised primarily of %-labeled material, assumed to be galactose since it was the input 3h label and would not be expected to bind to the column matrix. this assumption was verified by thinlayer chromatography of the flow-through material. standard galactose reproducibly migrated to a position corresponding to fraction 28 in this thin-layer system (fig. 6) . the carrier galactose present in the hydrolysate also migrated to fraction 28, as revealed by ammonium bisulfate staining. the distribution of 3h radioactivity after chromatography of the hydrolysate is shown in fig. 6 . two peaks were observed: a smaller peak at the point of sample application, and a larger peak which coincided with the position of galactose (fraction 28). the small amount of ?3-labeled material remaining in the hydrolysate was found at the origin; no %3 radioactivity comigrated with the galactose carrier. these results suggested that the majority of the 3h-labeled material recovered from purified t-ag was gala&se and provided direct evidence that t-ag is glycosylated. we noted, however, that different neutral monosaccharides were not particularly well-resolved in this tlc t-ag was isolated by immunoprecipitation and sds-page, then excised and eluted from the gel. after concentration, the sample was desalted by gel filtration, sds was extracted, and acid hydrolysis was performed in the presence of carrier galactose, as described under materials and methods. the sample was diluted, aliquote were removed for liquid scintillation spectroscopy, and the remainder was applied to a small column (about 5 ml packed volume) of dowex 5ow-x8. the flow-through was collected, and aliquots were again removed for liquid scintillation spectroscopy. *&s and ?i-i cpm were determined as described in the legend to table 1 . posthydrolysis = samples taken before application to the dowex column; post-ion exchange = samples taken from column flow-through. was purified, acid hydrolyzed, and subjected to cation-exchange chromatography as described in the legend to table 4 . the flow-through was neutralized, concentrated, and analyzed by thin-layer chromatography as described under materials and methods. the carrier and standard sugars (denoted by arrows in the figure) were visualized by staining with 30% aqueous ammonium bisulfate. the radioactivity profile was determined by dividing the lane containing the hydrolysate into 0.5-cm fractions and analyzing each fraction by liquid scintillation spectroscopy. system. since the peak of radioactivity in that area was somewhat broad, we cannot discount completely the possibility that a 0 5 10 15 67-galactose may have been converted to other sugars that were then incorporated into t-ag. experiments were designed to attempt to generate nonglycosylated t-ag that could be used to assess the role of glycosylation in t-ag function. tm and endoh treatments were chosen because their effects are well-characterized and both reagents are readily available in purified form. the success of such treatments depended, of course, upon the structural characteristics of the glycoprotein in question. to analyze the effect of endoh, sv40infected cells were labeled with [?3]methionine and t-ag was immunoprecipitated with haf and saci. immune complexes were washed, treated with tbs or various amounts of endoh, disrupted, and analyzed by sds-page (fig. 7a) . no change in the electrophoretic mobility of t-ag was observed after endoh treatment, suggesting that it was resistant to the effects of this endoglycosidase. control experiments utilizing a known endohsensitive glycoprotein, gp52 of mmtv (dickson and atterwill, 1980) that the treatment protocol was effective, even at the lowest concentration of enzyme used (fig. 7b) . it should be noted that endoh treatment did not alter the electrophoretic mobility of the endoh-resistant mmtv glycoprotein, gp36, or the nonglycosylated mmtv protein, ~28. to determine the effect of tm treatment, sv40-infected cells were treated with e-mem or various concentrations of tm starting at 15 hr pi., then were glucose-starved and labeled with ps]methionine or thlglucosamine from 21 to 24 hr p.i. after extraction, t-ag was immunoprecipitated and analyzed by sds-page (fig. 8a) . the mobility and intensity of labeling of t-ag with either isotope was unchanged in the presence of tm, suggesting that the glycosylation events i., then e-mem containing 0, 0.5, 1.0, or 1.5 rg/ml tm was added, and the cells were returned to 3'7". after 30 min starvation in glucose-free e-mem, cells were labeled from 21 to 24 hr p.i. with 100 pci/ml [86sjmethionine or ~-[l-*hjglucosamine. all solutions were supplemented with tm during starvation and labeling. cells were then extracted, and extracts were clarified, immunoprecipitated with haf and saci, disrupted, and analyzed by sds-page on 8% acrylamide gels. (b) vsv-infected tc-7 cells were tm-treated, glucosestarved, and labeled as in a, except that treatment began at 1 hr p.i., labeling was performed from 7 to 10 hr p.i., and vsv-infected cell lysates were immunoprecipitated using rhovsv. were resistant to this inhibitor. control experiments utilizing the tm-sensitive g glycoprotein of vsv (leavitt et ak, 1977) established that tm treatment was effective in tc-7 cells even at the lowest concentration of inhibitor used (fig. 8b ). in addition, incorporation of [shjglucosamine into total cellular protein was effectively inhibited by tm treatment (54-61%), with minimal effect evident upon overall protein synthesis (o-18%; table 5 ). this study has established that t-ag synthesized in sv40-infected cells is modified by glycosylation. metabolic-labeling experiments revealed that galactose and glucosamine could be incorporated into tag while mannose and fucose could not. the identity of t-ag was confirmed by its reactivity with a variety of t-ag-specific antisera. it should be noted, however, that t-ag was not strongly labeled with either galactose or glucosamine. many of our fluorograms required exposure times of l-4 weeks, and those were obtained only after we had optimized the conditions used for infections and labeling. it was crucial to infect cells at a high m.o.i., to prestarve cells with glucose-free medium, and to label with a high concentration of tritiated sugar added in the same medium at the maximal time of t-ag synthesis. in view of these limitations and of the resistance of glycosylated t-ag to treatments with endoh and tm (see below), the possibility of randomization of input labeled sugar posed a difficult problem in the interpretation of these results. we therefore rigorously explored that possibility. several different lines of evidence substantiated that randomization alone could not account for the monosaccharide labeling of t-a@;. first, only two of the four monosaccharides tested were incorporated into t-ag. mannose, which was not detectably incorporated, can enter glycolysis after conversion to fructose-6-phosphate, and could have generated labeled amino acids through the tricarboxylic acid cycle (lehninger, 1982) . second, only large tag was labeled with monosaccharides; small t-ag synthesized in the same cells was not. more direct evidence was obtained by labeling t-ag with thlgalactose in the presence of excess unlabeled amino acids or sugars. incorporation of the label was clearly inhibited by the presence of unlabeled sugars but was unaffected by the presence of unlabeled amino acids. in addition, treatment of doubly labeled tag with a mixture of glycosidases resulted in the preferential removal of 3h radioactivity, suggesting that 3h had been incorporated in the original input form of sugar. finally, we were able to remove and identify the sugar from purified tag. after acid hydrolysis of [3hjgalactoselabeled t-ag, the major peak of %i radioactivity cochromatographed with carrier galactose. taken together, these results strongly refute the possibility that t-ag was labeled with monosaccharides only after their metabolic conversion to some other form. thus, other explanations must be entertained for the relatively low level of incorporation of galactose or glucosamine into t-ag. one is probably the low specific activity of the tritiated monosaccharides we used for labeling. those were commer-cially available at only 2.4-25 ci/mmol, as compared to 1000-1500 ci/mmol for [%3]methionine. another possible explanation could be that only a specific subpopulation of t-ag is glycosylated. however, preliminary experiments from our laboratory suggest that this is not the case; rather, glycosylation appears to be a characteristic of t-ag in general (jarvis and butel, unpublished observations) . we are currently investigating other possible explanations. for example, the number of oligosaccharides attached to t-ag, or the number of sugar residues per oligosaccharide, or both could be quite small. the effects of endoh and tm on glycosylation of t-ag were analyzed for preliminary characterization of the glycosylation events involved. endoh cleaves highmannose n-linked oligosaccharides distal to the linkage sugar, n-acetylglucosamine (tarentino and maley, 1974) . tm is known to inhibit the initial glycosylation event in n-linked glycoproteins (takatsuki et a& 1975) . thus, these agents would exert their effects on t-ag only if it were an nlinked glycoprotein. this was possible, since there is a consensus n-glycosylation site (asn-arg-thr; hunt and dayhoff, 1970) at amino acid residues 156-158 of t-ag. however, treatment with neither agent changed the electrophoretic mobility of [%]methionine-labeled tag, even though control experiments verified the efficacy of both treatment protocols. because inhibition of glycosylation or removal of oligosaccharides might not be accompanied by a detectable change in electrophoretic mobility, tm experiments were also performed using phfglucosamine-labeled t-ag. no change in the intensity of labeling of t-ag was observed. these results suggest that t-ag is not an n-linked glycoprotein. this is consistent with our inability to label t-ag using mannose, since that sugar is a common constituent of n-linked glycoproteins. thus, t-ag provides another example of a protein in which a consensus n-glycosylation site is not used, suggesting that the site is necessary, but not sufficient, for nglycosylation (hunt and dayhoff, 1970) . t-ag is more probably an o-linked gly-coprotein. this interpretation is compatible with the apparent absence of mannose, with endoh resistance and with tm resistance. it is also compatible with the low level of monosaccharide incorporation, as discussed above, as the oligosaccharide moieties of many o-linked glycoproteins are small. for example, one of the oligosaccharides of the el glycoprotein of mouse hepatitis virus contains only a single residue each of sialic acid, galactose, and n-acetylgalactosamine; the other has only an additional sialic acid residue (niemann et c& 1984). we have attempted to verify the identification of t-ag as an olinked glycoprotein by mild alkaline /3elimination (spiro, 1966) . however, this approach has not been successful because the t-ag polypeptide appears to be degraded under those conditions (0.05m naoh, 1.0 m nabh4, 15 hr at 45"; jarvis and butel, unpublished observations). in any case, some controversy exists over the use of this method to distinguish between n-and o-linked glycoproteins (rasilo and renkonen, 1981; ogata and lloyd, 1982) . a better method would involve treatment of t-ag with acetylgalactosamine oligosaccharidase, which should cleave o-linked oligosaccharides. unfortunately, this enzyme is no longer commercially available. one of the most interesting characteristics of t-ag is its ability to mediate a large number of different functions in infected and transformed cells (tooze, 1980; martin, 1981; rigby and lane, 1983) . while it remains difficult to explain its multifunctional nature at a molecular level, the structural characteristics of tag reveal some intriguing possibilities. one explanation involves the presence of several unique structural domains on the polypeptide, with each domain performing one or more distinct functions. in fact, different t-ag functions have been localized to specific portions of the polypeptide (rigby and lane, 1983) . another possibility emphasizes the localization of t-ag to distinct subcellular compartments in the host cell. although the majority of t-ag is localized within the nucleus (pope and rowe, 1964, rapp et d, 1964) , small amounts are associated with the plasma membrane (tevethia et a& 1965; soule and butel, 1979; deppert et al, 1980; chandrasekaran et c& 1981; santos and butel, 1982; soule et d, 1982) and mitochondria 19'77) . t-ag species localized within different subcellular compartments might exhibit unique activities, due to putative structural differences and/or the influence of their local environments. a third model invokes the ability of t-ag to form different supramolecular complexes (prives et a& 1979; mccormick and harlow, 1980; fanning et ale, 1981) . these complexes could represent distinct subpopulations of t-ag capable of mediating different functions in the host cell. it is clear that some supramolecular forms exhibit distinct dna-binding and atpase properties (bradley et o& 1982; fanning et al, 1982; gidoni et ok, 1982) . the existence of discrete t-ag subpopulations is also suggested by the finding that certain monoclonal antibodies react with only a subset of the total t-ag present in host cells (gurney et al, 1980; scheller et cd, 1982) . thus, the functional diversity of t-ag might be explained by its structural diversity. structural variability might, in turn, be mediated by combinations of different chemical modifications. glycosylation could play an important role in generating structurally and functionally discrete forms of t-ag. the study of glycoproteins from other systems has established that glycosylation influences their structure and/or function. for example, n-glycosylation of fibronectin and acetylcholine receptor is crucial to their structural integrity (olden et al, 1982) . in the absence of glycosylation, those proteins were more susceptible to proteolytic degradation. in another example, the specific nuclear matrix association of certain high mobility group proteins was precluded in the absence of glycosylation (weintraub et ak, 1983) . this finding is particularly interesting, since some t-ag normally resides in association with the nuclear matrix of host cells (staufenbiel and deppert, 1983) . further, it has been shown that glycosylation is required for the proper supramolecular assembly of thyroid-stimulating hormone (reeves and chang, 1983) , suggesting that glycosylation might be important for the supramolecular assembly of t-ag subpopulations. it should be noted that o-linked glycoproteins remain relatively poorly characterized. in contrast to n-linked glycoproteins, little is known of their biosynthesis or function. it is possible that t-ag will provide a good model for the study of this class of macromolecules. relationship of oligomerization to enzymatic and dna-binding properties of the sv40 large t antigen surface proteins of simian-virus-40-transformed cells simian virus 40 t-antigen-related cell surface antigen: serological demonstration on simian virus 40-transformed monolayer cells in situ structure and processing of the mouse mammary tumor virus glycoprotein precursor pr73 glycoprotein encoded by the friend spleen focus-forming virus detection and characterization of multiple forms of simian virus 40 large t antigen subclasses of simian virus 40 large t antigen: differential binding of two subclasses of t antigen from productively infected cells to viral and cellular dna complete nucleotide sequence of sv40 dna separation and identification of monosaccharides from biological materials by thinlayer chromatography different forms of simian virus 40 large tumor antigen varying in their affinities for dna modification of sv40 t antigen by poly-adpribosylation monoclonal antibodies against simian virus 40 t antigens: evidence for distinct subclasses of large t antigen and for similarities among nonviral t antigens monoclonal antibodies specific for simian virus 40 tumor antigens identification and characterization of the avian erythroblastosis virus e&b gene product as a membrane glycoprotein a micromethod for complete removal of dodecyl sulfate from proteins by ion-pair extraction tunicamycin resistant glycosylation of a coronavirus glycoprotein: demonstration of a novel type of viral glycoprotein. fi-115 the occurrence in proteins of the tripeptides asn-x-ser and asn-x-thr and of bound carbohydrate structural comparisons of wild-type and nuclear transport-defective simian virus 40 large tumor antigens. fir-134 rapid isolation of antigens from cells with a staphylococcal protein a-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein a simian virus 40 large tumor antigen: a "rna binding protein acylation: a new post-translational modification specific for plasma membrane-associated simian virus 40 large t-antigen sv40 large t shares an antigenic determinant with a cellular protein of molecular weight 68,000 antigenic relationship of sv40 early proteins to purified large t polypeptide. fir-97 tunicamycin inhibits glycosylation and multiplication of sindbis and vesicular stomatitis viruses principles of biochemistry a simplified method of sample preparation for determination of tritium, carbon-14, or sulfur-35 in blood or tissue by liquid scintillation counting the transformation of ceil growth and transmogrification of dna synthesis by simian virus 40 enhancement of hexose entry into chick fibroblasts by starvation: differential effect on gala&se and glucose association of a murine 53,000 dalton phosphoprotein with simian virus 40 large t antigen in transformed cells characterization of the amino-terminal tryptic peptide of simian virus 40 small-t and large-t antigens the carbohydrates of mouse hepatitis virus (mhv) a59 structures of the 0-glycosidically linked oligosaccharides of glycoprotein el coronavirus glycoprotein el, a new type of viral glycoprotein characterization of simian cells transformed by temperature-sensitive mutants of simian virus 40 temperaturesensitive mutants of simian virus 40. i. isolation and preliminary characterization of b/c gene mutants mild alkaline borohydride treatment of glycoproteins-a method for liberating both n-and o-linked carbohydrate chains function of the carbohydrate moieties of glycoproteins tissue culture studies of mouse mammary tumor cells and associated viruses detection of specific antigen in sv40-transformed cells by immunofluorescence the product of the avian erythroblastosis virus e&b locus is a glycoprotein dna binding and sedimentation properties of sv40 t antigens synthesized in viro and in vitro virus-induced intranuclear antigen in cells transformed by papovavirus sv40. prm sot exp mild alkaline borohydride treatment liberates n-acetylglucosamine-linked oligosaccharide chains of glycoproteins the genome of simian virus 40 investigations of the possible functions for glycosylation in the high mobility group proteins. evidence for a role in nuclear matrix association structure and function of simian virus 40 large tumor antigen effect of cell chromosome number on simian virus 40 replication association of sv40 large tumor antigen and cellular proteins on the surface of sv40-transformed mouse cells antigenic structure of simian virus 40 large tumor antigen and association with cellular protein p53 on the surfaces of simian virus io-infected and -transformed cells a small subclass of sv40 t antigen binds to the viral origin of replication antigenic distinctions of glycoproteins in plasma and mitochondrial membranes of lymphoid cells neoplastically transformed by simian virus 40 new properties of simian virus 40 large t antigen expression of mammary tumor virus proteins in preneoplastic outgrowth lines and mammary tumors of balb/cv mice subcellular localization of simian virus 40 large tumor antigen detection of simian virus 40 surface-associated large tumor antigen by enzyme-catalyzed radioiodination characterization of carbohydrate unite of glycoproteins different structural systems of the nucleus are targets for sv40 large t antigen inhibition of biosynthesis of polyisoprenol sugars in chick embryo microsomes by tunicamycin purification and properties of an endo-&n-acetylglucosaminidase from streptomyces griseus modification of simian virus 40 protein a new surface antigen in cells transformed by simian papovavirus sv40 molecular biology of tumor viruses glycosylation of thyroid-stimulating hormone in pituitary tumor cells: influence of highmannose oligosaccharide units on subunit aggregation, combination, and intracellular degradation this investigation was supported in part by research grant ca 22555 and by national research service award ca 09197 from the national institutes of health. the authors thank dr. marion steiner for helpful advice on carbohydrate chemistry. key: cord-309015-t5v2sjus authors: york, joanne; agnihothram, sudhakar s.; romanowski, victor; nunberg, jack h. title: genetic analysis of heptad-repeat regions in the g2 fusion subunit of the junín arenavirus envelope glycoprotein date: 2005-12-20 journal: virology doi: 10.1016/j.virol.2005.08.030 sha: doc_id: 309015 cord_uid: t5v2sjus the g2 fusion subunit of the junín virus envelope glycoprotein gp-c contains two hydrophobic heptad-repeat regions that are postulated to form a six-helix bundle structure required for the membrane fusion activity of class i viral fusion proteins. we have investigated the role of these heptad-repeat regions and, specifically, the importance of the putative interhelical a and d position sidechains by using alanine-scanning mutagenesis. all the mutant glycoproteins were expressed and transported to the cell surface. proteolytic maturation at the subtilisin kexin isozyme-1/site-1-protease (ski-1/s1p) cleavage site was observed in all but two of the mutants. among the adequately cleaved mutant glycoproteins, four positions in the n-terminal region (i333, l336, l347 and l350) and two positions in the c-terminal region (r392 and w395) were shown to be important determinants of cell–cell fusion. taken together, our results indicate that α-helical coiled-coil structures are likely critical in promoting arenavirus membrane fusion. these findings support the inclusion of the arenavirus gp-c among the class i viral fusion proteins and suggest pharmacologic and immunologic strategies for targeting arenavirus infection and hemorrhagic fever. arenaviruses are endemic in rodent populations worldwide (salazar-bravo et al., 2002) and are transmitted to humans by exposure to infected animals. infection by old world arenaviruses such as lassa virus, or new world species such as the south american group including junín, machupo and guanarito viruses, are responsible for recurring and emerging outbreaks of viral hemorrhagic fevers with high mortality (mccormick and fisher-hoch, 2002; peters, 2002) . prophylactic and therapeutic options to combat arenavirus infection are limited, and the development of effective immunogens and antiviral agents to interfere with arenavirus entry may have broad public health benefits. the arenaviruses are enveloped bisegmented rna viruses whose genome consists of two single-stranded rna molecules clegg et al., 2000) . during biogenesis, arenaviral particles assemble and bud at the plasma membrane. the mature envelope glycoprotein complex of the arenavirus consists of three noncovalently associated subunits derived from the gp-c precursor by proteolytic cleavage events: a stable myristoylated 58 amino-acid signal peptide (ssp), the receptor-binding g1 subunit and the transmembrane g2 fusion protein (buchmeier, 2002; eichler et al., 2003; york et al., 2004) . the initial cleavage to yield ssp is likely mediated by the cellular signal peptidase, and the mature g1 and g2 subunits are subsequently generated by the cellular ski-1/s1p protease. both cleavage events, as well as the presence of the myristoylated ssp subunit, are required for envelope glycoprotein-mediated membrane fusion (york et al., 2004) . entry of virion particles into host cells is initiated by g1 binding to cell surface receptors followed by endocytosis of the virion into smooth vesicles (borrow and oldstone, 1994) . although adystroglycan serves as a binding receptor for the old world arenaviruses (cao et al., 1998) , the receptor utilized by the major new world group of arenaviruses is unknown (spiropoulou et al., 2002) . membrane fusion is ph-dependent and is activated upon acidification of the maturing endosome (borrow and oldstone, 1994; castilla et al., 1994; di simone and buchmeier, 1995; di simone et al., 1994) . the molecular basis for envelope glycoprotein-mediated membrane fusion in the arenaviruses is largely unknown, however, sequence analysis of the g2 ectodomain of lassa virus and lymphocytic choriomeningitis virus (lcmv) has revealed two heptad-repeat regions that can be represented to form amphipathic helices (gallaher et al., 2001) . this sequence motif is found among the large group of viral envelope glycoproteins that promote membrane fusion through the formation of a fusion-active core structure comprising a stable bundle of six a-helices. these class i fusion proteins include those of the retroviruses, orthomyxoviruses, paramyxoviruses, filoviruses and coronaviruses. in these proteins, the six-helix bundle structure involves a central coiled coil, formed by nterminal heptad-repeat regions of the trimeric fusion subunit ectodomain, surrounded by three anti-parallel helices of the cterminal heptad repeats which bind to the conserved hydrophobic grooves on the coiled-coil surface. formation of this thermodynamically favored six-helix bundle brings the viral and cell membranes into apposition and is thought to provide the driving energy to initiate membrane fusion (earp et al., 2005; eckert and kim, 2001; hughson, 1997; weissenhorn et al., 1999 and references therein) . in keeping with the proposal that the arenavirus g2 ectodomain might also form a fusionactive six-helix bundle, gallaher and colleagues have shown that a synthetic peptide derived from the n-terminal heptadrepeat region of lcmv is able to assume a-helical content (gallaher et al., 2001) . in this report, we have utilized alanine-scanning mutagenesis of the heptad-repeat regions of the new world junín arenavirus envelope glycoprotein to obtain genetic evidence in support of this model for membrane fusion in the arenaviruses. we show that mutations at predicted interhelical positions affect the ability of the envelope glycoprotein complex to mediate ph-dependent cell -cell fusion. these findings suggest that membrane fusion by the arenavirus envelope glycoprotein complex is promoted via a refolding into a highly stable sixhelix bundle structure characteristic of class 1 viral fusion glycoproteins. sequence analysis of the g2 ectodomain of the junín arenavirus the amino acid sequences of the heptad repeats of new world and old world arenaviruses were aligned to demonstrate the high degree of conservation within the family (fig. 1a) . one key consideration in assigning a register to the proposed helical coiled coil of the heptad repeats (i.e., positions a through g) was to maximize the degree of hydrophobicity at the interhelical a and d positions. upon inspection of the aligned sequences, a unique register of a and d positions was apparent (figs. 1a and b) . the a and d positions of the proposed n-terminal helix consist of hydrophobic residues common to the central interface of a trimeric coiled coil (e.g., leucine, isoleucine, methionine and valine). positions assumed to lie on the exterior face of the coiled coil (b, c and f positions) contain polar or charged residues. this pattern is repeated through the c-terminal heptad-repeat region and appears to end at the c-terminal-most d and a positions (k409 and d413, respectively). hydrophilic residues at two a positions in the new world viruses (r392 and s399) are replaced by less polar amino acids in the old world viruses (serine and alanine, respectively). the hydrophilic sidechains, if buried, may impart specificity to the process of coiled-coil folding at the expense of thermal stability (ji et al., 2000; lumb and kim, 1995) . to investigate the proposed a-helical structures and their role in promoting membrane fusion in the arenaviruses, we subjected the heptad-repeat regions of the junín virus envelope glycoprotein to scanning mutagenesis. each a and d position amino acid was individually changed to alanine, a small residue that is a good helix inducer yet contributes little to the hydrophobic forces that predominantly stabilize coiled-coil structures. alanine substitutions at these positions are likely to alter the stability of the proposed six-helix bundle, but not to disrupt its overall folding. we anticipated that alanine mutations at interhelical positions within the coiled-coil bundle may selectively affect the ability of the envelope glycoprotein to mediate membrane fusion. for these studies, we utilized the gp-c gene of the pathogenic junín virus isolate mc2 (york et al., 2004) . the wild-type and mutant plasmids were introduced in vero 76 cells, and the envelope glycoproteins were expressed using the t7 promoter of the plasmid vector and t7 rna polymerase provided by infection with the recombinant vaccinia virus vtf7-3 (fuerst et al., 1986) . cultures were metabolically labeled and subsequently solubilized. the envelope glycoproteins were immunoprecipitated using the mouse mab bf11 (sanchez et al., 1989) directed to the g1 subunit (york et al., 2004) . we have previously shown that the junín virus envelope glycoprotein complex isolated by immunoprecipitation retains the three noncovalently associated subunits: the mature g1 and g2 glycoproteins as well as the stable signal peptide, ssp (york et al., 2004 and see fig. 2) . cleavage by signal peptidase and the ski-1/s1p protease are incomplete upon recombinant expression such that two precursor glycoproteins (full-length gp-c and the g1 -g2 precursor lacking ssp) are observed. the glycoproteins isolated by immunoprecipitation of the wild-type envelope glycoprotein, and an ski-1/s1p cleavage-site mutant (cd-jgpc; york et al., 2004) , are illustrated in fig. 2a (far left). the two precursor glycoproteins migrate with molecular weights of 65 kda and 60 kda, metabolically labeled glycoproteins were immunoprecipitated using the g1-specific mab bf11 and separated on nupage 4 -12% bis -tris gels. the wild-type (wt) and ski-1/s1p cleavage-defective (cd) glycoproteins are shown for comparison with the n-and c-terminal heptad-repeat mutants. v326a indicates the glycoprotein in which v326 has been changed to alanine. the two bands labeled gp-c (i.e., the fulllength gp-c and the g1 -g2 precursor lacking ssp) as well as the heterodisperse smear of g1 are better resolved following deglycosylation using pngase f (b). respectively. the mature g2 glycoprotein appears as a band of å35 kda, whereas the heterogeneous smear of the g1 glycoprotein is difficult to discern. these bands are better resolved following deglycosylation using peptide:n-glycosidase f (pngase f) (fig. 2b, far left) . the g1 and g2 subunits are absent in the cleavage-defective cd-jgpc envelope glycoprotein. among the seventeen n-and c-terminal heptad-repeat mutants, all the gp-c glycoproteins were expressed, and all were cleaved by signal peptidase to generate the 5 kda ssp ( fig. 2a) . the relative efficiencies of ski-1/s1p protease cleavage, however, varied among the mutants ( figs. 2a and b) . in some mutants (e.g., y385a and l402a), ski-1/s1p cleavage was undetectable, similar to that in the cleavagesite-defective envelope glycoprotein. in other mutants, the amounts of cleavage ranged from markedly reduced to levels greater than those in the wild-type. to quantitate the relative extents of ski-1/s1p processing, we determined the amount of radioactivity present in the deglycosylated gp-c precursors and in the mature g1 and g2 polypeptides using a fuji 3000-g phosphorimager and image gauge software (fuji). our analysis of two independent experiments is shown in fig. 3 . several classes of mutants could be defined by this analysis. proteolytic maturation was essentially absent in the y385a and l402a mutants and significantly reduced in l350a and w395a (30 and 45% of wild-type levels, respectively). other mutants were cleaved much as the wildtype glycoprotein (l336a, n340a, i388a, r392a, s399a, m406a and k409a; from 65 to 100% of wild-type levels), whereas several mutations in the n-terminal heptad-repeat region appeared to enhance proteolytic cleavage up to 2-fold (v326a, m329a, i333a, m343a and i347a). shedding of the mature g1 subunit into the culture supernatant correlated with the extent of ski-1/s1p cleavage (not shown). although proteolytic processing varied among the mutants, all but two showed significant cleavage of the g1 -g2 precursor. transport of the mutant envelope glycoprotein complexes to the cell surface was determined by flow cytometry using the g1-specific mab be08 (sanchez et al., 1989) . cells from cultures transiently transfected to express the wild-type and cd-jgpc glycoproteins displayed two populations: cells expressing gp-c (!30 fluorescence channel) and those that escaped transfection (<30 fc) (fig. 4) . proteolytic maturation of the arenavirus envelope glycoprotein complex is clearly not required for transport to the cell surface. in fact, staining of the cleavage-defective mutant cd-jgpc was generally higher and more discrete than that of the wild-type envelope glycoprotein, likely due to shedding of the g1 subunit from the wild-type but not the cleavage-defective complex (york et al., 2004) . among the heptad-repeat mutants (fig. 4) , all patterns of staining were within this range and qualitatively similar. we conclude that transport of the mutant glycoproteins to the cell surface was not markedly affected. the precise pattern of cell-surface staining is expected to reflect the degree of transport and the relative extent of g1 shedding. our efforts to further characterize the surface gp-c complex biochemically were limited by the refractory nature of the gp-c complex to covalent modification by standard biotinylation reagents (unpublished). overall, however, successful entry into the golgi and transit to the cell surface suggests that the mutations do not globally perturb the structural integrity of the mutant gp-c complexes. the effects of the alanine mutations on the ability of the envelope glycoprotein to mediate ph-dependent cell -cell fusion were determined in vero 76 cells using the recombinant vaccinia virus-based lacz fusion reporter assay (nussbaum et al., 1994) as described (york et al., 2004) . in this assay, hgalactosidase expression initiated by fusion of the effector and target cells was determined using a chemiluminescent substrate (galactolite plus, tropix). the extent of ph-dependent cellcell fusion mediated by the wild-type and mutant envelope glycoproteins is shown in fig. 5 . five alanine substitutions at a and d positions reduced fusogenicity to less than 10% of the wild-type gp-c or comparably to the ski-1/s1p cleavage-site mutation in cd-jgpc: i333a in the n-terminal heptad repeat and y385a, r392a, w395a and l402a in the c-terminal heptad repeat (fig. 5) . although the lack of proteolytic cleavage in y385a and l402a is likely sufficient to account for the defect in fusion by these mutants, the other glycoproteins (i333a, r392a and w385a) showed significant levels of cleavage. three additional mutations in the n-terminal region (l336a, i347a and l350a) reduced cell -cell fusion to less than 50% fig. 3 . quantitation of ski-1/s1p cleavage and the formation of the mature g1 and g2 subunits. phosphorimages of the deglycosylated polypeptides represented in fig. 1b were analyzed using image gauge software (fuji) and the profile and background tools to quantitate the radioactivity (psl units). the efficiency of ski-1/s1p cleavage was defined as the ratio of psl units in the sum of the g1 + g2 peaks relative to the total number in both gp-c and g1 + g2 peaks. results were analyzed for two complete experiments, and the relative conclusions are consistent with those of additional partial studies. because the absolute efficiency of cleavage varied between experiments, each experiment was normalized to the average extent of cleavage in the entire 19member panel. this average ratio (16% and 24% in the two complete experiments shown here) was used to determine the relative degree of cleavage in each mutant, and these numbers were averaged to generate the values plotted. by this analysis, the wild-type envelope glycoprotein was cleaved 1.17-fold better than the arbitrary average of the panel; 100% and 50% of the wild-type level are shown as dashed lines. error bars represent t one standard deviation. of the wild type. in all cases, cell -cell fusion required exposure of the culture to acidic (ph 5.0) medium. in some mutants, clear defects in membrane fusion arose despite wild-type levels of ski-1/s1p cleavage (viz., i333a and i347a). in others, it was difficult to determine whether the deficiencies in cell -cell fusion were directly attributable to the envelope glycoprotein fusion machinery or to defects in ski-1/ s1p proteolytic processing. in several examples, mutants displaying reductions in cleavage efficiency were able to mediate cell -cell fusion much as the wild-type glycoprotein (viz., i388a and s399a), whereas other comparably cleavagedeficient mutants were defective in membrane fusion (viz., l336a, l350a and r392a). these observations suggested that the variable deficiencies in ski-1/s1p cleavage were not sufficient to account for the majority of the defects in cell -cell fusion under our assay conditions. in order to more fully examine this point, we reconstructed the variable extents of proteolytic cleavage using mixtures of the wild-type and ski-1/s1p cleavage-site mutant plasmids. in these studies, we determined the relative efficiencies of ski-1/ s1p cleavage by page and the extent of ph-dependent membrane fusion in the recombinant vaccinia virus-based lacz fusion reporter assay (fig. 6) . although the total amount of envelope glycoprotein remained constant as the wild-type plasmid was diluted 1:3 and 1:9 with cd-jgpc (90% and 85% of the undiluted wild-type level, respectively), the relative amounts of mature g1 and g2 decreased progressively, from 20% to 5% and 2%, respectively. despite a 90% decrease in overall ski-1/s1p cleavage, however, cell -cell fusion was unaffected. within these limits, our measurements of cell -cell fusion appear not to be sensitive to the relative amounts of proteolytically matured g1 and g2 glycoprotein, and we infer that mutants in which ski-1/s1p cleavage is retained to at least 10% of the wild type level can be informative in defining specific defects in membrane fusion. tertiary effects of the heptad-repeat mutations that may indirectly affect both proteolytic cleavage and fusogenicity, such as those that influence overall protein folding, trafficking fig. 4 . flow cytometric analysis of cell-surface envelope glycoprotein. vero cells expressing the envelope glycoproteins were stained using the g1-specific neutralizing mab be08 (sanchez et al., 1989 ) and subsequently stained using propidium iodide (1 ag/ml) to exclude dead cells in the population. cells were fixed using 2% formaldehyde and analyzed using a facscalibur flow cytometer (bd biosciences). background staining of mock-transfected cells is shown in the first panel; the non-expressing cell population within the gp-c transfected cultures (other panels) tend to pick up additional staining, possibly due to membrane exchange within the culture. the expressing cell populations were defined using a gate of !30 that included <0.1% of the mock-transfected population (arrow). or oligomer composition, would limit our ability to generalize from our simple reconstitution model. in the case of the cleavage-site defective glycoprotein, these considerations are likely moot as gp-c biogenesis appears to be otherwise blind to the ski-1/s1p site mutation. among the heptad-repeat mutants, we also observe no evidence for global defects in folding, assembly or transport and thereby extend the interpretation of the model experiment to these glycoproteins. thus, we conclude that the following a and d heptad-repeat positions contribute directly to promoting ph-dependent membrane fusion by the junín virus envelope glycoprotein: i333, l336, i347 and l350 in the n-terminal region and r392 and w395 in the c-terminal region. taken together, our studies identify a series of amino acid residues within the n-and c-terminal heptad-repeat regions of the g2 ectodomain that impair the ability of the junín virus envelope glycoprotein to mediate ph-dependent cell -cell fusion. the residues chosen for mutagenic analysis were modeled to lie at interhelical a and d positions, in keeping with the predominant role of these positions for coiled-coil formation. alanine substitutions at these positions are often benign (luo and weiss, 1998) , and the rich appearance of fusion-deficient phenotypes among our mutants is consistent with this model and with the important role of the a-helical coiled-coil bundle in promoting membrane fusion in the arenaviruses. several of the alanine mutations also affected ski-1/s1p cleavage of the g1 -g2 precursor glycoprotein, presumably through conformational effects that otherwise do not perturb envelope glycoprotein trafficking to the cell surface. these effects on proteolytic cleavage may signal a distinct role of the heptad-repeat regions in the folding of the native envelope glycoprotein during biogenesis. importantly, our studies suggest that these effects on proteolytic cleavage per se are unlikely to account for the specific deficiencies in membrane fusion activity. these genetic studies provide direct support to the proposal, originally based on amino acid sequence analysis (gallaher et al., 2001) , that membrane fusion in the arenaviruses is promoted by the formation of an a-helical coiled-coil structure similar to that described in the class i viral fusion proteins. biophysical evidence emerging from the laboratory of m. lu (weill medical college of cornell university) confirms that peptides derived from the n-and c-terminal heptad repeats of the junín virus g2 associate to form a six-membered trimeric structure with a high degree of helical content and thermal stability (unpublished). the precise limits of the n-and cterminal a-helices and the details of the interhelical interactions within the arenavirus six-helix bundle await atomic-level structure determination. the inclusion of the arenavirus envelope glycoprotein within this class of viral fusion proteins suggests several specific strategies towards the development of vaccines and therapeutic agents against arenavirus infection. peptides derived from the c-terminal helical region of the hiv fusion protein that are believed to irreversibly interfere with formation of the six-helix bundle are in clinical use in the treatment of hiv disease (kilby et al., 2002) , and small molecule inhibitors may be feasible (eckert et al., 1999) . similarly, broadly neutralizing antibodies that target the membrane-proximal region of the hiv gp41 ectodomain adjacent to the c-terminal heptad repeat (muster et al., 1993; stiegler et al., 2001; zwick et al., 2001) may likewise interfere in this process (follis et al., 2002) . further structural and immunochemical studies of the arenavirus envelope glycoprotein complex may point to conserved mechanisms and broadly applicable strategies towards the prevention and treatment of arenaviral hemorrhagic fevers. fig. 6 . reconstruction to determine the effect of variable ski-1/s1p cleavage efficiency on envelope glycoprotein fusogenic potential. vero 76 cells were transfected with 4 ag dna containing either the wild-type gp-c plasmid (wt), a 1:3 mixture of the wt and cd-jgpc plasmids, respectively, or a 1:9 mixture of the same plasmids. parallel cultures were metabolically labeled and immunoprecipitated, and pngase f-treated polypeptides were analyzed as described in figs. 2 and 3 (a) or subjected to the recombinant vaccinia virus-based fusion assay as described in fig. 5 (b) . 5 . fusogenic potential of wild-type and mutant envelope glycoproteins. the ability of the envelope glycoproteins to mediate ph-dependent cell -cell fusion was detected using the recombinant vaccinia virus-based h-galactosidase reporter assay (nussbaum et al., 1994) as described in materials and methods. h-galactosidase activity induced upon cell -cell fusion was quantitated using the chemiluminescent substrate galactolite plus (tropix). background levels of chemiluminescence obtained from cultures treated at neutral ph were subtracted from the relative light unit (rlu) measurement, and all rlu values were then normalized to that of the wild-type gp-c control. error bars represent t one standard deviation among 6 replicate fusion cultures. all conclusions were replicated using x-gal staining of parallel co-cultures (york et al., 2004) . the gp-c gene (ghiringhelli et al., 1991) of the pathogenic mc2 isolate of the argentine hemorrhagic fever (junín) virus was expressed using the bacteriophage t7 polymerase promoter in a pcdna3.1-based (invitrogen) expression plasmid (york et al., 2004) . the gp-c gene was modified to express an innocuous s-peptide (spep) affinity tag (kim and raines, 1993) at the cytoplasmic c-terminus to facilitate manipulations (york et al., 2004) . the h-galactosidase fusion reporter assay (nussbaum et al., 1994) was used to assess the ability of the envelope glycoprotein to mediate receptor-dependent cell -cell fusion. the recombinant vaccinia virus vcb21r-lacz expressing h-galactosidase under the control of the t7 promoter (nussbaum et al., 1994) was obtained from c. broder, p. kennedy and e. berger (nih) through the aids research and reference reagent program. mouse monoclonal antibodies (mabs) directed to the g1 subunit of gp-c (sanchez et al., 1989) were kindly provided by drs. tom ksiasek and tony sanchez (special pathogens branch, cdc, atlanta). the envelope glycoprotein was transiently expressed in vero 76 cells using the pcdna3.1-based t7 promoter and the recombinant vaccinia virus encoding t7 polymerase vtf7-3 (fuerst et al., 1986) . vero cells were infected with the recombinant vaccinia virus at a multiplicity of two in dulbecco's minimal essential medium (dmem) containing 2% fetal bovine serum (fbs). cytosine arabinoside (arac) was included at 10 am to limit vaccinia virus expression (hruby et al., 1980) . after 30 min, the cells were washed and transfected with the gp-c expression plasmid using lipofectamine 2000 reagent (invitrogen). metabolic labeling using 32 aci/ml of [ 35 s]-promix (amersham pharmacia biotech) was initiated 6 h post-transfection in methionine-and cysteine-free dmem containing 10% dialyzed fbs and 10 am arac and was continued for 12 -16 h. cultures were then washed in physiological buffered saline (pbs) and lysed using cold tris -saline buffer (50 mm tris -hcl and 150 mm nacl at ph 7.5) containing 1% triton x-100 nonionic detergent and protease inhibitors (1 ag/ml each of aprotinin, leupeptin and pepstatin). a soluble fraction was prepared by centrifugation at 15,000âg for 30 min at 4 -c. the expressed glycoproteins were isolated either by immunoprecipitation using the g1directed mab bf11 (sanchez et al., 1989) and protein a -sepharose (sigma) or by using the c-terminal (g2) spep affinity tag and s-protein agarose (novagen). in some experiments, the isolated glycoproteins were deglycosylated using peptide:n-glycosidase f (pngase f, new england biolabs). glycoproteins and polypeptides were resolved using nupage 4-12% bis -tris gels (invitrogen) and the recommended sample buffer containing lithium dodecyl sulfate and reducing agent. molecular weight markers included [ 14 c]-methylated rainbow proteins (amersham pharmacia biotech). radiolabeled proteins were imaged using a fuji fla-3000g imager and analyzed using imagegauge software (fuji). vero 76 cells transiently expressing the wild-type and mutant glycoproteins were labeled using the g1-specific mab be08 (sanchez et al., 1989 ) and a secondary fluorescein isothiocyanate (fitc)-conjugated goat anti-mouse antibody (jackson immunoresearch). cells were subsequently stained using propidium iodide (1 ag/ml) and then fixed in 2% formaldehyde. populations were analyzed using a facscalibur flow cytometer and cellquest software (bd biosciences). the h-galactosidase fusion reporter assay (nussbaum et al., 1994) was used to assess the ability of the envelope glycoproteins to mediate receptor-dependent cell -cell fusion. in these studies, vero cells infected with vtf7-3 and expressing the envelope glycoprotein were co-cultured with vero cells infected with vcb21r-lacz, a recombinant vaccinia virus expressing h-galactosidase under the control of the t7 promoter. these latter (target) cells were obtained by incubating vero 76 cells with a multiplicity of two vcb21r-lacz plaque-forming units per cell and allowing the infection to proceed overnight in the presence of 100 ag/ml of the vaccinia virus assembly inhibitor, rifampicin. after mixing of the envelope glycoprotein-expressing and target cells in medium containing both arac and rifampicin (hruby et al., 1980; york et al., 2004) , the co-cultures were continued for 5 h prior to being subjected to a 30 min pulse of neutral or acidic (ph 5.0) medium as previously described (york et al., 2004) . hgalactosidase expression is induced upon fusion of the effector and target cells and was detected, after 5 h of continued cultivation at neutral ph, in cell lysates (tropix) using the chemiluminescent substrate galactolite plus (tropix). cellcell fusion was quantified using a tropix tr717 microplate luminometer. mechanism of lymphocytic choriomeningitis virus entry into cells arenaviruses: protein structure and function arenaviruses and their replication identification of alpha-dystroglycan as a receptor for lymphocytic choriomeningitis virus and lassa fever virus the entry of junin virus into vero cells arenaviridae kinetics and ph dependence of acidinduced structural changes in the lymphocytic choriomeningitis virus glycoprotein complex acidic ph triggers lcmv membrane fusion activity and conformational change in the glycoprotein spike the many mechanisms of viral membrane fusion proteins mechanisms of viral membrane fusion and its inhibition inhibiting hiv-1 entry: discovery of d-peptide inhibitors that target the gp41 coiled-coil pocket identification of lassa virus glycoprotein signal peptide as a transacting maturation factor genetic evidence that interhelical packing interactions in the gp41 core are critical for transition to the fusion-active state of the hiv-1 envelope glycoprotein eukaryotic transientexpression system based on recombinant vaccinia virus that synthesizes bacteriophage t7 rna polymerase the viral transmembrane superfamily: possible divergence of arenavirus and filovirus glycoproteins from a common rna virus ancestor molecular organization of junin virus s rna: complete nucleotide sequence, relationship with other members of the arenaviridae and unusual secondary structures identification of a virus-specified protein in the nucleus of vaccinia virus-infected cells enveloped viruses: a common mode of membrane fusion? buried polar interactions and conformational stability in the simian immunodeficiency virus (siv) gp41 core the safety, plasma pharmacokinetics, and antiviral activity of subcutaneous enfuvirtide (t-20), a peptide inhibitor of gp41-mediated virus fusion, in hiv-infected adults ribonuclease s-peptide as a carrier in fusion proteins a buried polar interaction imparts structural uniqueness in a designed heterodimeric coiled coil roles in cell-to-cell fusion of two conserved hydrophobic regions in the murine coronavirus spike protein lassa fever a conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1 fusogenic mechanisms of enveloped-virus glycoproteins analyzed by a novel recombinant vaccinia virus-based assay quantitating cell fusion-dependent reporter gene activation human infection with arenaviruses in the americas mammalian reservoirs of arenaviruses junin virus monoclonal antibodies: characterization and crossreactivity with other arenaviruses new world arenavirus clade c, but not clade a and b viruses, utilizes alpha-dystroglycan as its major receptor a potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp41 of human immunodeficiency virus type 1 structural basis for membrane fusion by enveloped viruses the signal peptide of the junín arenavirus envelope glycoprotein is myristoylated and forms an essential subunit of the mature g1 -g2 complex broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41 we thank dr. min lu (weill medical college of cornell university, new york) for continued discussions throughout this work and for access to his unpublished data. we are grateful to drs. tom ksiasek and tony sanchez (special pathogens branch, cdc, atlanta) for monoclonal antibodies and to dr. meg trahey (the university of montana) for useful comments during preparation of the manuscript. technical assistance in plasmid preparation was provided by kimberly hardwick, and we thank kathryn follis for manuscript editing. v.r. holds a research career award from consejo nacional de investigaciones científicas y téchnicas (conicet), argentina. this project was supported by a grant to j.h.n. from the u.s. national institutes of health (ai059355). key: cord-310748-ao29zx1u authors: banner, lisa r.; mc lai, michael title: random nature of coronavirus rna recombination in the absence of selection pressure date: 1991-11-30 journal: virology doi: 10.1016/0042-6822(91)90795-d sha: doc_id: 310748 cord_uid: ao29zx1u abstract rna-rna recombination is thought to occur preferentially at certain selected sites and in only a few rna viruses; the mechanism for these restrictions is unknown. in this paper we report the development of a recombination assay for coronavirus, using polymerase chain reaction, in the absence of selection pressure. our results showed that within a 1-kb region of the peplomer gene, rna recombination occurred at almost every potential crossover site. thus, coronavirus rna recombination appears to be more random than previously realized. however, after serial passages of the recombinant viruses in tissue culture, the recombination sites among the progeny viruses became clustered in the region which contains the previously reported “hot spot” for coronavirus recombination. these results suggest that rna recombination is common and random in nature, but only certain recombinants can be selected. thus, the presence of recombinational “hot spots” for coronavirus or other rna viruses most likely resulted from selection of certain recombinant viruses and not restriction on the occurrence of rna recombination. the failure to detect recombinants in other rna viruses may therefore be due to unfavorable properties of recombinant viruses. this approach can be used to detect recombinants in these viruses. the ability to exchange genetic information may allow rna viruses to adapt to a changing environment and to overcome potential deleterious effects caused by the high error frequency of the rna polymerase. viruses with segmented genomes can undergo rna reassortment. however, the ability of rna viruses with nonsegmented genomes to exchange genetic elements is more limited. only a few rna viruses, including picornaviruses, coronaviruses, and a few plant viruses have been shown to undergo rna-rna recombination at various efficiencies (i-7). the failure of many other rna viruses and rna phages to recombine has been well documented (8, 9) although recent studies have identified additional viruses which undergo nonhomologous rna recombination under special circumstances (70, 1 i). nevertheless, homologous rna recombination remains rare despite the fact that nonhomologous rna rearrangement, in the form of defective-interfering (di) rna, has been widely demonstrated for most rna viruses. the attempted isolation of recombinants in rna viruses has usually been carried out by applying certain selection pressures and assaying the progeny virus harvested. this approach most likely detects only those recombinant viruses which do not lose their survival fitness under the selection pressures. by this approach, it has been shown that foot-and-mouth disease virus (fmdv) recombines in most genetic regions ' to whom requests for reprints should be addressed except the viral capsid protein vp1 and vp3 genes (1) . similarly, mouse hepatitis virus (mhv) and poliovirus have some apparent recombinational hot spots (72, 13). conceivably, the limitation andclusteringof recombination sites in these viruses and the #ailure to detect recombinants in other rna viruses coufd very welt be the result of selection for or against oertain recombinants, and may not necessarily reflect the actual mechanism of rna recombination or the lack thereof. to examine this issue, we initiated a study of rna recombination in coronavirus, in the absence of selection pressure. surprisingly, we found that the initial recombination events within a 1-kb region of the peplomer gene were almost entirely random. howevar, after only a few passages, the region in which recombination could be detected became restricted, such that the majority of crossover sites were locatized to a small area of the region examined. eventually, all the recombinants became undetectable. our results suggest that rna recombination is more common than previously recognized and that the failure to detect recombinants in certain rna viruses may be due to a negative selection against recombinant viruses. mhv, a prototypic member of the coronaviridae, was used as a model system for the study of rna recombination. mhv contains a positive-sense rna genome of 31 kb (14, 16) which has been shown to undergo recombination both in vitro (#, 5, 17, 18) and in viva ( 16) at a frequency approaching 25% for the entire genome (79). to study rna recombination in the absence of selection pressure, we developed a polymer-ase chain reaction (pcr) assay using two primers specific for the potential recombinant viruses which have a crossover site between the two primers. we first attempted to detect recombinant rna among the intracellular viral rnas in cells coinfected with two mhv strains. dbt cells, a murine astrocytoma cell line (20) were coinfected with the a59 and jhm-dl (21) strains of mhv at a multiplicity of infection (m.0.i.) of 5. at 7 hr postinfection, cells were lysed with 0.5% np-40; total rna was extracted and treated with proteinase k, and polya-containing rna was selected. we chose the peplomer (s) gene for this study, since several recombinants isolated using selection markers have been shown to contain crossover sites within this gene (12). two synthetic oligonucleotides specific for the recombinants were used as primers. a jhm-dl-specific primer (5'-cgcgatccgtgcacatccaagg-3') which bound to position 1554-1575 from the 5'-end of the peplomer gene, was used for first-strand cdna synthesis. this primer corresponds to a region which is deleted in a59 (12, 22) and, thus, does not bind to a59. the cdna was then used as template for amplification by pcr, along with an a59-specific second primer (5'-ggactgagctccgct-taatgttaatggctgatg-cat-3') which bound at position 581-604 of the peplomer gene. seven of eight nucleotides at the 3'-end of this primer differ from the corresponding region of jhm-dl. the region between these two primers spanned 950 nt and included a hypervariable area which is subject to frequent deletions (72, 22) and a previously identified recombinational "hot spot" between nucleotides 1148 and 1426 (12). only recombinant rnas which had a crossover between the two primers and contained a59-specific sequences on the 5'-side and jhm-dl-specific sequences on the 3'-side could be detected by this pcr approach. pcr conditions were chosen so that these two primers would bind only to recombinant rnas and would not generate nonspecific pcr products by reverse transcriptase or taq polymerase jumping. rna was amplified by a slight modification of the procedure of fugue et a/. (23) . briefly, 1 pg of rna was mixed with 50 ng of the two appropriate primers in a 1 00-~1 reaction mixture containing 10 mm tris-hci (ph 8.0) 50 mlm kci, 3 mm mgci,, 0.01 oh gelatin, 500 pm each of the four deoxyribonucleoside triphosphates, and 1 pg trna as a nonspecific competitor. the rna was denatured for 2 min at 94" and chilled quickly on ice. two units of amv reverse transcriptase (seikagaku, rockville, md) was added and the reaction incubated for 30 min at 42". after the addition of 2 units of taq dna polymerase (perkin-elmer cetus, norwalk, ct), the resulting cdna was amplified for 35 cycles, with each which binds at nucleotide 58 l-604. both primers were specific for jhm-dl. lane 3: a59 rna from a single infection was used as template for pcr with primers 109, which bound to nucleotides 1554-1575, and 152, which were specific for recombinants. lane 4: jhm-dl rna was used as template for pcr with primers 109 and 152. lane 5: equal amounts (0.5 ag) of a59 and jhm-dl rna were mixed together and amplified by pcr with primers 109 and 152. lane 6: intracellular rna from coinfected cells was amplified using primers 109 and 152. the lower 800~nt band was caused by incorrect binding of primer 109 at nucleotide 140 1 of the s gene on the a59 rna. lane 7: same as lane 5, except that the rna mixture was from purified virions derived from single infections. lane 8: same as lane 6. except that purified virion rna released from the coinfected cells was used. cycle consisting of 94" for 1'30", 58" for 2', and 72" for 3'. to confirm that the two primers were specific for the recombinants, each virus was grown separately, and the rna extracted and amplified by pcr using the pair of primers described. no product of expected size (950 bp) was detected (fig. 1, lanes 3 and 4) . however, when rna from the coinfected cells was used, a specific product of 950 bp (fig. 1, lane 6 ) most likely derived from the recombinant rna, was detected. several smaller bands were observed, but these represented nonspecific pcr products since they also were seen in control lanes. the presence of each parental rna in the coinfected cells was confirmed by using different primer pairs specific for each parent (fig. 1, lanes 1 and 2) . to rule out the possibility that this pcr product was generated by transcriptional jumping of reverse transcriptase or taq polymerase, rna from separate a59 and jhm-dl infections was mixed to-gether in equal amounts such that the total amount equaled that of the rna from the coinfection. when the rna mixture was used as a template for reverse transcription and pcr, the specific 950-bp product was not detected (fig. 1, lane 5) . thus, the pcr product present in lane 6 ( fig. 1 ) resulted from recombination events which occurred during the coinfection. to examine the possible sites of recombination, the pcr product was purified from low-melt agarose and directionally cloned into the vector ptz18u (u.s. biochemicals, cleveland, oh) through a sac1 restriction site present on the 5'-end primer and smal on the 3'-side. dna sequence analysis of 35 cloned pcr products showed that the crossover sites were almost randomly distributed throughout the nearly 1-kb region of the peplomer gene studied ( fig. 2a) . two additional recombinants had triple crossovers; one had three crossovers which occurred within a span of 98 nt (from 986 to 1084) and the other within 249 nt (from 897 to 1146). one recombinant rna had a 30-nt deletion, with each parental sequence flanking the deletion (fig. 3) . it is noteworthy that the deleted site was flanked by a 6-nt repeat, but only one copy of the repeat was retained in the recombinant. the structure of this recombinant was consistent with the interpretation that rna recombination occurs by a copy-choice mechanism as a result of polymerase jumping during transcription. we next sought to determine whether there were any selection advantages or disadvantages for certain recombination sites once the rna was packaged into the virion. cells were coinfected again with the same virus strains used above. after incubation for 13 hr, the medium containing released virus particles was harvested and the virions were pelleted through a 30% sucrose cushion at 26k in a beckman sw 28 rotor for 3.5 hr. viral genomic rna isolated by proteinase k treatment and phenol/chloroform extraction was used as a template for pcr using the same primers described above. upon applying the same controls as before, the 950-bp recombinant product was generated only from the viral rna derived from coinfection (fig. 1, lane 8) . the recombinant pcr product was cloned into plii18u and dna from individual clones was sequenced. analysis of 53 recombinant clones revealed that, similar to the intracellular recombinants, the crossover sites in the viral recombinant rnas were almost randomly distributed over the 1 -kb region of the peplomer gene (fig. 2b) . no triple crossovers or deletions were detected. we then determined whether the recombinant rna population could be altered by passage in tissue culture. dbt cells were infected with the virus harvested from the coinfected cells as described above. the supernatant from this infection, termed passage 1, was harvested and the progeny virions were purif&d. viral genomic rna was isolated and amp#ed by pcr using the same primers described previously. the specific 950-bp pcr product representing the recombinant rna was again detected (data not shown). sequence analysis of 34 recombinants showed that crossover sites were detected throughout the 1 -kb region; however, a large proportion (67%) were clustered within nucleotides 1000-l 350. this is in contrast to 40% in the same region for intracellular recombinant rna and 47.1% for the recombinants detected in &ion rna before passage (fig. 2c) . these data suggest that some fig. 3. diagram of sequences around the crossover site of the recombinant i-l 4, which had a deletion. both jhm-dl and a59 parental rnas were compared with clone l-l 4 in the region from nucleotides 1346 to 1407 from the 5'-end of the peplomer gene. thick lines underneath the sequences represent jhm-dl-specific sequences, and thin lines represent a59-specific sequences. boxed areas are 6-nt repeats flanking the deletion. placement of the boxed nucleotides in l-l 4 is arbitrary. dots in l-14 denote deleted area. recombinant rna molecules may have selective disadvantages during subsequent infection. after two additional passages, an even greater proportion (79.2%) of the crossover sites were localized within nucleotides 1000-l 350 (fig. 2d) . by the fourth passage, no recombinant rna molecules could be detected by pcr amplification (data not shown). this result suggests that the recombinant viral rnas which have crossovers within the peplomer gene may have a growth disadvantage under the culture conditions. one possible reason may be that one of the parental viruses, a59, had more efficient growth properties in cell culture. the other parental virus, jhm-dl, was affected much like the recombinants, in that no jhm-dl rna could be detected after passage 5. this study examined, for the first time, rna recombination occurring in the absence of artificial selection pressures. it was surprising that the recombination sites within the l-kb stretch of the peplomer gene appeared to be almost completely random under these conditions. recombination was detected at the majority of the potential crossover sites; thus, there were no apparent sequence motifs or repeated nucleotides required for recombination, except in the recombinant noted above, which had a deletion accompanying the crossover. all of the recombination events examined were the result of homologous recombination, which preserved the open reading frame of the peplomer gene. one particular recombinant had a crossover between two adjoining nucleotides which differ between the two parental rnas (fig. 4a ). a few more had crossover sites with only 1 or 2 nt separating the divergent nucleotides between the parental viruses (figs. 4b-4d). therefore, extensive sequence homology between the two parental rnas at the crossover sites does not appear to be required for recombination. the same conclusion has been reached with poliovirus rna recombination (3). these results further support the observation that coronavirus rna undergoes recombination at an extremely high frequency (5, 19) . however, after only three virus passages, crossover sites became clustered within a small region, suggesting that certain rna crossover events may lead to more favorable recombinants. this is consistent with our previous finding that, when selection pressures (neutralization by monoclonal antibodies and temperature-sensitivity in replication) were used, all of the recombinants obtained had crossover sites localized between nucleotides 1148 and 1426 of the peplomer gene, even though the potential crossover region extended more than 1.5 kb (72). one possible reason for this selective localization of crossover sites is that certain regions combining to make the hybrid peplomer protein may be structurally incompatible so as to interfere with normal peplomer functions, such as binding to cellular receptors. thus, the recombinational "hot spot" observed previously (12) most likely was the result of functional constraints on some of the recombinants, rather than a limitation of recombination events caused by rna structure. since the clustering site of recombination events corresponded to a hypervariable region (12, 22) it is logical to propose that this protein domain is more tolerant of structural modifications. it is interesting that recombinant rna containing a deletion was detected only in intracellular rna but not in rna isolated from virions, suggesting that these recombinants could not replicate under the culture conditions. also, the brackets indicate the region in which the crossovers occurred. thick lines underneath the sequences represent jhm-dl-specific sequences, and thin lines represent a59-specific sequences. even at passage 3, approximately 20% of the crossover sites still fell outside the nucleotide 1000-l 350 region. this was to be expected since recombination could still occur between the remaining parental a59 and jhm-dl rna upon each reinfection. it should be noted that this study examined only a small region of the mhv genome. when the entire mhv rna is considered, it is possible that recombination may occur at different rates in different regions. nevertheless, this study provided an interesting implication that the failure to detect homologous rna recombination in other rnaviruses may be due to a stringent structural requirement of the viral proteins such that hybrid proteins have an evolutionary disadvantage. this may explain why nonhomologous recombination (as exemplified by di rna generation) can be detected readily, whereas homologous recombination has not been demonstrated for most rna viruses. in addition, it explains why recombination events have not been detected in the capsid protein region of fmdv (1). the approach described in this report should provide a useful tool to study these possibilities. proc. natl. acad. sc;. usa 87 institute. key: cord-310967-15mv5yx7 authors: morris, vincent l.; tieszer, christina; mackinnon, joanne; percy, dean title: characterization of coronavirus jhm variants isolated from wistar furth rats with a viral-induced demyelinating disease date: 1989-03-31 journal: virology doi: 10.1016/0042-6822(89)90048-2 sha: doc_id: 310967 cord_uid: 15mv5yx7 abstract murine hepatitis virus (mhv) can cause neurological disease when inoculated intracerebrally (ic) into mice and rats. specifically the jhm strain of mhv (mhv-jhm) generally causes an acute encephalitis when inoculated is into 2-day-old wistar furth rats. in contrast, jhm generally produces a chronic demyelinating disease with resulting posterior paralysis when inoculated is into 10-day-old wistar furth rats. in addition, while jhm readily produces a productive infection in a mouse fibroblast cell line (l-2), it does not form syncytia or replicate well in a tissue cell line of glial origin (g26-24). we have isolated and characterized three mhv-jhm viral variants from the central nervous system of two wistar furth rats with a mhv-jhm-induced demyelinating disease. the pattern of viral-specific mrna for all three of these variants differed from what was observed for the wild-type parental mhv-jhm that had been passaged only in tissue culture. one of these variants, atiif cord virus, which induced a chronic demyelinating disease in 2or 10-day-old intracerebrally inoculated wistar furth rats, had a deletion in the coding region of the peplomer glycoprotein mrna. in addition, this variant formed massive syncytia and replicated well in g26-24 cells. we have not detected this deletion in the other two jhm variants, atiif brain virus and atiie brain virus. atiif brain virus and atiie brain virus primarily produced an acute encephalitis when reinoculated into 2-or 10-day-old wistar furth rats. in addition, these two variants did not form syncytia and had a reduced ability to replicate in g26-24 cells. it has been recognized for decades that the coronavirus, murine hepatitis virus (mhv), can cause neurological disease in murine species (cheever et a/., 1949) . when 2-day-old wistar furth rats are inoculated intracerebrally (ic) with the jhm strain of mhv, most of the rats die within one week of inoculation with an acute encephalitis (sorensen et al., 1980; parham et a/., 1986) . in these rats, grey matter lesions generally predominate in the central nervous system (cns). when wistar furth rats are inoculated ic at 10 days of age with jhm, they generally do not develop symptoms until 2-4 weeks postinoculation (jackson et al., 1984; parham et a/., 1986) . these rats develop a chronic demyelinating disease characterized by hind leg paralysis or paresis (sorensen et al., 1980; jackson et al., 1984; parham et a/., 1986) . those rats that survive for longer than 3 weeks postinoculation generally have predominately white matter lesions. wild-type mhv subgenomic rnas produced in mouse fibroblast (l-2) cells have molecular weights of approximately 0.8, 1.1, 1.4, 1.6, 3, and 4 x 10" da (cheley et a/., 1981 a,b) . by convention, these subgenomit mrnas are numbered consecutively with the 4 x 10" da mrna being designated mrna 2 and the 0.8 ' to whom requests for reprints should be addressed. x 1 o6 da mrna designated mrna 7; the genomic size mrna is called mrna 1 (spaan et al., 1981; wege et a/., 1981) . the mrnas form a 3'-coterminal nested set extending for different lengths in a 5' direction (stern and kennedy, 1980a,b; cheley et al., 1981a; lai and stohlman, 1981; leibowitz et al., 1981; spaan et al., 1983; weiss and leibowitz, 1983) . the 5' end of each mrna not present in smaller mrna species contains the coding sequence utilized during the infection (leibowitz et al., 1982; . the mrnas each contain a leader sequence of approximately 72 bases at the 5'termini (lai et al., , 1984 spaan et a/., 1983) . this leader is encoded only at the 5' end of the genomic rna (lai et al., , 1984 spaan et al., 1983) . the free leader rna species is synthesized initially, dissociates from the negative-stranded template, and rebinds to the template at the initiation sites of the mrnas. the leader rna thus takes part in a leader-primed transcription (baric et al., 1983; makino et al., 1986) . using in vitro translation, it has been shown that the lowest molecular weight mrna codes for the nucleocapsid protein (rottier et al., 1981; cheley et al,, 1981 a) . the 1.1 x 1 o6 da mrna codes for the el glycoprotein, and the 3 x 1 o6 da mrna species codes for the e2 glycoprotein (rottier et al., 1981) . the nucleocapsid (n) protein has a molecular weight of approximately 56 kda (anderson et al., 1979; . the el glycoprotein has a molecular weight of approximately 24 kda and likely functions as a matrix protein (cheley and anderson, 1981; sturman and holmes, 1983) . the e2 glycoprotein is a heterodimer with a molecular weight of 180 kda (sturman et a/., 1985) . this molecule forms the projecting peplomers of the virus and its functions likely include attachment to cells, induction of cell to cell fusion, and elicitation of neutralizing antibodies (collins et a/., 1982; siddell et al., 1982; sturman and holmes, 1984) . mhv has been shown to have a high rate of recombination (lai et al., 1985) . the recombinants are derived at a high rate by a mixed infection of dbt cells with temperature-sensitive mutants of mhv-a59 and mhv-jhm at the nonpermissive temperature (lai et a/., 1985) . recombinant virus also arises at a high frequency in mouse cns tissue that is infected with a mixture of ts mutants of a59 and jhm (keck eta/., 1988a) . we have previously reported a truncated version of the e2 glycoprotein mrna is present in the cns of wistar furth rats with a jhm-induced demyelinating disease (jackson et al., 1984) . further work demonstrated that jhm e2 glycoprotein can be detected in individual cells of jhm-infected cns tissue; however, the ratio of detectable e2 antigen to nucleocapsid antigen in the total cns tissue of infected rats is reduced by more than 13fold compared with jhm-infected tissue culture cells (parham et al., 1986) . in these studies, virus was not isolated from these rats and characterized, and we did not explore the possibility that changes in the isolated virus could be correlated with the biological properties of this virus. in this paper we report the isolation of jhm viral variants from the cns of wistar furth rats with a jhm-induced demyelinating disease. we found that differences in the subgenomic mrnas produced by the viral variants and wild-type jhm were accompanied by differences in the biological properties of these viruses. the jhm strain of mouse hepatitis virus (mhv) was obtained from the american type culture collection (rockville, md). the jhm virus was plaque purified three times. virus was propagated at 37" in 1 x eagle's minimum essential media (emem) in 5% fetal calf serum on l-2 murine fibroblast cells (rothfels eta/., 1959) or on the g26-24 murjne oligodendroglioma cell line (sundarraj eta/., 1975; lucas eta/., 1977; bignami and stoolmiller, 1979) . two-or ten-day-old wistar furth rat pups (sprague-dawley, indianapolis, in) were inocu-lated with approximately 5 x 1 o4 pfu of virus (jhm or viral variants) in a 20-~1 ic inoculation. a 1 o-day-old wistar furth rat pup (designated atllf) was inoculated ic with a cloned isolate of the murine hepatitis virus (mhv) strain jhm. at 14 days postinoculation, the rat was severely runted and developed hind leg paresis. virus was recovered independently from the brain and spinal cord and designated atllf brain virus and atiif cord virus, respectively. we thus could directly compare two virus isolates recovered from a single inoculated rat pup. a littermate of atllf (designated atlle) was also inoculated ic at 10 days of age with the same cloned stock of jhm virus. at 13 days postinoculation, rat atlie showed symptoms similar to those observed in atllf and was killed. virus was isolated from the brain of rat atlle and was designated atlie brain virus. results obtained with atlle brain virus were generally similar to those obtained with atiif brain virus. mhv-jhm viral variants were isolated from the brain or spinal cord of the inoculated wistar furth rats using a modified procedure of sorensen et al. (1980) . specifically the tissue was minced into 1 x emem supplemented with 10% fetal calf serum to form a 20% (w/ v) suspension. this suspension was homogenized with a motorized dounce (g. k. heller corp., floral park, ny), and passed first through an 18-gauge needle, and then through a 28-gauge needle. the cell debris was pelleted by centrifugation at 915 g for 10 min. the virus was then pelleted from the supernatant at 64,800 g for 1 hr. the pellet was resuspended in 1x emem with 10% fetal calf serum. atllf cord virus was three times plaque purified. similar results were obtained with virus preparations before or after plaque purification. the viral variant strains appeared to be stable in culture since the pattern of viral-specific mrna and proteins remained constant with passage in culture. when the cns tissue of a mock-infected littermate of atllf and atlie was homogenized and used to inoculate cell cultures using the same procedures as was described to isolate the viral variants, no virus was recovered. preparation of tissue and extraction of rna rats were killed and the brain and spinal cord were removed; samples were taken for histopathology as previously described (jackson et a/., 1984) . rna was extracted from tissue or from tissue culture cells using an urea-lici extraction procedure (auffray and rougeon, 1980) . plasmid g344 with a 1800-bp mhv-specific insert (budzilowicz et al., 1985) was provided by dr. s. weiss (university of pennsylvania, philadelphia, pa). the cloned dna maps from approximately 200 bp into the nonstructural gene 4 to 200 bp into gene 7 (nucleocapsid) (budzilowicz et al., 1985) . this dna was labeled by the procedure of feinberg and vogelstein (1984) . the labeled dna was purified using a spun column procedure (maniatis et a/., 1982) . northern transfer analysis was performed using the procedures of thomas (1980) . two loo-mm petri dishes of l-2 cells were infected with virus (multiplicity of infection, 1.8 pfu). at approximately 100% syncytia formation, the culture was labeled with [35s]methionine (10 &i/ml) for 30 min (cheley and anderson, 1981) . tunicamycin (4 fig/ml of media) was added at 10 or 50% syncytia and left on until 100% syncytial formation (duksin and mahoney, 1982) ; similar results were obtained when the drug was added at 10 or 50% syncytia. the viral specificity of the 180 kda envelope glycoprotein for the wild-type jhm virus, atile brain viral variant, and atllf brain viral variant and the 165 kda protein for the atiif cord viral variant were confirmed by immunoprecipitation (francoeur and mathews, 1982) with polyvalent jhm-specific antiserum (parham et al., 1986) . viral variants (atiif cord, atllf brain, and atlie brain) were isolated from the brains and spinal cord of wistar furth littermates with a murine hepatitis virus (strain jhm)-induced hind leg paresis (see materials and methods for details). all of the viral variants and wildtype parental jhm were capable of forming massive syncytia in mouse fibroblast l-2 cells ( fig. 1 ; data not shown for jhm virus). however, atllf brain virus, atlie brain virus, and wild-type jhm virus-infected oligodendroglioma cells (g26-24) resembled uninfected g26-24 cultures except individual cells "rounded up" and lifted off from the monolayer ( fig. 1 ; data not shown for jhm virus). these infected cultures only rarely contained viral-induced syncytia. in contrast, atiif cord virus formed massive syncytia equally well in mouse l-2 and g26-24 cells (fig. 1 ). starting with a single stock of atllf cord virus, the ratio of the titer in g26-24 cells to the titer in l-2 cells was 0.472 (table 1) . however, the same ratio for atllf brain virus was 0.008, for atlie brain virus was 0.011, and for wild-type jhm was 0.010 (table 1) . therefore, the ratio of the viral titer in g26-24 cells compared with l-2 cells was approximately 50fold higher for atllf cord virus than for the brain virus variants and wild-type jhm (table 1) . our data with the wild-type jhm are in good agreement with previously published results (lucas et al., 1977) . ten-day-old wistar fur-th rats were inoculated intracerebrally with the viral variants. generally some littermates were inoculated with one viral variant while the remaining littermates were inoculated with a different viral variants for comparison. in general, the atiif cord viral variant produced a different pattern of disease than was observed with atllf brain virus and atlie brain virus. in 17 rats injected with the atllf cord virus, a more chronic demyelinating disease typified by hind leg paralysis developed; these rats died in an average time of 20 days (fig. 2) . in 19 injected rats, atllf brain virus generally produced a rapid encephalitis, which killed the rats in an average time of 9 days (fig. 2) . results similar to those observed with atllf brain virus were obtained in eight rats injected with atlie brain virus. in addition, a litter of wistar furth rats was inoculated ic at 2 days of age with either atiif brain virus or atllf cord virus. the three atiif brain virus-injected rats died in an average time of 5 days; this time course is similar to what has been reported for wild-type jhm virus (sorensen et al., 1980) . for the four littermates injected with atllf cord virus, the average time of death was 13 days. the uninjected control rat showed no symptoms. histopathological examination indicated that, in general, the white matter lesions were more extensive in the spinal cord and brain stem region (metencephalon and mesencephalon) in rats inoculated at 10 days with atllf cord virus when compared with rats inoculated at 10 days with atllf brain or atlie brain virus (table 2) . forty-six percent of atllf cord virus-inoculated rats had moderate white matter lesions in the spinal cord; in contrast, 93% of atllf brain virus-infected rats had either no lesions or only minimal lesions in the white matter of the spinal cord. the number of atllf cord virusinjected rats with moderate or marked white matter lesions in the metencephalon and mesencephalon was at least twice that observed in atiif brain virus-injected rats. the prosencephalon, which includes the cerebral hemispheres and is the most anterior portion of the central nervous system, was the only part of the brain in which the severity of the white matter lesions was generally greater in atllf brain virus-injected rats than in rats injected with atiif cord virus. lesions in the optic nerve of injected rats were mainly minimal. in addition, the gray matter lesions were predominantly minimal in most samples. the histopathology on samples from wistar fur-th rat pups inatllf brain g26-24 7.0 x lo4 0.008 atllf brain l-2 9.0 x 10" atile brain g26-24 8.5 x lo4 0.011 atlle brain l-2 7.5x lo6 jhm g26-24 4.9 x lo5 0.010 jhm l-2 4.9 x 10' a for each virus the identical virus preparation was used to inoculate both l-2 and g26-24 cells. ' l-2 cells are a mouse fibroblast cell line. g26-24 are an oligodendroglioma cell line. jetted with atlle brain virus was similar to the results obtained with the atiif brain virus-injected rats. the white matter lesions were characterized by vacuolation, hypertrophy, and hyperplasia of astroglial cells, and minimal to moderate mononuclear cell infiltration. gray matter lesions were characterized by destruction of neurons and astroglial cells, proliferation of endothelial cells lining capillaries, and mononuclear and polymorphonuclear cell infiltrations. since the jhm variants caused different patterns of neurological diseases in wistar furth rats, we examined the mrna synthesized by these viruses. northern transfer analysis indicated that viral mrnas 4, 5, 6 (el envelope glycoprotein), and 7 (nucleocapsid) comigrated for all the viral variants and jhm wild-type viruses ( fig. 3 ; data not shown for atlie virus). however, the atllf cord virus produced a truncated mrna 3 (e2 envelope glycoprotein) with a molecular weight of 2.85 x 1 o6 da; the atiif brain, atlie brain, and wild-type jhm viruses all produced e2 glycoprotein mrna of 3.0 x 1 o6 da which comigrate. in addition, atllf brain and atlie brain viruses each produced two novel mrna species (3.3 and 3.6 x 10" da). these mrna species were distinct from the 4.0 x 10" da mrna species seen with the wild-type jhm virus. we observed a uniform deletion of approximately 1.5 x 1 o5 da from the e2 glycoprotein and higher molecularweight subgenomic mrnas of atllf cord virus when compared with atllf and atlie brain viruses. because the e2 mrna was the lowest molecular weight mrna with a deletion and because of the nested set arrangement of the coronavirus mrnas (stern and kennedy, 1980a,b; cheley et al., 1981 a; weiss and leibowitz, 1983) one can conclude that the deletion occurred in the coding region of the e2 glycoprotein mrna of the atiif cord virus. the genomic size rna from wild-type jhm and jhm variants appeared to comigrate (fig. 3) . therefore no size change was detected in this rna species. this result is in agreement with the jhm variants isolated from lewis rats (taguchi et a/., 1985) . fig. 1. cytopathic effect (cpe) resulting from infecting cells in culture with murine hepatitis virusjhm variants. panels labeled with (a) represent mouse l-2 cells; panels labeled (b) show murine oligodendroglioma cells (g26-24). panels la and 1 b are uninfected. panels 2a and 2b are infected with atlie brain virus. panels 3a and 3b are infected with atllf cord virus. panels 4a and 4b are infected with atllf brain virus. note the syncytia present in g26-24 cells infected with atllf cord virus (36) but absent in g26-24 cells infected with atllf brain virus (4b) or atile brain virus (2b). to further investigate the possibility of a deletion in the e2 glycoprotein mrna of the atllf cord virus, we examined the e2 protein synthesized by variant viruses and wild-type jhm. the e2 glycoprotein produced by atiif cord virus had an apparent molecular weight of 165 kda while the e2 glycoprotein made by atiif brain, atile brain and wild-type jhm virus had an apparent molecular weight of 180 kda (figs. 4a and b). to confirm the difference between the atllf cord virus e2 glycoprotein and that of the wild-type jhm virus, we mixed protein samples from cells infected with each virus and subjected them to electrophoresis. the difference in the electrophoretic mobility of the 180 and the 165 kda protein was quite apparent (fig. 4a) . since the apparent molecular weight difference could be due to differences in glycosylation, we also compared the sizes of the e2 polypeptides synthesized in the presence of tunicamycin. the differences in the sizes of the e2 polypeptides for atllf cord virus and the jhm virus and brain virus variants were still apparent even when the virus was grown in the presence of tunicamycin ( fig. 4c ; data not shown for atlie brain virus). again the differences were confirmed by mixing experiments, this time between protein extracted from atllf brain virus and atiif cord virus-infected cells (fig. 4c) . the atiif cord virus thus had a deletion in its e2 mrna of approximately 150,000 da (fig. 3) and a deletion of approximately 15,000 da from the e2 protein (figs. 3 and 4). these deletions corresponded to a loss of approximately 130 amino acids from the e2 glycoprotein and approximately 390 nucleotides from the e2 mrna. these results thus support the idea that the e2 glycoprotein mrna produced by the atllf cord virus contains a deletion in its coding region. mhv-jhm is capable of inducing neurological disease with two possible outcomes when inoculated intracerebrally into wistar furth rats. if rat pups are inoculated at 2 days of age, a rapid, acute encephalitis generally occurs within 1 week of inoculation (sorensen et al., 1980; parham et al., 1986) . gray matter cns lesions are usually more extensive in these animals. however, when wistar furth rats are inoculated at 10 days of age, they generally develop a chronic demyelinating disease characterized by hind leg paralysis at approximately 2-4 weeks postinoculation (sorensen et abcdef fig. 3 . northern transfer analysis of rna extracted from virus-infected and uninfected tissue culture cells. rna was extracted from infected and uninfected cells, denatured with glyoxal, separated by electrophoresis in 1.1% agarose gels, transferred to nitrocellulose paper, and hybridized with a ihm-specific probe. the jhm homologous rna species were visualized by autoradiography. lane , 1980; jackson eta/., 1984; parham et al., 1986) . in this report, we investigated viral variants that arose in the cns of rats with a jhm-induced demyelinating disease and studied the effect of alterations in their mrnas. when lo-day-old rats were inoculated with atllf brain virus or atlie brain virus, the rats developed a rapid encephalitis instead of the more chronic demyelinating disease that has previously been seen with wild-type parental jhm virus (sorensen et al., 1980; jackson et al., 1984) and was observed with atiif cord virus-injected rats. in contrast, when 2-day-old rats were inoculated with atllf cord virus, the more chronic cns disease resulted instead of the rapid encephalitis that has been reported for wild-type jhm (sorensen et a/., 1980; parham et a/., 1986) and was observed with the atiif brain virus variant. therefore, the alterations observed in the mrnas of viral variants appeared to be important in determining the course of the viral-induced cns disease. it has been reported that recombinant virus can be recovered from the brains of mice that were infected with two different strains of mhv (keck et al., 1988a) . the recombination frequency was very high and recombination occurred at multiple sites on the viral rna genome (keck et al., 1988a) . furthermore, keck and co-workers (198813) have shown that the replacement of a59 genetic sequences at the 5'end of the e2 glycoprotein gene with the fusion-negative mhv-2 sequences do not affect the fusion ability of the recombinant viruses. they thus suggest that the 3' end of the e2 glycoprotein may be crucial for the fusion-inducibility of the virus. our variant virus strains may be useful in exploring this question, since we found that a deletion in the e2 glycoprotein mrnaof atllf cord virus was associated with the ability of the variant to induce fusion in a cell line of glial origin (g26-24). even though we inoculated the rats with cloned virus, we were able to recover viral variants from the cns of these inoculated rats which differ in their patterns of a e2.. mrnas from the wild-type parental virus. in addition, even though a rat (designated atiif) was inoculated at one site intracerebrally, we were able to isolate two separate variants from the cns of this rat. the virus isolated from the brain (atllf brain virus) produced acute encephalitis when reinoculated ic into wistar furth rats, and the virus isolated from the spinal cord (atllf cord virus) produced a chronic demyelinating disease with predominantly white matter lesions. thus the site of infection in the cns may result in a selection of variants with different physical and biological properties. in our experiments, the alterations in the mrna of the variants occurred during the infection of the cns by the virus and did not involve any in vitro selection by antibodies or other means. the major difference that was apparent between these variants was the deletion in the e2 glycoprotein mrna that was present in the atiif cord virus. the atiif cord viral variant induced a chronic demyelinating disease in 2-or lo-day-old intracerebrally inoculated wistar furth rats. the other variants (atllf brain virus and atlie brain virus) produced an acute encephalitis in either 2-or 1 o-day-old intracerebrally inoculated wistar furth rats. these results confirmed and expanded our previous results that the jhm rna species present in the cns of wistar furth rats with a jhm-induced demyelinating disease differ from what is seen in tissue culture cells infected with wild-type jhm virus (jackson eta/., 1984) . our earlier work also suggests that a truncated version of the jhm e2 glycoprotein mrna is present in rats with a jhm-induced demyelinating disease (jackson et al., 1984) . work by fleming uses monoclonal antibodies to the e2 viral glycoprotein to select antigenic variant viruses that escape neutralization in vitro (fleming et a/., 1986) . variants selected with one of the e2 monoclonal antibodies are highly virulent and causes an encephalitis in inoculated mice. a second selected variant predominantly causes a subacute paralytic disease clinically and extensive demyelinating histology. thus the e2 glycoprotein appears to be important in determining jhm pathogenesis in different systems. however, one cannot rule out that other factors or undetected genomic changes could also be involved. future work will investigate this question. atllf brain virus and atlie brain virus produced two novel high-molecular-weight rnas (3.3 x 1 o6 and 3.6 x 1 o6 da) which were not present in the wild-type jhm. these rna species might represent deletions in mrna 2. however, since this mrna codes for a nonstructural protein whose antiserum is not available, we have not been able to determine if the protein coded by mrna 2 is altered. in addition, the nucieotide sequence of the coding region for mrna 2 has not been published; when this information is available it will assist in determining the nature of the novel rnas produced by the brain virus variants. polymorphism has been observed in the e2 glycoprotein of coronaviruses (talbot and buchmeier, 1985) . sequencing analysis has revealed that 89 amino acids are present in mhv strain a59 but are absent in jhm (schmidt et a/., 1987; luytjes et al., 1987) . this difference is similar to the number of amino acids that are deleted from the e2 glycoprotein of the variant atiif cord virus when compared with either wild-type jhm or atiif brain or atlie brain virus. since the e2 protein is involved in the adsorption of the virus to cells and the induction of cell to cell fusion (collins et al., 1982; siddell et a/., 1982; fleming et al., 1983; sturman and holmes, 1984) it seems logical that changes in this protein could alter the ability of the virus to infect certain cell types. since atllf cord virus infection was associated with greater white matter involvement and chronic demyelinating disease, one would predict that the variant atiif cord virus would have an increased ability to infect glial cells when compared with atllf brain and atlie brain viral variants. in fact, we observed that atiif cord virus showed a marked increase in syncytiogenesis in an oligodendroglioma cell line (g26-24). furthermore, the ratio of the titer in g26-24 cells over the titers in l-2 cells was approximately 50-fold higher for atllf cord virus than it was for atiif brain or atlie brain virus. viral variants have also been recovered from lewis rats with a jhm-induced acute encephalitis (taguchi et al., 1985) . taguchi and co-workers have reported that these variants produce mrnas 2 and 3 which are approximately 500 bases larger than is reported for wildtype jhm. these variants also produce an envelope glycoprotein that is 15,000 da larger than is seen with wild-type jhm. however, no alterations are detected in the genomic size rna. the wild-type jhm and one of the variant viruses (~1-2) produce cell fusion in a continuous cell line of mouse origin (dbt). the titer for the wild-type virus was approximately 1 o-fold higher than for ~1-2. both jhm and cl-2 produce acute encephalitis in intracerebrally inoculated rats; however, less infectious cl-2 virus is required to produce an acute encephalitis. some similarities and differences can be seen between these experiments and those reported in this paper. ourjhm variants were isolated from a wistar furth rat with a viral-induced demyelinating disease instead of an encephalitis. variant atllf cord virus contains an apparent deletion in the coding region of mrna 3 instead of an insertion of extra nucleotides. curiously, the size of the insertion in mrna 3 for the variant cl-2 is very similar to the size of the deletion in mrna 3 for atllf cord virus. the relative location of these alter-ations in mrna 3 may help determine the significance of this observation. in addition, cl-2 and the variant viruses we have isolated all induce cell fusion and replicate well in continuous cell lines in which jhm also induces syncytia and produces a high titer of virus. however, we have extended these observations to show that atllf cord virus can replicate much better in a cell line of glial origin (g26-24) than does wild-type jhm. ci-2 and atllf brain virus and atlie brain virus do not contain deletions in their e2 envelope glycoprotein and produce encephalitis when injected into rats. in contrast, atllf cord virus does have a deletion in the e2 glycoprotein and produces a demyelinating disease in rats. these results are consistent with the hypothesis that a deletion in the viral e2 glycoprotein is associated with the ability of the virus to produce a demyelinating disease in rats. further work with additional variants will be necessary to test this hypothesis. finally, no alterations are detectable in the genomic size rna produced by the variants isolated by both taguchi and coworkers (taguchi eta/., 1985) and ourselves; however, all of the variants produce alterations which are readily detectable in mrna 3 and/or mrna 2. the lack of detectable alterations in the genomic size rna raises the possibility that the variants may not arise via a simple deletion or insertion of bases in the viral genome. the viral mrnas are generated by a leader-primed transcription which involves the fusion of noncontiguous transcripts (baric et a/., 1983; makino et al., 1986) . therefore, a possible mechanism for the generation of the variants involves an alteration in either the leader coding sequences or in one or more of the primer binding sites. recent studies suggest that the binding of leader rna to template rna during the synthesis of subgenomic mrna may not be precise even for wildtype jhm (makino et a/., 1988) . therefore, small alterations in either the primer or primer binding sites could result in changes in the size of subgenomic mrnas. nucleotide sequencing studies should help determine how these variants arise. the presence of jhm variants in ic inoculated rats appears to be a general phenomenon. it is hoped our work and the work of others will determine their role in viral-induced cns disease. comparison of polypeptides of two strains of murine hepatitis virus purification of mouse immunoglobulin heavy-chain messenger rnas from total myeloma tumor rna. fur characterization of replicative intermediate rna of mouse hepatitis virus: presence of leader rna sequences on nascent 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the biology of coronaviruses. 1 ln vivo and in vitro models of demyelinating diseases. ill. jhm virus infection of rats coronavirus mrna synthesis involves fusion of noncontinuous sequences isolation and identification of virus-specific mrnas in cells infected with mouse hepatitisvirus (mhv-a59) coronavirus multiplication strategy. i. identification and characterization of virus-specific rna coronavirus multiplication strategy. ii. mapping the avian infectious bronchitis virus intracellular rna species to the genome synthesis and subcellular localization of the murine coronavirus nucleocapsid protein proteolytic cleavage of peplomeric glycoprotein e2 of mhv yields two 90k subunits and activates cell fusion the molecular biology of coronaviruses proteolytic cleavage of the e2 glycoprotein of murine coronavirus: activation of cell-fusing activity of virions by trypsin and separation of two different 90k cleavage fragments biochemically differentiated mouse glial lines carrying a nervous system specific cell surface antigen (ns-1) characterization of a variant virus selected in rat brains after infection by coronavirus mouse hepatitis virus jhm antigenic variation among murine coronaviruses: evidence for polymorphism on the peplomer glycoprotein hybridization of denatured rna and small dna fragments transferred to nitrocellulose coronavirus jhm: characterization of intracellular viral rna. 1 characterization of murine coronavirus rna by hybridization with virus-specific cdna probes we are grateful to dr. s. weiss for the plasmid 9344. we also thank dr. j. maclnnes for reading the manuscript. this work is supported by a grant from the medical research council of canada, grant mt 7321 awarded tov.l.m., and nserc grant a0071 awarded to d.p. key: cord-324054-d71rj29o authors: zhang, xuming; kousoulas, konstantin g.; storz, johannes title: the hemagglutinin/esterase gene of human coronavirus strain oc43: phylogenetic relationships to bovine and murine coronaviruses and influenza c virus date: 1992-01-31 journal: virology doi: 10.1016/0042-6822(92)90089-8 sha: doc_id: 324054 cord_uid: d71rj29o abstract the complete nucleotide sequences of the hemagglutinin/esterase (he) genes of human coronavirus (hcv) strain oc43 and bovine respiratory coronavirus (brcv) strain g95 were determined from single-stranded cdna fragments generated by reverse transcription of virus-specific mrnas and amplified by polymerase chain reaction. an open reading frame of 1272 nucleotides was identified as the putative he gene by homology to the bovine coronavirus he gene. this open reading frame encodes a protein of 424 amino acids with an estimated molecular weight of 47.7 kda. ten potential n-linked glycosylation sites were predicted in the he protein of hcv-oc43 while nine of them were present in brcv-g95. fourteen cysteine residues were conserved in the he proteins of both viruses. two hydrophobic sequences at the n-terminus and the c-terminus may serve as signal peptide and transmembrane anchoring domain, respectively. the predicted he protein of hcv-oc43 was 95% identical to the hes of brcv-g95 and other bovine coronaviruses, and 60% identical to the hes of mouse hepatitis viruses. phylogenetic analysis suggests that the he genes of coronaviruses and influenza c virus have a common ancestral origin, and that bovine coronaviruses and hcv-oc43 are closely related. coronaviruses possess a single-stranded, nonsegmented rna genome of positive polarity (1, 2) and are associated with a variety of diseases in man and animals (3-5). coronaviruses are divided into two major antigenic groups. the first group includes human coronavirus strain oc43 (hcv-oc43), bovine coronavirus (bcv), mouse hepatitis virus (mhv), and hemagglutinating encephalomyelitis virus of swine (hev) (2, 5). hcv-oc43 causes respiratory infection of man similar to those of influenza viruses (6). bcv causes enteritis of newborn calves and is also considered to be an etiological factor of respiratory diseases of calves (7, 8) . mhv can infect different organs, causing enteric, respiratory, and neurological diseases (5, 9) . a unique property of coronaviruses within this antigenic cluster is the presence of the hemagglutinin/esterase (he) gene. the genome of mhv-a59 contains an open reading frame (orf), which may code for an he protein. however, the he is not expressed in infected cells (10, 11, 23 binding) and acetylesterase (receptor-destroying) activities similar to the he (or hef) glycoprotein of influenza c virus (icv) (12) (13) (14) (15) (16) (17) (18) (19) (20) . it was shown that the he glycoprotein of bcv strain quebec induces neutralizing antibodies both in vitro and in viva and thus, is important in viral infectivity (21, 22) . it is evidently not required for viral infectivity in mhv-a59 and mhv-jhm (11). the role of the he gene and its protein in coronavirus evolution, replication, and pathogenesis remains unclear. the exact genomic organization of hcv-oc43 is not known. antigenic and nucleic acid hybridization studies indicate that the hcv-oc43 is closely related to bcv (23-25). by analogy to bcv, the order of the genes coding for the structural proteins probably is 5'-he-s-m-n-3'. recently, the n gene of hcv-oc43 was sequenced, and it was found to be similar to bcv n gene (97.59/o amino acid sequence homology) (26). the origin and evolutionary relationships among the he genes of hemagglutinating coronaviruses isolated from different species are poorly understood. to elucidate the molecular evolution of the coronavirus he genes, we sequenced the he genes of hcv-oc43, a bovine respiratory coronavirus (brcv), a virulent, and an avirulent bcv strains. we report here the complete nucleotide sequence of the he genes of hcv-oc43 and brcv-g95, and their phylogenetic relatedness to bcvs, mhv, and icv. hcv-oc43 was obtained from the american type culture collection (atcc, 759-vr) and propagated in human rectal tumor (hrt-18) cells as described previ-ously (27). a bovine respiratory coronavirus strain giessen 89-4595 (g95) was kindly provided by dr. w. herbst, institute of hygiene and infectious diseases of animals, justus liebig university giessen, germany. this virus was originally isolated from nasal swabs of a calf suffering from respiratory disorder, and propagated in hrt-18 cells. isolation and purification of viral rna, cdna synthesis, double-stranded (ds) cdna amplification and single-stranded (ss) cdna production by polymerase chain reaction (pcr), as well as dna sequencing were performed as described previously (28, 44) . primers were designed to generate cdna fragments from virus-specific mrnas by reverse transcription and pcr amplification, based on the high degree of genomic similarity between hcv-oc43 and bcvs (25, 26). these primers were previously used for amplification and sequencing of bcv s and he genes (28,44). pcrgenerated cdna fragments were directly sequenced in both directions. analysis of the sequences revealed that a large orf of 1272 nucleotides was identical in size to the he genes of bcvs (29, 30, 44) . this orf terminated 14 nucleotides upstream from the s gene (zhang eta/., unpublished data) , and encoded a protein of 424 amino acid residues with an estimated molecular weight of 47.7 kda (figs. 1 and 2). two identical sequences (ctaaac), similar to the consensus intergenie sequence upstream of the hcv-oc43 n gene (ctaaat) (26) and identical to the consensus sequence upstream of bcv he and s genes, were found 16 nt upstream of the predicted initiation codon (at nucleotides 16 to 18) for the he protein and 8 nt downstream from the termination codon, respectively. hydropathic analysis of the predicted amino acid sequence indicated that the putative he protein possessed the characteristics of a membrane protein. specifically, a hydrophobic stretch of 15 amino acids at the n-terminus may serve as signal peptide with a cleavage site between amino acids 15 and 16 (30, 31, 44) . another hydrophobic amino acid sequence near the c-terminus (amino acids 389 to 414) may serve as the transmembrane domain anchoring the protein in the viral envelope. a hydrophilic sequence of 10 amino acids at the c-terminus may serve as an intravirion-domain. ten potential a/-linked glycosylation sites were predicted in the he protein of hcv-oc43 while nine of them were present in that of brcv-g95. two internal orfs were predicted within the large orf extending from nt 107 to 517 and from 976 to 1228. by analogy to bcv he gene, these results suggest that the predicted large orf 2b represents mrna 2-l of hcv-oc43 and brcv-g95, encoding the he glycoprotein. the predicted amino acid sequences of the he genes from hcv-oc43 and brcv-g95 (fig. l) , bcv(29), bcv-l9, bcv-ly138 (44), mhv-a59 (io), mhv-jhm (11) were aligned using the programs of the university of wisconsin genetic computer group software package, version 6.1. the alignment revealed that the he gene of hcv-oc43 was more closely related to brcv-95 and bcvs than to mhvs. nucleotide and amino acid sequences among hcv-oc43, brcv-g95, and bcvs were 95.8 to 96.3% and 94.1 to 94.8% identical, respectively, while the amino acid sequence identity between hcv-oc43 and mhvs was approximately 60%. fourteen cysteine residues were strictly conserved in the he proteins of hcv-oc43, brcv-g95, bcvs, and mhvs. the mhv-jhm had 15 amino acids and two cysteine residues more than hcv-oc43 and brcv-g95. the alignment indicated that the eight he genes among coronaviruses can be divided into two groups. the first group includes hcv-oc43, brcv-g95, and all bcvs, and the second group includes mhv-jhm and mhv-a59. to identify a possible evolutionary pathway for the he gene of coronaviruses, we compared the coronavirus he genes with the icv he gene. an alignment of the predicted amino acid sequences is shown in fig. 2 . in this alignment, the icv he1 subunit shows a sequence identity of approximate 28.2% with the he protein of hcv-oc43, brcv-g95, and bcvs, and 26.3% with the he protein of mhvs. the alignment shows that several regions are completely identical. most importantly, the putative acetylesterase active site (f-g-d-s) (at amino acids 72 to 75 in fig. 2) is conserved in all he proteins of human, bovine, and murine coronaviruses and icv. ten of the 14 cysteine residue positions of hcv-oc43 are conserved among all he proteins compared. these data suggest that these proteins may be evolutionarily related to each other. dna sequences for each gene were optimally aligned based on the alignment of their respective amino acid sequences (fig. 2) . a maximum parsimony analysis was per-formed on the aligned dna sequences to predict possible phylogenetic relationships among coronaviruses (detailed methodology for the phylogenetic, computer-assisted analysis is described in the legend of fig. 3 ). cladistic analysis of the dna sequence data resulted in a single phylogenetic hypothesis (phylogram) with a total length of 1503 steps and a resealed consistency index of 0.962. this analysis suggested that all coronaviruses were divided in two clades. one clade included hcv-oc43, brcv-g95, and bcvs. the other clade consisted of mhv-jhm and mhv-a59. neither coronaviral clade is derived from the other. within the clades, all bcvs were closely related taxa to hcv-oc43, and the mhv-jhm and mhv-a59 were sister taxa. the phylogram sug-g tc ga ctaaactcagtgaaaa tgtttitgctl'cc!xgatltattctagttagctgcataattggtagcitaggttlttacaaccckctaccaatgttg'lltcgc ****** metpheleuleuproarqpheileleuvalsercysileileglyser~uglyphe~r~nprop~o~rasnvalvalserh """""""""""""""""""""""""""""""""""""""""""""""""""""" -ualaphephetrpalaleuarqleu---__""____"_"""""""_""""""""""""""""""""""""""""""-"-""""""""" the sequences for bcv-l9, bcvly138 were obtained from recent work (44). gested a common ancestor of this antigenic group of and assuming icv as outgroup. the highly variable recoronaviruses. the highly cell-adapted strains bcv-gions (positions 19-l 85, 235-365,451-l 046, 1255 -l9, bcv-quebec, and bcv-mebus are closely related 1295 , 1351 -1430 , and 1459 -1503 were excluded to the wild-types bcv-ly138 and brcv-g95. we ex-because they were not aligned with confidence. we cluded these strains in the final phylogenetic analysis, identified 125 phylogenetically informative sites (varibecause their close relationships resulted in collapsed able sites with at least two taxa potentially sharing a branches in the phylogenetic tree. we further at-derived base) from 439 aligned base positions. the tempted to analyze the relationship among the he phylogram shows an almost identical topology for the genes of selected coronaviruses and icv based on coronaviral ingroup obtained by the previous analysis these results using limited dna sequence information, (fig. 3) . (io), and bcv-ly138 (44) and a partial he gene sequence of icv (32) were used for this phylogram. the dna sequences were aligned based on their deduced amino acid sequence alignment as shown in fig. 2 since the hcv-oc43 and icv infect similar tissues in human subjects, the significant sequence homology between the he genes of the two viruses suggests that coinfection of an ancestral coronavirus with icv followed by recombination may have given rise to hcv-oc43. this was also proposed by luytjes et al. (10) . phylogenetic analysis also suggests that the he genes of coronaviruses and icv may originate from a common ancestor. it is worth noting that the he protein of icv contains receptor-binding, acetylesterase and fusion activities while that of coronaviruses contains only the receptor-binding and acetylesterase activities. the fusion function of icv is associated with the /v-terminal hydrophobic region of the he2 subunit of the he protein (17) (18) (19) 32) . a similar hydrophobic domain was not found in the coronavirus he protein. the high similarity between the he proteins of hcv-oc43 and bcvs (95% identity on the average) suggests that both viruses are very closely related. this hypothesis is also supported by the tree branch distance in the phylogenetic analysis shown in fig. 3 . interestingly, the he of hcv-oc43 is more closely related to those of brcvg95 and the wild-type, virulent strain bcv-ly138 than to that of the cell-culture adapted avirulent strain bcv-l9. the wild-type strain bcv-ly138 does not replicate in numerous bovine cells in vitro, but it grows readily in human cells (hrt-18) without requiring prior adaptation (27, 33, 34) . since these polarized human cells retain many features of primary epithelial cells, infection by bcv suggests that bcv may also infect humans, and therefore, it is a zoonotic virus (23, 35). we previously reported a case of human diarrhea caused by bcv-ly 138, in which the virus was identified from the infected patient (35). recently, we found that brcv-g95 exhibited almost identical cytopathology in vitro to the wild-type virulent strains bcv-ly 138 (data not shown). the hcv-oc43, bcvs, and brcv-g95 differed only in few amino acids in the he, and their putative acetylesterase active sites were conserved (see fig. 2 ). 0-acetylneuraminic acid was shown to be the major determinant for icv (36-38). hcv-oc43, bcvs, and brcv-g95 probably recognize this receptor on the surface of many different epithelial cells. they may be able to replicate in epithelial cells of both respiratory and intestinal tracts, and to cross species-barriers causing diseases in heterologous hosts. however, hcv-oc43 primarily causes respiratory diseases and bcvs cause enteritis. the ability of these viruses to replicate in different organs and to cause different clinical symptoms is probably due to multiple amino acid differences occurring within several viral proteins. the s protein of mhv was shown to be important in tissue tropism (39). recently, it was reported that turkey enteric coronavirus is antigenically and genomically closely related to bcvs (40) and similar functions were found in the he protein of hev (20). whether swines or turkeys may also serve as reservoir (mixing-vessel) for coronavirus recombination in nature, as it was proposed for influenza a viruses (41) remains to be elucidated. it is worth noting that icv was also isolated from pigs (42). it will be worthwhile to compare the he genes among these coronaviruses. comparison of the remaining genes with hcv-oc43 and bcvs will provide further insight into their evolution and host cell tropism. proc. nat/. acad. sci. usa proc. nat/. acad. sci. usa key: cord-315158-f6msh8od authors: taguchi, fumihiro; flemingt, john o. title: comparison of six different murine coronavirus jhm variants by monoclonal antibodies against the e2 glycoprotein date: 1989-03-31 journal: virology doi: 10.1016/0042-6822(89)90061-5 sha: doc_id: 315158 cord_uid: f6msh8od abstract we have examined six different jhmv variants, sp-4 (recloned wt jhmv), cl-2, cnsv, dl, ds, and jhm-x, in terms of the sizes of the mrna3 and e2 glycoprotein as well as their reactivity to a panel of monoclonal antibodies to the e2 glycoprotein. two of these variants, sp-4 and jhm-x, were found to have smaller mrna3 and e2 glycoprotein species compared with those of the other four variants. in addition, sp-4 and jhm-x were distinguished from the other four variants by their inability to bind to monoclonal antibodies recognizing two antigenic domains of the e2 molecule. thus, six jhmv variants could clearly be divided into two groups with respect to the size and antigenicity of their e2 glycoproteins. mouse hepatitis virus (mhv) belongs to the coronaviridae whose name is based on the characteristic morphology of petal-shaped spikes of these viruses (i). they are enveloped viruses with a large nonsegmented, positive-stranded rna genome (2, 3). mhv produces seven to eight discrete mrnas. they form a 3'-coterminal nested set extending different lengths in 5' direction and each mrna has a common 5'-leader sequence of about 70 bases (4, 5). in mhv-infected cells, three major viral proteins are detected. a nucleoprotein (n) of ca. 60,000 da is the most abundant and is translated from mrna7 (6-8). two other glycoproteins, el and e2, of ca. 23,000 and 120,000 to 160,000 da, are translated from mrnas 6 and 3, respectively (6-8). among these three viral structural proteins, the e2 or peplomer glycoprotein has been shown to have particularly interesting biological activities (9-11). it is believed that the fusion of cells infected with mhv is caused by the e2 glycoprotein, which is fusogenie only after it has been cleaved proteolytically (12, 13) . another important biological function of the e2 glycoprotein is thought to be its binding to receptors on the cell surface as an initial step in virus replication in cells (10). the e2 glycoprotein is also believed to be involved in the pathogenesis of mhv on the basis of studies on monoclonal antibodies ( 74-7 7) and variant viruses isolated from persistent infections (18). recently, we have shown that the highly virulent variant viruses with larger e2 glycoproteins were preferentially isolated from rat brain (19) and cultured astrocytes (20) after infection by wild-type (wt) jhmv which contains a small mrna3 and e2 glycoprotein. these re-' to whom requests for reprints should be addressed. sults suggest that there are at least two different types of jhmv with respect to the sizes of their e2 glycoproteins. it is of interest, therefore, to compare the e2 glycoproteins of various jhmv variants. in this communication, we report the comparison of six different jhmvs obtained from different laboratories with respect to the sizes of their mrna3 and e2 glycoproteins as well as their reactivities to a panel of monoclonal antibodies against the e2 glycoproteins. the variant viruses used for the present experiment were cl-2 and cnsv isolated by passaging wt jhmv in rat brain and primary glial cell cultures, respectively, as previously reported (79, 20) and kindly provided by dr. ter meulen, university of wurzburg: sp-4, which is a wt jmhv orginally maintained in germany and which had been subjected to four cycles of plaque-purification on dbt cells; dl and ds, which are plaque morphology variants of wt jhmv (21) kindly provided by dr. stohlman, university of southern california: and jhm-x (22), which is a derivative of wt jhmv maintained in japan and was kindly provided by dr. makino at the same university. first, a comparison of the mrna patterns of the six different jhmv variants was made, focusing on the size differences of mrna3. rna was extracted from dbt cells infected with each of the six jhmv variants, electrophoresed on agarose gels, and transferred onto nitrocellulose filters. virus-specific mrnas were detected by hybridization with 32p-labeled cdna prepared from mrna7 of jhmv as previously reported (23). as shown in fig. 1 , there were at least eight distinct mrnas with different mobilities on agarose gels. there were no substantial differences in the mrna patterns among the six different jhmv variants except for the sizes of mrnas 2, 2a, and 3. the variants sp-4 and jhm-x produced mrna 2, 2a, and 3 of smaller sizes (ca. 150,000 da) compared with those produced by other variant viruses. in addition to these differences in the sizes of particular mrnas, a striking difference in the amount of mrna2 produced by cnsv was observed. in all other variants, mrna2 was shown to be more abundant than mrna2a; however, cnsv produced an excess of mrna2a. next, we compared the sizes of the e2 glycoproteins produced by the six different jhmv variants. in addition, we examined the sizes of n proteins as control, because mrna7, encoding n protein, was not different in length among the six viruses. cell lysates prepared from dbt cells infected with each of variants were im3 . antigenic comparison of the structural proteins of the six different jhmv variants. major antigenic domains "a." "b," and "not-a, not-b" on the e2 glycoprotein were determined by competitive binding studies, as previously described (75, 26). antigenic relatedness of the different variants was determined by solid-phase elisa using the panel of monoclonal assays, each employing triplicate samples. binding is expressed as a percentage of the optical density relative to the dl strain, the virus used to produce the monoclonal antibodies. the blocks represent greaterthan 80% binding(m) or less than 15% binding (cl); in + an intermediate result was found. of the anti-e2 monoclonal antibodies, 1.2.2 is directed to site "b," j.7.1 to a "not-a, not-b," and all the rest are against site "a." munoprecipitated with a monoclonal antibody against the e2 glycoprotein and n protein, and these proteins were analyzed by sds-polyacrylamide gel electrophoresis as described elsewhere (24). as shown in fig. 2 , there was a clear difference in the mobility of the e2 glycoprotein among the six variants. e2 glycoproteins produced by sp-4 and jhm-x, both of which synthesized a small mrna3, were shown to be approximately 15,000 da smaller than those produced by the other variant viruses with larger mrna3.s. no significant differences were observed in the sizes of n proteins produced by the variants. the antigenic properties of the e2 glycoproteins of the variants were determined by testing their binding to a panel of monoclonal antibodies produced by immunization with the dl strain as previously reported (25). as shown in fig. 3 , the monoclonal antibodies uniformly had excellent binding to all the viruses tested, with the exception of sp-4 and jhm-x, the two variants with small mrna3s and e2 proteins. these two variants did not bind to monoclonal antibodies recognizing two major antigenic regions on the peplomer, which have been designated e2(a) and ez(b). monoclonal antibody j.7.1, recognizing a third site on e2, binds all viruses with essentially equal intensity, indicating that the e2 molecule is present in normal amounts in all variants tested. all monoclonal antibodies to n protein and one to e 1 protein bound strongly to all six strains. however, the other monoclonal antibody to the el protein, j.2.7, did not bind variants ~1-2, cnsv, and sp-4, showing that j.2.7 can distinguish variants maintained in west germany from other jhmvs. these results suggest that the determinants e2(a) and e2(b) are either antigenically altered or deleted in variants with small e2 glycoproteins. the second possibility seems more likely, in view of the uniformly high binding of the monoclonal antibodies to variants with large e2 proteins and the consistent lack of any binding of the antibodies to variants with small e2 glycoproteins. it seems very likely that these antigenic determinants reside in the domain consisting of a ca. 15,000-da protein region in large e2 glycoproteins which is missing in small e2 glycoproteins. it was recently reported (26) that the recombinant viruses having approximately two-thirds of the jhmv mrna3 coding region at the 5'-end and onethird of a59 mrna3 at the 3'-end have lost their reactivity to monoclonal antibodies recognizing e2(a) and e2(b) of the jhmv e2 moleucle, indicating that these two antigenic domains are likely encoded by one-third of the mrna3 of jhmv at the 3'-end. from such observations, it may be speculated that ca. 500 bases found only in larger mrna3s are located in one-third of the mrna3 at the 3'-end. at present, we are trying to obtain cdna which encode the larger e2 protein in order to localize the e2 domains specifically recognized by e2(a) and e2(b) monoclonal antibodies. it has recently been reported that only the viruses with the larger e2 glycoprotein were isolated from the rat brain after infection with wt jhmv with a small e2 glycoprotein (19). the isolated virus, ~1-2, was shown to replicate very well in the rat brain, causing severe encephalitis. this indicates that the viruses with the larger e2 glycoprotein may have a growth advantage in rat brain, resulting in acute and semiacute encephalomyelitis. experiments are now in progress to determine the relationship between the size of e2 glycoproteins and neurovirulence of jhmv for rats. proc. nat/. acad. sc;. usa 61 proc. nat/. acad. sci. usa we are grateful to dr. siddell for providing the cdna used for northern blot analysis and dr. nakanaga for the monoclonal antibod-ies used for immunoprecipitation.we are also grateful to drs. stohlman and lai for helpful suggestions and criticisms. key: cord-313091-ksrxsdpp authors: shirato, kazuya; kawase, miyuki; matsuyama, shutoku title: wild-type human coronaviruses prefer cell-surface tmprss2 to endosomal cathepsins for cell entry date: 2017-12-06 journal: virology doi: 10.1016/j.virol.2017.11.012 sha: doc_id: 313091 cord_uid: ksrxsdpp human coronaviruses (hcovs) enter cells via two distinct pathways: the endosomal pathway using cathepsins to activate spike protein and the cell-surface or early endosome pathway using extracellular proteases such as transmembrane protease serine 2 (tmprss2). we previously reported that clinical isolates of hcov-229e preferred cell-surface tmprss2 to endosomal cathepsin for cell entry, and that they acquired the ability to use cathepsin l by repeated passage in cultured cells and were then able to enter cells via the endosomal pathway. here, we show that clinical isolates of hcov-oc43 and -hku1 preferred the cell-surface tmrrss2 to endosomal cathepsins for cell entry, similar to hcov-229e. in addition, the cell-culture-adapted hcov-oc43 lost the ability to infect and replicate in air-liquid interface cultures of human bronchial tracheal epithelial cells. these results suggest that circulating hcovs in the field generally use cell-surface tmprss2 for cell entry, not endosomal cathepsins, in human airway epithelial cells. human coronaviruses (hcovs) are causative agents of human common colds, and most people experience initial infection during childhood (dijkman et al., 2008) . to date, four hcovs (229e, nl63, oc43 and hku-1) have been identified. the first two belong to alphacoronavirus and the latter two to betacoronavirus. hcov-229e was first reported in 1966 (hamre and procknow, 1966) and the isolate of the day is widely used as the laboratory strain of the american type culture collection (atcc, vr-740). hcov-229e enters cells via two distinct pathways: the endosomal pathway using cysteine protease to activate spike (s) protein and the cell-surface or early endosome pathway using extracellular proteases for activation. studies using the atcc isolate suggest that hcov-229e enters cells via the late endosome using cathepsin l to cleave s protein, although it can enter cells via the cell surface or early endosome in the presence of transmembrane protease serine 2 (tmprss2) or trypsin (bertram et al., 2013; kawase et al., 2009) . it has been thought that this is a general feature of hcov-229e entry. however, we recently reported that clinical isolates of hcov-229e preferred the cell-surface or early endosome pathway to the late endosome pathway for cell entry (shirato et al., 2017) . the ability of a clinical isolate of hcov-229e to use cathepsin l was originally low, but it became able to use cathepsin after repeated passage in hela cells (shirato et al., 2017) . this suggests that in natural situations hcov-229e enters respiratory epithelial cells via the cellsurface or early endosome route and that the character of atcc vr-740 has been changed by repeated passage in cultured cells. in recent years, a culture method for human bronchial tracheal epithelial (hbte) cells that uses an air-liquid interface (ali) has been developed and has come to be used as a model of the in vivo situation of human airway epithelium (hae) (fulcher et al., 2005) . using this system, hcov-hku1, which had previously been reported from sequence data, was first isolated (pyrc et al., 2010) , and it was later shown that all four hcovs could be isolated upon hbte-ali cell culture . in the present study, we found that field isolates of hcov-oc43 and hcov-hku1 could be isolated using hbte-ali cell culture, and we then used these clinical isolates to assess whether the mode of virus entry found in hcov-229e was also in play in other hcovs. for isolation of hcovs, nasal swabs were collected from outpatients who showed respiratory infection as a cardinal symptom when assessed at a hospital in tokyo, japan. specimens diagnosed as containing hcovs by real-time rt-pcr were used for virus isolation. recently, the circulating hcov strain in japan has been biased toward hcov-oc43 (hara and takao, 2015; matoba et al., 2015; yano et al., 2014) . therefore, we did not obtain specimens positive for hcov-229e or hcov-nl63; however, four hcov-oc43 and two hcov-hku1 isolates were finally isolated successfully (table 1 ). the sequences of s protein were analyzed and deposited in genbank (accession nos. lc315646 to lc315651). the number of cycles of real-time rt-pcr to which virusisolated specimens were subjected ranged from 18.25 to 26.71, and isolation was considered failed when more than 30 cycles were used (data not shown). this suggests that at least 10 4 copies of viral rna are required for successful isolation by hbte-ali culture. similar to hcov-229e, hcov-oc43 was identified between the late 1960s and 1970 (bruckova et al., 1970; mcintosh et al., 1967a mcintosh et al., , 1967b . the isolate at that time had been passed in suckling mouse brain and cell culture, and finally became widely used as a laboratory strain (atcc vr-1558). as previously described, the entry mechanism of the clinical isolate of hcov-229e differed from that of the cell-cultureadapted laboratory isolate, atcc-vr740; the clinical isolate preferred the cell-surface tmprss2 route to the endosomal cathepsin route preferred by the atcc isolate (shirato et al., 2017) . therefore, we evaluated differences in infectivity and replication in culture cells or hbte-ali culture using vr-1558 and the clinical isolate (sgh-36/ 2014) (fig. 1) . in hct-8 cells, vr-1558 infected and replicated well, as expected. on the other hand, the amounts of sgh-36/2014 virus entering hct-8 cells were much lower than the amounts of vr-1558, and replication of sgh-36/2014 was about 10 5 lower than that of vr-1558 (fig. 1a) . in hbte-ali culture, the result was completely reversed; sgh-36/2014 infected the cells and replicated well as expected. in contrast, vr-1558 could infect the hbte-ali culture. though the amount of entered virus was much lower compared to sgh-36/2014, no viral rna was detected in the cell wash 3 days after infection (fig. 1b) . this indicates that vr-1558 infected the hbte-ali culture with low efficiency, but it failed to produce progeny virus. in the previous report, a 20-times-passaged clinical isolate of hcov-229e exhibited changes that allowed it to replicate well in hela cells, but its ability to replicate in hbte-ali culture decreased relative to that of the original isolate (shirato et al., 2017) . the present study indicates that the phenomenon seen in hcov-229e is also present for hcov-oc43; namely, cell-culture-adapted virus cannot replicate in hbte-ali culture, which mimics human airway epithelial cells. in the case of hcov-229e, the clinical isolate enters cells via the cell-surface or early endosome route using tmprss2, and the atcc isolate vr-740 tends to enter cells via the late endosome pathway using cathepsin l (bertram et al., 2013; kawase et al., 2009; shirato et al., 2017) . the former route can be inhibited by the serine protease inhibitor camostat, and the latter by the cysteine protease inhibitor est (e64d) shirato et al., 2017 shirato et al., , 2013 . to evaluate the entry routes of clinical isolates of hcovs, viruses were inoculated onto hbte-ali in the presence of est or camostat (10 μm) and the amounts virus that entered were estimated by detecting subgenomic mrnas using real-time rt-pcr (fig. 2) . first, the effect of inhibitor treatment on cell viability was determined in all cells. the treatments did not affect survival of the cell cultures at this concentration (fig. 2a) . infection of hcov-oc43, vr-1558, was inhibited only by est and not by camostat (fig. 2b ). this indicates that vr-1558 enters cells via the late endosome pathway using cysteine proteases and it does not use tmrpss2 for cell entry in hbte-ali culture. in contrast, clinical isolates of hcov-oc43 were inhibited by camostat but were either not inhibited or were only partially inhibited by est ( fig. 2c-f ). in the case of sgh-36/2014, est increased entry (fig. 2c) . for clinical isolates of hcov-hku1 (similar to hcov-oc43), entry was inhibited only by camostat and est tended to increase entry ( fig. 2g and h) . as shown in fig. 2a , treatment with inhibitors did not affect cell viability. this suggests that the reduced infection of vr-1558 by est and clinical isolates by camostat were induced by the inhibition of proteases, not k. shirato et al. virology 517 (2018) 9-15 inhibitor treatment. these data are identical to those derived using hcov-229e (shirato et al., 2017) and suggest that the clinical isolates of hcovs that cause human infections enter cells via the cell-surface or early endosome pathway using cell-surface proteases such as tmrss2. the data also suggest that entry of cells via the late endosome using cathepsins is the case only for cell-culture-adapted or long-term-passaged isolates of hcovs. moreover, the increased entry of some clinical isolates in the presence of est suggests that entry of cells via the late endosomal pathway induces some anti-viral host immunity. the expression of tmrss2 in hbte-ali and hct-8 cultures was confirmed by mrna quantitation (fig. 3a) . the expression of tmrpss2 mrna was found in calu-3 and lovo cells, which are known to be susceptible to middle east respiratory syndrome (mers) coronavirus (shirato et al., 2013; tao et al., 2013) . tmrpss2 expression was highly up-regulated and was prominent among cells tested in differentiated hbte-ali cultures. in hct-8 cells, tmrpss2 mrna was detected, but the expression level was lower than in immature hbte cells. almost no tmrpss2 was detected in hela cells. this suggests that the reduced infection and replication of hcov-oc43 clinical isolates in hct-8 cells were caused by the low level of tmprss2 expression relative to hbte-ali cultures. that cathepsins use hcov-oc43 for entry into hct-8 cells was confirmed by an entry inhibition assay similar to that in hbte-ali cultures ( fig. 3b and c) . the entry of vr-1558 in hct-8 was inhibited only by est treatment at both 10 and 100 μm (fig. 3b) . in sgh-36/ 2014 infection, a high concentration (100 μm) of est inhibited entry (fig. 3c) . in contrast, camostat treatment did not show any inhibition in terms of vr-1558 and sgh-36/2014 in hct-8 infection; however, hctk. shirato et al. virology 517 (2018) 9-15 8 cells expressed tmprss2 mrna. these findings suggest that the expression of tmprss2 in hct-8 cells is insufficient for hcov-oc43 entry from the cell surface, and also suggest that cathepsins are used for hcov-oc43 entry into hct-8 cells. for many years, isolation of hcovs was considered difficult (peiris and poon, 2009; tyrrell and myint, 1996) . the first isolation of hcov-229e and hcov-oc43 was reported in the mid-1960s to 1970 using repeated passage in cultured cells or suckling mouse brains (bruckova et al., 1970; hamre and procknow, 1966; mcintosh et al., 1967a mcintosh et al., , 1967b . later, some studies reported isolation of hcovs using llc-mk cells, monkey kidney cells, and human embryo fibroblasts van der hoek et al., 2004) . our group and others reported that addition of an external protease, such as trypsin, and a transmembrane serine protease, tmprss2, enhanced the entry and replication of hcovs (bertram et al., 2013; kawase et al., 2009 kawase et al., , 2012 . for hcov-229e, addition of these proteases resulted in successful isolation (hirokawa et al., 2008; matoba et al., 2016) . however, no reliable cell line for isolation was available, unlike (for example) the mdck cells used to isolate influenza viruses. the results of our present study show that the reported difficulty in isolating hcovs is a natural phenomenon because hcovs circulating in the natural world do not enter cells via the late endosome pathway using cathepsins. it is difficult for the viruses to grow in commonly used cultured cells, which have only the late endosomal pathway. to date, some hcovs have been isolated using cell culture as described above, but it may be that these results were rather accidental. sometimes, particular features of the cultured cells may have affected the results, and, at other times, quasispecies may have been selected because of their ability to use cathepsins for cell entry. such forced selection does indeed produce hcovs, but the character of the viruses is changed from their natural state. in this study, inhibition of the late endosomal pathway by est increased the entry of some clinical virus isolates. this suggests that cell entry via the late endosomal pathway induces some anti-viral host immunity. indeed, bertram et al. (2013) reported that expression of interferon-induced transmembrane proteins (ifitms) that inhibit diverse enveloped viruses also inhibited the entry of pseudotyped virus bearing hcov-229e s protein, and that the reduced entry was rescued by co-expression of tmrpss2. it is thought that ifitms exert antiviral effects at the late stage of entry (desai et al., 2014; li et al., 2013; munoz-moreno et al., 2016) . thus, entering cells via endosomal cathepsins seems not to afford advantages for virus survival. therefore, hcovs circulating in the field may have evolved to not use the late endosomal route with cathepsins to avoid such unfavorable reactions by host cells. in addition, hcov-oc43 used cathepsins for entry into the cultured cell line hct-8. however, in this study est treatment could not inhibit viral entry completely, even at a high concentration (100 μm). previously, we reported that the sirna-mediated gene knockdown of cathepsin l could not inhibit the entry of hcov-229e completely, suggesting that other proteases are also used by hcov-229e in the process of infection (kawase et al., 2009) . moreover, treatment with est, camostat, and leupeptin in wi-38 and mrc-5 cells (human fetal pulmonary fibroblasts without tmprss2 expression), did not inhibit infection by mers-cov and hcov-229e. this suggested that proteases other than cysteine, serine, and threonine might be used for entry into these immature cells (shirato et al., 2013) . therefore, although cathepsins are used during the entry process of hcov-oc43 in hct-8 cells, proteases other than cathepsins and tmrpss2 are also used for the activation of s protein (like other human coronaviruses). development of the hbte-ali cell culture method allowed successful isolation of hcovs, probably in their natural state pyrc et al., 2010) . for the development of antiviral drugs, it is necessary to understand virus characteristics, and isolation of the virus is the first step. therefore, it is important to isolate a virus that has natural characteristics. madu et al. (2009) reported that a common motif (very highly conserved region, vhcr) is found in the s protein of coronavirus and may be a possible fusion peptide. it was thought that recognition sites for cleavable proteases might exist around the fusion peptide. in a previous study, we showed that the two amino acids that differ between laboratory and clinical isolates of hcov-229e (r642m and n714k) were the determinants of the difference in cell entry route. these sandwich the vhcr, and were therefore not thought to be protease recognition sites. however, substitution of these two amino acids between the laboratory and clinical isolates completely changes (inverts) their cathepsin l use during cell entry (shirato et al., 2017) . this implies that some structural modification of s protein caused by the two amino acid mutations affects the accessibility of cathepsin l to its true recognition site. as shown in fig. 4 , only two remarkable amino acid differences were seen between the laboratory and clinical isolates of hcov-oc43 (g766r and l981p). the g766r mutation is in the furin recognition site, and it may affect sensitivity to furin. park et al. (2016) reported that mers coronavirus had different entry processes dependent on whether s protein was cleaved by furin. if s protein was cleaved by furin before cell entry, entry was sensitive to cell-surface proteases and mers entered the cell by the early endosome. yet the virus that had un-cleaved s protein was trafficked through cellsurface proteases and into the late endosome. they also reported that these un-cleaved viruses were less infectious to human airway epithelial and calu-3 cell cultures (park et al., 2016) . indeed, all clinical isolates of hcov-oc43 retain the furin cleavage site (fig. 4) , and the data from this study show that their infectivity was dependent on the expression of tmprss2. this idea supports the findings in this study because the clinical isolates of hcov-oc43 with cleaved s protein selected the cellsurface tmrpss2 route for entry and were sensitive to cleavage by cell surface proteases. thus, although the mutation in the furin site may affect the use of cell-surface proteases, these two mutations sandwich the vhcr, similar to hcov-229e. as reported previously, adaptation to tmprss2-negative cell cultures altered the protease sensitivity of hcov-229e s protein from tmprss2 to cathepsin l (shirato et al., 2017) . additional studies are required to elucidate the role of mutations in s protein in the infection events of hcov-oc43. we showed that the clinical isolates of hcov-oc43 and -hku1 preferred the cell-surface tmrrss2 to endosomal cathepsins for cell entry, as did hcov-229e. in addition, the cell-culture-adapted hcov-oc43 lost the ability to infect and replicate in hbte-ali cell culture, which mimics the natural situation of hae. these results indicate that clinical isolates of hcovs from the field generally enter cells not via late endosomes with cathepsins but via the cell surface or early endosomes with tmprss2. in addition, this study suggests that virus adaptation in cultured cells after isolation alters the route of cell entry and that such modified isolates might not reflect the hcovs of the natural world. the strain hcov-oc43 (vr1558) was obtained from the atcc and propagated and titrated using rd cells (atcc ccl-136). clinical isolates of hcov-oc43 and hku1 were isolated using hbte-ali culture. nasal swabs were obtained from outpatients in a hospital in tokyo, japan between 2014 and 2016, after approval of the national institute of infectious diseases ethics review board for human subjects. the specimens were subjected to respiratory virus testing by real-time rt-pcr (kaida et al., 2014) . hcov-positive specimens were inoculated onto hbte-ali cultures, and after 4 h incubation were washed with dmem twice and incubated at 34°c. after incubation for 3 days, cells were washed four times with dmem containing 1% (v/v) fetal calf serum, which was then used as virus stocks. virus replication was confirmed by calculating rna copy numbers using real-time rt-pcr as described earlier (dare et al., 2007; van elden et al., 2004) and comparing them to those in the washings after virus adsorption. isolated viruses and the relevant rt-pcr results are listed in table 1 . the sequences of the s proteins were obtained using sanger's method and alignment analysis was performed using genedoc software (https:// www.nrbsc.org/gfx/genedoc/ebinet.htm). to evaluate the replication kinetics of clinical and atcc isolates of hcov-oc43, 10 8 copies of virus were inoculated onto hbte-ali cultures or hct-8 cells. after incubation for 4 h, the cells were washed twice and incubated at 34°c. after 1 day of incubation, the cells were collected and used for analysis of virus entry. cell supernatants or cell washings obtained after 3 days of incubation were used to evaluate virus replication as described below. the amounts of replicated virus were expressed as rna copy numbers in cell supernatants or cell washings minus those remaining in cell washings after virus adsorption. for the entry inhibition assay using hbte-ali cultures, 10 7 to 10 8 copies of viruses were inoculated onto cells that had been pre-treated with entry inhibitors [est (23,25)trans-epoxysuccinyl-l-leucylamindo-3-methylbutane ethyl ester) (330005: calbiochem, san diego, ca, usa)] or camostat mesylate (3193: tocris bioscience, bristol, uk) for 1 h. after incubation for 4 h in the presence of inhibitors, cells were washed twice and incubated at 34°c. after 1 day of incubation, the cells were collected. cell viability was determined using celltiter-glo and a glomax luminometer (promega, madison, wi, usa). total cellular rna was extracted using isogen (nippon gene, tokyo, japan) with ribonucleic acid from baker's yeast (r6750, sigma) as carrier rna. to assess virus entry, subgenomic rna containing nucleocapsid protein was amplified by real-time rt-pcr using the following primer and probe sets: for hcov-oc43, forward, 5′-tcactgatctcttgttagat ctttttgta-3′; reverse, 5′-tttgcttgggttgagctctt-3′; and probe, 5′-ggccgatcagtccgaccagttt-3′ (fam-labeled): for hcov-hku1, forward, 5′-tatcagcttacgatctcttgtcaga-3′; reverse, 5′-tggt cagcccaagaagtttt-3′; and probe, 5′-gaagctcctctggaaatcg ttcagga-3′ (fam-labeled). rna copy numbers were calculated from standard curves obtained with the aid of control rna templates. the data were statistically analyzed using the unpaired t-test and are presented as log rna copies per well. the expression level of tmprss2 mrna was examined by a realtime rt-pcr assay. total rnas were extracted by trizol (thermo fisher scientific, waltham, ma, usa) according to the manufacturer's instructions. real-time pcr was performed using fast virus 1-step master mix (thermo fisher scientific) and lightcycler96 (roche, basel, switzerland). commercial specific primers were used as follows: gapdh, 4326317e (thermo fisher scientific); and tmprss2, hs00237175_m1 (thermo fisher scientific). the data are presented as a relative value calculated based on each gapdh value. calu-3 cells (atcc, htb-55) and lovo cells (jcrb cell bank, japan, ifo50067) were used as the positive control for tmprss2 expression, and hela cells (atcc, ccl2.1) were used as the negative control. tmprss2 activates the human coronavirus 229e for cathepsin-independent host cell entry and is expressed in viral target cells in the respiratory epithelium the adaptation of two human coronavirus strains (oc38 and oc43) to growth in cell monolayers human coronavirus infections in rural thailand: a comprehensive study using real-time reverse-transcription polymerase chain reaction assays ifitm3 restricts influenza a virus entry by blocking the formation of fusion pores following virus-endosome hemifusion human coronavirus nl63 and 229e seroconversion in children isolation and characterization of current human coronavirus strains in primary human epithelial cell cultures reveal differences in target cell tropism well-differentiated human airway epithelial cell cultures a new virus isolated from the human 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"ibv-like" viruses from patients with upper respiratory tract disease recovery in tracheal organ cultures of novel viruses from patients with respiratory disease antiviral role of ifitm proteins in african swine fever virus infection proteolytic processing of middle east respiratory syndrome coronavirus spikes expands virus tropism coronaviruses and toroviruses culturing the unculturable: human coronavirus hku1 infects, replicates, and produces progeny virions in human ciliated airway epithelial cell cultures differences in neutralizing antigenicity between laboratory and clinical isolates of hcov-229e isolated in japan in 2004-2008 depend on the s1 region sequence of the spike protein middle east respiratory syndrome coronavirus infection mediated by the transmembrane serine protease tmprss2 clinical isolates of human coronavirus 229e bypass the endosome for cell entry bilateral entry and release of middle east respiratory syndrome coronavirus induces profound apoptosis of human bronchial epithelial cells university of texas medical branch at galveston frequent detection of human coronaviruses in clinical specimens from patients with respiratory tract infection by use of a novel real-time reverse-transcriptase polymerase chain reaction detection of human coronavirus oc43 in children with acute respiratory infections in mie we thank fumihiro taguchi (chungnam national university, daejeon, korea), for valuable suggestions. this study was supported by a grant-in-aid for scientific research (c: 26460563) from the japan society for the promotion of science and the japan agency for medical research and development (grant nos. 17fk0108313j0203 and 17fk0108103j0301). key: cord-325481-uzch2hwd authors: simmons, graham; bertram, stephanie; glowacka, ilona; steffen, imke; chaipan, chawaree; agudelo, juliet; lu, kai; rennekamp, andrew j.; hofmann, heike; bates, paul; pöhlmann, stefan title: different host cell proteases activate the sars-coronavirus spike-protein for cell-cell and virus-cell fusion date: 2011-05-01 journal: virology doi: 10.1016/j.virol.2011.02.020 sha: doc_id: 325481 cord_uid: uzch2hwd severe acute respiratory syndrome coronavirus (sars-cov) poses a considerable threat to human health. activation of the viral spike (s)-protein by host cell proteases is essential for viral infectivity. however, the cleavage sites in sars-s and the protease(s) activating sars-s are incompletely defined. we found that r667 was dispensable for sars-s-driven virus-cell fusion and for sars-s-activation by trypsin and cathepsin l in a virus-virus fusion assay. mutation t760r, which optimizes the minimal furin consensus motif 758-rxxr-762, and furin overexpression augmented sars-s-activity, but did not result in detectable sars-s cleavage. finally, sars-s-driven cell-cell fusion was independent of cathepsin l, a protease essential for virus-cell fusion. instead, a so far unknown leupeptin-sensitive host cell protease activated cellular sars-s for fusion with target cells expressing high levels of ace2. thus, different host cell proteases activate sars-s for virus-cell and cell-cell fusion and sars-s cleavage at r667 and 758-rxxr-762 can be dispensable for sars-s activation. a novel coronavirus (cov) has been identified as the causative agent of severe acute respiratory syndrome (sars), which claimed almost 800 lives in 2002-03 (drosten et al., 2003; ksiazek et al., 2003; peiris et al., 2004) . coronaviruses, including sars-cov, harbour three envelope proteins, spike (s), membrane (m), and envelope (e), which are required for virion assembly, release and infectious entry into target cells (masters, 2006) . the sars-cov-s-protein (sars-s) mediates infectious cellular entry (hofmann et al., 2004b; simmons et al., 2003; yang et al., 2004) and constitutes the major target of the neutralizing antibody response nie et al., 2004b nie et al., , 2004a . the carboxypeptidase angiotensin-converting enzyme 2 (ace2) is used by sars-cov as receptor for cell entry (li et al., 2003; wang et al., 2004) and is expressed on type ii pneumocytes, the major viral target cells (hamming et al., 2004; mossel et al., 2008; to et al., 2004) . several cellular c-type lectins augment or facilitate sars-s-driven entry (gramberg et al., 2005; jeffers et al., 2004; marzi et al., 2004; yang et al., 2004) . however, ace2 but not c-type lectin expression correlates with susceptibility to sars-s-driven infection (hofmann et al., 2004a; nie et al., 2004b) and is essential for sars-cov spread in experimentally infected mice (kuba et al., 2005) , indicating that ace2 is a major and likely the only receptor used by sars-cov in the infected host. collectively, sars-s interacts with host cell factors to mediate the first essential step in the viral life cycle, virus entry into target cells, and constitutes an attractive target for preventive and therapeutic approaches. the sars-s-protein is synthesized in the constitutive secretory pathway of infected cells. amino acid motifs in its cytoplasmic tail slow down transit through the golgi compartment (mcbride et al., 2007) where interactions with the m-protein facilitate virion incorporation (mcbride and machamer, 2010; voss et al., 2009) . the structural organization of sars-s is similar to that of several other viral envelope proteins, termed class i fusion proteins: the extracellular s1 domain facilitates binding to the receptor, ace2, while the membrane-anchored s2 domain harbours the functional elements required for fusion of the viral with a target cell membrane . viral class i fusion proteins are usually synthesized in an inactive form, and require activation by host cell proteases to transit into a fusion-active state (eckert and kim, 2001; harrison, 2008) . however, viral strategies to accomplish proteolytic activation can vary. for instance, the majority of strains of the murine coronavirus mouse hepatitis virus (mhv) contain sproteins that are cleaved by furin in infected cells, and these viruses are believed to enter target cells by receptor-dependent, ph-independent fusion with the plasma membrane (de haan et al., 2004; nash and buchmeier, 1997; qiu et al., 2006) , although some of these findings are controversial (eifart et al., 2007) . in contrast, the s-protein of the mhv type 2 strain is not cleaved by furin and the spike protein on incoming virions is activated in target cell vesicles by endosomal proteases of the cathepsin family (qiu et al., 2006) . similar to mhv-2, proteolytic activation of sars-s is mediated by cathepsins in target cells, most importantly by cathepsin l (simmons et al., 2005) . cleavage-activation by cathepsin l is thought to require previous binding of sars-s to ace2, which is believed to induce a conformational change in the s-protein (simmons et al., 2005) , and seems to involve at least two consecutive proteolytic processing steps (belouzard et al., 2009; simmons et al., 2005) . however, the cleavage sites in sars-s have been incompletely defined. arginine 667 was shown to be required for the robust augmentation of sars-s-driven cell-cell (belouzard et al., 2009; follis et al., 2006) and virus-cell fusion (kam et al., 2009 ) by trypsin and for the trypsin-dependent circumvention of the entry blockade imposed by lysosomotropic agents (belouzard et al., 2009) . in spite of these results, it is at present unknown if r667 is required for sars-s activation by cathepsin l. recent evidence suggests that r797 is a component of a second cleavage site (belouzard et al., 2009; watanabe et al., 2008) . thus, similar as for r667, residue r797 was demonstrated to be required for trypsin-dependent augmentation of sars-s-dependent membrane fusion and for trypsin-dependent resistance to lysosomotropic agents (belouzard et al., 2009) . nevertheless, efficient proteolytic processing of wild-type sars-s at this site, or at any other site, has so far not been demonstrated in cells (hofmann et al., 2004b; simmons et al., 2004; xiao et al., 2003; yang et al., 2004; yao et al., 2004) , with one exception (wu et al., 2004) . notably, the sars-s-protein contains a minimal furin cleavage site at position 758-761 (rntr), and a peptide comprising this sequence is efficiently cleaved by furin (bergeron et al., 2005) . however, the contribution of the rntr motif to proteolytic activation of sars-s is unknown. in order to explore the role of the minimal furin cleavage site at position 758-761 and to further investigate the importance of the protease sensitive site at position r667, we analyzed the sars-s mutants r667a and t760r. our analysis revealed the r667 was required for responsiveness to trypsin-treatment in some experimental systems but had no effect on sars-s activation by trypsin and cathepsin l in a virus-virus fusion assay, which adequately mimics, in isolation, the conditions required for sars-s-driven membrane fusion, suggesting that r667 might not play a major role in sars-s activation in target cells. mutation t760r, which optimized an existing minimal furin cleavage motif, increased sars-s activity but no evidence for cleavage of mutant t760r was obtained. finally, our results demonstrate the cathepsin l activates sars-s for virus-cell but not cell-cell fusion, which was dependent on the activity of a so far uncharacterized serine protease (for fusion with targets expressing high amounts of ace2) or addition of exogenous trypsin (for fusion with targets expressing low amounts of ace2). based on alignments with other coronaviruses, it has been suggested that residues r667 and k672 define a potential cleavage site for host cell proteases (bergeron et al., 2005; follis et al., 2006) . to determine the importance of these residues for sars-s sensitivity to proteolysis, we introduced mutations r667a or k672l into wt sars-s (fig. 1) . analysis of the sars-s mutants by lentiviral pseudotyping, an experimental approach which adequately models sars-s-driven entry into target cells (hofmann et al., 2004b; moore et al., 2004; simmons et al., 2004) , showed that the mutations r667a and k672l were both compatible with robust sars-s-driven virion incorporation and entry (figs. 2a and b) , albeit the entry efficiency of viruses harbouring the mutant s-proteins was somewhat reduced compared to pseudotypes bearing wt sars-s ( fig. 2a) . similar results were seen with a r667a and k672l double mutant (wt sars-s: 44,390 ± 3286 counts per second (c.p.s.), r667a/k672dl: 39,953 ± 1329 c.p.s.), demonstrating that these particular basic residues are not required for infectious entry in tissue culture. we next assessed if the exchanges r667a and k672l altered sensitivity of sars-s-bearing pseudotypes to trypsin treatment, which was previously shown to inactivate cell-free virions (simmons et al., , 2005 . indeed, pre-treatment of pseudotypes bearing wt sars-s and sars-s mutant k672l reduced viral infectivity in a dose-dependent manner (fig. 2c ). in contrast, sars-s mutant r667a was resistant to inactivation by trypsin (figs. 2b and c), indicating that r667 defines a trypsin-sensitive site and that cleavage at this site abrogates infectivity of free virions. to further characterize the importance of r667 for sars-s sensitivity to proteolysis, we compared pseudotypes bearing wt sars-s and sars-s mutant r667a for their sensitivities to inactivation by a panel of proteases. treatment with trypsin, plasmin factor xa and, to a lesser extent, thrombin reduced infectivity of viruses harbouring wt sars-s but not sars-s mutant r667a (fig. 2d ), further underlining that r667 defines a protease sensitive site. thermolysin and chymotrypsin treatment also diminished viral infectivity, but wt sars-s and sars-s mutant r667a were equally sensitive to inactivation by these proteases (fig. 2d ), indicating that they cleave sars-s at a site distinct from r667. collectively, r667 but not k672 defines a protease-sensitive site and proteolysis of cell-free virions at this site abrogates viral infectivity. lysosomotropic agents, such as ammonium chloride, interfere with endosomal acidification and block infectious entry of sars-cov (hofmann et al., 2004b; simmons et al., 2004; yang et al., 2004) , presumably by inhibiting cathepsins, which require low ph for optimal activity. treatment of cell-bound virus with trypsin was shown to allow infectious sars-s-driven entry into ammonium chloride-treated cells (simmons et al., 2005) , indicating that trypsin can functionally replace cathepsin l as a sars-s-activating protease under these conditions ("trypsin bypass"). we asked if r667 is required for a trypsin bypass of endosomal acidification. treatment of target cells with ammonium chloride markedly inhibited infectious entry driven by wt sars-s and sars-s mutant r667a, and infectivity of wt sars-s-bearing viruses could be fully restored by treatment of cell-bound virions with trypsin ( fig. 3) . in contrast, infectivity of r667a bearing virions was not rescued by trypsin treatment (fig. 3) , indicating that r667 is required for activation of cell-bound virions by trypsin. we next assessed the impact of r667a on sars-s proteolytic activation in a virus-virus membrane-fusion assay. in this assay, sars-s-bearing viruses (which also contain the avian sarcoma leukosis virus-a envelope protein, enva, in their membrane) are allowed to fuse with ace2 harbouring viruses. the fusion efficiency is then quantified by the addition of virions to leupeptin-treated (to exclude an impact of host cell proteases on sars-s activation) hela cells, which express the enva receptor tva, and which are not susceptible to sars-s-driven infection (simmons et al., 2005) . thus, the virus-virus fusion assay is a reductionistic model system, which allows the proteolytic activation of sars-s to proceed under cell-free conditions. efficient fusion of virions bearing wt sars-s with virions bearing ace2 was only observed upon treatment of particles with trypsin and recombinant activated cathepsin l (fig. 4) , in agreement with previously reported results (simmons et al., 2005) . strikingly, trypsin and cathepsin l treatment activated virions bearing wt sars-s and sars-s mutant r667a with similar efficiency (fig. 4) , demonstrating that r667 was dispensable for sars-s activation under these conditions. in summary, r667 defines a trypsin cleavage site, which is responsible for trypsin-dependent activation of cell-bound and inactivation of cell-free virions, respectively. however, r667 is dispensable in tissue culture infection and for sars-s activation by trypsin and cathepsin l in a virion-virion fusion assay, and might thus be dispensable for proteolytic activation of sars-cov in target cells. we hypothesized that the minimal furin cleavage site (rxxr) at positions 758-761 might contribute to sars-s-dependent membrane fusion by allowing sars-s cleavage, albeit with low efficiency. to investigate the role of this motif in sars-s-driven entry, we assessed if virus was allowed to bind, but not internalize into veroe6 cells pretreated with 20 mm ammonium chloride. subsequently, the cells were treated with tpck-trypsin (10 μg/ml) to activate sars-s for membrane fusion. results are presented as a percentage of no trypsin, no ammonium chloride control and are means and standard deviations of triplicate wells. similar results were seen on 293t-ace2 cells. optimization of the minimal furin cleavage site by exchange of t760r (resulting in the sequence rxrr, fig. 1 ) affects sars-s function. fluorescence-activated cell sorting analysis revealed that sars-s mutant t760r and wt sars-s were expressed to comparable levels at the surface of transfected 293t cells, and could thus be directly compared in functional studies ( supplementary fig. 1 ). we first determined if substitution t760r affected s-protein-driven infectious entry. in order to assess infectious entry facilitated by the s-protein variant, pseudotypes were used for infection of control or ace2transfected 293t cells (fig. 5 , left panel). the 293t cell line expresses endogenous ace2 and is therefore susceptible to sars-s-driven infection (hofmann et al., 2004b (hofmann et al., , 2004a li et al., 2003; yang et al., 2004) . accordingly, infectious entry of sars-sbearing pseudovirions into control transfected cells was detectable (fig. 5, left panel) . notably, entry driven by the t760r variant was about 2-fold more efficient than infection driven by sars-s wt, and a similar observation (4-fold increase) was made when infection of ace2transfected cells was examined (fig. 5, left panel) , suggesting that t760r augments s-protein activity. indeed, when sars-s-driven membrane fusion was assessed in a previously reported cell-cell fusion assay (hofmann et al., 2006) , which measures fusion of effector 293t cells expressing s-proteins with 293t target cells expressing ace2 or pcdna3, again the s-protein mutant was more active than sars-s wt (fig. 5, right panel) . thus, the introduction of an arginine residue at position 760 increases the membrane fusion activity of sars-s. exchange t760r optimizes a minimal furin cleavage site in sars-s and might increase sars-s activity by facilitating cleavage by furin or related proteases. we investigated whether furin can augment sars-s activity by determining the infectivity of p24-normalized sars-s pseudotypes produced in the absence and presence of overexpressed furin (fig. 6, left panel) . overexpression of furin moderately increased infectivity of pseudotypes bearing wt sars-s or mutant t760r. the most notable effect was observed for sars-s wt pseudotypes, which showed a~7-fold augmented infectivity (fig. 6 , left panel), albeit this increase was not statistically significant. comparable observations were made when s-driven cell-cell fusion was examined, and the increase in activity of wt sars-s and mutant t760r observed upon furin overexpression was statistically significant (fig. 6, right panel) . however, fusion driven by sars-s wt and t760r were augmented to similar degrees (about 2-3 fold) by furin overexpression. thus, high levels of furin enhance sars-s-protein driven cell-cell and virus-cell fusion. our functional data showed that mutation of t760r and overexpression of furin augmented sars-s activity, presumably by facilitating proteolytic processing of the s-protein. to assess sars-s cleavage, we conducted western blot analyses of lysates of s-protein transfected 293t cells, using a serum specific for the s2-portion of sars-s. expression of ß-actin served as loading control. our results revealed a prominent band of approximately 160 kda for both wt sars-s and mutant t760r (fig. 7) , which is expected for uncleaved sars-s (hofmann et al., 2004b) . treatment of cells with trypsin before lysis reduced the signal obtained for uncleaved sars-s, and a band of approximately 90 kda appeared, in accordance with the previously reported size of the s2-fragment of sars-s (bergeron et al., 2005; follis et al., 2006) . co-expression of furin with sars-s wt or the sprotein variant t760r did not result in the appearance of a s2-band, indicating that furin-mediated cleavage was inefficient or absent. we next investigated whether exchange t760r altered the sensitivity of sars-s-dependent membrane fusion to inhibition by the cathepsin l and b inhibitor mdl 28170. in agreement with published data (simmons et al., 2005) , mdl 28170 efficiently reduced infection by sars-s-bearing pseudotypes (fig. 8, left panel) . similar inhibition was observed with the sars-s mutant t760r, indicating that this change did not modulate cathepsin-dependence of viral entry. in stark contrast, mdl 28170 had no inhibitory effect on wt sars-s and t760r dependent cell-cell fusion (fig. 8, right panel) , fig. 4 . protease activation of virus-virus fusion mediated by wt sars-s and mutant r667a. pseudovirions bearing sars-s or ace2 were mixed and virion-fusion activated by trypsin (10 μg/ml) or cathepsin l (2 μg/ml). mock indicates the mock addition of protease to mixed particles. in addition to this control, further controls of spike-enva or ace-2 pseudotypes alone treated or untreated with protease are consistently below the level of detection, as previously described (simmons et al., 2005) . infection by luciferase-encoding particles was determined using luciferase substrate as described by the manufacturer (promega). results are presented as means and standard deviations of replicates of four wells. similar results were seen in 2 additional experiments. indicating the sars-s-protein-driven fusion of cellular membranes does not depend on cathepsin activity. since sars-s-driven cell-cell fusion was not dependent on cathepsinactivity, we asked if the activity of other proteases might be required. for this, we first sought to clarify to which extend sars-s-driven cell-cell fusion depends on the presence of exogenous trypsin, since previous studies reported that efficient sars-s-driven cell-cell fusion occurred only upon treatment of sars-s expressing cells with trypsin (howard et al., 2008; simmons et al., 2004) , while in our experiments trypsin was dispensable for robust cell-cell fusion (figs. 5, 6 and 8). when we examined fusion of sars-s or vsv-g transfected 293t cells with control transfected 293t cells (which express low levels of endogenous ace2 (hofmann et al., 2004a; li et al., 2003; simmons et al., 2004) ), we found that trypsin treatment was required for sars-s-but not vsv-g-driven cell-cell fusion, and that fusion driven by vsv-g but not sars-s was induced by low ph (fig. 9a) . however, when cell-cell fusion with ace2 transfected 293t cells was examined, robust sars-s-driven fusion was already observed in the absence of trypsin treatment, and the fusion activity was further augmented in the presence of trypsin (fig. 9a) . thus, sars-s-driven cell-cell fusion depends on trypsin-activation of sars-s only if receptor levels are limiting. if ace2 is expressed at high levels, robust fusion occurs in the absence of trypsin and is most likely due to sars-s activation by a host cell protease other than cathepsins. in order to investigate the need for a host cell protease for trypsinindependent sars-s-driven cell-cell fusion, we inhibited the wt sars-s-driven cell-cell fusion reaction by leupeptin, an inhibitor of cysteine and serine protease, aebsf, a serine protease inhibitor, and the cathepsin inhibitors e64c and mdl28170. all inhibitors were used at non-cytotoxic concentrations, as determined by a commercially available cytotoxicity assay (promega, madison, usa) and by the lack of inhibition of luciferase expression in cells cotransfected with the reporter plasmids employed to quantify cell-cell fusion (supplementary fig. 2 ). of all inhibitors tested, only leupeptin inhibited sars-sdriven cell-cell fusion, and inhibition was dose-dependent (fig. 9b) . finally, leupeptin, e64c and mdl28170 inhibited sars-s-driven virus-cell fusion (fig. 9c ), as expected from the results shown in fig. 8 and from previous work (simmons et al., 2005) . thus, sars-sdriven cell-cell fusion depends on the activity of a so far uncharacterized cysteine or serine protease, while virus-cell fusion requires cathepsin activity. the processing of sars-s by cellular proteases might determine route and efficiency of viral entry into target cells and might have important consequences for development of preventive and therapeutic strategies (belouzard et al., 2009; simmons et al., 2005; watanabe et al., 2008) . however, the sites in sars-s, which are recognized by host cell proteases, are incompletely defined. we show that r667 defines a trypsin sensitive site, which is required for inactivation of cell-free virus by trypsin and for trypsin-dependent infectious entry of cell-bound virus into targets pretreated with ammonium chloride. in contrast, the integrity of r667 was dispensable for infectious entry in cell culture and activation of sars-s-driven virus-virus fusion by trypsin and cathepsin l. optimization of an existing minimal furin cleavage site, 758-rntr-761, by mutation t760r augmented sars-s-driven cell-cell and viruscell fusion. however, no evidence for cleavage of sars-s at this motif was obtained. finally, differential blockade of sars-s-driven virus-cell and cell-cell fusion by protease inhibitors showed that these processes depend on different activating proteases, with a so far poorly characterized serine or cysteine protease being responsible for sars-sdriven cell-cell fusion. the spike protein of sars-cov contains several potentially protease sensitive sites, which have been implicated in proteolytic processing of sars-s (belouzard et al., 2009; bergeron et al., 2005; follis et al., 2006) . the most n-terminal motif, amino acids 657 to 676, shows some similarity to cleavage sites of other coronavirus s-proteins but does not comprise an rxxr motif recognized by proprotein convertases in the context of cleavable coronavirus s-proteins (follis et al., 2006) . nevertheless, mutation of r667 was reported to block the increase in sars-s membrane fusion activity observed for wt sars-s upon furin overexpression, and introduction of a furin consensus sequence at this site was shown to increase sars-s-driven cell-cell fusion (belouzard et al., 2009; follis et al., 2006) . our results demonstrate that this site is important for sars-s cleavage by trypsin, and that trypsin cleavage depends on the presence of r667 but not k672. thus, cell-free virions bearing wt sars-s but not sars-s mutant r667a were inactivated by trypsin, while cell-bound virions bearing wt sars-s but not mutant r667a were activated by trypsin for infection of ammonium chloride pretreated target cells (trypsin bypass). these findings are in agreement with published work (belouzard et al., 2009; follis et al., 2006) , and highlight that r667 defines a protease sensitive site in sars-s. unexpectedly, however, mutation of r667 was compatible with robust sars-s activation by trypsin and cathepsin l in a virion-virion fusion assay. the reasons for the differential requirement for r667 in the trypsin bypass and virion-virion fusion assays are at present unclear, but might relate to different properties of the target cell membrane relative to the virion membrane, in terms of receptor concentration and presence of cellular proteases. regardless of the underlying mechanism, the present data suggest that r667 might be dispensable for activation of sars-cov by trypsin and by cathepsin l. such a scenario would be in agreement with results by bosch and colleagues, who mapped the cathepsin l cleavage site in sars-s to amino acid t678 (bosch et al., 2008) . belouzard et al. suggested that proteolytic activation of sars-s might be a two-step process and might involve cleavage at r667 and at r797 (belouzard et al., 2009) . evidence for an important role of r797 in proteolytic activation of sars-s was obtained in cell-cell fusion and trypsin bypass experiments, in which the sars-s mutant r797n was found to be refractory to activation by trypsin (belouzard et al., 2009 ). in addition, the insertion of a furin consensus motif at this site increased sars-s activity in a cell-cell fusion assay (belouzard et al., 2009 ) and, as documented by an independent study, allowed cathepsin-independent infectious entry into target cells (watanabe et al., 2008) . notably, a minimal furin cleavage site, rxxr, is present in sars-s at amino acids 758 to 761, and a peptide spanning this motif was previously shown to be cleaved by furin, while peptides spanning r667 and r797 were not recognized by the protease (bergeron et al., 2005) . we found that optimizing the minimal furin site at position 758 (mutant t760r) significantly increased sars-s activity in cell-cell and virus-cell fusion assays, and we noted that the virus-cell fusion activity of wt sars-s but not mutant t760r was augmented by overexpression of furin in virusproducer cells, although this effect was not statistically significant. these results suggest that amino acids 758 to 761 might constitute an alternative processing site, which might be recognized by proprotein convertases or related enzymes. however, proteolytic processing of wt sars-s or variant t760r upon furin overexpression could not be demonstrated, indicating that cleavage was inefficient or absent. therefore, alternative scenarios for the role of amino acids 758 to 761 in sars-s activation must be considered. thus, it is possible that augmentation of sars-s activity upon furin overexpression and mutation t760r were separate effects, with t760r potentially modulating protease sensitivity of other sites in sars-s. cell to cell fusion assays are commonly used to functionally analyze sars-s, including the characterization of potential proteolytic processing sites in sars-s (belouzard et al., 2009; follis et al., 2006; hofmann et al., 2006; simmons et al., 2003) . in most studies, sars-s-driven cellcell fusion was examined upon activation of sars-s by exogenous trypsin. however, it has so far not been determined if sars-s-driven cell-cell fusion can also be activated by a host cell protease. for instance, it is unknown if the proteases responsible for proteolytic activation of virus-associated sars-s, cathepsins b and l, can also activate cellassociated sars-s. we found that trypsin-activation of sars-s was only required for fusion with cells expressing low amounts of ace2, while fusion with target cells expressing high levels of ace2 proceeded efficiently in the absence of trypsin, in agreement with a recent study (glowacka et al., 2011) . under the latter conditions, cell-cell fusion driven by sars-s was inhibited by leupeptin but not cathepsin inhibitors, indicating that a serine protease, or a cysteine protease other than cathepsins b and l, can activate sars-s for cell-cell fusion. the identity of the responsible protease(s) is at present unclear. a role for factor xa, a serine protease, has been suggested but the results await conformation (du et al., 2007) . the type ii transmembrane serine proteases (ttsps) tmprss2 and tmprss4 can activate the influenza virus hemagglutinin by cleavage (böttcher et al., 2006 ; chaipan et al., 2009) and these proteases were recently shown to also activate sars-s for membrane fusion (glowacka et al., 2011; matsuyama et al., 2010; shulla et al., 2011) . however, appreciable expression of tmprss2 or tmprss4 was not detected in 293t cells in a previous study (bertram et al., 2010) , and sars-s-driven cell-cell fusion was not inhibited by the serine protease inhibitor aebsf (present study), which was shown to be active against other ttsps (beliveau et al., 2009) . therefore, ttsps and related serine proteases are unlikely to account for sars-s activation under the conditions tested here, and future studies should focus on the role of cysteine proteases. expression plasmids pcaggs-sars-s, encoding the spike proteins of sars-cov strain frankfurt (hofmann et al., 2004b) or urbani (simmons et al., 2003) , and pcdna3-hace2, encoding the human ace2 receptor, have been described previously (hofmann et al., 2004b (hofmann et al., , 2004a . site-directed mutagenesis of the sars-s-protein was performed by overlap-extension pcr. for generation of mutation t760l the following overlapping primers were used: p5 sars-s t760r (5-ccgacgtgaagtgttcgctcaagtc-3) and p3 sars-s t760r (5-gacttgagcgaa. cacttcacgtcggttgcgatcctgttcagcagcaatacc-3). to facilitate generation of pcr fragments bearing the desired mutations, only the 3′ prime portion of the s-sequence was amplified using overlapping pcr. subsequently, the pcr-amplified fragments were introduced into a pcaggs variant harbouring the corresponding 5′ portion of the sars-s sequence. variants r667a, k672l and kptkr to epted were generated using quikchange site directed mutagenesis (stratagene), with sars-s in pcdna as template, and then transferred to pcaggs. all pcr amplified sequences were confirmed by automated sequence analysis. 293t cells were propagated in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal bovine serum (fbs), penicillin and streptomycin, and grown in a humified atmosphere of 5% co 2 . 293t cells stably expressing ace2 (293 t-hace2) were generated by transfection of plasmid pcdna3.1zeo-hace2 (hofmann et al., 2004a) into 293t cells followed by selection of resistant cells with zeocin (invitrogen) at 50 μg/ml. surface expression of ace2 on clonal cells was confirmed by facs analysis. for analysis of cell-cell fusion, 293t effector cells seeded in 6-well plates at 3 × 10 5 /well were capo 4 -cotransfected with plasmid pgal4-vp16, encoding the herpes simplex vp16 transactivator fused to the dna binding domain of the yeast transcription factor gal4 (stamminger et al., 2002) , and plasmids encoding sars-s-variants (or empty plasmid) and furin (or empty plasmid). in parallel, 293t target cells were seeded in 48well plates at 3 ×10 4 /well and transfected with pcdna3 or the hace2 expression plasmid together with plasmid pgal5-luc, in which luciferase reporter gene expression is controlled by five gal4 binding sites (stamminger et al., 2002) . the day after transfection, effector cells were diluted in fresh medium and added to the target cells. cell-cell fusion was quantified by determination of luciferase activities in cell lysates 48 h after cocultivation using a commercially available kit (promega, madison, usa) . results generated in the experimental system described earlier are shown in figs. 5, 6, 8 and 9b. alternatively, cell-cell fusion was assayed using α-complementation of β-galactosidase fragments, as previously described for hiv (holland et al., 2004) . for this, effector 293t cells were co-transfected with plasmids encoding sars-s or vsv-g and a plasmid encoding an n-terminal fragment of βgalactosidase (amino-acids 1-80; termed α peptide), while target 293t cells were co-transfected with a plasmid encoding ace2 or with the corresponding empty plasmid and a plasmid encoding the remaining cterminal portion of β-galactosidase (amino-acids 80-1023; termed ω fragment). the day after transfection, effector cells were diluted in fresh medium and added to the target cells at a ratio of 1:1. after one hour incubation for attachment and binding, cells were washed in serum-free medium and pulsed with medium adjusted to ph5.0, ph7.0, ph8.0 or containing tpck-trypsin at 15 μg/ml for 10 min at 37°c. ph or trypsin was neutralized by the addition of excess medium containing serum and trypsin inhibitor. cells were then incubated for a further 5 h. upon viral envelope-driven membrane fusion the α peptides and ω fragments of βgalactosidase trans-complement each other to give functional βgalactosidase enzymatic activity, which was detected in cell lysates employing a commercially available kit (galacton plus substrate, applied biosystems). results generated in this experimental system are shown in fig. 9a . for generation of lentiviral pseudoparticles, capo 4 transfections were performed as described (hofmann et al., 2004b; simmons et al., 2003) . in brief, 293 t cells were transiently cotransfected with pnl4-3 e-r-luc (connor et al., 1995) and expression plasmids for sars-s-variants or vsv-g. for some experiments, human furin was co-expressed during production of pseudoparticles. the culture medium was replaced at 16 h and harvested at 48 h post transfection. the supernatants were passed through 0.45 μm filters, aliquotted and stored at −80°c. capsid contents (p24) in harvested supernatants were determined using a commercially available kit (murex, wiesbaden, germany). for infection, 293t cells or 293t cells transiently transfected with pcdna3 or hace2 or 293t cells stably expressing hace2 were incubated with pseudotypes, normalized for infectivity or p24-capsid protein content, for three days before cells were lysed and luciferase activities determined using a commercially available kit (promega, madison, usa). 293t cells in 96-wells were pre-incubated with the cathepsin l and b inhibitor mdl 28170 (calbiochem, darmstadt, germany) for 30 min. thereafter, pseudotypes of comparable infectivity were added for 12-16 h, the culture medium was replaced and luciferase activities in cell extracts were determined after 72 h as described earlier. infectivity normalized pseudovirions bearing wild-type or mutant sars-s protein were incubated with varying amounts of protease for 30 min at room temperature. proteolysis was halted by the addition of an equal volume of medium containing 10% fbs, 100 μg/ml soy bean trypsin inhibitor (sti) and 100 μg/ml aprotinin. virus was then plated on 293t-ace2 cells, spin infected for 90 min at 2500 rpm and incubated at 37°c. after 4 h, the medium was replaced with fresh medium, and cells were incubated for 48 h before measurement of luciferase activity. all proteases were obtained from sigma, and final concentrations used in the assay were determined by preliminary experiments, or the maximal practical level achievable based on the stock solutions of 1 mg/ml. proteases were diluted in pbs. trypsin and chymotrypsin were used at a final concentration of 25 μg/ml, elastase and factor xa at 50 μg/ml, thermolysin and thrombin at 125 μg/ml and plasmin at 250 μg/ml. trypsin bypass experiments were performed as described (simmons et al., 2005) . briefly, 293t-ace2 cells were pretreated for 1 h with cold medium containing ammonium chloride (40 mm). an equal volume of diluted cold pseudovirion mixture (virus was ultracentrifuge-concentrated and resuspended in pbs to remove fbs) was added, and the cells were spin-infected at 4°c to allow virus-binding to cells. the medium was replaced with warm serum-free medium containing ammonium chloride (20 mm) and incubated at 37°c for 15 min. subsequently, the medium was removed, and fresh medium containing tpck-trypsin (10 μg/ml) was added for 10 min at 25°c. the trypsin-containing medium was then removed and medium supplemented with sti (75 μg/ ml) and ammonium chloride (20 mm) was added. after a 12 h incubation period, medium was replaced with fresh medium without ammonium chloride, and cells were incubated for a further 36 h before luciferase activity was measured. virus to virus fusion was assayed as described (simmons et al., 2005) . briefly, equal amounts of pseudovirions bearing either ace2 and encoding luciferase as a reporter (hiv-luc(ace2)) or both sars-cov spike (or mutants) and avian sarcoma leukosis virus-a envelope and encoding gfp (hiv-gfp(s+ e)) were mixed and incubated for 30 min on ice to allow binding. the temperature was then raised to 37°c for 15 min to allow induction of conformational rearrangements. particles were then either treated with 10 ug/ml tpck-trypsin (sigma) or the ph was lowered to ph 6 by the addition of 0.1 m citric acid, and preactivated recombinant cathepsin l was added to a final concentration of 2 ug/ml. proteolysis was halted after 10 min at 25°c by addition of soybean trypsin inhibitor and leupeptin. virus mixtures were then diluted and used to infect hela cells stably expressing tva that had been pretreated with 20 μg/ml leupeptin for 1 h. cells transiently expressing sars-s or vlps harbouring sars-s were lysed in sds-laemmli buffer and boiled for 15-30 min at 95°c. samples were separated via 12.5% sds-page and transferred onto nitrocellulose membranes (schleicher & schuell, dassel, germany). sars-s proteins were detected by staining with sars-s specific rabbit serum (imgenex, san diego, usa) at a 1:1000 dilution, followed by detection of bound antibodies by use of a peroxidase-conjugated anti-rabbit igg (dianova, hamburg, germany) at a dilution of 1:5000. for loading control, the stripped membranes were incubated with an anti-ß-actin antibody (sigma, deisenhofen, germany) at a 1:1000 dilution, followed by incubation with peroxidase-conjugated anti-mouse igg (dianova, hamburg, germany) at a dilution of 1:5000. chemiluminescence detection was performed employing a commercially available kit, according to the manufacturer's protocol (ecl western detection kit; amersham pharmacia biotech europe, freiburg, germany). statistical significance was calculated employing a two-tailed student's t-test for dependent samples. supplementary materials related to this article can be found online at doi:10.1016/j.virol.2011.02.020. probing the substrate specificities of matriptase, matriptase-2, hepsin and desc1 with internally quenched fluorescent peptides activation of the sars coronavirus spike protein via sequential proteolytic cleavage at two distinct sites implication of proprotein convertases in the processing and spread of severe acute respiratory syndrome coronavirus tmprss2 and tmprss4 facilitate trypsin-independent spread of influenza virus in caco-2 cells cathepsin l functionally cleaves the severe acute respiratory syndrome 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revealed by pseudotype virus bearing cleaved s protein the spike protein of severe acute respiratory syndrome (sars) is cleaved in virus infected vero-e6 cells the sars-cov s glycoprotein: expression and functional characterization ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign cleavage and serum reactivity of the severe acute respiratory syndrome coronavirus spike protein we thank b. fleckenstein, k. von der mark and t.f. schulz for constant support and k. korn for p24-elisa. this work was supported by grant r01ai074986 from the national institute of allergy and infectious diseases (g.s., j.a. and k.l.), r21 ai059172 (pb), u54 ai57168 (gs) marce, sfb 466/sfb 587 (h.h. and s.p.), graduiertenkolleg 1071 (c.c.), the center for infection biology at hannover medical school (i.s.) and bmbf (01ki 0703 to s.p.). key: cord-313906-fh85fzq9 authors: maruyama, junki; nao, naganori; miyamoto, hiroko; maeda, ken; ogawa, hirohito; yoshida, reiko; igarashi, manabu; takada, ayato title: characterization of the glycoproteins of bat-derived influenza viruses date: 2016-01-15 journal: virology doi: 10.1016/j.virol.2015.11.002 sha: doc_id: 313906 cord_uid: fh85fzq9 recently found bat-derived influenza viruses (bativs) have hemagglutinin (ha) and neuraminidase (na) gene segments distinct from those of previously known influenza a viruses. however, pathogenicities of these bativs remain unknown since infectious virus strains have not been isolated yet. to gain insight into the biological properties of bativs, we generated vesicular stomatitis viruses (vsvs) pseudotyped with the bativ ha and na. we found that vsvs pseudotyped with bativ has and nas efficiently infected particular bat cell lines but not those derived from primates, and that proteolytic cleavage with a trypsin-like protease was necessary for ha-mediated virus entry. treatment of the susceptible bat cells with some enzymes and inhibitors revealed that bativ has might recognize some cellular glycoproteins as receptors rather than the sialic acids used for the other known influenza viruses. these data provide fundamental information on the mechanisms underlying the cellular entry and host restriction of bativs. influenza a viruses (iavs), which belong to the family orthomyxoviridae, have 8 segmented negative sense rna genomes. iav is one of the most important zoonotic pathogens, with high morbidity in humans, pigs, horses, and poultry. iavs have two envelope glycoproteins, hemagglutinin (ha) and neuraminidase (na), and are divided into subtypes based on antigenicity. iavs of h1-16 ha and n1-9 na subtypes have been isolated from water birds such as migratory ducks, the natural reservoir of iavs (fouchier et al., 2005; kida and yanagawa, 1979; webster et al., 1992) . has are expressed as trimers on the virion surface (wilson et al., 1981) . ha is initially synthesized as an inactive precursor ha0 and subsequently cleaved into ha1 and ha2 subunits by trypsinlike proteases of host cells (sakai et al., 2014) . the proteolytic cleavage of the ha molecule is essential for iavs to acquire infectivity (lazarowitz et al., 1973; wiley and skehel, 1987) . ha1 is responsible for virus binding to sialic acid receptors on the cell surface, and ha2 mediates membrane fusion under acidic conditions in endosomes, thereby delivering the viral genomic rna into the cytoplasm of target cells (matlin et al., 1981; rust et al., 2004) . nas, expressed on the virion surface as tetramers, have sialidase activity that enables mature virus particles to be released from infected cells after budding (colman, 1994; webster et al., 1992) . recently, iav-like rna genomes were detected in succession from 2 frugivorous bat species, little yellow-shouldered bats (sturnira lilium) and flat-faced fruit bats (artibeus planirostris) in guatemala and peru, respectively. the nucleotide sequences of the ha and na of these bat-derived influenza viruses (bativs) were divergent from all previously known iavs and new subtypes, h17n10 and h18n11, have been proposed . however, infectious viruses have not been isolated yet. previous studies by others tried to rescue bativs using a reverse genetics approach, but failed to generate infectious bativs (juozapaitis et al., 2014; zhou et al., 2014) . thus, the information on the biological properties of bativs is mostly speculative and the possible functions of bativ has and nas are only hypothetical, based on structural analyses tong et al., 2013; zhu et al., 2012 zhu et al., , 2013 . in this study, we utilized a vesicular stomatitis virus (vsv) pseudotype system, enabling us to directly analyze the biological functions of the bativ glycoproteins, which presumably play important roles in the replication cycle and pathogenicity. we found some bat cell lines susceptible to vsvs pseudotyped with bativ has and nas. our data suggest that bativs do not use sialic acids as a viral receptor and may have a limited host range, at least considering receptor engagement. to investigate cellular entry mediated by bativ glycoproteins, vsvs pseudotyped with bativ has and/or nas (vsvδg*-h17n10, -h18n11, -h17, -h18, -n10, and -n11) were generated as described in materials and methods. we first observed the virions of these pseudotyped vsvs using transmission electron microscopy ( fig. 1) . we found that the virions of all of these pseudotyped vsvs showed characteristic morphology (i.e. a bullet-like shape) similar to parental vsvδg*-g. it was noted that vsvs pseudotyped with bativ ha and na ( fig. 1a and b), ha alone ( fig. 1d and e), and na alone ( fig. 1g and h) all had numerous spikes on their surfaces, as was the case with vsvs pseudotyped with iav ha (h3) and na (n2) (fig. 1c ), h3 ha alone (fig. 1f) , and n2 na alone (fig. 1i ). immune electron microscopy with anti-h17 ha and anti-n10 na antibodies revealed that both bativ ha and na were efficiently incorporated into vsv particles (fig. 1k and l) . no difference was found in the overall morphology among these vsv virions. these data indicated that bativ has and nas were efficiently incorporated into the vsv particles. since previous studies have suggested that cell lines commonly used for iav propagation are nonpermissive for bativs, we screened various cell lines, including bat-derived cells, for susceptibility to pseudotyped vsvs (table 1) (fig. 2) . vsvs pseudotyped with has and nas of bativs and well-characterized iav strains, a/wsn/1933 (h1n1) (wsn) and a/aichi/2/1968 (h3n2) (aichi), were generated and treated with trypsin before use, since bativ has, like wsn and aichi has, have a cleavage site potentially recognized by trypsin-like proteases . we found that vsvδg*-wsn, -aichi, and -vsv g infected all cell lines tested ( fig. 2a, b , and e). on the other hand, vsvδg*-h17n10 and -h18n11 infected bat cell lines yubfkt1, indfspt1, and subk12-08, but not the other cell lines tested, except mdck cells, which were much less susceptible than these bat cells. since indfspt1 cells showed the highest susceptibility to vsvδg*-h17n10 and trypsin requirement for the ha function and the dispensability of na in virus entry iav has are known to be cleaved into ha1 and ha2 subunits by trypsin-like proteases to acquire the ability to mediate membrane fusion (klenk and rott, 1988) . western blotting revealed that both h17 and h18 has were cleaved into ha1 and ha2 by trypsin treatment (fig. 3a) . thus, we investigated the requirement of ha cleavage for infectivity of pseudotyped vsvs. as expected, vsvs pseudotyped with bativ glycoproteins did not infect indfspt1 cells without trypsin treatment, in a manner consistent with other iavs (data not shown), whereas trypsin-treated viruses efficiently infected this cell line (fig. 2) . these data indicated that the ha cleavage was a prerequisite for bativ infectivity. next, to clarify whether bativ has was responsible for virus entry, vsvδg*-h17n10, -h18n11,-h17, -h18, -n10, and -n11 were inoculated to infectivities of pseudotyped vsvs in several cell lines. vsvδg*-wsn, -aichi, -h17n10, -h18n11, and vsvδg*-g were inoculated into several cell lines (table 1) . infectious units (ius) of each virus in different cell lines were determined by counting the number of gfp-expressing cells. each experiment was performed three times, and averages and standard deviations are shown. infectivities of vsvδg*-h17n10 and -h18n11 in some cell lines were under the limit of detection ( †). significant differences (student's t-test) were found between mdck and any of the bat cell lines (p o0.01). indfspt1 cells and their infectivities were compared (fig. 3b ). we found that vsvδg*-h17 and -h18 infected indfspt1 cells as efficiently as vsvδg*-h17n10 and -h18n11, whereas the infectivity of vsv pseudotyped with wsn or aichi ha alone was much lower than that of vsvs pseudotyped with both ha and na of the respective viruses. vsvδg*-n10 and -n11 showed no infectivity, similarly to vsvs pseudotyped with nas of wsn and aichi. these results suggest that bativ ha is the only glycoprotein mediating both virus attachment and membrane fusion and that bativ na is dispensable during the entry into cells. it is generally known that iav has mediate membrane fusion in a low ph-dependent manner (kida et al., 1983; rust et al., 2004) . to investigate the requirement of endosomal acidification for bativ ha-mediated membrane fusion, indfspt1 cells were treated with ammonium chloride, which is known to neutralize the ph of acidic intracellular compartments, and then infected with vsvδg*-wsn, -aichi, -h17n10, -h18n11, and vsvδg*-g. treatment of the cells with ammonium chloride markedly reduced the infectivity of vsvδg*-h17n10 and -h18n11, as was the case with vsvδg*-g, -wsn, and -aichi, in a dose-dependent manner, suggesting that bativ has require a low ph for membrane fusion, consistent with the other iav has (fig. 4a) . to obtain information on the biological characteristics of cellular receptors for bativs, indfspt1 cells were pretreated with tunicamycin, pronase, or neuraminidase (i.e., an n-linked glycosylation inhibitor, mixture of proteases, and sialidase, respectively), and then infected with pseudotyped vsvs (fig. 4b-d) . tunicamycin treatment markedly reduced the infectivities of vsvδg*-g, -h17n10, and -h18n11, but less significantly those of vsvδg*-wsn and -aichi (fig. 4b) . preincubation of cells with pronase reduced the infectivities of the pseudotyped vsvs, except for vsvδg*-wsn (fig. 4c ). neuraminidase treatment reduced vsvδg*-wsn and -aichi infectivities, but interestingly did not affect the infectivities of vsvδg*-h17n10 and -h18n11 (fig. 4d) . we confirmed that no remarkable cytotoxicity was observed during these treatments (fig. 4e) . these results suggest that bativ has do not recognize sialic acids which are critical components of the iav receptor and some other molecules such as glycoproteins may serve as bativ receptors. in recent years, particular attention has been paid to batderived viruses since some species of bats have been reported to be reservoirs of several viral zoonotic pathogens (e.g., lyssavirus, henipavirus, sars coronavirus, and marburgvirus) (calisher et al., 2006; smith and wang, 2013; wang et al., 2011 wang et al., , 2007 . although the zoonotic potential of bativs has not been fully evaluated yet, recent studies generated reassortant viruses that had ha and na gene segments of well-characterized iavs (i.e., h1, h3, and h7 has and n1, n2, and n7 nas) and the other gene segments derived from bativs, and demonstrated that the reassortant viruses replicated in cultured cells and caused severe disease in mice (juozapaitis et al., 2014; zhou et al., 2014) . however, characterization of bativ has and nas remains an open research problem, since reassortant viruses carrying the bativ ha and na gene segments have not been rescued due to the lack of information on cells susceptible to this novel virus. in this study, we first determined the potentially permissive bat cell lines using vsvs pseudotyped with bativ has and nas. we demonstrated that vsvδg*-h17n10 and -h18n11 efficiently infected the bat-derived cell lines indfspt1, yubfkt1, and subk12-08. while indfspt1 was derived from pteropus giganteus (family pterodidae), yubfkt1 and subk12-08 were prepared from bats belonging to the same species (miniopterus sp., family miniopteridae). based on a phylogenetic study of bats (agnarsson et al., 2011) , miniopteridae belongs to the same cluster as phyllostomidae, from which h17n10 and h18n11 bativs were detected, little yellow-shouldered bats (s. lilium) and flat-faced fruit bats (a. planirostris), respectively . thus, bativ has appear to recognize cell surface molecules shared among the bats at least in miniopteridae and phyllostomidae families. indfspt1 should also have such molecules since it showed the highest susceptibility to bativ ha-pseudotyped vsvs. it was noted that vsvδg*-h17n10 and -h18n11 also infected mdck cells, although less efficiently than these bat cell lines. this result might contradict a previous report that h17 ha did not bind to the surface of mdck cells (sun et al., 2013) . however, it is conceivable that the binding affinity of bativ ha to mdck cell surface molecules is quite low and thus below the level of detection in the assay used in the previous study. in the present study, mdck cells indeed showed much lower susceptibility to bativ ha-pseudotyped vsvs than yubfkt1, indfspt1, and subk12-08. nonetheless, it would be interesting to clarify whether mdck cells express some bativ receptor molecules shared with the bat cell lines. it is also noteworthy that vsvδg*-h17n10 and -h18n11 did not infect vero e6, hek293, sk-l, and qt6 cells. previous studies show that quails can act as an intermediate host in the interspecies spread of avian iavs (makarova et al., 2003; perez et al., 2003; uchida et al., 2011) . furthermore, pigs are thought to serve as "mixing vessels" for the production of reassortant viruses between avian and human iavs (chang et al., 2009; hinshaw et al., 1981; ito et al., 1998; kida et al., 1994; scholtissek et al., 1985) . our results suggest that bativs do not readily infect humans, pigs, or birds and support that notion that these viruses have limited zoonotic potential (juozapaitis et al., 2014; zhou et al., 2014) . it is known that vsv g protein and iav ha recognize ubiquitous cell surface molecules for virus entry. vsv g recognizes various cell surface molecules and thus vsv exhibits remarkably robust and pantropic infectivity (finkelshtein et al., 2013; johannsdottir et al., 2009; lichty et al., 2004; roche et al., 2008) . iav has recognize sialic acids typically occupying the terminal positions of glycoproteins or glycolipids (gambaryan et al., 2005; suzuki et al., 2000) . accordingly, vsvδg*-g, -wsn, and -aichi infected all cell lines used in this study, whereas we found that vsvδg*-h17n10 and -h18n11 infected only particular bat cell lines and that neuraminidase treatment did not affect the infectivities of vsvδg*-h17n10 and -h18n11. this result was in agreement with previous results based on the crystal structure analysis and surface plasmon resonance of sialylated glycans with α2,3-linkage or α2,6-linkage (sun et al., 2013; zhu et al., 2013) . glycan microarray analyses also showed that h17 ha did not display obvious avidity to any glycans (sun et al., 2013) . interestingly, we found that the infectivities of vsvδg*-h17n10 and -h18n11 were markedly reduced by the treatment of indfspt1 cells with tunicamycin, which , pronase (c), or neuraminidase (d) as described in materials and methods. treated cells were then infected with vsvδg*-wsn, -aichi, -h17n10, -h18n11, and vsvδg*-g appropriately diluted to yield 200-1000 ius. the percentages of infectivity were determined by setting the number of the untreated cells to 100%. each experiment was performed three times, and averages and standard deviations are shown. cell viabilities were measured by the alamar blue assay (e). the percentages of fluorescence were determined by setting the number of the untreated cells to 100%. each experiment was performed three times, and averages and standard deviations are shown. statistical significances compared to untreated cells were calculated using student's t-test (*po 0.01). inhibits n-linked glycosylation, leading to unfolding or misfolding of proteins and inhibition of glycoprotein expression. pretreatment of the cells with pronase also reduced the infectivities of vsvδg*-h17n10 and -h18n11. taken together, our data suggest that some particular glycoprotein(s) serve as receptors for bativs. previous studies indicated that n10 na did not have sialidase activity zhu et al., 2012) . it was also shown that most of the amino acid residues responsible for na activity were substituted, and proposed that n10 na protein should be termed an na-like protein (zhu et al., 2012) . in this study, we found that vsvs pseudotyped with bativ nas alone were not infectious, confirming that na did not play a central role in iav entry into cells. however, it should be noted that the production efficiency of pseudotyped vsvs bearing wsn and aichi has alone was much lower than that of vsvs pseudotyped with both has and nas, suggesting that na activity facilitated virus release from infected cells and/or increased the ha function (su et al., 2009 ). by contrast, no remarkable difference was found in the infectivity between vsvs pseudotyped with bativ glycoproteins (i.e., ha alone vs. ha and na). these data suggest that, unlike the other iavs, the target molecules of bativ has and nas are different and that the "ha-na balance" concept proposed for iavs does not be applied to bativs. because h17n10 and h18n11 bativs have never been isolated, their ability to infect humans and other mammals and the pathogenic potential for these hosts can only be hypothesized. in this study, the replication-incompetent vsv pseudotype system enabled us to investigate the cellular tropism controlled by the interaction between bativ ha and its cellular ligand, which might be some glycoproteins. although a reverse genetics approach and in vivo experiments for the infectious bativ are needed to provide direct evidence of its pathogenicity and host specificity, our data suggest that bativ may preferentially infect particular bat species. cells hek293, hek293t, and vero e6 cells were grown in dulbecco's modified eagle's medium (dmem) with 10% fetal calf serum (fcs) and penicillin-streptomycin. mdck cells were grown in dulbecco's modified eagle's medium (dmem) with 10% calf serum, lglutamine, and penicillin-streptomycin. bat cell lines bkt1, fbkt1, yubfkt1, indfspt1, demkt1, zfbk11-97, subk12-08, and zfbs13-76a were established as described previously (maeda et al., 2008; maruyama et al., 2014) . all bat cell lines were grown in rpmi-1640 medium with 10% fcs, l-glutamine, and penicillin-streptomycin. coding regions of the has and nas of bativs were synthesized in vector puc19 or pucfa, based on the nucleotide sequences of genbank (accession numbers for h17 ha, n10 na, h18 ha, and n11 na: cy103892, cy103894, cy125945, and cy125947, respectively) (fasmac). each coding region of the viral proteins was amplified by pcr with primers including restriction sites, the kozak sequence, and the stop codon. after digestion by restriction enzymes, each gene was cloned into the mammalian expression vector pcaggs (niwa et al., 1991) . h1 ha and n1 na of a/wsn/ 1933 (h1n1) (wsn), and h3 ha and n2 na of a/aichi/2/1968 (h3n2) (aichi) were cloned into pcaggs as described previously (muramatsu et al., 2013) . using vsv containing the green fluorescent protein (gfp) gene instead of the receptor-binding vsv g protein gene (vsvδg*-g), pseudotyped viruses with has and/or nas of bativs, wsn, and aichi were generated as described previously (takada et al., 1997) . vsvs pseudotyped with iav glycoproteins were pretreated with trypsin (final concentration 0.0005%) for 30 min at 37°c, followed by incubation with an anti-vsv g monoclonal antibody, vsv-g(n) 1-9, to abolish the background infectivity of parental vsvδg*-g (nakayama et al., 2011) . for virus titration, 10-fold diluted pseudotyped vsvs were inoculated into confluent monolayers of each cell line on 96-well plates, and the infectious unit (iu) in each cell line was determined 20 hours later by counting the number of gfp-expressing cells under a fluorescent microscope. transmission electron microscopy was carried out as described previously (maruyama et al., 2014) . pseudotyped vsvs fixed with 0.25% glutaraldehyde were adsorbed to collodion-carbon-coated copper grids and negatively stained with 2% phosphotungstic acid solution (ph¼ 5.8). for immune transmission electron microscopy, we used an anti-ha2 monoclonal antibody (3n12-6-4) broadly cross-reactive to group 1 ha subtypes, anti-n10 na mouse serum (fm0137) produced by immunization with a synthetic peptide corresponding to amino acid residues 328-343 (aqekgeggiqg-filde) of n10 na, and an immunogold-conjugated goat antimouse igg (hþ l) polyclonal antibody (bb international). samples were examined with an h-7650 electron microscope (hitachi) at 80 kv. pseudotyped vsvs were treated with or without trypsin (final concentration 0.0005%) for 30 min at 37°c and then mixed with sds-page sample buffer with 5% 2-mercaptoethanol and boiled for 5 minutes. after electrophoresis on 5-20% supersep (wako), separated proteins were blotted on a polyvinylidene difluoride membrane (millipore). the membrane was incubated with an anti-h3n2 chicken polyclonal antiserum or anti-ha2 monoclonal antibody 3n12-6-4, which reacts to h1, h2, h5, h6, h17, and h18 has, followed by incubation with peroxidase-conjugated rabbit anti-chicken igy (hþ l) or goat anti-mouse igg (hþ l) (jackson immunoresearch). the bound antibodies were visualized with immobilon western (millipore). indfspt1 cells were preincubated with the medium containing an endosomal acidification inhibitor, ammonium chloride (wako), at 37°c for 2 h in a co 2 incubator, and then infected with pseudotyped vsvs appropriately diluted to yield 200-1000 ius, followed by incubation in the presence of ammonium chloride. indfspt1 cells were also pretreated with pronase (a mixture of endo-and exoproteases from streptomyces griseus) (calbiochem) (narahashi et al., 1968) , for 20 min, an n-glycosylation inhibitor (tunicamycin from streptomyces sp.) (sigma) for 8 h, which blocks the reaction of udp-glcnac and dolichol phosphate in the first step of glycoprotein synthesis, thus inhibiting the synthesis of nlinked glycoproteins, or neuraminidase from vibrio cholerae (roche) (uchida et al., 1977) for 1 h at 37°c in a co 2 incubator. treated cells were washed with serum free rpmi-1640 medium 3 times, and then incubated with pseudotyped vsvs appropriately diluted to yield 200-1000 ius for 1 h. after adsorption of the virus, the inoculum was aspirated and the growth medium (10% fcs rpmi-1640 medium) was added. cells were incubated for 20 h, and infected cells were counted under a fluorescent microscope. cell viabilities were assessed by the alamar blue assay. after treatments of each enzyme and inhibitor, cells were incubated with fcs-free rpmi-1640 medium containing 10% alamar blue (biosource) for 2 h, and fluorescence with excitation wavelength at 530-560 nm was measured using envision (perkinelmer). a time-calibrated species-level 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a continuing source of emerging infections in humans characterization of uncultivable bat influenza virus using a replicative synthetic virus crystal structures of two subtype n10 neuraminidase-like proteins from bat influenza a viruses reveal a diverged putative active site hemagglutinin homologue from h17n10 bat influenza virus exhibits divergent receptor-binding and ph-dependent fusion activities this work was supported by kakenhi (15h01249), a grant-in-aid for scientific research from the ministry of education, culture, sports, science and technology (mext) and the japan society for the promotion of science (jsps) (14j00026), japan, and partly by the japan initiative for global research network on infectious diseases (j-grid) (15fm0108008h0001) and the japan science and technology agency (jst) and japan international cooperation agency (jica) within the framework of the science and technology research partnership for sustainable development (satreps) (15jm0110005h0004). funding was also provided by the program for leading graduate schools from mext, japan. key: cord-330907-srb8ac7l authors: leparc-goffart, isabelle; hingley, susan t.; chua, ming ming; jiang, xinhe; lavi, ehud; weiss, susan r. title: altered pathogenesis of a mutant of the murine coronavirus mhv-a59 is associated with a q159l amino acid substitution in the spike protein date: 1997-12-08 journal: virology doi: 10.1006/viro.1997.8877 sha: doc_id: 330907 cord_uid: srb8ac7l abstract c12, an attenuated, fusion delayed, very weakly hepatotropic mutant of mouse hepatitis virus strain a59 (mhv-a59) has been further characterized. we have previously shown that c12 has two amino acid substitutions relative to wild type virus in the spike protein, q159l (within a region of s1 shown to bind to viral receptor in anin vitroassay) and h716d (in the proteolytic cleavage recognition site). we have sequenced the rest of the 31-kb genome of c12 and compared it to wild type virus. only three additional amino acids substitutions were found, all encoded within the replicase gene. analysis of c12in vivoin c57bl/6 mice has shown that despite the fact that this virus replicates in the brain to titers at least as high as wild type and causes acute encephalitis similar to wild type, this virus causes a minimal level of demyelination and only at very high levels of virus inoculation. thus acute encephalitis is not sufficient for the induction of demyelination by mhv-a59. analysis of mutants isolated at earlier times from the same persistently infected glial cell culture as c12, as well as mutants isolated from a second independent culture of persistently infected glial cells, suggests that both the weakly demyelinating and the weakly hepatotropic phenotypes of c12 are associated with the q159l amino acid substitution. for the altered pathogenic properties of the mutant viruses, we compared the sequence of the spike (s) genes the murine coronavirus, mouse hepatitis virus strain of wild type and mutant viruses isolated from persistently a59 (mhv-a59), produces both hepatitis and neurologiinfected glial cells cultures. we focused on the s protein cal disease in susceptible mice. neurological disease because this protein mediates both binding to the viral includes both acute meningoencephalitis and chronic receptor (holmes and compton, 1995) and cell to cell demyelinating disease (lavi et al., 1984b) . in vitro, mhvfusion. this, in combination with the fact that viruses a59 causes a lytic infection in mouse fibroblast cells, with mutations in the s gene have altered pathogenic but a persistent productive, nonlytic infection of primary properties (fleming et al., 1986; gallagher et al., 1990 ) cultured glial cells (lavi et al., 1987) . the mechanism of suggest that s is a major determinant of viral tropism. demyelination is not well understood but it is thought to the fusion-delayed phenotype was shown to result from involve elements of direct viral infection as well as an the substitution of an aspartic acid residue for a histidine immune mediated component (houtman and fleming, (h716d) in the basic sequence that serves as a recogni-1996a). tion signal for proteolytic cleavage of the s protein (gom-we have been using a group of mhv-a59 mutants with bold et al., 1993) . this amino acid substitution prevents altered pathogenic properties to map the determinants of cleavage of the 180-kda precursor s protein into the two central nervous system pathogenesis and to understand approximately 90-kda subunits, s1 and s2 (luytjes et al., what features of infection of mice lead to demyelination. 1987; gombold et al., 1993) . aside from the cleavage we have previously characterized this group of attenurecognition site mutation (h716d), all the mutant viruses ated, fusion-delayed mutants, which were isolated from examined contained one, and only one, additional amino persistently infected glial cells. these mutants had acid substitution in s, q159l, located within the amino greatly reduced ability to replicate in the liver and cause terminal portion of the s1 subunit. analysis of fusion hepatitis (hingley et al., 1994) . competent revertant viruses, however, showed that atten-in order to begin to identify the mutations responsible uation and loss of hepatotropism were not linked to the fusion phenotype and did not map to the h716d amino acid substitution. an association between the q159l mu-hypothesis that this mutation may play a role in tissue fin and sectioned for staining with luxol fast blue to detect plaques indicative of demyelination. for each of five mice tropism. interestingly, q159l lies within the amino terminal 330 amino acids of s1, a region of the spike protein at each dose of virus, demyelination was quantitated by examining one spinal cord section (four quadrants) from demonstrated to bind to the viral receptor in an in vitro assay (kubo et al., 1994; suzuki and taguchi 1996) , sug-each of five levels of spinal cord; thus there were 100 quadrants examined for each dose of virus. the experi-gesting the possibility that this amino acid may influence the interaction of the spike protein with the viral receptor. ments were designed so that there would be five animals surviving acute infection even at high doses of virus rela-earlier studies (see table 1 ) also demonstrated that viruses with identical s gene sequences had different tive to the ld 50 ; however, in the case of the highest dose of c3 and all doses of b12, there were less than five virulence and hepatotropism phenotypes (hingley et al., 1994) . thus, to determine which additional genes may surviving animals (figs. 2 and 3) . demyelination was also verified in the brain by staining with luxol fast blue. influence pathogenesis, we have sequenced the entire genome of the c12 mutant of mhv-a59, one of the viruses isolated from persistently infected glial cells (gom-genome sequencing bold et al., 1993; hingley et al., 1994) . in addition to the two mutations found in the s gene, there were only five for sequencing of the viral genome, reverse transcriptase/polymerase chain reaction amplification (rt/ other mutations; of these, three resulted in amino acid substitutions, all within the viral replicase gene. pcr) was carried out, using as templates, cytoplasmic rna extracted from l2 cell monolayers infected with wild we describe here the further characterization of the c12 mutant. we have shown that despite the fact that c12 type mhv-a59, c12, c3, c5, c8, b11, or b12. complementary dna was synthesized using random oligomers as replicates in the brain and causes encephalitis similar to that of wild type virus, at a dose of 5000 pfus (2 wild primers. mhv-a59-specific primers were designed to amplify fragments of about 600 base pairs. double-type ld 50 ), at which wild type virus induces extensive demyelination in all infected animals, c12 does not in-stranded pcr products were gel purified and analyzed by automated sequencing using the taq dye terminator duce detectable demyelination. analysis of other mutants isolated at earlier time points from persistently infected procedure according to the manufacturer's protocol (taq dyedeoxy terminator cycle sequencing kit, applied bio-glial cell cultures (hingley et al., 1994) as well as mutants isolated from an independent culture of persistently in-systems) and the same primers used for amplification. each fragment was sequenced in both directions. the fected glial cells, suggests that the q159l mutation, and not the three replicase mutations, is associated with loss entire c12 genome was sequenced; fragments of each of the other viruses were sequenced as described under of the ability to demyelinate efficiently as well as the loss of the ability to induce hepatotropism. results. sequence comparison of the genomes of the c12 mutant and mhv-a59 mhv-a59 was obtained originally from dr. lawrence sturman (albany, ny). the mutants were plaque purified we have shown previously that the spike (s) protein from the supernatant of either the ''c'' culture of persisencoded by the c12 mutant has two amino acid substitutently infected glial cells at 1 week (c3), 6 weeks (c5), tions as compared with the wild type s protein. earlier 12 weeks (c8), and 16 weeks (c12) postinfection or from studies demonstrated that viruses with the identical s the independent ''b'' culture of persistently infected glial protein sequence had different virulence and hepatotropcells at 18 weeks postinfection (b11, b12) and characterism phenotypes (see table 1 ). for example the mutant ized as previously described (gombold et al., 1993; hing-b11 appeared to be blocked in its spread from the central ley et al., 1994, 1995) . nervous system to the liver, while the mutant c12 was inhibited in its ability to replicate in hepatocytes even demyelination when inoculated directly into the liver (hingley et al., 1994) . the b12 mutant, while nonhepatotropic like c12, four-to 6-week-old mhv-free c57bl/6 mice (jackson laboratories, bar harbor, me) were inoculated intracere-retained its virulence (hingley et al., 1994) . thus, we concluded that these pathogenic properties must also brally with 10-fold serial dilutions of wild type or mutant mhv-a59. at 30 days postinfection, mice surviving acute involve genes outside of s. this, in combination with the idea that it is difficult to assign phenotypes to specific infection were perfused with phosphate-buffered saline, followed by 10% buffered formalin. brains and spinal sequences in s without knowing if there are any other mutations outside of s, led us to sequence the rest of cords were removed and tissue was embedded in parafwith, the n protein (fischer et al., 1997) and the hemagglutinin esterase (he) protein, which is encoded, but not pathogenic phenotypes of mutants containing q159l expressed in mhv-a59 (luytjes et al., 1988 (luytjes et al., 1988) . needed to kill half the weanling c57bl/6 mice after intracerebral inoculation. a minus sign indicates the ld 50 is at least 2 log 10 greater than the deduced sequences of the nonstructural proteins wild type. these data were taken from hingley et al. (1994) . from the first amino acid in each orf.) of the two substitutions in orf 1a, p1699s is in the predicted papain-like proteinase-2 (plp-2) domain; the substitution in orf 1b, the c12 genome to identify other possible mutations that may play a role in these different pathogenicities. r1330s, is in the predicted helicase domain (lee et al., 1991; bredenbeek et al., 1990; bonilla et al., 1994) . there we have sequenced the entire 31-kb genome of c12. sequencing was carried out by rt/pcr of viral rna, is only one silent mutation in the genome of c12; nucleotide 2471 of orf 1a is u rather than c, which is in the present in infected cells, as described under materials and methods. this generates a consensus sequence wild type genome. we have also compared the noncoding regions of c12 because it is derived from the rna genome and not cloned cdnas; we compared the c12 sequence with the and wild type mhv-a59. we have found no differences between c12 and wild type in the 5 or 3 noncoding published sequence for wild type a59 (from our lab as well as from other labs) (budzilowicz et al., 1985; budziregions. with the exception of the conserved intergenic region preceding the m gene, there were also no differ-lowicz and weiss, 1987; zoltick et al., 1990; bonilla et al., 1994; weiss et al., 1993; bredenbeek et al., 1990 ; luytjes ences between c12 and wild type mhv-a59 intergenic sequences. there is one nucleotide change in the in-et , 1988 armstrong et al., 1983 armstrong et al., , 1984 . whenever we found a difference between c12 and the published tergenic region preceding the m gene. in c12 the sequence is aaucuaaac instead of aauccaaac present mhv-a59 wild type sequence, we made a new preparation of cdna from intracellular c12 rna, amplified the in wild type mhv-a59 (budzilowicz et al., 1985) . we examined the early mutants from the c culture for same double stranded dna fragment, and verified the c12 sequence. we then determined if our wild type se-the presence of the three replicase mutations as well as the intergenic nucleotide difference. these include the quence was the same as the published one. the c12 genome had surprisingly few mutations compared to the c3, c5, and c8 mutants isolated at various times after infection (1, 6, and 12 weeks, respectively). neither the wild type genome after 16 weeks of culture in glial cells. figure 1 shows the comparison of the wild type and c12 replicase mutations (table 2 ) nor the intergenic region mutation were present in any of the early mutants. this genomes. with the exception of the two previously discussed suggested that these mutations arose in the culture later than either of the s mutations. amino acid substitutions in s, h716d and q159l (fig. 1) , there are no amino acid substitutions in the deduced in order to determine if these replicase substitutions were a general characteristic of the attenuated, tropism sequences of any of the structural proteins; this includes spike (s) (luytjes et al., 1987; hingley et al., 1994) , matrix mutants that arise in persistently infected glial cells, we examined the genomes of two mutants, b11 and b12, (m) (armstrong et al., 1984) , nucleocapsid (n) (armstrong et al., 1983) , and small membrane (e) (budzilowicz and isolated from the independent (''b'') culture of persistently infected glial cells (gombold et al., 1993) . none of the weiss, 1987) proteins as well as the open reading frames (orfs) for the i protein, encoded within, but out of frame three replicase mutations were found in either of these fig. 1. sequence comparison of the c12 and wild type mhv-a59 genomes. the entire c12 genome was sequenced and the sequence compared to that of wild type mhv-a59 (as described in the text). (a) schematic diagram of the viral genome and the location of the five amino acid substitutions and the one nucleotide change in an intergenic region of c12. the structural proteins are spike, he (hemagglutinin esterase), m (matrix), n (nucleocapsid), e (small membrane), and i (internal orf). the nonstructural replicase proteins are encoded in orf 1a and orf 1b. the domains of the replicase plp-1 (papain-like proteinase-1) and plp-2 (papain-like proteinase-2), 3c (poliovirus 3c like proteinase), pol (polymerase), and hel (helicase) were all predicted by lee et al. (1991) . the nonstructural, nonessential protein products of orfs 2a, 4, and 5a are not shown. the one silent mutation (nucleotide 2471 is u rather than c, present in the wild type genome) is not shown. (b) genes and predicted functional domains which contain these amino acid substitutions. the amino acids are numbered from the beginning of each open reading frame (orf) (bredenbeek et al., 1990; bonilla et al., 1994; luytjes et al., 1987) . the cleavage site of s was mapped by luytjes et al. (1987) and the mapping of the receptor binding domain is from kubo et al. (1994) . the intergenic regions are thought to be involved in transcriptional regulation (van der most and spaan, 1995). genomes. we then sequenced a larger region around lab (bonilla et al., 1994; budzilowicz et al., 1985; budzilowicz and weiss, 1987; weiss et al., 1993; , the mutation in the predicted plp-2 domain (1100 nucleotides) and the mutation in the predicted helicase domain 1990, unpublished sequences of orf 1b). mutations encoding amino acid substitutions included two in orf 1a, (1800 nucleotides) to determine if there were other mutations within either of these predicted domains in b11 or two in orf 1b, one in s, one in n, and one each in orfs 4 and 5a. additional silent mutations included three in b12. b11 did have another amino acid substitution within the plp-2 domain, v1798m. orf 1a, two in orf 1b, and two in n. there were also two nucleotide changes and two insertions in the 3 non-we also found several mutations, both silent and coding, in the comparison of wild type mhv-a59 that we coding region of the genome. have used to derive the mutants (obtained from dr. l. sturman) as compared to the published sequences demyelination of c12 (bredenbeek et al., 1990; bonilla et al., 1994; luytjes et al., 1987; luytjes et al., 1988; hingley et al., 1994; armwe have shown previously that the c12 mutant of mhv-a59 has a fusion delayed phenotype in vitro (gom-strong et al., 1983 (gom-strong et al., , 1984 budzilowicz et al., 1985; budzilowicz and weiss, 1987; weiss et al., 1993; zoltick et bold et al., 1993) . furthermore, c12 replicates to titers equal to wild type virus in the cns and causes a similar al., 1990), some of which came from the sequencing of another mhv-a59 (obtained from dr. j. leibowitz) in our level of encephalitis. however, it is very weakly hepatoe fusion was defined as previously described gombold et al., (1993) . a negative sign signifies a delayed fusion phenotype in which fusion is negative when measured in l2 cells at 10 h postinfection but eventually does occur by about 24 h postinfection. tropic (hingley et al., 1994) . in an earlier study (gombold analysis of the mutants isolated earlier from persistently infected cultures (hingley et al., 1994) . we have used et al., 1995), we observed that c12 appeared to demyelinate strikingly less well than wild type virus. to assess the same mutants to ask whether demyelination is also associated with this mutation or perhaps with the repli-the difference in demyelination more quantitatively, we measured the amount of demyelination (as described case mutations that arise later during persistence. thus, the c3, c5, and c8 mutants were analyzed for the ability under materials and methods) induced by wild type and c12 viruses as a function of dose of virus inoculated. to induce demyelination. these results are shown in fig. 3 and summarized in table 2 . the c3 mutant was able figure 2 shows these results. for wild type mhv-a59, the amount of demyelination was approximately linear to induce demyelination with the same efficiency as wild type virus, while the c5 and c8 viruses exhibit a very with the log 10 of the amount of virus inoculated. with a dose of 5000 pfu (approximately 2 ld 50 s) of wild type weakly demyelinating phenotype, similar to c12. this suggests that the demyelination phenotype correlated virus, all the animals infected had demyelination (5/5) and demyelination was present in approximately 63% of with the appearance of the q159l mutation and before the h716d and the replicase amino acid substitutions in the spinal cord quadrants examined. we have analyzed demyelination by wild type mhv-a59 in four experiments. the evolution of the ''c'' culture. the range of demyelination was from 59 to 69% of the quadrants for a dose of 3700 to 5000 pfu; thus the discussion amount of demyelination is quite reproducible. at the we have previously shown that the group of fusion same dose c12 caused no detectable demyelination. delayed, nonhepatotropic mhv-a59 mutants isolated even at 100-fold higher dose of inoculation, demyelinfrom two independently infected glial cells had the same ation was observed in 3/5 of c12 infected animals and two, and only two, amino acid substitutions relative to in only 13% of the quadrants (fig. 2) . thus, despite the wild type in the gene encoding the spike glycoprotein s. fact that c12 replicates to high titer in the brain and the cleavage recognition site amino acid substitution causes encephalitis to a similar extent as wild type virus (h716d) clearly correlates with the delayed fusion pheno(hingley et al., 1994) , it is a poor inducer of demyelination. type, but not with loss of hepatotropism (hingley et al., similar results were obtained with the b11 and b12 mu-1994; gombold et al., 1993) . the other amino acid substitants, isolated from an independent culture of persistution, q159l, appeared to correlate with the hepatotroptently infected glial cells (fig. 2) . ism phenotype (hingley et al., 1994 (hingley et al., , 1995 see table 2 ). we concluded this because the q159l mutation first ap-association of demyelination with q159l peared in virus plaque purified from two independent persistently infected glial cell cultures at 6 weeks postin-an association between the q159l substitution in s and the loss of hepatotropism was demonstrated by the fection, at the same time that the altered hepatotropism phenotype became evident. the h716d mutation did not esis of this virus. since there is not yet available an infectious cdna clone for mhv, we were not able to appear until 12 weeks postinfection, along with the delayed fusion phenotype (hingley et al., 1994; table 2 ). introduce individual mutations into the viral genome. therefore, we used an alternative method to map muta-it was not possible to map unambiguously the hepatotropic phenotype to q159l when the sequence of the tions that alter pathogenic properties; we sequenced the entire 31-kb genome of c12. rest of the genome was not known. furthermore, the observation that c12, b11, and b12 have somewhat dif-surprisingly, there were only five amino acid substitutions in the entire 31-kb genome of c12 after 16 weeks ferent pathogenic properties while possessing the same sequence of s (table 1 ; hingley et al., 1994) suggested of persistent culture in glial cells. this suggests that the fidelity of the mhv polymerase is higher than commonly that mutations outside of s must influence the pathogenassumed (lai, 1990; adami et al., 1995) and/or that, dur-and for sequencing of genes 1, 2a, 4, and 5 (bonilla et al., 1994; budzilowicz and weiss, 1987; weiss et al., 1993; ing persistence in glial cell cultures, there is a strong selective pressure against mutation within the genome. zoltick et al., 1990) . the only amino acid substitutions in the c12 genome consistent with this observation was our finding that, in the comparison of our mhv-a59 lab strain with the other than the two encoded in the s gene were three amino acid substitutions in the replicase gene. there are published sequences, there were just 6 amino acid substitutions and 11 silent mutations. we do not think these two amino acid substitutions encoded in orf1a (p1699s and m2196k) and one in orf 1b, r1330s. the p1699s sequences differences between the wild type mhv-a59 parent of c12 and the published sequences are signifi-substitution is in the predicted plp-2 domain of orf 1a and the r1330s substitution is in the predicted helicase cant in terms of pathogenesis. the mhv-a59 used as our wild type in these experiments (obtained from dr. l. domain of orf 1b (bredenbeek et al., 1990; lee et al., 1991 ) (see fig. 1 ). it is difficult to predict whether the sturman) exhibits the same pathogenic properties as the mhv-a59 (obtained from dr. j. leibowitz) that we used mutation in plp-2 is likely to be significant. the b11 mutant also has a mutation, albeit a different one from for our previous pathogenesis studies (lavi et al., 1984b) c12, v1798m in plp-2. numerous attempts in our lab rather than the replicase mutations. the relationship of q159l to virulence is, however, not as clear. the obser-to detect an activity for plp-2 had been unsuccessful, suggesting that the plp-2 domain does not encode an vation that the b12 mutant retains its virulence (hingley et al., 1994, table 1 ) demonstrates that q159l is not active proteinase; if this were the case, then mutations in this region of the replicase gene would not be ex-sufficient for attenuation and furthermore that the loss of hepatotropism does not necessarily lead to attenuation. pected to have a phenotype. however, recently, we have found that a recombinant protein encoded in the prethe hepatotropic, demyelinating c3 mutant also retains its virulence while the nonhepatotropic, weakly demyelin-dicted plp-2 domain does indeed have a proteinase activity (h. teng and s. r. weiss, unpublished results) . ating c5 and c8 mutants are somewhat attenuated although probably not as attenuated as c12 (data not thus, if plp-2 is a necessary activity for replication, these mutations may be significant. the c12 mutation in orf shown). thus there may be other mutations in these viruses, absent in c12, that modify the virulence pheno-1b is in the predicted helicase domain. the recent demonstration of an atpase activity for a recombinant protein type. the data discussed above and shown in table 1 suggest there must be mutations outside of s that result encoded in the predicted helicase domain of orf 1b of the human coronavirus 229e (heusipp et al., 1997) in the differences in hepatotropism between b11 and c12, and differences in virulence between b12 and the supports the prediction that this is indeed a helicase domain. it is likely that such a polypeptide might interact other mutants. it is possible that the three replicase mutations in c12 modify the pathogenic properties. we cannot with host cell proteins and thus influence tropism. the third mutation is in orf 1a, m2196kl, in a region of the be sure of the effects of these mutations until we can analyze recombinant viruses containing only the q159l replicase that has not been assigned a function. the aaucuaaac sequence observed preceding the mutation; such work is in progress. the mechanism of mhv induced demyelination is to m gene of c12 (different from the wild type aauccaaac) is the sequence found in most of the intergenic regions this date still not well understood. there is mounting evidence that both immune mediated components as in the wild type genome (budzilowicz et al., 1985; bonilla et al., 1994; luytjes et al., 1987) . thus it is clear that this well as direct infection of virus are factors in the establishment of demyelination (houtman and fleming, sequence can function as an intergenic region. since the mechanism of generation of coronavirus mrna is still 1996a). our observation that for wild type mhv-a59 the amount of demyelination was proportional to the log 10 of not clearly understood, the role of coronavirus conserved intergenic regions is also not known. however, it is be-the virus inoculum (fig. 2) argues that the amount of virus in the cns is important in the development of demyelin-lieved that intergenic regions play a role in the control of subgenomic mrna transcription, perhaps serving as ation. this is consistent with the hypothesis that a direct infection mechanism plays a significant role in demyelin-promoters for subgenomic mrna synthesis (van der most and spaan, 1995) . thus, it is possible that this ation (houtman and fleming, 1996a) . alternatively, the larger virus inoculum could result in a larger number of nucleotide change could effect transcription of the c12 mrna encoding the m gene. we doubt that this is the persistently infected cells in the cns to be damaged by the immune response. case as the levels of m protein in cells infected with c12 is approximately the same as in wild type infected cells mhv acute infection includes encephalitis and virus replication at high titer in the cns. it has been shown (unpublished data). while there is no evidence suggesting that any of the mutations in the c12 genome, that while infectious virus cannot be recovered from the cns after the acute infection, viral rna persists in the either silent or encoding amino acid substitutions, can produce a change in replication or pathogenesis by ef-white matter of the cns probably throughout the lifetime of the mouse (lavi et al., 1984a; adami et al., 1995) . the fecting the secondary structure of the genome rna, we cannot completely rule out this possibility. group of mutants that we are working with all cause acute encephalitis, but at least in the case of the three we show here that the appearance of the q159l mutation within the ''c'' culture of persistently infected glial that we have examined carefully (c12, b11, b12), there are only minimal levels of demyelination and only at very cells, in addition to correlating with the loss of hepatotropism, also coincides with the development of the high levels of virus inoculated. thus encephalitis does not necessarily lead to demyelination. several other labs weakly demyelinating phenotype. further evidence that the q159l mutation is important in the demyelination have reported mutants of the jhm strain of mhv, in which encephalitis and demyelination are dissociated. for ex-phenotype comes from our observations that the b11 and b12 mutants, isolated from another parallel glial cell ample the attenuated jhm2.2-v-1 mutant (fleming et al., 1986 (fleming et al., , 1987 and the mhv-4 mutant 5a13.5 mutant (dalziel culture, are also weakly demyelinating (fig. 2) and contain this mutation in the s gene (hingley et al., 1994) . et al., 1986; fazakerley et al., 1992) cause little encephalitis but do induce significant demyelinating disease. both thus it appears that the demyelination and hepatotropism phenotypes of c12 are both associated with q159l, of these mutants were selected by resistance to mono-clonal antibodies directed against epitopes in the s gene; to a change in viral tropism, perhaps through receptor utilization. thus, the change in pathogenic phenotype is likely to we are currently using targeted recombination techmap to s. the mutants we have selected from persisnology developed by dr. paul masters (masters et al., tently infected glial cells (c12, b11, and b12 for example) fischer et al., 1997) to introduce q159l alone into are, to our knowledge, the first examples of mhv mutants the genome of wild type mhv-a59. this will allow us to in which encephalitis occurs without subsequent demyedetermine unambiguously the effects of this amino acid linating disease. substitution alone on pathogenesis and interaction of the the location of the q159l amino acid substitution mutant protein with the mhv receptor. within the predicted receptor binding domain (kubo et al., 1994) of s suggests that this mutation may play a acknowledgments role in the development of demyelination and hepatitis by affecting the interaction of s and the viral receptor. this work was supported by public health service grants ns-21954 the inability of c12 to interact with the receptor on one (s.r.w.) and ns-30606 (s.r.w.) as well as grants rg-2585a4/1 (s.r.w.) and rg-26151/2 (e.l.) from the national multiple sclerosis society. we or more liver cell types would be a reasonable explanathank drs. julian leibowitz and henry teng for reading and commenttion for the lack of hepatitis following infection with c12. ing on the manuscript. the difference in the ability of c12 and wild type virus to induce demyelination could also be related to the tro-references pism for a certain cell type. while we have shown that similar localization in the limbic system (hingley et al., virology 209, (337) (338) (339) (340) (341) (342) (343) (344) (345) (346) 1994), we are currently studying the spread of the two armstrong, j., smeekens, s., and rottier, p. (1983) . sequence of the nucleocapsid gene of the murine coronavirus mhv-a59. nucleic viruses to various cell types and the persistence in the acids res. 11, 883-891. cns after the acute disease and viral clearance occurs. armstrong, j., niemann, h., smeekens, s., rottier, p., and warren, g. the region defined as a receptor binding domain in(1984) . sequence and topology of a model intracellular membrane cludes the amino terminal 330 amino acids of s1 (kubo protein, e1 glycoprotein, from a coronavirus. nature (london) 308, 751-752. et al., 1994) . amino acid q159, and the region surhepatotropism and demyelination via another mecha-nucleic acids res. 18, 1825 res. 18, -1832 nism, perhaps due to a step in replication after binding budzilowicz, c. j., wilczynski, s. p., and weiss, s. r. (1985) . three inand entry. tergenic regions of coronavirus mouse hepatitis virus strain a59 contain a common nucleotide sequence that is homologous to the another possible explanation for the different pathopathogenicity of antigenic variants of murine of self reactive t cells after murine coronavirus infection interaction of immune and central nervous systems: tence of mhv-a59 rna in a slow virus demyelinating infection in contribution of thy-1/ cells to demyelination induced by coronavirus mice as detected by in situ hybridization experimental demyelination produced by the a59 strain of tion resistant variants of a neurotropic coronavirus are generated by mouse hepatitis virus fusion-defective tis virus (mhv)-a59 causes a persistent, productive infection in primutants of mouse hepatitis virus a59 contain a mutation in the spike mary glial cell cultures mouse hepatitis virus a59-induced demyelination can occur sequence of the murine coronavirus gene 1 encoding the putative in the absence of cd8/ t cells primary strucencoded in gene 1 of the human coronavirus 229e sequence of mouse hepatitis virus a59 mrna 2: virology 200, 1-10. indications for rna recombination between coronaviruses and influ analysis of the receptor binding site dissociation of demyelination transcription and rna recombination. in ''the coronaviradae'' (s. g. and viral clearance in congenitally immunodeficient mice infected siddell localization of neutralizmouse hepatitis virus (mhv), strain a59 contains two orfs and thus ing epitopes and the receptor binding site within the amino-terminal differs from ns4 of the jhm and s strains 5404-5410. mouse hepatitis virus orf 2a is expressed in the cytosol of infected mouse fibroblasts key: cord-307904-lnagg1uw authors: johnson, jennifer a; bragg, jennifer n; lawrence, diane m; jackson, andrew o title: sequence elements controlling expression of barley stripe mosaic virus subgenomic rnas in vivo date: 2003-08-15 journal: virology doi: 10.1016/s0042-6822(03)00285-x sha: doc_id: 307904 cord_uid: lnagg1uw barley stripe mosaic virus (bsmv) contains three positive-sense, single-stranded genomic rnas, designated α, β, and γ, that encode seven major proteins and one minor translational readthrough protein. three proteins (αa, βa, and γa) are translated directly from the genomic rnas and the remaining proteins encoded on rnaβ and rnaγ are expressed via three subgenomic messenger rnas (sgrnas). sgrnaβ1 directs synthesis of the triple gene block 1 (tgb1) protein. the tgb2 protein, the tgb2′ minor translational readthrough protein, and the tgb3 protein are expressed from sgrnaβ2, which is present in considerably lower abundance than sgrnaβ1. a third sgrna, sgrnaγ, is required for expression of the γb protein. we have used deletion analyses and site-specific mutations to define the boundaries of promoter regions that are critical for expression of the bsmv sgrnas in infected protoplasts. the results reveal that the sgrnaβ1 promoter encompasses positions −29 to −2 relative to its transcription start site and is adjacent to a cis-acting element required for rnaβ replication that maps from −107 to −74 relative to the sgrnaβ1 start site. the core sgrnaβ2 promoter includes residues −32 to −17 relative to the sgrnaβ2 transcriptional start site, although maximal activity requires an upstream hexanucleotide sequence residing from positions −64 to −59. the sgrnaγ promoter maps from −21 to +2 relative to its transcription start site and therefore partially overlaps the γa gene. the sgrnaβ1, β2, and γ promoters also differ substantially in sequence, but have similarities to the putative homologous promoters of other hordeiviruses. these differences are postulated to affect competition for the viral polymerase, coordination of the temporal expression and abundance of the tgb proteins, and constitutive expression of the γb protein. the synthesis of subgenomic messenger rnas (sgrnas) is a common strategy employed by positive-sense rna viruses to mediate expression and regulation of 3ј proximal open reading frames (orfs) on multicistronic genomic rnas. three general mechanisms have been proposed for sgrna synthesis (white, 2002) . the most commonly accepted mechanism, internal initiation from negative-sense rna templates (miller et al., 1985) , appears to be operating during sgrna synthesis by the bromoviruses (seigel et al., 1997 (seigel et al., , 1998 , alfamoviruses (van der kuyl et al., 1990) , turnip crinkle virus (tcv) (wang and simon, 1997) , and several other viruses . in contrast, a discontinuous mechanism of transcription that produces sgrnas with 5ј and 3ј sequences identical to the genomic rnas has been reported for the large coronaviruses (sawicki and sawicki, 1998) and arterioviruses (pasternak et al., 2001) . these sgrnas arise from minus-strand subgenomic templates produced via polymerase jumping during transcription from genomic rna templates. a third mechanism, premature termination, has been reported for flock house virus (fhv) (zhong and ruekert, 1993; lindenbach et al., 2000) , red clover necrotic mosaic virus (rcnmv) (sit et al., 1998) , and tomato bushy stunt virus (tbsv) (reviewed by white, 2002) . in these cases, truncated negative-sense rnas that had been generated by premature termination at regions of secondary structure of genomic rna are postulated to serve as templates for the positive-sense sgrnas. in the case of fhv and tbsv, cis-acting sequences within the genomic rna are postu-lated to mediate long distance interactions that contribute to premature polymerase termination during transcription of the negative-sense templates (white, 2002) , whereas in the case of rcnmv, termination during transcription from the rna 1 template requires base pairing of trans-activator sequences residing on rna 2 with trans-activator binding sequences on rna 1 (sit et al., 1998) . other less welldefined examples of premature termination appear to occur during synthesis of two classes of 5ј coterminal sgrnas appearing in citrus tristiza virus (ctv) infected plants (che et al., 2001) . although the possible functions of the ctv sgrnas have not been resolved, the synthesis of the two species appears to be regulated because they exhibit some differences in the timing of their appearance and they are the earliest and the most abundant sgrnas synthesized. the core sgrna promoters have been mapped and characterized in a variety of plant rna viruses that synthesize either single or multiple sgrnas . these promoters range in size from less than 30 nucleotides (nt) (johnston and rochon, 1995; wang et al., 1999) to nearly 150 nt (van der vossen et al., 1995; koev and miller, 2000) , and they normally reside upstream of, or encompass only a few nucleotides downstream of, the transcription initiation site. however, a few promoters include substantial regions downstream of the transcription initiation site (balmori et al., 1993; koev and miller, 2000) . for example, the beet necrotic yellow vein virus (bnyvv) rna3 sgrna promoter occupies more than 100 nt downstream of the transcription start site (balmori et al., 1993) and the barley yellow dwarf virus (bydv) sgrna2 promoter resides entirely within the sgrna. in addition to this complexity and variation among rna viruses, long distance interactions have also been noted in several viruses (miller and keov, 2000) . in the case of tobacco mosaic virus (tmv), the 3ј untranslated region (utr) contains three pseudoknot structures whose ectopic placement appears to redistribute polymerase activity to the closest upstream sgrna promoter (shivprasad et al., 1999) . in contrast, optimal activity of the tbsv sgrna2 promoter requires long distance cis interactions with complementary upstream sequences separated by more than 1000 nt (zhang et al., 1999; choi and white, 2002) . promoter elements within viruses expressing more than one sgrna can also vary considerably in their core sequences, and in their sizes and positioning relative to the transcription start sites of the sgrnas. in one well-studied case, the bydv sgrna1 promoter has been mapped from ϫ75 to ϩ21 relative to its transcription initiation site, while the sgrna2 and sgrna3 promoters map between positions ϩ1 and ϩ143, and ϫ6 and ϩ38, respectively . thus, the bydv sgrna1 and sgrna3 promoters occupy sequences that overlap the transcription initiation site, whereas the sgrna2 promoter resides entirely within the sgrna transcript. aside from a conserved hexanucleotide shared between the sgrna1 and sgrna2 promoters, little or no similarity exists between the three pro-moters. several lines of evidence suggest that both primary sequence and secondary structure, including two stem loop structures, function during the regulation of bydv sgrna1 synthesis. in contrast to the well-defined viruses described above, only rudimentary information is available to define sequences affecting expression of the sgrnas of a number of other viruses, including barley stripe mosaic virus (bsmv). bsmv is a hordeivirus whose genome is divided into three positive-sense, single-stranded rnas, designated ␣, ␤, and ␥ (fig. 1) . the ␣ and ␥ rnas are required for replication, while rna␤ is essential for cell-to-cell movement. the replicase proteins ␣a and ␥a are translated directly from their respective genomic rnas . in addition, rna␥ also encodes a small cysteine-rich protein, designated ␥b, that is translated from sgrna␥ and is dispensable for bsmv replication. the coat protein, ␤a, is translated directly from rna␤ and, following an intergenic region, the overlapping viral movement genes are arranged in a "triple gene block" (tgb). the three major tgb proteins (tgb1, 2, and 3) and one minor protein, tgb2ј, are translated from two sgrnas, designated sgrna␤1 and sgrna␤2. the tgb1 protein is expressed from sgrna␤1, while the other overlapping proteins tgb2, tgb2ј, and tgb3 are translated from sgrna␤2 (zhou and jackson, 1996b) . the tgb2ј protein is a translational readthrough product of the tgb2 orf, and the tgb3 protein is translated by leaky scanning of the tgb2 start codon (zhou and jackson, 1996b) . the transcription initiation sites of the three bsmv sgrnas have been mapped and their relative abundance has been determined (gustafson et al., 1987; zhou and jackson, 1996b) . northern blot analyses of nucleic acids extracted from bsmv-infected protoplasts have demonstrated that sgrna␥ and sgrna␤1 accumulate to high levels, whereas sgrna␤2 is present in much lower abundance jackson, 1996a, 1996b) . limited evidence from protoplasts also indicates that sgrna␤1 and ␤2 expression is regulated temporally and that sgrna␥ is expressed constitutively (zhou and jackson, 1996b) . to begin to determine how expression of the three bsmv sgrnas may be mediated, we have delineated the boundaries of the promoters based on the expression of sgrnas in infected protoplasts. in addition, we have mapped a cis-acting element adjacent to the sgrna␤1 promoter that is required for rna␤ replication (zhou and jackson, 1996b) . the three bsmv sgrna promoters have also been repositioned into ␤ and ␥ rnas that either contain or lack the native promoters. the results indicate that the analogous bsmv sgrna promoters have some sequence similarity to predicted promoter regions of other hordeiviruses. nevertheless, the three sgrna promoters do not share extensive sequence similarity, nor are recognizable common structural elements evident. although the bsmv core promoter sequences failed to function when inserted ectopically into rna␤ and rna␥, core promoters introduced with additional flanking sequences were active, but this activity was dependent on the context into which the sequences were inserted. in a previous study, we had determined that the sgrna␥ transcription start site begins at nt 2054 on rna␥ (gustafson et al., 1987) . this site resides within the 42-nt intergenic region between the ␥a and ␥b orfs (fig. 1) . to analyze the sgrna␥ promoter, by-2 protoplasts were transfected with in vitro transcripts, and the rnas synthesized during infection were evaluated by northern blot analyses. because the ␥a stop codon is located 18 nt upstream of the sgrna␥ transcriptional start site, we predicted that the sgrna␥ promoter would overlap the ␥a orf. the ␥a protein encodes the viral polymerase and hence is essential for viral replication. therefore, engineering deletions to map the sgrna␥ promoter could have interfered with the ␥a orf and might have affected replication. to circumvent this problem, protoplasts were transfected with two rna␥ derivatives. one rna, ␥kpni/hpai, provided a source of the ␥a replicase protein and also contained a 350-nt deletion (from positions 2112 to 2461 on rna␥) to remove the majority of the ␥b orf, and a second derivative designed to assess promoter activity contained deletions engineered within the putative sgrna␥ promoter ( fig. 2a) . the deletion present in ␥kpni/hpai rna al-lowed us to distinguish between the two rna␥ derivatives based on their sizes. protoplasts were transfected with rna␣, rna␥, and rna␥kpni/hpai, and nucleic acid was extracted from protoplasts at 20 h posttransfection. each of the three rnas replicated to wild-type (wt) levels in protoplasts and synthesized the predicted sgrnas (data not shown). therefore, in subsequent mapping experiments, fig. 1 . illustration of the bsmv genomic and subgenomic rnas. rna␣ serves as the mrna for the ␣a protein, which contains the capping and helicase subunits of the rna-dependent rna polymerase (rdrp). rna␤ encodes the ␤a protein (coat protein) and the "triple gene block" composed of the tgb1, tgb2, tgb2ј, and tgb3 proteins. the ␤a protein is translated from the genomic rna; the tgb1 protein is expressed from sgrna␤1 and the tgb2, tgb2ј, and tgb3 proteins are translated from sgrna␤2. the genomic rna␥ serves as a messenger for translation of the ␥a polymerase subunit of the rdrp and encodes the ␥b protein, which is expressed from sgrna␥. all genomic and subgenomic rnas are capped (black circle) at the 5ј terminus, contain an internal poly (a) tail (a n ), and possess a conserved trna-like structure (black rectangle) at the 3ј terminus. the schematic illustration shows the two ␥ rnas used in conjunction with rna␣ to transfect protoplasts. the ␥ kpni/hpai rna provided a source of the ␥a protein, while the second rna␥ was used to construct deletions within the region surrounding the sgrna␥ transcription start site (note arrow). tobacco by-2 protoplasts were cotransfected with the ␣ and ␥ kpni/hpai rnas plus rna␥ derivatives with deletions originating at either ϫ152 or ϩ57 relative to the sgrna␥ transcription start site. the deletions are shown above the blot. total nucleic acid was extracted 20 h posttransfection, separated on 1% agarose gels, and transferred to nylon membranes. northern blot analyses were conducted with a ␥-specific riboprobe designed to detect the presence of the genomic rna␥ and sgrna␥. the probe was derived from the kpni/hindiii (2111 to 2444 nt) fragment of rna␥ and hence does not hybridize to the genomic or sgrnas generated from the ␥ kpni/hpai rna. (b) analysis of rna␥ derivatives containing small deletions in the region spanning ϫ30 to ϩ10 relative to the sgrna␥ transcription start site. the sequence shows the intergenic region in the negative sense, with the arrow representing the sgrna␥ transcription start site. protoplasts were transfected with rna␣, rna␥kpni/hpai, and an rna␥ derivative that contained a deletion within the putative sgrna␥ promoter. to identify starting points for analysis of the sgrna␥ promoter region, two rna␥ derivatives were constructed. one contained a large-scale deletion in the ␥a orf from nt 593 to 1899 and the other had a smaller deletion from positions 2111 to 2262 in the ␥b orf. when these rnas were transfected individually into protoplasts along with rna␣ and rna␥kpni/hpai, both of the deletion derivatives were able to replicate and synthesize sgrna␥ at levels comparable to wt rna␥ (data not shown). these results indicate that the deleted regions are not required for sgrna␥ synthesis and that they do not contain regulatory elements that affect replication. therefore, a more refined analysis of the sgrna␥ promoter was initiated by evaluation of deletions beginning at position ϫ152 (position 1902 on rna␥) and extending to positions ϫ119, ϫ78, ϫ38, and ϫ18 upstream of the transcription start site. two additional deletions beginning at position ϩ57 and extending to ϩ27 and ϩ11 were also generated. to specifically visualize the replication of these rna␥ derivatives and the presence of sgrna␥, a 32 p-labeled riboprobe was used that is complementary to the kpni/hpai region that had been eliminated in the ␥kpni/hpai rna used to mediate replication of the test rnas. this probe thus recognizes only the rna␥ and sgrna␥ derivatives designed to assess promoter activity. as shown in fig. 2a , northern blot analysis revealed that each of the rna␥ test derivatives were able to replicate in trans and that the ␥rnas containing the deletions ϫ152/ ϫ119, ϫ152/ϫ78, ϫ152/ϫ38, ϩ57/ϩ27, and ϩ57/ϩ11 were able to synthesize sgrna␥. however, when the region between ϫ152 and ϫ18 was deleted, sgrna synthesis was not evident. therefore, these results indicated that the sgrna␥ promoter resides between positions ϫ38 and ϩ11 relative to the sgrna␥ transcriptional start site. to analyze the sgrna␥ promoter more precisely, smaller deletions were generated in the region between positions ϫ30 and ϩ10 (fig. 2b) . the ␥ rnas containing the deletions ϫ30/ϫ28, ϫ30/ϫ22, ϫ30/ϫ21, ϩ10/ϩ4, ϩ10/ϩ3, ϩ10/ϩ2, and ϩ10/ϩ1 were transfected into protoplasts along with the ␣ and ␥ kpni/hpai rnas. northern blot analysis demonstrated that the rnas containing deletions ϫ30/ϫ28 and ϫ30/ϫ22 replicated and were capable of synthesizing sgrna␥, but that removal of the residue at position ϫ21 eliminated sgrna␥ synthesis (fig. 2b ). our interpretation of these results is that the c at position ϫ22 is dispensable for sgrna␥ synthesis, but that the a at position ϫ21 is required for promoter activity. the rnas used for deletion analysis starting at position ϩ10 were all able to replicate, but only the ϩ10 to ϩ4 or ϩ3 deletions were able to synthesize sgrna␥ (fig. 2b ). when the next residue at ϩ2 was deleted, sgrna␥ was not evident, and hence, the a at position ϩ2 is required for sgrna␥ synthesis, whereas the a at ϩ3 is dispensable. therefore, these results indicate that the sgrna␥ promoter boundaries map to positions ϫ21 and ϩ2 relative to the sgrna␥ transcription start site. the sgrna␥ promoter thus overlaps the sgrna␥ transcription initiation site and the last two codons of the ␥a polymerase protein. rna␤ is dispensable for replication and therefore, substantial deletions could be engineered into the rna to facilitate analysis of the sgrna␤1 promoter. the sgrna␤1 transcription start site had previously been mapped to nt 789 in the 118-nt intergenic region between the ␤a and tgb1 orfs (zhou and jackson, 1996b) . in addition, we had identified a cis-acting element required for rna␤ replication within this intergenic region (zhou and jackson, 1996a) . therefore, careful consideration of the cis-acting element had to be taken into account because of the possibility that the sgrna␤1 promoter boundaries might reside within this region. hence, we devised a strategy to identify the cis element and to map the sgrna␤1 promoter by transfecting by-2 protoplasts with the ␣ and ␥ rnas and an rna␤ mutant to evaluate promoter activity. northern blot analysis with a ␤-specific probe was then used to assess replication and sgrna␤1 synthesis. identification of the sgrna␤1 promoter initially was complicated because the 18s rrna masked sgrna␤1 in northern blots (data not shown). to circumvent this problem, we constructed mutations in a cdna clone that contained a 1427-nt deletion (␤s/b) which removed the majority of the tgb orfs. this deletion did not affect rna␤ replication, in agreement with our previous results (zhou and jackson, 1996a) and also clearly resolved sgrna␤1 and the rrnas to permit detection of sgrna␤1 (data not shown). it is important to note that the ␤s/b rna deletion also eliminated the sgrna␤2 promoter to mitigate possible competition effects that might have affected synthesis of sgrna␤1. to define the starting points for mapping the sgrna␤1 promoter, we used two rna␤ derivatives. one of these, ␤⌬1.6, contained a deletion from position 295 to 633, and the other, ␤⌬2.0, eliminated nts 802 to 1375 on the genomic rna sequence. northern blot analysis of rna isolated from protoplasts transfected with the ␣ and ␥ rnas and the ␤⌬1.6 or ␤⌬2.0 rnas indicated that the deletions had no discernable effect on sgrna␤1 synthesis (data not shown). therefore, the bstbi site at ϫ156 (rna␤ nt 633) and the ncoi site at ϩ14 (nt 802) relative to the sgrna␤1 transcription start site were used as starting points for analysis. initially, deletions were generated from ϫ156 to ϫ107, ϫ74 and ϫ34 relative to the sgrna␤1 transcription initiation site. as shown in fig. 3a , the ϫ156/ϫ107 deletion mutant was able to replicate and to synthesize sgrna␤1, but the ϫ156/ϫ74 and ϫ156/ϫ34 deletions reduced replication to barely detectable levels. to confirm the replication of rnas ␣ and ␥, the blots were stripped and reprobed with ␣and ␥-specific probes (data not shown). these results indicated that the ϫ156/ϫ74 and ϫ156/ϫ34 deletions destroyed a cis-element within the intergenic region that is required for replication (zhou and jackson, 1996a) . to further define the rna␤ cis-acting element, two additional derivatives were constructed that contained deletions spanning ϫ107/ϫ74 and ϫ74/ϫ34. northern blot analysis using a ␤-specific probe revealed that replication of ϫ107/ϫ74, similar to that of the ϫ156/ϫ74 and ϫ156/ϫ34 mutants, was undetectable. however, the ϫ74/ϫ34 deletion mutant replicated to low levels and produced small amounts of sgrna␤1 (fig. 3a) . these results suggest that the cisacting element required for rna␤ replication resides within the 118-nt intergenic region between positions ϫ107 and ϫ74. however, sequences within the ϫ74 to ϫ34 region also contribute to efficient replication of rna␤. the sgrna␤1 promoter activity was examined in more detail by engineering 10-nt deletions in ␤s/b from positions ϫ60 to ϩ1 relative to the sgrna␤1 transcription start site. northern blot analyses revealed that the ϫ60/ϫ51, ϫ50/ ϫ41, and ϫ40/ϫ31 deletions did not affect genomic rna replication and abundant amounts of sgrna␤1 were synthesized. however, when the ϫ30/ϫ21, ϫ20/ϫ11, or ϫ10/ϩ1 regions were deleted, genomic rna replicated to wt levels but sgrna␤1 failed to accumulate (fig. 3b ). to further resolve the nature of the active sgrna promoter element, we engineered smaller deletions from positions ϫ30 to ϩ14. as shown in fig. 3c , when the a residue at ϫ30 was deleted, sgrna␤1 was evident, but deletion of the adjacent u at ϫ29 effectively eliminated promoter activity without obvious effects on replication of the genomic rnas. in contrast, deletions that spanned ϩ14/ϩ2, ϩ14/ ϩ1, and ϩ14/ϫ1 retained promoter activity (fig. 3c) . however, when the next residue, a u at ϫ2, was deleted, promoter activity was disrupted. these results suggest that the core promoter sequence required for sgrna␤1 synthesis resides immediately upstream of the sgrna␤1 transcription start site between positions ϫ29 and ϫ2. however, an alternative possibility that needs further exploration is that the ϩ14/ϩ1 deletion could have generated an alternative transcription initiation site at the upstream c residue. similar alternative sites have previously been identified in in vitro studies with the brome mosaic virus sgrna promoter (stawicki and kao, 1999) . the transcription start site of sgrna␤2 has previously been identified within the 3ј end of the tgb1 orf at position 2279 in rna␤ and is 1490 nt downstream of the sgrna␤1 transcription start site (zhou and jackson, 1996b) . since sgrna␤2 is expressed at very low levels in infected protoplasts jackson, 1996a, 1996b) , we attempted to increase its abundance to improve the ease and reproducibility of promoter analysis. for this purpose, the clone ␤1ϫ34/ϩ14 was created by removing nucleotides 755 through 802 (ϫ34/ϩ14 relative to the sgrna␤1 transcription start site) on rna␤ (fig. 4a ). as expected, this 48-nt deletion abrogated sgrna␤1 synthesis and also resulted in easily detectable levels of sgrna␤2 without affig. 3 . delineating the boundaries of the sgrna␤1 promoter and the rna␤ cis-acting element. protoplasts were transfected with wt ␣ and ␥ rnas plus rna␤ derivatives containing the 1427 nt s/b (␤ sali/bglii) deletion. total nucleic acid was extracted and blotted as described in fig. 2 , except that the blots were probed with a ␤-specific riboprobe (␤ ncoi/ sali). the numbers above the lanes correspond to deletions at positions relative to the sgrna␤1 transcription start site. (a) large-scale deletion mapping of the sgrna␤1 promoter and the rna␤ cis-acting element required for replication. (b) deletion mapping of the sgrna␤1 promoter using 10-nt deletions at positions corresponding to the transcription start site. (c) fine-scale mapping of the sgrna␤1 promoter. the sequence represents the negative-sense orientation of the intergenic region between the ␤a and tgb1 orfs which contains the sgrna␤1 promoter. the arrow represents the sgrna␤1 transcription start site. fecting rna␤ replication (data not shown). this result implies that competition between the two promoters has a major role in regulating the differential rates of synthesis of the two sgrnas. to initially define sequences flanking the sgrna␤2 promoter, two large deletions were generated in the ␤1ϫ34/ ϩ14 clone that eliminated rna␤ positions 1705 to 2109 and 2287 to 2434. rnas containing these deletions were individually transfected into protoplasts along with the ␣ and ␥ rnas, and nucleic acids were extracted at 20 h posttransfection. northern blot analyses using a ␤-specific probe revealed the presence of sgrna␤2 in both derivatives, indicating that neither of the deletions affected sgrna␤2 promoter activity (data not shown). therefore, we began to focus on the region between ϫ179 (nt 2109) and ϩ9 (nt 2288) relative to the sgrna␤2 transcription initiation site. analysis of four deletions extending from position ϫ179 to positions ϫ110, ϫ76, ϫ52, and ϫ28 relative to the transcription start site revealed that the mutant rnas were able to replicate in protoplasts, but only mutants containing the deletions ϫ179/ϫ110 or ϫ179/ϫ76 were able to direct synthesis of sgrna␤2 (fig. 4a ). more detailed analyses performed on the region between ϫ70 and ϫ52 demonstrated that the ϫ70/ϫ65 and ϫ58/ϫ53 mutants transcribed sgrna␤2, but that promoter activity was destroyed by the ϫ64/ϫ59 deletion (fig. 4a ). these results thus indicate that an upstream element residing between ϫ64 and ϫ59 relative to the sgrna␤2 transcription start site is required for sgrna␤2 promoter activity. however, in the presence of this element, deletions between positions ϫ40/ϫ32 and ϩ10/ϫ17 nearly eliminated sgrna␤2 synthesis (fig. 4b ). in contrast, northern blot analyses revealed that deletions between ϫ52/ϫ47, ϫ46/ϫ41, ϫ40/ϫ35, ϫ40/ϫ34, and ϫ40/ϫ33 had only minor effects on rna␤ replication and sgrna␤2 synthesis in transfected protoplasts ( fig. 4b and data not shown). similarly, the deletions ϩ11/ϫ9, ϩ11/ ϫ15, and ϩ10/ϫ16 were able to replicate and synthesize sgrna␤2. these results demonstrate that the sgrna␤2 promoter maps from the g at ϫ32 to the c at ϫ17 and hence does not encompass the transcriptional start site. the sgrna␤1, sgrna␤2, and sgrna␥ promoter fragments were initially inserted into rna␤ to determine whether they could function ectopically and to evaluate their competition with a native sgrna promoter present on the same rna. for this purpose, fragments encompassing each of the three sgrna promoters were pcr-amplified and inserted into rna␤ ␤ s/b or ␤1ϫ34/ϩ14 at position 1134 (344 nt downstream of the sgrna␤1 promoter). the ␤ s/b clone provided a background that contained the native sgrna␤1 promoter (fig. 5a) , while the ␤1ϫ34/ϩ14 clone lacked the sgrna␤1 promoter (fig. 5b) . two fragments that encompassed the sgrna␤1 promoter were inserted ectopically into the two rna␤ derivatives. the larger fragment ␤1a consisted of 266 nt amplified from positions 599 to 864 on the genomic rna and a smaller 150-nt fragment ␤1b was derived from positions 715 to 864. a 295-nt fragment (␤2) overlapping the sgrna␤2 promoter was derived from positions 2080 to 2374, and a 276-nt fragment (␥) was amplified from positions 1864 to 2139 to encompass the sgrna␥ promoter. northern blot analysis of rna isolated from protoplasts transfected with rnas ␣ and ␥, and rna␤ derivatives fig. 4 . defining the boundaries of the sgrna␤2 promoter. protoplasts were transfected with the ␣ and ␥ genomic rnas and rna␤ derivatives containing a 48-nt deletion (␤1ϫ34/ϩ14) to eliminate sgrna␤1 promoter activity. the deletions are numbered according to the sgrna␤2 transcription start site, which is illustrated by the arrow. total nucleic acids were extracted at 20 h posttransfection and processed as described in fig. 2 , except that hybridizations were performed with a ␤-specific riboprobe (␤ sspi/bglii) derived from sequence upstream of the sgrna2 promoter region. (a) large-scale deletion mapping of the sgrna␤2 promoter is shown on the blot to the left. to define the region between ϫ70 and ϫ53 further, 6-nt deletions were constructed and are shown on the blot to the right. (b) small-scale mapping of the sgrna␤2 promoter. the sequence represents the negative-sense region of the tgb1 orf that contains the sgrna␤2 promoter. containing the native sgrna␤1 promoter with the smaller sg␤1 (150 nt), sg␤2, or the sg␥ fragment insertions at position 1134 revealed that the ␤ rnas were able to replicate and synthesize sgrna␤1 from the native promoter (fig. 5a) . however, transcription from the ectopic sgrna␤1 and sgrna␤2 promoters could be detected only after protracted exposure of the blots (fig. 5a, ␤1 and ␤2 lanes, and data not shown). the rna␤ derivative containing the 48-nt deletion in the intergenic region was able to replicate and to express small amounts of the ectopic sgrnas from the smaller 150-nt sgrna␤1 fragment and sgrna␤2 (fig. 5b, ␤1 and ␤2 lanes) , but both the rna␤ derivatives failed to replicate when the larger 266-nt sgrna␤1 fragment was inserted (data not shown). in marked contrast, the 276-nt ectopic rna␥ fragment was active following insertion into both of the rna␤ derivatives and ectopic sgrnas of the appropriate sizes were easily detectable in both backgrounds (figs. 5a and b, ␥ lanes). these results demonstrate that the three bsmv sgrna promoters can function ectopically in rna␤, but that the extent of their activity varies depending on their flanking sequences and the genomic rna context into which they are inserted. the results also provide evidence that the ectopic sgrna␥ promoter competes for the bsmv replicase complex more efficiently than an ectopic sgrna␤1 or sgrna␤2 promoter when positioned downstream of the wt sgrna␤1 promoter. each of the four promoter fragments (␤1a, ␤1b, ␤2, and ␥) were also inserted into the ␥ cdna clone at position 2339, which is located 285 nt downstream of the transcription start site of the native sgrna␥ (fig. 5c ). as seen with rna␤, the presence of the sgrna␤2 fragment (␤2) and the sgrna␥ promoter derivative (␥) permitted abundant rna␥ replication and synthesis of native sgrna␥ (fig. 5c, ␤2 and ␥ lanes). both the sgrna␤2 and the sgrna␥ promoters were also able to direct synthesis of easily detectable amounts of ectopic sgrna. again, transcription from the ␤2 promoter was less than half of that from the native sgrna␥ promoter, and the ectopic sgrna␥ promoter also appeared to be substantially more active than the corresponding sgrna␤2 promoter. however, rna␥ replication fig. 5 . ectopic expression of the sgrna␥, sgrna␤1, and sgrna␤2 promoters in rna␤ and rna␥ derivatives. the designations on the genomic rnas are as described in the legend to fig. 1. n and e represent the native promoters and the ectopic promoter insertion sites, respectively. protoplast transfections and rna blots were carried out as described in expression of the sgrna␤1, sgrna␤2, and sgrna␥ promoters in the rna␤ s/b derivative (␤1ϫ34/ϩ14), which contains a 48-nt deletion inactivating the native sgrna␤1 promoter. (c) activity of the sgrna␤1, sgrna␤2, and sgrna␥ promoters inserted into the ␥b orf at nt 2339 of rna␥. protoplasts were coinoculated with rna␣ and rna␥ containing the promoter fragments and extracted at 20 hpi. rna blots were probed with a riboprobe that anneals to the conserved 3ј end of bsmv rnas and therefore should detect all genomic and sgrnas. asterisks along the side of a and b refer to a consistently observed band of unknown origin that originated from the sgrna␥ promoter insertions. was nearly eliminated when either the smaller 150-nt sgrna␤1 promoter (␤1, fig. 5c ) or the larger 266-nt sgrna␤1 promoter fragments were inserted (data not shown). these results suggest that the ectopic sgrna␤2 promoter competes less efficiently for the bsmv replicase complex than the wt sgrna␥ promoter. the deleterious effects of the sgrna␤1 fragment insertions in rna␥ also suggest that sequence, context effects, and perhaps competition for replicase can have drastic effects on replication. to determine if the three minimal sgrna promoters were sufficient for ectopic transcription, we inserted each of them into position 1134 of the rna␤ ␤ s/b derivative and the 48-nt deletion derivative (␤1ϫ34/ϩ14). similar insertions were made into rna␥ at position 2339. ectopic sgrna expression was not detected following protoplast transfection with any of the derivatives, although the genomic rnas were able to replicate (data not shown). this result suggests that although the minimal promoter sequences identified by deletion analysis are required for activity in their native context, each of the promoters requires additional flanking sequences to provide optimal promoter activity from the ectopic positions tested in these experiments. the comparisons shown in fig. 6a indicate that the three bsmv sgrna promoters share little obvious sequence relatedness. we also conducted a more refined gapped alignment and were still unable to detect major blocks of similarity (data not shown). additional comparisons of the 238 nt at the 3ј common termini of the genomic rnas and at the diverse 3ј ends of the three minus-strand rnas also indicated that the four replication promoters within the genomic rnas do not contain substantial regions of similarity to the three bsmv sgrna promoters (data not shown). however, comparisons of the three sgrna promoters of bsmv with the putative promoters of poa semilatent virus (pslv) and lychnis ringspot virus (lrsv) revealed that the analogous hordeivirus sgrna promoters share a number of blocks of identical sequence (fig. 6b) . furthermore, the bsmv and pslv sequences share a higher degree of similarity with each other than either does with lrsv. in the case of the bsmv sgrna␥ promoter, nearly 80% of the pslv residues are identical (19/24) to the bsmv sequence, whereas a gapped alignment between bsmv and lrsv indicated a considerably lower degree of similarity (14/24 residues) within the aligned region. these results buttress the existing strong evidence for a common origin of the hordeiviruses and support biological evidence, suggesting that bsmv and pslv are more closely related to each other than to lrsv (hunter et al., 1989) . to obtain additional information about the relationship of the hordeivirus sgrna promoters, sequence comparisons were conducted with other tgb-containing viruses. initial comparisons using the ncbi blast program failed to reveal significant matches with other viral promoter sequences. to obtain more definitive comparisons, we analyzed the 300 nucleotides surrounding the start codons of the tgb proteins of beet necrotic yellow vein virus, peanut clump virus (pcv), potato mop top virus (pmtv), potato virus m (pvm), and potato virus x (pvx). no obvious similarity was detected in comparisons of the sgrna␤2 promoter regions, and only short blocks of common sequence were noted in the regions surrounding the putative sgrna␤1 promoters. these common blocks primarily consisted of strings of a residues located at various positions upstream of the translational start site and hence they are unlikely to represent conserved promoter elements. thus, the results indicate that the sequence conservation noted among the tgb proteins of the hordeiviruses does not extend to the analogous tgb promoter regions of more distantly related viruses. we also conducted comparisons of the bsmv sgrna␥ promoter with putative promoter regions of other viruses. interestingly, the pcv p15, which encodes a cysteine-rich protein with some similarity to bsmv ␥b, and the bnyvv tgb1 sequences have a high degree of similarity to the bsmv sgrna␥ promoter. in these cases, 16 (pcv) or 14 (bnyvv) residues of 24 are identical to the bsmv sgrna␥ sequence. these regions of similarity include two blocks (accacuu and acua) within the 20 residues upstream of the transcriptional start site of the bsmv sgrna␥, and they surprisingly share more than 60% identity (15/24 residues) with a region of the tmv 30 k movement protein promoter (grdzelishvili et al., 2000) . despite these intriguing similarities, the significance, if any, of these common sequences is conjectural and additional analyses are needed to determine their importance in sgrna synthesis. during the bsmv infection cycle, the three sgrnas vary in abundance and in their timing of expression (zhou and jackson, 1996a) . early in infection, the predominant species are sgrna␥, which appears to be relatively constant throughout replication, and sgrna␤1, whose abundance decreases as infection progresses. in contrast, the abundance of sgrna␤2 is considerably lower than that of sgrna␤1, but both sgrnas appear to have similar patterns of temporal expression. these results suggest that the timing and the relative expression levels of the sgrnas may be important in regulating aspects of the infection cycle (zhou and jackson, 1996a) . therefore, understanding the nature of the sgrna promoters and the requirements for regulated expression of the mrnas under their control will contribute substantially toward elucidating events required for systemic invasion and disease development in host plants. as a fig. 6 . comparison of the bsmv sgrna promoter sequences. (a) sequence of the three bsmv sgrna promoters in the minus-sense orientation. the numbers above the sequence correspond to nucleotide positions relative to the respective transcription initiation sites. the underlined sequences are required for sgrna synthesis. (b) alignment of the sgrna␥, sgrna␤1, and sgrna␤2 promoter regions of the hordeiviruses bsmv, pslv, and lrsv. regions with the highest sequence similarity are shown in red. note that the transcription start sites for the pslv and lrsv sgrna promoters have not been defined experimentally, although their initiation sites have been predicted previously (savenkov et al., 1998) . alignments were performed using the megalign program associated with the lasergene software package. (c) alignment of the sgrna␤1, sgrna␤2, and sgrna␥ promoter regions of bsmv with mapped (tmv and pvx) or putative (bnyvv, pcv, pmtv, and pvm) sgrna promoter regions of other viruses. regions identified to have high sequence similarity are shown with residues identical to the bsmv sequence highlighted in red. alignments were performed using the megalign program associated with the lasergene software package. step toward such understanding, we have delineated the boundaries of the three bsmv sgrna promoters. the sgrna␥, sgrna␤1, and sgrna␤2 promoters map to positions ϫ21 to ϩ2, ϫ29 to ϫ2, and ϫ32 to ϫ17 relative to their transcription start sites, respectively. rna␤ also requires an enhancer-like element located between ϫ64 to ϫ59 for production of sgrna␤2. these three promoters are ͻ50 nt in size and are located in close proximity to their respective transcription initiation sites. the sgrna␥ promoter lies primarily upstream of its transcription initiation site but includes the ϩ1 nt that corresponds to the transcription start site and an additional nucleotide at the ϩ2 position. the bsmv sgrna␤1 and sgrna␤2 promoters, which reside upstream of their transcription initiation sites, differ from other plant virus sgrna promoters previously characterized because the sgrna␤1 and sgrna␤2 promoters appear not to overlap their transcription start sites . independent experiments have demonstrated the importance of the ϩ1 nt in the sgrna promoters of a number of well-characterized viruses. for example, in vitro studies using the bmv proscript system to elucidate the minimal requirements for replicase recognition of the bmv sgrna promoter have shown that changing the ϩ1 nt of the promoter from a c to a g decreases sgrna synthesis by more than 90% (siegel et al., 1997) . the importance of the ϩ1 nt has also been shown in another tricornavirus where changing the c to a u abolished amv sgrna promoter activity (van der vossen et al., 1995) . similarly, when the ϩ1 nt of the tmv mp sgrna promoter was changed from a c to a g, sgrna accumulation was below the limits of detection (grdzelishvili et al., 2000) . in contrast, transversion of the ϩ1 nucleotides of the bydv sgrna2 and sgrna3 promoters had no effect on sgrna accumulation, even though both promoters span this region . although the sgrna␥ promoter overlaps its transcription start site, we have not determined whether nucleotide substitutions at this position affect sgrna synthesis. unlike many rna viruses, the bsmv sgrna promoters do not share substantial blocks of sequence identity within their core regions (fig. 6a) . however, the sgrna␥ and sgrna␤2 promoters do appear to be more closely related to each other than either is to the sgrna␤1 promoter. this probably is an anomaly due to nucleotide composition within the promoters because the sgrna␤1 promoter sequence is particularly rich in a and u residues (73%), whereas the sgrna␤2 and sgrna␥ promoters have a more uniform distribution of bases with an a/u content of just over 50%. in analyses comparing the sgrna␤1 promoter with either the sgrna␤2 or the sgrna␥ promoter, we could align no more than two identical residues in a row. however, for the sgrna␤2 and sgrna␥ promoters, a block of five identical nucleotides (ccacu) can be aligned within the 26 residues upstream of their transcription start sites. the three bsmv sgrna promoters align well with the putative sgrna promoter regions of the less well-characterized hordeiviruses, pslv and lrsv, whose sgrna transcription initiation sites have not been mapped. comparison of the three bsmv sgrna promoters with the analogous promoters of the other hordeiviruses reveals over 75% sequence similarity ( fig. 6b and savenkov et al., 1998) . two blocks of identical residues were identified in the sgrna␤1 promoter regions (aaaaau and cuac). only one block of conserved sequence was observed for the sgrna␤2 promoter (ccacu), and this same block is present in bsmv and lrsv sgrna␥ promoters. among the three hordeiviruses, the sgrna␥ promoter displayed the greatest degree of similarity because it contained three blocks of identical sequence of three nucleotides or longer (gaagcu, acca, and acu) as well as individual conserved residues. additional analyses of the less conserved regions failed to indicate preferences for particular purine or pyrimidine residues. these results indicate that the abundance and timing of transcription of the three hordeivirus sgrnas, ␤1, ␤2, and ␥, may be regulated by promoter sequences that are only distantly related. alternatively, it is possible that short and/or long distance structural interactions that are not evident in our analyses may have profound roles in promoter timing and activity. it appears that the sgrna␤2 promoters of bsmv and pslv are sufficiently similar for recognition by the bsmv replicase because in vivo studies have revealed that bsmv can support synthesis of pslv sgrna␤2 (solovyev et al., 1999) . in these studies, a hybrid bsmv rna␤ containing pslv tgb1, tgb2, and most of the tgb3 sequence was able to replicate and move through the vasculature to uninoculated leaves in plants coinoculated with the ␣ and ␥ rnas (solovyev et al., 1999) . however, when a hybrid bsmv rna␤ containing the lrsv tgb1, tgb2, and tgb3 regions analogous to the plsv sequence was coinoculated with the ␣ and ␥ rnas, the hybrid virus was unable to infect the same common hosts. these results suggest that the bsmv replicase complex can recognize the related pslv sgrna␤2 promoter, but it either cannot recognize the lrsv sgrna␤2 promoter or the lrsv tgb proteins do not form sufficiently compatible interactions to mediate bsmv movement. in comparisons of the bsmv sgrna promoters with the analogous sequences of non-hordeivirus tgb promoters, we were unable to detect a strong correlation between the putative promoter sequences directing sgrnas encoding specific tgb proteins. for example, no obvious similarity was evident between the sgrna␤1 promoter and the regions upstream of the pmtv and pcv tgb1 genes, and only limited similarity was detected with the regions upstream of the tgb1 genes of bnyvv, pvm, and pvx. moreover, an equivalent degree of limited similarity was also observed in the regions upstream of the unrelated bnyvv p14 and the pcv tgb2 genes. likewise, the sgrna␥ promoter shares small blocks of common sequence with the region upstream of the pcv p15 gene, which encodes a protein closely related to the ␥b protein, but the promoter also has similarity to the regions upstream of the unrelated bnyvv tgb1 and tmv p30 genes. the bsmv, bnyvv, pvm, and pcv replicase proteins belong to the supergroup 3 tobamo lineage of rna-dependent rna polymerases (koonin and dolja, 1993) , so in these cases it is tempting to speculate that the promoter sequences coevolved in response to the polymerase specificity rather than as a block consisting of the promoter and its associated protein. however, the rdrp proteins of bmv and pvx are also classified in the supergroup 3 tobamo lineage, yet their sgrna promoter sequences share no apparent similarity to the bsmv promoters. therefore, it is difficult to make a case for promoter evolution based on either promoter protein constraints or polymerase similarity. we performed computer analyses to search for common secondary structural elements within or flanking the minimal bsmv core promoters, but were unable to identify common elements. this is surprising because in other viruses, sgrna promoter recognition by replicase complexes often requires primary sequence as well as secondary structural elements. these requirements may facilitate diverse interactions between replicase complexes and promoters with limited sequence similarity. in bydv, both rna sequence and secondary structure are essential for promoter activity (koev et al., 1999) . the bydv sgrna1 promoter folds into two stem loops downstream of its transcription initiation site, but comparisons with the predicted structures of the sgrna2 and sgrna3 promoters reveal that they possess very different sized stem loops downstream of their respective transcription initiation sites . the tmv mp sgrna promoter also requires a stem loop structure for promoter activity that will tolerate substantial variation in primary sequence without major effects on sgrna synthesis (grdzelishvili et al., 2000) . in addition, chemical and enzymatic probing of the 1.45-kb sgrna promoter of tcv has confirmed the presence of a 96-nt hairpin structure, although only a 21-nt hairpin and a 9-nt flanking single-stranded region comprise the minimal promoter (wang et al., 1999) . thus, our comparisons suggest that bsmv promoters differ from those of a number of other viruses in that conservation of primary sequence rather than conserved structure may have a predominant role in promoter activity. the available evidence indicates that sequences flanking the core promoter and positioning of the promoter contribute substantially to the transcription of the bsmv sgrnas. in the case of sgrna␤2, expression increased substantially upon removal of a 48-nt sequence required for sgrna␤1 promoter activity. this result suggests that competition for replicase has an important role in mediating the levels of expression of sgrna␤1 and sgrna␤2. the ectopic expression experiments support this hypothesis because both the ectopic sgrna␥ and the sgrna␤2 promoters directed transcription more efficiently in constructs lacking the wt promoters than in constructs with wt promoters. positioning also appears to have variable effects on promoter activity. accumulation of sgrnas transcribed from promoters located close to the 3ј terminus of the genomic-sense rna was less abundant than those of the wt sgrna from an upstream promoter. when the sgrna␥, sgrna␤1, and sgrna␤2 promoters were placed downstream of the wt sgrna␤1 promoter in rna␤, the ectopic sgrnas were considerably reduced in comparisons with the wt sgrna␤1 (fig. 5) . however, in rna␥, the ectopic sgrna␥ promoter strength appeared to be similar to that of the wt sgrna␥ promoter. on the other hand, when the ectopic sgrna␤2 promoter was placed downstream of the wt sgrna␥ promoter, the ectopic sgrna accumulated to much lower levels than the wt sgrna␥ (fig. 5c) . in contrast to the larger regions encompassing the bsmv sgrna promoters, the minimal bsmv promoter sequences were unable to function ectopically. these results suggest that positioning, flanking sequences, and, in the case of sgrna␤2, an upstream enhancer element may contribute substantially to promoter strength. in particular, the reduction in activity resulting from placement of the bsmv sgrna promoters near the 3ј end of the genomic rna contrasts with many examples reported from other rna viruses where more 3ј located ectopic promoters are preferentially transcribed (french and ahlquist, 1988; boccard and baulcombe, 1993; wang and simon, 1997; koev and miller, 2000) . the majority of viruses that contain multiple sgrna promoters on the same rna shows a strong correlation between the abundance of their sgrnas and their proximity to the 3ј end of the genomic rna (kelly et al., 1994; wang and simon, 1997; grdzelishvili et al., 2000) . however, bsmv sgrna␤1 and sgrna␤2 promoters appear not to share this correlation because sgrna␤2, which is proximal to the 3ј terminus of rna␤, accumulates to much lower levels than the upstream sgrna␤1. this pattern of synthesis for tgb sgrnas has also been demonstrated for potato virus x (verchot et al., 1998) and is likely the case for the multicomponent benyviruses, furoviruses, pomoviruses, and pecluviruses and the monopartite carlaviruses, allexiviruses, and foveaviruses (van regenmortel et al., 2000) , which contain related tgbs. during replication of bsmv, pvx, and bnyvv, two sgrnas are generated for translation of the overlapping tgb proteins. the relatively high abundance of sgrna1 reflects the accumulation of the tgb1 protein during the early stages of infection, whereas the 3ј proximal sgrna␤2 is often difficult to detect jackson, 1996a, 1996b; verchot et al., 1998) . it is possible that several evolutionary constraints may contribute to the low abundance of sgrna␤2 and the analogous sgrna in other tgb-containing viruses. one possibility is that the relative abundance of the proteins encoded by the sgrnas may be important for the movement complex function. alternatively, because the sgrna␤2 promoter resides within the tgb1 orf, optimal promoter strength may have been constrained by necessities for tgb1 protein function. due to the compact nature of plant rna viruses, se-quences residing within the same region of the genome often have evolved to perform multiple functions. this is especially evident in bsmv because the sgrna␤1 core promoter is proximal to a cis-acting element required for rna␤ replication, and also for the sgrna␥ and sgrna␤ promoters, which overlap regions encoding the ␥a and tgb1 orfs, respectively. since the bsmv sgrna promoter regions are multifunctional, it is likely that the replicase complex has coevolved with viral cis-acting elements to permit recognition of the three different sgrna promoters and to discriminate between the distinct elements at the 3ј ends of the positive-and negative-sense rnas. these varied interactions may also help explain the temporal regulation of the sgrna promoters in the same virus. for example, the diverse promoter-replicase interactions within a virus may be facilitated by varied complexes of replicase and host proteins with distinct promoter elements. such interactions may well mediate the strength and temporal activities of the bsmv sgrna promoters. the ␣, ␤ (␤42spi), and ␥ (␥42) cdna clones used in this study were derived from the bsmv nd18 strain (petty et al., 1988) . to define the starting points for mapping the sgrna␥ promoter, the ␥ cdna clone was digested with kpni and bsmi, the 3ј overhangs were removed with t4 dna polymerase, and the vector was religated to generate the clone ␥⌬kpni/bsmi. the ␥⌬e/e cdna clone constructed by zhou and jackson (1996a) was also used. to generate large-scale deletions in the sgrna␥ promoter, spei sites were engineered by site-directed mutagenesis (kunkel, 1985) at positions ϫ119, ϫ78, ϫ38, ϫ18, and ϩ11 relative to the sgrna␥ transcription start site at 2054 nt using the oligonucleotides listed in table 1 . the resulting clones, ␥spei-119, ␥spei-78, ␥spei-38, and ␥spei-18 were digested with clai (ϫ152) and spei, while the ␥speiϩ11 clone was digested with kpni (ϩ57) and spei. the digested clones were treated with t4 dna polymerase to generate blunt ends and religated. these clones were designated ␥ϫ152/ϫ119, ␥ϫ152/ϫ78, ␥ϫ152/ϫ38, ␥ϫ152/ϫ18, and ␥ϩ57/ϩ11. the clone ␥ϩ57/ϩ27 was constructed by digesting the ␥42bamhi clone with bamhi and kpni, blunting with t4 dna polymerase, and religating. smaller deletions within the sgrna␥ promoter region were engineered by site-directed mutagenesis using the ␥ cdna clone to create deletions ranging in size from 2 to 10 nts in the ϫ30 to ϩ10 regon surrounding the transcription start site. throughout the course of this work, deletions or site-directed mutations were sequenced to ensure that the desired changes had been introduced. to identify the starting points for mapping the sgrna␤1 promoter, we used the ␤⌬1.6 and ␤⌬2.0 clones . refined mapping of the sgrna␤1 promoter was performed using a ␤ cdna clone (␤s/b) containing a 1427-nt deletion from sali to bglii that removes part of the tgb1, tgb2, and tgb3 coding regions. this deletion was table 1 synthetic oligonucleotides used in this study primer sequence description engineered to increase the separation between the sgrna␤1 and the 18s ribosomal rna in agarose gels. kpni sites were introduced into the ␤s/b cdna clone at positions ϫ107, ϫ74, and ϫ34 relative to the sgrna␤1 transcription start site at 789 nt using site-directed mutagenesis and ␤1-specific oligonucleotides (table 1) . the resulting clones were digested with bstbi (ϫ156) and kpni, blunted with t4 dna polymerase, and religated to generate the ␤1ϫ156/ ϫ107, ␤1ϫ156/ϫ74, and ␤1ϫ156/ϫ34 clones. deletions were engineered in the sgrna␤1 promoter region of the ␤s/b clone by site-directed mutagenesis to produce the ␤1ϫ60/ϫ51, ␤1ϫ50/ϫ41, ␤1ϫ40/ϫ31, ␤1ϫ30/ϫ21, ␤1ϫ20/ϫ11, and ␤1ϫ10/ϩ1 clones with a series of sequential deletions from positions ϫ60 to ϩ1. more detailed resolution of the sgrna␤1 promoter region was obtained by site-directed mutagenesis of the ␤s/b cdna clone to generate the clones ␤1ϫ30, ␤1ϫ30/ϫ29, ␤1ϫ30/ϫ28, ␤1 ϩ14/ϩ2, ␤1,ϩ14/ϩ1, ␤1ϩ14/ϫ1, ␤1ϩ14/ϫ2, and ␤1ϩ14/ϫ3. to define the cis-acting element required for rna␤ replication, two kpni sites were created at ϫ107 and ϫ74, or ϫ74 and ϫ34, relative to the transcription start site using site-directed mutagenesis. these clones were digested with kpni and religated to produce the clones ␤1ϫ107/ϫ74 and ␤1ϫ74/ϫ34. the sgrna␤2 promoter region was analyzed in a ␤ cdna background that contained a deletion between ϫ34 and ϩ14 relative to the sgrna␤1 transcription start site and hence lacked the sgrna␤1 promoter. clone ␤1ϫ34/ϩ14 was constructed by digesting the ␤1ϫ34kpni clone with kpni and ncoi, blunting with t4 dna polymerase, and religating. since the sgrna␤2 transcription start site is located at nt 2279, the starting points for mapping the sgrna␤2 promoter were defined with the clone ␤⌬psti/ ecori, which lacks nts 1705 to 2109, and ␤2 ϩ 9/sspi, lacking nts 2290 to 2434. clone ␤⌬psti/ecori was generated from the ␤1ϫ34/ϩ14 clone by digestion with psti and ecori, blunting with t4 dna polymerase, and religation. clone ␤2 ϩ 9/sspi was engineered by introducing a smai site at position ϩ9 relative to the sgrna␤2 transcription start site by site-directed mutagenesis with the oligonucleotide ␤2 ϩ 9smai (table 1 ). the resulting derivative was partially digested with sspi, digested with smai, and religated to generate the ␤2 ϩ 9/sspi clone. for large-scale mapping of the sgrna␤2 promoter, the ␤1ϫ34/ϩ14 cdna clone was used in conjunction with the ␤2-specific oligonucleotides listed in table 1 to engineer unique kpni sites upstream of the sgrna␤2 promoter at positions ϫ110, ϫ76, ϫ52, and ϫ28. these clones were subsequently digested with ecori (ϫ179) and kpni, blunted with t4 dna polymerase, and religated to generate the clones ␤2ϫ179/ ϫ110, ␤2ϫ179/ϫ76, ␤2ϫ179/ϫ52, and ␤2ϫ179/ϫ28. six nucleotide deletions from ϫ70 to ϫ35 relative to the sgrna␤2 transcription start site were generated by sitedirected mutagenesis of clone ␤1ϫ34/ϩ14 to produce the clones ␤2ϫ70/ϫ65, ␤2ϫ64/ϫ59, ␤2ϫ58/ϫ53, ␤2ϫ52/ϫ47, ␤2ϫ46/ϫ41, and ␤2ϫ40/ϫ35. more refined mapping of the sgrna␤2 promoter was facilitated by generating the clones ␤2 ϫ40/ϫ35, ␤2ϫ40/ϫ34, ␤2ϫ40/ϫ33, ␤2ϫ40/ϫ32, ␤2ϫ40/ϫ31, ␤2ϩ11/ϫ9, ␤2 ϩ 10/ϫ15, ␤2 ϩ 10/ϫ16, ␤2 ϩ 10/ϫ17, and ␤2 ϩ 10/ϫ18 by sitedirected mutagenesis of the ␤1ϫ34/ϩ14 cdna clone. to determine if the three sgrna promoters can function ectopically, regions surrounding the sgrna␥ (276 nt), sgrna␤2 (295 nt), or the sgrna␤1 promoter (256 and 150 nt) were pcr amplified. these reactions were carried out with the oligonucleotides listed in table 1 to introduce bamhi sites at the 5ј and 3ј termini. a bamhi site was engineered in the ␤s/b clone at nt 1132-1137, by sitedirected mutagenesis with the ␤1134bamhi oligonucleotide ( table 1 ). the bamhi site was then introduced into rna␤ derivatives that either contained or lacked the sgrna␤1 promoter (␤1ϫ34/ϩ14). in addition, a bamhi site was engineered into the ␥ cdna clone (containing the sgrna␥ promoter) and the ␥ϫ30/ϫ21 cdna clone (lacking the wt sgrna␥ promoter) at nt 2338 -2343 with the ␥2239bamhi oligonucleotide (table 1) . these intermediate plasmids were then digested with bamhi and ligated to each of the amplified subgenomic promoters (sgrna␥, sgrna␤1, and sgrna␤2) to generate clones ␤1ϩsg␥, ␤1ϩsg␤1, ␤1ϩsg␤2, ␤ϩsg␥, ␤ϩsg␤1, ␤ϩsg␤2, ␥1ϩsg␥, ␥1ϩsg␤1, ␥1ϩsg␤2, ␥ϩsg␥, ␥ϩsg␤1 and ␥ϩsg␤2. since the sgrna␤1 promoter contains a mlui site, the ␥ ϩsg␤1 and ␥ ϩ sg␤1 clones were linearized for in vitro transcription reactions at a spei site introduced at their 3ј termini with the bsmv3јspei oligonucleotide (table 1) . protoplasts from the by-2 tobacco cell suspension line were prepared and transfected as described previously (watanabe et al., 1982) . to map the sgrna␥ promoter, protoplasts were transfected with in vitro transcripts (petty et al., 1988 ) generated from the cdna clones ␣ and ␥ kpni/hpai (zhou and jackson, 1996a) , plus one of the modified ␥ clones described above. for analysis of the sgrna␤1 and sgrna␤2 promoters, protoplasts were transfected with in vitro transcripts from the ␣ and ␥ cdna clones, plus the appropriate modified ␤ cdna clone. prior to use in in vitro transcription reactions, the ␤ cdna clones were linearized with spei, and the ␣ and ␥ cdna clones were linearized with mlui, except for the ␥1ϩsg␤1 and ␥ϩsg␤1 clones which were linearized with spei. rna was extracted from by-2 protoplasts at 20 h posttransfection as described previously (zhou and jackson, 1996a) . for northern blot analysis, 5 g of total nucleic acid was separated on 1% agarose tbe gels and blotted onto hybond nx (amersham pharmacia biotech) nylon membranes. prehybridization, hybridization, and washes were performed as recommended in the manufacturer's instructions. rna␤-and rna␥-specific [ 32 p]-utp-labeled riboprobes were generated by in vitro transcription reactions using t7 or t3 rna polymerase (zhou and jackson, 1996a) . portions of the ␤ (2434 to 2785 nt, 802 to 1375 nt) or ␥ (2111 to 2444 nt) cdna clones were introduced into pbluescript ksϩ (stratagene) or pgem5zfϩ (promega, madison, wi) plasmids and linearized prior to being used in in vitro transcription reactions. an additional rna␤-specific-riboprobe (2785-2985 nt) was generated by linearizing the ␤c-2785 clone (d.m. lawrence and a.o. jackson, unpublished results) with bamhi prior to use in in vitro transcription reactions. mapping the promoter for subgenomic rna synthesis on the beet necrotic yellow vein virus rna 3 mutational analysis of cis-acting sequences and gene function in rna3 of cucumber mosaic virus 5ј-coterminal subgenomic rnas in citrus tristeza virus-infected cells an rna activator of subgenomic mrna1 transcription in tomato bushy stunt virus characterization and engineering of sequences controlling in vivo synthesis of brome mosaic virus subgenomic rna mapping of the tobacco mosaic virus movement protein and coat protein subgenomic rna promoters in vivo nucleotide sequence and genetic organization of barley stripe mosaic virus rna ␥ relationship of lychnis ringspot virus to barley stripe mosaic virus and poa semilatent virus deletion analysis of the promoter for the cucumber necrosis virus 0.9-kb subgenomic rna characterization of the subgenomic rnas of an australian isolate of barley yellow dwarf luteovirus a positive-strand rna virus with three very different subgenomic rna promoters primary and secondary structural elements required for synthesis of barley yellow dwarf virus subgenomic rna1 evolution and taxonomy of positivestrand rna viruses: implications of comparative analysis of amino acid sequences rapid and efficient site-specific mutagenesis without phenotypic selection long-distance base pairing in flock house virus rna1 regulates subgenomic rna3 synthesis and rna2 replication synthesis of brome mosaic virus subgenomic rna in vitro by internal initiation on minus-sense genomic rna synthesis of subgenomic rnas by positivestrand rna viruses sequence requirements for rna strand transfer during nidovirus discontinuous subgenomic rna synthesis identification of barley stripe mosaic virus genes involved in viral rna replication and movement a novel strategy for one-step cloning of full-length cdna and its application to the genome of barley stripe mosaic virus mutational analysis of barley stripe mosaic virus rna ␤ genome sequences of poa semilatent and lychnis ringspot hordeiviruses a new model for coronavirus transcription heterologous sequences greatly affect foreign gene expression in tobacco mosaic virus-based vectors sequence-specific recognition of a subgenomic rna promoter by a viral rna polymerase moieties in an rna promoter specifically recognized by a viral rna-dependent rna polymerase rna-mediated transactivation of transcription from a viral rna movement of hordeivirus hybrids with exchanges in the triple gene block spatial perturbations within an rna promoter specifically recognized by a viral rna-dependent rna polymerase (rdrp) reveal that rdrp can adjust its promoter binding sites cis-acting elements involved in replication of alfalfa mosaic virus rna in vitro characterization of sequences controlling the synthesis of alfalfa mosaic virus subgenomic rna in vitro virus taxonomy: the taxonomy and nomenclature of viruses. the seventh report of the international committee on taxonomy of viruses in vivo translation of the triple gene block of potato virus x requires two subgenomic mrnas minimal sequence and structural requirements of a subgenomic rna promoter for turnip crinkle virus analysis of the two subgenomic rna promoters for turnip crinkle virus in vivo and in vitro virus multiplication in tobacco protoplasts inoculated with tobacco mosaic virus rna encapsulated in large unilamellar vesicular liposomes the premature termination model: a possible third mechanism for subgenomic mrna transcription in (ϩ)-strand rna viruses subgenomic mrna regulation by a distal rna element in a (ϩ)-strand rna virus flock house virus: down-regulation of subgenomic rna3 synthesis does not involve coat protein and is targeted to synthesis of its positive strand analysis of cis-acting elements required for replication of barley stripe mosaic virus rnas expression of the barley stripe mosaic virus rna ␤ "triple gene block we thank erin duncan for technical assistance and teresa rubio and sharon fodor for comments on the manuscript. this research was supported by usda competitive grant 97-35303-4572 and by a grant from torrey mesa research institute, syngenta research and technology. j.b. was the recipient of a national science foundation graduate research fellowship. key: cord-300372-h5g4z8ts authors: kelvin, alyson a.; degousee, norbert; banner, david; stefanski, eva; leon, alberto j.; angoulvant, denis; paquette, stéphane g.; huang, stephen s. h.; danesh, ali; robbins, clinton s.; noyan, hossein; husain, mansoor; lambeau, gerard; gelb, michael h.; kelvin, david j.; rubin, barry b. title: lack of group x secreted phospholipase a(2) increases survival following pandemic h1n1 influenza infection date: 2014-04-01 journal: virology doi: 10.1016/j.virol.2014.01.030 sha: doc_id: 300372 cord_uid: h5g4z8ts the role of group x secreted phospholipase a(2) (gx-spla(2)) during influenza infection has not been previously investigated. we examined the role of (reviewer 2 minor comment 2) gx-spla(2) during h1n1 pandemic influenza infection in a gx-spla(2) gene targeted mouse (gx(−/−)) model and found that survival after infection was significantly greater in gx(−/−) mice than in gx(+/+) mice. downstream products of gx-spla(2) activity, pgd(2), pge(2), ltb(4), cysteinyl leukotrienes and lipoxin a(4) were significantly lower in gx(−/−) mice bal fluid. lung microarray analysis identified an earlier and more robust induction of t and b cell associated genes in gx(−/−) mice. based on the central role of spla(2) enzymes as key initiators of inflammatory processes, we propose that activation of gx-spla(2) during h1n1pdm infection is an early step of pulmonary inflammation and its (reviewer 2 minor comment 2) inhibition increases adaptive immunity and improves survival. our findings suggest that gx-spla(2) may be a potential therapeutic target during influenza. influenza is a leading source of morbidity and mortality worldwide that is caused by ever changing and newly emerging influenza viruses including the introduction of the 2009 a/h1n1/2009 (h1n1pdm) and novel avian h7n9 viruses (fisher et al., 2005; groom and luster, 2011; widegren et al., 2011) . while effective vaccines and antiviral drugs have been developed for circulating strains of human influenza (santone et al., 2008) , continued antigenic drift and shift generate novel virus strains that pose a threat to immunologically naïve populations. the emergence of pandemic influenza h1n1pdm in the spring of 2009 led to hundreds of thousands of hospitalizations with significant numbers of fatalities in north america (update: influenza activity -united states, 2009-10). severe cases were characterized by viral pneumonia and uncontrollable pulmonary inflammation, and were similar to the inflammation observed in severe cases of sars, h5n1 and spanish influenza patients (baillie and digard, 2013; bermejo-martin et al., 2010; cameron et al., 2012; curfs et al., 2008) importantly there is little understood regarding the pathways driving the pulmonary inflammatory process for these diseases. host-defenses against influenza include anatomic barriers, mucociliary clearance, anti-microbial secretions and innate and adaptive immune responses. early host responses are characterized by the mobilization of leukocytes, such as alveolar and circulating macrophages, polymorphonuclear leukocytes (pmn) (world health organization, 2012) and nk cells which continues into the activation of adaptive immune cells such as t-cells, b-cells and dendritic cells. importantly, factors that lead to the activation of these cells and cell networks are increased after h1n1pdm infection (influenza activity -united states and worldwide, 2010; ohtsuki et al., 2006; ohtsuki et al., 2006; paquette et al., 2014) . these include inflammatory mediators such as chemokines, cytokines and lipid mediators like eicosanoids. the first step in the generation of eicosanoids, inflammatory mediators that participate in the regulation of the inflammatory response, is catalyzed by pla 2 enzymes, which release arachidonic acid (aa) from phospholipids (del et al., 2007) . to date, 11 secreted pla 2 enzymes (spla 2 ; ib, iia, iic, iid, iie, iif, iii, v, x, xiia and xiib) , six cytosolic pla 2 enzymes (cpla 2 s; α, β, γ, δ, ε, and ξ) (kim et al., 2007) , nine ca 2 þ -independent pla 2 enzymes (ipla 2 s; α, β, γ, δ 2 , δ, ε, ε2 ξ, θ, and η) , and two lysosomal pla 2 enzymes (femling et al., 2005) have been described. the spla 2 enzymes are structurally related, ca 2 þ -dependent proteins with unique biological properties, enzymatic activities against membrane phospholipids and tissue and cellular locations, suggest distinct roles for these enzymes in various pathophysiological events. spla 2 enzymes are implicated in lipid mediator release, degranulation, cellular proliferation, destruction of invading bacteria , viruses (kennedy et al., 1995; mazur et al., 2007) and activation of intracellular signaling cascades (kim et al., 2007) . gx-spla 2 is expressed in alveolar macrophages and epithelial cells in the lungs of patients with pneumonia (marshall et al., 2000) , neuronal cells (gaudreault and gosselin, 2008) , male reproductive organs (dennis, 1994) and atherosclerotic plaques , and is cleaved to its active form in inflamed tissues (lu et al., 2006) . arachidonic acid (aa) is the precursor of prostaglandins, thromboxanes, leukotrienes and lipoxins, eicosanoids that regulate pulmonary vascular and bronchial responses, leukocyte activation, adhesion and emigration (guan et al., 2013; henderson et al., 1995) . eicosanoids also regulate antigen presenting cell [apc] function (degousee et al., 2001; murakami et al., 2011; truchetet et al., 2012) , t cell maturation (myers et al., 2012) and th17 expansion (stephenson et al., 1988; zhao et al., 2011) . we found that il-17 and th17 cells are dysregulated during human h1n1pdm infection (baillie and digard, 2013; bermejo-martin et al., 2010; ohtsuki et al., 2006) . therefore, spla 2 enzymes and eicosanoids may play a central role in determining the outcome of pulmonary viral infection. the role of spla 2 enzymes in the immune responses to h1n1pdm infection in vivo has not been evaluated. gx-spla 2 has been highly implicated in the inflammatory response including pattern recognition receptor function, and displays the highest activity among all mammalian spla 2 s on phosphatidylcholine-rich liposomes in vitro (crooks and stockley, 1998; henderson et al., 1995) . recently, gx-spla 2 has been suggested as a signal amplifier in tlr4 stimulation which further suggests a role for gx-spla 2 in the regulation of the inflammatory response (schultz-cherry and jones, 2010) . considering the potential of gx-spla 2 in the inflammatory response, spla 2 enzymes may play a central role in determining the outcome of pulmonary viral infections, which cause uncontrolled inflammatory destruction of the respiratory tract (henderson et al., 1995) . we have developed a robust lethal mouse model of h1n1pdm infection to study innate host defense mechanisms and antiviral compound activity . we have shown that h1n1pdm infection in this mouse model leads to pulmonary inflammation, a histopathological picture similar to what is observed in fatal human cases, and over 90% lethality within 5-8 days . in this study, we document a marked increase in gx-spla 2 expression in lung following infection in gx þ / þ mice. to specifically evaluate the pathophysiological role of gx-spla 2 in our lethal influenza mouse model, we subjected gx þ / þ and gx à / à mice (henderson et al., 1995) to h1n1pdm infection in vivo. our results showed that, in two distinct mouse strains, targeted deletion of gx significantly increased survival following h1n1pdm infection in comparison with gx þ / þ mice. in addition, eicosanoid accumulation in bal fluid was attenuated and induction of t cell and b cell associated genes was higher in gx à / à than gx þ / þ mice after h1n1pdm infection. taken together, our data suggests a negative role for gx-spla 2 in the immune response to pulmonary infection with h1n1pdm influenza in vivo. furthermore, these findings implicate gx-spla 2 as a potential therapeutic target during severe influenza infection. airway epithelial cells and myeloid cells can both express gx-spla 2 (marshall et al., 2000) . previously, we have investigated the host immune responses to pulmonary viral infections, including infection with the influenza viruses h5n1 and h1n1pdm (baillie and digard, 2013; bermejo-martin et al., 2010; bhavsar et al., 2010; cameron et al., 2008; cameron et al., 2012; escoffier et al., 2010; huang et al., 2009; huang et al., 2012; kudo and murakami, 1999) . furthermore, we have also delineated the biology and molecular regulation of many of the enzymes that catalyze eicosanoid biosynthesis in vitro and in vivo degousee et al., 2006; degousee et al., 2008; degousee et al., 2002; degousee et al., 2003; leon et al., 2012; lu et al., 2006; saez de et al., 2011) . to begin to investigate the role of gx-spla 2 during h1n1pdm infection, we evaluated the pulmonary expression of gx-spla 2 in our h1n1 pandemic influenza mouse model. gx þ / þ mice were infected intranasally with a/mexico/4108/ 2009 (h1n1pdm) and lung tissues were harvested at baseline and on day 3, 6 and 14 post infection (pi). real-time pcr was performed on the extracted rna and identified a significant increase in the ratio of gx-spla 2 to gapdh mrna on day 3 and day 14, but not day 6 pi (fig. 1ai) . gx-spla 2 /gapdh mrna increased approximately four fold on day 3 and three fold on day 14 compared to baseline. furthermore, we also determined the regulation of cytosolic pla 2 (cpla 2 ) (fig. 1aii ) and the spla 2 family member gv-pla 2 (fig. 1aiii ) in both gx þ / þ and gx à / à mice. there was negligible total protein upregulation of cpla 2 and gv-pla 2 was not regulated throughout the infection time course. the absence of the change of total protein of cpla 2 was also confirmed by immunohistochemistry (data not shown). no statistical differences in mrna transcripts for cpla 2 and gv-pla 2 were noted between the gx þ / þ or gx à / à mice. these results demonstrated that h1n1pdm influenza infection stimulated a bimodal increase in pulmonary gx-spla 2 mrna expression which was /4108/2009) and the lungs were assessed for the mrna and protein expression and localization of plas during a 14 day time course. gx-spla 2 mrna (ai), cpla 2 mrna (aii) and gv-spla 2 mrna (aiii) expression quantified by real-time rt-pcr was normalized to gapdh, gx þ / þ (open bars) and gx à / à (filled bars) mice (c3h/hen background mice). gx þ / þ and gx à / à mouse lungs were perfusion fixed in situ with 4% paraformaldehyde, sectioned and subject to immunohistochemical analysis with the igg fraction of rabbit anti-mouse gx-spla 2 antiserum (1/100 dilution) (b). giia and gx-spla 2 protein expression determined by immunoblot analysis of lung tissue homogenates of wild type gx þ / þ (lane 1) and knockout gx à / à (lane 2) mice (c). for each blot, the corresponding recombinant spla 2 enzyme (rec spla 2 ) was run alone (lane 3) as a control. representative results for five separate experiments are shown. all the mice used in these experiments were genotyped littermates and grouped and analyzed by their genotype. a, p o 0.05 gx þ / þ or gx à / à vs. base; b, p o 0.05 gx þ / þ vs. gx à / à at any time point, anova followed by paired t-test, two tailed, assuming unequal variance. nz 8 per group; 400 â ; scale bar: 50 μm for immunohistochemistry. specific for this spla 2 since neither cpla 2 nor gv-spla 2 was upregulated. since gx-spla 2 mrna levels increased in response to h1n1pdm infection, we investigated the spatial and temporal expression of gx-spla 2 protein in mouse lungs after influenza infection. lungs from gx þ / þ and gx à / à mice infected with h1n1pdm were harvested at baseline, 3, 6 and 14 days pi and subjected to immunohistochemical analysis with anti-mouse gx-spla 2 antiserum. visualization by light microscopy revealed gx-spla 2 protein accumulation in the lungs of infected mice compared to baseline (fig. 1b) . gx-spla 2 protein was identified in inflammatory cells that had infiltrated in the alveolar space on day 3 and 6 in gx þ / þ mice infected with h1n1pdm (shown by arrows). gx-spla 2 protein was also clearly identified in epithelial cells lining the bronchioles on day 3, 6 and 14 in gx þ / þ mice infected with h1n1pdm (fig. 1b , upper right panels). no staining for gx-spla 2 protein was observed in gx à / à mice at baseline or at any time point after infection with h1n1pdm (fig. 1b , lower panel rows). similarly, no proteins cross reacting with the secondary antibody alone was identified in gx þ / þ or gx à / à mice (fig. 1b , left hand panels). we confirmed the loss of gx-spla 2 in the gx à / à mice by immunoblot analysis. we indeed observed a specific depletion of gx-spla 2 but no change in the expression of giia-spla 2 in the gx à / à mice compared to gx þ / þ mice (fig. 1c ). taken together, these results show that intranasal infection with h1n1pdm increases gx-spla 2 rna and protein expression in the lung that corresponds to the increase in lung inflammation associated with influenza infection. this suggests a possible role for gx-spla 2 in the pathogenesis of pulmonary h1n1pdm influenza infection. since gx-spla 2 was upregulated in the lung during h1n1pdm infection, we explored its role in the host response to pulmonary infection with h1n1pdm influenza. we first examined the clinical outcome of gx-spla 2 deletion by assessing weight loss and survival of g þ / þ and gx-spla 2 gene targeted mice à / à mice on two different genetic backgrounds following infection and assessed weight loss and survival. in the first series of infections, gx þ / þ (n¼25), gx þ /-(n¼32), and gx à /à (n¼24) mice on a c57bl/6j background were infected intranasally with h1n1pdm influenza a/mexico/4108/2009 ( fig. 2a ). mice on this background lack the giia-spla 2 gene (karabina et al., 2006) . animals were euthanized if their body weight decreased to less than 80% of baseline weight, or if the 14-day duration of the study was completed. survival 14 days after h1n1pdm influenza infection was 70% in gx à /à mice (blue line), 48% in gx þ /mice (green line) and 15% in gx þ / þ mice (red line). the difference in survival between gx à /à and gx þ /mice, and between gx à /à and gx þ / þ mice after h1n1pdm infection was statistically significant, pr0.01. to independently confirm these findings, we evaluated the survival of gx þ / þ (n¼ 71) and gx à / à (n ¼ 57) mice on a c3h/hen background (fig. 2b) which have a functional giia-spla 2 gene (karabina et al., 2006) . as with the studies with the c57bl/6j mice, animals were infected intranasally with a/mexico/4108/2009 and euthanized if their body weight decreased to less than 80% of baseline weight, or at the end of the study. survival of gx à / à mice on the c3h/hen background was again significantly higher (62%, blue line) following h1n1pdm infection than survival of gx þ / þ mice on a c3h/hen background (36%, red line). together, these studies showed that targeted deletion of gx-spla 2 in two different mouse models led to increased survival following h1n1pdm infection in vivo. furthermore, since the c3h/hen mice expressed endogenous giia-spla 2 , these results demonstrate that the ability to express giia-spla 2 does not compensate for the loss of gx-spla 2 during host immune responses to pulmonary h1n1pdm influenza infection. depletion of gx-spla 2 during h1n1pdm infection leads to a decrease in downstream phospholipid catalysis (aa) products but no difference in innate cell recruitment gx þ / þ and gx-spla 2 gene targeted mice à / à on a c3h/hen background were infected with a/mexico/4108/2009, and bal fluid was harvested 3 or 6 days post h1n1pdm infection. to assess the general inflammatory response and lung tissue destruction that typically occurs during h1n1pdm infection (ohtsuki et al., 2006; paquette et al., 2012) , we investigated the histopathology by h&e staining of lungs isolated from both gx à / à and gx þ / þ mice at baseline, day 3, 6 and 14 pi (fig. 3a) . the pulmonary pathology peaked quickly by day 3 pi in infected gx þ / þ and gx à / à animals. bronchiolitis and alveolitis with mononuclear cell and neutrophil infiltration were observed in several loci of the infected lungs of both groups. hemorrhage, edema, and necrotizing respiratory epithelia were also observed with similar severity among both groups. pathology persisted until day 7 pi where mononuclear cell and neutrophil infiltration were still profound and caused patches of consolidation in the lung tissue in both groups. it seemed by day 14 pi that the pulmonary pathology was slightly more minimal in the gx à / à mice with reduced level of leukocyte infiltration. in contrast, multi foci cell infiltration and tissue consolidation was still prominent in the gx þ / þ lungs by day 14 pi. to further examine the inflammatory cell types that may be recruited to the lung during h1n1pdm infection we analyzed lung homogenates from day 0, 3, 6 and 14 pi from both gx þ / þ and gx à / à mice by immunoblot for neutrophil and leukocyte cell markers, mpo and cd45 respectively. mpo was induced on day 3 and day 6 pi in both the mouse genotypes and returned to baseline on day 14 and cd45 was induced from baseline for all time points measured. neither mpo nor cd45 showed any variation in the lungs between gx à / à or gx þ / þ throughout the infection time course (fig. 3bi) ; densitometry did not reveal any statistical differences (fig. 3bii) . furthermore, we also analyzed gx à / à and gx þ / þ lungs for the presence and activation of macrophages by immunohistochemistry and real-time rt-pcr ( fig. 3ci , cii and d). here we found that the macrophage marker mac-3 was significantly increased and peaked at day 6 following infection as determined by immunohistochemistry staining which was further confirmed by quantifying the staining and quantification of the signal ( fig. 3ci and cii). furthermore the mrna for the inflammatory chemokine ccl2 was also significantly increased following infection (days 3 and 6) and decreased by day 14 (fig. 3d) . no difference was determined for mac-3 or ccl2 expression between gx à / à or gx þ / þ mice. taken together, these results suggest a similar inflammatory and innate response in both the gx þ / þ and gx à / à mice. to assess the role of gx-spla 2 in leukocyte infiltration into the bronchoalveolar space after h1n1pdm infection, we measured total leukocyte cell counts and the levels of different leukocyte cell types in the bal fluid of h1n1pdm infected in gx þ / þ and gx à / à mice (fig. 4a ). no significant difference in total cell counts ( fig. 4ai ) or the percentage of cd4 þ , cd8 þ , b or natural killer cells, or granulocytes were identified in the bal fluid of gx þ / þ and gx à / à mice 6 days after h1n1pdm infection (fig. 4aii ). in addition, targeted deletion of gx-spla 2 had no effect on lung viral titers 3 or 6 days pi (fig. 4b ). we next determined by elisa the levels of diferent aa metabolites including pgd 2 , ltb 4 , cysteinyl leukotrienes, pge 2 , a stable pge metabolite, and lipoxin a 4 which are known to regulate bronchiolar reactivity and inflammatory cell adhesion, migration and activation (henderson et al., 1995) were determined by elisa. levels of pgd 2 ( 5g ) were all significantly lower in the bal fluid from gx à / à mice (solid bars) on day 3 pi compared to gx þ / þ mice. conversely, on day 6 pi, the levels of these metabolites were similar in gx þ / þ and gx à / à mice ( fig. 5a-f) . in summary, these results show that deletion of gx-spla 2 in mice led to a transient but significant decrease in the levels of a panel of aa metabolites when mice were infected with a lethal h1n1pdm influenza virus that was not associated with alterations in inflammatory cell infiltration or viral clearance. increased expression of immunoglobulin chain, lymphocyte differentiation, antigen processing genes and presence of cd3 þ t cells in the lungs of mice lacking gx-spla 2 after h1n1pdm infection to increase our understanding of the molecular events leading to increased survival following h1n1pdm infection in gx à / à mice, we conducted microarray analysis of rna extracted from the lungs of gx þ / þ and gx à / à influenza infected animals. as previously reported by our group paquette et al., 2014; , influenza infection caused a progressive increase in the total number of upregulated genes in the lung tissue of gx þ / þ mice (1246 genes at 3 days pi and 2469 genes at 6 days pi). genes that belonged to different functional groups, such as immune response, inflammatory response and prostaglandin signaling pathways (figs. 6 and 7) showed a progressive increase that was parallel to the global evolution of gene expression. conversely, the expression of cytokine-related genes reached maximal levels 3 days pi and were maintained thereafter (fig. 6a) . at first sight, lack of gx-spla 2 did not modify the global evolution of gene expression in the lungs. similarly to gx þ / þ mice, gx à / à mice showed a progressive increase in the number of upregulated genes (1578 at 3 days pi and 2469 at 6 days pi). further analysis demonstrated that on day 3 pi, gx à / à mice showed significantly higher levels of the cytokines lta and ltb, the chemokines ccl19, cxcl9 and cxcl13 and the chemokine receptors cxcr3 and cxcr5 ( fig. 6b and c). in contrast, the expression pattern of cytokines and chemokines showed no differences between gx þ / þ and gx à / à mice 6 days pi (data not shown). interestingly, expression of 21 immunoglobulin chains, including heavy and light chains, was identified in gx à / à mice 3 days pi, while no expression of immunoglobulin chain genes was identified in gx þ / þ mice at this time point. in addition, the number of immunoglobulin chain related genes was higher in gx à / à than gx þ / þ mice 6 days after h1n1pdm infection (fig. 6b ). no differences were observed in the patterns of interferon regulated genes between gx þ / þ and gx à / à mice after h1n1pdm infection (data not shown). to determine which functional pathways are differentially enriched between gx þ / þ and gx à / à mice after h1n1pdm infection, we performed intersect analysis of the respective sets of upregulated genes (fig. 7) . at 3 days pi, expression of interferon regulated, inflammatory response and innate immune response genes were common to both gx þ / þ and gx à / à mice. a number of genes related with eicosanoid synthesis and their receptors were found to be regulated during influenza infection; however, gx-spla 2 deficiency did not cause any major alterations in their expression profiles (fig. s1) . the set of genes specifically enriched in the gx à / à mice at 3 day pi were those related to adaptive immune responses, such as immunoglobulin chains, lymphocyte differentiation and antigen processing and presentation. on the other hand, the set of genes more enriched in the gx þ / þ mice were genes involved in the tissue development category at 3 days pi (fig. 7a ). at 6 days pi (fig. 7b) , the enrichment profiles of upregulated genes in gx þ / þ and gx à / à mice were nearly identical. while immunoglobulin chain gene expression was identified in both gx þ / þ and gx à / à mice, expression of immunoglobulin chain genes remained elevated only in the gx à / à set of genes 6 days pi, while the gx þ / þ set of genes continued to show enrichment in the tissue development category at day 6 pi. to further evaluate the adaptive immune system of the gx à / à mice infected with h1n1pdm we investigated the t and b cell responses within the lung during infection. here we stained lung sections with anti-cd3 to assess infiltration of t cells using immunocytochemistry ( fig. 8a and b) . we found a significant increase of cd3 positive t cells in the lung on day 3 pi in the gx à / à mice compared to gx þ / þ mice (fig. 8a , upper right panels) by approximately 2 fold (fig. 8b) . interestingly, cd3 staining of the gx þ / þ animals had increased to similar levels seen in the gx à / à mice by day 6 and both genotypes had sustained levels of cd3 on day 14. moreover, we also investigated cd8a and igg (ighg) mrna levels in the lungs of the gx à / à mice throughout the time course and there was a slight trend for increase cd8a levels. taken together, the results from the cd3 and igg analysis supported the microarray studies where the adaptive immune system of the gx à / à had a faster and more robust initiation. gx-spla 2 has been highly implicated in various inflammatory diseases of the respiratory tract, including th2 cytokine-driven asthma (de jong et al., 2006; henderson et al., 2007) and lung injury (napolitani et al., 2009 ), but its role during influenza infection has not been previously investigated. here we evaluated the pathophysiological role of gx-spla 2 during severe influenza a h1n1pdm infection in the mouse. we found that gx-spla 2 expression was increased following infection, and that targeted deletion of gx-spla 2 led to increased survival in mice. lack of gx-spla 2 resulted in decreased levels of pgd 2 , ltb 4 , cysteinyl leukotrienes, pge 2 and lipoxin a 4 and increased adaptive immune responses at 3 but not 6 days following h1n1pdm infection. this demonstrates that gx-spla 2 plays an important role in the production of several biologically active inflammatory lipid mediators during the early phase of the inflammatory response that follows h1n1pdm influenza infection. human patients with a severe respiratory disease caused by influenza infection have a dysregulated inflammatory response that leads to lung pathogenesis associated with hypercytokinemia in most cases (bermejo-martin et al., 2010; curfs et al., 2008) . taken together with the previous findings showing a role of gx-spla 2 in inflammatory lung diseases, our work supports the further investigation of the therapeutic potential of attenuating gx-spla 2 during severe influenza infection as well as the interplay between eicosanoids and adaptive immunity. spla 2 has previously been implicated in pulmonary disease onset and progression putting it forth as a potential biomarker for severe respiratory diseases (henderson et al., 1995; henderson et al., 2007) . we show that gx-spla 2 protein and mrna expression increased in the lungs of gx þ / þ mice following h1n1pdm infection, suggesting that gx-spla 2 may be used as a possible biomarker of severe influenza infection. this is the first report of increased gx-spla 2 expression following influenza virus infection. both epithelial cells and leukocytes were found to be sources of gx-spla 2 during infection, and gx-spla 2 expression was detected in epithelial cells 3 days prior to the infiltration of leukocytes. in will be interesting to determine in future experiments whether the specific deletion of gx-spla 2 expression in epithelial cells vs. infiltrating leukocytes or both is responsible for the increased survival. here we observed a bimodal expression pattern of gx-spla 2 during the 14 day time course of infection. it is possible that this occurred due to the protein stability as it is used to regulate bioactive lipid mediator synthesis. if the protein does not remain stable throughout the course of infection and recovery, it may be important to have a second increase in gx-spla 2 in the later stages of infection to compensate for the loss of protein. it is in fact possible that the protein exerts distinct roles in the clearance of the virus and tissue remodeling in addition to the regulation of immune cells. such a scenario would require inductions at specific time points during infection. it will be important to further explore the local expression in the virus niche and the stability of protein gx-spla 2 during influenza infection to better understand how gx-spla 2 stability may influence influenza severity in the initiation of the innate immune response, adaptive maintenance, and recovery. furthermore, it would also be of value to investigate the source of gx-spla 2 by expression analysis of each cell type and also by investigating the role of hematopoietic gx-spla 2 compared to epithelial gx-spla 2 . the latter could be studied by employing bone marrow transplantation experiments from gx à / à mice into gx þ / þ and the reverse. while the association between gx-spla 2 and influenza related complications has not previously been investigated, ltb 4 , a downstream product of gx-spla 2 has been suggested to be a biomarker for pulmonary disease and respiratory complications following trauma (influenza activity -united states and worldwide, 2010; henderson et al., 2011; shridas et al., 2011) . multiple studies have implicated gx-spla 2 in the pathophysiology of pulmonary diseases onset and progression, suggesting gx-spla 2 might be a suitable therapeutic target in lung (henderson et al., 1995; morita et al., 2013) . deletion of gx-spla 2 in a th2 cytokine-driven mouse asthma model significantly impairs development of asthma (henderson et al., 1995) and accordingly, administration of a human gx-spla 2 selective inhibitor in a human gx-spla 2 knock-in mouse model led to a significant reduction in airway inflammation, mucus hypersecretion and airway hyperresponsiveness (henderson et al., 2007) . furthermore, although not specific for human gx-spla 2 , the indole-based spla 2 inhibitor varespladib has been shown to significantly inhibit spla 2 activity in the bal fluid of infants with post-neonatal ards (de jong et al., 2006) during induced asthma, suggesting the involvement of spla 2 among other spla 2 s. our results showing increased survival of the gx à / à mice after infection with h1n1pdm further support the notion that gx-spla 2 is a therapeutic target in pulmonary diseases due to viral infection and that infection with h1n1 might be better controlled by inhibiting this spla 2 . one of the main functions of gx-spla 2 is likely the generation of bioactive lipid mediators which play important roles in lung inflammatory diseases (gao et al., 2013; gaudreault and gosselin, 2007; van elssen et al., 2011) . although we did not see any major differences in the mrna analysis of the eicosanoid pathways between the gx à / à and gx þ / þ mice, measuring the mrna levels of these genes may have limited value to determine the level of activation of their signaling pathways. conversely, we observed decreased levels of pgd 2 , ltb 4 , cysteinyl leukotrienes, pge 2 and lipoxin a 4 in bal fluid 3 day pi in the gx à / à mice, indicating that gx-spla 2 acts upstream of these bioactive lipid mediators during influenza infection and thereby suggests a possible role of these bioactive mediators in pulmonary pathogenesis after influenza infection. in agreement with our findings, pgd 2 has been implicated during influenza a infection as pgd 2 expression in the lungs of older animals inhibits regulatory dendritic cells activity and t cell responses (zhang et al., 2000) . other eicosanoids have been implicated in different lung diseases, and the dysregulation of leukotrienes and lipoxins have been reported as contributing factors to the pathogenesis and severity of other respiratory diseases (bermejo-martin et al., 2009) . ltb 4 has been suggested to play a destructive inflammatory role in the lung by priming neutrophils for adhesion, chemotaxis and stimulation of granule release (cameron et al., 2007) . as well pgd 2 , pgd receptor, lipocalin-type pgd synthase and ltb 4 have been implicated in asthma pathogenesis (arima and fukuda, 2011; masuda et al., 2005; rusinova et al., 2012) . although asthma and pulmonary disease due to influenza infection differ in derivation, both are characterized by hyper-inflammation of the respiratory tract. taken together, our data supports a role of gx-spla 2 signaling and bioactive mediator production in the regulation of the pulmonary response to h1n1pdm infection. in the future it would be important to specifically determine whether pgd 2 , ltb 4 , cysteinly leukotrienes, pge 2 , lipoxin a 4 or another aa metabolite specifically modulates the response to h1n1pdm infection. the inflammatory response may be simultaneously beneficial and destructive during lung infection (baillie and digard, 2013; fig. 2 . increased survival of gx à / à vs. gx þ / à or gx þ / þ mice following a/mexico/ 4108/2009 infection. gx þ / þ (n¼ 25), gx þ / à (n ¼32), and gx à / à (n¼24) mice (c57bl/6j background, lacks giia-spla 2 ) were infected intranasally with a/mexico/ 4108/2009 and survival was assessed for a 14 day period (a). gx þ / þ (n¼71) and gx à / à (n¼ 57) mice (c3h/hen background, expresses giia-spla 2 ) were infected intranasally with a/mexico/4108/2009 and survival was assessed for a 14 day period (b). animals were sacrificed if their body weight decreased to less than 80% of baseline weight, or if the 14-day duration of the study was completed. log rank test, p o0.05 gx à / à vs. gx þ / þ and gx þ / à mice or po 0.05, gx à / à vs. gx þ / þ mice. all the mice used in these experiments were genotyped littermates and grouped and analyzed by their genotype. bermejo-martin et al., 2010) . although destructive killing of foreign pathogens is imperative for eradication and microbe clearing, the over production of inflammatory mediators leading to an overt inflammatory response may accentuate disease pathology, as is the case during severe influenza h5n1 and h1n1 infection (bermejo-martin et al., 2010; curfs et al., 2008; huang et al., 2009) . this illustrates the dual role of proinflammatory mediators, which has also been suggested for some gx-spla 2 downstream lipid mediators. for instance, ltb 4 has been shown to increase the activity of nasal neutrophil killing of human coronavirus, rsv, and influenza b virus (van elssen et al., 2011) and to induce the release of antimicrobial peptides in vivo in the lungs of fig. 3 . infection with h1n1pdm influenza induces similar pulmonary inflammation and recruitment of inflammatory cells in gx þ / þ and gx à / à mice. gx þ / þ and gx à / à mice (c3h/hen background mice) were infected with h1n1pdm (a/mexico/4108/2009) influenza and the lungs were perfusion fixed in situ with 4% paraformaldehyde on specific time points following infection, sectioned and subject to hematoxylin and eosin staining (a). mpo protein (neutrophil marker), cd45 protein (leukocyte marker) and gapdh protein (loading control) expression levels were determined by immunoblot analysis from lung tissue homogenates of gx þ / þ and gx à / à mice over a 14 day time course of h1n1pdm influenza infection (bi). densitometric analysis of mpo (bii) and cd45 (biii) protein levels normalized to gapdh levels in lung tissue of gx þ / þ (open bars) and gx à / à (filled bars) mice after h1n1pdm influenza infection, are presented. immunohistochemical analysis with specific rabbit primary antibody against mouse mac-3 antigen (marker for macrophages) is shown (ci). assessment of mac-3 positive cells (indicated by -) per high power field before, 3, 6 or 14 days after infection with h1n1pdm influenza is shown (cii). ccl2 mrna expression normalized to gapdh was determined by quantitative real-time pcr in lung tissue of gx þ / þ and gx à / à mice after h1n1pdm influenza infection (d). representative images ( â 200) from five independent experiments are shown. scale bar: 100 μm (a) or 50 μm (c). a, p o 0.05 gx þ / þ or gx à / à vs. base; b, p o0.05 gx þ / þ vs. gx à / à at any time point, anova followed by paired t-test, two tailed, assuming unequal variance. nz 8 per group. mice infected with viruses (gao et al., 2013; gaudreault and gosselin, 2007) . the lipid product protectin d1 has been implicated in influenza therapeutics (arima and fukuda, 2011; mitsuishi et al., 2006) . although these previous reports seem to suggest a conflicting role for gx-spla 2 in consideration of our data, it may be possible that ltb 4 and gx-spla 2 promote antiviral activity and are significant during a viral response but only at moderate levels. alternatively, it is possible that gx-spla 2 prevents h1n1 infection but also triggers excessive inflammation that is associated with lipid surfactant destruction. more work is needed to understand how the function of gx-spla 2 mediates both beneficial and deleterious roles during influenza infection. our survival data from gx gene targeted mice indicated that the loss of gx-spla 2 was beneficial to the host during influenza infection. the microarray mrna data from lungs of gx þ / þ infected mice were in agreement with our previously published data on pandemic h1n1 2009 virus infection, in mice including the progressive increase of immune and inflammatory responses and of the prostaglandin signaling pathway (paquette et al., 2014) . together, the results of microarray analysis, gene expression by real-time rt pcr and immunocytochemistry of the lungs suggested that gx à / à mice exhibited a more robust adaptive immune response than gx þ / þ mice. indeed, we observed significant differences in lymphocyte gene profiles at day 3 pi, associated with differences in the levels of lymphotoxin alpha and beta, b cell chemokines, t cell chemokine receptors and b cell immunoglobulin chains as measured which by immunofluorescence and rt-pcr. expression of b cell immunoglobulin chain genes were substantially increased on day 3 pi in the gx à / à mice but not in the gx þ / þ . b cell immunoglobulin gene expression was significantly greater on day 6 pi. and the expression of the t cell, b cell and dendritic cell chemokines and chemokine receptors, i.e., ccl19, cxcr3, etc., were significantly higher in the gx à / à samples than gx þ / þ . these results suggest that the downstream products of gx-spla 2 , such as pgd 2 , pge 2 , ltb 4 may inhibit the early adaptive immune responses of t and b cells during viral infection and this fits with the fact that aspirin, which attenuates eicosanoid production, can be an effective therapy for patients with influenza infection (matsuoka et al., 2000) . it would be of value in future studies to further investigate the effect of gx-spla 2 on the proliferation, activation and differentiation of t and b lymphocytes. consistent with this notion, the chemokines and chemokine receptors found to be upregulated in the h1n1pdm infected gx à /à mice are known to play significant roles in t and b cell migration and localization to the lymph nodes (goracci et al., 2010; muthuswamy et al., 2010) . cxcl13/cxcr5 signaling has been shown to activate b cells (sadik and luster, 2012) , which may explain the increased immunoglobulin chain gene expression observed in gx à /à mice. our data supports previous findings implicating pgd 2 in the inhibition of cell migration to lymph nodes (zhang et al., 2000) , and pge 2 in the inhibition of adaptive immune cellular events such as chemokine production by dcs and the attraction of naïve t cells (murakami et al., 2011; truchetet et al., 2012) . in conclusion, our findings provide new insights into the molecular pathophysiology of lethal influenza infection, highlighting a new role for gx-spla 2 during h1n1pdm infection. overall, the spla 2 appears as a negative effector but it may act at several steps during infection. we found that gx-spla 2 and its downstream products may have a role in the inhibition of adaptive immunity during viral infection in mice thereby contributing to pathogenesis. within this mechanism, it is in fact possible that t and b cell maturation and activation are initiated in mice lacking gx-spla 2 prior to virus infection, and that a more robust and earlier adaptive fig. 4 . lung viral titers and cell counts in bal fluid show similar cell numbers and cell population distributions following infection in gx þ / þ and gx à / à mice. gx à / à and gx þ / þ mice infected with a/mexico/4108/2009 were investigated for lung cell numbers, populations and viral load. bal fluid was harvested from infected gx þ / þ (open bars) and gx à / à (filled bars) mice (c3h/hen background mice) on day 0 and 6 and the cell numbers (ai) and cell population distributions (aii) were assessed by facs. viral load was determined on day 0, 3 and 6 pi of gx þ / þ (open bars) and gx à / à (filled bars) mice by real-time rt-pcr vrna quantification (b). all the mice used in these experiments were genotyped littermates and grouped and analyzed by their genotype. a, p o0.05 gx þ / þ vs. base, anova followed by paired t-test, two tailed, assuming unequal variance. nz 7 per group. immune response increased the survival of gx à / à mice after h1n1pdm infection. since gx-spla 2 may contribute to inflammatory response dysregulation during influenza infection and contribute to the morbidity and mortality associated with hospitalized influenza patients, this work may shed important insight into the molecular mechanisms of severe influenza infection. our findings further support the notion that gx-spla 2 is an interesting therapeutic target in lung inflammatory diseases. whether inhibition or attenuation of gx-spla 2 activity during severe influenza infection has a therapeutic effect remains to be demonstrated. to dissect the role of gx-spla 2 in the molecular regulation of pulmonary infection with h1n1pdm influenza, mice that lack gx-spla 2 (gx à / à mice) on the c57bl/6j background previously described were used (matsuoka et al., 2000) . this mixed background strain has a naturally occurring mutation in the gene encoding giia-spla 2 (karabina et al., 2006) , which has been fig. 5 . decreased eicosanoid levels in the bal fluid 3 but not 6 days after infection with a/mexico/4108/2009 in gx à / à vs. gx þ / þ mice. the eicosanoid levels in the bal fluid of gx à / à and gx þ / þ mice (c3h/hen background) were investigated at baseline and following infection with a/mexico/4108/2009. gx þ / þ (open bars) and gx à / à (filled bars) mice (c3h/hen background mice) bal fluid was harvested by instilling ice cold nacl (1 ml) five times and pooled. levels of pgd 2 mox (a), ltb 4 (b), cysteinyl leukotrienes (c), pge 2 (d), stable pge metabolite (e), pge 2 plus pge metabolite (f) and lipoxin a 4 (g) were assessed by elisa. these results are the mean of 5 independent studies. all the mice used in these experiments were genotyped littermates and grouped and analyzed by their genotype. gx þ / þ (open bars) and gx à / à (filled bars). results are expressed in pg/ml. a, p o0.05 gx þ / þ or gx à / à vs. base; b, p o0.05 gx þ / þ vs. gx à / à at any time point, anova followed by paired t-test, two tailed, assuming unequal variance. n z7 per group. implicated in bacterial phospholipid hydrolysis . furthermore, we generated gx à / à mice on the c3h/hen background (fig. 1c ) by backcrossing the c57bl/6j gx à / à mice for 10 generations which had functional giia-spla 2 . mice were maintained on standard animal feed and water ad libitum in the conventional environmental conditions and controlled temperature and humidity with a 12 h light and dark cycle. for infection studies, animals were housed in hepa-filtered cage racks adherent to absl2 þ conditions (toronto general hospital, animal resource centre, toronto, canada). all animal procedures were performed in a certified class ii biosafety cabinet (baker company, sanford, nc, usa). housing and experimental procedures were approved by the animal care committee of the university health network, and were in accordance with the guide for the care and use of laboratory animals research statutes, ontario (1980) . all infection experiments were conducted with h1n1pdm strain, a/mexico/4108/2009 (h1n1pdm), provided by the centers for disease control and prevention (atlanta, ga, usa). virus was propagated and titrated in embryonated eggs and titrated prior to animal challenge. viral stocks were stored in liquid nitrogen and thawed prior to use. mice were weighed and randomly assigned for sample collection, and were infected through intranasal instillation with 50 ml phosphate-buffered saline (mock infection) or 50 ml a/mexico/4108/2009 (h1n1pdm) at 1 â 10 5 or 1 â 10 4 50% egg infectious dose (eid) 50 . virus dosage were 1 â 10 4 eid 50 and â 10 5 eid 50 for host response profiling in c57bl/6j mice and 1 â 10 4 eid 50 for comparing disease severity between gx þ / þ and gx à / à mice. throughout infection experiments, animal survival, clinical signs, and weights were recorded daily. in accordance with animal care committee recommendation, mice were euthanized when recorded body weight fell below 80% of original body weight. at day 0, 3 and 6 pi, 3 gx þ / þ and 3 gx à / à mice were euthanized and lung homogenates collected for viral load determination by either madin-darby canin kidney (mdck) cell growth determination or real-time rt-pcr (rna analysis methods and table s1 ). for mdck determination lungs were homogenized (10% w/v) in high glucose (4.5 g/l) dulbecco's modified eagle medium (dmem), supplemented with 1% bovine serum albumin, 50 mg/ml gentamycin, 100 u/ml penicillin, 100 mg/ml streptomycin, and 1 mg/ml tpck-trypsin (vdmem). homogenates were then serially diluted (0.5 log 10 ) in quadruplicate over madin-darby canine kidney cells, cultured at 2.0 â 10 4 cells/well in 96-well plates. cells were incubated for 2 h at 37 1c and 5% co 2 . homogenates were then removed and replaced with fresh vdmem. cells infected were incubated for 6 days at 37 1c and 5% fig. 6 . effect of gx-spla 2 deficiency in the mrna expression levels of cytokines, chemokines and their receptors and immunoglobulin chains in the lung tissue of mice during influenza infection. gx þ / þ and gx à / à mice (c3h/hen background mice) were infected with influenza a/mexico/4108/2009 and the gene expression profiles were analyzed in the lung tissue at day 0, 3, and 6 days after infection by microarray analysis (n¼ 4 per group). evolution of gene enrichment (fisher's exact test) of the kegg category "cytokine-cytokine receptor interaction" (a). differences in the expression levels of cytokines (b) and chemokines (c) and their receptors at 3 days pi. the heatmaps show the genes that are significantly upregulated with respect to the control group and the blue boxes indicate that the expression levels of those genes are significantly higher in the gx à / à than in the wild type mice at the same time-point. evolution in the expression levels of immunoglobulin genes: total number of regulated genes (d) and overview of different experimental groups and time-points (e). all the mice used in these experiments were genotyped littermates and grouped and analyzed by their genotype. fig. 7 . intersect analysis of the genes up-regulated in the lung tissue of gx à / à and gx þ / þ mice during influenza infection and functional classification of the resulting gene subsets. venn diagrams are representative of the total number of genes that are significantly up-regulated with respected to the uninfected mice. david annotation tool was used to classify the genes of each subset, and the fold enrichment is shown for each category. all the mice used in these experiments were genotyped littermates and grouped and analyzed by their genotype. * the "immunoglobulin chains" category was manually curated and contains 84 genes. nn the "interferon responses category". fig. 8 . gx-spla 2 deficiency increases t cell recruitment and immunoglobulin heavy chain mrna expression in the lung tissue of mice during influenza infection. day 0 and 3 pi with h1n1pdm (a/mexico/4108/2009) influenza the lungs from gx þ / þ and gx à / à mice (c3h/hen background mice) were perfusion fixed in situ with 4% paraformaldehyde, sectioned and subject to immunofluorescence analysis with specific rabbit primary antibody against mouse cd3 antigen (marker for t-cell) (ai). representative images (at â 400 with 3.4 zoom factor) from five independent experiments are shown. assessment of cd3 positive cells per high power field for day 0, 3, 6 and 14 days following infection with h1n1pdm influenza is shown (aii). igg (b) and cd8a (c) mrna expression normalized to gapdh were determined by quantitative real-time pcr in lung tissue of gx þ / þ and gx à / à mice after h1n1pdm influenza infection. scale bar: 10 μm. a, po 0.05 gx þ / þ or gx à / à vs. base; b, p o 0.05 gx þ / þ vs. gx à / à at any time point, anova followed by paired t-test, two tailed, assuming unequal variance. nz 8 per group. all the mice used in these experiments were genotyped littermates and grouped and analyzed by their genotype. co 2 , after which cell culture supernatants were tested for the presence of virus by hemagglutination assay using 0.5% (v/v) turkey red blood cells (lampire biological laboratories, pipersville, pa, usa). viral loads were determined as the reciprocal of the dilution at which 50% of wells were positive for viral infection. viral loads were reported as tcid 50 per gram of lung tissue. limit of detection of 10 1 tcid 50 /g. lung tissues from both gx à / à and gx þ / þ mice were collected at 3 and 6 days post infection (pi) and from uninfected controls (four mice per group). rna was purified from lung tissue using tripure (roche, indianapolis, in, usa). purified rna was then reverse transcribed using improm-ii reverse transcription system (promega, madison, wi, usa). real time rt-pcr was performed using the abi-prism 7900ht sequence detection systems (applied biosystems, foster city, ca, usa). data was collected with applied biosystems sequence detection systems version 2.3 software. each reaction well contained 4 μl of 0.625 ng/μl cdna, 0.5 μl each of forward and reverse primers (final concentration of 200 nm), and 5 μl sybr green master mix, for a total reaction volume of 10 μl and run in quadruplicate. host gene expression was normalized to the glyceraldehyde-3-phosphate dehydrogenase (gapdh) housekeeping gene, and quantified by relative standard curve method. viral load was quantified by the absolute standard curve method, normalized to gapdh housekeeping. primer sequences are listed in table s1 . histology, immunohistochemistry, immunofluorescence and immunoblotting gx þ / þ and gx à / à mice were euthanized at baseline, day 3, day 6 and day 14 pi (n 45 mice) and the mouse whole body was vascular perfused by cardiac puncture in situ with a fixative solution of 10% buffered formalin by a continuous release pump under pressure and volume-controlled conditions. fixed lung tissues were paraffin wax embedded for histology and immunohistochemistry. for h&e, tissue slides were then stained with hematoxylin-eosin for histopathology assessment. rabbit antimurine gx-spla 2 (degousee et al., 2008) was used to assess the tissue distribution of the gx-spla 2 protein. sections were counterstained with hematoxylin and eosin and observed under light microscope (accu-scope s , commack, ny, usa). images were captured using a digital camera and se premium software (micrometrics tm , londonderry, nh, usa) . for mac-3 tissue expression, rat anti-mouse mac-3 was used (bd biosciences, mississauga, on). for cd3 immunofluorescence, following heat-induced antigen retrieval, lung tissue sections were blocked with donkey serum and stained with primary antibodies rabbit anti-cd3 (dako, burlington, on). donkey anti-rabbit cy3 was used as secondary antibodies (millipore, billerica, ma) and dapi (sigma) for nuclear counterstain. images were recorded with an olympus fluo view 1000 confocal laser scanning microscope (olympus, tokoyo, japan). immunoblots for gx-spla 2 were carried out as described by our group (degousee et al., 2008) . for immunoblotting detection antibodies against mpo (upstate, lake placid), cd45 (bd biosciences, mississauga, on), and gapdh (santa cruz biotechnology, dallas, tx). lungs were lavaged at 0 and 6 days post h1n1 infection with 5 ml of normal saline. the bal fluid was centrifuged at 250g for 10 min and the supernatant was used for estimation of pgd 2 , pge 2 , ltb 4 , cysteinyl leukotriens and lipoxin a 4 content. analysis of pgd 2 in bal fluid 0.5 ml of bal fluid was mixed with 0.5 ml of ice-cold acetone, incubated on ice for 5 min and centrifuged for 10 min at 3000g at 4 1c. after supernatant aspiration, the pellet was extracted with 1 ml of ice-cold acetone and centrifuged again. the acetone extracts were combined and the acetone evaporated under nitrogen. all the samples were then methoximated (pgd 2 -mox eia kit (cayman chemical), and purified on oasis hlb columns (waters corporation) equilibrated with methanol/0.2% formic acid. methanol eluants were evaporated in a savant speed vac concentrator and samples dissolved in cayman eia buffer before eia analysis, according to the manufacturer's instructions. analysis of pge 2 , ltb 4 and cysteinyl leukotriens in bal fluid 1.2 ml of bal fluid was mixed with 2.4 ml of methanol containing 0.2% formic acid, incubated on ice for 5 min and centrifuged at 3000g for 10 min at 4 1c. after adjustment of methanol to 15%, supernatants were loaded on oasis hlb column equilibrated with methanol/0.2% formic acid (waters corporation). columns were processed in a vacuum manifold (waters corporation). after wash with water/0.03% formic acid, the samples were eluted with methanol/0.2% formic acid, methanol eluants were evaporated in a savant speed vac concentrator and samples dissolved in cayman eia buffer before eia analysis for pge 2 , ltb 4 and cysteinyl leukotrienes (eia kits, cayman chemical) according to the manufacturer's instructions. 0.6 ml of bal fluid was extracted with 1.2 ml of ice-cold methanol, incubated on ice for 5 min and centrifuged at 3000g for 10 min at 4 1c. the supernatants were diluted with water to achieve 11% methanol concentration and adjusted to ph 3.5 with 1n hcl. samples were purified on c18 sep-pak columns (waters corporation) preconditioned with methanol. after column wash with water followed by hexane, samples were eluted with methyl formate. the eluants were evaporated under nitrogen and the samples reconstituted in eia buffer and assayed for lipoxin a 4 content (neogen corporation) according to the manufacturer's protocol. lung tissues from both gx à / à and gx þ / þ mice were collected at 3 and 6 days pi and from uninfected controls (four mice per group) as with the real time rt-pcr. rna was purified from lung tissue using tripure (roche, indianapolis, in, usa) and amplified with illumina totalprep rna amplification kit (ambion, austin, tx, usa). 1.5 mg of crna was labeled and hybridized to mouse wg-6 v2.0 expression beadchip (illumina, san diego, ca, usa) and scanned on illumina beadstation 500gx. raw data was processed with illumina genomestudio v2010.3 software. the data sets were subjected to quantile normalization, variance stabilization and log 2 transformation. genes were considered significantly regulated if the expression levels with respect to the uninfected controls were z1.5fold different and the student t-test's p value was o0.05. david bioinformatics resource v6.7 (http://david.abcc.ncifcrf.gov/home.jsp) (hicks et al., 2007) was used to perform functional classification of differentially expressed genes. additionally, interferon regulated genes were selected by using the interferome (v2) database (http://interfer ome.its.monash.edu.au/interferome/home.jspx) (rubin et al., 2005) . immunoglobulin chains and prostaglandin-related gene categories were defined by searching for relevant keywords in the annotated microarray datasets. multiexperiment viewer v4.7.2 (http://www.tm4. org/mev/) was used to perform complete hierarchical clustering and generate heatmap representations of selected genes. data are presented as mean 7sem. analyses of data recorded at one time point were performed by 2-tailed, unpaired, student t-tests. analyses of data recorded at several time points for two groups (gx þ / þ and gx à / à mice) were performed by 2-way anova (to evaluate the effect of group, time and group-time interactions); if significant, a bonferroni correction for multiple comparisons was applied for post-hoc analysis between different time points or between different groups at the same time point. survival after h1n1pdm influenza infection was assessed by a logrank test. a value of p o0.05 was accepted as statistically significant. the authors had full access to and take full responsibility for the integrity of the data. all authors have read and agree to the manuscript as written. prostaglandin d(2) and t(h)2 inflammation in the pathogenesis of bronchial asthma influenza -time to target the host? th1 and th17 hypercytokinemia as early host response signature in severe pandemic influenza host adaptive immunity deficiency in severe pandemic influenza corticosteroid suppression of lipoxin a4 and leukotriene b4 from alveolar macrophages in severe asthma 12 gene expression analysis of host innate immune responses during lethal h5n1 infection in ferrets interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome lack of innate interferon responses during sars coronavirus infection in a vaccination and reinfection ferret model2 leukotriene b42 macrophage secretory phospholipase a 2 group x enhances anti-inflammatory responses, promotes lipid accumulation, and contributes to aberrant lung pathology ex vivo effect of varespladib on secretory phospholipase a 2 alveolar activity in infants with ards p38 mapk regulates group iia phospholipase a 2 expression in interleukin-1b stimulated rat neonatal cardiomyocytes groups iv, v, and x phospholipases a 2 s in human neutrophils: role in eicosanoid production and gram-negative bacterial phospholipid hydrolysis map kinase kinase 6-p38 map kinase signaling cascade regulates cyclooxygenase-2 expression in cardiac myocytes in vitro and in vivo c-jun n-terminal kinase-mediated stabilization of microsomal prostaglandin e 2 synthase-1 mrna regulates delayed microsomal prostaglandin e 2 synthase-1 expression and prostaglandin e 2 biosynthesis by cardiomyocytes microsomal prostaglandin e2 synthase-1 deletion leads to adverse left ventricular remodeling after myocardial infarction regulation of dendritic cell migration and adaptive immune response by leukotriene b4 receptors: a role for ltb4 in up-regulation of ccr7 expression and function1 diversity of group types, regulation, and function of phospholipase a 2 fatal outcome of human influenza a (h5n1) is associated with high viral load and hypercytokinemia group x phospholipase a 2 is released during sperm acrosome reaction and controls fertility outcome in mice seasonal h1n1 infection induces cross protective pandemic h1n1 immunity through a cd8 independent, b cell dependent mechanism synergy between extracellular group iia phospholipase a 2 and phagocyte nadph oxidase in digestion of phospholipids of staphylococcus aureus ingested by human neutrophils altered lung phospholipid metabolism in mice with targeted deletion of lysosomal-type phospholipase a 2 human infection with a novel avian-origin influenza a (h7n9) virus leukotriene b4-mediated release of antimicrobial peptides against cytomegalovirus is blt1 dependent leukotriene b4 induces release of antimicrobial peptides in lungs of virally infected mice low molecular weight phospholipases a 2 in mammalian brain and neural cells: roles in functions and dysfunctions4 cxcr3 ligands: redundant, collaborative and antagonistic functions4 h7n9 incident, immune status, the elderly and a warning of an influenza pandemic the arachidonate-activable, nadph oxidase-associated h þ channel. evidence that gp91-phox functions as an essential part of the channel importance of group x-secreted phospholipase a 2 in allergen-induced airway inflammation and remodeling in a mouse asthma model blockade of human group x secreted phospholipase a 2 (gx-spla 2 )-induced airway inflammation and hyperresponsiveness in a mouse asthma model by a selective gx-spla 2 inhibitor leukotriene b4 receptor antagonists as therapeutics for inflammatory disease: preclinical and clinical developments comparative analyses of pandemic h1n1 and seasonal h1n1, h3n2, and influenza b infections depict distinct clinical pictures in ferrets differential pathological and immune responses in newly weaned ferrets are associated with mild clinical outcome of pandemic 2009 h1n1 infection1 systematic and integrative analysis of large gene lists using david bioinformatics resources atherogenic properties of ldl particles modified by human group x secreted phospholipase a 2 on human endothelial cell function a natural disruption of the secretory group ii phospholipase a 2 gene in inbred mouse strains lysis of human immunodeficiency virus type 1 by a specific secreted human phospholipase a 2 diverse functional coupling of prostanoid biosynthetic enzymes in various cell types sequencing, annotation and characterization of the influenza ferret infectome1 disruption of jnk2 decreases the cytokine response to plasmodium falciparum glycosylphosphatidylinositol in vitro and confers protection in a cerebral malaria model involvement of cytosolic phospholipase a 2 and secretory phospholipase a 2 in arachidonic acid release from human neutrophils expression of secretory phospholipase a 2 enzymes in lungs of humans with pneumonia and their potential prostaglandin-synthetic function in human lungderived cells prostaglandin d2 as a mediator of allergic asthma5 acetylsalicylic acid (asa) blocks influenza virus propagation via its nf-kappab-inhibiting activity group v and x secretory phospholipase a 2 prevents adenoviral infection in mammalian cells the lipid mediator protectin d1 inhibits influenza virus replication and improves severe influenza different functional aspects of the group ii subfamily (types iia and v) and type x secretory phospholipase a(2)s in regulating arachidonic acid release and prostaglandin generation. implications of cyclooxygenase-2 induction and phospholipid scramblase-mediated cellular membrane perturbation57 recent progress in phospholipase a(2) research: from cells to animals to humans pge(2) transiently enhances dc expression of ccr7 but inhibits the ability of dcs to produce ccl19 and attract naive t cells11 lymphotoxin alpha(1) beta (2) expression on b cells is required for follicular dendritic cell activation during the germinal center response1 prostaglandin e2 enhances th17 responses via modulation of il-17 and ifn-gamma production by memory cd4þ t cells3 transgenic expression of group v, but not group x, secreted phospholipase a 2 in mice leads to neonatal lethality because of lung dysfunction interleukin-6 is a potential biomarker for severe pandemic h1n1 influenza a infection2 pandemic h1n1 influenza a directly induces a robust and acute inflammatory gene signature in primary human bronchial epithelial cells downstream of membrane fusion in vivo ribavirin activity against severe pandemic h1n1 influenza a/mexico/4108/2009 modeling host responses in ferrets during a/california/07/2009 influenza infection cytosolic phospholipase a 2 -a is necessary for platelet-activating factor biosynthesis, efficient neutrophil-mediated bacterial killing, and the innate immune response to pulmonary infection: cpla 2 -a does not regulate neutrophil nadph oxidase activity interferome v2.0: an updated database of annotated interferon-regulated genes1 lipid-cytokine-chemokine cascades orchestrate leukocyte recruitment in inflammation2 cxcl13/cxcr5 signaling enhances bcr-triggered b-cell activation by shaping cell dynamics1 mast cell stabilization improves cardiac contractile function following hemorrhagic shock and resuscitation influenza vaccines: the good, the bad, and the eggs group x secretory phospholipase a 2 enhances tlr4 signaling in macrophages1 increased concentrations of leukotrienes in bronchoalveolar lavage fluid of patients with ards or at risk for ards prostaglandin i(2) analogues enhance already exuberant th17 cell responses in systemic sclerosis3 update: influenza activity -united states, 2009-10 season inflammation-restraining effects of prostaglandin e2 on natural killer-dendritic cell (nk-dc) interaction are imprinted during dc maturation1 ltb4 increases nasal neutrophil activity and conditions neutrophils to exert antiviral effects2 global influenza programme. (ref type: online source) innate immunity and pulmonary host defense age-related increases in pgd (2) expression impair respiratory dc migration, resulting in diminished t cell responses upon respiratory virus infection in mice1 supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2014.01.030. key: cord-329819-dpgexphf authors: hu, weiwei; zhang, shuai; shen, yumeng; yang, qian title: epidermal growth factor receptor is a co-factor for transmissible gastroenteritis virus entry date: 2018-06-04 journal: virology doi: 10.1016/j.virol.2018.05.009 sha: doc_id: 329819 cord_uid: dpgexphf transmissible gastroenteritis virus (tgev) causes severe diarrhea and high mortality in newborn piglets. it is well established that porcine intestinal epithelium is the target of the tgev infection, however the mechanism that tgev invades the host epithelium remains largely unknown. aminopeptidase n (apn) is a known receptor of tgev. this study discovered that the extracellular receptor binding domain 1 pertaining to epidermal growth receptor (egfr) interact with tgev spike protein. apn and egfr synergistically promote tgev invasion. tgev promotes apn and egfr clustering early in infection. furthermore apn and egfr synergistically stimulate pi3k/akt as well as mek/erk1/2 endocytosis signaling pathways. tgev entry is via clathrin and caveolin mediated endocytosis in ipec-j2 cells. tgev binds with egfr, and subsequently promotes egfr internalization by a clathrin-mediated endocytosis pathway. these results show that egfr is a co-factor of tgev, and that it plays a synergistic role with apn early in tgev infection. procine transmissible gastroenteritis virus (tgev) is a member of the enteropathogenic alpha-coronavirus (αcov) family. tgev infects intestinal epithelial cells resulting in severe and frequently fatal diarrhea in newborn pigs, with mortality rates reaching 100% (doyle and hutchings, 1946) . tgev is an enveloped cov, with a large positivesense single-stranded rna genome, about 28.5 kb in length. it has a diameter ranging from 80 to 120 nm, including surface projections. porcine intestinal columnar epithelial cells (ipec-j2) offer a practical model for studying porcine enteric pathogens (brosnahan and brown, 2012) . we will use this model to study the entrance mechanism of tgev. aminopeptidase n (apn), also known as cd13, is a typeⅱtransmembrane glycoprotein, about 150 kda, belonging to a membrane-bound metalloprotease family (delmas et al., 1994) . most alpha coronavirus use apn as cellular receptors for virus entry, such as human coronavirus 229e (hcov-229e), feline infectious peritonitis virus (fipv), canine coronavirus (ccov), porcine epidemic diarrhea virus (pedv), and transmissible gastroenteritis virus (tgev) (delmas et al., 1992 (delmas et al., , 1993 kolb et al., 1996; li et al., 2007; tresnan et al., 1996) . the high degree of tropism of tgev for the villous enterocytes of newborn pigs is well established and has been suggested as being a factor in age sensitivity of newborn pigs to the virus (schwegmann-wessels and herrler, 2006) . there has been some confusion around the question that if apn is the only receptor for tgev entry, why older piglets are not susceptible to tgev, especially since apn was found to be highly expressed in villous enterocytes of both newborn and older piglets. research suggest a known protein, approximately 200-kda in size, only expressed in the upper villi of newborn piglets, it has high affinity for tgev (weingartl and derbyshire, 1994) . it has also been demonstrated that apn is not essential for pedv cell entry (ji et al., 2018; li et al., 2017) . it is most likely that tgev do have more than one receptor. many cell surface components have been identified as virus receptors, including: chemokine receptors (feng et al., 1996) , fibroblast growth factor receptors (qing et al., 1999) , the tumor necrosis factor receptor family (terry-allison et al., 1998) , and integrin (wang et al., 2005) . pidermal growth factor receptor (egfr), a member of the receptor tyrosine kinases (rtk) family, is widely expressed on many cells including epithelial and mesenchymal cells (wells, 1999b) . it has been demonstrated that many viruses interact with egfr to facilitate viral entrance, including: influenza a virus (iav), hepatitis c virus (hcv), herpes simplex virus 1 (hsv), and human cytomegalovirus (hcmv) (chan et al., 2009; eierhoff et al., 2010a; lupberger et al., 2011; zheng et al., 2014b) . ligand binding to egfr induce receptor dimerization and cross phosphorylation, which in turn actives the intracellular signaling cascades critical for cellular protein synthesis, cytoskeleton reorganisation, apoptotic inhibition, transcriptional activation, and cell motility. the ligand binding to egfr results in rearrangement of the cytoskeleton network through egfr-mediated signaling, and subsequently ligand-egfr complexes are internalize through clathrin-coated pits (zheng et al., 2014a) . many researchers have shown that numerous viruses utilize egfr endocytosis to mediate virus internalization (mercer et al., 2010a) . the interactions between viruses and their receptors are specific, but the affinity is low. many multiple receptor binding sites exist on virus particles which are likely to cluster receptor proteins. it is known that multiple viruses use more than one type receptor to aid uptake into host cells (marsh and helenius, 2006) . egfr also has been identified as a co-receptor for many viruses, such as human cytomegalovirus (hcmv), hepatitis c virus (hcv), and adenoassociated virus serotype 6 (aav6) (lupberger et al., 2011; wang et al., 2003; weller et al., 2010) . previous studies that we have conducted demonstrated that tgev spike protein interacts with egfr, and stimulates phosphorylation of cofilin as well as stimulating polymerization of f-actin through the pi3k-rac1/cdc42-pak-limk signaling pathway. this is required for efficient tgev entry (hu et al., 2016) . we further demonstrate whether egfr is another co-factor for tgev. egfr is a transmembrane protein with two dimer forms, it can be divided into extracellular, transmembranal, and intracellular regions. its extracellular region contains two receptor-binding domains receptor 1 (57-168 aa) and receptor 2 (361-481 aa). its intracellular protein associated with tyrosine kinase pi3k/akt and ras/raf/erk1/2 pathways are activated by phosphorylated tyrosine located in egfr cytoplasmic tails (fig. 1a) . the objective of our present study was also to study the relationship between apn and egfr in the early stage of tgev infection. ipec-j2 cells are porcine intestinal columnar epithelial cells that are isolated from the middle jejunum of neonatal piglets. ipec-j2 cells were purchased from dsmz (germany). hek293t cells and swine testis (st) cells were purchased from atcc (united states). ipec-j2, st, and hek293t cells were cultured in dulbecco's modified eagle's medium (dmem) with high glucose, hepes containing with 10% fetal bovine serum (fbs, gibco), 1% penicillin-streptomycin (invitrogen) at 37°c in a 5% co 2 incubator (thermofisher scientific). transmissible gastroenteritis virus (strain shxb) was isolated in shanghai, china. the complete genome sequence for tgev shxb is available in genbank (kp202848.1) (weiwei et al., 2014) . to analyze viral entry, cells were incubated with tgev at a multiplicity of infection of 2 (moi = 2) for 1 h at 4°c. subsequently, the cells were washed with phosphate-buffered saline (pbs), and maintained in a maintenance medium (dmem supplemented with 2% fbs and 1% penicillin-streptomycin) for 1 h at 37°c in a 5% co 2 incubator. for viral labeling, viruses were filtered with 0.22 µm filter, and then clarified by centrifugation at 10,000g for 2.5 h, followed by ultra-centrifugation using 20%, 40%, and 60% sucrose gradient at 10,000g for 2.5 h. viruses were labeled with the fluorescent probe dylight 488, 633, and 594 nhs ester (thermofisher scientific, waltham, usa), according to the manufacturer's instruction. unincorporated dye was removed by using commercial fluorescent dye removal columns (thermofisher scientific). flow cytometry analysis for the entry of tgev was performed as follows: fluorescent probe labeled "tgev particles" were incubated with ipec-j2 cells for 1 h at 4°c. subsequently, the cells were washed with pbs and maintained in dmem for 1 h at 37°c in a 5% co 2 incubator, the cells were harvested by 0.25% trypsin, and then washed with pbs three times. cells acquisition was performed by facs (becton dickinson), and the date was analyzed using flowjo software. antibodies used in the present study were obtained from commercial sources. these antibodies included: rabbit anti-human egfr, rabbit anti-human phospho-egfr (tyr1068), rabbit anti-human akt, rabbit anti-human phospho-akt, rabbit anti-human phospho-erk1/2, and rabbit anti-human erk1/2 (cell signaling technology, danvers, usa). mouse anti-procine apn antibody was donated by prof. zhu guoqiang in yangzhou university. mouse monoclonal antibodies to ha and gfp (cmctag, milwaukee, usa). goat anti-rabbit igg (h + l) secondary antibody, dylight 594 conjugate, goat anti-mouse igg (h + l) secondary antibody, dylight 488 conjugate (thermofisher scientific). anti-gapdh monoclonal antibody, hrp-conjugated goat anti-rabbit igg (h + l), and hrp-conjugated goat anti-mouse igg (h + l) (vazyme, nanjing, china). ipec-j2 cells were washed with pbs and lysed in an ice-cold cell lysis buffer, phosphatase inhibitor and protease inhibitor (thermofisher scientific) were added in the cell lysis buffer according to the manufacturer's instructions. the supernatant of lysates were obtained by centrifugation at 12,000g for 10 min at 4°c, and subsequently equal protein levels of the prepared lysates were fractionated by sds-page (10-12% gradient). the separated proteins were transferred to pvdf (merck millipore), and the membranes were blocked for 2 h in tris-buffered saline (tbs), containing 5% nonfat dry milk. after which they were reacted with indicated primary antibodies at 4°c overnight. membranes were exposed to species-specific horseradish peroxidase (hrp)-conjugated secondary antibodies, (dilution 1:5000) followed by enhanced chemiluminescence (ecl, thermofisher scientific) detection by use of autoradiography. western blotting was quantified by quantity one (quantity one 1-d analysis software 170-9600, bio-rad). the intensity of the bands in terms of density was measured and normalized against gapdh expression. all data were expressed as means ± sd of three independent experiments. the plvx-dsred-monomer-n1 is an hiv-1-based, lentiviral expression vector that expresses the gene of interest fused to the dsred-monomer (clontech, palo aito, ca). egfr and apn sequences were inserted into the ecori/bamhi site. egfr receptor1 and egfr re-ceptor2 sequences were cloned into a pacgfp1-c vector (sali/bamhi) (clontech), and tgev spike1 sequence was cloned into pcmv-c-ha (bamhi/xbai) (d2639, beyotime, china). table 2 showed the primers used for cloning. all constructs were verified by dna sequencing. hek 293 t cells were optimized for lentivirus production, we transfected apn or egfr lentiviral overexpression vector and lenti-x htx packaging mix (vsv-g, plp1, plp2) into hek 293t cells using the x-treme-gene hp dna transfection reagent (roche, switzerland), according to the manufacturer's instructions. ipec-j2 cells were treated with apn, egfr overexpressing lentiviral particles (moi = 1), and after 24 h of incubation, infected cells were maintained with fresh dmem, and continued for extra 12-24 h to allow the overexpressing lentiviral particles to achieve their maximum effect. his-egfr receptor1 and his-egfr receptor2 were cloned into and expressed in escherichia coli bl-21, then purified using ni-nta resin. the purified proteins were eluted with elution buffer containing 8 m urea, which was removed using a dialysis bag against buffer (50 mm nah 2 po 4 , 300 mm nacl) with gradual reduction in the concentration of urea (6 m, 3 m, buffer alone). the concentrations of the purified proteins were measured with the bca assay. the two purified proteins were verified by sds-page and western blot, respectively. plvx-shrna2 is an hiv-1-based, lentiviral expression vector designed to express a small hairpin rna (shrna) used for rna interference (rnai) studies (clontech) ( table 1 ). the best silencing efficiency was observed with clone kf280271 (porcine apn), nm_214007 (porcine egfr), nm_001146127.1 (procine clathrin), nm_214438 (procine caveolin). these shrna oligonucleotides were inserted into the bamhi/ecori site. all constructs were verified by dna sequencing. his-tagged egfr extracellular receptor binding domain 1 or 2 expressed in e.coli bl21 and purified in ni-nta columns, the purified products were separated using sds-page and stained with coomassie brilliant blue. (c) the purified egfr extracellular receptor-binding domain 1 or 2 were verified by western-blot. (d) tgev (moi = 2) was incubated in dmem containing his-32a, his-egfr receptor 1 or his-egfr receptor 2 at 37°c for 2 h, then incubated with ipec-j2 cells and cultured for 1 h. the invasion of tgev was detected by rt-pcr. (e) tgev (moi = 2) was incubated in dmem containing his-32a, his-egfr receptor 1 and his-egfr receptor 2 at 37°c for 2 h, then incubated with ipec-j2 cells, and cultured for 1 h, the viral titers of intracellular tgev were analyzed by tissue culture infectivity dose 50 tcid 50 . (f) ipec-j2 cells were pretreated with his-egfr receptor 1 at different concentrations at 37°c for 2 h, then incubated with ipec-j2 cells, and cultured for 1 h. the invasion of tgev was detected by rt-pcr. (g) intracellular tgev were analyzed by viral plaque morphology in st cells. (h) the lysates of tgev-infected ipec-j2 cells were immunoprecipitated with rabbit anti-egfr or normal rabbit igg. immunoblotting was then performed to determine the presence of egfr and tgev in the egfr immunoprecipitate. (i and j) 293t cells were co-transfected with a ha-tagged tgev s1 expression plasmid together with gfp-tagged egfr receptor 1 or gfp-tagged egfr receptor 2 expression plasmid, cell lysates were immunoprecipitated with an anti-ha antibody or an anti-gfp antibody, the resulting precipitates were examined by immunoblotting using an anti-ha or an anti-gfp antibody to examine the interaction between ha-tgev s1 and gfp-tagged egfr. (** p < 0.01). ipec-j2 cells were treated with apn, egfr interference lentiviral particles (moi = 1), after 24 h of incubation, infected cells were maintained with fresh dmem and continued for 12-24 h to allow the interference lentiviral particles to achieve their maximum effect. in regards to detecting the interaction between tgev s1 and egfr, cells were lysed in a lysis buffer after infection of tgev (moi = 100). cell lysates were cleared by centrifugation at 12,000g for 10 min, and the supernatant was immune-precipitated with rabbit anti-human egfr monoclonal antibody (4267s, cell signaling technology, danvers, usa) or normal rabbit igg (a7016, beyotime, china) at 4°c for 8 h, and fresh protein a/g magnetic beads (b23201, bimake, usa) were added and incubated with cells at 4°c for another 3 h. magnetic beads containing protein complexes were washed five times with pbs and incubated with concentrated tgev at 4°c for 5 h. magnetic beads containing egfr proteins and tgev complexes were washed five times with pbs. complexes were subsequently boiled for 10 min in 1 × protein loading buffer (takara, janpan), then analyzed by western blotting with specific primary and secondary antibodies. the primary antibodies used to probe membranes were rabbit anti-egfr mab and rabbit anti-tgev n pab. table 2 primers used for cloning. primer sequence (5′-3′) vector tgev infection causes the co-localization of apn and egfr. ipec-j2 cells were infected with tgev (moi = 2) and cultured for 30 min, then stained for fluorescence microscopy using mouse anti-apn pab, followed by dylight 488-conjugated goat anti-mouse igg and rabbit anti-p-egfr mab, followed by dylight 594-conjugated goat anti-rabbit igg. mock-infected cells served as controls. the data shown are from two independent experiments. for detecting the binding domain of tgev s1, hek293t cells lysates were prepared by ice-cold np-40 lysis buffer (p0013f, beyotime, china), and protease inhibitor (thermofisher scientific) was thereafter added according to the manufacturer's instructions. cell lysates were cleared by centrifugation at 12,000g for 10 min, and supernatant was incubated with protein a/g magnetic beads (b23201, bimake, usa) and normal igg (from the same species as that of the immunoprecipitating antibody) at 4°c for 1 h, to eliminate nonspecific binding to the magnetic beads or igg. after centrifugation at 1000g for 5 min, the supernatant was incubated with ha (at0024, cmctag) or gfp (at0028, cmctag) antibody at 4°c for 8 h. fresh protein g plus-magnetic beads were added and incubated with cells at 4°c for another 3 h. magnetic beads, containing protein complexes, were washed five times with pbs, and complexes were boiled for 10 min in 1 × protein loading buffer (takara, janpan). at this point they were analyzed by western blotting with specific primary and secondary antibodies. tgev-infected and mock-infected cells were seeded onto 6-well plates (5 × 10 5 ) and cultured for 24 h. tgev was collected by freezing and thawing the plates three multiple times, and were determined by the tissue culture infectious dose 50 (tcid 50 ) in st cells. tgev plaque assays were performed on monolayers of st cells seeded in 12-well plates. cells were inoculated with ten-fold dilutions of stock virus, and incubated for 1 h at 37°c. these cells were overlaid with dmem containing 2% fbs and 1% low melting point agarose (sigma-aldrich). after which they were incubated for about 72 h until plaques were visible. plaques were stained with 1% crystal violet in methanol. ipec-j2 cells were grown on coverslips in 24-well tissue culture plates, after tgev infection, at room temperature, cells were fixed in 4% paraformaldehyde in pbs for 15 min. they were washed three times with pbs, and incubated with 0.1% triton x-100 in pbs for 5 min, washed three times with pbs. at this point, cells were blocked in 5% bovine serum albumin (bsa) for 20 min. for studies analyzing the colocalization of apn and p-egfr, cells were incubated with apn and p-egfr primary antibodies (1:1000) at 4°c, overnight. for studies analyzing the internalization of egfr, cells were incubated with egfr primary antibody (1:1000) at 4°c, overnight, and were followed by three washes with pbs, then subsequently incubated with fluorochrome-conjugated secondary antibodies (1:500) at room temperature for 1 h. cells were washed three times with pbs and the nucleus were stained using 1 μg/ml dapi (4′,6′diamidino-2-phenylindole dihydrochloride)-pbs for 5 min at room temperature. images were captured with a zeiss fluorescence microscopy system and a 40× objective lens. the co-localization of the two channels was detected using zen 2012 (zeiss) software. cells were incubated with tgev at moi = 2 for 1 h at 4°c and washed with pbs (ph7.2 at 4°c) three times to remove unbound virus, then maintained in maintenance medium (dmem supplemented with 2% fbs and 1% penicillinstreptomycin) at 37°c in a 5% co2 incubator, after the indicated time (5 min, 15 min, 30 min, 60 min), cells were washed with acidic pbs (ph 3.0 at 4°c) to remove the virus bound to the cell membrane (not enter the cell), then cell membrance protein was performed according to the manufactures'instructions. ligand egf was used as a positive control, egf (100 ng/ml) was added to the cell culture medium for 1 h at 4°c, and washed with pbs (ph7.2 at 4°c) three times to remove unbound egf, then maintained in maintenance medium (dmem supplemented with 2% fbs and 1% fig. 4 . apn and egfr synergistically activate pi3k/akt and mek/erk1/2 signaling pathways. (a) ipec-j2 cells were transfected with overexpression vector plvx-dsred, plvx-apn-dsred, plvx-egfr-dsred, or plvx-apn-dsred + plvx-egfr-dsred through lentiviral supernatant. cells were infected with tgev at an moi of 2 and cultured for 30 min. normal cells and tgev infected-normal cells served as controls. the activation of downstream signaling pathways analyzed by westernblot with anti-p-egfr, anti-egfr, anti-p-akt, anti-akt, anti-p-erk1/2, anti-erk1/2, and anti-gapdh antibodies. (b) ipec-j2 cells were transfected with interference vector plvx-shrna-apn, plvx-shrna-apnctrl, plvx-shrna-egfr, plvx-shrna-egfrctrl, or plvx-shrna-apn + plvx-shrna-egfr through lentiviral supernatant. cells were infected with tgev at an moi of 2, and cultured for 30 min normal cells and tgev infected-normal cells served as controls. the activation of downstream signaling pathways analyzed by western-blot with anti-p-egfr, anti-egfr, anti-p-akt, anti-akt, anti-p-erk1/2, anti-erk1/2, and anti-gapdh antibodies. (c-h) the ratio of p-egfr, egfr, p-akt, akt, p-erk1/2 or erk1/2 to gapdh was normalized to control conditions. data shown are the mean results ± sd from three independent experiments. (* 0.01 < p < 0.05, ** p < 0.01). penicillinstreptomycin) at 37°c in a 5% co2 incubator, after the indicated time (5 min, 15 min, 30 min, 60 min), then cell membrance protein was performed according to the manufactures'instructions (mem-per™ plus membrane protein extraction kit, catalog number: 89842,thermo). human transferrin-fitc labele was obtained from nanocs (usa). cholera toxin beta subunit (ct-b)-fitc labele was purchased from sigma-aldrich (usa). ipec-j2 cells were prepared on coverslips in 24well tissue culture plates, and incubated with dylight 594-tgev with 50 μg/ml of fitc-transferrin or 10 μg/ml of fitc-ctxb for 30 min at 4°c to synchronize entry. at this point they were then shifted to 37°c for 30 min. cells were fixed in 4% paraformaldehyde for 15 min, and nucleus were stained using 1 μg/ml dapi for 5 min. images were captured with a zeiss lsm710 confocal laser-scanning microscopy system and a 40× objective lens. top view images were prepared as compacted z-stack images of non-permeabilised cells. x-y plane and z-axis views of confocal images were prepared using zen 2012 le software from zeiss, germany. all results were presented as means ± standard deviation (sd) from three independent experiments and performed using graphpad prism 5 software (sandiego, ca), p values for all data were determined using student's t-test and one-way analysis of variance (anova) (* 0.01 < p < 0.05, ** p < 0.01). purified escherichia coli-expressed fusion protein his-tagged receptor binding protein 1 and 2 were verified by sds-page and western blot, respectively ( fig. 1b and c) . tgev was incubated with with interference vector plvx-shrna-clathrin, plvx-shrna-clathrinctrl, plvx-shrna-caveolin, or plvx-shrna-caveolinctrl through lentiviral supernatant. normal cells served as controls. cells were infected with tgev at an moi of 2 and cultured for 1 h. the invasion of tgev was detected by rt-pcr. (e-g) ipec-j2 cells were transfected with interference vector plvx-shrna-clathrin, plvx-shrna-clathrinctrl, plvx-shrna-caveolin, or plvx-shrna-caveolinctrl through lentiviral supernatant. cells were infected with tgev at an moi of 2, and cultured for 1 h. the invasion of tgev was detected by flow cytometry (e and f). the viral titers of intracellular tgev were analyzed by tcid 50 (f). (h and i) the co-localization of transferrin and cholera toxin with tgev, (scale bar = 20 µm), the immunofluorescence experiment was repeated two times, every time there are three groups of parallel samples. the data shown are the mean results ± sd from three independent experiments. (* 0.01 < p < 0.05, ** p < 0.01). receptor 1 (200 ng/ml) and receptor 2 proteins (200 ng/ml) at 37°c incubator for two hours. receptor 1 protein was able to bind with tgev and inhibit tgev invasion ( fig. 1d and e). receptor 1 protein was able to inhibit tgev invasion in a dose dependent manner (fig. 1f ). receptor 1 protein inhibition tgev invasion was proven by plaque assays (fig. 1g) . to investigate the direct interaction between tgev s1 and egfr, co-immunoprecipitation was performed on lysates from tgevinfected cells precipitated with antibody against egfr. egfr and tgev n protein were detected in the precipitates of tgev-infected cells, indicating that tgev interacts simultaneously with egfr in infected cells (fig. 1h) . the interaction between tgev s1 and egfr receptor 1 / receptor 2 were validated in precipitates from 293t cells co-transfected with plasmids expressing tgev s1-ha and egfr receptor 1 / receptor 2-gfp ( fig. 1i and j) . this further confirmed that tgev s1 directly interacts with egfr receptor 1 but not egfr receptor 2. apn is a receptor of tgev, we wanted to determine whether egfr had any interaction with apn. hence, the localization of apn and egfr the protein of the cell membrane was extracted. cell membrane egfr was analyzed by westernblot using rabbit anti-egfr pab. (b) the ratio of egfr to the mean of e-cadherin and gapdh was normalized to control conditions. the data shown are the mean results ± sd from three independent experiments. was assessed by fluorescent microscopy. in mock-infected cells, it was noticed that apn and p-egfr distributed evenly on the surface of the cell membrane. in tgev-infected cells, egfr was activated. the level of p-egfr increased, apn and egfr were co-localization significantly (fig. 2) . to determine the role of egfr and apn in mediating tgev infection, lentivirus interference methods were used to reduce the expression of egfr and apn. ipec-j2 cells were transfected with lentivirus constructs that expressed apn or egfr targeting shrnas. apn or egfr expression level reduced significantly ( fig. 3a and b) . the results of real-time reverse transcription pcr (rt-pcr) for detection of tgev invasion revealed that apn-targeting shrna, egfr-targeting shrna, and apn + egfr-targeting shrnas, significantly inhibited tgev entry. the inhibition of apn + egfr-targeting shrnas was seen to be more significant (fig. 3e) . these results were further verified by flow cytometry, tgev particles were labeled with fluorescent probe dylight 488, apn-targeting shrna, and egfr-targeting shrna inhibited the invasion of tgev with the similar results. apn+egfr-targeting shrnas showed a more significant inhibitory effect ( fig. 3d and e) . tgev infection was measured in the supernatant of infected ipec-j2 cells by tcid50 (fig. 3f) . plaque formation in st cells by the intracellular of infected ipec-j2 cells showed that apn + egfr-targeting shrnas inhibited tgev entry more significantly (fig. 3g) . all of these results indicated to us that egfr and apn synergistically promote tgev invasion. the activation of phosphatidylinositol-3 kinase (pi3k) and the extracellular signal-regulated kinase 1/2 (erk1/2) has been identified in many viruses as part of their entry mechanism. egfr is an upstream mediator of pi3k/akt signaling pathway and erk1/2. to determine whether egfr can induce the endocytosis signaling pathway to support tgev entry, the activation of egfr, pi3k/akt and erk1/2 were investigated. cells were incubated with tgev at 4°c for 1 h, and unbound viruses were removed. then cells were incubated at 37°c for 30 min, and the phosphorylation level of egfr was increased in the early infection stage of tgev. in apn overexpressed, egfr overexpressed, and apn+egfr overexpressed cells, overexpression of egfr and apn were confirmed in figs. 4a and s1, tgev infection caused an a comparably increased phosphorylation level of egfr, akt, as well as erk1/2 than that of the tgev infection control group ( fig. 4a and c) . in apn-targeting shrna, egfr-targeting shrna, and apn+egfr-targeting shrnas cells, tgev infection resulted in the decreased phosphorylation level of egfr, akt, and erk1/2 significantly in comparison to the tgev infection control group (fig. 4f-h) . apn-targeting shrna inhibited tgev-induced egfr activation more significantly than egfr-targeting shrna (fig. 4b and f) . these data demonstrated that apn is required for tgev-induced egfr activation. hence, tgev binding to apn induces egfr activation and is required for viral entry. to explore whether the endocytosis pathway supports tgev entry into ipec-j2 cells, ipec-j2 cells were transfected with lentivirus that expressed clathrin or caveolin targeting shrnas. the clathrin or caveolin expression level was reduced significantly (fig. 5a and b) . rt-pcr for detection tgev invasion results showed that both clathrin and caveolin targeting shrnas significantly inhibited tgev entry (fig. 5c and d) . this data was reinforced by flow cytometry, tgev particles were labeled with fluorescent probe dylight 633, clathrin-targeting shrna, and caveolin-targeting shrna which inhibited the invasion of tgev ( fig. 5e and f) . the viral titers of intracellular tgev were also analyzed by tcid 50 (fig. 5g ). our previous studies have found that nystatin, an cholesterol removing agent which also functions by inhibiting the lipid/caveolin pathway can in fact inhibit tgev binding and entry. this result also show us that caveolin-mediated endocytosis is a method that can be utilized in tgev internalization. to verify and confirm our results, clathrin and caveolin mediated endocytosis specific markers were used to provide direct evidence in an attempt to prove the tgev endocytosis pathway. we found that both fitc-transferrin and fitc-ct-b were co-localized with dylight 594-tgev ( fig. 5h and i) . taken together, we confirmed that both clathrin and caveolin mediated endocytosis are important for tgev entry. to explore the role of egfr in tgev internalization, the internalization of egfr was investigated by confocal laser-scanning microscopy. in normal ipec-j2 cells, most egfr was located at the cell surface membrane, and egfr was internalized upon egf stimulation. tgev infection also promotes egfr internalization ( fig. 6a and b) . we also detected cell surface membrane egfr expression levels ( fig. 6c and d). egfr ligands (egf and tgf-α) induced receptor dimerization, activation, and internalization (wells, 1999a) . ipec-j2 cells were incubated with tgev for different times at 4°c for 1 h, and ligand egf was used as a positive control. when egf-stimulated cells were transferred at 37°c, egfr internalized rapidly. at 15 min post-infection (mpi), most cell membrane egfr was internalized into the cytoplasm. at 60 mpi, egfr was recruited to cell membrane. tgev infection caused egfr internalization in a time dependent manner. egfr was internalized into the cytoplasm from 15 mpi to 60 mpi. in normal cells, most egfr resided on the cell membrane. this data suggests that tgev particles cause the internalization of egfr early in tgev infection. to explore the role of caveolin and clathrin in egfr internalization early in tgev infection, we reduced clathrin or caveolin down in normal ipec-j2 cells through targeting shrnas, and investigated cell membrane egfr expression levels during tgev invasion. clathrin targeting shrna significantly inhibited egfr internalization. in caveolin targeting shrna cells, egfr was internalized from 15 mpi to 30 mpi, and recruited to the cell membrane at 60 mpi. egfr circulation was faster than that of normal cell groups ( fig. 7a and b) . these results indicate that tgev causes egfr internalization through the clathrinfig. 8 . apn and egfr synergistically promote tgev invasion. egfr is a cofactor for tgev invasion. tgev s1 protein interacts with egfr extracellular receptor binding domain 1. tgev infection induces egfr internalization and causes apn and egfr clustering. apn and egfr synergistically promote tgev invasion. apn and egfr synergistically activate pi3k/akt and mek/erk1/2 signaling pathways. clathrin and caveolin mediate the endocytosis of tgev and egfr internalization through clathrin endocytosis pathway. mediated endocytosis pathway. in this study, we demonstrated that egfr is an another co-factor of tgev, and the tgev spike protein can interact with egfr extracellular receptor binding domain 1. tgev infection causes the co-localization of apn and egfr. furthermore apn is required for tgev-induced egfr activation. pi3k/akt and erk1/2 signaling pathways are involved in tgev internalization. in addition to this, tgev particles and egfr internalize through clathrin-mediated endocytosis. egfr has also been demonstrated to participate in the invasion of other viruses, including: adeno-associated virus serotype 6, influenza a, hepatitis c virus, and human cytomegalovirus (chan et al., 2009; eierhoff et al., 2010a; lupberger et al., 2011; weller et al., 2010) , suggesting that egfr activation and internalization may be common mechanisms utilized in virus invasion. the activation of pi3k and erk1/2 has been found in many virus entry mechanisms, such as: human cytomegalovirus, influenza a, and herpes simplex virus (eierhoff et al., 2010b; johnson et al., 2001a johnson et al., , 2001b zheng et al., 2014b) . pi3k activation has been demonstrated to regulate vesicular uptake, and trafficking of ebola virus (saeed et al., 2008) . ligand binding to egfr on the cell surface induces receptor dimerization and cross phosphorylation, which leads to the activation of downstream signaling cascades of which include: mapk, pi3k, jak/ stat, and plcγ signaling pathways (zheng et al., 2014a) . in this study, we found that early in the tgev infection process, apn and egfr synergistically stimulate pi3k/akt and mek/erk1/2 signaling pathways, and promoted tgev entry. many pathogens enter the host cell by endocytosis which results in cell surface receptor, ligand, and membrane component internalization (mosesson et al., 2008) . enveloped viruses are capable of entering directly through the cell membrane surface receptors, or are able to be internalizing via endocytosis via fusion taking place in the endosomal compartment (belouzard et al., 2012) . our previous published research found that tgev can be internalized into ipec-j2 cells, however the mechanism of tgev entry is still not known. it has been confirmed that pedv invades via clathrin-mediated endocytosis independent of caveolin-mediated endocytosis (park et al., 2014) . our results suggest that tgev infection reduces in clathrin or caveolin targeting shrnas cells. furthermore, co-localization between endocytosed fitc-transferrin/ fitc-ctxb and fluorochrome-labeled tgev support the conclusion that both clathrin and caveolin mediated endocytic uptake are the pathways of tgev entry. typically for particle sizes internalized through caveolin ranging from 50 to 100 nm and through clathrin less than 200 nm (aleksandrowicz et al., 2011) . tgev particles sizes are between 100 and 150 nm, some are smaller tgev particles that are internalized through caveolin-mediated endocytosis. the actin cytoskeleton has been thought of as participating in the formation of the clathrin-mediated endocytosis structure, as well as providing mechanical force to enable complete endocytosis (kaksonen et al., 2006; smythe and ayscough, 2006) . our previous research has also found that tgev infection induces actin cytoskeleton rearrangement through cofilin, and actin surround tgev particles in the spatial part of the virus co-localize with the actin, which indicates that clathrin and actin are involved in the early invasion of tgev. receptor clustering is an actin-dependent process that uses rho-family gtpase signaling, and the actin regulatory proteins filamin and cofilin (jimenezbaranda et al., 2007; yoder et al., 2008) . the co-localization of apn and egfr is also found in the early infection of tgev, suggesting that egfr phosphorylation and pi3k-rac1/cdc42-limk-cofilin signaling pathways participate in the regulation of actin cytoskeleton. tgev particles movement towards entry sites, the co-localization of apn and egfr are all mediated by actin cytoskeleton re-modeling. in the resting state, most egfr reside on the cell membrane surface. upon ligand binding, egfr is activated and internalized via clathrin-coated pits (wells, 1999a; zheng et al., 2014b) . numerous viruses utilize egfr endocytosis to mediate virus internalization and trafficking to the site of replication during the infection of the host cell (mercer et al., 2010b) . some previous studies have suggested clathrin-coated pits can form naturally at 0°c in the presence of egf, but are not internalized into the cytoplasm (brown and petersen, 1998; jiang et al., 2003) . clathrin-coated pits connected to the cell surface and clathrin-coated vesicles are intracellular membranal structures. lipid rafts function as platforms to active intracellular egfr effector signals and egfr internalization (puri et al., 2005) . in this study we found that when egf was incubated with cells at 4°c for 1 h, egf formed clathrin-coated pits connected to the cell surface and egfr became localized in the coated pits. when cells were transferred at 37°c, egfr was rapidly internalized into the cytoplasm through clathrin-coated vesicles. for tgev infected cells, tgev particles bound with cells at 4°c. as soon as cells were transferred at 37°c, tgev stimulated clustering of lipid rafts and activated egfr and effector signals. this process took comparatively more time, hence at 15 mpi, egfr was found to begin internalization of tgev particles. in this study, we found that tgev infection caused egfr internalization, and clathrin-targeting shrna inhibited egfr internalization. egfr also was identified as an another receptor for tgev. we can get to the conclusion that in the early infection stage of tgev, tgev particles bound with apn and egfr, the virus-receptors complex are subsequently internalized by clathrin. caveolin targeting shrna has no effect on the internalization of egfr. however further research is needed to specify the mechanism of tgev particles internalized by caveolin. all our data confirms that egfr is a co-factor for tgev invasion, and that tgev s1 protein interacts with egfr extracellular receptor binding domain 1. tgev infection induces egfr internalization and causes apn and egfr clustering. plus, apn and egfr not only synergistically promote tgev invasion, but they also active pi3k/akt and mek/erk1/2 signaling pathways. clathrin and caveolin mediate endocytosis of tgev and egfr internalization through the clathrin endocytosis pathway (fig. 8) . these findings are conducive to enhancing our understanding of the entry mechanism of tgev, and for providing a potential target for the development of new anti-tgev therapies. ebola virus enters host cells by macropinocytosis and clathrin-mediated endocytosis mechanisms of coronavirus cell entry mediated by the viral spike protein porcine ipec-j2 intestinal epithelial cells in microbiological investigations an image correlation analysis of the distribution of clathrin associated adaptor protein (ap-2) at 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sequence of the coronavirus transmissible gastroenteritis virus shxb isolated in china epidermal growth factor receptor is a co-receptor for adeno-associated virus serotype 6 egf receptor egf receptor hiv envelope-cxcr4 signaling activates cofilin to overcome cortical actin restriction in resting cd4 t cells viruses exploit the function of epidermal growth factor receptor epidermal growth factor receptor-pi3k signaling controls cofilin activity to facilitate herpes simplex virus 1 entry into neuronal cells this work was supported by 31772777 from the national science grant of china and a project funded by the priority academic program development of jiangsu higher education institutions (papd). supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2018.05.009. key: cord-331916-n744pymd authors: liu, jue; chen, isabelle; chua, huikheng; du, qingyun; kwang, jimmy title: inhibition of porcine circovirus type 2 replication in mice by rna interference date: 2006-04-10 journal: virology doi: 10.1016/j.virol.2005.12.006 sha: doc_id: 331916 cord_uid: n744pymd porcine circovirus type 2 (pcv2) is the primary causative agent of an emerging swine disease, postweaning multisystemic wasting syndrome (pmws) for which no antiviral treatment is available. to exploit the possibility of using rna interference (rnai) as a therapeutic approach against the disease, plasmid-borne short hairpin rnas (shrnas) were generated to target the pcv2 genome. transfection of these shrnas into cultured pk15 cells caused a significant reduction in viral rna production that was accompanied by inhibiting viral dna replication and protein synthesis in infected cells. the effect was further tested in vivo in a mouse model that has been developed for pcv2 infection. mice injected with shrna before pcv2 infection showed substantially decreased microscopic lesions in inguinal lymph nodes compared to controls. in situ hybridization and immunohistochemical analyses showed that shrna caused a significant inhibition in the level of viral dna and protein synthesis detected in the lymph nodes of the treated mice relative to the controls. taken together, these results indicate that shrnas are capable of inhibiting pcv2 infection in vitro as well as in vivo and thus may constitute an effective therapeutic strategy for pcv2 infection. postweaning multisystemic wasting syndrome (pmws) is a new emerging disease affecting pigs primarily at 5 to 18 weeks of age. this disease was first identified within high-health herds in western canada in 1991 (clark, 1997) and now is considered to be endemic in many swine-producing countries of the world (allan and ellis, 2000) . pigs affected with pmws show weight loss, dyspnea, tachypnea, anemia, diarrhea, and jaundice. characteristic macroscopic findings are enlargement of lymph nodes and noncollapsed lungs with tan mottling. microscopic lesions can be detected in a number of tissues, the most characteristic being those of lymphoid tissues. these lesions consist of lymphocyte depletion of follicular and interfollicular areas together with macrophage infiltration of lymphoid tissues. other common lesions described for pmws include interstitial pneumonia, periportal to diffuse hepatitis, and interstitial nephritis (clark, 1997; rosell et al., 1999) . isolation of virus from tissues of affected pigs led to the identification of a porcine circovirus type 2 (pcv2), considered to be of etiological importance in pmws ellis et al., 1998; hamel et al., 1998; meehan et al., 1998; morozov et al., 1998) . porcine circovirus type 1 (pcv1) was originally identified as a contaminant of porcine kidney cell cultures (pk15 atcc ccl-33) (tischer et al., 1982) and is nonpathogenic to pigs (allan et al., 1995; tischer et al., 1982) . porcine circovirus (pcv) is classified in the genus circovirus of the circoviridae family (pringle, 1999) . the pcv virion is icosahedral, non-enveloped, and 17 nm in diameter. the genome of pcv is a single-stranded circular dna of about 1.76 kb. the overall dna sequence homology within the pcv1 or pcv2 isolates is greater than 90%, while the homology between pcv1 and pcv2 isolates is 68-76%. two major open reading frames (orfs) have been recognized for pcv, orf1, called rep gene, which encodes a protein of 35.7 kda involved in virus replication (mankertz et al., 1998) , and orf2, called cap gene, which encodes the major immunogenic capsid protein of 27.8 kda (cheung, 2003a; nawagitgul et al., 2000) . in addition to the replicase orf1 and 0042-6822/$ -see front matter d 2005 elsevier inc. all rights reserved. doi:10.1016/j.virol.2005.12.006 the capsid protein orf2, a novel protein, orf3, has been detected in pcv2 productive infection and is not essential for pcv2 replication in cultured cells but is involved in virusinduced apoptosis (liu et al., 2005) . orf2 protein has been demonstrated to stimulate protective responses to injected pigs with baculovirus-expressed product or dna prepared from the gene (blanchard et al., 2003; kamstrup et al., 2004) . furthermore, other studies have shown that chimeric pcv1 -2 virus with the immunogenic orf2 capsid gene of pathogenic pcv2 cloned into the nonpathogenic pcv1 genomic backbone as well as an attenuated pcv2 after 120 passages could induce specific antibody responses (fenaux et al., 2004a (fenaux et al., , 2004b . however, there is a great need for the development of a new therapy for pcv2 infection. rna interference (rnai) is a natural process by which double-stranded rna directs sequence-specific post-transcriptional gene silencing (fire, 1999; hammond et al., 2001a,b; sharp, 2001) . specific inhibition of endogenous or pathogen mrna by rnai can be triggered by the introduction of 21 -23 nucleotide (nt) duplexes of rna (sirna) or by transcription of dna precursor into short hairpin rnas (shrna) homologous to target sequences (brummelkamp et al., 2002; elbashir et al., 2001a; paddison et al., 2002) , opening up possibilities for controlling replicative processes of pathogens. this discovery promoted the use of rnai for specifically inhibiting gene expression and replication of infectious viruses. over the past years, rnai approaches have shown to be effective against a variety of viruses in cell culture, among these are prominent viruses such as human immunodeficiency virus 1 (capodici et al., 2002) , influenza virus (ge et al., 2003) , poliovirus (gitlin et al., 2002) , severe acute respiratory syndrome virus (wang et al., 2004) , herpes virus (jia and sun, 2003) , human papillomavirus (hamel et al., 1998) , dengue virus (caplen et al., 2002) , and west nile virus (mccown et al., 2003) . synthetic sirnas as well as shrnas transcribed in vivo from dna templates have been shown to be potent inhibitors of gene expression in adult mice (mccaffrey et al., 2003) . studies in mice injected with shrnas or sirnas directed against hepatitis b virus have raised expectations about the use of rnai as an antiviral strategy (giladi et al., 2003; mccaffrey et al., 2003) . furthermore, sirnas or dna vectors produced shrnas have also been shown to reduce the influenza viral load in murine lungs and increase survival (ge et al., 2004; tompkins et al., 2004) . recently, there is a report that administration of sirnas to mice before west nile virus infection has reduced the viral load and offered partial protection from lethal infection (bai et al., 2005) . pcv2 has been demonstrated to cause lymphoid lesions of infected balb/c mice characterized by expansion of germinal centers in lymphoid organs with large numbers of histiocytic cells and lymphoblasts, apoptosis of histiocytic cells, and lymphoid depletion (kiupel et al., 2001) , which is consistent with that of pcv2-infected pigs. therefore, this model system offers the opportunity to test the ability of rnai to inhibit replication of pcv2 in vivo. here, we report that shrnas directed against the pcv2 orf1 and orf2 regions are capable of efficiently inhibiting viral replication and protein synthesis both in pk15 cell culture system as well as in the mouse model. inhibition of pcv2 viral protein synthesis in cell culture by shrna is sequence-specific and dose-dependent to test whether rnai could inhibit pcv2 in cell culture, we generated six shrna-expressing plasmids targeting the orf1 and orf2 regions of the pcv2 genome (fig. 1a) for their ability to suppress expression of pcv2 viral proteins. the pk15 cells were transfected with the shrna-expressing plasmids and then infected with pcv2, and the levels of pcv2 viral protein production were determined by ifa at 48 h after infection. the time points for measuring the effects of shrna in cells were chosen following preliminary optimization experiments (data not shown). to confirm the specificity of the inhibition, pvp2, an shrna-expressing plasmid directed against porcine parvovirus viral sequence, was used as a negative control in the experiments. the results showed that all shrna-expressing plasmids targeting the pcv2 genome caused a decrease in pcv2 antigen levels to varying degrees (figs. 1b and c). among these specific shrnas, psir3 and psir6 exhibited the highest level of inhibition (figs. 1b and c). therefore, we chose these two shrna-expressing plasmids for the following experiments. in contrast to the pcv2-specific shrnas, the control pvp2 had no effect on pcv2 antigen level in the treated cells, indicating that inhibition by the pcv2-targeted shrnas was specific and due to rnai. a dose -response analysis ( fig. 2a ) was conducted by transfecting pk15 cells with increasing amounts of psir3, psir6, or negative control pvp2-expressing plasmid followed by measuring the numbers of pcv2 antigen cells by ifa 72 h post-transfection. the results showed that, at concentrations of 1500 to 2500 ng shrna-expressing plasmid per 5 â 10 4 cells, the highest level of inhibition (fivefold) of viral antigen was obtained with psir3 and psir6 plasmids, whereas pvp2 had no effect ( fig. 2a) . at higher doses of shrna, we observed a slight nonspecific inhibitory effect of pvp2 on viral protein production ( fig. 2a ). in addition, kinetic study results (fig. 2b ) revealed a continuous trend in the specific inhibition of pcv2 viral production by psir3 and psir6 from 48 to 120 h after transfection. the inhibition of pcv2 viral production was maximal at 72 h but thereafter started to decline by 96 h after transfection. to confirm that the effect of psir3 and psir6 on pcv2 viral production was through the reduction of pcv2 rna levels, we performed real-time rt-pcr analysis with rna extracted from pk15 cells 72 h after transfection with psir3, psir6, and pvp2-expressing plasmid, respectively. the amount of each viral mrna was normalized to that of gapdh mrna in the same sample. the abundance of orf1 and orf2 mrnas was significantly reduced in pcv2-infected cells transfected with psir3-or psir6-expressing plasmids (fig. 3a) . shrnas are known to be incorporated into an rnainduced silencing complex and to direct rna-induced silencing complex-mediated sequence-specific mrna degradation (hammond et al., 2000) . orf1 and orf2 of pcv2 have been demonstrated to contain different mrna, but their mrna levels were reduced in the pcv2-infected cells regardless of orf1 or orf2 shrna transfection. one possible explanation for the effects of this coordination is that the target mrna bound with a shrna might affect the other non-target mrna of the virus through a downstream effect on all the viral transcripts because viral replication is inhibited, but the detailed mechanism of this process remains unclear. to further study the inhibitory effect of psir3-or psir6expressing plasmids on pcv2 dna replication, the supernatants of infected pk15 cells after being transfected with the shrna plasmids at 72 h post-transfection were directly assayed by pcr amplification with primers 1446f and 420r. the results showed that reduced level of newly synthesized viral replicative form (rf) dna was observed in the treated cells with psir3-or psir6-expressing plasmids (fig. 3b ). in contrast, the viral rf dna was detected in the treated cells with the control pvp2-expressing plasmid as seen in that of pcv2-alone-infected cells (fig. 3b ). to further examine the ability of orf1 shrna to suppress viral gene expression in pcv2-infected pk15 cells, single nucleotide mutations were introduced into psir3 sequence as shown in fig. 4a . cells were infected with pcv2 after being transfected with these shrna-expressing plasmids, and total proteins were extracted and examined by an immunoblot analysis with mouse polyclonal antibody against orf1 or orf2 protein at 48 h post-infection. the cells transfected with psir3-or psir6-expressing plasmid manifested a marked depletion of viral proteins compared with cells transfected with pvp2-expressing plasmid (fig. 4b ). while extinctions of the orf1 and orf2 protein expression were observed in the psir3 plasmid-treated cells, a significant suppression of orf2 protein expression and noticeable decreases in the level of the orf1 protein were detected after the psir6 treatment. in addition, drastic suppression or not was detected in the mutant shrna-expressing plasmids dependent on the location of mutation in the rnai sequence. as shown in fig. 4b , a mutant with one nucleotide mutation in the 5v-end of the sense strand (psir3-m1) still had an effective reduction in the synthesis of orf1 and orf2 proteins, whereas with a nucleotide substitution at the center (psir3-m2) or 3v-end (psir3-m3) of the sense sequence, the resultant shrnas failed to significantly suppress expression of pcv2 orf1 and orf2 in the transfected cells, further verifying the specificity of rnai as demonstrated (amarzguioui et al., 2003; harborth et al., 2003) . neither viral infection nor treatment with shrna alone influenced the level of gapdh production (fig. 4b) . we also examined the effects of psir3 and psir6 as well as mutated shrnas by facs with porcine antibody raised against pcv2 72 h after shrna-expressing plasmids transfection. as shown in fig. 4c , pcv2 viral production was significantly reduced in pk15 cells that were pretreated with psir3-or psir6-expressing plasmids. in addition, the psir3-m1 significantly suppressed pcv2 protein synthesis, whereas the two other shrna mutants (psir3-m2 and psir3-m3) had no effect (fig. 4c) , indicating that the effects of pcv2 shrnas are indeed highly specific. as expected, the pvp2 shrna also did not inhibit pcv2 viral expression (fig. 4c ). to determine whether shrnas exert an antiviral response in vivo in balb/c mice, we administered the psir3 or psir6 plasmids 1 day before viral inoculation. no clinical signs or gross lesions were observed in shrna-expressing plasmids transfected or mock-transfected mice that received pcv2 at any time during the 12-day observation period. microscopic alterations in pcv2 alone mice were first noticed at day 5 postinfection, slight germinal centers developed in the parenchyma of the inguinal lymph nodes (data not shown). at 11 days post-infection, the mice developed prominent germinal centers that consisted of large lymphoblastic cells and histiocytic cells, and the paracortex had loss of lymphocytes and was infiltrated by large numbers of histiocytes (fig. 5a ). in contrast, the lymph nodes showed normal microscopic morphology in treated mice with psir3 or psir6 at 5 days post-infection as seen in that of mock-infected mice (data not shown). at 11 days postinfection, mice treated with psir3 or psir6 showed significantly lower ( p < 0.05) lesion scores than that of untreated mice and showed a considerable reduction in inguinal lymph node lesions, as evidenced by decreases in the lymphocytes and in the number of infiltrating histiocytes compared to controls (figs. 5b and c). in addition, the nonspecific pvp2 shrna did not attenuate the lymph node lesions induced by pcv2 infection at 5 and 11 days post-infection (data not shown). no microscopic lesions were observed in mock-infected mice (fig. 5d) . the results suggest that pcv2 shrna can significantly attenuate pcv2-induced lymph node pathology in mice. to further study the effect of psir3 or psir6 on viral dna replication in vivo, we performed an in situ hybridization analysis with inguinal lymph nodes of mice treated with shrnas at 6 and 12 days after injection. replicated pcv2 genomic dna was present in 2.0 t 0.5% of infected cells in inguinal lymph node sections of mice that received pcv2 alone at 6 days after injection, with slight inhibitory effect observed in the psir3 or psir6-treated mice (data not shown). at 12 day post-injection, pcv2 nucleic acid was detected in 8.2 t 1.5% of infected cells such as macrophages, histiocytes, and occasional lymphocytes in inguinal lymph node tissue sections from mice that were inoculated with pcv2 alone. positive cells typically exhibited a dark purple to black reaction product in the cytoplasm as well as in the nucleus (figs. 6a and b) . lymph node sections from mice receiving psir3 had significantly reduced numbers of positive cells ( p < 0.05), with 1.2 t 0.5% of stained cells (fig. 6c) . the number of stained cells in sections from psir6-treated mice was also significantly reduced ( p < 0.05, 1.6 t 1.2% of infected cells) (fig. 6d) . in contrast, the number of positive cells in sections of mice treated with pvp2 rnai did not show any reduction ( p > 0.05, 8.5 t 2.3%) (fig. 6e) . no staining was seen in sections from mockinfected mice (fig. 6f) . these results demonstrate that pcv2 shrna can inhibit the pcv2 dna replication in vivo. we also assessed the effect of shrna on pcv2 protein synthesis by immunohistochemical staining for pcv2 in inguinal lymph node sections from mice treated in the experiment described above. at 6 days after injection of the shrnas followed by inoculation of pcv2, the numbers of cells stained positively for pcv2 in pcv2-alone-infected group were 2.5 t 0.6%, with slight inhibitory effect observed in the psir3or psir6-treated mice (data not shown). as shown in figs. 7a and b, the numbers of pcv2 antigen-positive cells increased significantly in the inguinal lymph nodes of the virus-inoculated cells at 12 days after injection ( p < 0.05, 8.9 t 2.1%), but mice treated with psir3 or psir6 exhibited small increase in pcv2 antigen-positive cells (1.5 t 0.4% for psir3 or 1.8 t 0.7% for psir6 treatment) (figs. 7c and d) and, thus, overall had a significant inhibition ( p < 0.05) compared to the virusinoculated alone mice. pcv2 staining was detected mainly in the cytoplasm but for some in the nucleus as well. as a control, mice treated with pvp2 showed similar pcv2-positive cells (8.7 t 2.2%) to the virus control at 12 days after injection (fig. 7e) . no staining was seen in sections from mice that were not inoculated with pcv2 (fig. 7f) . the results emphasized the significant antiviral activity of the psir3 and psir6 and their ability to suppress pcv2 protein synthesis in vivo. rnai operates at the post-transcription level to suppress gene expression and has developed into a powerful method to downregulate the expression of endogenous as well as exogenous sequences of the cell (mcmanus and sharp, 2002; morris et al., 2004) . the ability to silence mammalian gene expression using sirna opens new and exciting routes to the understanding of mammalian cell biology and its pathology. there are many precedents for rnai being effective against a wide range of viral pathogens in vitro. in addition, rnai has been demonstrated to be functional in mice and can inhibit viral replication and expression when injected with specific sirnas against hepatitis b and influenza viruses (ge et al., 2004; giladi et al., 2003; mccaffrey et al., 2003; uprichard et al., 2005) . these studies have shown that rnai can potentially be used as a therapeutic or prophylactic mechanism against viruses. here, we have provided evidence that shrnas targeting the orf1 and orf2 regions of pcv2 are capable of inhibiting all the steps of pcv2 infection in cultured cells, which support viral production, and in vivo in a mouse model. in pcv2, three orf regions have been recognized. in this study, to test which region of the pcv2 genome is the most effective site for sirna targeting, six different anti-pcv2 shrnas were selected at the orf1 and orf2 genes that have important functions in viral replication and production, respectively. after testing the effect of these vector-based sirnas on the inhibition of pcv2 replication in cultured pk15 cells, we found that all six shrnas could cause a decrease in pcv2 replication to varying degrees, with two of them being more effective. these results may be due to different positional accessibility caused by steric hindrance by a secondary or tertiary structure and/or protein binding (kamstrup et al., 2004) . we also noticed that the nonspecific pvp2-expressing plasmid has some inhibitory effect on pcv2 replication after being transfected at higher concentration. this could be due to the nongene-specific suppression of sirna. higher concentrations of sirna have been demonstrated to exhibit more measurable nonspecific gene suppression activity (persengiev et al., 2004) . therefore, identifying highly active sirnas and decreasing sirna concentrations can alleviate some nonspecific effects while maintaining efficient silencing. it has been thought that sirnas target mrnas containing the same sequences and induce their cleavage. in this study, we used real-time rt-pcr to detect orf1 or orf2 mrna level after being transfected with psir3-or psir6-expressing plasmids and found that not only can shrna significantly reduce its corresponding mrna level but inhibit other viral gene transcription as well (fig. 3a ). this could be related to the life cycle of the virus. it can be assumed that the pcv2-specific shrna first recognizes the corresponding sequence of the viral mrna and initiates the degradation of this mrna. as a consequence, the degradation of the mrna results in blockage of the corresponding protein synthesis, i.e., silencing of viral gene expression. thus, the amount of newly synthesized mature virions might have been reduced in the subsequent virus infection. therefore, significant inhibition in other mrna level could be due to reduction of newly synthesized mature virions induced by shrna when it does not directly target this mrna. this hypothesis has been further confirmed by the data that viral dna was reduced in supernatants of transfected cells (fig. 3b ) and pcv2 protein synthesis was inhibited in transfected cells (fig. 4c) . the roles of sirna target recognition have been examined employing diverse sirnas with nucleotide substitutions (amarzguioui et al., 2003; chiu and rana, 2003; elbashir et al., 2001b; harborth et al., 2003; pusch et al., 2003) . introducing a single nucleotide change to the sirna sequence could abrogate sirna-mediated silencing. to clarify this question, we performed evaluations using a series of psir3 mutants containing point mutations at different locations. the data (figs. 4b and c) suggest that point mutations in the middle and 3v-end sequence of psir3 sense strand could eliminate the anti-pcv2 activity probably due to a decrease in the affinity of the sirna for the target mrna (schwarz et al., 2003) , whereas the point mutation of the 5v-end sequence did not interfere with the antiviral activity. sequence specificity of sirna is very stringent as single base pair mismatches between the sirna and its target sequence can dramatically reduce the silencing capability (elbashir et al., 2001a (elbashir et al., , 2001b yuan et al., 2005) . however, the molecular mechanism of this phenomenon needs to be further studied. in addition, it has been demonstrated that escaping mutants were generated following sirna or shrna treatment gitlin et al., 2002 gitlin et al., , 2005 , this can be prevented by treatment with multiplex specific sirnas or targeting long portions of the viral genome. in this study, we also performed three passages of pcv2 on newly psir3-expressing plasmidtransfected cells to investigate whether escaping pcv2 mutants were generated, but did not detect any mutants (data not shown), suggesting that the psir3 targeting sequence is a critical site for the fitness of the virus. therefore, this kind of targeting sequence as psir3 can be used as targets for designing sirna in pcv2 therapy. a few studies have addressed the potential of sirna-based therapeutics in vivo using model animal systems. interference with transgene and endogenous gene expression in mice following administration of sirna duplexes has been reported (mccaffrey et al., 2002; song et al., 2003) . to prolong the expression of sirna, a plasmid or an integrating viral vector expressing endogenous sirna can be used to achieve this purpose (mccaffrey et al., 2003; uprichard et al., 2005) . such vector-based strategies with generation of sirna may be used to test inhibition or suppression of viral gene expression and replication in animal models and permit greater understanding of the role and activity of viral genes in vivo. by contrast to that pcv2 was capable of replicating in balb/c mice and caused microscopic lesions (kiupel et al., 2001) , quintana et al., (2002) reported that no microscopic lesions compatible with pcv2 infections in pigs were detected in inoculated mice, but it might be due to the inoculum of the dosage and administration route (quintana et al., 2002) . in the present study, we observed enlargement of germinal centers by infiltration with large numbers of lymphoblastic and histiocytic cells, apoptosis of histiocytes, and lymphocytic depletion in the paracortex of lymph nodes in pcv2-inoculated balb/c mice ( fig. 5a and data not shown) which resembled early lesions in lymph nodes in pcv2-inoculated gnotobiotic pigs. furthermore, pcv2 replication was confirmed by the amounts of viral dna and protein synthesis in the lymph nodes (figs. 6a and 7a) as well as other tissues (data not shown) in the pcv2inoculated mice. therefore, we used balb/c mice as an animal model for inhibiting pcv2 infection by rnai in vivo. the study described herein shows that rnai induced by plasmid-borne small interfering rnas can be used to inhibit replication of a porcine pathogen in a mouse model system for 12 days after injection. the antiviral treatment decreased viral expression in the inguinal lymph node and attenuated lymph node lesions in pcv2-infected mice and thus may be applicable as a therapeutic strategy for naturally occurring pcv2 infection. in conclusion, we show that shrna can be designed to induce an antiviral effect on pcv2 dna replication and protein synthesis both in cultured cells and in animal model. these data demonstrate that rnai is a potential prophylaxis and therapy for pcv2 infection. the permanent pk15 cell line, which was free of pcv, was maintained in minimal essential medium (mem, gibco) supplemented with 5% heat-inactivated fetal bovine serum (fbs), 5% l-glutamine, 100 u/ml of penicillin g, and 100 al/ ml streptomycin at 37 -c in a humidified 5% co 2 incubator. the pcv2 virus strain bjw (liu et al., 2005) was used in the study. the mammalian expression vector rnai-ready psiren (psir) (clontech, bd) was used for the expression of sirna. six specific anti-pcv2 shrna expression plasmids were constructed to target orf1 and orf2 genes of the pcv2 strain bjw (genbank accession no. ay847748) genome: psir1 (targeting sequence, nt f527 to f545), 5v-aatgcagaagcgtgattgg-3v; psir2 (nt f590 to f608), 5v-aagcaaatgggctgctaat-3v; psir3 (nt f624 to f642), 5v-accacatactggaaaccac-3v; psir4 (nt r1612 to r1594), 5v-aaatggcatcttcaacacc-3v; psir5 (nt r1429 to r1411), 5v-ggttgaattctggccctgc-3v; and psir6 (nt r1307 to r1289), 5v-actactcctcccgc-cattc-3v (fig. 1a) . the suffix (f or r) of the oligonucleotide indicates the orientation of the targeting sequence. f indicates forward direction from nt 0 to 1767, while r indicates reverse direction from nt 1767 to 0. a nonspecific shrna expression vector, pvp2 (5v-cctacctgagctggcctaa-3v), targeting capsid protein vp2 gene of porcine parvovirus, was constructed as a negative control. these sequences were all analyzed by a blast search of the genbank database to avoid similar sequences found in any other genes but share 100% homology within the published pcv2 strains as well. oligonucleotides were synthesized and inserted into the psir vector in the bamhi and ecori sites according to the manufacturer's instructions. the transfection of shrnas was performed under optimal conditions. briefly, pk15 cells were seeded and grown at 37 -c overnight. when cells reached 60 to 70% confluency, they were transfected with shrna expression plasmids with lipofectamine reagent (invitrogen). following 24 h of transfection, cells were washed and infected with pcv2 strain bjw at the indicated multiplicity of infection (moi) for 1 h. the cells were then overlaid with complete medium and were incubated at 37 -c in 5% co 2 . cells were additionally treated with 300 mm d-glucosamine at 24 h after infection as described previously (tischer et al., 1987) . at 48 h postinfection, they were analyzed by ifa, and supernatants and cell lysates were collected for pcr analysis and western blotting, respectively. at 24 h of post-transfection with shrna expression plasmids, pk15 cells grown on 24-well plate were incubated with the pcv2 for 60 min at 37 -c at an moi of 1 tcid 50 and added mem for incubation. following the incubation at 37 -c, cells at 48 h post-infection were washed with pbs and fixed for 30 min at room temperature (rt) with 4% paraformaldehyde (pfa) in pbs. after fixation, the cells were blocked by pbs with 3% bsa at rt for 1 h. primary antibody, porcine anti-pcv2 antibody, was diluted in pbs with 3% bsa and incubated with the cells for 1 h at 37 -c. after washing with pbs, the cells were incubated with rabbit anti-porcine fluorescein isothiocyanate (fitc)-conjugated antibody (sigma) diluted in pbs with 3% bsa for 1 h at 37 -c. the cells were washed three times with pbs and examined using a fluorescence microscope. cells positive for pcv2 viral antigens were counted in six fields of view. pk15 cells were harvested and fixed for 30 min in a 2% pfa solution. nonspecific binding was reduced by exposure to 10% equine serum for 30 min and three washes with fluorescence-activated cell sorter (facs) wash buffer (phosphate-buffered saline with 1% fetal calf serum and 0.1% sodium azide) prior to incubation with a 1:200 dilution of porcine against pcv2 antibody for 30 min at rt. the cells were then washed three times with facs buffer and incubated at rt with a 1:100 dilution of fitc-conjugated anti-swine igg (sigma) for 30 min. after incubation, the cells were washed three times with facs buffer and resuspended in 400 al of facs buffer before acquisition and analysis on a facscalibur instrument (becton dickinson) using winmdi 2.8 software (purdue university cytometry laboratories). western blot analysis was performed after the cells were harvested by centrifugation at 500 â g for 8 min. the pellets were lysed for 10 min on ice in 150 al lysis buffer [20 mm tris -hcl, ph 7.5, 150 mm nacl, 5 mm edta, ph 8.0, 1% triton x-100, 10% glycerol, 1 mm pmsf, 5 mm dtt, and 20 ag/ml of the proteinase inhibitor cocktail (novagen)]. lysates were then collected by centrifugation at 14,000 â g for 10 min at 4 -c and the concentration was measured. twenty micrograms of total cellular protein from each sample was resolved on 10 or 12% sds-polyacrylamide gel electrophoresis (sds-page) and blotted onto nitrocellulose (nc) membranes (stratagene) with a semidry transfer cell (bio-rad trans-blot sd). the membranes were blocked for 2 h at rt in blocking buffer tbst (20 mm tris -hcl [ph 7.4], 150 mm nacl, 0.1% tween-20) containing 5% skim milk powder to prevent nonspecific binding and then incubated with mouse anti-orf1, anti-orf2 antibody (liu et al., 2005) , or monoclonal antibody against human glyceraldehyde-3-phosphate dehydrogenase (gapdh) (chemicon international, inc., ca, usa) at rt for 2 h. then, the membranes were washed three times with tbst and incubated for 1 h at rt with horseradish-peroxidaseconjugated anti-mouse secondary antibody (dako) diluted in blocking buffer (1:2000). after washing, the membrane was reacted with 3,3v-diaminobenzidine tetrahydrochloride (pierce, rockford, ill., usa) substrate and then stopped with distilled water. newly synthesized viral dna in cell culture was assayed by pcr as elsewhere described (cheung, 2003b) . briefly, the supernatants of infected pk15 cells after being transfected with shrna-expressing plasmids 72 h post-transfection were used as templates for pcr amplification. the sense primer 1446f: 5v-tgtagtattcaaagggtatagaga-3v and antisense primer 420r: 5v-tcacagcagtagacaggtcactccg-3v were used for amplifying pcv2 dna. the pcr consisted of an initial enzyme activation step at 94 -c for 15 min followed by 35 cycles of denaturation at 94 -c for 45 s, annealing at 52 -c for 45 s, extension at 72 -c for 1 min, and a final extension at 72 -c for 10 min. the pcr product was electrophoresed in 1% agarose gel and photographed. total cell rnas were prepared from virus-infected pk15 cells 72 h after being transfected with shrna-expressing plasmids by using trizol rna extract reagent (invitrogen) for reverse transcription (rt)-pcr. the rna samples were incubated with dnase i for 60 min at 37 -c to remove any contaminating viral dna. the following primers were used: f223, 5v-tccaggggttcgctaa-3v, and r412, 5v-gtagacaggt-cactccg-3v, for pcv2-orf1; r1566, 5v-atcaagcgaac-cacag-3v, and f1317, 5v-ggtcataggtgagggc-3v, for pcv2-orf2; and sense, 5v-catcactgccacccagaaga-3v, and antisense, 5v-gctgtagccaaattcgttgt-3v, for gapdh. cdnas were reverse transcribed from total rnas by the use of antisense primers and the first-strand synthesis system (amv reverse transcriptase kit, roche). quantitative real-time pcrs were performed on a lightcycler (roche). the amplification protocol followed the instructions of a taqman gold pcr kit. each sample was run in triplicates. the relative amount of target gene mrna was normalized to that of gapdh mrna in the same sample. fifty balb/c mice of 8 weeks of age were randomly assigned into five rooms of 10 animals each. mice in group 1 were uninoculated and served as negative controls. psir3, psir6, and pvp2 expression plasmids (10 ag per mouse) were injected via both tail vein and intramuscular routes to mice in groups 2 to 4, respectively. mice in group 5 was served as positive controls. after 24 h, all mice in groups 2 to 5 were each inoculated intranasally and interperitoneally with about 10 5 tcid 50 of the pcv2 strain bjw. the animals were monitored daily for clinical signs of disease. at 5 and 11 days post-infection, five mice were randomly selected from each group and necropsied. inguinal lymph nodes were collected during necropsy and processed for histological examination and in situ hybridization (ish) as well as immunohistochemical (ihc) staining. for histological examination, inguinal lymph nodes were collected and fixed by immersion in 4% pfa. fixed samples were dehydrated, embedded in paraffin wax, and sectioned at 5 am then stained with hematoxylin and eosin (he). lymph node scores were assessed for depletion of lymphoid cells, infiltration by histocytic cells, and/or the presence of multinucleated cells. for ish, a pcv2-specific oligoprobe was used for demonstration of pcv2 nucleic acid. briefly, a 253 bp dna fragment from orf2 of pcv2 was amplified using a pair of primers: 5v-atcaagcgaaccacagtcaaaac-3v and 5v-ggtcataggtgaggctgtggcc-3v. the specific oligoprobe was labeled with digoxigenin (dig) (roche). after deparaffinization and rehydration, the lymph node sections were proteolytically digested with 10 ag/ml of proteinase k in pbs buffer for 15 min at 37 -c and then post-fixed with 0.4% pfa in pbs for 30 min at 4 -c. after acetylation, the slides were prehybridized in hybridization buffer (50% formamide, 5â ssc, 50 ag/ml denatured salmon sperm dna) for 120 min at 37 -c followed by overnight hybridization at 42 -c with the dig-labeled dna probe at a concentration of 300 ng/ml. after post-hybridization washes, the slides were incubated with anti-dig (roche) (150 al of 1:5000 diluted in buffer [10% fetal calf serum; 100 mm tris -hcl; 150 mm nacl, ph 7.5]) overnight at 4 -c. the slides were washed, incubated with nitro blue tetrazolium-bcip (5-bromo-4-chloro-3-indolyphosphate) mixture for color development, and then mounted for examination under the microscopy. for ihc, a polyclonal anti-pcv2 antibody was used to detect the presence of pcv2 viral antigen. briefly, tissue sections were deparaffinized and rehydrated through graded alcohol. endogenous peroxidase activity was inhibited by flooding the slides with 3% h 2 o 2 in 1% triton x-pbs for 10 min. after being blocked in 5% normal mouse serum for 1 h at rt, the slides were incubated with purified pcv2 antibody (1:100) overnight at rt followed by incubation with a secondary biotinylated anti-swine igg (1:200) for 1 h at rt. an avidin-biotin peroxidase (abc) method (pierce, il, usa) diluted in 1:100 was applied for 1 h at rt. the sections were finally incubated in diaminobenzidine (dab) -hydrogen per-oxide solution for 5 min and counterstained with 1% methyl green, dehydrated and mounted with permount (fisher scientific inc.), and examined microscopically. results are presented as averages t standard deviations or standard errors of the means, as indicated. statistical comparisons are made by using student's t test, and differences between groups were considered significant if the p value was <0.05. porcine circoviruses: a review pathogenesis of porcine circovirus: experimental infections of colostrum deprived piglets and examination of pig foetal material experimental reproduction of severe wasting disease by co-infection of pigs with porcine circovirus and porcine parvovirus tolerance for mutations and chemical modifications in a sirna use of rna interference to prevent lethal murine west nile virus infection protection of swine against postweaning multisystemic wasting syndrome (pmws) by porcine circovirus type 2 proteins human immunodeficiency virus type 1 escape from rna interference a system for stable expression of short interfering rnas in mammalian cells inhibition of viral gene expression and replication in mosquito cells by dsrna-triggered rna interference inhibition of hiv-1 infection by small interfering rna-mediated rna interference transcriptional analysis of porcine circovirus type 2 the essential and nonessential transcription units for viral protein synthesis and dna 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disease characterization of a previously unidentified viral protein of porcine circovirus type 2-infected cells and its role in virusinduced apoptosis identification of a protein essential for replication of porcine circovirus rna interference in adult mice inhibition of hepatitis b virus in mice by rna interference the utility of sirna transcripts produced by rna polymerase in down regulating viral gene expression and repication of negative-and positive-strand rna viruses gene silencing in mammals by small interfering rnas characterization of novel circovirus dnas associated with wasting syndromes in pigs detection of a novel strain of porcine circovirus in pigs with postweaning multisystemic wasting syndrome small interfering rna-induced transcriptional gene silencing in human cells open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein short hairpin rnas (shrnas) induce sequence-specific silencing in mammalian cells nonspecific, concentrationdependent stimulation and repression of mammalian gene expression by small interfering rnas (sirnas) virus taxonomy at the xith international congress of virology nucleotide sequence homology requirements of hiv-1-specific short hairpin rna experimental inoculation of porcine circoviruses type 1 (pcv1) and type 2 (pcv2) in rabbits and mice pathological, immunohistochemical and in-situ hybridization studies of natural cases of postweaning multisystemic wasting syndrome (pmws) in pigs asymmetry in the assembly of the rnai enzyme complex rna interference rna interference targeting fas protects mice from fulminant hepatitis a very small porcine virus with circular single-stranded dna replication of porcine circovirus: induction by glucosamine and cell cycle dependence protection against lethal influenza virus challenge by rna interference in vivo clearance of hepatitis b virus from the liver of transgenic mice by short hairpin rnas inhibition of severe acute respiratory syndrome virus replication by small interfering rnas in mammalian cells inhibition of coxsackievirus b3 replication by small interfering rnas requires perfect sequence match in the central region of the viral positive strand we would like to thank prof. ling eng ang and mdm ng geok lan, anatomy department of the national university of singapore for their generous help. this work was supported by a grant from the temasek life sciences laboratory, singapore. key: cord-353748-y1a52z8e authors: bhattacharya, rajarshi; gupta, aayatti mallick; mitra, suranjita; mandal, sukhendu; biswas, swadesh r. title: a natural food preservative peptide nisin can interact with the sars-cov-2 spike protein receptor human ace2 date: 2021-01-02 journal: virology doi: 10.1016/j.virol.2020.10.002 sha: doc_id: 353748 cord_uid: y1a52z8e nisin, a food-grade antimicrobial peptide produced by lactic acid bacteria has been examined for its probable interaction with the human ace2 (hace2) receptor, the site where spike protein of sars-cov-2 binds. among the eight nisin variants examined, nisin h, nisin z, nisin u and nisin a showed a significant binding affinity towards hace2, higher than that of the rbd (receptor binding domain) of the sars-cov-2 spike protein. the molecular interaction of nisin with hace2 was investigated by homology modeling and docking studies. further, binding efficiency of the most potent nisin h was evaluated through the interaction of hace2:nisin h complex with rbd (receptor-binding domain) of sars-cov-2 and that of hace2:rbd complex with nisin h. here, nisin h acted as a potential competitor of rbd to access the hace2 receptor. the study unravels for the first time that a globally used food preservative, nisin has the potential to bind to hace2. the ongoing global outbreak of covid-19, a severe life-threatening infectious respiratory disease caused by a recently discovered severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has drastically affected human life with over eighteen millions of cases of infection globally (https://coronavirus.jhu.edu/map.html). until now, no specific antiviral medication is available for covid-19, but extensive efforts are underway worldwide. although vaccines are thought to be the most powerful weapon to fight against virus invasion, it may take quite a long time to go from the lab to successful applications in humans. considering the acute crisis of covid-19 pandemic, there is an urgent need for developing effective antiviral therapeutics for the prevention and treatment of covid-19. it is well accepted that the spike protein on the outer surface of sars-cov-2 is a crucial recognition factor for its attachment and entry to the host cells (shang et al., 2020) . the viral infection in humans is initiated by binding of rbd (receptor binding domain) of spike protein to human angiotensin-converting enzyme 2 (hace2) receptor (wang et al., 2020) . therefore, a therapeutic agent that blocks hace2 might prevent the interaction of spike protein of sars-cov-2 and thereby could reduce the establishment of infection. although small non-proteinaceous molecules are commonly preferred as therapeutics, they are not effective in blocking protein-protein interactions (ppis) particularly, where a deep binding pocket may be missing at the interface (arkin et al., 2014) . on the contrary, peptides are more suitable for disrupting ppis by specifically interacting with the interfaces. more importantly, small peptides have reduced immunogenicity (sorolla et al., 2020) . hence, peptides are potentially the ideal candidates for application as novel therapeutics. the recently described peptides are all small, synthetic and costly, and have not produced promising results against sars-cov-2 (du et al., 2005) . the peptides recently designed computationally (han and král, 2020) against the sars-cov-2 has to be synthesized prior to practical application, hence such peptides are not natural and food-grade. the present study attempts to investigate the ability of food-grade nisin a and its natural variants to block the interaction between hace2 and the spike protein of sars-cov-2, a key step of covid-19 disease initiation. nisin, a pentacyclic antibacterial peptide with 34 residues, is produced by certain strains of food-grade lactococcus lactis, widely used for cheese manufacturing (fox and wood, 1971; lubelski et al., 2008; juncioni et al., 2009) . nisin belongs to a group of cationic peptide antimicrobials collectively called type a (i) lantibiotics (smith and hillman, 1016) . it was first identified in fermented milk cultures and is now globally used as a natural and safe food preservative in a variety of food products around the world, such as processed cheese, dairy desserts, milk, fermented beverages, meat and canned foods (hurst, 1981; fons et al., 2009; mitra et al., 2011) . it has been approved by the european union (e234), world health organization (who) as well as by the us food and drug administration (fda). currently, nisin is licensed in over 50 countries (shin et al., 2015) . because of the high safety profile over the past 40 years of usage and its strong antimicrobial action against a wide range of food spoilage and pathogenic bacteria, nisin has been extensively studied. it also has multiple applications in biomedicine including bacterial infections, cancer, oral diseases and other veterinary and research field (shin et al., 2015) . since the discovery of nisin a, eight natural variants of nisin have been discovered which include nisin a, z, f, q, h, u, u2 and p (garcia-gutierrez et al., 2020) . nisin z producing organisms are very common in nature (mitra et al., 2011; vos et al., 1993) . the structures of eight variants of nisin were analyzed in the present study. all nisin peptides were aligned to show their identity and modeled on swiss-model web server. hace2 and rbd domain of 2019-cov-2 were also modeled on the same platform to increase the acceptability of the structures. all the peptides and rbd were docked with hace2 using haddock server. the binding affinity of the peptides was examined by docking analysis based on z-score, binding affinity and buried surface area. structurally, nisin is a unique molecule containing unusual amino acids including dehydroalanine and dehydrobutyrine, formed by dehydration of serine and threonine residues, respectively. these two residues are stereo-and regio-specifically coupled to the thiol group of the cysteines to form lanthionine and β-methyl lanthionine introduced enzymatically at post-translational level (cotter et al., 2012) . nisin is thus a thioether-bridged pentacyclic peptide. the crystal structure of nisin has not been developed. the peptide molecule adopts different conformations depending on the environment. the structure of nisin cannot be described in terms of regular secondary-structure elements, due to the presence of the ring systems in which 65% of the residues are incorporated. however, the nmr structure is available in pdb database, which was used in this study as template to generate the model structures of the nisin variants. the nmr structure of nisin has determined two structured domains: an n-terminal domain (residues 3-19) containing three lanthionine rings, a, b and c; and a c-terminal domain (residues 22-28) containing two intertwined lanthionine rings numbered d and e (hilbers, 1996) . these domains are flanked by regions showing structural flexibility. the four-residue rings b, d and e of nisin all show a β-turn structure, which is closed by the thioether linkage. the backbones of the rings b and d form type i1 β-turns. the c-terminal domain consists of three consecutive β-turns. the nmr data will help us to locate residues in nisin interacting with hace2. the present study attempts to evaluate the potential of nisin variants to interact with hace2 by predicting nisin binding site using nisin-hace2 docking computation with the nmr structure of nisin in the pdb database. this is the first report on the potential of widely used food-grade antibacterial peptide nisin to bind with hace2 and predicting the possibility of nisin as therapeutic against covid-19. the work is significant in finding a solution to prevent the infection by novel coronavirus sars-cov-2. amino acids sequences of eight nisin variants: nisin z ( (park et al., 2019) . esprit 3 software (robert and gouet, 2014) was used to represent the msa using blosum 62 algorithm. homology models of all nisin variants were done using the swissmodel web server (waterhouse et al., 2018) using nisin z (smtl id:1wco.1) as template. the steriochemical property of each of the models was evaluated by ramachandran plot using volume, area, dihedral angle reporter (vadar) server (willard et al., 2003) (fig. s1) . similarly, the rbd (receptorbinding domain) of spike protein of sars-cov-2 and hace2 receptor was modeled using smtl id: 6lzg.1 and smtl id: 6m18.1, respectively. all the models of nisin variants were superimposed together to determine their structural differences using read scoring matrix in pymol software. (the pyolecular graph). molecular docking was performed to test the binding affinity of all nisin variants towards hace2. in order to understand the comparative binding strength, multi-body docking were done between hace2:nisin h complex with the rbd of sars-cov-2 and hace2:rbd complex with nisin h. the solvated docking software, haddock (melquiond et al., 2016) was used without defying any restraints for such study. most reliable model was selected by lowest haddock score value. the score is calculated as where evdw is the intermolecular van der waals energy, eelec the intermolecular electrostatic energy, edesol represents an empirical desolvation energy. active site residues of hace2 (k31, e35, d38, m82) responsible of rbd spike binding were selected for docking. the residues surrounding the active loci were considered as passive. the interacting residues were visualized using discovery studio. (systèmes, 2017) prodigy@bonvin lab web server (xue et al., 2016) was used to calculate δg to predict the affinity of nisin h for hace2 at 25 • c with other parameters remained under default condition. grand average of hydropathy score of hace2 was calculated with exapassyprotparam webserver. (gasteiger et al.walker, 2005) in multiple sequence alignment (fig. 1) of amino acid residues of eight nisin variants (nisin a, z, q, h, p, u, u2 and f), nisin z shared 82.35% amino acid sequence similarity with nisin h, whereas nisin p, u, u2, q and f shared only 70.97%, 67.74%,67.74%, 76.47% and 79.41%, respectively with nisin h (table s2) . nisin a was found to be closely related to nisin z (97.06% identity) with only a single amino acid difference (his27asn). in contrast, nisin h differs from nisin a by five different amino acids at positions 1, 6, 18, 21 and 31 with 85.29% identity. nisin p is shorter than nisin h (34 residues) by three residues from the c-terminus. nisin h differs from nisin f by 7 residues, f1i, m6l, t18g, y21 m, h27 n, i30v and k31h. nisin q is different from nisin h due to the presence of isoleucine, leucine, valine, glycine, leucine, asparagine, valine and histidine at positions 1, 6, 15, 18, 21, 27, 30 and 31, respectively. nisin u and u2 differed from nisin h by ten amino acids. the residual surface accessibility is present at the bottom of the alignment (fig. 1) . the model structures of all nisin variants, hace2, rbd of spike protein built on using swiss-model web server were validated for steriochemical properties using ramachandran plot (fig. s2) . we considered the number of amino acids in the disallowed regions except for glycine and proline because of their chirality and imino group, respectively. homology model of nisin p and u2 had no disallowed amino acids. nisin h and u had only one residue in disallowed region, whereas two residues were found in the disallowed region for nisina, f, q and z. the rmsd (c-alpha) from all the superimposed variants of nisin was found 0.191. these signify that all the nisin models were structurally similar to one another. the binding efficiency of nisins with hace2 was further evaluated from docking studies. best haddock model of nisin variants in complex with hace2 was analyzed for three parameters viz. z-score, buried surface area, and binding affinity. the z-score indicates how many standard deviations from the average of the cluster is located in terms of score (the more negative the better). z-score of hace2-sars-cov-2 rbd, hace2-nisin a, hace2-nisin z, hace2-nisin h, hace2-nisin q, hace2-nisin u, hace2nisin u2, hace2-nisin f, and hace2-nisin p was predicted as − 1.5,-1.6,-1.9,-2.1,-1.4,-1.7,-0.8,-1.4, and − 1.5. hence, both nisin h and nisin z were lowest than rest of the nisin variants as well as rbd of spike protein. burried surface area of nisin z and nisin h with hace2 were found higher, 2332.4 å 2 and 2395.1 å 2 , respectively in contrast to 2092 å 2 for the rbd. this suggests that nisin h and nisin z had better binding efficiency for hace2. the binding affinity of docked structures of all eight variants of nisin in complex with hace2 was calculated as δg derived from analysis with prodigy for each complex in comparison with the rbd of spike protein of sars-cov-2. δg of hace2-sars-cov-2, hace2-nisin a, hace2-nisin z, hace2-nisin h, hace2-nisin q, hace2-nisin u, hace2-nisin u2, hace2nisin f, and hace2-nisin p was − 11 kcal/mol, − 10.6 kcal/mol, − 10.8 kcal/mol, − 11.3 kcal/mol, − 10.5 kcal/mol, − 10.5 kcal/mol, − 12.3 kcal/mol, − 12.5 kcal/mol, and − 11.4 kcal/mol, respectively. thus δg of hace2-nisin z and hace2-nisin h are much higher conferring strong binding affinity than that of hace2-rbd.gravy score of nisin a, z, h, q, u, u2, f, p and rbd-sars-cov-2 was calculated as 0. 415, 0.406, 0.185, 0.524, 0.542, 0.439, 0.171, 0.185, − 0.258, respectively (table 1) . from the gravy score of all nisin variants, nisin h turned out to be more hydrophilic than nisin a and nisin z and will thus more potent to interact with the hydrophobic groove of hace2 than others variants of nisin. from the docking analysis it is evident that nisin z and nisin h interacts to hace2 more efficiently. the interacting residues and atoms are given in (table s1 ). the hydrogen bonds (k31:c19, k31:t13, k31: k12, e35:k12, e35:c19, e35:n20, d38:k22, d38:c27, m82:c7, k353: n27) and hydrophobic bonds (m82:i4, m82:c7, k31:c19, y83:c7, k353: c28) are the major binding force for hace2-nisinz interaction. interacting residues of nisin z was predicted as i4, c7, k12, t13, c19, n20, k22, and c27. all interacting residues of nisin z were hydrophilic in nature. the residues in nisin h interacting with the hace2 include hydrogen bond of t13:k31, c19:k31, k12:k31, t8:k31, p9:k31, k12: e35, k22:d38, n20:e35, c26:d38, h27:d38, c28:k353, t23:k353 (fig. 2) and hydrophobic bond of c19:k31 and y21:k31, c7:m82, a24: k353, c26:k353, c28:k353. among all these interacting residues, t8, p9, c11, k12, t13, c19, k22, c26 were highly conserved among all the nisin variants. like rbd, surface accessible hydrophilic residues, t8, p9, c11, k12, t13, k22, and c26 were found to be involved in binding to hydrophobic groove of hace2. it was found that nisin z and nisin h recognized five common residues (k31, e35, d38, m82, k353) in hace2 that were also recognized by rbd of spike. the binding efficiency of preformed hace2:nisin h complex was performed by competitive tertiary docking with rbd of sars-cov-2 (fig. 3) . as, nisin h had already occupied the active site residues of hace2 with strong hydrogen bond and hydrophobic interactions (table s3) , rbd of sars-cov-2 could not get access the active residues of hace2 with reasonable efficiency by overcoming the binding strength of hace2:nisin h interaction with δg of − 11.3 kcal/mol (binding affinity of rbd:hace2 complex is − 11 kcal/mol). on the contrary when nisin h was allowed to interact with the hace2:rbd complex, it was found that nisin h could be able to interact with active residues (k31 and m82) of hace2 from the hace2:rbd complex (table s4) . nisin h, being more potent candidate could able to interfere in the interaction between rbd-hace2. there is high possibility that nisin would be able to competitively displace bound sars-cov-2 because of its higher binding affinity towards the ace2 receptor compared to that of the virus. furthermore nisin being a non-synthetic molecule and smaller in size, will ensure high bioavailability. based on such study, we hypothesize that nisin h, z, a and u could be an eligible competitor of rbd of sars-cov-2 for having the same binding patch in hace2. recently, several peptides computationally designed to target the spike protein of sars-cov-2 have been reported (han and král, 2020; baig et al., 2020) as a strategy to prevent their interaction with ace 2 receptor for tackling covid-19 infection. from an application perspective, it would be advantageous of using nisin as an effective treatment option over the reported designed peptides for several reasons, including its natural occurrence, food-grade status, extreme stability and ease of manufacturing through microbial fermentation, cost effectiveness, delivery at high concentration, etc. however, further experimental validation is required to confirm nisin binding to hace2. among all analyzed nisin variants, nisin z, nisin a, nisin u and nisin h were most effective in interacting with human endothelial cell surface-receptor hace2, the site where rbd of spike of sars-cov-2 binds to initiate infection. compared to the rbd of viral spike protein, nisin binds with the hace2 receptor with higher affinity. nisin being a low molecular weight peptide and readily bioavailable in the system, its binding to hace2 is expected to over-rule the interaction possibility of the rbd of spike of sars-cov-2 and could essentially exclude the virus entry to the host cell. since nisin is a heat stable natural food grade peptide, can be produced cost effectively, even in large quantity through microbial fermentation, the present work will create greater interest among researchers to develop a new nisin-based treatment strategy for covid-19, either through oral or nasal applications. however, further experimental validation is necessary to determine its doses and mechanistic application to check the competition of nisin and spike protein of sars-cov-2 for accessing the human. the authors declare that they have no conflict of interest. small-molecule inhibitors of protein-protein interactions: progressing toward the reality identification of a potential peptide inhibitor of sars-cov-2 targeting its entry into the host cells bacteriocins -a viable alternative to antibiotics? molecular modeling and chemical modification for finding peptide inhibitor against severe acute respiratory syndrome coronavirus main proteinase microbial ecology in health and disease mechanisms of colonisation and colonisation resistance of the digestive tract part 2: bacteria/bacteria interactions mechanisms of colonisation and colonisation resistance of the digestive tract first evidence of production of the lantibiotic nisin protein identification and analysis tools on the expasy server computational design of ace2-based peptide inhibitors of sars-cov-2 surface location and orientation of the lantibiotic nisin bound to membrane-mimicking micelles of dodecylphosphocholine and of sodium dodecylsulphate introduction, i. a. hurst nisin biotechnological production and application: a review biosynthesis, immunity, regulation, mode of action and engineering of the model lantibiotic nisin the haddock2.2 web server: user-friendly integrative modeling of biomolecular complexes potential application of the nisin z preparation of lactococcus lactis w8 in preservation of milk the embl-ebi search and sequence analysis tools apis deciphering key features in protein structures with the new endscript server structural basis of receptor recognition by sars-cov-2 biomedical applications of nisin therapeutic potential of type a ( i ) lantibiotics , a group of cationic peptide antibiotics precision medicine by designer interference peptides: applications in oncology and molecular therapeutics dassault syst mes biovia properties of nisin z and distribution of its gene , nisz , in lactococcus lactis structural and functional basis of sars-cov-2 entry by using human ace2 swiss-model: homology modelling of protein structures and complexes vadar: a web server for quantitative evaluation of protein structure quality structural bioinformatics prodigy: a web server for predicting the binding affinity of protein-protein complexes we most sincerely acknowledge the research grant (bt/pr16246/ get/119/73/2016) received from dbt, department of biotechnology, govt. of india, new delhi. supplementary data to this article can be found online at https://doi. org/10.1016/j.virol.2020.10.002. rb curated, analyzed and interpreted the data. amg helped in docking studies. s mitra, s mandal and srb supervised the work. all the authors write, review and edited the manuscript. key: cord-331807-ooym5eh3 authors: wu, tao; ge, yiyue; zhao, kangchen; zhu, xiaojuan; chen, yin; wu, bin; zhu, fengcai; zhu, baoli; cui, lunbiao title: a reverse-transcription recombinase-aided amplification assay for the rapid detection of n gene of severe acute respiratory syndrome coronavirus 2(sars-cov-2) date: 2020-07-29 journal: virology doi: 10.1016/j.virol.2020.07.006 sha: doc_id: 331807 cord_uid: ooym5eh3 the current outbreak of coronavirus disease 2019 (covid-19), caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) was reported in china firstly. a rapid, highly sensitive, specific, and simple operational method was needed for the detection of sars-cov-2. here, we established a real-time reverse-transcription recombinase-aided amplification assay (rt-raa) to detect sars-cov-2 rapidly. the primers and probe were designed based on the nucleocapsid protein gene (n gene) sequence of sars-cov-2. the detection limit was 10 copies per reaction in this assay, which could be conducted within 15 min at a constant temperature (39 °c), without any cross-reactions with other respiratory tract pathogens, such as other coronaviruses. furthermore, compared with commercial real-time rt-pcr assay, it showed a kappa value of 0.959 (p < 0.001) from 150 clinical specimens. these results indicated that this real-time rt-raa assay may be a valuable tool for detecting sars-cov-2. rt-raa assays were manually designed based on the sequence of n gene (table 1) . nuclease-free water was used as a negative control. the sensitivity of rt-raa assay for sars-cov-2 was determined using a panel of 124 serially diluted recombinant plasmid ranging from 10 1 to 10 5 copies per reaction. the 125 repeatability of the method was evaluated by testing each dilution 8 times (table 2 ). the minimum detection limit of real-time raa assay was 10 copies / reaction. as as showed in table 3, pathogen genomics in public health use of a rapid 207 reverse-transcription recombinase aided amplification assay for respiratory syncytial virus detection epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in 211 china: a descriptive study detection of 2019 novel coronavirus (2019-ncov) by real-time 214 rt-pcr clinical features predicting mortality 216 risk in patients with viral pneumonia: the mulbsta score genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus 221 origins and receptor binding assessment of the quantitative real-time polymerase chain reaction using a cdna standard for 224 human group a rotavirus notice on novel coronavirus pneumonia diagnosis and treatment plan (trial version fifth 226 revision) a new arenavirus in a cluster of fatal transplant-associated diseases dna detection using recombination 231 proteins 2019 novel coronavirus of pneumonia in wuhan, 233 china: emerging attack and management strategies a rapid 235 and sensitive recombinase aided amplification assay to detect hepatitis b virus without dna 236 extraction multiple-centre clinical evaluation of an ultrafast single-tube assay for sars-cov-2 rna clinical microbiology and infection : the official publication of the european society of clinical 241 microbiology and infectious diseases far-eastern subtype of tick-borne encephalitis virus who, 2020. coronavirus disease 2019 (covid-19) situation report -35 development of a reverse 248 transcription recombinase-aided amplification assay for the detection of coxsackievirus a10 and 249 coxsackievirus a6 rna rapid detection 251 of salmonella with recombinase aided amplification a novel 254 coronavirus from patients with pneumonia in china key: cord-321265-il9vbbgk authors: den boon, johan a.; spaan, willy j.m.; snijder, eric j. title: equine arteritis virus subgenomic rna transcription: uv inactivation and translation inhibition studies date: 1995-11-30 journal: virology doi: 10.1006/viro.1995.0009 sha: doc_id: 321265 cord_uid: il9vbbgk abstract the expression of the genetic information of equine arteritis virus (eav), an arterivirus, involves the synthesis of six subgenomic (sg) mrnas. these are 5′ and 3′ coterminal since they are composed of a leader and a body sequence, which are identical to the 5′ and 3′ ends of the genome, respectively. previously, it has been suggested thatcis-splicing of a genome-length precursor rna is involved in their synthesis. this was reevaluated in a comparative analysis of the sg rna synthesis of eav, the coronavirus mouse hepatitis virus (mhv), and the alphavirus sindbis virus. uv transcription mapping showed that the majority of the eav sg rnas made at later stages of infection is not derived from a genome-length precursor. however, complete independence of sg rna synthesis from that of genomic rna was never observed during the course of infection. the possibility that this resulted from uv irradiation-induced effects on the synthesis of the viral replicase was investigated by inhibiting translation using cycloheximide. for eav, ongoing protein synthesis was found to be more important for the synthesis of sg rna than for that of genomic rna. in general, mhv transcription was extremely sensitive to translation inhibition, whereas eav genomic rna synthesis became independent ofde novoprotein synthesis late in infection. as in the case of coronaviruses, the arterivirus sg rnas are composed of sequences which are not contiguous equine arteritis virus (eav) is the type member of a in the genomic rna (de vries et al., 1990) . a common 5 recently reclassified group of enveloped positiveleader sequence of 206 nt, derived from the 5 end of stranded rna viruses, the arteriviruses (for a review: the genome, is fused to mrna body sequences which plagemann and moennig, 1992) . other arteriviruses are are colinear with the genomic 3 end. for eav sg rnas lactate dehydrogenase-elevating virus of mice (godeny 6 and 7 the so-called leader-body junction site is a penet al., 1993) , porcine reproductive and respiratory syntanucleotide sequence (5 ucaac 3), present at the 3 drome virus (meulenberg et al., 1993a) , and simian hemend of the leader sequence and at the 5 end of the orrhagic fever virus (godeny et al., 1995) . mrna body (de vries et al., 1990) . the sequence analysis of the eav genome previously despite the structural similarities between arterirevealed that arteriviruses are evolutionarily related to and coronavirus sg rnas, their modes of synthesis corona-and toroviruses (den boon et al., 1991) . their were reported to be different. stern and sefton (1982) , common ancestry is illustrated by the presence of a num-jacobs et al. (1981) , and yokomori et al. (1992) have ber of homologous replicase domains and striking simiused uv transcription mapping analyses to investigate larities in genome organization and expression. the eav coronavirus sg rna synthesis. they showed that late genome is a 12.7-kb rna molecule which is structurally in infection the uv sensitivity of the synthesis of the polycistronic (fig. 1) . from this rna open reading frames sg mrnas is not equal to that of the genomic rna. (orfs) 1a and 1b are translated into two large replicase instead, uv target sizes of subgenomic transcripts polyproteins, 1a and 1ab, which are n-terminally identical were concluded to be proportional to their physical but, due to ribosomal frameshifting, c-terminally different size. in contrast, van berlo et al. (1982) reported ap-(den boon et al., 1991) . the more distally located orfs proximately identical uv target sizes for the genomic 2-7, which encode four structural proteins (de vries et rna and all sg rnas of the arterivirus eav. although al., 1992) and two proteins of unknown function, are exa slight deviation was observed for the smallest sg pressed from a 3 coterminal nested set of subgenomic rna, currently known as rna 7, the data suggested a (sg) rnas (van berlo et al., 1982; de vries et al., 1990) . posttranscriptional processing mechanism that generates all sg rnas from a genome-length precursor. nisms would be essentially different. upon reexamination by medium containing 10 mg/ml of dactinomycin to block host rna synthesis. after 30 -60 min, cultures of the experimental details of the previous eav uv transcription mapping analysis (van berlo et al., 1982) , we were incubated for 60 -90 min in medium containing 100 -500 mci of [ 3 h]uridine and 10 mg/ml of dactino-found the applied uv doses to be remarkably low: a maximum dose of 300 ergrmm 02 was used. for compari-mycin. rna lysates were prepared as described previously (spaan et al., 1981) . son, the inactivation of the synthesis of sg rna 2 of the coronavirus mouse hepatitis virus (mhv), which is only 3 kb smaller than the eav genome, required uv doses rna electrophoresis of up to 3000 ergrmm 02 (jacobs et al., 1981) . in view of denaturing rna electrophoresis was carried out using these observations, we decided to repeat the experi-1-1.5% agarose gels containing 10 mm mops (morpholiments. a comparative analysis on the basis of new uv nepropanesulfonic acid) and 2.2 m formaldehyde. for transcription mapping data for eav, mhv, and the analysis of 3 h-labeled rna samples, agarose gels were alphavirus sindbis virus (sin) is described in this paper. fixed in methanol, impregnated with 3% ppo (2,5-diphe-the latter was chosen as a reference because the genyloxazole) in methanol, rinsed with water, and dried at nome sizes of eav and sin are similar and because the 60њ. after fluorography, incorporation of label into individsynthesis of the sg 26s rna of sin is known to be fully ual rna species was quantified by excision of bands independent from that of the genome (brzeski and kenfrom dried gels and scintillation counting. nedy, 1978). in the context of these experiments, we have also studied the effect of translation inhibition by cyclo-uv transcription mapping heximide on the synthesis of viral rna. were infected with an m.o.i. of 50. at 6 1 2 hr p.i., the me-cells and viruses dium was removed, and cells were uv-irradiated for various intervals with a dose rate of 40 ergrs 01 rmm 02 equine arteritis virus (bucyrus strain) and sin (hr strain) were grown in baby hamster kidney cells (bhk-(the total dose was between 0 and 3600 ergrmm 02 ). of rna synthesis after t sec of irradiation, n 0 that of the dose of 2400 ergrmm 02 at 4, 5, or 6 hr p.i., and metabolically labeled rna was subsequently analyzed as de-unirradiated control, and k is a constant (sauerbier and hercules, 1978) . curves were fitted using linear regres-scribed. sion analysis. inhibition of protein synthesis in a separate experiment, the effect of uv irradiation at different time points in infection was investigated. eav-protein synthesis was inhibited by replacing the medium with medium containing 100 mg/ml of cyclohexi-infected bhk-21 cells were uv-irradiated with a single spaan et al. (1988) , and strauss et al. (1984) . such as the sg rnas found in eav-infected cells, are derived from the same precursor molecule. if so, the sensitivity of their synthesis to uv irradiation (uv target the well-characterized alphavirus sin, using the same size) will for all sg rnas be identical to the target size irradiation doses and the same bhk-21 cells used for of that precursor. alternatively, when the transcription the eav analysis. the length of the 49s sin genomic of sg rnas is fully independent from that of the ge-rna (11.7 kb) is comparable to that of eav (12.7 kb) (fig. nome, a uv target size which is proportional to its physical size should be measured for each sg rna. 1). the replication of sin involves the synthesis of a eav-infected bhk-21 cells were uv-irradiated at 6 1 2 hr single 4.1-kb sg rna (26s) from a well-defined internal promoter on the genome-sized minus-strand template p.i., when rna synthesis approaches its maximum (van rna (ou et al., 1982; levis et al., 1990) . as shown in fig. berlo et al., 1982 , and data not shown). subsequently, viral rna synthesis was monitored by [ 3 h]uridine incor-2 and table 1 , the relative physical and uv target sizes poration. in parallel, 3 h-labeled rna was isolated from of the genomic and sg sin transcripts were in good mhv-infected cells which were uv-irradiated at 6 hr p.i. agreement, as was previously shown by brzeski and ken-to determine the uv inactivation kinetics for individual nedy (1978) . in addition to this difference with the corona-rna species, 3 h-labeled rnas were separated in denaand arterivirus systems, sin transcription in general was turing agarose gels. representative fluorographs for both less sensitive to uv irradiation than eav and mhv tranviruses are shown in fig. 2a . for both eav and mhv, scription. we restricted our analysis to the genomic rna and three sg rnas (2, 6, and 7). bands were cut from four agarose uv irradiation has differential effects early and late in gels, derived from two independent experiments. in fig. eav infection 2b the average percentages of remaining rna synthesis have been plotted against the uv dose. these graphs yokomori et al. (1992) showed that during the early clearly show that for both viruses the sg rna synthesis stages of mhv infection the uv target sizes of sg rnas is not directly dependent on the synthesis of a genomeapproach that of the genomic rna. this observation length precursor rna. on the other hand, the uv target prompted us to compare the uv target sizes of the eav sizes of the rnas, which are reflected by the slopes of transcripts at three different time points in infection. in the curves, are not proportional to their physical sizes fig. 3 the reduction in the synthesis of rnas 1, 2, 6, and either (table 1) . compared to the genomic rna, the syn-7 is compared after uv irradiation with a single dose thesis of sg rnas of both eav and mhv is more sensitive (2400 ergrmm 02 ) at 4, 5, or 6 hr p.i. and subsequent to uv irradiation than would be expected on the basis metabolic labeling for 1 hr. quantitative analysis on the of their lengths. basis of two experiments showed that, similar to the situation in coronavirus-infected cells, uv sensitivities sindbis virus uv transcription mapping during early and late transcription were different. upon irradiation at 4 or 5 hr p.i. the relative decrease in the to investigate whether the discrepancy between relasynthesis of the genomic and sg rnas was similar, tive physical sizes and uv target sizes of genomic and whereas after irradiation at 6 hr p.i. sg rna synthesis sg rnas is typical of arteri-and coronavirus transcription, a uv transcription mapping analysis was carried out for was less affected than that of rna 1 (fig. 3b) . sis. this was irrespective of the stage of infection. as previously reported by sawicki and sawicki (1986) , this transcription differential dependence was not observed for mhv. the uv transcription mapping data showed that the third, the overall mhv transcription was significantly eav sg rnas were not produced by processing of a more dependent on de novo protein synthesis than that genome-length precursor rna. however, unlike the case of eav and sin. even during the late phase of the mhv of the sin sg rna, their uv target sizes were larger than infection cycle, when all infected cells were fused into expected on the basis of their physical sizes, even when one large syncytium, both genomic and sg rna synthesis sg rna synthesis had reached its maximum. the genowere severely impaired by the addition of cycloheximide. mic rna encodes the viral replicase, which is involved in contrast, eav genomic rna synthesis was nearly in both genomic and sg rna synthesis. thus, its inactivatranslation-independent late in infection. in the case of tion as a transcription unit by uv irradiation could have sin, the transcription of 49s genomic rna was less afan important side effect: reduction of the amount of fected than that of 26s sg rna during the earlier stages mrna for replicase synthesis. depending on the requireof infection, but the synthesis of both became largely ment of de novo replicase synthesis for sg rna transcriptranslation-independent as infection progressed. late in tion, it was feasible that the uv inactivation of genomic infection, 26s rna transcription was even less affected rna synthesis indirectly influenced the production of sg by translation inhibition than 49s transcription. rnas, which would therefore never become completely independent. discussion we investigated to what extent eav rna synthesis depended on continuous protein synthesis and whether the eav uv inactivation experiments described in this paper show that at the peak of sg rna synthesis conven-the uv transcription mapping results could be explained by a combined effect on transcription and replicase tional cis-splicing, if at all, is not a major mechanism in the production of the eav sg mrnas. however, in a translation. protein synthesis was inhibited by the addition of cycloheximide to eav-infected bhk-21 cells at fully independent transcription system the slopes of the curves in fig. 2b , which reflect the uv target sizes, should different time points after infection. one hour later, the effect of translation inhibition on rna synthesis was be directly proportional to the physical sizes of the transcription units. in that case, the transcription of e.g., the measured by metabolic labeling. rnas were isolated and analyzed by denaturing gel electrophoresis (fig. 4) . for eav genome (12.7 kb) would be expected to be 18 times more sensitive to uv irradiation than the synthesis of the comparison, similar analyses with mhv and sin were carried out. smallest sg rna, rna 7 (0.7 kb). it is clear that the relative rna sizes and relative uv sensitivities (table 1 ) on the basis of these experiments a number of conclusions could be drawn. first, ongoing protein synthesis do not support the conclusion that the transcription of the eav genomic and sg rnas are fully independent. was most important during the early phase of infection. second, compared to genomic rna synthesis, eav sg a similar analysis of our mhv data (table 1 ) also revealed that the transcription of the sg rnas of coronavi-rna synthesis was more dependent on protein syntheruses is not fully independent from that of genomic rna. been published by jacobs et al. (1981) and yokomori et al. (1992) . both groups reported differences between the as for eav, there are clear differences in uv sensitivity between larger and smaller rnas, but the quantitative uv target sizes of the various mhv rnas which are in the same range as our observations. for example, the mhv analysis shows differences between relative physical sizes and relative uv sensitivities. our results are in uv target size of mhv rna 7 calculated by jacobs et al. (1981) is only about eight times smaller than that of geno-general agreement with those which have previously mic rna, whereas the physical size ratio is about 1:18. due to the fact that at that time the length of the mhv genome was assumed to be only 16 kb, instead of the currently known 31 kb, jacobs et al. (1981) concluded that their data supported fully independent transcription of mhv genomic and sg rnas. yokomori et al. (1992) , who recently repeated the mhv uv transcription mapping experiments, did not describe a detailed quantitative analysis of the data they obtained late in infection, but concluded that uv sensitivities roughly paralleled mrna sizes. however, a calculation using the graphs published by yokomori et al. (1992) revealed only a sevenfold difference between the genomic rna and rna 7. similar observations were made when we reevaluated the data obtained with another coronavirus, infectious bronchitis fig. 5 . the synthesis of sg rnas 2, 6, and 7, relative to that of the genomic rna, during different time intervals throughout infection. data virus (stern and sefton, 1982) . sulted in an almost perfect correlation between physical rna sizes and uv target sizes ( fig. 2; table 1 ). these results demonstrate that the experimental approach was models have been put forward, which are not necessarily mutually exclusive (jeong and makino, 1994; sawicki and appropriate and that our observations for eav cannot be explained by technical imperfections. apparently, the sawicki, 1990; van der most et al., 1994) . leader-primed transcription could, for example, generate a first genera-intrinsic properties of coronavirus-like rna transcription are different from those of alphaviruses. tion of sg mrnas, which could subsequently function as templates for sg minus-strand production. these could although cis-splicing events cannot be ruled out as an early-stage mechanism to produce sg rnas, another in turn be used to transcribe a second generation of positive-strand sg mrnas. the recent analysis of the transcription mechanism must be operating in eav-infected cells and is most likely similar to that of coronavi-transcriptional activity of a number of mhv ts-mutants indicates that sg minus strands are likely to be used ruses. the so-called leader-primed transcription model has been proposed to explain the synthesis of coronavi-as transcription templates late in infection (schaad and baric, 1994) . like the uv transcription mapping results rus sg rnas (baric et al., 1983; spaan et al., 1983 ) and a substantial amount of data in its favor has been re-described for mhv (yokomori et al., 1992) , our data on eav indicate that different mechanisms of sg rna tran-ported (for a review: spaan et al., 1988; lai, 1990) . according to this model, sg rnas are synthesized either scription might be operating simultaneously. the differences in uv sensitivity of sg rna synthesis early and by an intrinsic ability of the viral leader/polymerase complex to dissociate from its template and subsequently late in infection (fig. 3) can be interpreted to reflect the levels at which each of these mechanisms participate in resume synthesis at a more distal position or by priming by free leader transcripts at subgenomic promoter se-transcription, at a given stage in infection. we have considered the possibility that replicase syn-quences (the complements of the so-called intergenic sequences on the positive strand). the conserved thesis was affected by uv irradiation and that genomic and sg rna synthesis were inhibited as a result. differen-leader-body junction sequences in eav sg rnas (de vries et al., 1990; den boon et al., manuscript in preparatial effects of translation inhibition early vs late in infection (fig. 4 ) may be based on the properties as well as tion) possibly represent such complementary promoter sequences. similar sequence elements have been de-the absolute amount of the replicase. our results and previously published data from sawicki and sawicki scribed for other arteriviruses (godeny et al., 1993; conzelmann et al., 1993; chen et al., 1993; meulenberg et (1986) on mhv rna transcription indicated that ongoing protein synthesis is an absolute requirement during the al., 1993b; zeng et al., 1995) . a single genome-length minus-strand rna could be the template used for the early stages of infection and that both mhv genomic and sg rna synthesis at later stages were still significantly synthesis of genomic as well as sg mrnas. nevertheless, anti-leader-containing subgenomic minus strands reduced when translation was blocked. in contrast, the eav data indicated that the synthesis of sg rnas was in coronavirus-infected cells have been demonstrated (hofmann et al., 1990; sethna et al., 1989 sethna et al., , 1991 . their more impaired by inhibition of translation than was the case for the production of genomic rna. in addition, the presence in double-stranded replicative intermediates suggests that these might function in mrna transcription data presented in figs. 3 and 4 revealed that sg rna synthesis is down-regulated relative to the synthesis of (sawicki and sawicki, 1990 ). on the basis of these and other observations several coronavirus transcription genomic rna late in infection (fig. 5) . coronavirus minus-strand synthesis and effect of cycloheximide on coronavirus rna synthesis of replicative intermediate rna of mouse hepatitis virus: presence of leader rna sequences on nascent chains coronavirus transcription: subgenomic mouse hepatitis virus replicative intermediates function ruses: structure and genome expression genetics of mouse hepatitis kinetics of inactivation of infectious bronchitis virus rna synthesis virus transcription: evidence that subgenomic negative strands are by uv light 92-copies of replicating coronavirus mrnas contain anti-leaders 491-562. mic minus-strand rnas and the potential for mrna replicons 5755-5764. mic rna synthesis directed by a synthetic defective interfering rna spaan coronavirus mrna 424-434. transcription: uv light transcriptional mapping studies suggest an early requirement for a genomic-length template sis of simian hemorrhagic fever virus (shfv) subgenomic rnas, junction sequences, and 5 leader brzeski, h., and kennedy, s. i. t. (1978) . synthesis of alphavirus-speci-whether our findings are indicative of a functional polyfied rna. j. virol. 25, [630] [631] [632] [633] [634] [635] [636] [637] [638] [639] [640] merase switch, from synthesizing sg rna to producing chen, z., kuo, l., rowland, r. r. r., even, c., faaberg, k. s., and plagegenomic rna, remains to be investigated. similar to the mann, p. g. w. (1993) . sequence of 3 end of genome and of 5 regulatory function of nonstructural polyprotein processing end of orf 1a of lactate dehydrogenase-elevating virus (ldv) and common junction motifs between 5 leader and bodies of seven in alphavirus replication (for a review: strauss and strauss, subgenomic mrnas. j. gen. virol. 74, [643] [644] [645] [646] [647] [648] [649] [650] [651] [652] [653] [654] [655] [656] [657] [658] [659] [660] 1994), proteolytic processing may influence the transcripconzelmann, k. k., visser, n., woensel, p. v., and thiel, h. j. (1993) . an alternative explanation for the observed difference in 3339-3346. sensitivity to translation inhibition between genomic and lai, m. m. c. (1990) . coronavirus-organization, replication and expression of genome. annu. rev. microbiol. 44, sg rna synthesis could be a demand for one or more levis, r., schlesinger, s., and huang, h. v. (1990) . promoter for sindbis relatively instable host protein factors involved in sg rna virus rna-dependent subgenomic rna transcription. j. virol. 64, transcription, the synthesis of which might be affected key: cord-345088-krb1eidw authors: shen, s; law, y.c; liu, d.x title: a single amino acid mutation in the spike protein of coronavirus infectious bronchitis virus hampers its maturation and incorporation into virions at the nonpermissive temperature date: 2004-09-01 journal: virology doi: 10.1016/j.virol.2004.06.016 sha: doc_id: 345088 cord_uid: krb1eidw the spike (s) glycoprotein of coronavirus is responsible for receptor binding and membrane fusion. a number of variants with deletions and mutations in the s protein have been isolated from naturally and persistently infected animals and tissue cultures. here, we report the emergence and isolation of two temperature sensitive (ts) mutants and a revertant in the process of cold-adaptation of coronavirus infectious bronchitis virus (ibv) to a monkey kidney cell line. the complete sequences of wild type (wt) virus, two ts mutants, and the revertant were compared and variations linked to phenotypes were mapped. a single amino acid reversion (l(294)-to-q) in the s protein is sufficient to abrogate the ts phenotype. interestingly, unlike wt virus, the revertant grows well at and below 32 °c, the permissive temperature, as it carries other mutations in multiple genes that might be associated with the cold-adaptation phenotype. if the two ts mutants were allowed to enter cells at 32 °c, the s protein was synthesized, core-glycosylated and at least partially modified at 40 °c. however, compared with wt virus and the revertant, no infectious particles of these ts mutants were assembled and released from the ts mutant-infected cells at 40 °c. evidence presented demonstrated that the q(294)-to-l(294) mutation, located at a highly conserved domain of the s1 subunit, might hamper processing of the s protein to a matured 180-kda, endo-glycosidase h-resistant glycoprotein and the translocation of the protein to the cell surface. consequently, some essential functions of the s protein, including mediation of cell-to-cell fusion and its incorporation into virions, were completely abolished. the coronaviridae family contains causative agents of a wide spectrum of diseases affecting humans, mammals, and birds. it is the etiologic agent of severe acute respiratory syndrome (drosten et al., 2003; fouchier et al., 2003) . the molecular basis of host specificity and tissue or cell tropism of coronavirus partially resides in the specific interaction between the surface spike protein and cellular receptor(s). this interaction would determine the outcome of infection: whether it is acute or chronic and persistent (rowe et al., 1997a (rowe et al., , 1997b , which organs or tissues are targeted, what symptoms are shown (ballesteros et al., 1997; leparc-goffart et al., 1997; navas et al., 2001) , and even whether host specificity is changed (kuo et al., 2000) . coronaviruses are a group of enveloped rna viruses with positive-sense, single-stranded genomes of 27 to 32 kb that are packaged into helical structures by the nucleocapsid protein (n). the viruses acquire their envelopes by budding of the structural components into the intermediate compartment (ic) between the endoplasmic reticulum (er) and the golgi apparatus (krijnse-locker et al., 1994; lai and cavanagh, 1998; tooze et al., 1984) . the membrane glycoprotein (m) and the envelope protein (e) mediate the formation of the envelope, as they are involved in the induction of virion assembly and incorporation of other structural components into virus particles (vennema et al., 1996) . the spike (s) glycoproteins incorporate into the envelope (de haan et al., 1999) in the form of homooligomeric, 20 nm projections, giving the distinctive morphology of the coronavirus virion. some coronaviruses also contain another envelope protein, the hemagglutinin esterase (he). the largest structural s protein is a type i integral membrane glycoprotein and spans the viral envelope once. in some cases, the s protein is proteolytically cleaved into the n-terminal s1 and the c-terminal s2 subunits of equal size by a host proteinase . the s1 and s2 subunits are noncovalently associated to each other. a hydrophobic transmembrane domain (tm) of s2 anchors on the envelope with a short cytoplasmic-tail inside. one leucine-zipper (heptad repeats) domain overlaps with the tm domain and another extends outside the envelope, folding into a-helices and coiled-coil structures involved in oligomerization (de groot et al., 1987) . it was found that the tm domain and endodomain mediated the incorporation of s into virions but the details of the s/m interaction remain to be elucidated. although s protein is dispensable for the formation of virus-like-particles (vlps), its incorporation into virion is critical to the assembly of infectious virus particles. the s protein is cotranslationally n-glycosylated in the er and also forms stable complex with m in the pre-golgi membrane (holmes et al., 1981; opstelten et al., 1995; sturman et al., 1985) . it was then transported to the golgi apparatus where its high mannose side chains are subsequently trimmed and further modified. the matured s protein is an endo-glycosidase-h-resistant (endo-h), 180-kda form (luytjes et al., 1997) . at the trans-golgi, the matured virions are encapsulated into vesicles of the constitutive secretory pathway and released from infected cells. a portion of s protein might be transported to the plasma membrane and responsible for the cell-to-cell membrane fusion, resulting in the formation of typical syncycia and spread of virus infection to neighboring cells. the tm, cysrich and leucine-zipper domains of s2 and the disparate region of s1 are associated with the membrane fusion activity of the protein (chang et al., 2000; gallagher et al., 1991; krueger et al., 2001; luo et al., 1999; tsai et al., 1999) . cleavage of the s protein enhances the membrane fusion, though not necessarily required. the s protein contains receptor-binding domains in the s1 subunit (kubo et al., 1994; godet et al., 1994; saeki et al., 1997; suzuki and taguchi, 1996) . upon binding of the s protein to the receptor(s), conformation changes induce the virus -cell membrane fusion and subsequently unloading of the ribonucleocapsid inside the cell. it has been demonstrated that the cell-to-cell spread of mhv infection does not need the presence of the receptor, but the s protein is required to mediate the cell-to-cell fusion (gallagher et al., 1992; nash and buchmeier, 1996) . for coronaviruses, however, the detail of molecular mechanisms of membrane fusion, especially those linked to the s1 region, is yet to be fully elucidated. in this study, ts mutants were generated by growing the beaudette strain of ibv at progressively lower temperatures from 35 to 28 jc in vero cells. two ts mutants were isolated from passages grown at 29 jc (ts291602) and 28 jc (ts282902). a revertant was also obtained by growing the ts mutant at the nonpermissive temperature (40 jc). sequence comparison revealed that mutations in the s1 subunit were responsible for the ts phenotype. comparative studies of viral protein synthesis and growth properties of the wt, ts mutant, and revertant viruses demonstrated that the ts mutants were extremely unstable at the nonpermissive temperature. if they were allowed to enter cells by absorption for 1 h at the permissive temperature (32 jc), the s protein would be core-glycosylated and partially modified at the nonpermissive temperature, but was unable to assemble into virions. the membrane fusion activities of the ts mutants were totally abolished in virus-infected cells and in cells overexpressing the mutant s protein at the nonpermissive temperature. the beaudette strain of ibv, grown in vero cells at 37 jc, was plaque-purified and was initially adapted to grow at 35 jc. after 4 passages, the virus was subsequently adapted to grow at progressively lower temperatures by culturing at 34 jc for 5 passages, 33 jc for 7 passages, 32 jc for 44 passages, 30 jc for 12 passages, 29 jc for 21 passages, and 28 jc for 29 passages. when the virus was initially adapted to a lower temperature, no cpe was observed within 72 -96 h postinfection. apparent cpe appeared usually after 2 to 5 passages. the virus was continuously passaged at the same temperature until cpe became visible within 24 -48 h postinfection, and then shifted to a lower temperature. plaque assays of different passages were performed at both 32 and 40 jc. it was found that viruses from early passages grown at 32 jc could form plaques at both temperatures at similar levels. but passages grown at 29 jc produced much fewer plaques at 40 jc than at 32 jc, suggesting the emergence of ts mutants during cold-adaptation. these ts mutants dominated quickly at 28 jc. two ts mutants, designated ts291602 and ts282902, were plaque-purified from passage 16 grown at 29 jc and passage 29 grown at 28 jc, respectively. plaque assays showed that both ts291602 and ts282902 formed plaques only at the permissive temperature, but not at all at the nonpermissive temperature. by growing the ts mutant at 37 jc overnight and then at 40 jc for 2 days, a revertant, rev-1, was obtained which produced plaques at both 32 and 40 jc at a similar level ( table 1 ). the titers of rev-1 were much higher than that of the wild-type virus wt6501 (isolated from passage 65 at 37 jc) at 32 jc, as it was derived from the cold-adapted virus (table 1) . to map the defects responsible for the ts phenotype described above, the complete nucleotide sequences of wt6501, ts291602, ts282902, and rev-1 were determined using rt-pcr products and cdna clones from each virus. comparison of the sequence of wt6501 with ts291602 revealed 22 point mutations, a 5-base-deletion, a 1-base-, 3-base-and a 9-base-insertion (table 2 ). these alterations caused 15 amino acid substitutions in eight mature viral proteins, a single amino acid insertion in the 3a protein, a truncated 3b (compared with early passages of wt virus) caused by a single base insertion in the 3b of ts291602, and a 3-amino acid insertion in the n protein. in ts282902, similar point mutations, deletion and insertion were found ( table 2 ). the most different feature of the two ts mutant is the insertion in the 3b gene. in ts291602, a single a insertion was found at a poly-a stretch at positions 24075 to 24080, causing frameshift of the 3b gene and resulting in a c-terminally truncated 3b protein (shen and liu, 2003) . however, a three-a-insertion at the same poly-a stretch was found in ts282902, which does not affect the open reading frame of the 3b gene. comparison of the sequence of rev-1 with the two ts mutants showed only one amino acid change (q 294 -to-l 294 ) in the s protein and one amino acid deletion in the nonstructural protein 3a (i 28 ), which exists in both ts291602 and ts 282902 but not in rev-1 and wt virus. the genotype of the rev-1 at these two positions (q 294 and i 28 ) are therefore the same as that of wt6501, but different from the two ts mutants. as both wt virus and the revertant could form plaques at 32 and 40 jc, these two mutations might be responsible for the ts phenotype. a blast search of genbank clearly showed that the amino acid at position 294 of the s protein was a conserved glutamine. the leu 294 residue in the two ts mutants is located immediately downstream of a hypervariable region and the upstream of a conserved region of the s1 subunit. one additional mutation, d 709 -n 709 , was found in ts291602 only, and an i 769 -m 769 mutation in the two ts mutants and rev-1. after mapping the potential determinants responsible for the ts phenotype, we would like to explore the mechanisms by which these mutations affect the propagation of the ts mutants at the nonpermissive temperature. synthesis of the s protein in cells infected with wt virus, ts mutants, and revertant was analyzed. two sets of experiments were carried out. first, vero cells were infected with wt6501, ts291602, ts282902, and rev-1 at 32 and 40 jc, respectively, and lysates were immunoprecipitated with anti-ibv antibodies. it was obvious that synthesis of the ts mutant s intergenic region between m and 5a 25,346 table 1 titers of wild type, mutant and revertant viruses at 32 jc and 40 jc (pfu/ml) titer 32 jc 4 0 jc wt6501 8.5 â 10 3 5.5 â 10 6 ts291602 2.0 â 10 6 0 ts282902 3.2 â 10 6 0 rev-1 7.0 â 10 6 5.5 â 10 6 protein was detectable at 32 jc but not at 40 jc (fig. 1a , lanes 3, 4, 5, and 6) under these conditions. in contrast, expression of the s protein was observed in cells infected with wt6501 and rev-1 at both temperatures ( fig. 1a , lanes 1, 2, 7, and 8), though the level of the proteins detected in cells infected with wt6501 was much lower at 32 jc. this was consistent with the lower titers of wt virus at 32 jc. in the second set of experiments shown in fig. 1b , cells were absorbed with each virus at 32 jc for 1 h and one of the duplicates was shifted to 40 jc. under these conditions, the two ts mutant s proteins were synthesized at both 32 and 40 jc (fig. 1b, lanes 3 , 4, 5, and 6), like those of the wt virus and revertant (fig. 1b , lanes 1, 2, 7 and 8). these results indicated that the viral structural protein could be synthesized at nonpermissive temperature if the mutants were allowed to enter cells. the viral subgenomic rna and one of the processed proteins, the 3c-like proteinase, were also detected in the same ways (data not shown), and the results were consistent with those described above. comparison of the nucleotide sequences and the s protein synthesis of wt, ts mutants and revertant indicate that the single amino acid mutation (q 294 -l 294 ) in the s protein may be responsible for the temperature sensitivity of the mutant virus. this possibility was studied by biochemical and functional characterization of the s protein from the revertant and a ts mutant. the s gene of rev-1 and ts291602 was cloned under the control of a t7 promoter to investigate whether membranefusion activity was affected by the mutations in the s protein. cells were infected with recombinant vaccinia/t7 virus and were transfected with plasmids containing the s gene from either ts291602 or rev-1. the expression of the s proteins was analyzed by western blotting using anti-ibv antibodies. as shown in fig. 2a , the s protein of both revertant and ts mutant was expressed at 32 and 40 jc (lanes 3, 4, 5, and 6), though the s protein of ts291602 was expressed at a relatively lower level than the rev-1 at 40 jc (compare lanes 4 and 6). the fusion activity of the s protein derived from the revertant and ts291602, respectively, was then examined. as shown in fig. 2b , membrane fusion was observed in cells expressing the s gene of ts291602 at 32 jc (panel c) but not at 40 jc (panel f), at 48 h posttransfection. in contrast, membrane fusion was observed in cells expressing the s gene of revertant at both 32 and 40 jc (panels b and e). interestingly, membrane fusion appeared at 72 h posttransfection, after the cells expressing the s gene of ts291602 were shifted from 40 to 32 jc at 48 h posttransfection (panel i). the recombinant s genes of the ts mutant and revertant were transfected into cells in 35 mm dishes at 32 jc and one of the duplicates was shifted to 40 jc at 4 h posttransfection. radiolabeled proteins were immunoprecipitated and treated with endo-h at 37 jc. as shown in fig. 3a , the ts mutant s protein, synthesized at 40 jc, was sensitive to endo-h digestion, as no 180-kda form was observed (lane 12). in contrast, a portion of the ts mutant s protein, synthesized at 32 jc (lanes 10), was resistant to endo-h digestion. the revertant s protein, synthesized at both 32 and 40 jc, was also resistant to endo-h digestion (lanes 6 and 8). these a b fig. 1 . analysis of the expression of the s protein from wt (wt6501), ts mutants (ts291602 and 282902) and revertant (rev-1) viruses. (a) cells were infected with each virus at 32 jc and 40 jc as indicated on the top for 1 h and were maintained at the same temperatures. (b) two dishes of cells were infected with each virus at 32 jc for 1 h. one of the duplicates was maintained at 32 jc (lanes 1, 3, 5, and 7) and the other one was shifted to 40 jc (lanes 2, 4, 6, and 8). radiolabeled cell lysates were immunoprecipitated with anti-ibv antibodies. the proteins were separated on 12.5% polyacrylamide gels and detected by autoradiography. numbers on the left indicate molecular mass in kilodalton and the position of the s protein is indicated on the right. results indicate that the ts mutant s protein synthesized at 40 jc was not a mature form of the glycoprotein. in addition, a novel glycosylated form of the s protein, migrating between the endo-h treated 130-kda and the matured 180-kda forms, was observed. it might represent trimming of an initial, core-glycosylated form of the s protein in the er and the cis-golgi. interestingly, this band was also observed in ts291602-infected cells (fig. 3b, lane 2) , when the incubation time at 32 jc was extended before the culture was shifted to 40 jc. this band is much stronger than the 180-kda form (lane 2), giving direct evidence that the mutations in the s protein of ts291602 dramatically hampered the maturation process of the s glycoprotein. compared with ts282902, the s protein of ts291602 contains another amino acid substitution, d 709 -n 709 (table 1) , which is not present in the s protein of rev-1 and wt6501. as this band was not observed in ts281602-infected cells (data not shown), it is not clear at this point if this additional mutation may also play a role in the maturation of the s protein. it was also noted that, at 40 jc for all viruses, the s1 and s2 subunits were hardly detected in cell lysates but abundant in the supernatants (fig. 3b) . at the nonpermissive temperature, the s protein was not cleaved at all (lanes 2 and 6). these results may suggest that only the mature form of the s glycoprotein could be cleaved into s1 and s2 and fig. 2 . membrane fusion activity of the s protein expressed from wt and ts291602. (a) vero cells were transfected with plasmids without insert (lanes 1 and 2) or with the s gene from the wt (lanes 3 and 4) and ts291602 (lanes 5 and 6), and were cultured at 32 jc (lanes 1, 3, and 5) or 40 jc (lanes 2, 4, and 6). the expression of the s protein was examined in western blot using anti-ibv antibodies, and h-tubulin was immunostained as loading controls. (b) the membranefusion activity of the s protein from the wt (panels b, e, and h) and ts mutant (panels c, f, and i) at 32 jc (panels b and c) and 40 jc (panels c and f) 2 days post-transfection was compared. panels h and i show induction of membrane fusion on cells transfected with the wt and ts291602 by shifting cells shown in panels e and f to 32 jc for 1 day. panels a, d, and g show cells transfected with empty plasmids. reinforce the conclusion that the majority of the s protein of the ts mutants synthesized at the nonpermissive temperature may represent a pre-mature form of the s glycoprotein. to detect whether the ts mutant s protein would be transported to the plasma membrane at the nonpermissive temperature, the s protein was transiently expressed in vero cells using the vaccinia virus-t7 expression system. the transfected cells were treated with cycloheximide at 3.5 h posttransfection for 30 min to inhibit further protein synthesis. the cells were then incubated at 32 and 40 jc, respectively. the subcellular localization of the s protein was analyzed by indirect immunofluorescent staining at 4, 8, 16, and 24 h postinfection, respectively. the confocal microscopy images of transfected cells are shown in fig. 4 . plasma membrane staining of the ts mutant s protein was not observed at any time points posttransfection at 40 jc, but was clearly seen at 32 jc at 16 and 24 h postinfection (fig. 4) . the revertant s protein was expressed on the cell surface at both temperatures at 16 and 24 h posttransfection. these results may explain the lack of membrane fusion activity of the ts mutant s protein in both infected and transfected cells. endo-h treatment of the s protein from rev-1 and ts291602. the s protein derived from rev-1 (lanes 5, 6, 7, and 8) and ts291602 (lanes 9, 10, 11, and 12) were expressed in vero cells at 32 jc (lanes 5, 6, 9, and 10) and 40 jc (lanes 7, 8, 11, and 12), using a t7-vaccinia expression system. the radiolabeled proteins were immunoprecipitated with anti-ibv antibodies, the eluted proteins were endo-h-(lanes 6, 8, 10, and 12) or mock-treated (lanes 5, 7, 9, and 11) and analyzed by sds-page. (b) detection of the less matured s protein from ts291602 at 40 jc and the defect in cleavage of the mutant s protein. cells were infected with ts291602 and rev-1 for 5 h at 32 jc, one of the duplicates was maintained at 32 jc (lanes 1, 3, 5, and 7), and the other one was shifted to 40 jc (lanes 2, 4, 6, and 8). cells were radiolabeled and viral proteins were immunoprecipitated with anti-ibv antibodies and analyzed by sds-page. lanes 1, 2, 3, and 4 refer to viral products detected from cell lysates and lanes 5, 6, 7, and 8 refer to viral proteins detected from virus particles released to the cultured media. quantitative analysis of surface expression of the wild type and ts mutant s protein was carried out by immunofluorescent staining with anti-s protein antiserum and the positive staining cells were sorted by flow cytometry. as shown in fig. 5 , 5.5% of nonpermeabilizing (panel a) and 0.7% of permeabilizing (panel f) cells expressing the empty plasmid showed background staining. when the cells were incubated at the permissive temperature, 3.8% (9.2 -5.5) of hela cells expressing the wt s protein (panel b) and 4.1% (9.6 -5.5) of cells expressing the ts mutant s protein (panel c) displayed surface staining. after permeabilizing with 0.1% saponin, 11.8% (12.5 -0.7) of cells expressing the wt s protein (panel g) and 10.9% (11.6-0.7) of cells expressing the ts mutant s protein (panel h) showed positive staining. when the cells were incubated at the nonpermissive temperature, 10.4% (15.9 -5.5) of cells expressing the wt s protein (panel d) and 2.3% (7.8 -5.5) of cells expressing the ts mutant s protein (panel e) exhibited surface staining. after permeabilizing with 0.1% saponin, 9.3% (10 -0.7) of cells expressing the wt s protein (panel i) and 9.7% (10.4 -0.7) of cells expressing the ts mutant s protein (panel j) showed positive staining. these results confirm that the ts mutant s protein could not be efficiently translocated to the cell surface at the nonpermissive temperature. to investigate whether the s protein of ts mutant was assembled into virion or not, ts291602 and rev-1-infected cells were radiolabeled and the virus particles were purified through sucrose gradients twice. immunoprecipitation of the purified virions using anti-ibv antibodies showed that the s protein of ts291602 was not detected at 40 jc (fig. 6 , lane 2), but was observed at 32 jc (fig. 6, lane 1) . the s protein was detected at both temperatures for rev-1 (fig. 6, lanes 3 and 4) . these results render support that the spike protein of the ts mutant may not be assembled into virus particles at 40 jc. coronavirus s protein is responsible for receptor binding and membrane fusion. it also induces neutralizing antibodies and bears determinants for virulence. a considerable diversity in s protein among coronaviruses exists, which contributes to host specificity, cell and organ tropisms, and pathogenesis. characterization of mutants with point or deletion mutations in the s protein has helped to establish links between the variations and the functions or altered antigenicity and virulence of viruses. through sequence analysis of wt virus, two ts mutants, a revertant and different passages of a cold-adapted ibv, data presented in this study not only mapped the defects of the ts mutants to the s gene, but also revealed the molecular events occurred during evolution of the ibv s gene. compared with the parental wt virus, mutations that are identical in the two ts mutants and the revertant might be associated with the cold-adaptation phenotype, while those identical only in the two mutants but different from the revertant and wt virus were considered to be linked to the ts phenotype. according to this criterion, the q 294 -to-l 294 mutation in the s protein that exists in the two ts mutants, and interestingly, occurred at a highly conserved domain among ibv viruses immediately downstream a variable domain in the s1 subunit may be responsible for the ts phenotype. the mutants with this mutation accumulated and became dominant under selective pressures, in this case, changes in the hosts and temperatures. the i 769 -m 769 mutation emerged earlier than the q 294 -l 294 mutation, which among variations in other gene products may be associated with cold-adaptation, suggesting potential segregation of mutations responsible for either cold-adaptation or ts phenotypes. further confirmation of the possibility that the q 294 -to-l 294 mutation may cause the ts phenotypic changes is currently being carried out by introducing the mutation to an infectious ibv clones and isolation of a recombinant virus containing this mutation only. the s protein is co-translationally n-glycosylated in the er, oligomerized (luytjes et al., 1997) if folded properly, and associated with the m protein in pre-golgi membrane (opstelten et al., 1995) . one of the essential steps in nlinked glycosylation is transfer of a preformed, 14-coreunit-oligosaccharide to a specific asn residue in the sequence asn-x-ser/thr (where x is any residue except pro, asp, or glu). the oligosaccharide chain is donated by dolichol-pyrophosphate-oligosaccharide to the protein chain in the er. it is trimmed down in the er and cis-golgi, and different external sugars are then added to the trimmed chain in the medial-and trans-golgi. glycoproteins with high mannose oligosaccharides in the er and cis-golgi remain sensitive to endo-h. they become endo-h resistant after being processed by the medial-and trans-golgi resident enzymes to glycoproteins with complex oligosaccharides. the acquisition of endo-h resistance is therefore an indication that viral n-linked glycoproteins are properly processed and transported to the golgi apparatus (luo and weiss, 1998; luo et al., 1999; luytjes et al., 1997) . examination of the amino acid sequence of ibv s protein showed that there are 13 and 9 potential glycosylation sites in the s1 and s2 subunits, respectively. the calculated molecular weight of the s protein is 128 kda. if all 21 sites are used, the initially transferred oligosaccharides account for 50 kda. in cells overexpressing s protein, core-glycosylation of the s protein in the er resulted in a 180-kda product as expected. trimming of the s glycoprotein in the er and cis-golgi led to a partially processed 155-kda form, larger than the endo-h-treated 130-kda form which only has one sugar residue left at each site. both the trimmed 155-and core-glycosylated 180-kda forms were sensitive to endo-h digestion. at the permissive temperature, further modification of the mutant s protein in the medial-and trans-golgi by addition of sugars to the trimmed chains resulted in the maturation of s, which co-migrates with the core-glycosylated 180-kda form but is resistant to endo-h digestion. at the nonpermissive temperature, however, no mature form of the fig. 6 . analysis of the structural proteins on purified ts291602 and rev-1 virions. immunoprecipitations were performed using virions, purified from ts291602-(lanes 1 and 2) and rev-1-infected (lanes 3 and 4) vero cells at 32 jc (lanes 1 and 3) and 40 jc (lanes 2 and 4) . the viral proteins were separated on 12.5% polyacrylamide gels and detected by autoradiography. numbers on the left indicate molecular masses in kilodalton and the positions of the three structural proteins are indicated on the right. mutant s protein was produced. in virus-infected cells, the endo-h sensitive, 155-kda form of ts291602 was abundant at the nonpermissive temperature, clearly indicating that the mutant s protein was trimmed in the er and cis-golgi but was not transported to the trans-golgi. furthermore, the mutant s protein was not cleaved into s1 and s2 at the nonpermissive temperature, which usually occurs in the trans-golgi shortly before the release of virions from cells (frana et al., 1985) . as only spikeless virions were produced, these results demonstrated that the core-glycosylated or the trimmed forms of s were unable to incorporate into virions. in addition, the mutant s protein fails to induce cell -cell fusion at the nonpermissive temperature. as the q 294 -to-l 294 mutation is not in the fusogenic domain of s, it renders support to the conclusion that the mutation causes the defect in its modification and intracellular transport, instead of having direct impact on the fusion process. taken together, these studies clearly indicate that the mutant s protein could not reach the site where the s protein is incorporated into virions. the reasons for the retention of the mutant s protein in the er and cis-golgi are yet to be explored. previous studies show that the retention of monomeric s protein in the er is due to misfolding induced by disruption of disulfide bonds (opstelten et al., 1993) , and also caused by lack of oligomerization of endo-h-sensitive s protein of ts mutants though the locations were not determined (luytjes et al., 1997) . it is not clear whether mutations in ibv s protein have an effect on its folding or oligomerization. nevertheless, in both circumstances, the intracellular transport, modification of the s protein and its incorporation into virion would have been affected. studies with some of the other enveloped viruses demonstrated that molecular chaperones (like calnexin and calreticulin) in the er transiently and specifically interact with partially trimmed, monoglycosylated form of viral n-linked glycoproteins (tatu and helenius, 1997) . for instance, calnexin interacts with the influenza hemagglutinin and vesicular stomatitis virus g protein (hebert et al., 1996; hammond and helenius, 1994) . these and other proteins could facilitate proper folding and ensure quality control for viral glycoproteins. proteins that fail to fold and oligomerize are prevented from transport and ultimately degraded. with the ts mutants, it is possible to explore the interaction of the s protein with molecular chaperones and folding enzymes. this approach would provide new insights into the maturation and assembly of the coronavirus s protein. the ts mutant quickly lost its infectivity at the nonpermissive temperature. this low thermostability indicated that even if the mutant s protein was synthesized, fully modified and assembled into virions at the permissive temperature, the resulting particles were relatively unstable and lost infectivity at the nonpermissive temperature. it suggests that the q 294 residue in the conserved s1 domain of ibv may also play an essential role in maintaining the thermal stability of the spike in the virion, resembling the s 287 residue in the s1 region of an mhv ts mutant (ricard et al., 1995) . in addition, if cells were incubated with the ts mutants at the nonpermissive temperature, viral proteins were hardly detected, indicating that at the nonpermissive temperature, the ts mutant s protein may be unable to either bind efficiently to the receptor or induce virus -cell membrane fusion in contrast to the s protein of the revertant and wt virus. this observation suggests that the mutation in the s protein might induce conformation changes of the spikes on virions at the nonpermissive temperature and hamper either process. vero cells were maintained in complete dmem medium (gibco brl), supplemented with newborn calf serum (10%), streptomycin (1000 ag) and penicillin (1000 units/ml). the beaudette stain of ibv was purchased from atcc and propagated in chicken embryonated eggs for three passages. the virus was then adapted to grow and passage on vero cells for 65 times at 37 jc. in this study, the viruses were further passaged on vero cells at progressively lower temperatures for 121 passages (at 35, 34, 33, 32, 30, 29, and 28 jc for 4, 5, 7, 44, 12, 21, and 29 passages) . virus stocks were used for plaque assays, plaque-to-plaque purification and viral rna extraction as described previously (shen et al., 1994) . the wild-type virus, wt6501, was plaque-purified from passage 65 grown at 37 jc, while ts mutants, ts291602 and ts282902, were purified from passage 16 grown at 29 jc and passage 29 grown at 28 jc, respectively. the revertant, rev-1, was plaque-purified from cells infected with ts 291602 for 12 h at 37 jc and then shifted to 40 jc for 2 days. recombinant vaccinia/t7 (v/t3) virus was propagated and was tittered on vero cells. virus stocks were kept at à80 jc until use. confluent monolayers of vero cells were infected with viruses at a multiplicity of infection (moi) of 0.1. after 2h absorption, the viruses were radiolabeled by replacing medium with methionine-free dmem supplemented with 30 aci/ml [ 35 s]-methionine. after 16-h incubation at temperatures appropriate for each virus, cells were harvested and virus stock was prepared by freezing and thawing three times. cell debris was removed by centrifugation at 5000 rpm for 15 min (beckmen, 25.50). the supernatant was centrifuged through a 20% sucrose cushion and the resulting pellet was resuspended in tne buffer (50 mm tris -hcl, ph 7.4, 100 mm nacl, 1 mm edta). the viruses were further purified by centrifugation through a 20-55% su-crose gradient in tne buffer at 45,000 rpm (beckmen, sw50) twice. fractions containing the virus were pooled together and used for protein analysis. viral structural proteins were labeled with [ 35 s]-methionine, immunoprecipitated and separated on 12.5% polyacrylamide gels as described previously (liu et al., 1998) . the anti-ibv serum was raised in rabbit against purified ibv virions and was used in immunoprecipitation and western blot as previously described (liu and inglis, 1991) . a portion of cell lysates was immunoprecipitated with anti-ibv serum. the pellets were washed three times with standard ripa buffer, dissolved in 20 al of digestion buffer (50 mm tris, ph 6.8, 0.25% sds) and incubated at 95 jc for 5 min. ten microliters of the supernatants were mixed with 10 al of digestion buffer with or without endo-h (0.2 mu, boehringer mannheim) and incubated for 4 h at 37 jc. confluent monolayers of vero cells were infected with recombinant vaccinia/t7 viruses at a moi of 0.1. after 1h absorption, cells (2 â 10 7 ) were trypsinized, centrifuged at 250 g and resuspended in 2 ml of pbs. the cells (0.3 ml) were mixed with 5 ag plasmid and electroporated (easyject, equibio) at 600 v in a 0.4-cm cuvette (biorad). the cells were then plated in a 35-mm dish containing 3 ml of dmem with 2% of newborn calf serum and incubated at temperatures indicated. viral rna was extracted from the purified viruses using the rneasy mini kit (qiagen) according to the manufacturer's instructions. reverse transcription and polymerase chain reaction (rt-pcr) were performed using the expand reverse transcription and high fidelity pcr kits (boehringer mannheim). annealing and extension times of pcr were optimized for amplification of pcr products with different sizes using different primers. more than 100 specific primers were used for amplification, sequencing and cloning. automated sequencing was carried out using pcr products or cdna clones and specific primers as previously described (shen et al., 1999) . sequence analysis was carried out using the gcg and blast suite of programs as described previously (shen and liu, 2000) . hela cells were infected with vaccinia/t7 virus, transfected with constructs encoding wt and ts mutant s protein using qiagen effectene transfection reagent, and incubated at the permissive and nonpermissive temperatures, respectively, for 18 h. cells were harvested, washed once with pbs, resuspended in blocking buffer (20% fbs and 1% bsa in pbs), incubated on ice for 30 min. a half of the cells were permeabilized with 0.1% saponin in facs washing buffer (2.5% fbs and 0.05% sodium azide in pbs) incubated for 10 min at room temperature, and stained with 1:100 diluted primary antiserum, the rabbit anti-ibv s protein. the other half was stained directly with the same primary antibody. cells were then washed two times with the facs washing buffer, stained with 1:20 diluted fitc conjugated swine anti-rabbit antibody (dako). after washing two times with the facs washing buffer, cells were then fixed with 1% ice cold paraformaldehyde and analyzed by flow cytometry. two amino acid changes at the n-terminus of transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism coronavirus-induced membrane fusion requires the cysteine-rich domain in the spike protein evidence for a coiled-coil structure in the spike of coronaviruses mapping of the coronavirus membrane protein domains involved in interaction with the spike protein identification of a novel coronavirus in patients with severe acute respiratory syndrome aetiology: koch's postulates fulfilled for sars virus proteolytic cleavage of the e2 glycoprotein of murine coronavirus: host-dependent differences in proteolytic cleavage and cell fusion alteration of the ph dependence of coronavirus-induced cell fusion: effect of mutations in the spike glycoprotein cell receptor-independent infection by a neurotropic murine coronavirus major receptorbinding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein folding of vsv g protein: sequential interaction with bip and calnexin calnexin and calreticulin promote folding, delay oligomerization and suppress degradation of influenza hemagglutinin in microsomes tunicamycin resistant glycosylation of coronavirus glycoprotein: demonstration of a novel type of viral glycoprotein characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the rer to the golgi complex requires only one vesicular transport step variations in disparate regions of the murine coronavirus spike protein impact the initiation of membrane fusion localization of neutralizing epitopes and the receptor-binding site within the amino-terminal 330 amino acids of the murine coronavirus spike protein retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier the molecular biology of coronaviruses altered pathogenesis of a mutant of the murine coronavirus mhv-a59 is associated with a q159l amino acid substitution in the spike protein association of the infectious bronchitis virus 3c protein with the virion envelope proteolytic mapping of the coronavirus infectious virus 1b polyprotein: evidence for the presence of four cleavage sites of the 3c-like proteinase and identification of two novel cleavage products roles in cell-to-cell fusion of two conserved hydrophobic regions in the murine coronavirus spike protein amino acid substitutions within the leucine zipper domain of the murine coronavirus spike protein cause defects in oligomerization and the ability to induce cell-tocell fusion characterization of two temperature-sensitive mutants of coronavirus mouse hepatitis virus strain a59 with maturation defects in the spike protein spike glycoprotein-mediated fusion in biliary glycoprotein-independent cell-associated spread of mouse hepatitis virus infection murine coronavirus spike protein determines the ability of the virus to replicate in the liver and cause hepatitis disulfide bonds in folding and transport of mouse hepatitis coronavirus glycoproteins envelope glycoprotein interactions in coronavirus assembly a conditional-lethal murine coronavirus mutant that fails to incorporate the spike glycoprotein into assembled virions evolution of mouse hepatitis virus: detection and characterization of spike deletion variants during persistent infection generation of coronavirus spike deletion variants by high-frequency recombination at regions of predicted rna secondary structure identification of spike protein residues of murine coronavirus responsible for receptor-binding activity by use of soluble receptor-resistant mutants determination of the complete nucleotide sequence of a vaccine strain of porcine reproductive and respiratory syndrome virus and identification of the nsp2 gene with an unique insertion emergence of a coronavirus infectious bronchitis virus mutant with a truncated 3b gene: functional characterization of the 3b protein in pathogenesis and replication rearrangement of the vp6 gene of a group a rotavirus in combination with a point mutation affecting trimer stability sequence analysis and in vitro expression of genes 6 and 11 of an ovine group b rotavirus isolate, kb63: evidence for a non-defective, c-terminally truncated nsp1 and a phosphorylated nsp5 proteolytic cleavage of the e2 glycoprotein of murine coronavirus: activation of cell-fusing activity of virions by trypsin and separation of two different 90k cleavage fragments analysis of the receptor-binding site of murine coronavirus spike protein interactions between newly synthesized glycoproteins, calnexin and a network of resident chaperones in the endoplasmic reticulum replication of coronavirus mhv-a59 in sac-cells: determination of the first site of budding of progeny virions a 12-amino acid stretch in the hypervariable region of the spike protein s1 subunit is critical for cell fusion activity of mouse hepatitis virus nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes this work was supported by the biomedical research council, agency for science technology and research, singapore. key: cord-345630-bam3pa70 authors: lee, han-jung; shieh, chien-kou; gorbalenya, alexander e.; koonin, eugene v.; la monica, nicola; tuler, jeremy; bagdzhadzhyan, anush; lai, michael m.c. title: the complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and rna polymerase date: 1991-02-28 journal: virology doi: 10.1016/0042-6822(91)90071-i sha: doc_id: 345630 cord_uid: bam3pa70 abstract the 5′-most gene, gene 1, of the genome of murine coronavirus, mouse hepatitis virus (mhv), is presumed to encode the viral rna-dependent rna polymerase. we have determined the complete sequence of this gene of the jhm strain by cdna cloning and sequencing. the total length of this gene is 21,798 nucleotides long, which includes two overlapping, large open reading frames. the first open reading frame, orf 1 a, is 4488 amino acids long. the second open reading frame, orf 1 b, overlaps orf 1 a for 75 nucleotides, and is 2731 amino acids long. the overlapping region may fold into a pseudoknot rna structure, similar to the corresponding region of the rna of avian coronavirus, infectious bronchitis virus (ibv). the in vitro transcription and translation studies of this region indicated that these two orfs were most likely translated into one polyprotein by a ribosomal frameshifting mechanism. thus, the predicted molecular weight of the gene 1 product is more than 800,000 da. the sequence of orf 1 b is very similar to the corresponding orf of ibv. in contrast, the orf 1 a of these two viruses differ in size and have a high degree of divergence. the amino acid sequence analysis suggested that orf 1 a contains several functional domains, including two hydrophobic, membrane-anchoring domains, and three cysteine-rich domains. it also contains a picornaviral 3c-like protease domain and two papain-like protease domains. the presence of these protease domains suggests that the polyprotein is most likely processed into multiple protein products. in contrast, the orf 1b contains polymerase, helicase, and zinc-finger motifs. these sequence studies suggested that the mhv gene 1 product is involved in rna synthesis, and that this product is processed autoproteolytically after translation. this study completes the sequence of the mhv genome, which is 31 kb long, and constitutes the largest viral rna known. mouse hepatitis virus (mhv), a murine coronavirus, contains a single-stranded, positive-sense rna genome (lai and stohlman, 1978; wege eta/., 1978) . the genomic organization is well understood lai, 1990) . it contains 8 genes, each of which is expressed from the 5'-end of a polycrstronic mrna species. these mrnas have a 3'-coterminal, nestedset structure (lai et al., 1981) . starting from the 5'-end of the genome, the genes are named 1, 2a, 2b, 3, and so on until gene 7 (cavanagh eta/., 1990) . genes 2b, 3, 6, and 7 encode the four known viral structural proteins, i.e., he (hemagglutinin-esterase), s (spike), m (membrane), and n (nucleocapsid) proteins, respectively. the remaining genes presumably encode nonstructural proteins, most of which are yet to be identified in the virus-infected cells. the nucleotide sequences of genes 2 to 7 have been determined for two strains, a59 and jhm, of mhv (armstrong eta/., 1983 (armstrong eta/., , 1984 skinner and siddell, 1983, sequence data from this article have been deposlted with the embugenbank under accession no. m55148 . ' to whom correspondence should be addressed. 1985; schmidt et a/., 1987; luytjes et a/., 1987 luytjes et a/., , 1988 shieh et al., 1989) . altogether these seven genes account for roughly 9.5 kb. the remaining gene, gene 1, which is the 5'-most gene, has been estimated to be longer than the size of all of the other genes combined (pachuk eta/., 1989; baker et al., 1990) . only the 5'-terminal 5.3 kb in jhm strain and the 3'-terminal 8.4 kb of this gene in a59 strain have so far been sequenced baker et al., 1989; pachuk et al,, 1989; bredenbeek et al., 1990) . the corresponding gene of an avian coronavirus, infectious bronchitis virus (ibv), has been completely sequenced and shown to be 20 kb long . this ibv gene consists of two open reading frames (orfs), which can be translated into a polyprotein via a ribosomal frameshifting mechanism (brierley et a/., 1987 (brierley et a/., , 1989 . again, the gene products have yet to be detected in the virus-infected cells. the size of mhv gene 1 has not been determined. from the approximate sizes of the cdna clones, it has been estimated to be roughly 22-23 kb (pachuk et al,, 1989; baker et al,, 1990) . comparison of the published partial sequences of gene 1 showed that ibv and mhv share sequence similarity in the 3'-terminus of the gene ( , 1990) , and yet their 5'-ends are diverged (soe eta/., 1987; baker et al., 1989) . thus, the evolutionary relationship of these two viruses in gene 1 is not clear. several pieces of evidence suggest that gene 1 may encode proteins which are directly involved in viral rna synthesis: first, since mhv does not contain rna polymerase (brayton et al., 1982) , this enzyme has to be synthesized from the incoming virion genomic rna. this translation is only possible if the gene is located at the 5'-end of the genome. second, rna recombination studies using temperature-sensitive (ts) mutants indicated that the ts lesions affecting rna synthesis are localized within the gene 1 region (keck et al., 1987) . this conclusion has been confirmed by rna recombination mapping studies (baric et a/., 1990) . third, the 3'-half of the gene 1 sequences of ibv and mhv-a59 contains the sequence motifs for rna polymerase and helicase, which are the activities expected to be involved in rna synthesis gorbalenya et a/., 198913; bredenbeek et a/., 1990) . however, these postulated functions have not been directly demonstrated. at least one enzymatic activity, i.e., an autoprotease , has been associated with the gene product. the presence of the protease activity suggests that the gene 1 product is likely to be processed into multiple proteins. the properties of the rna polymerase of coronavirus are of considerable interest since the coronavirus rna synthesis utilizes an unusual mechanism of discontinuous transcription, probably involving a free leader rna species (lai, 1988) . the understanding of the rna polymerase should shed further light on the mechanism of rna synthesis. to this end, we have obtained the complete sequence of gene i of the jhm strain of mhv. this gene is nearly 22,000 nucleotides long and contains two overlapping orfs, similar to the corresponding ibv gene. sequence analysis shows that the mhv gene may have undergone extensive divergence from the ibv gene, particularly at its 5'-half. several functional domains were identified, which may be important for the processing and the enzymatic activities of its gene product. virus and cells. the plaque-cloned jhm strain of mhv (makino et a/., 1984) was used throughout this study. the virus was propagated on dbt cells (hirano et a/., 1974) at m.o.i. of 1. virus was harvested and purified from the medium, and viral rna was prepared as previously described (makino et a/., 1984) . cdna cloning. the cdna c\ones encompassing , i i i i , i i i / , i i i i i 1 2000 2500 3000 3500 3 orf la/lb 0 orf lb , i i i i , i i i i , i i i i i ' ' 1000 1500 2000 2500 fig. 2. hydropathy profiles of the predicted amino acid sequences of orf 1 a and orf 1 b. values above the lrne are hydrophobic and values below the line are hydrophilic. the hydropathicrty was calculated using a moving window of 40 amino acids, with a value plotted every 16 residues (kyte and doolittle, 1982) . gene 1 were obtained by using specific synthetic oligo-gonucleotides were derived from rna sequence analynucleotides as primers and purified virion genomic sis of the rnase tl-resistant oligonucleotides which rna as template. initially, the sequences of these olihad been mapped to either gene 1 or 2 (shieh et al, , 13, 600 13,650 nucleotide number 13,700 fig. 3. dragram of the codon preference in the region between orf 1 a and orf 1 b. the codon usage patterns for the three reading frames of the predicted amino acid sequences at the junction between the orf 1 a and orf 1 b are shown. the two stop codons at 13600 (tag) and 13679 (taa) are marked. the codon usage table was generated for genes 3, 6, and 7, which encode the viral structural proteins, of mhv-ihm (schmidt er a/., 1987; skinner and siddell, 1983) and used for comparison with orfs 1 a and 1 b. the parameters used are a window length of 25 and a maximum scale of 1.1 (gribskov et al., 1984) . l bv-m42 5' 12337 gauaagaauuauuuaaacggguacgggguagcagug----aggcucggcugauaccccuugcuagugg 3' ii i lllll 11111111111 lllllli iiii i ii ii 11-11111 iii illll mhv-jhm 5' 13643 gacacgaauuuuuuaaacggguucgggguacaaguguaaaugcccgucuuguacccugugccagugg 3' mhv-a59 5' 284 llllllll 11111111111 iiiiiiiiiiiiiiiltriiiiiiiiiiiiiiiiiiiiiiiiiiii gacacgaacuuuuuaaacggauucgggguacaaguguaaaugcccgucuuguacccugugccagugg 3 comparison of the rna sequences and the proposed secondary structure of the mhv-jhm, mhv-a59 and ibv rnas at the junction between orf 1 a and orf 1 b. (a) alignment of nucleotide sequences. the first nucleotides are numbered according to boursnell era/. (1987) for ibv, and bredenbeek ef al. (1990) for mhva59, and termrnation codons are underlrned. (b) tertiary rna structure at the region of ribosomal frameshifting. the potential signal for ribosomal frameshifting is boxed, and the stop codon is underlined. arrows indicate the differences in the rna sequence of mhv-jhm in comparison with that of ibv (boldfaced) and mhv-a59 (outlined). soe et al., 1987) . cdna synthesis was were trimmed with t4 dna polymerase and ligated to performed by the general method of gubler and hoff-ptz18u (united states biochemical corp.) either by man (1983) . the double-stranded cdna molecules blunt-end ligation or ecorl linker ligation. the recombi4, and 7) . generating a 0.5.kb rna. translation was performed in a rabbit reticulocyte lysate system using [35s]methionine. translation products were analyzed directly (lanes l-3) or after immunoprecipitation using the orf 1 a-specific antiserum (lanes 4-6) or rabbrt preimmune serum (lanes 7-9). m indicates molecular weight markers in kilodaltons; lanes 3. 6, and 9, translation of ptz(orfaug). nant dnas were transformed into escherichia co/i strain mvl 190 competent cells (dagert and ehrlich, 1979) . homopolymer dc tailing to the 3'-end of the cdnas using terminal transferase were also used to anneal to pstl-linearized pbr322 with oligo(dg) tails and transformed into e. co/i strain mc1 061. specific cdna clones were identified using 5'-end-labeled oligonucleotides as probes and confirmed by subsequent hybridization to viral mrna . once the sequences of the cdna clones were obtained, oligonucleotides complementary to the 5'-ends of these clones were synthesized to serve as primers for addi-tional cdna cloning to obtain overlapping cdna clones. dna sequencing. sequencing was performed as previously described . both chemical modification (maxam and gilbert, 1980) and dideoxynucleotide chain termination @anger et a/., 1977) methods were used directly on plasmid dna (chen and seeburg, 1985) . construction of recombinant plasmids for the frameshifting analysis. subcloning and mutagenesis of cdna clone t-l 2 was accomplished using synthetic oligonucleotides and polymerase chain reaction (pcr). briefly, oligomer #166 (5'-gatcgaattcctttacat-ggtgaaggggtg-3') which extends from nucleotide 13,147 to 13,167 of gene 1 and contains mismatches at both nucleotides 13,154 and 13,156, and oligomer #199 (5'-catatgacacaggatcctttatgcc-3') which is complementary to nucleotides 13,529 to 13,553 and includes the bamhl site at nucleotide 13,537, were used for dna amplification by pcr according to the standard procedures (saiki et al., 1988) . the resulting pcr dna product encompasses sequences from nucleotide 13,147 to 13,537 with a specific mutation (t to a) at nucleotide 13,154 and another (t to g) at nucleotide 13,156, resulting in the introduction of an atg codon. the dnawas then digested with ii i iill iii iiii lllll ir iii1 iiiii iii qts~gvcvvcnspti the overall alignment was generated by combining segments aligned by programs optal (gorbalenya et al., 1989a) and multalin (corpet, 1988) . it consists of four distinct pieces separated by regions that could not be aligned with certainty. for the latter regions, only the total numbers of amino acid residues are indicated. the amino acid numbers of the first and the last residues of each aligned segment are indicated in parentheses. two dots, identical residues; single dots, similar residues. conserved cys residues are highlighted by boldface. asterisks, putative catalytic residues of proteases; arrows, putative cleavage sites for bc-like proteases. box, the putative cleavage site for 3clpr0 in ibv substituted by a kr dipeptide in mhv-jhm. the ibv sequence was from boursnell et al. (1987) . mhva: orf la of mhv. ibvfl: orf la of ibv. previously described . the resulting in vitro transcription and translation. recombinant rna was translated in the mrna-dependent rabbit replasmids ptz(orfaug) and ptz (frsh) shin and morrison (1989) and analyzed by electrophoresis on 7.5 to 15% polyacrylamide gel. computer analysis of nucleotide and amino acid sequences. sequence data were analyzed on a vax 1852 using the gcg sequence analysis software package developed by genetics computer group of university of wisconsin. detailed comparative analyses of coronavirus protein sequences were done by programs multalin (corpet, 1988 ) optal (gorbalenya et a/., 1989a), dothelix (leontovich et al., 1990) , and site (koonin et a/., 1990) . the programs dothelix and site are parts of the genbee program package for biopolymer sequence analysis. to clone the gene i region, which represents more than two thirds of the mhv genome, a synthetic oligonucleotide (oligo 30; 5'-ctgaatftgggg-gttggg-3') was initially used as a primer for cdna synthesis . the sequence of this oligonucleotide was based on the sequence analysis of the rnase tl-resistant oligonucleotide no. 30, which had previously been mapped to gene 2 (makino et al., 1984) . the resulting cdna clones contained inserts ranging from 0.5 to 3 kb in size. these cdna clones detected only the genomic rna on northern blots of intracellular rnafrom mhv-infected cells (data not shown). based on the nested-set structure of mhv 9 . a schematic presentation of the relationship between the orf 1 a of mhv-jhm and ibv. the two orf 1 a are shown to scale. the designation of regions, for which specific functional predictions could be made, and of regions of similarity between the two viruses are shown in the bottom of the figure. high similarity, statistical significance over 10 sd (standard deviation), when aligned by the program optal (gorbalenya eta/., 1989a,b) ; moderate similarity, significance of 3 to 10 sd. the alignments in the regions, with predicted functions, were significant at the level of at least 5 sd. regrons of similarity between the two viruses are joined. vertical arrows, putative cleavage sites for 3clpr0'). horizontal arrows, putative papain-like proteases (two copies in mhv-jhm, and one copy in ibv). mrnas (lai et a/., 1981) , this result indicated that these cdna clones represent part of gene 1. the 5'ends of these dnas were sequenced, and synthetic oligonucleotides complementary to these sequences were generated to prime further cdna synthesis for walking toward the 5'-end of gene 1. in this way, overlapping dna clones which encompass about 11 kb at the 3'-end of gene 1 were obtained (fig. 1) . cdna clones representing the 5'-terminal 6.2 kb of gene 1 were derived as described . the cdna clones spanning the gap between the two cdna groups were obtained by using specific primers representing both the sequences downstream and upstream of the gap as primers for first-strand and second-strand cdna synthesis, respectively. the overlap of these cdna clones was determined by southern blotting and confirmed by dna sequencing. the complete cloning of jhm gene 1 indicated that the size of gene 1 is approximately 22 kb in length (fig. l) , longer than that of ibv , and agrees with the previous estimate for the gene 1 of the a59 strain of mhv (pachuk et al., 1989) . analysis of the nucleotide sequence and the predicted amino acid sequence. the complete mhv-jhm gene 1 sequence was obtained from the cdna clones as indicated in fig. 1 . this sequence has been deposited with genbank (accession no. m55148), and will not be duplicated in this publication. the complete sequence of gene 1 contains 21,798 nucleotides preceding the ucuauac, which is the transcriptional initiation site for gene 2 . analysis of the sequence revealed two large, overlapping open reading frames (orfs), orf 1 a and orf 1 b (fig. 1 a) . orf 1 a is 4488 amino acids long and has a predicted molecular weight of 499,319, which includes the coding region for p28 protein at its n-terminus . the hydropathy plot (kyte and doolittle, 1982) shows that orf 1 a has several long stretches of hydrophobic regions at the carboxy-terminal region, which indicate potential membrane-spanning domains (fig. 2) . orf 1 b, which overlaps orf 1 a for 75 nucleotides but is located at a different reading frame, is 2731 amino acids long with a predicted molecular weight of 308,483. the orf 1 b sequence is very similar to that of mhv-a59 in both nucleotide and predicted amino acid sequences (bredenbeek et al., 1990) . only minor substitutions were noted between the two strains (data not shown). the orf 1 b starts with cug instead of aug. the first potential initiator codon aug is located 399 nucleotides downstream of the first amino acid the numbers of amino acid residues to the known or postulated termini of the respective viral 3clpro and between the aligned segments are indicated. for mhv 3clpro, the postulated n-terminus is at amino acid residue 3350 ( fig. 8 gorbalenya et a/. (1989b) , except bwyv (veidt et a/., 1988) and sbmv (wu et al., 1987) . riore. in the aa position columns, the amino acid positions of the respective q residues are indicated. the arrows show the predicted cleavage sites, abbreviations: mpl , mp2, putative membrane proteins flanking the 3clpr0 at the n-and c-sides, respectively. pol: polymerase motif. hel: helicase motif. gfl: growth factor-like domain. the data on ibv was obtained from gorbalenya ef al. (1989b) . the sequence analysis was performed using the computer program as described under materials and methods. codon in orf 1 b. nevertheless, the codon preference plot suggests that the 399 nucleotides upstream of the first aug are most likely translated together with the downstream sequences using the same reading frame (fig. 3) . in light of the corresponding sequences of ibv and mhv-a59 bredenbeek et a/., 1990) , this result suggests that this region could be translated via a ribosomal frameshifting mechanism (brierley et al,, 1989) . comparison of tertiary structure of rna in the frameshift regions. it has been proposed that the nucleotide sequences in the overlapping regions between orf 1 a and orf 1 b in ibv and mhv-a59 rnas are able to fold into a pseudoknot tertiary structure, which is essential for efficient frameshifting and, thus, expression of the downstream orf 1 b (brierley et a/., 1989; bredenbeek et al., 1990) . comparison of the primary sequence revealed that the corresponding region of mhv-jhm contains a "slippery" sequence, uuuaaac, similar to that of ibv (fig. 4a) . the possible folding of rna in this region into a pseudoknot tertiary structure is similar among ibv, mhv-a59, and mhv-jhm (fig. 4b) . it is interesting to note that the nucleotide changes between mhv-jhm and ibv in either the stem or loop regions are compensated by mutations at the complementary positions (fig. 48) . this suggests the significance of the putative tertiary structure in ribosomal frameshifting. only two nucleotides differ between mhv-jhm and mhv-a59 in this region; they are located at the regions immediately upstream and downstream of the uuuaaac sequence. ribosomal frameshifting in vitro. to confirm that the orf 1 a and 1 b of mhv-jhm could be translated into one polypeptide by ribosomal frameshifting, we cloned the region spanning from nucleotide 13,147 to 14,164 of gene 1 into an expression vector under the control of the t7 promoter for in vitro translation studies. because of the lack of a translational initiation codon, an atg codon was introduced by pcr-mediated mutagenesis at nucleotide 13,154-l 3,156. if the translation of this transcript terminates at the uaa stop codon in orf 1 a, a 19-kda protein will be produced. however, if the -1 translational frameshift occurs, a 37-kda protein will be synthesized. as shown in fig. 5 , the in vitro translation of this rna yielded both proteins (lane 2). the 37-kda protein was heterogeneous; the smaller proteins may represent aberrant translational initiation or specific processing of the translation products. the addition of protease inhibitors in the rabbit reticulocyte lysates did not alter this translation pattern (data not shown). the antiserum prepared against the amino acid sequence just upstream of the frameshift (unpublished) precipitated both proteins (lane 5). surprisingly, the major products precipitated by this antiserum migrated faster than the respective primary translation products, suggesting that protein processing had occurred. none of the proteins was immunoprecipitated by the preimmune serum. as controls, the transcripts containing either the 5'-or the 3'-halves [ptz(orf""g)] of the orf did not yield the 37-kd protein. as predicted, only the products of the 5'-half were precipitated by this antibody (fig. 5b, lane 4) . these results are in agreement with the results obtained with ibv (brierly et a/., 1987) and mhv-a59 (bredenbeek et a/., 1990) . analysis of sequence homology among mhv-jhm, mhv-a59, and ibv. the comparison of nucleotide and predicted amino acid sequences between mhv-jhm and ibv revealed considerable similarity between the two. the dot matrix comparison of the amino acid sequences shows that orf 1 b is very similar between mhv and ibv (fig. 6) . overall, there are 47.7% similarity at nucleotide level and 52.8% at amino acid level. similar to the orf 1 b of ibv, the mhv orf contains the polymerase and helicase motifs at the corresponding positions (gorbalenya et al., 1989b ) (data not shown). the putative zinc-binding domain is also largely conserved between the two viruses. on the other hand, two of the residues implicated in metal binding for ibv (gorbalenya et al., 198913) are replaced in mhv, suggesting that the specific structures of the putative "fingers" may differ (fig. 7) . the orf 1 b of mhv-jhm and mhv-a59 are also very similar (95.9% at nucleotide level, and 94.9% at amino acid level) (data not shown). in contrast, the orf 1 a is more diverged (fig. 8) . the mhv orf 1 a is longer than the corresponding ibv orf by 537 amino acids. the c-terminal half of the orf 1 a is relatively conserved between mhv-jhm and ibv, while the n-terminal half is very diverged (fig. 6) . the alignment of amino acids in orf la of mhv-jhm and ibv showed that there are four possible stretches of moderate homology which are separated by highly diverged sequences (fig. 8) . analysis of the functional domains of orf la. although orf la is highly diverged between mhv-jhm and ibv, common functional domains could be identified in this orf of both viruses by detailed amino acid sequence analysis (see materials and methods) (fig. 9 ). two hydrophobic, potentially membrane-anchoring regions are present in the c-terminal half. there are three cysteine-rich domains, one of which contains a segment distantly resembling growth factors and their receptors (gorbalenya et al., 1989b) . in both coronaviruses, homologous domains of about 300 residues each have been identified to be related to the putative 3clike proteases (3clpro) of picorna-, coma-, nepo-, poty-, sobemo-and luteoviruses (gorbalenya et al., 1989b) . the sequences of the putative coronavirus 3c-like proteases possess certain unusual features distinct from that of other viral 3clike proteases (fig. 10 , and see discussion). the search for sequences resembling the cleavage sites for the 3c-like proteases revealed six conserved putative target sites for the mhv and ibv 3c-like proteases (table 1 ) (see discussion). these potential cleavage sites are localized in the orf 1 b and the c-terminal half of the orf la. interestingly, the n-terminal one of these cleavage sites marks the n-end of the putative 3clike protease itself. finally, there is a region of moderate conservation between mhv and ibv, which contains short segments resembling those around the catalytic cys and his residues of papain-like proteases (fig. 11) . this region is duplicated in the mhv genome, but not in ibv, at an upstream site in the orf la. this upstream papain-like cysteine protease has been identified as the one responsible for the cleavage of p28 from the n-terminus of the gene 1 protein . a domain of considerable conservation between mhv and ibv (x domain in fig. 9 ) has been found next to the putative coronavirus papain-like proteases. interestingly, a homologous conservative domain also flanks the putative thiol proteases of alpha-and rubiviruses (a. e. gorbalenya, unpublished observations) . the complete sequence of gene 1 of mhv presented in this paper shows that this gene is probably the largest known viral gene among rna viruses. evidence was presented suggesting that the two orfs in this gene may be translated into a large polyprotein. this interpretation is consistent with the lack of the transcriptional initiation signal (ucuaaac) in the entire gene 1 sequence except at the extreme 5'-end. although the putative "slippery" sequence (uuuaaac) between the orf 1 a and 1 b (brierly et a/., 1989) is similar to the transcriptional initiation signal, no major subgenomic mrnas have been detected within this gene. thus, this gene most likely encodes a single polyprotein of at least 800 kda. the total size of the rna genome of mhv is approximately 3 1 kb, which is considerably larger than any of the other known viral rna. the evolution of the coronavirus rna genome into such a large rna may have reflected the unusual mechanism of coronavirus rna synthesis. the complexity of the discontinuous mode of coronavirus rna synthesis (lai, 1988) suggests that the coronavirus rna polymerase needs a variety of different enzymatic activities. the amino acid sequence of gene 1 of mhv shows considerable similarity to that of ibv. the orf 1 b is particularly conserved. its degree of conservation between mhv and ibv is higher than that for any of the other genes in the coronavirus genomes. the orf 1 b contains the polymerase, helicase, and metal-binding motifs (gorbalenya el a/., 1989b) , suggesting that this region may be directly involved in rna synthesis. these structural features are conserved between these viruses. the proposed pseudoknot structure which is important for the ribosomal frameshifting for cotranslation of orf 1 a and orf 1 b (brierley et a/., 1989) is also highly conserved. this fact has previously been recognized in the partial sequence of gene 1 of mhv-a59 (bredenbeek eta/., 1990) . the sequence differences between mhv-a59 and mhv-jhm within this junction region are located at the nucleotides which do not affect the putative pseudoknot structure. in contrast, orf la is much more diverged. it is nearly 2 kb longer than the orf la of ibv, and contains several stretches of sequence which are not present in the ibv genome. these nonhomologous stretches of sequence are interspersed between the conserved regions. furthermore, a papain-like protease domain, which is present once in the ibv genome, is duplicated in the 5'-half of the orf la of mhv. the n-terminal sequence including ~28, which is cleaved by the papain-like protease of mhv , is also highly diverged between mhv and ibv. thus, it appears that the 5'-end of orf 1 a has undergone considerable sequence rearrangement and possibly recombination, while the remaining sequences in gene 1 are almost colinear between mhv and ibv. in contrast to the orf 1 b which contains sequence motifs related to the synthesis of rna, the orf la contains several domains suggestive of other functions. first of all, there are two long stretches of hydrophobic domains, which are conserved between ibv and mhv. the presence of these domains suggests that the gene 1 products may be anchored to the membrane. this possibility is consistent with the finding that mhv rna synthesis occurs on the membrane fractions in the infected cells (brayton et al., 1982) . second, there are three cysteine-rich regions, which are also homologous between mhv and ibv. the function of the cys-rich domains is still not clear. however, it has been noted with ibv that the c-terminal cys-rich domain is related to that of the growth factors and their receptors (gorbalenya et al., 1989b) . third, there is a 3c-like protease domain (3clpro) in the 3'-half of orf 1 a, which is also conserved in ibv. the putative catalytic his and cys residues previously predicted in ibv have also been observed in mhv (fig. 10) . however, the putative coronavirus proteases remain unique in that they do not contain a conserved asp(glu) residue that could serve as the third catalytic residue as suggested for the other 3c-like proteases (gorbalenya et al., 1989b) . furthermore, the unusual substitution of tyr for gly in the putative substrate-binding region, described previously in ibv, is also observed in the putative mhv 3clp" (fig. 10) . the potential cleavage sites for this 3c-like protease have been identified to be mainly in orf 1 b and the c-terminus of orf 1 a (gorbalenya et al., 1989b) . these sites (qs) are either conserved or converted to qa in mhv (table 1 ). the potential cleavage at q/s and q/a sites by picornavirus 3clp" has been demonstrated previously (parks and palmenberg, 1987) . two qg dipeptides proposed to be cleaved in ibv were substituted in mhv by qc in one case, and by kr dipeptide in another (table 1) . substitution of a c (unlike several other residues) for g in a cleavage site for encephalomyocarditis virus protease did not abolish processing in an in vitro system (parks et a/., 1989) . dibasic dipeptides are cleaved in the polyproteins of flaviviruses (strauss and strauss, 1988) . thus, these postulated cleavage sites are potentially cleavable by mhv 3clpro despite the divergence. these cleavages could separate different functional domains of the gene 1 polyprotein into distinct protein products. whether these sites are indeed cleaved in mhv-infected cells remains to be studied. fourthly, the n-terminal portion, which is the most diverged region, contains a papain-like protease domain as pointed out previously for ibv (gorbalenya et al., 1989b) . the papain protease domain is duplicated in the mhv orf 1 a (fig. 1 1) and is homologous with the known proteases (fig. 12) . this protease is probably involved in the cleavage of the n-terminus of the gene 1 polyprotein (baker eta/., 1989) , which has been demonstrated in mhv-infected cells (denison and perlman, 1987) . site-specific mutagenesis studies demonstrated that this protease has cys and his at its active site (unpublished observation). the possible presence of the protease domains suggests that the gene 1 polyprotein is processed into many proteins. it has been shown that there are at least five to six complementation groups involving mhv rna synthesis, five of which have been mapped within gene 1 (leibowitz eta/., 1982; baric eta/., 1990) . these proteins conceivably participate in various aspects of mhv rna synthesis. none of the proteins have been detected so far. sequence of the nucleocapsid gene from murine coronavirus mhv-a59 sequence and topology of a model intracellular membrane protein, el glycoprotein, from a coronavirus murine coronavirus gene 1 polyprotein contains an autoproteolytic activity identification of a domain required for autoproteolytic cleavage of murine coronavirus gene a polyprotein. 1. viroi establishing a genetic recombination map for murine coronavirus strain a59 complementation groups completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus characterization of two rna polymerase activities induced by mouse hepatitis virus the primary structure and expresslon of the second open reading frame of the polymerase gene of the coronavirus mhv-a59; a highly conserved polymerase is expressed by an efficient rlbosomal frameshifting mechanism an efficient ribosomal frame-shifting signal in the polymerase-encoding region of the coronavirus ibv characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot recommendations of the coronavirus study group for the nomenclature of the structural proteins, mrnas and genes of coronavirus supercoil sequencing: a fast and simple method for sequencing plasmid dna multiple sequence alignment with hierarchical clustering prolonged incubation in calcium chloride improves the competence of escherychia co/i cells identification of putative polymerase gene product in cells infected with murine coronavirus a59 a comprehensive set of sequence analysis programs for the vax purification and amino acid sequence of chicken liver cathepsin l an ntp-binding motif is the most conserved sequence in a highly diverged monophyletic group of proteins involved in positive strand rna viral replication coronavirus genome: prediction of putative functional domains in the nonstructural polyprotein by comparative amino acid sequence analysis the codon preference plot: graphic analysis of protein coding sequences and prediction of gene expression a simple and very efficient method for generating cdna libraries replication and plaque formation of mouse hepatitis virus (mhv-2) in mouse cell line dbt culture multiple recombination sites at the 5'-end of murine coronavirus rna a method for localization of motifs in amino acid sequences. biopolim. kletka a simple method for displaying the pathic character of a protein replication of coronavirus rna coronavirus: organization, replication and expression of genome mouse hepatitis virus a59: messenger rna structure and genetic localization of the sequence divergence from the hepatotropic strain mhv 3 the rnaof mouse hepatitis virus genetic analysis of murine hepatitis virus strain jhm. 1. viral a method for generation of complete local similarity maps between two amino acid sequences. dothelix program of the genbee package primary structure of the glycoprotein e2 of coronavirus mhv-a59 and identification of the trypsin cleavage site sequence of mouse hepatitisvirus a59 mrna 2: indications for rna recombination between coronavirus and influenza c virus analysis of genomic and intracellular viral rnas of small plaque mutants of mouse hepatitis virus sequencing end-labeled dna with base-specific chemical cleavages. ln evolutionary origin of a calcium-dependent protease by fusion of genes for a thiol protease and a calcium-binding protein cloning and characterization of a mouse cysteine proteinase molecular cloning of the gene encoding the putative polymerase of mouse hepatitis coronavirus strain a59 proteolytic cleavage of encephalomyocarditis viral capsid region substrates by precursors to the 3c enzyme site-specific mutations at a picornavirus vpb/vpl cleavage site disrupt in vitro processing and assembly of capsid precursors primer-directed enzymatic amplification of dna with a thermostable dna polymerase dna sequencing with chain-terminating inhibitors nucleotide sequence of the gene encoding the surface projection glycoprotein of coronavirus mhv-jhm identification of a new transcriptional initiation site and the corresponding functional gene 2b in the murine coronavirus rna genome the 5'.end sequence of the murine coronavirus genome: implications for multiple fusion sites in leaderpnmed transcription production and properties of chimeric antibody molecules coronavirus mhv-jhm mrna 5 has a sequence arrangement which potentially allows translation of a second, downstream open reading frame. 1. gem viral coronavirus jhm: nucleotide sequence of the mrna that encodes nucleocapsid protein coding sequence of coronavirus mhv-jhm mrna 4 sequence and translation of the murine coronavirus y-end genomic rna reveals the n-terminal structure of the putative rna polymerase. 1. viral coronaviruses: structure and genome expression. 1. gem viral replication of the rnas of alphaviruses and flaviviruses nucleotide sequence of beet western yellows rna genomic rna of the murine coronavirus jhm. 1 sequence and organization of southern bean mosaic virus rna we thank dr. susan baker for advice throughout the course of the study. we also thank lisa banner and daphne shimoda for editorial assistance. a.e.g. and e.v.k. are most grateful to professor v. i, ago1 for constant support, to dr. l. i. brodsky for supply of the gen-bee program package, and to dr. key: cord-322904-9mta0aem authors: neu, ursula; stehle, thilo; atwood, walter j. title: the polyomaviridae: contributions of virus structure to our understanding of virus receptors and infectious entry date: 2009-02-01 journal: virology doi: 10.1016/j.virol.2008.12.021 sha: doc_id: 322904 cord_uid: 9mta0aem this review summarizes the fields major findings related to the characterization of polyomavirus structures and to the characterization of virus receptors and mechanisms of host cell invasion. four members of the family that have received the most attention in this regard are the mouse polyomavirus (mpyv), the monkey polyomavirus sv40, and the two human polyomaviruses, jcv and bkv. the structures of both the mpyv and sv40 alone and in complex with receptor fragments have been solved to high resolution. the majority of polyomaviruses recognize terminal sialic acid in either an α 2,3 linkage or an α 2,6 linkage to the underlying galactose. studies on virus structure, receptor utilization and mechanisms of entry have led to new insights into how these viruses interact in an active way with cells to ensure the nuclear delivery and expression of their genomes. critical work on virus entry has led to the discovery of a ph neutral endocytic compartment that accepts cargo from caveolae and to novel roles for endoplasmic reticulum (er) associated factors in virus uncoating and penetration of er membranes. this review will summarize the major findings and compare and contrast the mechanisms used by these viruses to infect cells. the family polyomaviridae are classified as group 1 viruses in the baltimore classification scheme as they contain double stranded dna genomes. there is a single genus designation within the virus family (polyomavirus) and this genus contains 14 different species that infect at least 8 different mammalian species. in addition to containing dsdna genomes all members of this virus family are non-enveloped. the members of the family typically display restricted species and cell type specificity for lytic infection but many can induce the transformation of cells that do not support lytic infection. host cell and species-specific transcription and replication factors contribute significantly to the restricted specificity displayed by these viruses (feigenbaum et al., 1987; lynch and frisque, 1991; tada et al., 1989) . virus-receptor interactions are also known to contribute to cell type specificity and more importantly to pathogenesis in vivo (caruso et al., 2007; chen and atwood, 2002; dubensky et al., 1991; freund et al., 1991a; freund et al., 1991b; fried et al., 1981) . like many other virus families the polyomaviruses use multiple and often distinct receptors and entry mechanisms to infect cells. at high enough multiplicities many of these viruses can infect cells using alternative often poorly described mechanisms. the structures of two major members of the family have been solved in complex with receptor fragments and this has given insight into how these viruses recognize host cells (liddington et al., 1991; neu et al., 2008; stehle and harrison, 1997; stehle et al., 1994) . a major area of current investigation by several labs is focused on understanding the intracellular trafficking of polyomaviruses in host cells and the mechanisms involved in virus uncoating and delivery of the dsdna genomes to the nucleus. this paper will overview the field of virus structure and entry from its beginnings to the present. the focus will be on the mouse polyomavirus, on sv40, and on the two major human polyomaviruses, jcv and bkv. the mouse polyomavirus (mpyv) was first described by ludwig gross in 1953 in his studies on the transmission of mouse leukemias (gross, 1953) . he noticed that mice inoculated with cell free extracts from leukemias were capable of not only transmitting leukemia to naïve animals but that some of the animals also developed parotid tumors. in classic experiments he found that the "parotid" agent could be distinguished from the mouse leukemia virus by filtration. filters with larger pore sizes allowed both viruses to pass but filters of smaller pore size selectively trapped the leukemia agent indicating virology 384 (2009) [389] [390] [391] [392] [393] [394] [395] [396] [397] [398] [399] that the parotid agent was smaller than the leukemia virus. in the mid to late 1950s stewart and eddy followed up on these observations and found that the "parotid" agent was capable of inducing many different types of tumors when inoculated into mice and renamed the virus "poly"oma from the greek for many tumors stewart et al., 1958) . sv40 was discovered as a contaminant of formalin inactivated polioviruses that were grown in rhesus monkey kidney cells (sweet and hilleman, 1960) . the subsequent characterization of this virus as a papovavirus capable of transforming cells in vitro and inducing tumors in experimental animals was a cause for great concern as several hundred thousand individuals were inoculated with the contaminated vaccine (girardi et al., 1962) . this launched several decades of intensive research on sv40. this work on sv40 led to the discovery of many important cellular processes described elsewhere in this issue of the journal. jcv and bkv were both identified in 1971 from immunosuppressed patients. jcv was islolated in human fetal brain cultures following transfer from the brain of a patient with pml (padgett et al., 1971) . it was given the name jcv after the patient's initials. bkv was isolated in vero cells (african green monkey kidney cells) from the urine of a renal transplant recipient (gardner et al., 1971) . it was also named after the initials of the patient from whom it was isolated. the first studies of polyomavirus host-cell interactions followed closely behind their initial discovery and isolation in mammalian tissue culture. shortly after virus cultivation a number of groups began examining the interaction of mpyv with host cells using both biochemical and electron microscopy (em) techniques. infection of cells by the mpyv was envisioned as a multi-step process that included virus adsorption, virus penetration, and virus uncoating. morphological studies documented that whole virions were taken up into cells by phagocytosis as membrane bound single particles and membrane bound aggregates (mattern et al., 1966; mattern et al., 1967) . virus particles then appeared between the nuclear membrane but viral particles were not seen within the nucleus at these early times. this led to the conclusion that viral particles uncoat between the nuclear membranes and before they reach the nucleus (as we will see later this interpretation is largely correct with some minor modifications). in the early 1970s it became apparent that polyomavirus preparations contained significant numbers of defective interfering particles. the ability to plaque purify virus preparations allowed for an enrichment of infectious virions and for their separation from empty capsids by density gradient centrifugation. detailed analyses of these individual populations by em indicated that empty capsids and light populations of virions (pseudovirions) were endocytosed by two different pathways (bolen and consigli, 1979; mackay and consigli, 1976) . virions and pseudovirions were predominantly endocytosed as single membrane-bound particles that trafficked intact to the nucleus. empty capsids were endocytosed as aggregates into large vesicles that later appeared to fuse with lysosomes, apparently destined for degradation. a model was developed showing these two opposing endocytic routes in the cell (fig. 1) . the fact that virions and pseudovirions behaved differently than empty capsids led consigli and colleagues to carefully examine the viral particles for subtle differences in protein composition. using radiolabeled preparations they identified six species of vp1 (a-f) by isolelectric focusing (bolen et al., 1981) . only virions and pseudovirions were found to contain species e leading the group to hypothesize that species e was critical for delivering virions and pseudovirions to the nucleus. antibodies raised against virions that were found to be neutralizing specifically recognized species d, e, and f supporting this hypothesis (bolen et al., 1981) . the group then went on to attempt isolation of cellular receptors for mpyv by preparing anti-idiotypic antibodies to vp1 (marriott and consigli, 1985; marriott et al., 1987) . these antibodies blocked infection and pulled down several proteins associated with cell surfaces. the identities of these proteins were never determined and none were found to act as receptors for the virus. the mpyv and the two human polyomaviruses, jcv and bkv are known to require sialic acid for binding to host cells as "receptor destroying enzyme" or neuraminidase inhibits the viruses' ability to agglutinate red blood cells and to infect cells (table 1) . sv40 was thought not to attach to sialic acid as it did not agglutinate red blood cells and neuraminidase treatment of host cells did not reduce infectivity (clayson and compans, 1989) . however, sv40 does attach to a sialic acid containing receptor, ganglioside gm1, with sialic acid being the major contact point, but the narrow specificity of sv40 for gm1 prevents it from binding to carbohydrates on red blood cells. in fig. 1 . early model of polyomavirus attachment, penetration, and nuclear entry. open virions represent empty capsids that are found to be internalized in large endosomal vesicles that presumably lead to degradation. closed virions represent infectious particles and unlike empty capsids these are internalized into monopinocytotic vesicles. some virions were found in large tubular structures that likely represent the pinching off of these structures from caveosomes described years later by helenius (see text for detailed descriptions). modified from information in mackay and consigli, 1976 and maul et al., 1978. addition, the neuraminidase used for treating cells before challenge with sv40 does not cleave the sialic acid off gm1 so that it leaves the receptor intact (miller-podraza et al., 1982) . it can even cleave sialic acid off higher order gangliosides to yield gm1. therefore, neuraminidase treatment did not reduce sv40 receptors on cells and did not abolish infectivity. early reports in the mpyv system suggested that the sialic acid interactions were largely due to non-specific electrostatic interactions between the virus and these negatively charged cell surface carbohydrates. subsequent studies by the paulson group proved this hypothesis to be incorrect (cahan and paulson, 1980; fried et al., 1981) . they showed that mpyv recognized specific sialic acid structures on red blood cells and on mouse cells containing α2,3linked sialic acid. small and large plaque binding variants of mouse polyomavirus were subsequently shown to have different affinities for a branched compound that additionally contained a second, α2,6linked sialic acid. large plaque strains bound less well to the branched structure than did the small plaque strain (cahan et al., 1983) . these data correlated well with an earlier study showing that small plaque viruses adsorb to cells much better than the large plaque strains (diamond and crawford, 1964) . in more recent studies from the benjamin lab these differences were exploited and the plaque phenotypes genetically mapped to a single amino acid difference in the major capsid protein of the virus (freund et al., 1991b) . more importantly these differences in plaque behavior directly translated to their behavior in mouse models of infection (dubensky et al., 1991; freund et al., 1991a) . the large plaque strain spread more efficiently in mice and induced a higher frequency of tumors and the tumors were more widespread than those caused by the small plaque strains. this led to the hypothesis that the branched structures act as pseudoreceptors that are bound by the virus, but do not result in productive infection. the analysis of virushost cell interactions was thus important for understanding spread and pathogenicity of this group of viruses and generated increased interest in identifying specific receptors. the benjamin lab also attempted to isolate specific proteinaceous receptors for mpyv by producing and screening several thousand mononclonal antibodies raised against permissive mouse cell surfaces (bauer et al., 1999) . none of the antibodies were found capable of inhibiting infection leading them to conclude that no single molecule on the surface of the cells served as a specific receptor. the first hint of a proteinaceous receptor for mpyv came from studies in the amati lab where antibodies directed against natural integrin ligands as well as antibodies directed at α4β1 integrin inhibited a post-attachment step in infection (caruso et al., 2003) ( table 1 ). this was found to be mediated by an ldv integrin binding motif present in mpyv vp1 as a specific mutation in this motif reduces jcv is unique in that it initially enters cells by clathrin dependent endocytosis. the virus then traffics to early endosomes and then to caveosomes. bkv, sv40, and mpyv are reported to use caveolae dependent mechanisms of entry that traffic these viruses to caveosomes. sv40 and mpyv can also use non-caveolar but cholesterol dependent mechanisms of entry to access the caveosome and the mechanism used depends on cell type. once in the caveosome sv40 and mpyv traffic to the endoplasmic reticulum in tubular structures that bud off the er membrane. it is presumed that jcv and bkv use the same pathway but data on this is lacking. adapted from marsh and helenius, 1989. infectivity (caruso et al., 2007) . interestingly, when inoculated into newborn mice, the mpyv displays an altered pattern of infection when compared to wild type (caruso et al., 2007) . previous receptor hunting experiments described above would have missed this ligand as the virus apparently needs to undergo a conformational change after binding to sialic acid that then allows it to bind to the integrin receptors as a second step in the entry process. it is not certain whether infection of all cells requires this second step but it is clear that in some cases it is utilized. however, the ldv motif is buried under the receptor binding loops deep within vp1, and the mutation could have affected the structure of the binding site indirectly. recent studies from both the benjamin and rapoport groups clearly delineated a role for the gangliosides gd1a and gt1b in mpyv infection and for the ganglioside gm1 in sv40 infection (gilbert and benjamin, 2004; gilbert et al., 2005; tsai et al., 2003) (table 1 ). the rapoport group used sucrose flotation assays to initially ask whether mpyv would float when mixed with plasma membrane preparations from human erythrocytes, from bacterial membranes or from yeast membranes. they found that when mpyv was incubated with buffer or with either bacterial or yeast membranes the virus fractionated in the bottom third of the gradient. when mpyv was incubated with plasma membrane from human erythrocytes the virus floated to the top of the gradient indicating an association with plasma membrane lipids. upon treatment of the plasma membranes with neuraminidase the virus failed to float and fractionated at the bottom of the gradient. specific gangliosides were then tested for their ability to interact with polyomaviruses. mpyv only floated to the top of the gradient in the presence of gd1 and gt1b, and sv40 only floated to the top of the gradient in presence of gm1. the group then showed that rat cells cell deficient in complex ganglioside biosynthesis are poorly susceptible to mpyv and sv40 infection. however, when preincubated with gd1a or gt1b they become highly susceptible to mpyv and gm1 restores susceptibility to sv40. fluorescent co-localization studies indicated that the virus traffics directly to the er with the gangliosides (fig. 2) . this mode of internalization is strikingly similar to the one used by er directed bacterial toxins of the ab 5 family, such as cholera toxin, whose known receptors are gangliosides. in the case of the toxins, however, trafficking proceeds through the golgi. thus far there is no evidence that mpyv or sv40 traffics through the golgi and instead they appear to traffic through caveosomes to eventually reach the er where partial uncoating of the viruses take place. once the mpyv reaches the er it takes advantage of an unusual member of the protein disulfide isomerase family, erp29 (magnuson et al., 2005) (fig. 2) . pdi family members are oxidoreductases that contain one to several cxxc motifs in their thioredoxin domains (hebert and molinari, 2007) . they function to both reduce and oxidize disulphide bonds in newly synthesized proteins. they also isomerize disulphide bonds assisting in the formation of native protein structures. erp29 is structurally related to pdi proteins but it contains only a single cysteine in its fig. 3 . mechanisms used by polyomaviruses to exit the er and target their genomes to the nucleus. the mouse polyomavirus and sv40 both traffic to the er and it is presumed but not formally proven that jcv and bkv do the same. once in the er mpyv utilizes the unusual pdi family member, erp29 to induce a conformational change in its coat protein. this may lead to cleavage of the c-terminal arm of vp1 allowing the particle to bind to lipid membranes. the er retrotranslocation protein derlin 2 is involved in er exit. sv40 utilizes the canonical er oxidoreductases, pdi, and erp57, to unravel 12 of the vertex pentamers in the virus shell. this unfolded protein is then recognized by components of the er associated degradation (erad) pathway (derlin 1 and sell1) to exit the er. the viruses encounter a low calcium environment in the cytosol that may allow for their further disassembly. once disassembled the viral protein-mini-chromosome complex is transported across an intact nuclear pore for genome delivery to the nucleus. entire sequence and hence can only function in the isomerization reaction. a role for erp29 in normal cellular functions has not been established. the tsai group identified erp29 as the pdi family member responsible for inducing a conformational change in mpyv vp1 that exposes a trypsin sensitive site in vp1 (tsai et al., 2003) (fig. 3) . this conformational change is thought to result in the proteolytic cleavage of the c-terminal arm of vp1. as the c-terminal arm is involved in linking and stabilizing pentamer-pentamer interactions its cleavage would be predicted to destabilize the particle. the cleavage also led to the formation of a virion particle capable of binding and penetrating a lipid bilayer. this is predicted to facilitate the release of a partially disassembled and de-stabilized virion into the cytosol where nuclear localization signals in the vp1 protein could direct it across the nuclear pore (fig. 3) . work from the benjamin lab identified another er protein as being critical for mpyv infection. in this study shrnas directed at a protein involved in retrotranslocation of misfolded proteins out of the er, derlin 2, was found to significantly impair mpyv infection (lilley et al., 2006) (fig. 3) . in related work helenius and colleagues found that several er associated factors played critical roles in sv40 disassembly and release from the er (schelhaas et al., 2007) (fig. 3) . inhibition of pdi, erp57, and derlin 1 by sirna knockdown all inhibited sv40 infection of cells. knockdown of erp29, calnexin or calreticulin had no effect on sv40 infection. they found that erp57 uncoupled the 12 five-coordinated vp1 pentamers of sv40. this led to complete uncoating in the presence of egta, which removes the calcium ions that help to anchor the incoming c-terminal arms. the group also demonstrated a role for derlin 1 and sell1, two proteins involved in the erad retrotranslocation pathway in sv40 infection (fig. 3) . the model that emerges is one where the virion is delivered to the er where it is recognized by components of the er quality control machinery and is subjected to pdi related proteins that isomerize specific disulfide bonds involved in linking pentamers together within the capsid. the destabilized virion is then recognized essentially as a misfolded protein and is retrotranslocated out of the er where it encounters a low calcium environment in the cytosol. calcium is critical for viral particle stability and low levels of calcium in the cytosol likely contribute to further destabilization of the virion. exposure of nuclearlocalization signals in viral capsid proteins that likely remain bound to the mini-chromosome then transport the mini-chromosome across the nuclear pore (ishii et al., 1996; kasamatsu and nakanishi, 1998; nakanishi et al., 1996; yamada and kasamatsu, 1993) (fig. 3) . it thus appears that at least two polyomaviruses, mpyv and sv40, utilize er associated degradative pathways for uncoating. studies on sv40 entry mechanisms proceeded similarly and often in parallel with the work on mpyv described above. hummeler and colleagues followed the fate of sv40 particles in cells by electron microscopy (hummeler et al., 1970) . they describe the majority of particles becoming rapidly enveloped at the plasma membrane into monopinocytotic vesicles. sv40 particles could be seen as early as 1 h post-infection in the nucleus. maul and colleagues subsequently investigated the uptake and trafficking of sv40 in permissive and nonpermissive cells (maul et al., 1978) . no qualitative differences were seen between the two cell types indicating that these steps did not restrict virus infection of cells. their findings were very similar to those of hummeler but they described several intervening steps in the entry process including a prominent association of viral particles in tubular membrane structures and of particles associated with rough er. they did observe particles in the nucleus but these particles were not surrounded by membrane. they proposed a model where the entry of sv40 followed a series of membrane fusion and fission steps eventually liberating virions in the nucleus for uncoating. as they observed some free virions in the cytoplasm and some in the nucleus they proposed that the nucleus must contain an uncoating enzyme not present in the cytosol. as it turns out it appears that the er contains the necessary factors for uncoating. the tubular structures they observed were likely virions pinching off in large tubular structures from caveosomes as described by helenius many years later (pelkmans et al., 2001) . these tubular vesicles are transported to the er in a microtubual dependent manner (pelkmans et al., 2001) (fig. 2) . we now appreciate that most if not all polyomaviruses are trafficked to the er where uncoating occurs or at least begins. the single virus particles being enclosed in membranous vesicles at the plasma membrane described in the maul paper were the first demonstration of caveolae which were only later appreciated as being critical organelles for sv40 entry into cells (anderson et al., 1996) (fig. 2) . caveolae are membrane microdomains rich in sphingolipids and signaling molecules. sv40 exploits these domains most likely by virtue of its interaction with the ganglioside gm1. entry is relatively slow and proceeds in a ph neutral manner from caveolae to caveosomes to the er (pelkmans et al., 2001) . in addition to the ganglioside gm1, sv40 was also reported to use mhc class i molecules to infect permissive cells (atwood and norkin, 1989) (table 1) . the virus can infect some cells lacking mhc i and in these cases gm1 is the principle receptor although infection is inefficient. the relationship between these receptors remains unclear. the human polyomaviruses, jcv and bkv were discovered more recently and difficulties in isolating receptors for the mouse polyomavirus and sv40 probably precluded any interest in pursuing this with jcv and bkv as both are somewhat more difficult to propagate than sv40 or the mouse polyomavirus. with the introduction of new tools and techniques these issues have begun to be explored by several groups. both of the human polyomaviruses require sialic acid to infect cells. jcv can use either an α2,3or an α2,6-linked sialic acid to infect permissive glial cells (dugan et al., 2007; liu et al., 1998 ) and a ganglioside (gt1b) may also be involved (komagome et al., 2002) (table 1) . bkv uses only the α2,3-linkage to infect cells (dugan et al., 2005) (table 1) . a prominent role for the gangliosides gd1b and gt1b has been observed for bkv infection of kidney cells (low et al., 2006) (table 1) . bkv entry into cells is also similar to that of sv40, using caveolae mediated endocytosis to presumably deliver the virus to the er for uncoating (fig. 2) . jcv behaves differently than any of the described polyomaviruses and rather than being internalized directly into caveolae the virus utilizes clathrin dependent mechanisms to infect the cell (pho et al., 2000) (fig. 2) . virus entry proceeds by the normal clathrin dependent pathway to early endosomes (fig. 2) . once in the early endosome, however, the virus is transported to caveosomes in a ph dependent manner (querbes et al., 2006) . from here the virus is most likely transported to the er, presumably the common site of all polyomavirus uncoating events in the cell. in addition to utilizing sialic acid as a receptor jcv has been shown to require the serotonin receptor, 5ht2ar, to infect glial cells (fonseca-elphick et al., 2004) (table 1 ). the 5ht2a receptor is sufficient to confer susceptibility to cells lacking this receptor (hela and hek293a). infection of 5ht2ar expressing cells is still neuraminidase sensitive indicating a prominent role for cellular carbohydrates in infection. the 5ht2ar protein contains several potential glycosylation sites but it is unclear whether the sialic acid moieties responsible for infection reside on this protein or on other sialic acid containing molecules such as gangliosides. one group has found that jcv can infect human brain microvascular endothelial cells that lack the 5ht2a receptor (chapagain et al., 2007) . infection is very inefficient but the results indicate that virus infection can proceed by alternative mechanisms on some cell types. the polyomavirus capsid consists of 12 five-coordinated and 60 sixcoordinated morphological units that assemble into a t = 7d icosahedral capsid (klug, 1965) . icosahedral capsid symmetry allows the virus to construct a container for its dna from a large number of identical subunits, eliminating the need to encode many different components (crick and watson, 1956) . strict icosahedral symmetry, in which all subunits as well as their interactions with each other are identical, can only apply to capsids consisting of no more than 60 identical subunits. some exceptionally simple isocahedral virus capsids are indeed constructed from 60 identical subunits and therefore possess strict icosahedral symmetry (tsao et al., 1991) . however, the need for more complex structures that can package larger genes as well as internal proteins requires for many viruses a capsid that cannot be formed with such a small number of subunits. therefore, most viruses, including polyomaviruses, build larger capsids constructed from multiples of 60 subunits. in these capsids, subunits must be joined in a quasiequivalent manner, meaning that they do not form the same contacts with their neighbors throughout the capsid, but exhibit flexibility or alternative binding modes to accommodate local symmetry mismatches (caspar and klug, 1962) . the quasiequivalence principle postulated that the pentavalent and hexavalent morphological units of such larger icosahedral capsids were pentamers and hexamers of the same subunit. in some cases, this has been proven correct, e.g. for herpesvirus capsids (zhou et al., 2000) . the t = 7d icosahedral capsids of polyomaviruses were therefore also thought to consist of 420 subunits, grouped into 12 pentamers and 60 hexamers of the major capsid protein vp1 (finch, 1974) . however, the crystal structure of the mpyv capsid at 22.5 å resolution revealed that both the fiveand sixcoordinated morphological units of the capsid were in fact pentamers, a surprising result that appeared to challenge the concept of quasiequivalence as the same pentameric module is able to form both pentavalent and hexavalent contacts (rayment et al., 1982) . the crystal structure of sv40 at 3.8 å resolution, which was determined almost 10 years later finally provided the molecular basis for the unexpected capsid construction of polyomaviruses (liddington et al., 1991) . it revealed that the interpentamer contacts are primarily mediated by c-terminal extensions of vp1, termed arms, that protrude in different directions from each vp1 monomer and contact neighboring pentamers. both pentavalent and hexavalent pentamers donate and accept five c-terminal arms to tie the capsid together. the vp1 pentamer cores and the contacts between incoming arm and acceptor monomer are identical throughout the capsid, demonstrating that the polyomavirus capsid also uses a form of quasiequivalence as the basis for its construction. monomeric vp1 is formed by two antiparallel β-sheets: a sheet formed by strands b, i, d and g (the bidg sheet) and a sheet formed by strands c, h, e and f (the chef sheet). the two β-sheets are organized into a jelly-roll fold, a structural motif that is quite often observed in virus capsid proteins but rare in non-viral proteins. each incoming arm adds one strand (j′) to the edge of the bidg sheet of a monomer. the j′ strand is then fixed by the n-terminus of the accepting monomer, which acts as a clamp by adding a short helix and a final β-strand (a) to the sheet next to the j′ strand. the assembled structure is further stabilized by two calcium ions. these findings were confirmed for mpyv and are thought to hold true for the other members of the polyomavirus family as well because of conservation of the key contact residues (stehle et al., 1996; stehle et al., 1994) . structural studies of unassembled, or "free", pentamers that lack their c-terminal arms provide insight into one aspect of capsid assembly (neu et al., 2008; stehle and harrison, 1997) . the only structural change between free vp1 and vp1 in the context of the capsid is a rearrangement at the n-terminus, which forms a helix fixing the incoming arm in the capsid. in free pentamers, this part of vp1 forms a β-strand that aligns with the g strand and contacts residues at the beginning of the c-terminal arm. one likely interpretation of this finding is that this arrangement serves to guide the c-terminal arm away from its own subunit, thereby preventing misassembly of the polyomavirus capsids. receptor binding by mpyv was structurally investigated early on for whole virions as well as for vp1 pentamers (stehle et al., 1996; stehle and harrison, 1997; stehle et al., 1994) as it was known that mpyv bound α2,3-linked sialic acid (cahan et al., 1983; fried et al., 1981) . the sialylated oligosaccharides used as receptor mimics bound to vp1 in shallow pockets on the outer surface of the pentamers. these sialylated oligosaccharides were later found to be portions of the gd1a and gt1b gangliosides, which are the physiologic receptors for mpyv (tsai et al., 2003) . the same group also identified the ganglioside gm1 as a receptor for sv40 (tsai et al., 2003) . the structural and functional characterization of the interaction between sv40 and gm1 was made possible because a wide variety of glycans were available for defining receptor specificity and as the oligosaccharide portion of gm1 was available in amounts sufficient for structural analysis (campanero-rhodes et al., 2007; neu et al., 2008) . as an increasing amount of structural information on viral attachment proteins in complex with sialylated carbohydrate receptors has become available in recent years, we will focus the remainder of this chapter on those findings and their general implications. sialic acids are present on virtually every cell type in higher vertebrates (varki, 2008) . sialylation is a terminal modification on glycolipids and glycoproteins, and sialic acids can be attached to a variety of monosaccharides via different glycosidic linkages. physiological functions include cell-cell attachment via specific receptors, general charge repulsion of blood cells as well as roles in neuronal plasticity, immune regulation and glomerular filtration (reviewed in (varki, 2008) . many pathogens attach to sialic acid-containing receptors to enter cells, such as a number of viruses as well as bacterial toxins belonging to the clostridium neurotoxin and ab 5 families. the most common sialic acid in humans is n-acetyl neuraminic acid (neunac). it forms a six-membered glycosidic ring, to which several groups are attached, some in substitution for the typical hydroxyl groups of carbohydrates: a carboxylate group at the anomeric carbon c2, an n-acetyl group at the c5 carbon, and a glycerol chain at carbon c6 (fig. 4a) . the overall structure of neunac is rather rigid. in solution, the sugar ring prefers the energetically most favorable chair conformation, positioning its three distinctive groups in three different directions that serve as "handles" with which attachment proteins can interact. while the glycerol chain with three freely rotating bonds is quite flexible, the other two groups possess limited degrees of rotational freedom. high-resolution structural information on the interaction of viral attachment proteins with sialylated carbohydrates is available for the following systems: influenza virus a haemagglutinin (ha) in complex with oligosaccharides containing α2,3-linked and α2,6linked neunac (eisen et al., 1997; gamblin et al., 2004; ha et al., 2001; ha et al., 2003; russell et al., 2006; sauter et al., 1992; stevens et al., 2006; stevens et al., 2004; weis et al., 1988) , mpyv vp1 with a fragment of ganglioside gd1a (stehle and harrison, 1997) , rhesus, swine and human rotavirus vp8⁎ with methyl-α2,3-sialoside (blanchard et al., 2007; dormitzer et al., 2002a) , adenovirus ad37 fiber knob (ad37) with α2,3and α2,6-sialyllactose (burmeister et al., 2004) and sv40 vp1 with ganglioside gm1 (neu et al., 2008) . paramyxoviruses bind to their host cells with their haemagglutininneuraminidase protein that uses the same binding site for attachment to neunac and cleavage of its glycosidic bond (crennell et al., 2000) . we will, however, focus here on viral proteins that solely neunac is depicted in orange, the protein residues forming hydrogen bonds to neunac are colored green and the residues making van der waals contacts are colored grey. the viral surface is shown in grey. this figure was prepared with pymol (delano scientific, inc.). mediate attachment and do not possess enzymatic activity. sialic acid binding by catalytically active viral neuraminidases is probably not directly comparable to binding by attachment proteins, as neuraminidase function requires high affinity as well as additional binding energy to distort the sialic acid for catalysis. neuraminidases thus feature deeply buried binding sites with a higher number of specific contacts than are found in attachment proteins. although most of the attachment proteins listed above are not homologous and are found on unrelated enveloped as well as nonenveloped viruses, there are some general similarities in the modes of oligosaccharide binding. first, in all viral complexes, neunac is the major, and in the case of ad37 and vp8⁎ the only, point of contact. this distinguishes these complexes from, for example, bacterial toxins, which can also recognize sialylated receptors but form more contacts with carbohydrates attached to sialic acid than with the sialic acid itself (fotinou et al., 2001; merritt et al., 1994; stenmark et al., 2008) . second, all viral attachment complexes bind neunac in shallow depressions rather than deep grooves on the protein surface. the affinities for these interactions are only in the millimolar range, which is consistent with the exposed binding surfaces (burmeister et al., 2004; dormitzer et al., 2002b; eisen et al., 1997; neu et al., 2008; stehle et al., 1996) . a relatively low affinity may in fact be crucial for a productive viral infection as a very tight attachment to the host cell will prevent progeny virus from spreading. third, the attachment protein complexes all feature neunac in its chair conformation, which is the preferred conformation in solution. fourth, as detailed in the next section, there are several parallels in the strategies used by attachment proteins to bind sialic acid. the listed similarities observed in complexes of viral attachment proteins and sialylated receptors do not extend to other proteins that also bind the same type of receptors, indicating that they do represent characteristic properties of viral proteins that mediate engagement of host cell receptors. for example, the low affinity binding by viral attachment proteins is in contrast to bacterial toxins, which also bind sialylated oligosaccharides but employ deeper pockets and generally feature significantly higher affinity (fotinou et al., 2001; merritt et al., 1994; stenmark et al., 2008) . in contrast to the numerous binding sites on viruses, bacterial toxins have only one or at most five carbohydrate binding sites, and the interactions between these and cellular receptors must be sufficient to provide cell entry. the charged carboxylate group of sialic acid is arguably the most uniquely identifiable feature of this carbohydrate when compared to similar, uncharged structures. it is therefore perhaps not surprising that all of the viruses or viral proteins investigated here engage this group. however, they do so by employing two different strategies. the mpyv and ad37 form salt bridges from the positively charged side chains of arg and lys residues to the negatively charged carboxylate group (figs. 4e and c, respectively). by contrast, the other three viruses engage the carboxylate group with two parallel hydrogen bonds. in each case, the carboxylate group accepts two hydrogen bonds from two specifically spaced hydrogen bond donors in the proteins. in the influenza ha, sv40 vp1 and rotavirus vp8⁎ complexes, these are either the hydroxyl groups of ser and thr side chains or backbone amido groups (figs. 4b, d, e) . these parallel hydrogen bonds also have similar geometry. for each of them, the angle between the carboxylate c1, the carboxylate o that is engaged and the hydrogen bond donor approaches 120°. recognition of the negatively charged carboxylate group of neunac alone would not distinguish it from other molecules bearing a negative charge. the n-acetyl group, situated on the opposite side of the sugar ring as the carboxylate group, can serve as additional "handle" on neunac and it provides specificity by additional favourable contacts with the correct ligand. the n5 atom of the n-acetyl group is contacted by a hydrogen bond in each of the complexes. the binding partners are hydrogen bond acceptors in the protein, such as backbone carbonyl groups (figs. 4b-d) , the side chain carbonyl group of an asn residue (fig. 4f ) or the hydroxyl group of a tyr side chain (fig. 4e) . the mpyv vp1 also forms an additional hydrogen bond by an asn side chain, which contacts the carbonyl group of the acetyl function. with the sole exception of mpyv vp1, the binding sites of all proteins also feature a depression into which the methyl group of the n-acetyl moiety inserts. this depression is shallow and rather hydrophobic in the case of ad37, influenza ha and rotavirus vp8⁎, as it is lined with hydrophobic side chains (figs. 4b-d) . interestingly, sv40 vp1 does not feature such a shallow depression to accommodate the methyl group. rather, it has a substantially deeper pocket that is formed by hydrophobic residues such as leu65 and phe75 and lined with hydrophilic side chains of residues gln62 and gln278 at its rim (fig. 4f) . the natural hosts of sv40 are monkeys, in which n-glycolyl neuraminic acid (neungc) is the predominant sialic acid. in contrast to the human neunac, neungc has an additional hydroxyl group attached to the methyl group. the big, partly hydrophilic and likely water-filled cavity in sv40 could likely accommodate the more polar neungc better than neunac (campanero-rhodes et al., 2007) , which might account for its shape. even though contacts with the carboxylate and n-acetyl groups are sufficient to position neunac correctly on the protein surface, most attachment complexes feature contacts with the polar and flexible glycerol moiety. this group protrudes away from the protein in the mpyv vp1 and ad37 complexes (figs. 4c and e), but is recognized by a number of contacts in the other complexes. there are hydrogen bonds to o8 and o9 by each of sv40 vp1, influenza ha and rotavirus vp8⁎ that are formed by ser, tyr and arg side chains (figs. 4b, d, f) . in addition, the glycerol chain lies in a shallow pocket in all of these complexes and makes van der waals contacts to residues that confer shape specificity. in summary, while all of the binding sites recognize the carboxylate group and form a hydrogen bond to the n-acetyl group, only some (sv40 vp1, influenza ha and rotavirus vp8⁎) contact the glycerol chain both by hydrogen bonds and shape complementarity. in addition, most also feature a depression that accommodates the methyl group of the n-acetyl moiety. since all binding surfaces are exposed, the interactions determining neunac binding lie in the rather polar environment of the protein surface. the polar interactions are therefore probably quite weak. hydrogen bonds between sialic acid and protein atoms can be replaced by contacts between water molecules and protein atoms without much difference in energy. salt bridges in aqueous solution also possess less energy than salt bridges that are located in a partially hydrophobic, solvent-inaccessible environment. likewise, the non-polar van der waals interactions between carbohydrate and protein are also not very strong as such interactions increase in strength with the area of surface buried in the contact. most of the contacts described above exclude rather small areas from solvent compared to the extensive interfaces found in protein-protein complexes. the observed, relatively weak interactions are consistent with the experimentally-determined millimolar affinities for the proteinglycan complexes. the shallow binding sites of attachment proteins do not exclude binding of incorrect ligands by steric clashes. instead, specificity is provided by favorable interactions that are spaced in a way that only the correct ligand can engage in all contacts. in the case of influenza ha, mpyv vp1 and sv40 vp1, sialic acid by itself is not sufficient for binding, but additional sugars are also needed for the interaction. two of the viruses mentioned before, rhesus rotavirus and ad37, appear to bind neunac relatively indiscriminately, although it is possible that additional carbohydrates may contribute to binding. mpyv, sv40 and different strains of influenzaviruses, however, have distinct specificities for the structural contexts of the sialic acid. mpyv vp1 binds carbohydrates containing a neunac-α2,3-gal motif that is unbranched at the gal position (cahan et al., 1983) . the gal moiety is recognized by a hydrogen bond between the backbone carbonyl group of gly78 and the distinctive axial o4 hydroxyl group of gal (stehle and harrison, 1997) . the lack of a side chain in gly78 also confers binding specificity since the presence of even a small side chain in this position would cause sterical clashes with the gal and abolish binding. the architecture of the binding site accepts only one conformation of the neunac-α2,3-gal glycosidic linkage. this conformation is stabilized by an internal hydrogen bond and also occurs in solution, but it could not be adopted by compounds that are branched at the gal residue due to steric clashes. sv40 vp1 possesses quite narrow specificity for the oligosaccharide part of the ganglioside gm1, which is a branched compound of the following structure: gal-β1,3-galnac-β1,4-[neunac-α2,3-]-gal-β1,4-glc. while sv40 still binds the same compound with neungc substituted for neunac, binding is abolished if one of the terminal sugars, i.e. gal or neunac, are removed or if one of them bears an additional sugar moiety (campanero-rhodes et al., 2007; neu et al., 2008) . both the gal-β1,3-galnac and neunac branches directly contact the protein, providing binding affinity. the core structure of gm1, i.e. the branching gal with galnac and neunac attached to neighboring atoms is sterically constrained as clashes between galnac and neunac could occur in many conformations of the glycosidic linkages. the moiety adopts one dominant conformation in solution (poppe et al., 1994) , which is bound by sv40 vp1. specificity therefore arises from sv40 recognizing the correct placement of the two binding arms by a rigid spacer. different strains of influenza a virus infect different species because their ha molecules bind sialic acid in different contexts. the adaptation of avian has to human hosts occurs by a switch in binding specificity from avian-like α2,3-sialylated glycans to the longer α2,6-sialylated glycans that are present in the upper respiratory tract epithelia of humans (russell et al., 2006; skehel and wiley, 2000; stevens et al., 2006; van riel et al., 2007) . neunac is bound in the same orientation and by the same residues of both avian and human has so that specificity is conferred by the residues in contact with additional monosaccharides. recent data indicate that the different glycan types are not discriminated by contacts with the sugar residue to which the neunac is linked, but that the different overall topology of the glycans is recognized (chandrasekaran et al., 2008) . given the preferred torsion angles of the neunac-α2,3-gal-β1,3/4-glcnac motif typical of the recognized α2,3-linked glycans, the gal-β1,3/4-galnac part is fairly linear and can occupy the space delineated by a cone with neunac at its tip. sugar moieties farther away from the neunac mostly do not interact with the protein in this conformation. the glycans containing α2,6-linked neunac, for which neunac-α2,6-gal-β1,4-glcnac is typical, can also adopt this coneshaped topology. however, these also sample a topology, in which the gal is relatively fixed with respect to neunac and there is a kink at the gal position, forcing the glcnac and subsequent sugars to point back towards the protein. the space that can be sampled by the glycan therefore resembles an umbrella, with neunac-α2,6-gal as the stick and the rest of the glycan as the umbrella. for α2,6-linked trisaccharides that can sample both cone-and umbrella-shaped topologies, the cone-shaped one is preferred. however, the longer α2,6-linked glycans that confer infectivity in humans prefer the umbrella-shaped topologies, leading to additional contacts with the protein. if the ha of an influenza strain cannot accommodate these long, umbrella-shaped α2,6-linked glycans, it cannot spread in humans (chandrasekaran et al., 2008) . several of the structural and functional studies described above required the use of correct ligands. the use of model glycans such as α2,6-linked sialyllactose would not have revealed the difference in topology that is crucial for glycan recognition by has, as these short glycans do not adopt the umbrella shape that is bound by human influenzaviruses. in the case of sv40, only the correct gm1 receptor bound to the protein. smaller, unbranched components of the gm1 structure would not have given insight into sv40 receptor binding as they do not bind with detectable affinity (neu et al., 2008) . several other viruses, e.g. strains of coronavirus and reovirus, bind sialylated glycans, but structural information about these interactions is currently lacking. the availability of complex glycans and the use of glycan screens to define binding specificity have proven of great importance and will undoubtedly advance the understanding of glycan binding by these other viruses. the original studies describing polyomavirus host cell interactions depicted infection as involving a series of discrete steps that included virus adsorption, virus entry into membrane bound organelles, and virus trafficking by sequential membrane fusion and fission events. although there was controversy regarding whether these viruses could penetrate the nuclear envelope intact an early pivotal study demonstrated that the virus capsid only penetrates the outer nuclear envelope. recent studies on these viruses describe largely the same series of steps but in significantly more detail. we now know and appreciate that the majority of these viruses recognize specific linkages of sialic acid on gangliosides and glycoproteins. these viruses also recognize other cellular components such as the 5ht2a receptor for jcv, α4β1 integrin for mpyv, and the mhc class i protein for sv40, likely as part of a receptor complex with gangliosides or other sialic acid containing structures. there are clear cell type specific and viral strain differences that determine what components of the receptor complex are required to initiate infection. once bound to cells all of these viruses induce molecular signals that initiate infectious entry. the mpyv, sv40, and bkv all infect cells by caveolae dependent endocytosis. non-caveolar cholesterol dependent mechanisms can also be exploited by some of these viruses depending on cell type. downstream of both caveolae and raft dependent mechanisms the viruses traffic to a ph neutral caveosome that lacks markers of other cellular organelles. high resolution video microscopy of virus entry has found that these viruses are trafficked out of the caveosomes in elongated tubular structures that fuse with the er membrane. this trafficking step is dependent on microtubules. the human polyomavirus, jcv, appears to be an exception and rather than utilizing caveloae to enter cells this virus exploits clathrin dependent mechanisms to initially traffic to early endosomes. in the early endosomes jcv is found to be tightly opposed to the inner leaflet of the endosome and in a ph and rab5 dependent step traffics from the early endosome to caveosomes. at this point the virus likely follows the fate of other polyomaviruses and travels to the er. once in the er the viruses take advantage of protein disulfide isomerases to rearrange their capsid structure. in the case of sv40, pdi and erp57 rearrange the 12 five-coordinated vp1 pentamers. the now misfolded structure is recognized by components of the erad pathway (derlin 1, sell1) and exported to the cytosol. calcium levels in the cytosol of resting cells are significantly lower (0.1 μm) than levels in the er (100-400 μm). as calcium is required to stabilize polyomavirus virions the low levels of calcium encountered as the destabilized virus exits the erad pathway are likely sufficient to further distort and disassemble the virion and prepare it for transport through the nuclear pore. in the case of mpyv a novel pdi like protein, erp29 acts to unfold vp1 exposing a protease sensitive site in the cterminal arm of the protein. once cleaved the destabilized particle can insert into a lipid membrane. the virus may directly escape the er via this mechanism or it might utilize the retrotranslocator derlin 2 as inhibition of this protein reduces mpyv infection. it is likely that the two human polyomaviruses use similar mechanisms to escape the er but this has not been formally demonstrated. early structural studies on mpyv and sv40 challenged the quasiequivalence theory of capsid assembly as there were 72 vp1 pentamers arranged that formed both pentavalent and hexavalent contacts. subsequent high resolution structural studies provided the molecular basis for this unexpected capsid construction. it revealed that the interpentamer contacts are primarily mediated by c-terminal extensions of vp1, termed arms, that protrude in different directions from each vp1 monomer and contact neighboring pentamers. both pentavalent and hexavalent pentamers donate and accept five c-terminal arms to tie the capsid together. the vp1 pentamer cores and the contacts between incoming arm and acceptor monomer are identical throughout the capsid, demonstrating that the polyomavirus capsid also uses a form of quasiequivalence as the basis for its construction. recent high resolution structural studies on viral capsid proteins in complex with receptor fragments has provided a detailed picture of how these viruses engage this component of the receptor complex. this level of detail may provide the basis for the design of small molecular weight compounds capable of antagonizing polyomavirus host cells receptor interactions and may in time lead to novel therapeutic approaches to treat patients with polyomavirus induced disease. bound simian virus 40 translocates to caveolin enriched membrane domains, and its entry is inhibited by drugs that selectively disrupt caveolae class i major histocompatibility proteins as cell surface receptors for simian virus 40 discrimination between sialic acid containing receptors and pseudoreceptors regulates polyomavirus spread in the mouse insight into host cell carbohydrate-recognition by human and porcine rotavirus from crystal structures of the virion spike associated carbohydrate-binding domain (vp8⁎) differences in the sub-populations of the structural proteins of polyoma differential adsorption of polyoma virions and capsids to mouse kidney cells and guinea pig erythrocytes crystal 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inoculation, in early infancy, with filtered (berkefeld n), or centrifugated (144,000 ×g), ak-leukemic extracts x-ray structures of h5 avian and h9 swine influenza virus hemagglutinins bound to avian and human receptor analogs x-ray structure of the hemagglutinin of a potential h3 avian progenitor of the 1968 hong kong pandemic influenza virus in and out of the er: protein folding, quality control, degradation, and related human diseases morphological aspects of the uptake of simian virus 40 by permissive cells analysis of a nuclear localization signal of simian virus 40 major capsid protein vp1 how do animal dna viruses get to the nucleus? 424-431. the manuscript entitled "structure, attachment and entry of polyoma-and papillomaviruses oligosaccharides as receptors for jc virus structure of simian virus 40 at 3.8 a resolution murine polyomavirus requires the endoplasmic reticulum protein derlin-2 to initiate infection infection of glial cells by the human polyomavirus jc is mediated 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gm1 pentasaccharide biosynthesis and localization of gangliosides in cultured cells association with capsid proteins promotes nuclear targeting of simian virus 40 dna structural basis of gm1 ganglioside recognition by simian virus 40 cultivation of papovalike virus from human brain with progressive multifocal leukoencephalopathy caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the er jc virus enters human glial cells by clathrindependent receptor-mediated endocytosis carbohydrate dynamics at a micellar surface: gd1a headgroup transformations revealed by nmr spectroscopy a jc virus induced signal is required for infection of glial cells by a clathrin and eps15 dependent pathway invasion of host cells by jc virus identifies a novel role for caveolae in endosomal sorting of noncaveolar ligands polyoma virus capsid structure at 22.5 a resolution avian and human receptor binding by hemagglutinins of influenza a viruses binding of influenza virus 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vacuolating virus, s.v. 40 cell type-specific expression of jc virus early promoter is determined by positive and negative regulation gangliosides are receptors for murine polyomavirus and sv40 the three-dimensional structure of canine parvovirus and its functional implications human and avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals sialic acids in human health and disease structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid role of nuclear pore complex in simian virus 40 nuclear targeting seeing the herpesvirus capsid at 8.5 a we would like to thank wendy virgadamo for designing figs. 1-3. work in the atwood lab is supported by nih grants ca71878 and ns43097. work in the stehle lab was supported by the deutsche forschungsgemeinschaft (sfb-685). key: cord-337976-c2auspti authors: weiss, susan r. title: coronaviruses sd and sk share extensive nucleotide homology with murine coronavirus mhv-a59, more than that shared between human and murine coronaviruses date: 1983-04-30 journal: virology doi: 10.1016/s0042-6822(83)80022-1 sha: doc_id: 337976 cord_uid: c2auspti a cdna probe representing the genome of mouse hepatitis virus (mhv) strain a59 (mhv-a59) was used to measure nucleotide sequence homologies among murine and human coronaviruses and the sd and sk coronaviruses isolated by burks et al. since sd and sk were isolated by inoculation of multiple sclerosis (ms) central nervous system (cns) tissue into mice or cultured mouse cells, it is important to determine their relationships to other murine and human coronavirus isolates. our results indicate that sd and sk share almost complete nucleotide homology (approximately 90%) with mhv-a59 and generate subgenomic rnas of the same sizes as mhv-a59. the human coronavirus (hcv) strains tested show less homology with mhv-a59. the immunologically unrelated hcv-229 e shows no nucleotide homology with mhv-a59. the immunologically crossreactive hcv-oc43 shows nucleotide homology with mhv-a59 by blot hybridization but not when hybridized in solution and assayed by s1 nuclease digestion. coronaviruses have been associated with acute and chronic neurological diseases in many species of animals (mcintosh, 1974) . infection of rodents with the murine coronavirus, mouse hepatitis virus mhv strain jhm, has been used as a model system to study virus-induced demyelination (weiner, 1973; nagashima et al., 1978; stohlman and weiner, 1981) . after initial panencephalitis caused by mhv-jhm, this virus produces a persistent infection with primary demyelination with some evidence for remyelination (weiner, 1973) . thus persistent mhv-jhm infection of rodents has been cited as a model to study the human demyelinating disease multiple sclerosis (ms). human coronaviruses (hcv) are ubiquitious in nature with a large portion of the human population possessing neutralizing antibodies (mcintosh, 1974) . these viruses were isolated usually as respiratory, and occasionally as enteric viruses. to whom reprint requests should be addressed. they are estimated to be responsible for 15% of common colds (mcintosh, 1974) . there are no reports thus far of involvement of human coronaviruses with persistent neurological disease. some strains of hcv such as 0c43, are antigenically related to murine coronaviruses such as mhv strain jhm (mcintosh, 1974; gerdes et al., 1981a, b) and may be grown in the brains of suckling mice (mcintosh et al., 1967) . others such as hcv-229e are unrelated antigenically to mhv or hcv-0c43 (mcintosh, 1974; pederson et al., 1978) . because (1) murine coronaviruses are associated with chronic demyelinating disease in rodents (weiner 1973; nagashima et al., 1978) , (2) antibody against hcv is very common in the human population (mcintosh, 1974) , and (3) there is evidence suggesting that ms may be caused by a virus, various workers have undertaken comparisons of human and murine coronaviruses and have started to search for coronaviruses in central nervous system (cns) tissue from ms patients. there is one report of particles with coronavirus-like morphology seen by electron microscopy in brain tissue from an ms patient (tanaka et al., 1976) . more recently burks and co-workers have isolated two coronaviruses, designated sd and sk, by intracerebral inoculation of unfrozen ms cns autopsy tissue either into weanling mice or into 17cl-1 mouse cells in culture (burks et al., 1980) . gerdes et al. (1981a, b) and we (this manuscript) have examined the relationship between these viruses and other known murine and human coronaviruses. gerdes et al. (1981a, b) showed that sd and sk are antigenically related to mhv-a59 and to hcv-oc43 but not hcv-229e. they were inconclusive about which strains their isolates were more related to. we have further compared murine and human coronaviruses and sd and sk by using molecular hybridization of virus-specific rna with cdna probes. our results show extensive nucleotide homology between sd and sk and mhv-a59, more than that between the human viruses and mhv-a59. viruses and cells. mhv-a59 was grown in 17cl-1 cells as previously described (weiss and leibowitz, 1983) . sd and sk viruses (burks et al., 1980) were obtained from dr. j. gerdes and were also grown in 17cl-1 cells. hcv-229e was obtained from the american type culture collection (atcc) and grown in human embryonic lung (l-132) cells also obtained from the atcc. these viruses were plaque purifled two times and grown in dulbecco's medium with 10% fetal calf serum (robb and bond, 1979) . hcv-oc43 was obtained as a 20% suckling mouse brain suspension from dr. j. hierholzer at the center for disease control (cdc), atlanta. it was inoculated intracerebrally into c57bl/6 suckling mice, harvested 2 days later, and a 10% brain suspension was made in phosphate-buffered saline (pbs) containing 0.75% bovine serum albumen. the mothers of the suckling mice were obtained from jackson labs as mhv-free animals. all were negative for antibodies against mhv-a59 (as de-termined by an enzyme-linked immunosorbent assay) and hcv-oc43 (as shown by a lack of 0c43 hemagglutination inhibiting activity in the sera of these animals (hierholzer et al., 1969) ) and thus were considered uninfected by these coronaviruses. virus in brain homogenates was assayed by hemagglutination of chicken red blood cells at room temperature (hierholzer et al., 1969) . virus was further verified as hcv-oc43 since hemagglutination was inhibited by an anti-0c43 reference antisera obtained from cdc. hcv-oc43 was also grown in human rectal tumor (hrt) cells (laporte et al., 1980) obtained from dr. david brian. infected mouse brain homogenates were adsorbed onto monolayers of hrt cells for 1 hr at room temperature. the cells were extensively washed, medium added, and the cells incubated at 33 ~ virus in the supernatant was titered at various times postinfection by hemagglutination (hierholzer et al., 1969) . mock-infected cells were adsorbed with a brain homogenate from uninfected suckling mice. cdna probes, cdnas were synthesized using purified genome rna as template, oligomers of calf thymus dna as primers, and reverse transcriptase (taylor et al., 1976) . cdnas were labeled with [32p]dctp to specific activity of approximately l0 s cpm/#g. when used for liquid hybridization, cdna was synthesized in the presence of actinomycin d and was >95% single stranded. such cdnas were validated to be highly virus specific and to represent the majority of the genome rna as previously described in detail leibowitz, 1981, 1983) . rna extraction. mhv-a59, sd, sk, and hcv-229e virus infections were carried out with a multiplicity of infection of between 0.1 and 1 plaque-forming units per cell. rna was extracted 18 hr after infection with a59, sd, and sk viruses, when massive syncytia were present. 229e-infected cells were labeled with [3h]uridine in the presence of 10 ttg/ml actinomycin d from 18 to 24 hr postinfection when rna was extracted. rna was extracted from oc43-infected hrt cells at 3 days post-infection. rna was extracted from the cytoplasm of infected cells by sds-proteinase k treatment followed by phenol extraction as previously described (weiss and leibowitz, 1983) . rna was extracted from suckling mouse brain homogenates by sds-proteinase k treatment followed by phenol extraction (weiss, varmus and bishop, 1977) . rna analysis. (1) gel electrophoresis. rna was electrophoresed in 1% agarose gels, in the presence of methyl mercury hydroxide (bailey and davidson, 1976) or formaldehyde (lehrach et al., 1977) as denaturant. gels were either fluorographed with sodium salicylate (chamberlain, 1979) or blotted onto nitrocellulose (thomas, 1980) . (2) blots. dot blots. rna was adsorbed onto nitrocellulose filters in various amounts (as designated in figure legends), dried, and the filters were baked and hybridized with cdna (thomas, 1980) . northern blots: rna was electrophoresed in gels, blotted onto nitrocellulose filters, and hybridized with cdna (alwine et al., 1977; thomas, 1980) . (3) hybridization in solution was carried out at 68 ~ 0.6 mnac1, and assayed by $1 nuclease digestion as previously described (leong et al, 1972) . in crt curves, increasing amounts of rna were hybridized with a fixed amount of cdna to achieve increasing crt values where crt = concentration of rna • time of hybridization. hcv-oc43 has been difficult to grow and assay in cell culture and this has impaired the study of viral nucleic acids. this virus is usually grown in the brains of suckling mice and titered either by infection of suckling mice or by hemagglutination (hierholzer et al., 1969) . schmidt et al. (1979) have reported growing and plaquing hcv-oc43 on human rhabdomyosarcoma (rd) cells. although we had difficulty with growing the virus in rd cells, we have had some success with growth in human rectal tumor (hrt) cells (laporte et al., 1980) . we have used hemagglutination to detect and quantitate hcv-oc43 in both infected suckling mouse brain homogenates and hrt cell supernatants. as shown in table 1 brain homogenates from infected mice contained 40,000 hau/ml of 0c43 and homogenates from control mock-infected animals had none. this activity could be specifically inhibited by anti-oc43 reference antisera but not by a59 antisera or preimmune sera (data not shown). also shown in table 1 after infection of hrt cells with oc43-infected mouse brain homogenates (800 hemagglutinating units/ 106 cells), hemagglutinating activity could be measured in the medium. as expected pretreatment of brain homogenates with antisera directed against oc43 prevented hemagglutinating units/ml of oc43. one hau is defined as the amount of virus present in 0.05 ml of the highest dilution of brain homogenate or supernatant capable of agglutinating 0.05 ml of 0.5% chicken erythrocytes in the standard assay described by hierholzer et al. (1969) . b mock-infected mice or cells are mock-infected with a 10% homogenate or uninfected suckling mouse brains. c oc43-infected suckling mouse brain homogenate was incubated with anti-oc43 reference antiserum for 1 hr at room temperature before infecting cells. 1981b), the reciprocal e x p e r i m e n t using hcv-229e cdna was also c a r r i e d out. as i l l u s t r a t e d in fig. 2 , c d n a r e p r e s e n t i n g the hcv-229e g e n o m e h y b r i d i z e d only to its homologous r n a a n d not to hcv-oc43 r n a or to mhv-a59 rna. the blot e x p e r i m e n t s i l l u s t r a t e d in the above sections show the h o m o l o g y between the nucleic acids of c o r o n a v i r u s s t r a i n s mhv-a59 and sd, sk, a n d hcv-0c43 a n d the lack of homology w i t h hcv-229e. these techniques, however, do not q u a n t i t a t e the p e r c e n t a g e of the g e n o m e sequences t h a t are homologous. to be more q u a n t i t a rna from infected or mockinfected cells or mouse brain homogenates was spotted onto nitrocellulose filters. in the case of intracellular rnas, 5.0, 0.5, and 0.05 ttg amounts were used. in the case of purified genome rna, 0.1, 0.01, and 0.001 #g were used. filters were hybridized to 106 cpm (108 cpm/#g) of a59 [sup]cdna and autoradiographed (alwine et al, 1977; thomas, 1980) . similar hybridizations were carried out with rnas from cells infected with the other viruses and the plateau values for the percentage cdna hybridized are summarized in table 2 . sd and sk are almost completely homologous to mhv-a59, more so t h a n another mhv strain, jhm. hcv-229e showed no homology with a59 cdna as predicted from the blot experiments. hcv-oc43 showed no homology with the cdna by this assay. this is probably due to stringency of hybridization and s1 nuclease assay for hybridization (see discussion). to f u r t h e r compare the rna of the murine and h u m a n strains the intracellular subgenomic rnas were examined by gel electrophoresis. as illustrated in fig. 4 , cells infected with mhv-a59 contain six m a j o r subgenomic rnas as well as genome-sized rna (band 1) (cheley et al., 1981a; leibowitz et al, 1981; spaan et al., 1981) . these range in molecular weight from 0.63 • 106 daltons for rna 7 to 6.1 • 106 daltons for r n a 1. intraeellular rnas extracted from eells infected with sd or sk, were eleetrophoresed in parallel with mhv-a59 rna, blotted onto nitrocellulose and the virusspecific species detected by hybridization with mhv-a59 edna. seven rna bands were observed, all comigrating with the m a j o r mhv-a59 rnas. rnas 2, 4, 5, and, in the ease of sk, rna 3, are less abundant than with mhv-a59 and the extra band between rnas 2 and 3 is more prominent. the extra bands between rnas 3 and 4 are also more p r o m i n e n t in the sd rna. the intraeellular rnas of hcv-229e were also compared to those of mhv. (in this experiment mhv-3 rna was used instead of mhv-a59. the genome of mhv-3 is 95% homologous to mhv-a59 and mhv-3 generates the same size intraeellular rnas as mhv-a59 (cheley et al., 1981b, weiss and ). since hcv-229e rna does not cross-hybridize fig. 2, dot blot hybridizations of hcv-229e edna with coronavirus rnas. rna from infected or mockinfected cells or brain homogenates or from purified virions was spotted onto a nitrocellulose filter. in the case of intracellular rna, 5.0, 0.5, and 0.05 ~g were used and in the case of purified genome rna 0.1, 0.01, and 0.001 ug were used. the filter was hybridized with 106 cpm (10 s cpm/#g) of 229e [32p]cdna and autoradiographed (alwine et al, 1977; thomas, 1980) . with a59 edna, 229e intracellular rna was labeled with [3h]uridine in the presence of actinomycin d (to inhibit host dna-dependent r n a synthesis). as shown in fig. 5 , 229e also generates six subgenomic rnas, but they are of different sizes from mhv-3 and hence from sd and sk. (genome rna was difficult to observe in this experiment probably due to some rna degradation). m a m m a l i a n coronaviruses have been divided into two antigenic groups (pederson et al, 1978) . one includes mhv, hcv-oc43, a rna from virions, infected cells, or brain homogenates was hybridized with 2000 cpm (10 s cpm/ t~g) of a59 [azp]cdna to completion, the plateau portion of a crt curve. hybridization was assayed by $1 nuclease digestion. these values have been normalized to 100% hybridization of a59 cdna with its homologous a59 rna. the actual values of hybridization of a59 cdna with a59 rna ranged from 85 to 100%. gastroenteritis virus of swine, and canine coronavirus. gerdes et al. (1981a, b) have shown that the sd and sk viruses fall into the first group. they showed that all a59 intracellular proteins are immunoprecipitable with antisera directed against sd, sk, or hcv-oc43. from these experiments, however, it was impossible to determine whether sd and sk were more related to human (0c43) or murine (a59) viruses. this is important in determining the origin of sd and sk and the possible link to ms. we have used nucleic acid hybridization with cdna to further explore the relationship among these viruses. our cdnas are representative of most if not all of the genome rna sequences leibowitz, 1981, 1983) and thus are appropriate reagents for quantitating sequence homologies. the relationship between sd and sk and mhv-a59 as determined by molecular hybridization experiments basically agrees with immunological studies. the sd and sk viruses show extensive homology in nucleotide sequence (approximately 90%) to the a59 strain of mhv even when assayed by the stringent $1 nu-clease assay. this is more homology than that shared between mhv-a59 and another mhv strain, jhm leibowitz, 1981, 1983) . furthermore, the pattern of intracellular rnas generated by sk and sd is very similar to that of mhv-a59. this is not surprising, considering that gerdes et al., (1981a, b) showed that cells infected by sd or sk have patterns of viral proteins similar to a59-infected cells (gerdes et al., 1981a, b) . an extra polypeptide of 42,000 daltons molecular weight was observed in , or sk (lane c) were electrophoresed in a 1% agarose gel containing formaldehyde as a denaturant (lehrach et al, 1977) . after electrophoresis the gel was stained with ethidium bromide to locate ribosomal rnas, blotted onto nitrocellulose (thomas, 1980) , hybridized with l0 s cpm of a59 [32p]cdna, and autoradiographed (alwine et al., 1977) . arrows designate the position of 18 s and 28 s ribosomal rnas. viral rnas are numbered according to (bailey and davidson, 1976) . after electrophoresis the gel was stained with ethidium bromide to locate ribosomal rnas and fluorographed (chamberlain et al, 1979) . the positions of 18 s and 28 s ribosomal rnas are designated by arrows. there is no extra major rna band seen in the sd and sk samples. the molar ratios of some of the rnas are different in sd-and sk-infected cells, specifically, rnas 2 and 5 are less prominent and the extra rna band between 2 and 3 is quite prominent. the extra bands between rnas 3 and 4 are also more prominent in the sd sample. however, there is similar variation among the mhv strains (cheley et al., 1981b; leibowitz et al., 1981; weiss and leibowitz, 1981 ) and we do not understand its significance. perhaps this is due to a small amofint of degradation in the extraction of these very large rnas. comparison of the human coronaviruses oc43 and 229e with mhv-a59 reveals less homology than between mhv-a59 and sk and sd. rna extracted either from brain homogenates of oc43-infected suckling mice or from hrt cells infected with oc43 shows homology with a59 cdna when assayed by blot hybridization. this homology is not detected when liquid hybridization followed by the more stringent s1 nuclease assay is used. there is precedence for this in at least two other systems. homology between murine and human papovaviruses (howley et al., 1979) and murine and human rotaviruses (schroeder et al, 1982) is detected only under relaxed hybridization and assay conditions. these viruses are antigenically related; this immunological cross-reactivity is detected without difficulty as is the immunological cross-reactivity between mhv-a59 and hcv-oc43. there is no homology detected between hcv-229e and mhv-a59 or hcv-oc43 in cross-hybridizations using dot blots and cdna representing either virus. it is unlikely that there is homology betwen hcv-229e and sd and sk since the latter viruses are so closely related to mhv-a59. this is not surprising considering these viruses fall into different antigenic groups (pederson et al., 1978) . however, there are reports of immunological cross-reactivity between the nucleocapsid proteins of these viruses (gerdes et al., 1981b) . these sequences if related, are probably too diverged to be detected by our assay. perhaps under less stringent blotting conditions, homology could be detected. these results show that sd and sk are more closely related to mhv-a59 than are the human viruses 0c43 and 229e. because sd and sk are almost completely homologous (90%) to mhv-a59 it is unlikely that they share much homology with either hcv-oc43 or hcv-229e. it is possible that the 10% of sd and sk sequences that are not homologous to mhv-a59 are homologous to either hcu or hcv-229e. however, if that were the case, it would still suggest that these isolates are more related to murine than human coronaviruses. this, in addition to the fact that sd and sk grow only in murine and not human cells (gerdes et al., 1981b) , suggests that these viruses are not of human, but murine origin. method for detection of specific rnas in agarose gels by transfer to diazobenzyloxymethyl paper and hybridization with dna probes methylmercury as a reversible denaturing agent for agarose-gel electrophoresis two coronaviruses isolated from central nervous system tissue of two multiple sclerosis patients fluorographic detection of radioactivity in polyacrylamide gels with the water soluble fluor, sodium salicylate intracellular murine hepatitis virus-specific rnas contain common sequences rna and polypeptide homology among murine coronaviruses antigenic relationships of coronaviruses detectable by plaque neutralization, competitive enzyme linked immunosorbent assay, and immunoprecipitation coronavirus isolates sk and sd from multiple sclerosis patients are serologically related to murine coronaviruses a59 and jhm and human coronavirus oc43 but not to human coronavirus 229e standardized viral hemagglutination and hemagglutination-inhibition tests ti. description and statistical evaluation a rapid method for detecting and mapping homology between heterologous dnas some characteristics of hemagglutination of certain strains of "ibv-like" virus mouse hepatitis virus a59: mrna structure and genetic localization of the sequence diversion from hepatropic strain mhv-3 comparative analysis of rna genomes of mouse hepatitis virus une lign6e cellulaire particuli~rement sensible a la r6p-lication du coronavirus ent6ritique: les cellules hrt 18 rna molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination the virus-specific intracellular rna species of two murine coronaviruses: mhv-a59 and mhv-jhm virus-specific ribenucleic acid in cells producing rous sarcoma virus: detection and characterization growth in suckling mouse brain of "ibvlike" viruses from patients with upper respiratory tract disease: prec coronaviruses: a comparative review coronavirus induced subacute demyelinating encephalomyelitis in rats: a morphological analysis antigenic relationship of the feline infectious peritonitis virus to coronaviruses of other species pathogenic murine coronaviruses. l characterization of biological behavior in vitro and virus specific intracellular rna of strongly neurotropic jhmv and weakly neurotropic a59v viruses plaque assay and improved yield of human coronaviruses in a human rhabdomyosarcoma cell line sequence relationship between the genome segments of human and animal rotavirus strains isolation and identification of virus-specific mrnas in cells infected with mouse hepatitis virus (mhv-a59) chronic central nervous system demyelination in mice after jhm virus infection intracisternal virus-like particles in the brain of a multiple sclerosis patient efficient transcription of rna into dna by avian sarcoma virus polymerase hybridization of denatured rna and small dna fragments transferred to nitrocellulose. prec pathogenesis of demyelination induced by a mouse hepatitis virus (jhm virus) comparison of the rnas of murine and human coronaviruses characterization of murine coronavirus rna by hybridization with virus-specific cdna probes the size and genetic composition of virus-specific rnas in the cytoplasm of cells producing avian sarcoma-leukosis viruses this research was supported by nih grant a117418 and grant rg 1421 from the multiple sclerosis society. i thank maureen highkin and david petcu for help with some of the experiments and roseann femia for typing the manuscript. key: cord-332356-au7s3dmp authors: strandin, tomas; hepojoki, jussi; wang, hao; vaheri, antti; lankinen, hilkka title: the cytoplasmic tail of hantavirus gn glycoprotein interacts with rna date: 2011-09-15 journal: virology doi: 10.1016/j.virol.2011.06.030 sha: doc_id: 332356 cord_uid: au7s3dmp we recently characterized the interaction between the intraviral domains of envelope glycoproteins (gn and gc) and ribonucleoprotein (rnp) of puumala and tula hantaviruses (genus hantavirus, family bunyaviridae). herein we report a direct interaction between spike-forming glycoprotein and nucleic acid. we show that the envelope glycoprotein gn of hantaviruses binds genomic rna through its cytoplasmic tail (ct). the nucleic acid binding of gn-ct is unspecific, as demonstrated by interactions with unrelated rna and with single-stranded dna. peptide scan and protein deletions of gn-ct mapped the nucleic acid binding to regions that overlap with the previously characterized n protein binding sites and demonstrated the carboxyl-terminal part of gn-ct to be the most potent nucleic acid-binding site. we conclude that recognition of the rnp complex by the gn-ct could be mediated by interactions with both genomic rna and the n protein. this would provide the required selectivity for the genome packaging of hantaviruses. rodent-and insectivore-borne hantaviruses (genus hantavirus) are enveloped viruses that together with four other genera (orthobunya-, nairo-, phlebo-and tospovirus) constitute the family bunyaviridae (elliott et al., 2000; nichol et al., 2005) . the asymptomatic carrier rodents and insectivores spread hantaviruses in their excreta, which is considered the main route for transmission to humans kariwa et al., 2007; vaheri et al., 2011) . when transmitted to man hantaviruses cause two severe diseases: hemorrhagic fever with renal syndrome (hfrs) and hantavirus cardiopulmonary syndrome (hcps). old world hantaviruses include causative agents of hfrs, whereas the hantaviruses found in the new world have been associated with hcps (mir, 2010; c. schmaljohn and hjelle, 1997) . additionally hantaviruses cause nephropathia epidemica, a mild form of hfrs, that occurs mainly in northern europe vapalahti et al., 2003) . the single-stranded, negative-sense rna genome of hantaviruses is segmented into small (s), middle (m) and large (l) segments encoding nucleocapsid (n) protein, glycoproteins (gn and gc) and the rna-dependent rna polymerase (l protein), respectively (plyusnin et al., 1996) . the rna genome is encapsidated via oligomerization of n protein to form rnp complexes (alfadhli et al., 2001; alfadhli et al., 2002; alminaite et al., 2006; alminaite et al., 2008; kaukinen et al., 2005; wang et al., 2008) . the rnps interact with the spike complex, formed of four units of both gn and gc glycoproteins (battisti et al., 2010; hepojoki et al., 2010a; huiskonen et al., 2010) , by binding to the cytoplasmic tails (cts) of both gn and gc (hepojoki et al., 2010b; wang et al., 2010) . for enveloped viruses it is common that the contacts between rnp and envelope proteins are mediated by a matrix protein (timmins et al., 2004) . however, members of the family bunyaviridae do not contain a separate matrix protein and therefore interactions between the spike complex and rnps are assumed to initiate assembly of the virions. of the two hantavirus glycoprotein cts, gn-ct is considerably larger and capable of forming domain structures (~110 amino acids) while gc-ct represents an intraviral insertion of a transmembrane anchor (~10 amino acids). the gn-ct harbors a tandem zinc finger (zf) fold, the structure of which was resolved by nuclear-magnetic resonance (nmr) (estrada et al., 2009) . gn-ct carries also a conserved endocytosis signal yxxl, functionally characterized in many cell surface proteins (owen and evans, 1998) , but its role in hantaviruses remains elusive. this motif resembles a class of late domain (l-domain) motifs (bieniasz, 2006; groseth et al., 2010) , that are commonly utilized by matrix proteins to drive the budding process of viruses. taken together, the gn-ct is assumed to operate as a surrogate matrix protein of hantaviruses. we previously reported that both gn-ct and gc-ct are able to bind isolated rnp, and interact with purified recombinant n protein (hepojoki et al., 2010b; wang et al., 2010) . we showed that the gn-ct is fundamental to the rnp-spike interaction in the case of native proteins. additionally we found that there are at least three binding sites in the gn-ct towards n protein which are located on either side adjacent to the zf domain and on the very c-terminal part of the protein. recent studies suggest that gn-cts of rift valley fever virus (rvfv, genus phlebovirus) and crimean-congo hemorrhagic fever virus (cchfv, genus nairovirus) are able to interact with genomic rna (estrada and de guzman, 2011; piper et al., 2011) . for cchfv this interaction was shown to involve a zf domain of gn-ct that closely resembles the one found in hantaviruses. however, in the same study the zf domain of hantaviruses could not bind rna when expressed without the flanking regions. in this report we aimed to investigate the ability of the full gn-ct of hantaviruses to interact with genomic rna. we show an interaction between puuv gn (but not gc) and exogenous 32 p-labeled genomic rna by cross-linking and co-immunoprecipitation experiments. the nucleic acid binding site is pin-pointed to gn-ct using gst-fused recombinant proteins and synthetic peptides. the results indicate that the binding ability of gn-ct towards nucleic acids is rather unspecific since in addition to genomic rna, also unrelated rna as well as single-stranded dna was able to interact with gn-cts of puuv and tulv. the nucleic acid binding activity of hantavirus gn-ct resided predominantly at the c-terminal part of the protein. in conclusion we hypothesize the rna-gn-ct interaction to be linked to the assembly process of hantaviruses. we assessed biochemically, whether one or both of the glycoproteins of puuv would directly bind genomic rna. purified, lysed and micrococcal nuclease (mnase)-treated virus preparation and in vitro transcribed and radiolabeled genomic puuv s segment were allowed to form rna-protein complexes prior to uv cross-linking. samples were subsequently treated with rnase a in order to facilitate separation of proteins according to their molecular size in sds-page. proteins were detected by western blotting using n, gn and gc antibodies. the rna binding to viral proteins was studied in parallel by separating identically treated sample in sds-page and using autoradiography for detection. firstly, the successful transcriptions of the s segment (~2 kilobases {kb}) together with an unrelated marker rna (unrna of 2 kb) were verified on a tbe-urea gel (fig. 1a) . from fig. 1b it is evident that the bands migrating above 250 kda and at 50 kda remained radiolabeled after rnase a-treatment, indicating cross-linking of rna to proteins of the indicated sizes. interestingly, the most intense radioactivity (above 250 kda) overlapped with the band recognized by anti-gn immunoblot which suggested that the genomic rna interacts with gn. the band detected by autoradiography at 50 kda overlapped with the band recognized by n protein immunoblot. this was expected since the rnabinding properties of n protein are well-characterized (gott et al., 1993; mir and panganiban, 2004; severson et al., 1999) . next we performed similar experiments as before using tulv as the source of viral proteins ( fig. 2a ). in addition to the genomic s segment rna of puuv, we used an excessive amount of radiolabeled unrelated rna (unrna) as a non-genomic rna substrate to analyze the rna-binding specificity of viral proteins. similarly to what was observed using puuv, the radioactivity after rnase a-treatment was retained in the bands that by migration represent gn and n proteins. the same pattern of rna-bound proteins was observed with exogenous hantavirus s segment and the unrna. the capacity of both n and gn protein to bind both viral and unrna suggests that the rna-binding activity of these proteins did not absolutely require a specific sequence or structure of the rna substrate. however, our experimental set-up was not optimized to study the binding affinity of proteins to different rna sequences since the amounts of added rnas differed and thus we cannot rule out the possibility that viral proteins was incubated with purified mnase-treated puuv lysate, uv cross-linked, treated with rnase a and the proteins were separated in 6% sds-page (b). as indicated, the puuv proteins were detected by immunoblotting using gn-or gc-specific pab and n-specific mab 5e1, a combination of appropriate irdye-conjugated secondary antibodies and odyssey infrared imaging. protein-bound nucleotides were detected by autoradiography. m indicates the marker lane. fig. 2 . cross-linking of exogenous 32 p-rna to tulv proteins. tulv rna-binding proteins were detected by western blotting (using gn-or gc-specific pab and n-specific mab 3c11) and autoradiography as described in fig. 1b (a) . two different autoradiography exposures (long and short) emphasize the difference in the concentrations of the applied exogenous s-rna vs. unrna (500 vs. 2500 cpm; specific activities of the probes were similar). reduced mobility of gn in sds-page due to uv-induced cross-linking is shown (b). tulv preparations were treated and immunoblotted as described in fig. 1b but without exogenous rna. would prefer genomic rna over unrelated. we further observed that while n and gc proteins migrated mainly as monomers, the mobility of puuv and tulv gn were drastically reduced after rna-binding and uv-induced cross-linking (figs. 1b and 2a) . to assess whether this phenomenon is due to cross-linking alone we treated tulv as before but without the addition of exogenous rna (fig. 2b ). the mobility of gn was clearly reduced by uv cross-linking thereby showing that the exogenously applied rna is probably not responsible for the reduced mobility of gn in sds-page. next we wanted to confirm the ability of gn to bind rna and chose to use co-immunoprecipitation with the well-described puuvspecific monoclonal antibodies (mabs) 5a2 (gn-specific), 4g2 (gcspecific) and 5e1 (n-specific) in parallel with rabbit polyclonal antibodies (pabs) directed to puuv gn, gc or n. since these pabs are known to cross-react with tulv proteins in western blot, we firstly wanted to verify their ability also to immunoprecipitate the envelope proteins of tulv. for this purpose we used metabolically radiolabeled lysates of both puuv and tulv as the source of viral proteins in immunoprecipitation with gn-and gc-specific pabs. immunoprecipitated proteins were separated in sds-page and detected by autoradiography according to their typical migration pattern (fig. 3a) . the results demonstrated that the gn-and gc-specific pabs cross-react between puuv and tulv and in both cases precipitate, in addition to their respective target antigens, also a complex formed of gn and gc. unexpectedly the puuv gc pab seemed to precipitate tulv gc more efficiently than its native antigen. this suggests differences in the accessibility between gc of puuv and tulv towards this pab. both pabs favor their target antigens over the gn-gc complex which is in contrast to the puuv gn-and gc-specific mabs 5a2 and 4g2 that both precipitate close to equimolar amounts of gn and gc (hepojoki et al., 2010a; hepojoki et al., 2010b; strandin et al., 2011) . the capability of structural proteins to bind rna was analyzed by incubating purified, lysed and micrococcal nuclease (mnase)-treated virus preparations with radioactive puuv s segment rna in the case of puuv ( the samples were immunoprecipitated with mabs or pabs specific to n, gn or gc and retained radioactivity on protein g beads after washing was measured by scintillation counting. the amount of precipitated rna is reported as a fold of increase compared to the value obtained with negative pab. the gn-specific antibodies precipitated the highest amount of rna in the case of both puuv and tulv, and thus the rna-binding activity of gn was further solidified. the substantially higher amount of rna precipitated by tulv gn compared to puuv gn is at least partially explained by the different amounts of applied rna (approximately 5 times more of unrna than s segment rna). the amount of viral proteins in precipitates of puuv and tulv was nearly the same ( fig. 3a) suggesting that the maximum rna-binding capacity of puuv gn could be considerably higher than detected with the amount of exogenous s segment rna used in this experiment. surprisingly, the antibodies against n protein precipitated exogenous rna-n protein complexes very weakly if at all. the likely explanation to this is a nuclease-induced aggregation or modification of the n protein, thus rendering it unrecognizable by antibodies. another possibility could be inefficiency of nucleases to degrade the endogenous n protein-bound genomic rna. this would also explain the fairly low amount of exogenous rna cross-linked to n protein as compared to gn in the cross-linking experiments (figs. 1b and 2a) . the fact that gc-specific mab 4g2 precipitated more rna than gcspecific pab is probably due to effective co-immunoprecipitation of the gn-gc complex by this mab, as mentioned earlier. the nucleic acid binding activity of puuv and tulv gn is retained in their cytoplasmic tails based on our results using native viral proteins, the gn of both puuv and tulv can bind rna. in the native virion or in infected cells the gn protein can only encounter nucleic acids via its cytoplasmic tail and therefore the rna-binding activity should reside in this part of the protein to be biologically relevant. the ability of gn-ct of puuv and tulv to bind nucleic acids was studied with gst-fused gn-cts purified, lysed and mnase-treated puuv or tulv proteins, respectively, are shown. the radioactivity retained on protein g sepharose-beads after immunoprecipitation was measured by scintillation counting and the fold of change as compared to negative serum is indicated. the mabs 5a2, 4g2 and 5e1 are specific for puuv gn, puuv gc and puuv n, respectively. the mab indicated ha is specific for hemagglutinin-tag and used as a negative control. purified using reduced glutathione (gsh)-beads. the expression of the recombinant proteins eluted from gsh-beads was analyzed by ponceau s staining (fig. 4a) . the rna-binding of bead-bound gn-cts of either puuv s segment or unrelated rna, which were applied in similar radiolabeled amounts, was measured by scintillation counting. in parallel, we also tested with single-stranded 42-mer dna whether the gn-cts would bind dna. the sequence of this irdye800-labeled dna was derived from the 3′-end of the puuv genomic s segment and the binding of this dna was detected by odyssey infrared imaging. we compared the amounts of gn-ct bound nucleic acids to plain gst and report the results as fold change (fig. 4b ). the results indicated that the gn-cts of puuv and tulv bind to both rna and dna. the observed difference in nucleic acid binding between gn-ct of tulv and gn-ct of puuv is likely due to a higher level of expression of tulv gn-ct as compared to puuv gn-ct as demonstrated in fig. 4a . it seems that there is also more plain gst in the puuv gn-ct preparation as compared to tulv gn-ct. however, since plain gst as negative control did not show significant binding to nucleic acids, this should not influence the obtained results. the fact that the unrelated rna bound both cts more efficiently than the genomic rna and the ability of gn-ct to bind also relative short dnas further solidifies the unspecific nature of the nucleic acid recognition of gn-ct. however, due to the different detection methods used, we could not directly compare the binding strength of rna vs. dna. to define the regions in gn-ct that bind nucleic acids, we synthesized the gn-ct of puuv as overlapping 20-mer peptides in a 3 residue shift in celluspot™ format. the celluspot™ array was probed with the irdye800-conjugated dna shown to interact with the gst-tagged gn-ct of puuv (fig. 4b) . the mapping indicated that the same peptides that were previously shown to interact with rnp and n protein (hepojoki et al., 2010b) were capable of binding also nucleic acids (fig. 5a ). next we wanted to study the nucleic acid binding capacity of the three binding sites individually using biotinylated soluble peptides gn n , gn m and gn c , which represent the n-binding sites 1, 2 and 3 of puuv gn-ct as described earlier (hepojoki et al., 2010b) . schematic presentation of the peptides with respect to the primary sequence of puuv gn-ct is shown in fig. 6 . we also used the synthetic peptide corresponding to gc-ct, which in the earlier study was also shown to bind n protein (hepojoki et al., 2010b) . the peptides were pre-bound to avidin beads through biotin and analyzed for rna-or dna-binding activity similarly as was done for gst-fused proteins (fig. 4) . the gn c showed the strongest binding of the analyzed peptides irrespective of the applied nucleic acids (fig. 5b) . also gn m showed significant rna-binding capacity but only very little dna-binding activity. the binding levels of gn n and gc-ct were above control (biotin-bound avidin beads) but clearly lower than those of gn m or gn c . in addition, in the case of gc-ct, this observed interaction might not have any biological relevance since we did not detect any cross-linking of rna to native gc ( figs. 1 and 2) . finally we were interested in mapping the nucleic acid binding sites of tulv gn-ct and thus performed a similar celluspot™ assay as with puuv gn-ct, shown in fig. 5a . the strongest dna-binding peptides of tulv gn-ct (fig. 7a) located to the c-terminal part of the protein and overlapped with the corresponding sequence of puuv gn c peptide. this result suggested that the main nucleic acid binding site in gn is conserved among hantaviruses and locates to the cterminal part of the gn-ct which contains the putative late domain motif yrtl and is adjacent to the membrane-spanning signal sequence for gc. to confirm the significance of this region to nucleic acid binding we used a previously described (wang et al., 2010) truncated gst-fusion protein of tulv gn-ct lacking the c-terminal dna-binding sequence (figs. 6 and 7b) . when comparing the rnaand dna-binding activities of this mutant (gn-ctdel13) to the fulllength gn-ct of tulv in a similar gst pull-down as before, we observed that the rna-binding capacity of the mutant was completely abolished (fig. 7c) . however, the dna-binding activity was seen only to diminish due to the deletion of the c-terminal part, and thus we conclude that while the whole gn-ct containing the zf domain might be important in regulating nucleic acid binding it is the c-terminal part that is mainly responsible for this activity. in this report we show using several techniques that the gn protein of hantaviruses is able to bind nucleic acids and map this interaction to its ct. furthermore, by using peptide array we were able to pin-point this interaction to the very c-terminus of gn-ct. in our previous study we demonstrated that the cts of both gn and gc interact with the native rnp consisting of n protein and n proteinencapsidated genomic rna (hepojoki et al., 2010b; wang et al., 2010) . we also showed recombinant n protein to be able to mediate binding to gn-and gc-ct; however, we were unable to rule out the involvement of nucleic acids in these interactions. the results in this report show that the n protein encapsidated genomic rna could be directly involved in the interaction between glycoprotein cts and rnp. this is supported by the fact that the binding sites of nucleic acids and n protein either overlap or are contiguous in the primary sequence of gn-ct. thus amino acid residues of n protein and nucleotides from genomic rna would form the interaction surface in rnp that mediates the binding to glycoprotein cts. our results corroborate recent findings suggesting that gn-cts of other bunyaviruses could bind genomic rna. for rvfv of phleboviruses it was demonstrated that genomic rna is required for the efficient release of infectious vlps and the authors hypothesized that this would occur through direct recognition of viral rna by gn-ct (piper et al., 2011) . furthermore a zf domain in gn-ct of cchfv (nairovirus), closely resembling the one found in hantavirus gn-ct (estrada et al., 2009) , was found to bind viral rna and again a role for this interaction in virus assembly was suggested (estrada and de guzman, 2011) . specific recognition of the rnp structure is a crucial step in the egress of enveloped viruses. during infection the cells may contain various types of nucleic acids encapsidated by the viral nucleoproteins; however, mainly the genomic rna is incorporated into virions and this requires accentuated regulation of genome packaging. several enveloped viruses encode a matrix protein that bridges between the rnp and the viral envelope. hantaviruses do not possess an individual matrix protein and based on recent evidence, instead, the relatively large gn-ct substitutes for this activity (battisti et al., 2010; hepojoki et al., 2010a; huiskonen et al., 2010; wang et al., 2010) . the matrix protein of both family bornaviridae (neumann et al., 2009) and orthomyxoviridae (elster et al., 1997; wakefield and brownlee, 1989; ye et al., 1989) viruses have been shown to bind, in addition to the viral core proteins, also the genomic rna. thus our results suggest by analogy the gn-ct of hantaviruses to be equivalent of a matrix protein in the family bunyaviridae. prior to recent reports for cchfv and rvfv (estrada and de guzman, 2011; piper et al., 2011) , interaction involving a viral envelope protein and its genomic rna has only been shown for the family coronaviridae, where the envelope protein m recognizes the viral rna packaging signal and incorporates itself in to the nascent virions independently of the core protein (narayanan et al., 2003) . however, the m protein of coronaviruses possesses only a tiny extracellular domain, thus differing from the spikeforming glycoprotein gn of hantaviruses. the segmented, negative-sense rna viruses are divided in three families: orthomyxoviridae, bunyaviridae and arenaviridae. in the case of fig. 5 . mapping of the nucleic acid binding sites in the gn-ct of puuv. the gn-ct (amino acids 525-638) was synthesized in celluspot™ format as partially overlapping 20-residue peptides with a 3-residue shift. the celluspot tm slide was probed with the irdye800-conjugated 42-mer single-stranded dna (a). the signal detected in odyssey infrared imaging for each individual peptide is shown on the left side next to the amino acid sequence of the respective peptide. the indicated binding sites 1-3 were determined for n protein using spot peptide assay as previously described (hepojoki et al., 2010b; wang et al., 2010) , and are colored grey. puuv gn-ct peptides were used to pull-down nucleic acids (b). monomeric avidin beads preloaded to saturation by the biotinylated peptides gn n , gn m , gn c and gc-ct were used to pull-down rna ( 32 p-s-rna in 400 cpm and 32 p-unrna in 500 cpm) or irdye800-conjugated dna (irdye800conjugated 42mer 3′-end of hantavirus s-segment, 100 nm) and detections were done by scintillation counting and odyssey infrared imaging, respectively. results are indicated as a fold increase in respect to biotin-saturated avidin beads. fig. 6 . scheme of gn-ct peptides and proteins used to map nucleic acid-binding sites in puuv and tulv. the full length cytoplasmic tails of puuv and tulv gn (111 amino acids) and tulv gn-ct with 13 residue c-terminal deletion (gn-ctdel13) were expressed as n-terminal gst-fusion proteins (wang et al., 2010) . biotinylated peptides (gn n , gn m and gn c ) derived from puuv gn-ct was synthesized as described previously (hepojoki et al., 2010b) . the amino acid numbers in the image refer to the puuv or tulv glycoprotein precursors. influenza viruses of the family orthomyxoviridae, the eight-segmented genome is recognized via packaging signals formed of secondary structures in rna (hutchinson et al., 2010) . although the exact mechanism of genome incorporation in to virions is not known, it has been shown that the matrix protein m1 interacts with rnp (baudin et al., 2001; elster et al., 1997; noton et al., 2007; ye et al., 1999) . the m1 protein harbors a zf and a nuclear localization signal with rna-binding abilities (elster et al., 1997; wakefield and brownlee, 1989; ye et al., 1999; ye et al., 1989) . interestingly, the influenza m1 has been shown to repress endogenous viral transcription through its rna-binding domains (perez and donis, 1998; watanabe et al., 1996; ye et al., 1989) . this activity could be biologically relevant in the late stages of infection where it would halt viral mrna production and thereby trigger the packaging of viral genome eventually leading to the egress of virions. similarly to influenza m1, the matrix protein z of arenaviruses also acts as a transcriptional repressor (cornu and de la torre, 2001; cornu and de la torre, 2002; lopez et al., 2001) , and a zf domain is required for this activity. curiously, most bunyavirus gn-cts also contain a zf domain (estrada et al., 2009 ) the functions of which are currently unknown. unlike the zf domain of cchfv, the zf-domain of hantavirus gn-ct is unable to bind rna directly (estrada and de guzman, 2011) . however, according to the results of the present study there are other motifs in gn-ct capable of binding rna and thus a regulatory role in viral transcription for the hantavirus zf cannot be ruled out. since the matrix proteins of some non-segmented negativesense rna viruses have also been described to regulate transcription through interaction with the rnp (ghildyal et al., 2002; iwasaki et al., 2009; suryanarayana et al., 1994) , it is intriguing to speculate that hantavirus gn-ct would, through binding to rnp, mediate regulatory functions in gene expression and in virion packaging. further studies regarding the functions of gn-ct in hantavirus assembly or replication would greatly benefit from reverse genetics or infectious virus-like particle generation systems that are unfortunately still lacking today for these viruses. hantavirus cultivation, radiolabeling and purification puuv sotkamo strain and tulv moravia strain 5302 were propagated in vero e6 cells (green monkey kidney epithelial cell line; atcc: crl-1586) in which they have been isolated and to which they are adapted producing titers of 10 4 to 10 8 ffu/ml in to conditioned medium (schmaljohn et al., 1985; strandin et al., 2008; vapalahti et al., 1996) . vero e6 cells, grown at 37°c in a humidified atmosphere containing 5% co 2 , were propagated in minimal essential medium supplemented with 10% heat-inactivated fetal calf serum, 2 mm glutamine, 100 iu/ml of penicillin and 100 μg/ml of streptomycin. cell cultures in 75-cm 2 flasks were inoculated for 1 h at 37°c with virus suspension and conditioned medium collected 7-10 days post infection (d.p.i.) for tulv and 12-21 d. p.i. for puuv. conditioned medium containing virus (stored in aliquots at −70°c) was used as an inoculum. for radiolabeling of viral proteins (fig. 4) . the fold of change to gst control is indicated. infected vero e6 cell cultures were starved for 1 h at 37°c with medium depleted of methionine and cysteine, and propagated with a 1 mci mixture of 35 s-cysteine and 35 s-methionine (wallac perkin-elmer) for 3 days at 37°c. for purification and concentration of viruses, cell culture medium, passed through a 0.22-μm filter (millipore), was concentrated by pelleting through a 30% (w/v) sucrose cushion (beckman sw28 rotor, 27,000 rpm, 2 h, 4°c) and suspended in 25 mm 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (hepes) ph 7.4; 150 mm nacl (hbs). neutralizing bank vole monoclonal antibodies (mabs) directed to puuv gn (5a2) and gc (4g2) and to puuv n (5e1) and tulv n (3c11) (lundkvist and niklasson, 1992; lundkvist et al., 1993; lundkvist et al., 1996a; lundkvist et al., 1996b) were kindly provided by prof. åke lundkvist (swedish institute for infectious disease control). polyclonal antisera (pabs) raised against glutathione-stransferase (gst) fusion proteins of puuv gn, gc and n have been described previously (vapalahti et al., 1995) . the anti-hemagglutinin (ha) mab was from abcam. the irdye800-and alexafluor680conjugated secondary antibodies were from li-cor and invitrogen, respectively. micrococcal nuclease (mnase) and rnase a were from mbi fermentas. the genomic puuv s segment was in vitro transcribed (tran-scriptaid™ t7 high yield transcription kit; mbi fermentas) from a pgem-t plasmid containing the respective sequence information under a t7 promoter. the puuv s segment together with an unrelated rna (unrna) of similar size (included in transcriptaid™ t7 high yield transcription kit; mbi fermentas) were radioactively labeled with 32 p-utp (wallac perkinelmer) during transcription and purified using tripure (roche). the specific activity of these rnas was assumed similar since both contained approximately 1/3 of uridine residues. successful rna synthesis was verified on a denaturing polyacrylamide gel electrophoresis (page) in 5% gel containing 8 m urea in tbe buffer (22.5 mm tris base, 22.5 mm boric acid, 2.5 mm edta). the gel was dried and rna visualized by autoradiography. the 42-mer single-stranded dna represented the 3′-end of the genomic puuv s segment: 5′-tccagactttctcgtagtagcttttcaaggagtc-tactacta-3′. an irdye800-moiety was conjugated to the 5′-end of the oligonucleotide which was synthesized by oligomer oy, finland. the purified puuv or tulv, lysed in 0.5% tritonx-100, were treated with 0.1 u/μl mnase in 50 mm tris-hcl ph 7.5; 5 mm cacl 2 for 1 h at 37°c. mnase was inactivated with 10 mm ethylene glycol tetra-acetic acid (egta) for 15 min at room temperature prior to addition of 32 p-labeled rna, that was added in amounts of 500 counts per minute (cpm) of puuv s segment to both puuv and tulv extracts or 2500 cpm of unrelated rna (unrna) to tulv. after 2-h incubation at rt the rnas were cross-linked to proteins under a uv lamp for 30 min at rt and rna cleaved with 10 μg rnase a for 1 h at 37°c. the enzyme was inactivated by addition of laemmli sample buffer, samples boiled and proteins separated on 6% sds-page. to visualize the rna-interacting proteins, the gel was either subjected to autoradiography or proteins were transferred to nitrocellulose and immunoblotted with gn-or gc-specific pabs and n-specific 3c11 or 5e1 mabs. after probing with infrared dye-conjugated secondary antibodies the results were visualized by odyssey infrared detection system (li-cor). the puuv or tulv preparations, incubated with radioactive rna prior to cross-linking as described earlier, were subjected to immunoprecipitation in binding buffer (bb; 50 mm tris-hcl, 150 mm nacl, 0.1% triton x-100). firstly, 10 μg of mabs (5a2, 4g2, 5e1 or ha) or 20 μl of pabs (gn-, gc-, n-specific or negative serum) were incubated with the samples for 1 h at rt. then 10 μl of protein g sepharose beads (ge healthcare) were added and incubation continued for 1 h under end-over-end rotation in eppendorf tubes at rt. the beads were then washed two times with bb and liquid removed prior to scintillation counting (cerenkov-32 p; wallac 140 microbeta trilux liquid scintillation counter). the results were calculated as a fold change in respect to a negative control sample. the efficiency of gn-, gc-or negative pab to bind viral proteins was monitored with metabolically radiolabeled virion lysates of puuv or tulv by immunoprecipitation similarly as described earlier. the protein g bound protein complexes were eluted to laemmli sample buffer, boiled and separated in 10% sds-page. the gel was dried and proteins visualized by autoradiography. the gn-cts of puuv and tulv were expressed as glutathione stransferase (gst) fusion proteins in e. coli according to protocols of the manufacturer (gst gene fusion system handbook, amersham bioscience 18-1157-58 edition aa). the expression plasmids for gsttagged gn-cts were constructed by inserting respective gn-ct fragments (residues 526-637 of puuv and 521-632 of tulv) into the pgex-4t-3 vector (ge healthcare). protein production was induced by addition of 0.5 mm isopropyl β-d-1-thiogalactopyranoside (iptg) to cultures at 0.5 od 600 , protein expression was performed under vigorous shaking over 4 h at 30°c, bacteria were lysed and the expressed gst and gst-fusion proteins were purified by glutathione (gsh) sepharose 4b beads (amersham pharmacia, ge healthcare) using hbs as washing buffer. the gst-fusion protein with gst as the negative control (eluted from 10 μl of gsh beads to reducing laemmli sample buffer) was separated in 6% sds-page in tris/tricine/sds running buffer (bio-rad) or in conventional 12% sds-page, and the proteins were transferred to nitrocellulose for recording of the relative protein amounts by ponceau s staining. the synthesis of biotin-conjugated peptides gn n , gn m , gn c and gc-ct has been described earlier (hepojoki et al., 2010b) and contained the following amino acid sequences: gn n = biotin-kvkkeyqktmgsmv-cevc-oh, gn m = biotin-qahfkvckltsrfqenlkk-oh, gn c = biotin-epmqgcyrtlslfryrs-oh and gc-ct = biotin-cprrpsykkdhkp-oh. these peptides or biotin as a negative control were bound on monomeric avidin beads (pierce) at saturating amounts. the gstfusion protein coated on gsh and biotinylated peptide coated on avidin beads were used in the pull-down experiments of the radiolabeled s segment rna (400 cpm) or unrna (500 cpm). after 1 h incubation under shaking in bb at rt the beads were washed twice with bb and the amount of bound rna detected by scintillation counting. for the binding of dna, the irdye800-conjugated singlestranded oligonucleotide (100 nm) was incubated for 1 h under shaking at rt and the bound dna was eluted after two washes with bb by boiling in laemmli sample buffer. 1 μl of the lsb eluate (corresponding to 1 μl bead volume) was dot-blotted on to nitrocellulose, cross-linked by uv and the amount of irdye800-conjugated oligonucleotide quantified using odyssey. the results are presented as a fold change as compared to negative control. gn-cts of tulv and puuv were synthesized in celluspot™ format as 20-residue long peptides on multipep synthesizer (intavis ag) and the slides printed with slidespotter (intavis ag), all according to the manufacturer's instructions as described (beutling et al., 2008) . prior to probing, the peptide array slides were rinsed with ethanol and incubated in 10 mm tris-hcl ph 8.0, 100 mm nacl, 1 mm ethylenediaminetetraacetic acid (edta) and 0.0005% tween-20 (tent) for 5 min. the 5′-irdye800-conjugated oligonucleotide was applied in blocking buffer for 1 h at rt, unbound dna washed from slide with tent and bound dna detected with odyssey. hantavirus nucleocapsid protein oligomerization hantavirus nucleocapsid protein coiled-coil domains oligomerization of hantaviral nucleocapsid protein: charged residues in the 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influenza a virus matrix protein mapped with anti-idiotypic antibodies and synthetic peptides this work was supported by the helsinki university nanoscience key: cord-322084-gkg1059v authors: jeong, yong seok; repass, john f.; kim, young-nam; hwang, sun-min; makino, shinji title: coronavirus transcription mediated by sequences flanking the transcription consensus sequence date: 1996-03-01 journal: virology doi: 10.1006/viro.1996.0118 sha: doc_id: 322084 cord_uid: gkg1059v abstract in our studies of murine coronavirus transcription, we continue to use defective interfering (di) rnas of mouse hepatitis virus (mhv) in which we insert a transcription consensus sequence in order to mimic subgenomic rna synthesis from the nondefective genome. using our subgenomic di system, we have studied the effects of sequences flanking the mhv transcription consensus sequence on subgenomic rna transcription. we obtained the following results. (i) insertion of a 12-nucleotide-long sequence including the ucuaaac transcription consensus sequence at different locations of the di rna resulted in different efficiencies of subgenomic di rna synthesis. (ii) differences in the amount of subgenomic di rna were defined by the sequences that flanked the 12-nucleotide-long sequence and were not affected by the location of the 12-nucleotide-long sequence on the di rna. (iii) naturally occurring flanking sequences of intergenic sequences at gene 6–7, but not at genes 1–2 and 2–3, contained a transcription suppressive element(s). (iv) each of three naturally occurring flanking sequences of an mhv genomic cryptic transcription consensus sequence from mhv gene 1 also contained a transcription suppressive element(s). these data showed that sequences flanking the transcription consensus sequence affected mhv transcription. intergenic consensus sequence, which marks the start of the gene (joo and lai et al., 1984 ; ma-mouse hepatitis virus (mhv), a coronavirus, is an envekino et al., 1988b; shieh et al., 1987; spaan et al., 1983) . loped virus with a single-stranded, positive-sense rna mhv subgenomic rnas are detected in mhv-infected genome of approximately 31 kb (lee et al., 1991; pachuk cells but not in mhv virion stohlman, 1978). et al., 1989) . mhv-infected cells synthesize genomic-therefore, subgenomic-sized rnas must be synthesized length virus-specific mrna and six or seven species of from a genomic-sized rna. synthesis of subgenomic virus-specific subgenomic mrnas. the viral mrnas mrnas involves a discontinuous transcription step; durhave a 3-coterminal nested-set structure (lai et al., 1981; ing subgenomic-sized rna synthesis independently leibowitz et al., 1981) and are numbered 1 to 7, in detranscribed leader rna species possessing a trans-actcreasing order of size (lai et al., 1981; leibowitz et al., ing property fuse with the body sequences of subgeno-1981) . none of the mrnas are packaged into mhv virimic-sized rna (jeong and makino, 1994; zhang et al., ons, except for mrna 1, which is efficiently packaged 1994). there are at least two stages in coronavirus subdue to the presence of a packaging signal (fosmire et genomic rna synthesis: we call the first stage primary al., 1992). the 5 ends of the mhv genomic rna and the transcription, during which subgenomic-sized rna is subgenomic mrnas start with a leader sequence that is synthesized from a genomic-sized template rna; the approximately 72 to 77 nucleotides long other stage is called secondary transcription, during 1984; spaan et al., 1983) . the leader sequence is enwhich subgenomic-sized rna serves as template (jeong coded only once in the genomic rna at the 5 end. the and . mhv-specific genes, which are downstream from the when an intergenic region from mhv is inserted into leader, are separated from one another by an intergenic a location in an mhv defective interfering (di) rna, a region. each intergenic region, located upstream of a novel subgenomic di rna is transcribed in helper virusgene essential for mhv replication, includes the unique infected cells (makino et al., 1991) . we used this subgeconsensus sequence of ucuaaac, or a very similar senomic di rna system to study how mhv transcription quence (lai et al., 1984; spaan et al., 1983) . on the subis flexible enough to recognize a mutated transcription genomic mrnas, the leader sequence is fused to the consensus sequence. we constructed a series of mhv di rnas that contain one ucuaaac consensus se-rnas showed that the mhv transcription mechanism is dna construction flexible enough to recognize mutated transcription con-mhv disse-specific cdna clone de5-w3 (makino and sensus sequences; subgenomic di rnas are synthe) was used as a parental clone for dna consized from most of the mutated consensus sequences struction. conventional methods of dna manipulation (joo and . in another study we showed that (sambrook et al., 1989) were used. by using pcr-based sequences flanking the same intergenic region between site-directed mutagenesis, a 12-nucleotide-long segenes 6 and 7 do not affect the efficiency of subgenomic quence, tctaatctaaac, was inserted into di cdna di rna transcription (makino and joo, 1993) . these two (joo and makino, 1992 , and these recombinant pcr was also employed for construction regions are not utilized for transcription. of plasmid di-d20 and di-ta7 (higuchi, 1990) . for the one of the possible reasons why transcription does construction of fdi-1/2wt, fdi-2/3wt, fdi-6/7wt, fdi-m1, not occur in these 19 mhv genomic regions is that the fdi-m2, and fdi-m3, mhv-jhm-specific cdna was iniflanking sequences of these regions suppress transcriptially synthesized by incubating mhv-jhm genomic rna tion. in both of the previous studies we used di rnas with specific primers (makino et al., 1988a) , the desired which contain the intergenic region between genes 6 mhv-jhm-specific rt-pcr products were obtained after and 7; we used that intergenic region because mrna 7, incubating mhv-jhm cdna with two specific oligonuclewhich is synthesized from this intergenic region, is the otides, as described previously (makino and joo, 1993) . most abundant mhv mrna species and we expected for construction of fdi-1/2m, fdi-2/3m, and fdi-6/7m that a large amount of subgenomic di rna would be the 12-nucleotide-long sequence, tctaatctaaac, was synthesized from this inserted intergenic region. howinserted into the rt-pcr products using the recombinant ever, characterization of only this intergenic region may pcr procedure. the recombinant pcr procedure was overlook the possibility that the sequences flanking these also used to insert a tcttaac sequence into fdi-m5. 19 regions may suppress transcription from these rethe resulting rt-pcr product was inserted into the afliigions. sacii site of de5-w3. for all of the constructs used in deletion analysis of those mhv di rnas that contain this study we sequenced the inserts that were derived the intergenic sequence from gene 6-7 with its naturally from pcr products to confirm the presence of specific occurring flanking sequences showed that reducing the mutations and the absence of extraneous mutations. number of base pairs between the genomic leader sequence and the intergenic region decreases the tran-rna transcription and transfection scription efficiency (makino et al., 1991) . however, when van der most et al. (1994) used another mhv di rna, plasmid dnas were linearized by xbai digestion and those authors reported that the extent of base pairing transcribed with t7 rna polymerase as previously debetween the leader rna and the intergenic sequence scribed (makino and lai, 1989) . the lipofection procedoes not control subgenomic rna abundance; they used dure (makino et al., 1991) was used for rna transfection di rnas which lacked the naturally occurring sequences into dbt cells. that flank that intergenic sequence. if the flanking sequences of the transcription consensus sequence affect preparation of virus-specific intracellular rna and transcription, then the different results from these two northern (rna) blotting studies may be explained by the differences in the nature of sequences flanking the inserted intergenic sequence. virus-specific rnas in virus-infected cells were extracted as previously described (makino et al., 1984) . for we have studied the possible influence of the flanking sequences on transcription initiated at a transcription each sample, 1.5 mg of intracellular rna was denatured and electrophoresed through a 1% agarose gel con-consensus sequences by using a subgenomic di system. our data indicated that indeed some flanking se-taining formaldehyde, and the separated rna was blotted onto nylon filters as described previously (jeong and quences affected transcription. . in some experiments poly(a) containing rnas that were selected by oligo (dt)-cellulose column viruses and cells chromatography were used (makino et al., 1984) . the nylon filter was soaked in a prehybridization buffer, and the plaque-cloned a59 strain of mhv (mhv-a59) (lai et al., 1981) was used as a helper virus. mouse dbt northern blot hybridization was performed (jeong and makino, 1992) . the 32 p-labeled probes were prepared by cells (hirano et al., 1974) were used for growth of viruses. a random-priming procedure (sambrook et al., 1989) . for the densitometric analysis, autoradiograms were scanned using a scanner (reli 4816 scanner, relysis) and the intensity of each band was quantitated using scan analysis program (biosoft, cambridge, uk). primer extension products were purified from the gel and amplified by pcr under the same conditions described above. the gel-purified rt-pcr products were separated by agarose gel electrophoresis. direct pcr sequencing was performed according to the procedure established by winship (1989) . we examined whether sequences outside of the consensus sequence could affect subgenomic rna transcription efficiency by using the subgenomic di rna system. we initially constructed eight different mhv di cdna clones, each with a 12-nucleotide-long tctaatctaaac insert (12-nt sequence) placed at a different site; that inserted 12-nt sequence is perfectly complementary to the 3 region of the genomic leader sequence and in(makino et al., 1988a) and intide numbering begins from the 5-end of de5-w3, and the inserted 12cludes cis-acting replication signals, which are essential nucleotide sequences are boxed. for mhv-jhm di rna replication; these regions were the 5-most 0.47 kb, the 3-most 0.46 kb, and about 60 nucleotides near the 5 end of domain ii (kim et al., 1993; labeled by random-priming with 32 p. this probe detects only helper virus genomic rna, genomic di rnas, and kim and makino, 1995b; lin and lai, 1993) (fig. 1a) . we used pcr-based site-directed mutagenesis to insert the subgenomic di rnas ( fig. 2a) . for the detection of the remaining di mutants, we used the eagi-sphi fragment 12-nt sequence into de5-w3. the locations of the 12-nt sequences in these eight clones, di-afliii, di-stui, di-of de5-w3 as a probe (fig. 2c) , and a probe corresponding to a region 18 to 262 nucleotides from the 3-end of eagi, di-sphi, di-spei, di-d20, di-nrui, and di-ta 7, are shown in fig. 1b . ; the former probe detects only helper virus mrna 1 and genomic di rnas and the latter we examined replication and transcription of these di rnas in di rna-transfected, mhv-infected cells. di binds to all mhv rna species. to directly compare the efficiency of subgenomic di rna transcription, we used rnas were synthesized in vitro and transfected by lipofection into dbt cell monolayers that were infected with the same membrane in the experiments documented in figs. 2b and 2c. all eight di rnas replicated efficiently mhv-a59 helper virus 1 hr prior to transfection. at 7 hr postinfection, intracellular rnas were extracted, sepa-and synthesized a subgenomic di rna of expected size; however, the amounts of the subgenomic di rnas dif-rated by formaldehyde-agarose gel electrophoresis, and analyzed by northern blot. for the analysis of di-afliii, di-fered (fig. 2) . subgenomic di rna was most efficiently transcribed from di-spei di rna. di-sphi and di-d20 stui, and di-eagi, we electrophoresed poly(a)-containing intracellular rnas and probed northern blots with the also supported subgenomic di rna transcription. di-nrui supported a low level of subgenomic di rna tran-eagi-sphi dna fragment of de5-w3 (fig. 1a ) that was scription, which was not immediately apparent in one quence insertion differed. if the sequences flanking the 12-nt sequence are important for subgenomic di rna experiment, as shown in fig. 2b , in which it could only transcriptional regulation, then the efficiency of subgenobe detected after prolonged exposure of the gel (data mic di rna transcription from the newly constructed di not shown); in another experiment, di-nrui subgenomic rnas should be similar to that of their parental di rnas. di rna synthesis was observed more readily, because we examined synthesis of subgenomic di rna in di the autoradiogram had a lower background (fig. 2d) . a rna-transfected, mhv-a59-infected cells by northern lower level of subgenomic di rna synthesis was also blot analysis in which we applied only poly(a)-containing observed in di-stui and di-eagi. although synthesis of rnas onto the gels. we used probe 1 (see fig. 3 ) to di-afliii and di-ta 7 subgenomic di rnas was not eviestimate the ratio of genomic to subgenomic di rna for dent by northern blot analysis, synthesis of both subgemost of the di rnas; the exception was that we used nomic di rnas was detected by rt-pcr analysis (jeong probe 2 (see fig. 3 ) for the analysis of fdi-d20. probe 1 and makino, 1994) (data not shown). direct sequencing was not suitable for analysis of fdi-d20, because while of genomic di rna-specific rt-pcr products demonthis probe hybridizes with genomic fdi-d20 rna at two strated that the inserted 12-nt sequence and its flanking different sites (one is downstream of the aflii site and regions was maintained for all the mutants in di rnathe other is downstream of the sacii site), it hybridizes replicating cells (data not shown). with the subgenomic di rna at just a single site. all the tested di rnas replicated efficiently in the di rna-flanking sequences of the 12-nt sequence affected transfected, mhv-a59-infected cells (fig. 4) . a more transcription slowly migrating band appeared in fdi-spei di rna-to test whether the flanking sequences of the inserted replicating cells, in fdi-d20 di rna-replicating cells and 12-nt sequence affected the efficiency of transcription, in fdi-nrui di rna-replicating cells (fig. 4 , lanes 5-7); we constructed another set of di cdnas. from di cdnas, this band probably represented a di rna that was newly di-stui, di-eagi, di-sphi, di-spei, di-d20, and di-nrui, generated in di rna-transfected cells. all the di rnawe removed a 0.4-kb-long pcr fragment, which carried replicating cells synthesized subgenomic di rna with the 12-nt sequence and 0.2 kb of its upstream and downdifferent transcription efficiencies. the difference in the stream flanking sequences, and inserted this into the radioactivity ratios of subgenomic di rna to genomic di aflii-sacii site of de5-w3 to produce fdi-stui, fdi-eagi, rna for these five di rnas was roughly comparable to fdi-sphi, fdi-spei, fdi-d20, and fdi-nrui, respectively those of the parental di rnas (figs. 2 and 4) . the 0.2(fig. 3) . these six cdnas were the same size, and each kb-long upstream and downstream sequences flanking carried the same 12-nt sequence in the middle of a samethe 12-nt sequence clearly affected subgenomic di rna transcription efficiency. length insertion; only the regions flanking the 12-nt sefig. 3 . schematic diagram of the structure of de5-w3 and de5-w3-derived insertion mutants with flanking sequences. fragments for fdis contained a 0.4-kb-long pcr product consisting of the 12-nucleotide sequence (solid box) and 0.2 kb from an upstream and a downstream flanking sequence; the flanking sequences were derived from di-stui, di-eagi, di-sphi, di-spei, di-d20, and di-nrui genomic fragments. the 0.4-kb pcr fragment of di-stui, di-eagi, di-sphi, di-spei, di-d20, and di-nrui was inserted into the aflii-sacii site of the de5-w3 to produce fdi-stui, fdi-eagi, fdi-sphi, fdi-spei, fdi-d20, and fdi-nrui, respectively. solid boxes represent the 12-nucleotide sequence. locations of probe 1 and probe 2 used for northern blot analysis (see fig. 4 ) are also shown. effect of the 12-nt sequence location on transcription probe 1 (fig. 5a ). fdi-stui/sp, fdi-eagi/sp, fdi-sphi/sp, fdi-aflii/sp, and fdi-sacii/sp replicated and transcribed we next examined whether the location of the 12-nt (fig. 5b) . the molar ratio of the genomic to subgenomic sequence could affect transcription by constructing a new series of di cdnas with the 12-nt sequence positioned at different sites in de5-w3; each construct was similar to the others in that they all contained the 0.4-kb inserted region that carried the 12-nt sequence and its upstream and downstream 0.2-kb sequences; each construct differed only in the location of the insertion within the di genome. we chose the 0.4-kb fragment used for construction of fdi-spei, because fdi-spei showed the most efficient subgenomic di rna transcription (fig. 4) . we inserted this 0.4-kb fragment at the afliii, stui, eagi, sphi, aflii, sacii, and nrui sites of de5-w3 to produce fdi-afliii/sp, fdi-stui/sp, fdi-eagi/sp, fdi-sphi/sp, fdi-aflii/sp, fdi-sacii/sp and fdi-nrui/sp, respectively (fig. 5a) . we expected that this set of di constructs would overcome the transcriptional suppressive effect that structure of de5-w3 and de5-w3-derived insertion mutants with flanking sequences. fragment for fdis contained a 0.4-kb-long pcr product consisting of the 12-nucleotide sequence (solid box) and 0.2 kb from an upstream and a downstream flanking sequence of di-spei genomic fragments. this 0.4-kb pcr fragment was inserted into the afliii site, stui site, eagi site, sphi site, aflii site, sacii site, and nrui site of the de5-w3 to produce fdi-afliii/sp, fdi-stui/sp, fdi-eagi/sp, fdi-sphi/sp, fdi-aflii/sp, fdi-sacii/sp, and fdi-nrui/sp, respectively. solid boxes represent the 12-nucleotide sequence. location of probe 1 for northern blot analysis is also shown. (b) northern blot analysis of fdi rnas. intracellular rnas were extracted from di rna-transfected, mhv-infected cells (lanes 6-10) or di rna-transfected, mock-infected cells (lanes 1-5), and poly(a)-containing rnas were analyzed by northern blot analysis. lane 11 represents rna from mhv-infected cells. 32 p-labeled probe 1 (see a) was used as a probe. arrow represents genomic di rnas. di rna from these replication-competent di rnas was inserted within the cis-acting replication signals in these di rnas (kim et al., 1993) (fig. 1) . probably the insertion essentially the same (fig. 5b) ; the ratio was approximately 0.3, a value that was very close to that of fdi-of the 0.4 kb disrupted the structure of the cis-acting replication signals in regions that were essential for di spei. we did not observe replication of fdi-afliii/sp and fdi-nrui/sp (data not shown). the 0.4-kb region was rna replication, resulting in failure of the di rna to replicate. these data indicated that the location of 12-nt 2 and genes 2-3 demonstrated subgenomic di rna transcription activity that was similar to the 0.4-kb-long sequence on de5-w3 was not crucial for the regulation of subgenomic di rna transcription. flanking sequences of the intergenic region of genes 6-7, indicating that no transcriptionally suppressive element existed in the naturally occurring flanking se-effect of the naturally occurring sequences that flank quences adjacent to the intergenic regions at genes 1the mhv intergenic regions on transcription 2 and 2-3. interestingly, sequence alteration from a wildtype intergenic sequence to the 12-nt sequence affected the data presented above demonstrated that some sequences flanking the 12-nt sequence could suppress transcription efficiency only in the di rna containing the intergenic region from genes 6-7, but not in di rnas subgenomic di rna transcriptional efficiency. this data contrasted with our previous observation that the effi-containing intergenic regions from genes 1-2 and 2-3. these data demonstrated that the sequence(s) sur-ciency of subgenomic di rna transcription is not regulated by sequences flanking the genes 6-7 intergenic rounding the intergenic region of genes 6-7 contained a transcriptionally suppressive element(s) that suppressed sequence (makino and joo, 1993) . here we present that not all sequences flanking the 12-nt sequence inhibited transcription from the 12-nt sequence, but not from the naturally occurring 18-nucleotide-long intergenic se-the transcription; some sequences flanking the 12-nt sequence did not suppress transcription (see fig. 4 ). we quence. less subgenomic di rna synthesis in fdi-6/ 7m than in fdi-6/7wt was consistent with our previous interpreted these data as indicating that nonnaturally occurring sequences flanking the 12-nt sequence could study; in that study a mutant like fdi-6/7m, with the same deletion in the intergenic region between genes 6 and suppress subgenomic di rna transcription, whereas naturally occurring sequences flanking the genes 6-7 7, synthesized significantly less subgenomic di rna than did a di rna with an intact intergenic region (makino intergenic sequence do not suppress transcription. we examined naturally occurring flanking sequences and joo, 1993). from other mhv intergenic regions. for this analysis we chose sequences surrounding the genes 1-2 intergenic sequences surrounding genomic cryptic consensus sequence and the genes 2-3 intergenic sequence. the sequences suppressed transcription amounts of mrna 2 and mrna 3, synthesized from the intergenic regions between genes 1-2 and genes 2-3, mhv transcription regulation is flexible enough to recognize altered consensus sequences in which just one respectively, are about 50 and 30 times lower than the amount of mrna 7 in mhv-infected cells (leibowitz et of seven consensus sequence nucleotides is changed to any of the three possible alternative bases (joo and al., 1981) . currently we do not know why the amount of mrna 2 and mrna 3 is significantly lower than mrna . indeed some mhv sequences that differ only by one nucleotide from the consensus sequence 7. we constructed six different di cdnas, all of which had an insertion of a 0.4-kb fragment at the aflii-sacii are transcriptionally active (schaad and baric, 1993) . however, many genomic cryptic consensus sequences site of de5-w3 (see figs. 3 and 6) . the inserted 0.4-kb fragment of fdi-1/2wt, fdi-2/3wt, and fdi-6/7wt, was a that also differ from ucuaaac by only one nucleotide do not act as sites for initiation of subgenomic rna syn-rt-pcr fragment of the mhv-jhm sequence at genes between 1-2, 2-3, and 6-7, respectively (fig. 6) . the thesis (joo and . because some of the sequences flanking the inserted 12-nt sequence negatively 0.4-kb fragment consisted of the intergenic sequence and its natural occurring 0.2-kb flanking sequences. fdi-affected subgenomic di rna transcription, subgenomic rna transcription from a sequence that differs slightly 1/2m, fdi-2/3m, and fdi-6/7m had structures that, respectively, were very similar to fdi-1/2wt, fdi-2/3wt, and from ucuaaac may possibly be suppressed by the sequences flanking that sequence; this could explain the fdi-6/7wt, except that these di cdnas contained the 12nt sequences instead of the naturally occurring in-flexibility of the consensus sequence. to test this possibility we constructed a new series of tergenic sequences (fig. 6) . we examined synthesis of genomic di rna and sub-mutant di rnas, all of which contained an insertion of a 0.4-kb fragment at the aflii-sacii site of de5-w3. the genomic di rna in di rna-transfected, mhv-a59-infected cells by northern blot analysis using the 32 p-la-inserted 0.4-kb fragment of fdi-m1, fdi-m2, and fdi-m3 corresponded to mhv-jhm sequences at about 0.7-1.1 beled sacii-spei fragment of de5-w3 as a probe (probe 2) (see fig. 3 ). the molar ratio of genomic di rna to kb, 7.9-8.3 kb, and 10.7-11.1 kb from the 5-end, respectively. the center of these 0.4-kb fragments had a natu-subgenomic di rna in the tested di rnas was approximately the same, except that fdi-6/7m synthesized less rally occurring ucuuaac sequence, which differed by only one nucleotide from the ucuaaac consensus se-subgenomic di rna (fig. 7) . in spite of the significantly lower amounts of mrna 2 and mrna 3 relative to mrna quence. these naturally occurring ucuuaac sites in the mhv genome are not transcriptionally active. we also 7 that are inherent in mhv-infected cells, the 0.4-kb-long sequences flanking the intergenic region of genes 1-constructed fdi-m5 as a control clone; fdi-m5 was similar to fdi-1/2wt except that fdi-m5 contained a ucu-we examined the synthesis of genomic di rna and subgenomic di rna in di rna-transfected, mhv-a59-uaac sequence (fig. 8) in place of the fdi-1/2wt aau-cuauac sequence (see fig. 6b ). fdi-m5 deleted the 5 infected cells by northern blot analysis using the 32 plabeled sacii-spei fragment of de5-w3 as a probe two as of the intergenic sequence of fdi-1/2wt; fdi-m5 was a more appropriate control than fdi-1/2wt, because (probe 2, see fig. 3 ). all the di rnas replicated efficiently, whereas subgenomic di rna synthesis occurred only in the presence of two as immediately upstream of the consensus sequence sometimes increases the level of fdi-m5 (fig. 9) . replication of fdi-m5 was lower than that of the other di rnas, which was consistent with subgenomic di rna transcription (makino et al., 1991) . our previous study showing that di rnas that do not synthesize subgenomic di rna replicate more efficiently than those that synthesize subgenomic di rna (jeong and makino, 1992) . here we clearly showed that flanking sequences of ucuuaac in fdi-m1, fdi-m2, and fdi-m3 suppressed subgenomic di rna transcription, whereas the flanking sequences of ucuuaac in fdi-m5 did not. transcription from some of the genomic cryptic consensus sequences was suppressed by the sequences flanking these regions. we have investigated whether different sequences flanking a 12-nt constructed sequence that carried an mhv transcription consensus region might affect subsus sequence suppressed transcription. we found that transcriptionally active when its flanking sequences were some synthetic sequences flanking the 12-nt sequence replaced with the flanking sequences of the intergenic did suppress subgenomic di rna transcription. placesequence at genes 1-2, indicating that transcription from ment of the 12-nt sequence with fixed flanking sethe ucuuaac sequence in mhv was suppressed by its quences within different regions of the di genome did flanking sequences. these assembled data indicated not significantly affect subgenomic di rna transcription. that the influence the flanking sequence(s) exert on tran-naturally occurring intergenic sequences from genes 1scription from a transcription consensus sequence is a 2, 2-3, and 6-7 inserted into mhv di-rna along with point of regulatory control in coronavirus transcription. their flanking sequences did not suppress transcription, although our present study clearly showed that some whereas a di rna containing the 12-nt sequence plus flanking sequences of the transcription consensus sethe genes 6-7 naturally occurring flanking sequences quence suppressed transcription, we do not know how showed reduced transcription efficiency. subgenomic di that suppression occurred. mhv transcription generally rna synthesis did not occur from a ucuuaac cryptic occurs from the ucuaaac sequence or very closely retranscription consensus sequence with its naturally oclated sequence and some nucleotide curring flanking sequences, whereas that sequence was changes within the ucuaaac affect transcription efficiency (joo and . noncoronavirus rnas containing negative sense transcription consensus sequence of coronavirus transmissible gastroenteritis virus (tgev) serve as templates for transcription in tgev-infected cells (hiscox et al., 1995) . these data suggest that the coronavirus transcription machinery most probably recognizes transcription consensus sequences for transcription. recognition of the intergenic region by mhv transcription machinery may involve host proteins (zhang and lai, 1995) . one possible mechanism of flanking-sequence-mediated transcription-suppression is that the transcription consensus sequence and its flanking sequences may form a stable rna structure that may prevent accessibility of the mhv transcription mechanism to the consensus sequence. many of the first series of fig. 9 . northern blot analysis of di rnas. intracellular rnas were extracted from di rna-transfected, mhv-infected cells (lanes 6-9) or di rnas had 12-nt sequences flanked by mhv cis-acting di rna-transfected, mock-infected cells (lanes 2-5) and analyzed by replication signals (fig. 1a) . these cis-acting replication northern blot analysis. lane 1 represents rna from mhv-infected cells. signals may form stable secondary or tertiary struc-32 p-labeled probe 2 (see fig. 3 ) was used as a probe. arrow and ture(s), which are essential for the recognition by viral arrowhead represent genomic di rnas and subgenomic di rna, respectively. polymerase and host factors (kim et al., 1993) . rna sec-ondary (or higher) structure of the internal cis-acting rep-ference in the flanking sequences of the inserted intergenic regions. probably flanking sequences of the in-lication signal is indeed important for viral rna synthesis (kim and makino, 1995b) . the first set of di rnas which serted transcription consensus sequence in the di rnas that were used by van der most et al. (1994) do not contained the 12-nt sequence within or proximal to these cis-acting replication signals replicated efficiently, indi-contain a transcription suppressive element; in these di rnas minor changes in the intergenic sequence would cating that the putative structures made by the cis-acting replication signals were probably maintained during di not significantly affect transcription. we use di rnas with an inserted intergenic region from genes 6-7 that rna replication. to maintain active rna structure for di rna replication, rna structures formed by a 12-nucleo-contained the transcription suppressive element; in these di rnas, when the intergenic sequence contained tide sequence located within or near the predicted stable rna structures of the cis-acting replication signal may nucleotide deletions, the transcription suppressive element suppressed transcription. in yet another case, di not be transcriptionally optimal; the mhv transcription machinery may not have had easy access to a 12-nucleo-rnas constructed to carry only the 18-nucleotide-long intergenic sequence of genes 6-7, show altered tran-tide sequence located within a stable rna structure, resulting in lower levels of subgenomic di rna synthe-scription efficiency when nucleotides within this sequence are deleted (makino and joo, 1993) . in that study sis. fdi-6/7m synthesized a lower amount of subgenomic we chose the insertion site of the intergenic region arbitrarily, so that the sequences surrounding the inserted di rna than fdi-1/2m, and fdi-2/3m, indicating that flanking sequences of the intergenic sequence at genes intergenic sequence possibly may have contained a transcription suppressive element(s); this is similar to the 6-7 could suppress subgenomic di rna transcription. the presence of a transcription suppressive element(s) way that the sequence around the genes 6-7 intergenic sequence suppresses transcription. this may be the rea-in the flanking sequences of the intergenic sequence at genes 6-7 was unexpected, because previous deletion son why synthesis of subgenomic di rna is susceptible to the sequence changes within the 18-nucleotide-long analysis of this region did not reveal a transcription suppressive element (makino and joo, 1993) . as shown in intergenic sequence in these di rnas. the naturally occurring flanking sequences of the in-this study and previous studies (makino et al., 1991; makino and joo, 1993) , if di rnas contained the natural tergenic regions at genes 1-2 and 2-3 did not suppress subgenomic di rna transcription. the ns 30 protein and occurring 18 nucleotide-long intergenic sequence, then the sequences flanking this region did not suppress sub-the s protein are translated from mrna 2 and mrna 3, respectively. the function of ns 30 is not clear, yet this genomic di rna transcription; the suppressive effect was only obvious when the intergenic region contained is a conserved gene for many mhvs, except for one isolate that lacks most of this gene (schwarz et al., 1990) . the 12-nt sequence. most of di rnas we studied in our previous studies contained the 18-nucleotide-long natu-s protein is essential for mhv replication; s protein binds to the cell receptor to initiate infection (dveksler et al., rally occurring intergenic sequence from genes 6-7 with its natural occurring flanking sequence, thus we could 1991). expression of both of those mhv genes seems to be crucial for replication and pathogenesis of mhv. that not detect the transcriptional suppressive effect of flanking sequences at genes 6-7 (makino et al., 1991; makino the flanking sequences of the intergenic regions of these genes did not suppress transcription indicated that tran-and joo, 1993). for mutants fdi-1/2wt, fdi-1/2m, fdi-2/3wt, and fdi-scriptionally suppressive sequences were eliminated during evolution of mhv. during the evolution of mhv, 2/3m that lacked the transcription suppressive element found in the flanking sequences, the sequence comple-many mutations must have occurred within the sequences flanking the intergenic region of these genes; mentarily between the intergenic sequence and the 3end of leader sequence did not directly correlate with variant viruses whose flanking sequences did not suppress transcription might have had a selective advan-transcription efficiency. in a similar report, van der most et al. (1994) concluded that the extent of base pairing tage. we showed that flanking sequences of the intergenic between the leader rna and the intergenic sequence does not control subgenomic rna abundance; they ana-sequence at genes 6-7 contained the transcription suppressive element. mhv mrna 7 synthesized from the lyzed several mhv-a59-derived di rnas with inserted transcription consensus sequences. on the contrary, de-intergenic region at genes 6-7 encodes the n protein that is essential for mhv replication; n protein forms creasing complementarily between the intergenic sequence and the 3-end of the leader sequence results in helical nucleocapsid and is most likely required for viral rna synthesis (compton et al., 1987; kim and makino, a decreasing level of subgenomic di rna transcription in di rnas with an inserted genes 6-7 region (makino 1995a) . probably sequences that correspond to the transcription suppressive element encode an important func-et al., 1991; makino and joo, 1993) . we speculate that the different conclusions between van der most et al. tion for either m protein or n protein, thus this transcription suppressive element was not eliminated during mhv (1994) and our previous studies probably reflect the difevolved to contain the 18-nucleotide-long intergenic se-analysis of intracellular small rnas of mouse hepatitis virus: eviquence that can overcome the transcription suppression dence for discontinuous transcription in vitro replication of mouse hepatitis one curious, unanswered question from this study is virus strain a59 cloning of the 0.4-kb region from genes 1-2, 2-3, and 6-7 synthesized mouse hepatitis virus (mhv) receptor: expression in human and similar amounts of subgenomic di rna. this inconsishamster cell lines confers susceptibility to mhv identification and mic di system and actual mhv transcription. the major characterization of a coronavirus packaging signal rna for subgenomic rna synthesis; mhv genomic rna recombinant pcr. in ''pcr protocols'' (m. a. innis, is 31 kb, whereas genomic di rna is only 2.6 kb replication kb from the 3-end of genome, respectively, whereas our and plaque formation of mouse hepatitis virus (mhv-2) in mouse experimental intergenic regions were located only 1.2 kb cell line dbt culture investi-rna has multiple intergenic regions, whereas genomic gation of the control of coronavirus subgenomic mrna transcription by using t7-generated negative-sense rna transcripts mechanism of coronavirus transcripbetween the amounts of subgenomic di rna and real tion: duration of primary transcription initiation activity and effect of mhv mrnas? coronavirus rna synthesis is considered subgenomic rna transcription on rna replication characterization of a murine coronavirus defective interfering rna internal cis-acting replication sig-1995). the transcription factor may dissociate from the nal template during scanning is longer, the chances of the loss of a factor during scan-characterization of leader rna sequences on the virion and mrnas ning should be higher. furthermore some scanning facof mouse hepatitis virus, a cytoplasmic rna virus mouse hepatitis virus a59: mrna structure intergenic regions, each of which is separated by about and genetic localization of the sequence divergence from hepato-125 nt, into di rna results in synthesis of two subgenotropic strain mhv-3 which is transcribed from the upstream in-presence of leader sequences in the mrna of mouse hepatitis virus. tergenic region (on the positive-sense rna) rna of mouse hepatitis virus. of the smaller subgenomic di rna (joo and makino mhv genomic rna contains many intergenic re gions, many transcription scanning factors may fall off the complete the template during scanning and this may result in the sequence (22 kilobases) of murine coronavirus gene 1 encoding the lower level of synthesis of the larger subgenomic putative proteases and rna polymerase the virus-mrnas. specific intracellular rna species of two murine coronaviruses: acknowledgments mhv-a59 and mhv-jhm deletion mapping of a mouse hepati-this work was supported by public health service grants ai29984 and ai32591 from the national institutes of health. tis virus defective interfering rna reveals the requirement of an internal and discontinuous sequence for replication evidence for new transcription units encoded at the 3 end of the mouse hepatitis virus genome. flanking sequences on coronavirus transcription a system for study of coronavirus mrna synthesis: a regulated, expressed subgenomic rus nonstructural protein ns2 is not essential for viral replication in transformed cells the 5-end sequence of the murine coronavirus genome: implications for multiple fusion sites in leader-primed transcription coronavirus switching during coronavirus defective interfering rna replication subgenomic rna synthesis directed by a synthetic defective interfering rna rna of murine coronavirus uous transcription generates heterogeneity at the leader fusion sites of coronavirus mrnas an improved method for directly sequencing pcr material using dimethyl sulfoxide interaction between the cytoplasmic proteins and the intergenic (promoter) sequence of mouse hepatitis mouse hepatitis virus and virus rna: correlation with the amounts of subgenomic mrna transcribed molecular cloning of the gene encoding the putative polymerase of mouse hepatitis virus, strain molecular cloning key: cord-329245-6tj2k1yn authors: corse, emily; machamer, carolyn e title: the cytoplasmic tails of infectious bronchitis virus e and m proteins mediate their interaction date: 2003-07-20 journal: virology doi: 10.1016/s0042-6822(03)00175-2 sha: doc_id: 329245 cord_uid: 6tj2k1yn abstract virus-like particle (vlp) formation by the coronavirus e and m proteins suggests that interactions between these proteins play a critical role in coronavirus assembly. we studied interactions between the infectious bronchitis virus (ibv) e and m proteins using in vivo crosslinking and vlp assembly assays. we show that ibv e and m can be crosslinked to each other in ibv-infected and transfected cells, indicating that they interact. the cytoplasmic tails of both proteins are important for this interaction. we also examined the ability of the mutant and chimeric e and m proteins to form vlps. ibv m proteins that are missing portions of their cytoplasmic tails or transmembrane regions were not able to support vlp formation, regardless of their ability to be crosslinked to ibv e. interactions between the e and m proteins and the membrane bilayer are likely to play an important role in vlp formation and virus budding. enveloped viruses acquire their lipid membranes from those of the host cell in a process known as budding, in which the virus envelope proteins accumulate at the appropriate membrane and cooperate with the nucleocapsid and other viral components to induce membrane curvature and pinching (garoff et al., 1998) . most well-studied enveloped viruses, such as influenza and retroviruses, bud from the plasma membrane of cells, which results in the release of virus particles from the host cell as soon as the pinching process is complete. however, the envelopes of other viruses are derived by budding into the lumen of intracellular secretory compartments, such as the endoplasmic reticulum (er) and golgi apparatus (griffiths and rottier, 1992) . intracellular budding is a seemingly more complex way of obtaining a lipid envelope, since virus particles are contained within intracellular membrane-bound compartments as a result of budding. the newly budded viruses presumably exploit the cellular secretory pathway to exit the cell for a new round of infection. the reasons that some enveloped viruses have evolved to bud inside the cell are not known. several clinically important viruses bud into intracellular membranes. viruses of the family flaviviridae, which include hepatitis c virus and kunjin virus, derive their lipid envelopes by budding into the lumen of the er (dubois-dalcq et al., 1984; mackenzie and westaway, 2001) . as another example, the hemorrhagic fever-causing hantaviruses, members of the family bunyaviridae, bud into the membranes of the golgi complex (hung et al., 1985; pettersson, 1991) . the advantage provided by intracellular budding remains an interesting and important question in the biology of these and other viruses that obtain their envelopes from intracellular membranes. coronaviruses are positive-stranded rna viruses that acquire their membrane envelope by budding into the lumen of a pre-golgi compartment or cis-golgi network (cgn) (klumperman et al., 1994) . accumulating evidence indicates that the e and m proteins are instrumental in this process. rottier and colleagues made the initial observation that mouse hepatitis virus (mhv) e and m proteins are sufficient to produce membrane-bound particles very similar to virions in size and shape (vennema et al., 1996) , and this result has been confirmed with the e and m proteins of several other coronaviruses (baudoux et al., 1998; corse and machamer, 2000; godeke et al., 2000) . these observations suggest that coronavirus budding is directed by envelope proteins alone, which is an unusual type of enveloped virus assembly (garoff et al., 1998) . the coronavirus e protein appears to play a critical role in the budding step, since mutations introduced into the cytoplasmic tail of mhv e by targeted rna recombination result in elongated virions (fischer et al., 1998) . indeed, it has been shown that both the mhv e and the infectious bronchitis virus (ibv) e proteins are sufficient for formation of the virus-like particles (vlps) described above (corse and machamer, 2000; maeda et al., 1999) , although the efficiency probably varies with cell type and protein expression system. we are interested in understanding the mechanisms by which the avian coronavirus ibv selects a bud site and assembles in cgn membranes. we have previously shown that the ibv m and e proteins independently localize to the golgi region of transfected cells (corse and machamer, 2000; machamer and rose, 1987) and characterized the golgi targeting signals in the two proteins that direct this localization (corse and machamer, 2002; machamer et al., 1993) . in the study described here, we examined interactions between the ibv e and m proteins in transfected ost7-1 cells using the chemical crosslinker dithiobis[succinimidyl propionate] (dsp). using mutant e and m proteins that are correctly targeted to the golgi complex, we found that the cytoplasmic tails of both proteins are required for their interaction. we found that ibv e and m proteins can be efficiently assembled into vlps in ost7-1 cells, and we investigated which domains of ibv e and m are required for this process. interestingly, the interaction of m and e, as measured by crosslinking, was not sufficient for vlp formation. it is likely that interactions of e and m with each other as well as the membrane bilayer are important for vlp formation and virus budding. although the ibv e protein is distributed throughout the golgi stack when expressed alone machamer, 2000, 2001) , the ibv e and m proteins exactly colocalize in ibv-infected vero cells examined by indirect immunofluorescence at early times postinfection (fig. 1a) . we wanted to investigate the possibility that this precise colocalization was indicative of a physical interaction between the two proteins. since attempts to coimmunoprecipitate e and m proteins from ibv-infected vero cells in a variety of detergent conditions were unsuccessful, we employed the cellpermeable chemical crosslinker dsp, which is a thiol-cleavable molecule that contains two amine-reactive groups separated by a 12 å spacer arm. ibv-infected vero cells were radiolabeled at 45 h postinfection and treated with dsp prior to lysis and immunoprecipitation with anti-e or anti-m antibodies. when we analyzed the immunoprecipitates by sds-page, we found that m was present in the anti-e immunoprecipitations and vice versa (fig. 1b, lanes 4 and 8) . this association was dependent on the presence of dsp since it was not seen in samples that were mock-treated (lanes 2 and 6). immunoprecipitation with irrelevant antibodies yielded no visible bands (not shown). in the absence of ␤-mercaptoethanol, which cleaves the disulfide bond of dsp, the unseparated crosslinked e and m proteins were present as high molecular weight aggregates and migrated at a high position on the gel (not shown). the crosslinking and coimmunoprecipitation was not quantitative, since only a fraction of the total e protein (as measured by the amount of e in the anti-e immunoprecipitation) was found in the anti-m immunoprecipitation. this could be due to the inherent inefficiency of chemical crosslinking. alternatively, this result could reflect the small e:m ratio in virions (liu fig. 1 . ibv e and m colocalize and interact in ibv-infected vero cells. (a) ibv-infected vero cells were fixed for immunofluorescence at 6 h postinfection and double labeled with rat anti-e antibody (a) and rabbit anti-m antibody (b). secondary antibodies were fluorescein-conjugated goat antirat igg and texas red conjugated donkey anti-rabbit igg. panel c is a differential interference contrast (dic) image of the labeled cells. bar, 15 m. (b) ibv-infected vero cells were labeled with [ 35 s]methionine-cysteine at 45 h postinfection, treated with dsp as indicated, lysed, and immunoprecipitated with anti-e or anti-m antibodies as described under materials and methods. the immunoprecipitates were analyzed by sds-15% page, in the presence or absence of ␤-mercaptoethanol (␤me) as indicated, and fluorography. ibv e is coprecipitated with ibv m after crosslinking and vice versa. these data are representative of at least three independent experiments. and inglis, 1991), which is presumably determined by the stoichiometry of envelope protein interactions. we conclude that the ibv e and m proteins interact in infected cells. ibv-infected cells contain other viral proteins which might be required for interaction of e and m. to determine if the ibv e and m proteins could interact directly, we coexpressed them in ost7-1 cells. we used indirect immunofluorescence to verify coexpression and correct intracellular localization of the two proteins (fig. 3a , panels a-c). as expected, the ibv e and m proteins colocalized in a compact perinuclear region characteristic of the golgi complex in this cell type, as seen previously in bhk-21 cells (corse and machamer, 2000) . when we treated ost7-1 cells coexpressing ibv e and m with dsp and analyzed anti-e and anti-m immunoprecipitates as described above, we found that m protein was present in the anti-e immunoprecipitate (fig. 2, lane 4) and that e protein was present in the anti-m immunoprecipitate (fig. 2, lane 8) , suggesting that the two proteins directly interact in transfected cells. detection of the interaction required crosslinking (compare lanes 3 and 7 to lanes 4 and 8) and antisera specific for e or m. even though less e was expressed in transfected cells (fig. 2 ) compared to ibv-infected cells (fig. 1b) , the ratio of e:m crosslinks (as seen in the relative amounts of e and m in the anti-m and anti-e immunoprecipitations, respectively) was similar, suggesting that there is a precise stoichiometry of interaction. to investigate which domains of the e and m proteins interact, we made use of a panel of mutant e and m proteins that were generated to study the golgi targeting signals present in the two proteins (corse and machamer, 2002; machamer and rose, 1987) . although many mutant e and m proteins have been generated, in this study, we used only those that are properly targeted to the golgi region. since these mutant proteins had previously been localized in bhk-21 or cos-7 cells, we first confirmed that they were properly targeted in ost-7 cells. cells coexpressing wildtype m protein and e mutants were analyzed by indirect immunofluorescence microscopy (fig. 3a) . eg3 (panels d-f) contains the transmembrane domain from the plasma membrane protein vesicular stomatitis virus (vsv) g; cte (panels g-i) is an n-terminal truncation of ibv e consisting only of its cytoplasmic tail, and get (panels j-l) is a chimeric protein containing the lumenal and transmembrane domains of vsv g and the cytoplasmic tail of ibv e. in all cases, the e mutant proteins were correctly localized to the golgi region of ost7-1 cells (including the partial transmembrane replacement mutants eg1 and eg2, not shown), consistent with our previous studies in bhk-21 cells (corse and machamer, 2002) . fig. 3b shows cells that are coexpressing wild-type e protein and the m protein deletion mutants mct⌬1 (panels a-c), which is missing cytoplasmic tail amino acid residues 103-203, mct⌬2 (panels d-f), which is missing cytoplasmic tail residues 119 -203, mct⌬3 (panels g-i), which is missing cytoplasmic tail residues 103-118, or m⌬m2,3 (panels j-l), which is missing the second and third transmembrane domains (amino acid residues 43-101). the m mutants, especially mct⌬3 (fig. 3b , panel h) and m⌬m2,3 (fig. 3b , panel k), were incompletely localized to the golgi region, since some er staining was observed. however, they all reached the golgi to some extent, as indicated by the juxtanuclear spot. we asked whether the e transmembrane replacement mutants eg1, eg2, and eg3 (in which the first third, the first two-thirds, or the entire e transmembrane domain, respectively, were replaced with the corresponding regions of the vsv g transmembrane domain) (corse and machamer, 2002) could be crosslinked to wild-type m protein. the appropriate proteins were coexpressed and immunoprecipitated with anti-e or anti-m antibodies after treating the cells with dsp as described above (fig. 4a ). all three e transmembrane replacement mutants were crosslinked to m protein, suggesting that the e transmembrane domain is not required for e and m interaction. however, there was less m in the anti-e immunoprecipitation of eg3 (fig. 4a , lane 4), suggesting that the interaction of m with this mutant could be decreased. perhaps the e and m transmembrane domains interact, or the conformation of the cytoplasmic fig. 2 . ibv e and m interact in transfected ost7-1 cells. ost7-1 cells expressing e and m proteins alone or together were labeled with [ 35 s]methionine-cysteine, treated with dsp as indicated, lysed, and immunoprecipitated with anti-e or anti-m antibodies as described under materials and methods. all immunoprecipitates in this and the following figures were deglycosylated by treating with n-glycanase to collapse the m protein to a single band. the immunoprecipitates were analyzed by sds-17.5% page in the presence of ␤me, and fluorography. the asterisk indicates a band that corresponds to m protein dimers. these data are representative of at least three independent experiments. tail of e is altered when the complete transmembrane domain is replaced (see below). to evaluate the role of the e cytoplasmic tail in interaction with m, we tested whether the chimeric protein get (which consists of the cytoplasmic tail of ibv e and the transmembrane and lumenal domains of vsv g; see eg3 and wild-type m protein (d-f), cte and wild-type m protein (g-i), or get and wild-type m protein (j-l) were fixed for immunofluorescence and double labeled with rat anti-e antibody (a, d, g, and j) and rabbit anti-m antibody (b, e, h, and k). secondary antibodies were fluorescein-conjugated goat anti-rat igg and texas red conjugated donkey anti-rabbit igg. the third image in each row (c, f, i, and l) is a dic image of the labeled cells. in the diagrams of the e mutant and chimeric proteins, ibv e sequence is shown in black and vsv g sequence is shown in gray. bar, 10 m. (b) ost7-1 cells expressing wild-type e protein and mct⌬1 (a-c), mct⌬2 (d-f), mct⌬3 (g-i), or m⌬m2,3 (j-l) were fixed for immunofluorescence and double labeled with rat anti-e antibody (a, d, g, and j) and rabbit anti-m antibody (b, e, h, and k). secondary antibodies were fluorescein-conjugated goat anti-rat igg and texas red conjugated donkey anti-rabbit igg. the third image in each row (c, f, i, and l) is a dic image of the labeled cells. in the diagrams of the deletion mutant m proteins, the thin lines indicate deleted sequence. bar, 10 m. ost7-1 cells expressing wild-type m protein and wild-type e protein or the e transmembrane replacement mutant proteins eg1, eg2, or eg3 were labeled with [ 35 s]methionine-cysteine, treated with dsp, lysed, and immunoprecipitated with anti-e or anti-m antibodies as described under materials and methods. the immunoprecipitates were analyzed by sds-17.5% page in the presence of ␤me, and fluorography. the asterisk indicates a band that corresponds to m protein dimers. (b) ost7-1 cells coexpressing the get chimera and wild-type m protein were labeled with [ 35 s]methionine-cysteine, treated with dsp, lysed, and immunoprecipitated with anti-e or anti-m antibodies as described under materials and methods. the immunoprecipitates were analyzed by sds-15% page in the presence of ␤me, and fluorography. the asterisk indicates a band that corresponds to m protein dimers. (c) ost7-1 expressing cte protein alone or with m protein were labeled with [ 35 s]methionine-cysteine, treated with dsp, lysed, and immunoprecipitated with anti-e antibodies as described under materials and methods. the immunoprecipitates were analyzed by sds-17.5% page in the presence of ␤me, and fluorography. these data are representative of at least three independent experiments. the e cytoplasmic tail and thus the get chimera. the reciprocal immunoprecipitation with anti-m antibody contained get protein (lane 3). we conclude that the cytoplasmic tail of ibv e protein is sufficient for interaction with m protein. indeed, the n-terminal truncation mutant cte, which consists only of the cytoplasmic tail of ibv e (corse and machamer, 2002) , was coprecipitated with m after crosslinking (fig. 4c, lane 2) . we previously found that partial or complete removal of a 50 amino acid region within the 80 amino acid e cytoplasmic tail resulted in transport of the get chimera to the cell surface (corse and machamer, 2002) . therefore, we did not attempt to find a specific region within the ibv e cytoplasmic tail that interacts with ibv m, because colocalization of the ibv e and m proteins is likely to be required for their interaction. to determine which regions of the ibv m protein were involved in interaction with ibv e, we tested how well the m deletion mutants mct⌬1, mct⌬2, mct⌬3, and m⌬m2,3 could be crosslinked to wild-type e protein compared to wild-type m protein (fig. 5) . m⌬m2,3 is missing the second and third transmembrane domains of ibv m; since it was crosslinked to e protein as well as wild-type m (lanes 5 and 10), we conclude that these membrane-spanning regions are not involved in interactions with e. a large portion of the m cytoplasmic tail is deleted in mct⌬1 (see fig. 3b ), and this protein was not crosslinked efficiently to e, compared to wild-type m protein (fig. 5, lanes 2 and 7) , although a small amount of e protein is visible in the mct⌬1 immunoprecipitation (fig. 5, lane 7) . this result indicates that the m cytoplasmic tail is involved in interaction with e. the portion of the m cytoplasmic tail removed by the mct⌬1 deletion is split into parts by the mct⌬2 and mct⌬3 deletions. note that the mct⌬1 and mct⌬2 mutants are not radiolabeled as efficiently as wild-type m and the mct⌬3 and m⌬m2,3 mutants (compare lanes 7 and 8 with lane 6) because they have fewer methionines and cysteines due to their deletions, which could explain why a reciprocal amount of mct⌬1 is not seen in the anti-e immunoprecipitation (fig. 5, lane 2) . mct⌬3, which is missing amino acids 103-118 of the m cytoplasmic tail, was crosslinked to e (fig. 5, lanes 4 and 9) , while mct⌬2, which is missing amino acids 119 -203 of the m cytoplasmic tail (see fig. 2b ), was not (fig. 5, lanes 3 and 8) . this indicates that the amino acids removed by the mct⌬2 deletion (119 -203) are required for crosslinking of the ibv m protein to the ibv e protein. the e and m proteins of several coronaviruses are released from cotransfected cells in membrane-bound particles that are morphologically similar to virions (vlps), suggesting that interactions between these proteins are an integral part of coronavirus assembly (baudoux et al., 1998; corse and machamer, 2000; godeke et al., 2000; vennema et al., 1996) . we initially attempted to study vlps formed in bhk cells from ibv e and m proteins expressed by coinfection with recombinant vaccinia viruses encoding each protein. however, we found that vlp formation was extremely inefficient under these conditions and estimated that only 0.01% of the cellular e and m proteins were released into the supernatant as vlps (corse and machamer, 2000) . since we were interested in using vlp formation to delineate portions of the ibv e and m proteins that are important in virus assembly, we employed the expression system used by rottier and colleagues in their initial description of mhv vlps (vennema et al., 1996) . these authors infected ost7-1 cells (elroy-stein and moss, 1990) , which are osteosarcoma cells that stably express phage t7 rna polymerase, with a vaccinia virus encoding t7 polymerase (vtf7-3) (fuerst et al., 1986) to provide higher levels of t7 rna polymerase. they then transfected the cells with plasmids encoding mhv e and m behind the t7 promoter. when we expressed the ibv e and m proteins this way, we found that vlps were formed efficiently (fig. 6, lanes 3 and 14) . the supernatant side of the gel (lanes 12-22) shown in this figure was exposed 20 times longer than the cell side to allow the visualization of e and e transmembrane mutant proteins (lanes 1-11). we have consistently observed at least 10%, and frequently more, of the cellular m protein being released into the supernatant during the 3 h chase (data not shown). this level of vlp release was dependent on e protein, since less than 1% of the cellular m protein was found in the supernatant in the absence of e. e protein was not visualized in the supernatants when expressed alone (fig. 6, lane 12) . we believe that a small amount of e is released into the supernatant when it is expressed alone, and that it is simply not visualized unless a large amount of supernatant is loaded on the gel, as in our previous experiments (corse and machamer, 2000) . our results differ from the data of makino and colleagues, who observed a substantial amount of mhv e being released into the supernatant when expressed by itself (maeda et al., 1999) ; this discrepancy could be explained by the different expression system used to produce the e protein in their study. we favor the hypothesis that vlp formation involves a lattice of m protein within the membrane with curvature induced by small amounts of the e protein (see discussion). this would account for the small amount of ibv e protein relative to ibv m protein that is found in vlps. we found that the partial or complete replacement of the ibv e transmembrane domain (mutants eg1, eg2, and eg3) had no effect on e's ability to induce vlps as measured by the amount of ibv m protein released into the supernatant (fig. 6 , compare the m protein bands in lanes 14, 16, 18, and 20). we did notice that more eg2 and eg3 proteins were incorporated into vlps compared to wildtype e and eg1 proteins (fig. 6 , compare the e protein bands in lanes 18 and 20 with those in lanes 14 and 16). it is possible that conformational changes induced by replacement of the transmembrane domain facilitates increased incorporation of these mutants into vlps. the eg2 and eg3 mutants did not induce vlps more efficiently than wild-type e, because an increased amount of m protein was not released into the supernatant. we examined the ability of the e cytoplasmic tail to induce vlps by using the chimeric protein get. when expressed by itself, we observed that a negligible amount of this protein was released into the supernatant (fig. 6 , lane 21; recall that the supernatant side of the gel was exposed 20 times longer than the cell side) and that expression of get with ibv m did not increase the amount of m that was released when m was expressed alone (fig. 6, lanes 22 and 13) . we conclude that the get chimera does not induce vlps. since both the eg3 and the get chimeras contain the transmembrane domain of vsv g and the cytoplasmic tail of ibv e, it seems likely that the vsv g lumenal domain is incompatible with vlp formation, either because it is too large or because it cannot be incorporated into the m protein lattice for some other reason (see discussion). thus, it is difficult to predict if the e cytoplasmic tail alone is capable of inducing vlps. we also did not observe vlp formation with the ibv e n-terminal truncation mutant cte, which consists only of the e cytoplasmic tail (data not shown). however, this result is inconclusive since the cte protein is less stable than wild-type e protein and thus accumulates to significantly lower levels (corse and machamer, 2002) . we evaluated the incorporation of the ibv m deletion mutants mct⌬1, mct⌬2, mct⌬3, and m⌬m2,3 into vlps. as shown in fig. 7 , none of the deletion mutants were able to support vlp formation at a detectable level, regardless of whether they could be crosslinked to ibv e. the mct⌬1 and mct⌬2 mutants are not radiolabeled as efficiently as wildtype m and the mct⌬3 and m⌬m2,3 mutants (compare lanes 5,6,7, and 8 with lanes 2 and 3) because they have fewer methionines and cysteines due to their deletions. however, we would still expect to see a detectable signal in the supernatants after a long exposure if these mutants supported vlp formation at a level comparable to wild-type m. since we have never seen such a signal, we conclude that these mutants do not support vlp formation. these results indicate that both the cytoplasmic tail and the transmembrane regions of ibv m are involved in vlp formation, which is consistent with the results of rottier and colleagues, who found that mutations in all domains of the mhv m protein abrogated vlp formation (de haan et al., 1998) . these results suggest that a direct interaction between ibv e and m proteins is not sufficient for vlp formation and that this process may depend on how the two proteins interact with the membrane bilayer, both separately and together. we have demonstrated an interaction between the ibv e and m proteins using both in vivo chemical crosslinking and vlp formation as assays. using ibv e and m protein mutants that were generated in the course of golgi targeting studies, we examined the domains in the ibv e and m proteins that are involved in their interaction. we found that the cytoplasmic tails of both proteins mediate their interaction, as measured by crosslinking. interestingly, however, we observed that reciprocal crosslinking of ibv e and m protein mutants did not correlate with vlp formation, suggesting that interaction between ibv e and m is not sufficient for vlp formation. here we compare our results to those reported by other groups and discuss the implications of our findings for coronavirus assembly. lim and liu previously used a coimmunoprecipitation assay to detect ibv e and m protein interactions (lim and liu, 2001) . they also concluded that the cytoplasmic tail of ibv e is important for interaction with ibv m, since dele-tion of a 20 amino acid region within the e cytoplasmic tail (amino acids 37-57) prevented coimmunoprecipitation of the e and m proteins and vlp formation. as mentioned above, we did not attempt to find a region within the e tail that mediates its interaction with ibv m, because we previously found that partial or complete replacement of a 50 amino acid region in the ibv e tail results in transport of a reporter chimera from the golgi complex to the cell surface, which indicates that the cytoplasmic tail of e contains golgi targeting information (corse and machamer, 2002) . our subcellular localization results differ from those of lim and liu, who reported that ibv e is localized to the endoplasmic reticulum of transfected cells (lim and liu, 2001) , possibly because they examined cells overexpressing epitope-tagged ibv e protein. the m protein is by far the most abundant envelope protein of coronaviruses and has been estimated to account for ϳ40% of the mass of ibv and mhv particles (stern et al., 1982; sturman et al., 1980) . thus the physical nature of the coronavirus envelope must be largely determined by the characteristics of the m protein. interestingly, however, the m protein cannot drive coronavirus budding by itself, but displays a strict requirement for a small amount of e protein for vlp formation (baudoux et al., 1998; corse and machamer, 2000; godeke et al., 2000; vennema et al., 1996) . we have observed this low e protein:m protein ratio in ibv vlps (fig. 6, lane 14) ; measurements of the amount of e and m proteins in ibv virions also reveal a relatively low amount of e protein (liu and inglis, 1991) . we favor the hypothesis that vlp formation involves a specific membrane curvature that is induced by the proper ratio of m and e proteins. the mechanism by which a small amount of e protein interacts with a large amount of m protein to induce invagination and pinching of the membrane to form vlps fig. 7 . cytoplasmic and transmembrane region deletion mutants of ibv m do not support vlp formation. ost7-1 cells expressing the indicated proteins were labeled with with [ 35 s]methionine-cysteine for 1 h and chased for 3 h, and the supernatants and cells were harvested and immunoprecipitated with anti-e and anti-m antibodies as described under materials and methods. the immunoprecipitates were analyzed by sds-17.5% page and fluorography. the supernatant samples (lanes 12-22) were exposed to film 13 times longer than the cell samples (lanes 1-11) . the mct⌬1 and mct⌬2 mutants are not radiolabeled as efficiently as wild-type m and the mct⌬3 and m⌬m2,3 mutants because they have fewer methionines and cysteines due to their deletions. these data are representative of at least three independent experiments. remains unclear, but its elucidation is likely to be essential for a complete understanding of coronavirus assembly. several studies have addressed the question of how the coronavirus e and m proteins interact in vlp formation. rottier and colleagues (de haan et al., 1998) studied the primary sequence requirements for incorporation of the mhv m protein into vlps. they found that all regions of mhv m seem to be important for vlp formation, since mutations in the short lumenal domain, the transmembrane region, and the cytoplasmic tail all impaired vlp formation to some extent. these results are consistent with our observation that deletions in both the cytoplasmic and the transmembrane domains of ibv m prevent vlp formation. interestingly, the extreme c-terminus of the cytoplasmic tail mhv m was found to be especially important, since deletion of only the c-terminal threonine residue prevented vlp formation (de haan et al., 1998) . we were unable to examine the role of the extreme c-terminus of the ibv m protein in vlp formation because our antibody to ibv m recognizes this sequence. laude and colleagues (baudoux et al., 1998) showed that coexpression of the e and m proteins of transmissible gastroenteritis virus (tgev) and of bovine coronavirus (bcv) resulted in vlp production. in this study vlps were also produced with a bcv-tgev chimeric m protein. interestingly, the chimeric m protein was able to form vlps with tgev e, bcv e, and bcv-tgev chimeric e proteins. since tgev and bcv are in different coronavirus groups based on serological and genomic sequence relationships, this suggests that vlp formation does not depend on interactions between e and m proteins that are based on strict sequence requirements. well-conserved three-dimensional characteristics of the e and m proteins may be instrumental in vlp formation. our results suggest that vlp formation involves more than an interaction between e and m proteins, since m protein mutants that could be crosslinked to e protein (mct⌬3 and m⌬m2,3; see fig. 5 ) are not incorporated into virus-like particles. it is possible that vlp formation requires interactions between m proteins, as proposed by rottier and colleagues . these workers demonstrated homotypic interactions among mhv m protein molecules by coimmunoprecipitation and incorporation of vlp assembly-incompetent mhv m mutants into vlps via their interaction with assembly-competent mhv m proteins. these authors hypothesize that the vlp (and virion) envelope mainly consists of an m protein lattice, with a few e proteins interspersed within this lattice. although we have not examined whether homotypic interactions between ibv m proteins exist, we think it possible that such interactions may be important in ibv vlp assembly. we did observe a small amount of sds-resistant ibv m protein dimers in our anti-m immunoprecipitations (indicated by asterisks in figs. 2, 4 , 5, and 6), although we have no direct evidence that these dimers are physiologically relevant. we also showed that the get chimera could be crosslinked to ibv m protein (fig. 4b ), but did not induce vlp formation (fig. 6 ). since the get chimera contains the same transmembrane domain and cytoplasmic tail as the transmembrane replacement mutant eg3, which was able to induce vlp formation as well as wild-type e protein (fig. 6) , a likely explanation is that the vsv g ectodomain prevents induction of vlps. one possibility is that this large ectodomain does not fit well into the lattice of m protein in the membrane. a similar observation was reported by rottier and colleagues , who showed that foreign proteins such as wild-type vsv g protein, equine arteritis virus m protein, and cd8 were effectively excluded from mhv vlps. likewise, it is possible that the m deletion mutants we tested in the vlp assay are not in the right conformation to fit into such a lattice of m protein, and thus, do not support vlp formation. as mentioned above, we have previously shown that the cytoplasmic tail of the ibv e protein is sufficient to localize it to the golgi complex and to mediate its redistribution with the golgi scaffold proteins gm130 and p115 during brefeldin-a treatment (corse and machamer, 2002) . since these and other golgi scaffold proteins are known to be part of a complex which is thought to mediate vesicle tethering during intra-golgi transport (barr et al., 1998; seemann et al., 2000a,b) , this led us to hypothesize that the ibv e protein may be targeted to the golgi apparatus via a direct interaction with one or more golgi scaffold proteins. furthermore, interaction of ibv e with the golgi scaffold could result in modulation of vesicular traffic, which might be advantageous for collecting coronavirus envelope proteins in a specific golgi compartment for assembly (corse and machamer, 2002) . since we have shown here that the cytoplasmic tail of ibv e is also important for interactions with ibv m, this raises the question of whether the e cytoplasmic tail can interact with m and the golgi scaffold simultaneously. if not, then it is possible that two functional pools of ibv e protein exist in an infected cell. one pool could interact with ibv m and the membrane at the assembly site to induce virus budding, while the other could associate with the golgi scaffold to influence vesicular traffic through the golgi complex. the latter interaction might aid in collecting viral membrane proteins for assembly at golgi membranes. since the small amount of ibv e protein found in virions (liu and inglis, 1991) and vlps (shown here) suggests that not much e protein is required to induce virus budding, maybe a second function of e protein, such as modulation of golgi transport, accounts for the apparent excess of ibv e protein in infected cells (corse and machamer, 2000) . ost7-1 cells (elroy-stein and moss, 1990) and vero cells were maintained in dulbecco's modified eagle's medium (dmem) containing 10% fetal calf serum (fcs) and antibiotics. the adaptation of ibv (beaudette strain) to vero cells has been described (machamer and rose, 1987) . the recombinant vaccinia viruses encoding phage t7 polymerase (vtf7-3), ibv m (vvibvm), and wild-type and mutant versions of ibv e (vvibve, vveg3, vvcte) have been described machamer, 2000, 2002; fuerst et al., 1986; machamer and rose, 1987) . growth and titering of recombinant vaccinia viruses were done as described (weisz and machamer, 1994) . the pbs/ibv e, pbs/eg1, pbs/eg2, pbs/eg3, pbs/ get, par/m and par/m⌬m2,3 plasmids have been described machamer, 2000, 2002; machamer and rose, 1987) . ibv m was subcloned into the bamhi site of pt7/t3-18 (brl) to create pt7/m. the m cytoplasmic tail deletion mutant plasmids par/mct⌬1, par/mct⌬2, and par/mct⌬3 were constructed by oligonucleotide-directed mutagenesis as described (machamer and rose, 1987) . mct⌬1 is missing amino acids 103-203, mct⌬2 is missing amino acids 119 -203, and mct⌬3 is missing amino acids 103-118. all three of these cytoplasmic tail deletion mutants contain the ibv m c-terminal 22 amino acids that are recognized by the polyclonal anti-m antibody (see fig. 3b ). par/e and par/cte were made by pcr addition of bamhi sites at both ends of the e and cte inserts. pbs/ibv e and pbs/cte (corse and machamer, 2002) , respectively, were used as templates for pcr. the bamhi-digested pcr products were cloned into the bamhi site of par2529, and subclones were screened for correct orientation by indirect immunofluorescence. the pt7/mc⌬1, pt7/mct⌬2, and pt7/mct⌬3 plasmids used in vlp experiments were made by pcr addition of ecori and xbai restriction sites, with the corresponding ar plasmids as templates. the t7/ m⌬m2,3 plasmid used in vlp experiments was made by subcloning the par/m⌬m2,3 bamhi fragment into the the bamhi site of pt7/t3-18. the polyclonal anti-m antibody and its affinity purification for use in immunofluorescence has been previously described (machamer and rose, 1987) . the rat and rabbit polyclonal antibodies to the c-terminal 14 amino acids of ibv e have been reported (corse and machamer, 2000) . the rabbit anti-vsv polyclonal antibody used to immunoprecipitate radiolabeled g and get proteins has been described (weisz et al., 1993) . texas red conjugated donkey anti-rabbit immunoglobulin g (igg) and fluorescein-conjugated goat anti-rat igg were from jackson immunoresearch laboratories, inc. (west grove, pa). vero cells were plated in 35-mm dishes 1 day before being infected with ibv (passage 12), as previously described (machamer and rose, 1987) . the cells were fixed in 3% paraformaldehyde in phosphate-buffered saline (pbs) for 20 min at room temperature, permeabilized with 0.5% triton x-100, and stained as previously described (swift and machamer, 1991) with rat anti-e antibody and rabbit anti-m antibody. ost7-1 cells were plated on cover slips in 35-mm dishes 2 days before infection with vtf7-3, vvibve, vveg3, or vvcte at a multiplicity of infection of 20. adsorption was for 1 h at 37°c, and cells infected with vtf7-3 were transfected with 5 g of each expression plasmid using 20 l of lipofectin (invitrogen, carlsbad, ca) as directed by the manufacturer. at 3.5 to 4 h postinfection the cells were fixed and stained as described above. ost7-1 cells were plated in 35-mm dishes 2 days before being infected with vtf7-3 and transfected with the appropriate constructs as described above. in samples transfected with only a plasmid encoding ibv e or ibv m alone, an equal amount of empty vector plasmid was added so that an equal amount of plasmid dna was added to all samples. at 4 h postinfection, the cells were labeled with 100 ci of 35 s-promix (amersham pharmacia biotech, piscataway, nj) in 0.5 ml methionine-and cysteine-free medium for 1 to 1.5 h at 37°c. vero cells were plated in a 10-cm dish 1 day before being infected with ibv and labeled at 45 h postinfection for 1 h at 37°c with 500 ci of 35 s-promix. after being rinsed twice in room temperature pbs, cells were scraped into pbs, transferred to microfuge tubes, and the thiol-cleavable crosslinker dsp (pierce, rockford, il) was added to 1 mm after being prepared just before use as a 20 mm stock solution in dmso. for mock dsp treatments an equivalent amount of dmso was added. the cells were incubated with dsp for 10 min at room temperature, and the dsp was quenched by adding glycine to 40 mm. the cells were lysed in detergent solution [62.5 mm edta, 50 mm tris (ph 8), 0.4% deoxycholate, 1% np-40] containing protease inhibitors, and the postnuclear supernatants were immunoprecipitated with the appropriate antibodies in the presence of 0.2% sds as described (machamer and rose, 1987) . treatment of immunoprecipitates with n-glycanase was as previously described (machamer et al., 1990) . the samples were subjected to sds-page in the presence of ␤mercaptoethanol (except for the indicated samples in fig. 1b ) and the labeled proteins were visualized by fluorography. appropriate constructs were expressed in ost7-1 cells by infection with vtf7-3 and transfection as described above. at 3 h postinfection the cells were shifted from 37 to 32°c for 1 h and then radiolabeled at 32°c with 35 s-promix for 1 to 1.5 h as described above. after labeling the cells were chased in 1 ml of growth medium for 3 h at 32°c. the medium was microcentrifuged at 14,000 rpm for 20 min at 4°c to remove cellular debris, and the supernatants were immunoprecipitated with rat anti-e and rabbit anti-m antibodies in the presence of 1% triton x-100 and 0.2% sds. the cells were harvested by rinsing in pbs and lysing in detergent solution. the postnuclear supernatants were immunoprecipitated with anti-e and anti-m antibodies in the presence of 0.2% sds. the immunoprecipitates were treated with n-glycanase as described (machamer et al., 1990) . mapping the interaction between grasp65 and gm130, components of a protein complex involved in the stacking of golgi cisternae coronavirus pseudoparticles formed with recombinant m and e proteins induce alpha interferon synthesis by leukocytes infectious bronchitis virus e protein is targeted to the golgi complex and directs release of virus-like particles infectious bronchitis virus envelope protein targeting: implications for virus assembly the cytoplasmic tail of infectious bronchitis virus e protein directs golgi targeting coronavirus particle assembly: primary structure requirements of the membrane protein assembly of the coronavirus envelope: homotypic interactions between the m proteins assembly of enveloped rna viruses cytoplasmic expression system based on constitutive synthesis of bacteriophage t7 rna polymerase in mammalian cells analysis of constructed e gene mutants of mouse hepatitis virus confirms a pivotal role for e protein in coronavirus assembly eukaryotic transient expression system based on recombinant vaccinia virus that synthesizes bacteriophage t7 rna polymerase virus maturation by budding. microbiol assembly of spikes into coronavirus particles is mediated by the carboxy-terminal domain of the spike protein cell biology of viruses that assemble along the biosynthetic pathway morphology and morphogenesis of viruses of hemorrhagic fever with renal syndrome (hfrs). i. some peculiar aspects of the morphogenesis of various strains of hfrs virus coronavirus m proteins accumulate in the golgi complex beyond the site of virion budding the missing link in coronavirus assembly. retention of the avian coronavirus infectious bronchitis virus envelope protein in the pre-golgi compartments and physical interaction between the envelope and membrane proteins association of the infectious bronchitis virus 3c protein with the virion envelope retention of a cis golgi protein requires polar residues on one face of a predicted alpha-helix in the transmembrane domain the e1 glycoprotein of an avian coronavirus is targeted to the cis golgi complex a specific transmembrane domain of a coronavirus e1 glycoprotein is required for its retention in the golgi region assembly and maturation of the flavivirus kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively release of coronavirus e protein in membrane vesicles from virus-infected cells and e protein-expressing cells protein localization and virus assembly at intracellular membranes matrix proteins can generate the higher order architecture of the golgi apparatus the role of the tethering proteins p115 and gm130 in transport through the golgi apparatus in vivo structural analysis of virion proteins of the avian coronavirus infectious bronchitis virus isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid a golgi retention signal in a membrane-spanning domain of coronavirus e1 protein nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes use of recombinant vaccinia virus vectors for cell biology oligomerization of a membrane protein correlates with its retention in the golgi complex this work was supported by national institutes of health grants gm42522 and gm64647. we thank the members of the machamer lab for critical reading of the manuscript. key: cord-340983-w219g6qs authors: smith, mary ellen; koser, martin; xiao, sa; siler, catherine; mcgettigan, james p.; calkins, catherine; pomerantz, roger j.; dietzschold, bernhard; schnell, matthias j. title: rabies virus glycoprotein as a carrier for anthrax protective antigen date: 2006-09-01 journal: virology doi: 10.1016/j.virol.2006.05.010 sha: doc_id: 340983 cord_uid: w219g6qs live viral vectors expressing foreign antigens have shown great promise as vaccines against viral diseases. however, safety concerns remain a major problem regarding the use of even highly attenuated viral vectors. using the rabies virus (rv) envelope protein as a carrier molecule we show here that inactivated rv particles can be utilized to present bacillus anthracis protective antigen (pa) domain-4 in the viral membrane. in addition to the rv glycoprotein (g) transmembrane and cytoplasmic domains, a portion of the rv g ectodomain was required to express the chimeric rv g anthrax pa on the cell surface. the novel antigen was also efficiently incorporated into rv virions. mice immunized with the inactivated recombinant rv virions exhibited seroconversion against both rv g and anthrax pa, and a second inoculation greatly increased these responses. these data demonstrate that a viral envelope protein can carry a bacterial protein and that a viral carrier can display whole polypeptides compared to the limited epitope presentation of previous viral systems. until recently infection with bacillus anthracis, the causative agent of anthrax had posed only a low risk, with minimal natural occurrence in humans. however, the development of b. anthracis as a bioterrorism tool with the death of five people and the infection of at least seventeen others in the u.s. (jernigan et al., 2002) in 2001, has underscored the danger of an anthrax attack. existing prophylactic and therapeutic strategies for combating anthrax are insufficient. the most lethal form of the disease, inhalation anthrax, presents with general, flu-like symptoms, so that early diagnosis is difficult. prompt recognition of symptoms is required for effective therapeutic treatment using antibiotics or passive immunization following unknown exposure (sternbach, 2003) . while passive immunization is effective (sawada-hirai et al., 2004) , the availability of immune sera is highly limited and no approved therapeutic antibodies against anthrax are currently available. several antibiotics care effective against b. anthracis, but, antimicrobial resistance engineered into future bioterrorism-grade anthrax can be envisioned. moreover, antibiotics can eliminate the bacteria but not the toxins that may already have been released. thus, vaccination is a highly practical alternative to post-exposure treatment and eliminates the risk of infection. virology 353 (2006) 344 -356 www.elsevier.com/locate/yviro anthrax disease is caused by the toxins. the anthrax toxins act as a tripartite system in which transport of the active components, lethal and edema factors into the cell, is facilitated by the binding component, protective antigen (pa) . pa binds to receptors on the cell surface and is cleaved by cellular furin (molloy et al., 1992 ) into a small subunit and the larger 63-kda constituent, pa63. seven receptor-bound pa63 units oligomerize to form a complex that binds with lethal factor and edema factor and that passes into the cytoplasm. two receptors, atr/tem8 (bradley et al., 2001) and cmg2 (scobie et al., 2003) , bind pa and mediate toxicity. pa is comprised of four distinct domains, with receptor interactions occurring at sites within pa domain-4 (d4) and -2. the structure of the receptor binding ridge is highly conserved, suggesting that atr/tem8 and cmg2 interact with pa in the same manner (santelli et al., 2004) . passive and active immunization indicates that protection is mediated by humoral immune responses (enserink, 2002; pitt et al., 2001; welkos et al., 2001) and binding of pa to the cell surface receptors can be prevented by anti-pa antibodies. of note, antibodies elicited by pa immunization can confer protection against b. anthracis challenge (beedham et al., 2001; ivins et al., 1992 ivins et al., , 1998 kobiler et al., 2002; little et al., 1997) , and antibodies against epitopes within pa d4 also show protective capacity (flick-smith et al., 2002b; kasuya et al., 2005; maynard et al., 2002) . an fda-licensed vaccine against anthrax exists consisting of a cell culture filtrate adsorbed onto aluminum hydroxide (bioport, 2002) . however, several aspects of this product point to need for development of a novel vaccine: (i) it is indicated only for use in at-risk individuals; (ii) the material is difficult to characterize (turnbull, 2000) ; (iii) it requires six immunizations within the first 1.5 years with annual boosters thereafter (bioport, 2002) ; and (iv) reactogenicity is reported in up to 35% of patients (bioport, 2002) . additional vaccine strategies, most of which focus on pa, are being pursued. these include dna vaccines encoding pa (ferrari et al., 2004) , as well as approaches using various microorganism vectors to express pa, such as influenza (li et al., 2005) , vaccinia virus (iacono-connors et al., 1991) , adenovirus (tan et al., 2003) , venezuelan equine encephalitis virus-based replicons (lee et al., 2003) and a salmonella enterica serovar typhimurium live vaccine encoding domain four of pa (galen et al., 2004) . the best-developed vaccine candidate is a recombinant pa (rpa) adsorbed to aluminum hydroxide (keyserling et al., 2004) . efforts to enhance the immunogenicity of rpa include immunogenic formulations such as copolymers (coeshott et al., 2004) or microsphere encapsulation (flick-smith et al., 2002a) or addition of adjuvants (berthold et al., 2005; ivins et al., 1992 ivins et al., , 1998 mcbride et al., 1998) . rabies virus is a promising vaccine vector able to induce humoral and cellular immune responses efficiently to foreign antigens (mcgettigan et al., 2001a (mcgettigan et al., , 2001b schnell et al., 2000) . because of low seroprevalence in the human population, rv is an excellent viral vector candidate. methods for engineering the virus are well established, up to two foreign genes totaling 6.5 kb have been incorporated thus far, and foreign sequences are stably maintained. rv grows to high titers in cell lines approved for human vaccine production and manufacture is economical. our current rv vaccine vector is based on the vaccine strain sad-b19 used since 1983 as a live oral immunogen to vaccinate wild life in europe and asia (schneider et al., 1988) . to further increase safety, several attenuating mutations have been introduced into these live viral vectors (mckenna et al., 2003) . inactivated rv has been used to vaccinate humans in the u. s. since 1978 (plotkin, 1980) and thus has a significant history of safety (haupt, 1999) . potent and long-lasting neutralizing antibodies to the rv glycoprotein are generated by immunization with killed particles (thraenhart et al., 1994) . strong humoral responses to heterologous glycoproteins such as hiv-1 envelope (mckenna et al., 2004; schnell et al., 2000) and hcv e1/e2 (siler et al., 2002) are elicited by killed rv particles incorporating the foreign proteins. the highly repetitive and rigid organization of viruses and virus-like particles is thought to maximally stimulate b cells (fehr et al., 1997; jegerlehner et al., 2002) . cd4+ t helper epitopes provided by virus particles can also provide necessary signals for b cell activation (hooper et al., 1994) . here, we generated several rv g-pa chimeras in which either pa63 or pa d4 was fused to ectodomain (ed) truncation mutants of rv g. one of these chimeras, d4-e51, properly trafficked to the cell surface and was efficiently incorporated into virus particles. mice immunized with killed spbn-d4-e51 particles mounted high antibody titers against b. anthracis pa. the response was dose-dependent and was skewed toward th2-associated antibodies. lymphocyte proliferation was also observed, in response to viral antigens and anthrax pa. these results suggest that inactivated rabies virus can serve as an immunostimulatory carrier for immunization against pathogens such as anthrax. anthrax pa is an excellent target for an anthrax vaccine because particular anti-pa antibodies can prevent entry of the toxin into cells. therefore, anthrax pa63, optimized for human codon usage to ensure high expression levels, was cloned into the rv vaccine vector pspbn resulting in pspbn-pa63 (fig. 1) . recombinant rv was recovered by standard methods and expression of pa63 was confirmed by immunostaining with anti-pa monoclonal antibody (fig. 2 , panel b′). to analyze the immunogenicity of the recombinant rv spbn-pa63, five balb/c mice were inoculated i.m. with 10 6 ffu and sera from immunized mice were analyzed by a pa-specific elisa 14 days after immunization. however, no pa-specific antibodies were detected in any of the immunized animals (data not shown). to determine whether surface expression of anthrax pa might increase its immunogenicity in the vaccinated host, we used the rv g er translocation ss in addition to the rv g tm and cd (fig. 1) . the gene encoding the chimeric rv g-anthrax pa was introduced into the rv vector pspbn, resulting in pspbn-pa63-tc. bsr cells were infected with spbn ( fig. 2, antibody directed against pa ( fig. 2 panels a′′, b′′, c′′) or permeabilized with triton x-100 for internal staining with an antibody against anthrax pa ( fig. 2 panels a′, b′, c′) or rv n protein ( fig. 2 panels a , b, c). all cells were infected with rv as indicated by a bright signal with an rv n-specific antibody. moreover, anti-pa monoclonal antibody detected the internal expression of pa63 in both spbn-pa63-or spbn-pa63-tcinfected cells. however, no expression of pa63 on the cell surface was detected for either of the viruses expressing pa63 independent of the use of the rv g signal sequence. to test the hypothesis that a lack of transport through the er and golgi apparatus accounted for the lack of surface expression, and that a portion of the rv g might support such transport, we utilized different portions of the rv g ed in addition to the tm and cd of rv g. we constructed five different plasmids containing increasing portions of the rv g ed in addition to anthrax pa. based on previous research indicating that d4 of anthrax pa is the most potent antigen to induce protective antibodies, we used this 140-aa fragment comprising the carboxy-terminal region of pa. use of this fragment allowed us to examine whether d4 flanked by the rv g ss, tm and cd fails to be transported to the cell surface as observed for pa63. the genes encoding rv ganthrax pa d4 fusion proteins containing 0, 51, 127, 210, or 439 amino acids of the rv ed were pcr-amplified and introduced into an expression plasmid under the control of the t7 rna polymerase promotor (fig. 3) . the resulting plasmids were designated pd4-δed, pd4-e51, pd4-e127, pd4-e210, and pd4-e439. to evaluate the cell surface expression of the recombinant rv g-d4 fusion proteins, bsr cells were infected with recombinant vaccinia virus expressing t7 rna polymerase for one h, transfected with 5 μg of each of the five plasmids and 24 h later fixed in paraformaldehyde, permeabilized, and immunostained with anti-pa monoclonal antibody. a second set of transfected cells was treated similarly but was not permeabilized. as shown in fig. 4a , all proteins were successfully expressed (internal staining) in transfected cells, whereas significant surface expression was detected only for recombinant protein d4-e51 (surface staining). more quantitative analysis by facs (fig. 4b ) confirmed that only d4-e51 was expressed at high levels on the cell surface, while surface expression for pd4-pd4-e127 and pd4-e439 was detectable but at lower levels than observed for pd4-e51. western blotting analysis of transfected cell lysates using antibodies directed against anthrax pa or rv g cd ( fig. 4c ) revealed chimeric rv g-d4 proteins of the expected sizes of 21 kda, 29 kda, 37 kda, or 74 kda for d4-δed, d4-e51, d4-e127, and pd4-e439 with both the pa-and the rv g cd-specific antibodies. interestingly, the rv g cd-directed antibody failed to detect the d4-e210 fusion protein suggesting cleavage or degradation of the carboxy-terminus of this protein. the antibody directed against the amino-terminal-located d4 of d4-e210 detected the protein but at a smaller than predicted size, further indicating degradation. all recombinant fusion proteins showed a diffuse migration pattern over a range of up to 20 kda. sequence analysis of pa indicates several potential n-and o-glycosylation sites within d4, which might contribute to the observed retarded migration. fig. 3 . construction of plasmids containing d4 fused to various rv g ed fragments. panel a shows rv g protein is shown in panel a. the three potential sites of nlinked glycosylation are indicated. to construct chimeric rv g/anthrax pa fusion proteins, the rv g ed was replaced with domain 4 of pa (d4-δed, b). the membrane-proximal 51, 127 or 210 amino acids of the rv g ectodomain containing one, two or three potential n-linked glycosylation sites, respectively, were reintroduced into d4-δed to generate d4-e51, d4-127 and d4-210, respectively (c, d, e). the full-length rv g ectodomain was inserted into d4-δed resulting in d4-e439 (f). the results above indicated the highest level of surface expression for d4-e51, suggesting its suitability for incorporation into the rv envelope. thus, the gene encoding d4-e51 was cloned into pspbn resulting in pspbn-d4-e51. recombinant . as expected, the rv g cd-specific antibody detected rv g for both viruses. an additional protein migrating at the expected size for the d4-e51 was detected in the case of spbn-d4-e51-infected cells or spbn-d4-e51 virions (fig. 5 , lanes 2 and 6). the successful incorporation of d4-e51 into virions enabled us to study the induction of anti-pa immune responses using both inactivated and live spbn-d4-e51 in an animal model. three groups of five female swiss webster mice were inoculated i.m. with 3 × 10 6 ffu spbn-d4-e51, 50 μg inactive spbn-d4-e51, 3 × 10 6 ffu spbn or 50 μg inactive spbn. blood samples were obtained from all mice at 14 days post-immunization and sera were analyzed by elisa on plates coated with rpa or rv g. as shown in fig. 6b all immunized mice seroconverted against rv g. moreover, all mice primed with inactivated or live spbn-d4-e51 also mounted anthrax pa-specific antibodies, which were not detected in sera from mice immunized with spbn. a second inoculation increased the rv g-specific titers about 3-fold, whereas the anthrax paspecific elisa titers were increased 10-fold, with no significant differences in antibody titers between mice immunized with live rv or inactivated rv virions (fig. 6) . several vaccine approaches that appear promising in mice are difficult to reproduce in larger animals or humans due to the large amount of antigen needed for immunization. thus, we performed a dose-response analysis using antigen in amounts fig. 5 . expression and incorporation of d4-e51 into rv particles. bsr cells were infected with spbn or spbn-d4-e51 at an moi of 1 and after 48 h cell lysates were prepared, separated by sds-page, and transferred to a nitrocellulose membrane (lanes 1-4). to analyze incorporation of d4-e51 into rv virions, sucrose-purified spbn or spbn-d4-e51 particles were separated by sds-page and transferred to a nitrocellulose membrane (lanes 5-8). blots were probed with either sera directed against the rv g cd (α-g-cd) or anti-pa monoclonal antibody (α-pa). to analyze surface expression of the fusion proteins, transfected cells were fixed in 3% paraformaldehyde and either permeabilized (internal staining) with 1% triton x-100 or left untreated (surface staining). cells were stained with mouse anti-pa monoclonal antibody followed by fitc-labeled donkey anti-mouse and examined by fluorescence microscopy (a). for facs analysis of transfected cells was stained with mouse anti-pa followed by fitc-labeled donkey anti-mouse, fixed with formaldehyde and analyzed by flow cytometry (b). for western blot analysis, lysates of transfected cells were prepared, separated by sds-page, and transferred to a nitrocellulose membrane. blots were probed with either sera directed against the rv g cd (right panel) or anti-pa monoclonal antibody (left panel). ranging from 50 μg to 5 ng. a mixture of 100 ng recombinant pa and 100 ng of rv g was included as a control. groups of three mice were immunized with the respective antigen on days 0 and 21, bled 14 days post-immunization, and sera were analyzed by rv g-and anthrax pa-specific elisas (table 1) . all mice immunized with killed spbn-d4-e51 in a range of 50 μg to 50 ng showed strong responses to rv g and anthrax pa. moreover, immunization with as little as 5 ng resulted in antigen-specific seroconversion. in response to immunization with rpa and rv g, mice mounted titers similar to the 5 ng dose of spbn-d4-e51. a second inoculation with the same vaccine used for priming significantly increased the pa and rv g responses. in addition, antibodies levels in sera of mice immunized twice with recombinant pa and rv g were comparable to those in sera of mice immunized twice with 50 ng spbn-d4-51. a separate experiment (experiment b in table 1 ) using the same antigens and schedule but with 5-fold increased amounts of recombinant anthrax pa and rv g showed similar results with the exception that the increased amount of rv g protein induced higher antigen-specific seroconversion. however, priming with 500 ng pa still resulted in low pa-specific titers after priming. to determine whether were differences in igg subclasses induced by our immunization approach, sera from the doseresponse immunization regimen used in experiment b above were analyzed by a pa-specific elisa for igg1 and igg2a. as shown in fig. 7 , immunization with the killed particles elicited predominantly igg1, whereas immunization with "protein only" resulted in a high level of igg2a compared to igg1. although th2 cells are known to facilitate antibody production it remains whether such cells underlie the potent seroconversion after priming with inactivated virions. antigen-specific proliferative response after immunization with spbn-d4-e51 to analyze the memory immune responses elicited by vaccination with this novel vaccine carrier, spleens of four 6to 8-week-old swiss webster mice either left unimmunized (fig. 8, open bars) or immunized i.m. with 50 μg of inactivated spbn-d4-e51 (fig. 8 , shaded bars) on days 0 and 21 were harvested 4 weeks post-boost and splenocytes were stimulated with rv g, rv rnp, inactivated virions (spbn) or recombinant pa (see materials and methods). in three out of four immunized mice, the proliferative responses were greatly increased after restimulation with rv-specific antigens, whereas one mouse mounted no detectable response. comparison of the two groups showed that such responses differed 11fold for restimulation with inactivated rv particles (p < 0.07) and 15-fold for rv rnp (p < 0.02). the immunized group also responded to rv g (p < 0.15). note the very substantial proliferative responses (11-fold, p < 0.03) detected after restimulation with recombinant anthrax pa, indicating that such responses can also occurs in the absence of the rv antigens. this report describes the development of a novel approach to create a potential vaccine against anthrax using inactivated rv as a carrier. virus particles present surface antigens in a dense and repetitive array that maximally stimulates b cell proliferation and antibody production (zinkernagel, 2003) . additionally, the virus particle can provide cd4+ t helper epitopes that supply the required second signal for b cell activation (baumgarth, 2000) . to incorporate these properties into vaccine against anthrax, we generated an rv particle that displays the two independent experiments (a and b) were performed. experiment a: groups of three mice were immunized i.m. with 50 μg, 5 μg, 500 ng, 50 ng or 5 ng of inactivated spbn-d4-e51 or with 100 ng rv g + 100 ng rpa at 0 and 21 days. mice were bled 2 weeks after each immunization. sera for each group were pooled placed in triplicate elisa wells and the geometric mean determined. experiment b: mice were immunized and bled as in experiment a except that the protein group was immunized with 500 ng rv g + 500 ng rpa. sera from individual mice were run in elisa and the geometric mean determined. * all three mice yielded titers >109,350. antigenic component of b. anthracis, engineering the rv g such that pa is expressed on the surface of rv particles. while virus particles expressing chimeras of heterologous glycoproteins have been produced, the generation of viruses expressing viral glycoprotein-bacterial toxin fusion proteins is recent as exemplified by the insertion of pa d4 in the influenza virus hemagglutinin (li et al., 2005) . rv g is a type i transmembrane protein and exists in virus particles as a homotrimer (gaudin et al., 1992) . the glycoproteins of rv and vesicular stomatitis virus (vsv), closely related rhaboviruses, share 20% amino acid homology and their ectodomains are predicted to have similar three-dimensional structure (whitt et al., 1991) . studies table 1 were evaluated for anti-anthrax-pa-specific igg1 and igg2a responses by elisa. because no seroconversion was detected for 5 ng dose after priming for spbn d4-e51, these sera were not analyzed (n/a). of vsv demonstrate the requirement for trimer formation in the endoplasmic reticulum for transport of vsv g to the cell surface (doms et al., 1987 (doms et al., , 1988 . consistent with these observations, our initial construct, pa-tc, as well as the d4-δed lacking any of the rv g ectodomain, were not transported to the cell surface. additionally, it has been shown that glycosylation of at least one site on the rv g ectodomain is required for trimer formation and for cellsurface expression of wild-type rv g (shakin-eshleman et al., 1992) . for vsv g, only the 12 membrane proximal amino acids of the ed are required for trimer formation, cell surface expression and subsequent virus incorporation (robison and whitt, 2000) . however, this cannot explain why rv g chimera d4-e51 was successfully trafficked to the cell surface because this region of the rv g ed does not contain potential glycosylation sites. however, trimerization of the d4-e51 molecules or between wild type rv g and d4-e51 might also be required for transport, a possibility that awaits biochemical analyses. while we were succeeded in expressing a chimeric rv g/anthrax pa fusion protein on the cell surface, a concern remains regarding transport of the chimeric glycoproteins depending on the size of the foreign polypeptide. however, our preliminary results using the entire pa63 (428 aa) as well as heavy-chain 50 of botulinum neurotoxin a (436 aa) fused to the e51 construct indicate excellent cell surface expression (koser and schnell, unpublished) . together, our results show that rv g is permissive for the fusion of large foreign protein fragments, that virus particles can incorporate such a chimeric glycoprotein, and that such rv-based vectors are replication-competent. it is well accepted that pa is the major the antigenic component of the current anthrax vaccine (bioport, 2002) and most anthrax vaccines under development focus on pa. the lack of a humoral response to our initial vaccine expressing pa63 by live rv is not entirely surprising since the protein is produced intracellularly and availability to stimulate b cells is therefore probably low. we previously reported that the nucleoprotein of sars-cov was not immunogenic in mice when expressed by a similar rv vaccine vector, but high titers of neutralizing antibodies were detected when the sars-cov spike protein was expressed by rv (faber et al., 2005) . even though the reason for this might lie in the different immunogenicity of the two sars-cov proteins, it appears that proteins expressed on the cell surface and incorporated into rv virions are more potent inducers of humoral responses than are internally expressed proteins. this motion is supported by the finding that a single immunization with rv induces strong humoral responses against rv g but only modest responses against the internal rv proteins . in contrast, pa expressed on the surface of virus particles induced potent humoral responses with either live or killed spbn-d4-e51 particles stimulating antibody production after only a single dose. note that several other approaches to anthrax vaccination require multiple doses for induction of antibody responses (aulinger et al., 2005; galen et al., 2004; rhie et al., 2003) . this is consistent with our expectation that spbn-d4-e51 particles maximally stimulate b cells. rv g in wild type virus comprises 24% of the virus mass (flamand et al., 1993; wunner, 1991) . the chimeric glycoprotein d4-e51 was incorporated, at best, equivalently to rv g. thus, attributing 12% of the virus particle mass to the d4-e51 glycoprotein, a dose of 7.5 μg of spbn-d4-e51 contains an antigen dose equivalent to 500 ng of rpa. we speculate that this 150-fold decrease in spbn-d4-e51 dose requirement is due to presentation by the virus particle, consistent with studies comparing the immunogenicity of proteins presented in a soluble form disordered form versus proteins complexed to viruses or virus-like particles (eckhart et al., 1996; fehr et al., 1997 fehr et al., , 1998 . in addition to maximally stimulating b cells upon prime, the virus particle is expected to be a strong inducer of t cell help, especially as compared to a monomeric recombinant protein. the rv g and nucleocapsid possess cd4+ t helper epitopes (bunschoten et al., 1989; ertl et al., 1991; macfarlan et al., 1984) , and exposure of mice to rv ribonucleoprotein significantly augments virus-neutralizing antibodies upon subsequent introduction of rv (hooper et al., 1994) . for anthrax, immunization with pa at antibody-sub-stimulating levels primes for a more robust and rapid humoral response upon boost as compared to without prime (marcus et al., 2004) . as expected, boosting with a second dose of live or killed spbn-d4-e51 resulted in an increase in anti-pa and anti-rv g titers. high antibody titers also resulted following boost with rpa, but there was a clear difference in the igg subclass. antibodies to pa, either induced by vaccination or passively administered, confer protection against challenge (beedham et al., 2001; ivins et al., 1998) , so that stimulation of a th2 type response is desirable. analogous to the case with ava (semenova et al., 2004) and with rpa formulated in aluminum hydroxide (tan et al., 2003) the subclass distribution of sera from mice immunized with 50 ng or more of spbn-d4-e51 was primarily igg1 indicating a th2-biased response. by contrast, the 5 ng dose of spbn-d4-e51 as well as rpa stimulated th1-associated antibodies consistent with the notion that igg subclass response is dependent upon dose and context of antigen presentation. we have demonstrated that killed rv particles can serve as immunostimulatory carrier molecules for display of foreign b cell antigens. spbn-d4-e51 recombinant particles were potent inducers of humoral immune responses after even a single inoculation, which might be especially important for preexposure treatment, where rapid responses are required. the observed immune responses are th2-dominated, and immunized mice showed potent memory responses. our results establish proof-of-concept in the design of the particle and the ability to stimulate b-cell responses. the quality of the antibody response in terms of in vitro protection and challenge awaits subsequent studies. pgem pa63 (genbank accession number dq190737) contains the sequence encoding the newcastle disease virus (ndv) transmembrane (tm) and cytoplasmic domain (cd) followed by the sequence encoding the translocation-deficient form [k397d, d425k (sellman et al., 2001) ] of the 63-kda fragment of b. anthracis pa (anthrax pa, genbank accession number m22589, (welkos et al., 1988) . the pa63 gene was optimized for human codon usage to ensure high expression levels. all sequences were synthesized by non-template pcr and inserted into the pgem-t vector (promega inc.) at the xbai site. the following pcr reactions were performed using vent polymerase (biolabs inc.) and the primers listed in table 1 (see supplementary material). pa63 was pcr-amplified from pgem pa63 using primers rp187 and rp188. the rv vector pspbn (morimoto et al., 2001) and pcr product were digested with bsiwi and nhei and ligated, resulting in plasmid pspbn-pa63. to construct a plasmid encoding pa63 flanked by the rv g signal sequence (ss), cd and tm, the n-terminal 22 amino acids of the rv g including the rv ss (19 aa) was pcr amplified from ptit-g (mcgettigan et al., 2001b) using primers rp77 and rp125 (pcr1) . in a second pcr reaction, the sequence encoding rv g tm and cd was amplified from ptit-g using the primers rp79 and rp80 (pcr2). pcr1 and pcr2 products were digested with spei and ligated. the ∼300 base pair (bp) product was digested with xbai and psti, and cloned in the xbai-and psti-digested pbs-skii+ (stratagene inc.). the resulting plasmid was designated pbs-sts. the pa63 sequence was pcr-amplified from pgem pa63 using primers rp189 and rp190, digested with spei and hpai, and cloned into spei-and hpai-cleaved pbs-sts. the resulting plasmid was designated ppa63-tc. pa63-tc was digested with bsiwi and xbai and cloned into pspbn, yielding pspbn-pa63-tc. anthrax pa d4 was amplified from pgem pa63 using primers rp293 and rp310, digested with avrii and hpai, and cloned into pbs-sts to yield pd4-δed. to generate the d4-rv g chimeric glycoproteins containing different portions of the rv g ed (see fig. 3 ), a series of rv g ed fragments was amplified from ptit-g using the reverse primer rp299 and forward primers rp295, 296, 297 and 298 and plasmid ptit-g. the pcr products, as well as plasmid pd4-δed, were digested with styi and hpai, and the different ed fragments were ligated into pd4-δed, resulting in pd4-e439, pd4-e210, pd4-e127 and pd4-e51. to construct the recombinant rv expressing d4-e51, pd4-e51 and pspbn were digested with bsiwi and nhei and the d4-51 fragment ligated to pspbn, resulting in pspbn-d4-e51. all recombinant viruses were recovered using published methods . bsr cells were infected at a multiplicity of infection (moi) of 0.1 in dulbecco's modified eagle's medium (dmem, mediatech, inc.) supplemented with 10% fetal bovine serum (fbs) and incubated for 2 h with frequent gentle agitation. cells were washed three times with phosphate-buffered saline (pbs) and optipro medium (invitrogen, inc.) was added. virus was harvested at 3 and 6 days post-infection. supernatants from the two harvests were pooled and clarified by ultracentrifugation using an sw28 rotor at 22,000 rpm for 1 h at 4°c. pelleted material was resuspended in pbs overnight at 4°c. beta-propiolactone (bpl, sigma, inc.) diluted 1:100 in ice-cold dh 2 o was added to the virus solution to a final bpl dilution of 1:2000 and incubation continued overnight at 4°c. inactivated virus was then layered over a 20% sucrose gradient and pelleted by ultracentrifugation as described above. the pelleted virus was resuspended in pbs and stored in aliquots at −80°c. the concentration of a virus standard was determined by bca kit (pierce, inc.). the bsa standard and the virus were each prepared in detergent solution (final concentration 0.002% np-40, 0.0007% deoxycholate, 11 mm edta, 2 mm tris-hcl, 0.0002% protease inhibitor, 0.0002% sds), incubated at 95°c for 5 min and placed on wet ice immediately prior to analysis. subsequent lots of virus were analyzed by sds-page and concentration relative to the virus standard was determined. expression of pa by the viral constructs spbn-pa63 and spbn-pa63-tc was analyzed in bsr cells infected at an moi of 0.01 and incubated for 48 h, while expression of the d4-rv g chimeras was analyzed in bsr cells infected at an moi of 5 for 2 h with vaccinia virus expressing the t7 promoter (vvt7) (fuerst et al., 1986 ) followed by transfection with 5 μg pd4-e439, pd4-e210, pd4-e127, pd4-e51 or pd4-δed using calcium phosphate (stratagene, inc.). after 2 h incubation, transfected cells were washed twice in dmem and incubated overnight in dmem supplemented with 10% fbs. cells were fixed by incubation in 3% paraformaldehyde for 30 min at room temperature followed by two washes in 10 mm glycine in pbs (pbs-glycine). for internal staining, cells were incubated in 1% triton x-100 for 5 min at room temperature followed by two washes in pbs-glycine. cells were immunostained with mouse anti-pa monoclonal antibody m2-v116 (oem, inc.) followed by fitc-labeled donkey anti-mouse igg (jackson immunoresearch, inc.) or fitc-labeled anti-rv nucleoprotein (centocor, inc.). bsr cells were transfected with bluescript skii (stratagene, inc.), pd4-e439, pd4-e210, pd4-e127, pd4-e51, or pd4-δed as described above, washed once with pbs, detached from the plate by treatment with 50 mm edta for 5 min at room temperature, and washed twice in facs buffer (2% fbs in pbs). cells were incubated with a 1:200 dilution of anti-pa antibody m2-v116 in facs buffer for 1 h at room temperature, washed twice in facs buffer, and further incubated with a 1:200 dilution of fitc-labeled donkey anti-mouse igg (jackson immunoresearch, inc.). after two washes in facs buffer, cells were fixed in formaldehyde (caltag gas004 fix and perm) for 15 min at room temperature, washed twice in facs buffer and analyzed by facscan (beckman coulter xl analyzer with a 488-nm argon ion laser). bsr cells transfected overnight with plasmids pd4-e439, pd4-e210, pd4-e127, pd4-e51 or pd4-δed as described above, were washed twice with pbs and incubated with lysis buffer (1% np-40, 0.4% deoxycholate, 66 mm edta, 10 mm tris-hcl, protease inhibitor, 0.1% sds) for 5 min on wet ice. lysates and purified virus were prepared for western analysis by adding protein-loading solution to a final concentration of 2% beta-mercaptoethanol and heating for 5 min at 95°c. proteins were separated by 8% sds polyacrylamide gel electrophoresis (sds-page) and transferred to a polyvinyl difluoride membrane (osmonics, inc.). blots were blocked with 5% low-fat milk powder in pbs followed by incubation with either a 1:2000 dilution of anti-pa m2-v116 or a 1:25,000 dilution of anti-rv g cd (siler et al., 2002) in pbs for 1 h at room temperature, and washed twice in 0.1% tween-20 in pbs (pbs-tween). blots were probed with a 1:5000 dilution of hrp-conjugated donkey anti-mouse igg (jackson immunoresearch, inc.) or donkey anti-rabbit igg (jackson immunoresearch, inc.) and washed twice as described above. chemiluminescence was applied as instructed by the manufacturer (sigma, inc.). to generate rv g, virus was purified as described above. octyl β-d-glucopyranoside (sigma) was added to a final concentration of 1%. the material was centrifuged at 100,000×g at 15°c for 1 h. rv g contained in the supernatant was removed, diluted to 1 mg/ml in pbs and stored at −70°c. viral ribonucleoprotein (rnp) was prepared as described (koser et al., 2004) . all antigens were quantified by bca kit according to manufacturer's protocol (pierce). recombinant pa was obtained from the biodefense and emerging infections research resources repository (beiresources). for comparison of live and killed vaccines, groups of five 6to 8-week-old swiss webster mice were immunized intramuscularly (i.m.) in the gastrocnemius with 50 μg of inactivated spbn-d4-e51, 3 × 10 6 focus-forming units (ffu) of live spbn-d4-e51, 50 μg of inactivated spbn, or 3 × 10 6 ffu of live spbn. mice were boosted as for primary inoculation 3 weeks after first immunization and blood samples were obtained at 2 weeks after each inoculation. for dose-response studies, groups of three 6-to 8-week-old swiss webster mice were immunized i.m. in the gastrocnemius with 50 μg, 5 μg, 500 ng, 50 ng or 5 ng of inactivated spbn-d4-e51 or with 100 ng rv g + 100 ng rpa (list biological laboratories, inc.). mice were boosted 3 weeks later and bled 2 weeks after each inoculation. in a second experiment, mice were immunized as above with the same doses of inactivated spbn-d4-e51 or with 500 ng rv g + 500 ng rpa. for lymphocyte proliferation analysis, groups of four 6-to 8week-old swiss webster mice were either left unimmunized or immunized i.m. in the gastrocnemius with 50 μg of inactivated spbn-d4-e51 on days 0 and 21. at 4 weeks post-boost, mice were sacrificed and spleens harvested. all animal experiments were performed under iacucapproved protocols (animal welfare assurance no. a3085-01). elisa plates (96-well) were coated with 200 ng/well of rpa (beiresources) or 100 ng/well rv g in coating buffer (5 mm na 2 co 3 , ph 9.6) overnight at 4°c. plates were washed four times in pbs-tween and blocked with 5% low-fat milk in pbs for 1 h at room temperature. 100 μl sera diluted in pbs as indicated in the figures were added to wells and incubated for 1 h at room temperature. after washing plates 4 times in pbs-tween 100 μl hrp-conjugated donkey antimouse igg (jackson immunoresearch, inc.) diluted was added per well and incubation continued at 37°c for 30 min. plates were washed four times with pbs-tween before opd substrate prepared according to the manufacturer's instructions (sigma, inc.), was added. incubation was continued for 15 min at room temperature in the dark and the reaction was stopped by adding 2 m h 2 so 4 . optical density at 490 nm was determined. gamma-globulin subclass-specific elisas were performed using one dilution series prepared for each of the three mice and distributed to duplicate plates. assays were performed as above, using the secondary antibodies igg1 (southern biotechnology, inc.) and igg2a (southern biotechnology, inc.) at 1:1500 and 1:1000 dilutions, respectively. single-cell suspensions from mouse spleens harvested 4 weeks post-boost were prepared by homogenization and passage through a 100-μm cell strainer. erythrocytes were lysed and cells were washed and diluted in iscove's modified dmem supplemented with 5% fcs. for each mouse, 2.5 × 10 6 cells/well were seeded in 24-well plates. cells in triplicate wells were stimulated with 0.3 μg rv g, 1 μg rv rnp, 1 μg spbn or 10 μg rpa. one set of wells without antigen served as the medium control. at various time points, 100 μl of cells was removed to a 96-well plate and pulsed with 1 μci of [3h]-thymidine. cells were harvested 24 h later onto glass fiber filter mats using a cell harvester (skatron), placed in scintillation vials with 3 ml scintillation fluid and assessed for radioactivity in a scintillation counter. average from triplicate wells was calculated in counts per minute (cpm) and cpm of the medium control subtracted for each antigen. combining anthrax vaccine and therapy: a dominant-negative inhibitor of anthrax toxin is also a potent and safe immunogen for vaccines a two-phase model of b-cell activation passive transfer of protection against bacillus anthracis infection in a murine model immunogenicity in mice of anthrax recombinant protective antigen in the presence of aluminum adjuvants identification of the cellular receptor for anthrax toxin (see comment rabies virus-specific human t cell 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administration of an adenovirus-based vaccine encoding humanized protective antigen. hum long-term humoral and cellular immunity after vaccination with cell culture rabies vaccines in man current status of immunization against anthrax: old vaccines may be here to stay for a while sequence and analysis of the dna encoding protective antigen of bacillus anthracis the role of antibodies to bacillus anthracis and anthrax toxin components in inhibiting the early stages of infection by anthrax spores membrane fusion activity, oligomerization, and assembly of the rabies virus glycoprotein the chemical composition and molecular structure of rabies viruses on natural and artificial vaccinations recombinant anthrax pa was obtained from the biodefense and emerging infections research resources repository (beiresources). this study was supported by internal funds from thomas jefferson university and nih grant ai062807 to mjs. supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.virol.2006.05.010. key: cord-333525-67bbmo4m authors: yao, qianqian; masters, paul s.; ye, rong title: negatively charged residues in the endodomain are critical for specific assembly of spike protein into murine coronavirus date: 2013-07-01 journal: virology doi: 10.1016/j.virol.2013.04.001 sha: doc_id: 333525 cord_uid: 67bbmo4m coronavirus spike (s) protein assembles into virions via its carboxy-terminus, which is composed of a transmembrane domain and an endodomain. here, the carboxy-terminal charge-rich motif in the endodomain was verified to be critical for the specificity of s assembly into mouse hepatitis virus (mhv). recombinant mhvs exhibited a range of abilities to accommodate the homologous s endodomains from the betacoronaviruses bovine coronavirus and human sars-associated coronavirus, the alphacoronavirus porcine transmissible gastroenteritis virus (tgev), and the gammacoronavirus avian infectious bronchitis virus respectively. interestingly, in tgev endodomain chimeras the reverting mutations resulted in stronger s incorporation into virions, and a net gain of negatively charged residues in the charge-rich motif accounted for the improvement. additionally, mhv s assembly could also be rescued by the acidic carboxy-terminal domain of the nucleocapsid protein. these results indicate an important role for negatively charged endodomain residues in the incorporation of mhv s protein into assembled virions. coronaviruses are a family of enveloped, positive-sense singlestranded rna viruses (spaan et al., 2005) . since the first disease caused by a coronavirus, feline infectious peritonitis, was described one century ago, these viruses have come to be recognized as important pathogens that cause a variety of diseases in respiratory, digestive, and nervous systems of avian and mammalian hosts (siddell, 1995) . before the identification of a new human coronavirus, severe acute respiratory syndrome-associated coronavirus (sars-cov) in 2003, there were only two known human coronaviruses, both associated with the common cold (peiris et al., 2003; lai, et al., 2007) . in the past decade numerous new coronaviruses have been isolated, including three additional human coronaviruses and numerous previously unknown bat coronaviruses. a revised and updated taxonomy has divided the family into four genera-the alpha-, beta-, gamma-, and deltacoronaviruses (adams and carstens, 2012) . the coronavirus spike (s) protein, a glycosylated class i viral fusion protein, is critical for viral infectivity, species and tissue tropism, and pathogenesis (gallagher and buchmeier, 2001) . in many coronaviruses, including the prototype betacoronavirus mouse hepatitis virus (mhv), most s molecules incorporated into virions are cleaved by a cellular furin-like enzyme into two equal-sized subunits, s1 and s2 (de haan et al., 2004) . the receptor binding domain is located in the n-terminal subunit, s1, while components involved in membrane fusion, such as the fusion peptide and heptad repeats, are located in the ectodomain portion of the c-terminal subunit, s2 (cavanagh, 1995; holmes et al., 2001; masters, 2006) . the carboxy terminus of s is composed of a hydrophobic transmembrane (tm) domain and a hydrophilic endodomain (endo), and virus-like particle studies originally mapped to these two domains the ability of s protein to be recruited by the membrane (m) protein for virion assembly (de haan et al., 1999 godeke et al., 2000) . endo is further divided into two regions of roughly equal size: a membraneproximal cysteine-rich motif and a carboxy-terminal charge-rich motif (bos et al., 1995; chang et al., 2000; godeke et al., 2000) . the cysteine-rich segment of endo is the target for multiple modifications by s-palmitoylation. a minimum number of cysteine residues is required for viral viability (yang et al., 2012) , and the cysteine-rich motif appears to be principally required for cell-cell fusion (bos et al., 1995; chang et al., 2000; ye et al., 2004; petit et al., 2007; shulla and gallagher, 2009; mcbride and machamer, 2010a) . the charge-rich motif, on the other hand, has been shown to be the major determinant for s protein incorporation into assembling virions (ye et al., 2004; bosch et al., 2005) . however, some evidence also suggests an effect of the cysteine-rich motif on assembly (thorp et al., 2006) . targeted rna recombination is a reverse genetics system for coronaviruses that has been efficiently used to study the interactions of coronavirus structural proteins (masters and rottier, 2005; masters et al., 2006) , as well as for the expression of foreign genes engineered to replace the nonessential genes 2 and 4 of mhv (das sarma et al., 2002; hurst et al., 2005; yang et al., 2011 yang et al., , 2012 ye et al., 2004) . we previously combined both of these properties to develop a method to dissect the tm and endo domains of s protein and to distinguish between the effects of mutations on the assembly of s into virions versus other functions of s (ye et al., 2004) . in this strategy, one set of recombinants was created in which mutations were introduced directly into the s protein tm or endo and their effects on virus viability and growth properties were evaluated (fig. 1a) . a second set of recombinants was also generated in which wild-type s protein remained unaltered, and the same mutations were moved into the tm and endo domains of a heterologous membrane protein, designated hook (hk), which was expressed in place of the nonessential genes 2a/he. hk is small (38-kda), epitope-tagged synthetic protein composed of a signal peptide, ectodomain, tm, and endodomain from various cellular sources (chesnut et al., 1996; ye et al., 2004) . the effects of the mutations on the incorporation of hk protein into purified virions could then be directly assayed, independent of whether they had an impact on s protein (fig. 1b ). in the current study, we analyzed the effects of the endo charge-rich motif on virion incorporation of mhv s protein through substitutions of the homologous regions from the alphacoronavirus porcine transmissible gastroenteritis virus (tgev), the betacoronaviruses bovine coronavirus (bcov) and sars-cov, or the gammacoronavirus avian infectious bronchitis virus (ibv). the results showed that the ability of recombinant mhvs to accommodate tm and endo domains from other virus species depended on their phylogenetic relatedness to mhv. thus, the bcov and sars-cov substitutions were completely allowed, while the tgev substitution retained only partial functionality, and the ibv substitution was lethal. further analysis of tgev substitutions and revertants obtained there from revealed the importance of negatively charged endo residues for the incorporation of mhv s protein into assembled virions. finally, we were able to replace the mhv endo charge-rich motif with the acidic carboxy-terminal domain of the nucleocapsid (n) protein. there are currently some twenty to thirty species of coronaviruses, which are now classified into four genera (formerly groups), designated the alpha-, beta-, gamma-, and deltacoronaviruses (adams and carstens, 2012; spaan et al., 2005) . although the s2 portions of coronavirus s proteins show some degree of conservation, the tm and endo domains are highly divergent, with the exception of a conserved cluster of seven hydrophobic residues (wpwyvwl) at the start of tm. to evaluate the functionality of different c-terminal sequence motifs in the mhv s protein, we constructed two sets of mutants in which the ectodomain of either s protein or hk protein was fused to the tm and endo domains from tgev (an alphacoronavirus), bcov (a betacoronavirus), sars-cov (a betacoronavirus), or ibv (a gammacoronavirus) ( fig. 2a) . substitution of the c-terminal sequence from bcov s did not significantly hinder the assembly of mhv s into virions. as shown in fig. 2b , the chimeric s recombinant formed slightly smaller plaques than wild-type mhv. correspondingly, the bcovsubstituted hk protein was incorporated into the virions at a lower level than the mhv version of hk protein (2b). more significant assembly was observed with the c-terminal replacement from sars-cov s. the chimeric s recombinant formed plaques similar in size and morphology to the mhv wild type, and much more sars-cov-substituted hk protein became incorporated into purified virions. by contrast, substitution of the c-terminal sequence from ibv s protein was lethal to recombinant mhv; consistent with this finding, ibv-substituted hk protein was not incorporated into purified virions. intermediate between these extremes, an mhv s mutant containing the c-terminus of the tgev s protein was markedly debilitated but viable. this recombinant had a much lower titer and formed smaller, heterogeneous plaques compared to those of wild-type mhv (larger plaques in fig. 2b were later found to be revertants). however, there was no detectable incorporation of the tgev-substituted hk protein into virions. in general, the toleration of mhv s protein for replacement of its tm and endo domains by the homologous regions of other s proteins was highest within the same genus (betacoronaviruses) and marginal for the somewhat more closely related alphavirus genus. a substitution originating from the more distantly related gammacoronavirus genus was unallowed. we previously demonstrated, through the construction of deletion and point mutations, that the charge-rich motif of endo has a dominant role in the incorporation of s protein into virions (ye et al., 2004) . we thus hypothesized that this motif might be the critical element allowing or precluding the substitution of c-termini from other coronavirus s proteins in place of that of mhv. to test this notion, we constructed two groups of recombinant mhvs with chimeric c-terminal sequences (fig. 3a ), in each case as separate s protein and hk protein mutants. in mut-ssm and mut-ttm, the tm and the cysteine-rich motif were from sars-cov s or tgev s, respectively, while the charge-rich motif was from mhv s. in mut-mms and mut-mmt, the tm and the cysteine-rich motif were from mhv s, and the charge-rich motif was from either sars-cov s or tgev s, respectively. for negative controls, we used the truncation mutant mut-30 (mutant δ20 in ye et al., 2004) , which essentially lacks the entire mhv charge-rich motif. mut-30 is minimally viable as an s protein mutant and its hk protein counterpart fails to be incorporated into virions. as we anticipated, recombinants bearing the charge-rich motif of mhv s (mut-ssm and mut-ttm) or the charge-rich motif of sars-cov (mut-mms) produced wild type-like plaques (fig. 3b) and their corresponding hk proteins were incorporated into virions (fig. 3c) , irrespective of the source of their tm and cysteine-rich motifs. conversely, the s protein recombinant containing the tgev charge-rich motif (mut-mmt) produced tiny and irregular plaques (fig. 3b ) that were similar to the mutant with the entire c-terminus of tgev s (fig. 2b) , despite that fact that both the tm and cysteine-rich motif of mut-mmt were derived from mhv s. moreover, the mut-mmt hk protein exhibited no incorporation into virions (fig. 3c) . these results confirmed that the charge-rich motif in endodomain plays a more important role in the specific assembly of mhv s into virions than does the tm domain or the cysteine-rich motif. reverting mutations in tgev chimeras improved s assembly by eliminating positively charged residues in the endodomain mhv s protein mutants containing the entire carboxy terminus or just the charge-rich motif of tgev s (mut-tgev and mut-mmt) produced irregular plaques (figs. 2b, 3b, 4a). most of these plaques were very small and morphologically similar to those produced by the c-terminal truncation mutant, mut-30 (ye et al., 2004) . larger plaques arose following multiple passages of the tgev chimeric mutants, while plaques produced by mut-30 maintained a stable small morphology after passage under the same conditions. a number of larger plaques were randomly picked and subjected to multiple rounds of plaque purification, during which their large-plaque morphology remained stable (fig. 4a) . rt-pcr and sequencing analyses of the relevant segment of the s gene and downstream genes showed that each of the isolated revertants had acquired a deletion in endo (fig. 4b) . moreover, the observed deletions fell into two classes. in one class (revertants tgev-r1 and mmt-r1), the charge-rich motif of tgev s was totally deleted, and a new carboxyterminal sequence of seven residues, tenlnnl, was created through juxtaposition of a normally untranslated open reading frame beginning 24 nucleotides downstream of the s gene. in the second class (revertants tgev-r2 and mmt-r2), the tgev s protein carboxy terminus was retained, but a portion of the charge-rich motif containing two adjacent positively-charged arginine residues was deleted. no other mutations were observed in these revertants in the s tm domain or in the structural proteins e, m, or n (data not shown). significantly, these reverting mutations displayed a tendency to delete positively-charged residues, either two arginines (rr) from the charge-rich motif of tgev s or two lysines (kk) from the cysteine-rich motif of mhv s (fig. 4b ). this suggested that positively-charged residues in those positions were harmful to the assembly of mhv s. additionally, the heptapeptide tenlnnl, created by two of the reverting mutations, introduced one negatively-charged glutamic acid (e) plus four polar residues (t and n) that might be beneficial to the assembly of s. to directly test the role of the heptapeptide tenlnnl in mhv s protein incorporation, three further sets of mutants were generated (fig. 5a ). mut-70 was designed to reconstruct the revertant mmt-r1. mut-69 was a mimic of revertant mmt-r1, but did not contain the heptapeptide. finally, mut-71 maintained just three residues (ten) of the heptapeptide, to check the importance of the negatively-charged e residue. the chimeric s recombinant of mut-70 formed homogenous plaques (fig. 5b ) whose morphology and size relative to the wild type was very similar to that of revertant mtt-r1 (fig. 4a ). in accord with this, the mut-70 hk protein was incorporated into virions almost as well as wild-type hk protein (fig. 5c ). this confirmed that the identified deletion in revertant mtt-r1 was indeed responsible for its observed gain of s protein function. the chimeric s recombinant of mut-71 produced plaques that were smaller than those of mut-70 but were still larger and clearer than those of the mut-69 s-mutant control (fig. 5b) . however, both the mut-71 and mut-69 hk proteins displayed weak incorporation into virions (fig. 5c) . thus, the tripeptide ten is not sufficient to fully recapitulate the advantage conferred by the heptapeptide tenlnnl. curiously, the weak incorporation into virions of mut-69 hk protein was still stronger than the consistently undetectable incorporation of mut-30 hk protein, despite the fact that the mut-30 endodomain was 7 residues longer than that of mut-69. we speculate that, in the absence of multiple negatively charged residues elsewhere in endo, the deletion of two adjacent positively-charged lysines (kk) in mut-69 removed a charge and spatial hindrance, thereby allowing some limited enhancement of s (or hk) protein incorporation into virions. the coronavirus m protein is the central organizer of virion assembly and budding through interactions that it carries out with both the s and the n proteins (masters, 2006) . m-s interactions encompass the carboxy-terminal endodomain of m protein but do not involve the extreme carboxy terminus of m protein (de haan et al., 1999; mcbride and machamer, 2010b) . conversely, m-n interactions are predominantly or solely mediated by the extreme carboxy terminus of m protein and the carboxy-terminal domain 3 of n protein (kuo and masters, 2002; hurst et al., 2005; verma et al., 2007; hurst et al., 2010) . to determine if it was possible to replace the m-interacting region of the s protein with the m-interacting region of the n protein, we created s protein and hk protein mutants in which the charge-rich motif was replaced with mhv n protein domain 3 (mut-mn) (fig. 6a) . we also made recombinants substituting the related domain 3 of the bcov n protein (mut-bn) or else the short endodomain of the mhv hemagglutinin-esterase (he) protein (mut-he). he is a membrane envelope structural protein that is expressed in a subset of betacoronaviruses (lissenberg et al., 2005) ; the m-interacting region of he has not been mapped. the chimeric s recombinant of mut-mn formed plaques that were smaller than those of the wild type but slightly larger than those of mut-bn or mut-he (fig. 6b) . the mut-mn hk protein displayed weaker incorporation into virions than wild-type or mut-70 hk protein, but incorporation of mut-mn hk protein was substantially stronger than that of mut-bn hk protein (fig. 6c ). from these results we conclude that the mhv n protein domain 3 could partially function to replace the s protein endodomain charge-rich motif. however, it is unlikely that this transplanted n domain 3 appropriated the normal m-n interaction, since the bcov domain 3 in mut-bn was unable to play the same role. in the mhv m-n interaction, domain 3 of bcov n protein is entirely functionally interchangeable with its mhv counterpart (hurst et al., 2010) . additionally, our results show that the he protein endodomain could not replace the s protein charge-rich motif. the coronavirus s protein is the largest described class i viral fusion protein, comprising as many as 1450 amino acid residues. its ectodomain, which contains the elements of receptor recognition and membrane fusion, accounts for almost the entirely of the protein. a small tm domain and endo region, at most some 70 amino acid residues, governs membrane anchoring and incorporation of s into virions. at the boundary between these two regions, either proximal to, or within the outer leaflet of the membrane, there is a cluster of 7 aromatic hydrophobic residues (wpwyvwl) that is highly conserved among all coronaviruses (bos et al., 1995; sainz et al., 2005) . in contrast to this conservation, many of the other parts of s protein show considerable divergence, both across genus boundaries and even within a given genus. the functional division between the s protein ectodomain and the tm plus endo was first demonstrated in virus-like particle studies . it was later shown that exchanging the s ectodomain at the wpwyvwl motif could switch the species specificity of coronaviruses, provided that the native transmembrane domain and endodomain were retained. this served as the basis to establish a strong host-range-based selection for targeted rna recombination as a reverse genetics system (kuo et al., 2000; haijema et al., 2003) . we have used this system to dissect the requirements for s incorporation into virions through construction of mutations in the mhv s protein tm plus endo region. since such mutational alteration of s may possibly affect s maturation, trafficking, or membrane fusion, we also constructed the same mutations in an epitope-tagged artificial membrane protein, hk, which was derived from heterologous components (originally in phook1, chesnut et al., 1996) . this strategy previously allowed us to map the assembly competence of s to endo (ye et al., 2004) , a conclusion also reached by others (bosch et al., 2005) . in the present work, we further probed s protein incorporation by engineering interspecies chimeras in which the tm and endo segments of mhv s were replaced with those of closely or distantly related coronaviruses. we observed that these domains taken from the betacoronaviruses bcov and sars-cov could readily substitute for their mhv counterparts (fig. 2) . the functionality of the bcov components in mhv is not surprising, given the generally high sequence homology between many proteins of these two viruses, and the interchangeability of other domains between them hurst et al., 2010) . the ability of the sars-cov s protein tm and endo to operate in mhv further underscores the phylogenetic relatedness of these two viruses. although initial characterizations of sars-cov placed it in a unique grouping, it has since become firmly established that this virus falls in the betacoronaviruses and that mhv and bcov are among its closer relatives (snijder et al., 2003; weiss and navas-martin, 2005) . s protein tm and endo domains derived from other genera of the coronavirus family were much less effective in mhv s. the most far-reaching substitution, that of ibv, was lethal. the tgev substitution, however, was marginally functional. construction of additional chimeras within the tm plus endo region more finely mapped the major determinant of virion incorporation to the charge-rich motif of endo (fig. 3) , consistent with our previous results (ye et al., 2004) . examination of allowed and unallowed substitutions suggested that the number and location of negatively-charged residues in the charge-rich motif was critical for s protein incorporation into virions. additional work provided strong support for this surmise. first, we were able to obtain revertants of tgev chimeras that restored some of the functionality of the chimeric endo segments. analysis of these mutants showed that the quality they had in common was the gain of negatively-charged residues and the loss of positively-charged residues in the charge-rich motif (figs. 4 and 5) . second, replacement of the charge-rich motif of s protein with the acidic carboxy-terminal domain 3 of the mhv n protein at least partially restored endo function (fig. 6) . the evidence suggests that this came about through mimicking the essential attributes of the charge-rich motif and not through the interaction that n domain 3 normally makes with the extreme carboxy terminus of m protein. the bcov n domain 3 was unable to replace the endo charge-rich motif of mhv s, even though bcov n domain 3 is completely active as a replacement for the mhv n domain 3 (hurst et al., 2010) . the region of the mhv m endodomain that interacts with s protein has been previously localized to be upstream of the extreme carboxy terminus of m (de haan et al., 1999) . our data suggests that this s-interacting surface must have one or more key positively-charged residues that contact the negatively-charged charge-rich motif of the mhv s endo. it also appears that endodomain interactions with m protein are not the sole means by which viral membrane proteins can partition into assembling virions. the failure of the mut-he mutant to become incorporated into virions suggests that there exist other types of interactions that lead to the effective inclusion of he protein in the viral envelope. additionally, it is possible that the constraints on s protein endodomain composition reflect its more complex range of interactions with membranes. thus, a charge-rich region of a certain length may be uniquely required by s protein for virion assembly, in order to counterbalance the properties of other elements required for the fusion function of s, such as the wpwyvwl motif and the cysteine-rich motif. the m protein endodomain has thus far been resistant to structural determination. thus, there is a large gap in our understanding of the complexity of its interactions with multiple proteins, including other molecules of m. the identification and characterization of all m protein interactions within the coronavirus virion remains an important future challenge. mouse 17 clone 1 (17cl1) cells were used for propagation of wild-type mhv-a59 and recombinant mhvs. mouse fibroblast l2 cells were used for plaque assays and plaque purification of wildtype and recombinant mhvs. felis catus whole fetus cells (fcwf) were used to grow the interspecies chimeric virus fmhv.v2 in which the ectodomain of the s gene of mhv is replaced with that of fipv (goebel, et al., 2004; kuo, et al., 2000) . the three cell lines were cultured in dmem (gibco, invitrogen) supplemented with 10% fbs and 0.37% sodium bicarbonate in a humidified 5% co 2 atmosphere at 37 1c. for the generation of recombinants in which mutations were introduced directly into the s protein tm and endo region, donor rna transcription vectors were derived from plasmid pmh54 (kuo et al., 2000) . dna fragments with deletion or replacement mutations were created as described previously (yang et al., 2011; ye et al., 2004) using primer-pair pcr and splicing overlap extension pcr. mutated fragments were then transferred to pmh54 by replacement of the unique mlui-sbfi fragment running from the s ectodomain to the intergenic region downstream of s. for the generation of recombinants in which mutations were introduced into the hk protein tm and endo region, donor rna transcription vectors were derived from the previously described vector phkp1 (ye et al., 2004) . the pmh54-derived plasmids were used as the templates for the amplification of pcr fragments of the mutated tm and endo regions, which were then used to replace the unique sali-asci fragment of phkp1 that contains the hk protein tm and endo region. all constructs were identified by restriction digest analysis, and cloning sites and junctions in plasmids were confirmed by sequencing. mhv mutants were produced by targeted rna recombination (masters and rottier, 2005) . briefly, fcwf cells were infected with fmhv and digested with trypsin at 2 h post-infection (hpi) and washed twice with mg ++ -and ca ++ -free pbs. for preparation of donor rna transcription, the constructed pmh54 or phkm plasmids were digested with paci and transcribed into mrna using mmessage mmachine t7 ultra kit (ambion). approximately 10 μl of each recombinant transcript was used for transfection of ∼10 7 infected fcwf cells. rna transfection was performed using a gene pulser xceii electroporation system (bio-rad), with one pulse at settings of 975 μf and 0.3 kv. the co-transfected fcwf cells were loaded onto l2 monolayers grown in 6-well plates. cytopathic effects were monitored and the supernatants were collected at 24, 48, and 72 hpi. recombinant mhvs were purified by two rounds of plaque purification on l2 cells, and all mutations were verified by rt-pcr and sequencing. l2 cells were grown in 60-mm dishes to 70-80% confluence and infected with 1 ml of media containing viruses at dilutions ranging from 10 −3 to 10 −6 . seven ml of 0.95% agar (amresco) in dmem with 5% fbs was overlaid onto cells at 2 hpi. plaques were picked between 48 and 72 hpi. for plaque staining, 3 ml of agar with 0.02% neutral red (sigma-aldrich) was overlaid onto cells. six to eight hours later, the stained plaques were counted or photographed using a white light transilluminator (upland). mhv recombinants expressing wild-type and mutant hk proteins were purified as described previously (ye et al., 2004) . briefly, viral supernatants were precipitated by polyethylene glycol, and resuspended virus was isolated through two cycles of gradient centrifugation with 0 to 50% potassium tartrate contrasting 30 to 0% glycerol in tme buffer (50 mm tris-maleate, ph 6.5 and 1 mm edta). gradients were centrifuged at 111,000 â g in a beckman sw41 rotor at 4 1c for 16-24 h. after centrifugation, viral bands were collected from the gradients and diluted with tme with 100 mm nacl. virions were pelleted through a glycerol cushion by centrifugation for 2 h at 151,000 â g and resuspended in pbs. infected 17cl1 cells were washed twice with ice-cold pbs and collected in ipp buffer (50 mm tris-hcl, ph 8.0, 150 mm nacl, 1.0% nonidet p-40) containing completer protease inhibitor cocktail (roche). cell lysates were incubated on ice for 5 min and clarified by centrifuging at 12,000 â g for 5 min at 4 1c. protein samples from either lysates or purified virions were mixed with an equal volume of 2 â sample buffer and heated at 95 1c for 5 min. one set of cell lysates and two identical sets of purified virions samples were separated by sds-polyacrylamide gel electrophoresis. after electrophoresis, one gel with a set of purified virions was stained with coomassie blue to illustrate n protein. another gel with a set of purified virions and the gel with cell lysates were transferred onto pvdf membranes using a criterion blotter (bio-rad). membranes were blocked for 1 h in 5% non-fat milk and incubated with 0.4 μg/ml mouse mab to ha tag (roche) overnight at 4 1c, followed by incubation with horseradish peroxidase-conjugated secondary antibody (ge, amersham). the signal was developed using ecl system (ge, amersham). ratification vote on taxonomic proposals to the international committee on taxonomy of viruses mutational analysis of the 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endodomains in regulating coronavirus entry the coronaviridae unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage order nidovirales palmitoylations on murine coronavirus spike proteins are essential for virion assembly and infectivity importance of the penultimate positive charge in mouse hepatitis coronavirus a59 membrane protein coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. microbiol replication of murine coronavirus requires multiple cysteines in the endodomain of spike protein partial deletion in the spike endodomain of mouse hepatitis virus decreases the cytopathic effect but maintains foreign protein expression in infected cells genetic analysis of determinants for spike glycoprotein assembly into murine coronavirus virions: distinct roles for charge-rich and cysteine-rich regions of the endodomain this study was supported in part by grants from the national institutes of health (national institute of allergy and infectious diseases), usa (r01 ai064603-p.s.m.) and the national natural science foundation, china (31170786-r.y.). key: cord-326027-58whwspe authors: hernaez, bruno; escribano, jose m.; alonso, covadonga title: visualization of the african swine fever virus infection in living cells by incorporation into the virus particle of green fluorescent protein-p54 membrane protein chimera date: 2006-06-20 journal: virology doi: 10.1016/j.virol.2006.01.021 sha: doc_id: 326027 cord_uid: 58whwspe many stages of african swine fever virus infection have not yet been studied in detail. to track the behavior of african swine fever virus (asfv) in the infected cells in real time, we produced an infectious recombinant asfv (b54gfp-2) that expresses and incorporates into the virus particle a chimera of the p54 envelope protein fused to the enhanced green fluorescent protein (egfp). the incorporation of the fusion protein into the virus particle was confirmed immunologically and it was determined that p54-egfp was fully functional by confirmation that the recombinant virus made normal-sized plaques and presented similar growth curves to the wild-type virus. the tagged virus was visualized as individual fluorescent particles during the first stages of infection and allowed to visualize the infection progression in living cells through the viral life cycle by confocal microscopy. in this work, diverse potential applications of b54gfp-2 to study different aspects of asfv infection are shown. by using this recombinant virus it was possible to determine the trajectory and speed of intracellular virus movement. additionally, we have been able to visualize for first time the asfv factory formation dynamics and the cytophatic effect of the virus in live infected cells. finally, we have analyzed virus progression along the infection cycle and infected cell death as time-lapse animations. many aspects of the infection cycle of african swine fever virus (asfv) remain still poorly understood. some events of the infection such those viral proteins involved in the virus attachment, the intracellular transport to perinuclear areas of the nucleus for virus replication, as well as morphogenesis and transport events of the intracellular virus away from the factories have been biochemically defined. however, a clear understanding of these processes, how and when they occur, has been hampered by the inability to directly observe these events in infected cells. asfv assembly occurs in viral factories that contain high levels of viral structural proteins, viral dna, and amorphous membranous material used to produce viral envelopes. the 170-kb genome of asfv encodes some 150 open reading frames, and as many as 50 viral proteins are incorporated to the viral particle (esteves et al., 1986) . approximately 35% of the mass of the virion is provided by p72, the major capsid protein, while the structural proteins p150, p37, p34 and p14, all of them derived from polyprotein p220, provides another 25% of the virion mass (andres et al., 1997) . asfv particles assemble within cytoplasmic viral factories from endoplasmic reticulum-derived viral membranes (andres et al., 1997 rouiller et al., 1998) at perinuclear sites (nunes et al., 1975) that contain fully assembled virions seen as 200-nm-diameter hexagons in cross-section, and a series of one to six-sided assembly intermediates (rouiller et al., 1998) . morphological evidence indicates that viral membranes become icosahedral particles by the progressive construction of the outer capsid layer, which is composed mainly of viral protein p72 . biochemical data also suggest that protein p72 is assembled in a time-dependent fashion into large membrane-bound complexes that may correspond to capsid-like structures (cobbold and wileman, 1998) . concomitantly, the core shell is formed underneath the viral envelope, and subsequently the viral dna and nucleoproteins are packaged and condensed to form the electrondense nucleoid (andres et al., 1997 (andres et al., , 2002 brookes et al., 1998) . intracellular mature virions made at the assembly sites are infectious (andres et al., 2001) . a fraction of them, however are transported by microtubule-mediated transport (alonso et al., 2001; de matos and carvalho, 1993) to the plasma membrane, where they are released by budding (breese and pan, 1978) to give rise to the infectious extracellular enveloped virions. because of the high viscosity of the cytoplasm, movement of asfv particles by diffusion is likely to be very limited (for review see (luby-phelps, 2000) . some viruses overcome this obstacle by hijacking cytoplasmic motors to utilize the cellular cytoskeleton as a roadway. for instance, herpes simplex virus hsv-1, adenovirus, asfv and rabies virus are thought to use dynein motors to travel along microtubule network for intracellular transport (alonso et al., 2001; raux et al., 2000; sodeik et al., 1997; suomalainen et al., 1999) . asfv interacts with the dynein motor complex through the structural virus protein p54 (alonso et al., 2001) . to further characterize the intracellular trafficking of some of these viruses, several fluorescence-based methods have been developed allowing the detection and characterization of intracellular complexes of viral origin. importantly, several of these labeling methods allow the visualization of individual virions in living cells (smith and enquist, 2002) . these studies suggest that the intracellular trafficking of these viruses is a highly ordered process using cellular motor pathways. similarly, with tagged viruses it would be possible to visualize virus morphogenesis and cytopathic effects produced in the cells as infection progresses. in this report, we describe the construction of an asf recombinant virus, in which we have exchanged the single copy of the p54 open reading frame in the asfv genome, gene e183l (rodriguez et al., 1994) with a chimeric gene encoding p54-egfp. interestingly, this virus is fully viable and exhibits growth kinetics similar to those of its parental virus. moreover, p54-egfp is incorporated into the virus particle with the same efficiency as p54. the presence of p54-egfp in the virion results in fluorescent particles which are readily visualized with a fluorescence microscope, allowing visualization of p54-egfp within live cells. as a consequence of such sensitive detection of p54-egfp throughout infection, we have been able to use time lapse confocal microscopy to monitor the trafficking of p54-egfp within individual cells. we present those results as subsequent images and time lapse animations. thus, we have generated a reagent which will enable the visualization of several aspects of asfv infection in live cells, including virus entry, assembly, trafficking and apoptotic cell death. the main goal of the molecular manipulations described below was to generate a tagged asf virion that allowed us monitoring the infection in living cells. we selected the asfv envelope protein p54, encoded by the essential e183l gene (rodriguez et al., 1994) , to drive the egfp incorporation into the virus envelope. then, we replaced the protein p54 in the virus particle by p54-egfp fusion chimera in two steps. first, we introduced in the tk gene of the virus the sequence encoding the fusion protein p54-egfp under the control of p54 promoter (fig. 1a) . recombinant virus plaques exhibiting green fluorescence were picked and individual recombinant viruses were further plaque purified by limiting dilution and screened by pcr and southern blot to confirm the presence of the p54-egfp coding sequence in the tk locus, as expected (fig. 1b) . the recombinant virus obtained was designated as b54gfp-1 and used to infect cells to visualize the fluorescence properties of individual virus clones (figs. 1c and d). moreover, the use of egfp as marker gene allowed a faster detection of recombinant viral plaques when compared to wild-type virus (data not shown). egfp fusion to a specific protein often modifies its inherent properties, so we first determined whether the addition of egfp to the c-terminus of p54 could affect the usual subcellular distribution of this protein. then, vero cells were infected with b54gfp-1 or wild-type virus (ba71v) and subcellular distributions of p54-egfp and p54 were examined by conventional immunofluorescence microscopy (figs. 1e-j). no differences were observed when compared, demonstrating that typical perinuclear accumulation described for p54 in asfv infection was not altered by the gfp fusion. p54 is an essential protein and then to study p54-egfp functionality in the virus morphogenesis and to increase the number of copies of the tagged protein in the virions, the wildtype e183l gene was interrupted by insertion of the βgalactosidase coding sequence. this second step in recombinant virus generation was performed by homologous recombination with vector pδp54 which contains the β-galactosidase coding sequence under the control of p72 promoter and flanqued by 424 pb and 888 pb from p54 e183l right and left extremes, respectively. resulting recombinant virus plaques exhibiting β-gal activity were picked and analyzed by pcr as well as southern blot to confirm the interruption of gene e183l in the genome of asfv (fig. 1b) . after isolation by several rounds of plaque purification, all virus plaques exhibited both gfp and β-galactosidase activities. the double recombinant virus finally obtained was designated b54gfp-2 and when used to infect cells, no trace of the wild-type p54 protein could be detected by western blot in cell extracts obtained at different times of infection using a specific anti-p54 serum (fig. 3a) . (a) schematic representation of the ecorik and ecorie fragments in the genome of the wild type asfv (ba71v) and the recombinants viruses generated (b54gfp-1 and b54gfp-2) after insertion of chimera p54-egfp in tk locus by homologous recombination and disruption of the p54 gene by insertion of the β-gal marker gene, as described in methods. e: ecori site. (b) genomic analysis, after ecori digestion, of parental and recombinant asf viruses by southern blot using a digoxigenin-11-dutp labeled dna probe corresponding to the p54 complete coding sequence. size of the fragments probed was as expected after genome manipulations. (c and d) after insertion of p54-egfp recombinants viral plaques formed in vero cells monolayers exhibited fluorescence and allowed and easy detection after 5 dpi. (c) a single recombinant fluorescent plaque was photographed in a fluorescence microscope (original magnification ×100) and correspondent transmitted light image is shown below (d). fluorescence microscopy analysis of b54gfp-1 (e-g) or ba71v infected cells at 14 hpi (h-j). p54-egfp was performed by direct fluorescence (e), while p54 was detected with an anti-p54 serum and secondary antibody conjugated to alexa 488 (h). in both cases viral and cellular dna were counterstained with hoechst to visualize viral factories, indicated by arrows (f and i). as it can be observed, in the merged images (g and j), p54-egfp and p54 showed similar perinuclear distribution coincident with viral factories. scale bar, 18 μm. to determine that protein p54, a major component of the external envelope of asfv, fused to egfp protein remains incorporated to the viral particle, ba71v and b54gfp-2 virions were percoll purified and analyzed by sds-page and western blotting using specific antibodies (fig. 2a) . no reactivity of anti-gfp antibody with ba71v purified virus was found, while a 51-kda band corresponding to the expected size of p54-egfp protein was detected in b54gfp-2 purified virus. however, when an anti-p54 serum was probed, a 25-kda band appeared with ba71v virus and with anti-gfp serum, a 51-kda band was only reactive with the recombinant virus, demonstrating the incorporation of p54-egfp fusion protein into the virus particles. the typical doublet detected with anti-p54 serum in ba71v samples was not present in those from b54gfp-2 when detection of p54-egfp was carried out with monoclonal antibody anti-gfp. it is noteworthy that the levels of p54-egfp and p54 incorporated into their respective particles were apparently equivalent, since the amount of total virus protein loaded in sds-page was similar in both cases, as judged by coomasie blue staining. in order to further confirm this, we analyzed by western blotting the presence of major structural asfv protein p72 in these highly purified virions (fig. 2a) . the proportion of p54-egfp relative to p72 in b54gfp-2 virions was roughly the same as p54/p72 rate inba71v virions, indicating that the fusion protein was incorporated to virus particle at similar rates than p54. to ascertain the growth properties of the b54gfp-2 virus, a time course of infection was carried out for both, wild-type and recombinant viruses. monolayers of vero cells were infected with ba71v or b54gfp-2 at a multiplicity of infection (moi) of 5 pfu/cell and harvested at different postinfection times. total cell lysates were analyzed by sds-page and western blotting, and the kinetics of synthesis of virus proteins were assessed (fig. 3a ). western blotting with an anti-p54 serum allowed the identification of a 51-kda band, the expected size for p54-egfp, only in b54gfp-2 infected cell extracts from 8 to 24 hpi. at the same times postinfection, p54 was detected as a doublet in ba71v infected cell extracts while a single 51 kda band was detected with a monoclonal antibody against gfp, only in recombinant virus infected cell extracts at these time points. p54-egfp apparently remained intact throughout the course of the infection and sample preparation, since no doublet was observed in wb. as expected, fusion protein was synthesized late after infection (detectable from 8 hpi by wb), in contrast to the early protein p30 used as a control, which was detected from 4 hpi. to demonstrate that p54-egfp is synthesized during the late viral expression phase under the control of p54 promoter, we examined the synthesis of fusion protein in the presence and absence of a dna replication inhibitor, such as cytosine βarabinofuranoside (ara-c). in the presence of ara-c, we could detect neither p54-egfp nor p54 with anti-p54 serum, while early asfv protein p30 expression was confirmed by wb with a monoclonal antibody against p30 in the presence and absence of inhibitor (fig. 3b) . these results demonstrated that virus gene expression was similar in b54gfp-2 and wild-type virus infected cells, but it was conceivable, since p54-egfp is a structural component of the virus particle, that assembly and/or release from the cell of the double recombinant virus could be in some way restricted. to assess the rate of virus assembly and egress throughout several infection cell cycles, growth curves were carried out for both b54gfp-2 and the ba71v virus. vero cells were infected at a multiplicity of 0.1 pfu/cell and harvested every 10 h for 72 h, and both extracellular and intracellular virus yields were calculated for both viruses. the results showed that the growth curves for extracellular and intracellular virus were similar for wild-type and recombinant virus infections, not only in total infectious virus production after 72 hpi, but at every time point we examined (fig. 3c) . these results imply that the rates of both virus assembly and virus egress from the cell were the same for the two viruses. cell monolayers were infected with ba71v or b54gfp-2 at 5 pfu/, in presence (+) or absence (−) of inhibitor cytosine βarabinofuranoside (ara-c) to discriminate late from early viral protein synthesis. cells were harvested at 14 hpi and analyzed by western blot with a serum anti-p54 that recognized p54-egfp and p54 only in absence of ara-c. early asfv protein p30 was detected in presence and absence of inhibitor using monoclonal antibody anti-p30. (c) analysis of the replicative phenotype of b54gfp-2. cells were infected with 0.1 pfu/cell of ba71v or b54gfp. every 12 hpi (for a maximum of 72) extracellular and intracellular infectious virus progeny was estimated by plaque assays. growth curve obtained for recombinant virus resulted similar to the parental virus ba71v. the incorporation of p54-egfp into virions of the asfv b54gfp-2 makes possible that these particles can be detectable by fluorescence. to test if this was the case, virions purified by sedimentation through sucrose cushion were used to infect vero cells grown onto a coverslip at a multiplicity of 10 pfu/cell. after synchronizing the infection at 4°c for 2 h, cells were incubated for 30 min at 37°c and subsequently examined by fluorescence microscopy. fluorescent particles, visualized as point sources of gfp fluorescence, were readily detected inside cell cytoplasm (fig. 2b) . when target cells were washed of free virus and allowed to incubate further at 37°c, a significant proportion of the gfp signal accumulated in the perinuclear region, in a short time after infection 4 h (additional file 1). images were acquired at 30 min and 4 h postinfection, before late viral protein expression phase onset to avoid that observed gfp signal corresponds to newly synthesized p54-egfp. some obvious applications of a virus expressing a fluorescent structural protein would be to localize that protein within the cell and follow its trafficking during the infection cycle and ultimately, to visualize the pathway of virus assembly in live cells. as previously shown, wild-type p54 and p54-egfp distribution at late times in wild type and b54gfp-1 infection was shown in perinuclear viral factories (figs. 1e-j). we examined this distribution for b54gfp-2 infected cells at 12-16 hpi by confocal fluorescence microscopy in fixed cells . protein p54-egfp distribution coincided with assembly sites in the viral factories, which is characterized by a great accumulation of newly synthesized viral dna. to further confirm that p54-egfp distribution could be used as a novel viral factory marker in asfv infection we showed that the golgi complex in living b54gfp-2 infected cells was located surrounding, but not coincident with the viral factory where a great accumulation of p54-egfp is found (figs. 4e-g). the ultimate elegance of a gfp-incorporating virus is the potential for monitoring virus infection in individual living cells. the abundant incorporation of p54-egfp into asfv particles indicated that it should be possible to visualize the intracellular movement of virions in real time by laser-scanning confocal microscopy of live cells. approximately 50% confluent vero cells were infected with more than 10 pfu of b54gfp-2 virus per cell. virus infection was synchronized by incubation of cells at 4°c for 90 min and images were collected, after incubation at 37°c for nearly 2 h, to analyze the first steps of infection at high magnification. individual virus particles were readily observed and images were acquired with the confocal microscope setup at a rate of 1 frame/3s for a maximum of 2 min. with this short interval of time, we discerned that intracellular movement in the proximity of perinuclear areas was composed of frequent stops and starts, and often without any apparent direction. fig. 5 shows an example of this intermittent or saltatory movement of an individual virion. over the times and distances measured in following frames acquired from 10 different virions, an average speed ranged from 0.2 to 0.5 μm/s at this stage of infection was calculated. in order to test the role of microtubules at this stage of infection, disruption of microtubules with 10 μm nocodazole was achieved prior to infection with b54gfp-2. under these conditions we could not track any virion movement examined at 1 hpi or 4 hpi in live cells. interestingly, at mentioned times postinfection most gfp signal was localized far from perinuclear areas in contrast to infected cells with intact microtubules (additional file 2). to determine the stage of infection at which newly synthesized p54-egfp could initially be detected by direct fluorescence microscopy, and to visualize virus factory constitution, vero cells were infected with b54gfp-2 at a multiplicity of infection of 1 pfu/cell. after synchronization of the infection, cells were examined for egfp fluorescence every fig. 6 . protein p54-egfp was first observed in the majority of cells as early as 7-8 hpi. the protein was mainly localized as small, cytoplasmic and fluorescent aggregates around the nucleus (2 or 3 spots were usually observed within single cell). from this point, all of these perinuclear spots finally aggregated along the next 2 h to give rise to a unique brilliant spot close to the nucleus (fig. 6b ). this accumulation of p54-egfp, which was previously shown coincident to the viral factory, gradually increased in size and fluorescence intensity as infection proceeded. it should be mentioned that aggregation of initial perinuclear spots of p54-egfp could not be observed in every infected cell, but in most of them, probably due to changes in the optical section acquired during the monitoring of the infection. at late times postinfection, in most of the cells, it was usually to observe violent changes in viral factories location (fig. 6a) , however these changes seem to correspond to movements of infected cell prior to detachment from the dish, while factory location is conserved. in another set of experiments, we investigated the relationship, of mitochondria status and viral factory formation in a time course manner. at initial infection steps mitochondria were observed as an almost continuous succession of organelles extending throughout the cytoplasm. nevertheless, time lapse experiments with image acquisitions every 5 min in vero cells infected at a moi of 1 pfu/cell, showed the progressive alteration of the normal mitochondrial pattern and mitochondrial irregular clumping and aggregation around viral factory was observed (fig. 8) . this aggregation started at 8 h after infection, coincident with p54-egfp synthesis, suggesting the high energy requirements of the virus assembly process. apoptosis has been described to play an important role in asfv pathogenesis (ramiro-ibanez et al., 1996) , so we decided to analyze the very late stages of asfv infection in living cells infected with b54gfp-2. fig. 7 shows in detail the characteristic morphological changes that occur during programmed cell death of an infected cell. since 12 hpi an early cytopathic effect, consisting in cytoplasmic vacuolization, rounding and reduction in the size of the infected cell was be observed. from this time point onwards, the cell lost contacts with neighboring cells and detached from the monolayer, undergoing nuclear fragmentation and final cell death. this process included typical membrane blebbing of the infected cell that led to the formation of numerous vesicles containing large amounts of p54-egfp. vesicle formation has been considered a very characteristic feature of asfv infection in cultured cells and these are viruscontaining vesicles. completion of the process from the first morphological features of apoptosis lasted around 6 h. successful african swine fever virus life cycle is dependent on several and critical interactions with host cell which remain poorly understood. some of these events include virus internalization into cellular cytoplasm, transport to perinuclear areas where viral dna replication occurs, egress of viral progeny from assembly sites to cell periphery and virus induced cell death. recent improvements in optical imaging techniques combined with protein fusion to gfp variants, open new fig. 5 . analysis of the intracellular movement of b54gfp-2 viral particles. vero cells were b54gfp-2 infected with 20 pfu/cell and examined by confocal microscopy at 1 hpi, acquiring images every 3 s corresponding to 0.5 μm optical sections from the z axis. virus particles were detected as egfp point sources, and correspondent transmitted light image provided points of reference information. movement of 10 virions were individually analyzed. upper image shows an example of the trajectory followed by one of the virions analyzed (white discontinuous line) and selected area is enlarged below. total distance covered by this virion was 11.74 μm in 30 s, with an average speed of 0.5 μm/s. as can be observed in the figure, virion velocity was not constant, with a maximum speed from 12 to 15 s. scale bar, 8μm as indicated. possibilities for the direct analysis of these dynamic processes. this new methodology has been successfully applied to study different aspects of the infection of adenoviruses (lux et al., 2005) , herpesviruses (glotzer et al., 2001; sampaio et al., 2005) , poxviruses (geada et al., 2001; hollinshead et al., 2001; ward and moss, 2001) , rhabdoviruses (finke et al., 2004) or retroviruses (mcdonald et al., 2002; muller et al., 2004) . we reported here the generation of a fluorescently tagged and viable asfv expressing the structural protein p54 as an egfp fusion protein. expression of p54-egfp enables easy identification of infected cells by direct fluorescence analysis, which results useful when immunodetection of diverse antigens in the same sample is desirable. selection of p54 as the viral fusion protein was supported by several reasons. since p54 is located in the inner viral envelope (rodriguez et al., 1994) , fluorescent p54 would allow the detection of individual viral particles upon infection. as the carboxi-terminal tail of p54 is facing the surface of mature virions, it was expected to be a suitable target for egfp fusion. on the other hand, newly synthesized p54 accumulates within perinuclear areas where morphogenesis takes place at late times postinfection. thus, p54-egfp could become a very helpful tool to analyze viral factories assembly. the strategy to generate tagged virus b54gfp-2, consisted in two sequential steps. after initial insertion of p54-egfp coding sequence under the control of p54 promoter, it was found that fusion of egfp to the c-terminus of p54 did not affect the perinuclear subcellular distribution of the viral protein, as it was directly observed in b54gfp-1 infected cells. then, we interrupted e183l gene in b54gfp-1 genome by insertion of β-galactosidase coding sequence as second selection marker, to ensure that every p54 molecule that accommodates in virion inner envelope would be egfp tagged. previously, p54 has been shown to be essential for virus viability, playing and important role in asfv morphogenesis (rodriguez et al., 1996 (rodriguez et al., , 2004 , so the recovery of recombinant virus b54gfp-2 after wild type p54 interruption indicated that fusion did not apparently affect the normal function of p54. furthermore, growth curves obtained with recombinant virus indicated identical replication behavior and infectious efficiency when compared to the parental strain. furthermore, fusion to egfp did not alter the expression pattern determined by wb analysis for p54 in infection as p54egfp expression remained under control of p54 natural promoter in b54gfp-2. p54-egfp was synthesized and detected during late viral expression phase, from 8 hpi, as expected. this observation was confirmed by wb analysis in the presence of inhibitor ara-c, which demonstrated that there was not chimera protein expression when viral dna replication was inhibited. also, expression levels of p54-egfp in infection, determined by wb, did not apparently differ from those observed for p54. in order to track single fluorescent asfv particles, it is an essential requirement that p54-egfp is incorporated into viral particles. previously, p54 has been successfully used as a target to generate asfv chimeras incorporating short foreign viral epitopes such as the antigenic site a from foot-and-mouth disease virus vp1 protein and the da3 antigenic determinant from transmissible gastroenteritis coronavirus nucleoprotein n (brun et al., 1999) . in both cases, chimeric p54 proteins were successfully incorporated into the viral particles, supporting selection of p54 as the fusion protein. as expected, western blot with percoll purified b54gfp-2 particles demonstrated the association of p54-egfp with asfv virions. as deduced from detection of control protein p72 and p54 in percoll purified samples, inclusion of p54-egfp into virions appeared to occur with same effectiveness than p54. such tagged virus will enable live analysis of various stages in the asfv replicative cycle using confocal microscopy. one of these potential applications of b54gfp-2, shown in present work, would be the analysis of intracellular movements of fluorescent particles during the initial phase of infection immediately after internalization into the host cell. when collecting confocal microscopic images every 3 s, from 2 hpi, it was found that most of the viral particles exhibited a special kind of movement defined as saltatory, intermittent and apparently disorganized, making difficult the estimation of fig. 7 . live-cell analysis of the last stages of b54gfp-2 infection. series of egfp and transmitted light simultaneous acquisitions of the same b54gfp-2 infected vero cell by confocal microscopy from 10 hpi (when viral factory is already constituted) and every 5 min (see additional file 3 to complete animation sequence). rounding of the infected cell is followed by membrane blebbing and subsequent formation of p54-egfp (green) containing vesicles. at 18 hpi, detachment and final cell destruction is observed. scale bar, 20 μm. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) speed rates. this type of movement has been described as characteristic of microtubule associated movement. from the analysis of movement of 10 individual virions an average speed ranging from 0.2 to 0.5 μm/s was calculated with the applied resolution. these data are in agreement with those obtained for other large enveloped viruses. using fluorescence videomicroscopy, which allows a resolution of 1 frame/s, speed rates from 0.3 to 1 μm/s have been determined during the late phase of infection with labeled vaccinia virus (geada et al., 2001; ward, 2005; ward and moss, 2001) . recently, human cytomegalovirus has been shown to move with apparent velocity of 0.7 to 0.8 μm/ s (sampaio et al., 2005) at very early stages in infection. moreover, hiv virions transport towards host cell nucleus linked to microtubules occurs at 1 μm/s (mcdonald et al., 2002) . the saltatory movements of tagged asfv virions and the speed rates obtained are consistent with a microtubular based transport (king, 2000) and association of this virus with microtubules has been described (de matos and carvalho, 1993) . previously, we identified the lc8 subunit of cytoplasmic dynein, a minus end directed microtubule-associated motor protein, as an interacting protein with p54 (alonso et al., 2001) , resulting this interaction critical in the first stages of asfv infection. collectively, these data suggested a role for microtubules in the intracellular transport of asfv after internalization. this role is supported by data showing that nocodazole disruption of microtubules prior to infection results in stop of virion movement and lack of perinuclear accumulation of viral particles at 4 hpi that remained dispersed in the cytoplasm. also, there was no subsequent constitution of the characteristic virus factory and finally a reduction in asfv progeny was found (additional files 1 and 2 and alonso et al., 2001) . also, during the late phase of asfv infection, transport of newly assembled virions from viral factory to cell periphery along microtubules has been recently demonstrated (stefanovic et al., 2005) , similar to the exit transport described for vaccinia virus (carter et al., 2003) . further live studies involving labeled b54gfp-2 that are now in course will provide useful and detailed information to clarify the whole asfv penetration and egress process into the host cell. p54 has been previously described to localize in membrane structures within the virus factories and in immature asf virions, as judged by immunogold electron microscopy (rodriguez et al., 1994) . p54-egfp, just as p54, showed an intracellular distribution fully coincident with asfv factory at late postinfection times. this allowed us, to follow the viral factory formation process in live cells throughout the infection cycle for first time. animations obtained show that p54-egfp was first detected in cytoplasm as several discrete and bright spots representing initial viral assembly sites. as infection progresses these initial assembly sites migrated to a perinuclear area, fused and finally formed a single large accumulation of p54-egfp per cell. similar assembly sites were previously described as viral antigens inclusions containing viral precursors which remained tightly associated to microtubules (carvalho et al., 1988) . indeed, microtubule associated motors are required to transport these initial assembly sites towards the microtubule organization center (mtoc), where final asfv factory is constituted (alonso et al., 2001; heath et al., 2001) following viral replication and viral assembly takes place. first steps in asfv assembly consist in the modification of er membranes rouiller et al., 1998) . protein p54 has been involved in the recruitment to the viral factories and transformation of the er-derived envelope precursors that are finally acquired by virions (rodriguez et al., 2004) . this new role for p54 in morphogenesis supports the selection of p54 as viral fusion protein and suggests that studies about p54-egfp trafficking during infection in live cells would be helpful to analyze the acquisition of asfv envelopes from er during virus assembly. the formation of the viral factory often involves not only host cytoplasmic membranes and cytoskeletal components, but also cell organelles such as mitochondria. these are thought to play an important role in several large dna enveloped virus infections (murata et al., 2000; novoa et al., 2005; rojo et al., 1998) , supplying the energy required in the morphogenetic and assembly processes. our results, obtained with live asfv infected cells have confirmed these previous observations in fixed cells (hernaez et al., 2004a; rojo et al., 1998) , which showed that asfv infection induce the migration of mitochondria to the perinuclear assembly sites, producing the clustering of this actively respiring organelle around viral factory. this migration occurs since 8 hpi, before the definitive viral factory is fully constituted, suggesting that energy contribution from mitochondria is required since first stages of asfv morphogenesis. finally, we analyzed the destruction of the asfv infected cell. this process started at 12 hpi and usually lasted about 6 h, being identical to apoptotic cell death, which has been demonstrated to play a relevant role in asfv pathogenesis (hernaez et al., 2004a (hernaez et al., , 2004b ramiro-ibanez et al., 1996) . in live cells, it was possible to observe host cell membrane blebbing, resulting in the formation of p54-egfp containing vesicles. this finding raised the possibility that these vesicles could contain virus particles. previous publications reported that the abundant vesicle fraction released in last asfv infection stages contains virus (carrascosa et al., 1985) . then, it is conceivable that the fluorescence detected in the vesicles formed at the end of apoptosis (blebbing), could correspond to gfp labeled virus particles. this vesicle formation observed in live cells is rarely observed using common fixation methods, then it is not possible to observe it with the parental unlabeled virus. in summary, we have generated a fluorescently tagged asfv, which could be a powerful tool since it enables live analyses of diverse aspects of asfv live cycle, including viral particles intracellular transport, morphogenesis and apoptosis of the infected cell. we presented here, for the first time, live images of some of these stages in asfv infection and more detailed information will arise from future and novel studies of virus entry and egress. vero cells were obtained from ecacc and grown at 37°c in a 5% co 2 atmosphere in dulbecco's modified eagle's medium (dmem) supplemented with 5% foetal bovine serum (fbs). asfv isolate ba71v, adapted to grow in vero cells, was used in infection experiments carried out at 37°c and 5% co 2 . when synchronization of infection was required, this was performed for 90 min at 4°c after viral inoculum was added. in most cases, asfv stocks from culture supernatants were semipurified from vesicles by ultracentrifugation at 40.000 × g through a 20% (w/v) sucrose cushion in pbs for 1 h at 4°c. when needed, asfv particles were highly purified from the extracellular medium by percoll equilibrium centrifugation as previously described (carrascosa et al., 1985) . after centrifugation, percoll gradients were fractionated and fractions 2 to 8 from percoll gradients, containing virus particle essentially free of vesicles and contaminant membranes, were collected after sedimentation and gel filtered through sephacryl s-1000 (amersham) to remove percoll. the construction of the insertion vector pins-54egfp involved several steps. the expression plasmid pegfp-n1 (clontech) was used as template to obtain the egfp coding sequence by pcr with specific primers (5′-cgcggatccgc-agtaaaaaaatg-3′ and 5′-cgcggaattcatggtgag-caagggc-3′). a fragment of 800 pb corresponding to p54 promoter and complete p54 coding sequence was also obtained from ba71v genome by pcr with specific primers (5′-gcgggatcccgttgtctaggtaa-3′and 5′-gcggaatt-ccaaggagttttct agg-3′). in order to generate the vector pins-54gfp, previous pcr products were digested with ecori and bamhi and cloned into the 4.7 kpb fragment resulting from the digestion of plasmid pins72gal (gomez-puertas et al., 1995; rodriguez et al., 1992) with bamhi. the construction of the vector pδp54, used to perform the interruption of e183l gene, was described previously (rodriguez et al., 1996) . briefly, this vector contains β-galactosidase coding sequence under control of p72 promoter, flanked by two fragments from e183l that allow homologous recombination with this orf in ba71v genome. these flanking sequences correspond to 424 pb and 888 pb from right and left extremes from p54 gene, respectively. an african swine fever virus recombinant expressing an enhanced version of the gfp gene (egfp) fused to the cterminus of the p54 coding sequence was constructed in two steps: first, the insertion of the p54-egfp coding sequence into thymidine kinase locus (tk) in ba71v genome was achieved by homologous recombination after transfection of vero cells with pins-p54egfp in the presence of fugene (roche), according to manufacturer's instructions. eight to 16 h after transfection, cells were infected with ba71v at a moi of 0.1 pfu/ cell. when the cytophatic effect was complete (approximately 72 hpi) the cultures were harvested and sonicated. these transfection-infection mixtures were used to infected monolayers of vero cells seeded onto 100 mm dishes (nunc) at different dilutions. after 5-6 dpi, the recombinant virus generated was isolated from progeny virus by three rounds of plaque purification on vero cells, during which recombinant virus plaques were screened for egfp direct fluorescence. the resulting recombinant virus was named b54gfp-1. additional second step consisted in the inactivation of the original gene of p54 (e183l) in the b54gfp-1 genome to finally generate the recombinant virus b54gfp-2. the transfection of vero cells with pδp54 and following infection with b54gfp-1 allowed the insertion of the β-galactosidase coding sequence downstream of the strong p73 promoter within e183l gene. isolation of b54gfp-2 was performed as described above, screening recombinant virus plaques for egfp fluorescence and expression of β-galactosidase (gomez-puertas et al., 1995) . the genomic structure of these recombinant viruses was confirmed by dna hybridization analysis and could be observed in figs. 1a and b . briefly, dna from parental and recombinant viruses were obtained and purified as previously described (esposito et al., 1981) . resulting fragments, after eco ri digestion, were resolved by agarose gel electrophoresis and then blotted onto uncharged nylon membranes (amersham). membranes were probed with a digoxigenin-11-dutp labeled dna fragment corresponding to the complete p54 coding sequence. final detection of digoxigenin was performed with cspd (roche) after probing the membrane with specific monoclonal antibody anti-digoxigenin (roche). preconfluent monolayers of vero cells were infected with either recombinant b54gfp-2 or parental ba71v at 0.1 pfu/ cell. after 90 min, inoculum was removed and cells were washed with fresh dmem and overlaid with dmem supplemented with 2% fbs. infected cells with their culture supernatants were harvested at different times postinfection (0, 12, 24, 48 and 72 hpi) and centrifuged at 3000 × g for 10 min. cell pellets was resuspended in dmem. both the cellular fraction and the supernatant were sonicated and separately titrated by plaque assay to determine the intracellular and extracellular virus production respectively. plaques assays were performed as described previously (gomez-puertas et al., 1995) . when isolation of recombinant virus plaques was desired, detection of egfp fluorescence and expression of β-galactosidase were performed at 4 and 6 days postinfection respectively. vero cells preconfluent monolayers seeded on 6-well plates were infected with ba71v or b54gfp-2 at a moi of 5 pfu/cell. when inhibition of late viral protein synthesis was required, cells were incubated with cytosine β-arabinofuranoside (ara-c) 50 μg/ml at 3 hpi. in all cases, cells were washed with pbs and harvested at 0, 4, 8, 12 and 24 hpi and lysed in ripa buffer. after centrifugation at 10,000 × g for 20 min, proteins from cleared lysates were electrophoresed in 12% sds-polyacrylamide gels and transferred to nitrocelluose membranes (biorad). membranes were incubated for 2 h at room temperature in pbs containing 5% non-fat dry milk and then probed with corresponding primary antibodies: monoclonal antibody anti p30 diluted 1:500, monoclonal antibody anti-gfp diluted 1:2500 (clontech), and specific serum against p54 raised in rabbit diluted 1:1000, for 1 h at room temperature in pbs containing 0.05% tween 20 (pbs-t). after extensive washing with pbs-t, membranes were incubated with anti mouse igg antibody (diluted 1:5000) conjugated to horseradish peroxidase (amersham) or anti rabbit igg (diluted 1:4000) conjugated to horseradish peroxidase (amersham). in other experiments, proteins from asfv highly purified particles were analyzed by western blotting in the same way, but membranes were probed with monoclonal anti-gfp antibody, monoclonal anti-p72 antibody and polyclonal specific antibody anti-p54 raised in rabbit (diluted 1:1000). anti mouse igg antibody (diluted 1:5000) and anti rabbit igg antibody (diluted 1:4000) conjugated to horseradish peroxidase (amersham) were used as secondary antibodies respectively. in all cases, final detection on membranes was performed with ecl western detection reagent (amersham) and exposure to x-ray films (kodak). vero cells were grown on coverslips at 50-60% confluency and then infected with ba71v or recombinant viruses at different moi depending on the experiment. at desired time postinfection cells were rinsed twice with pbs and fixed with pbs containing 4% paraformaldehyde for 10 min at room temperature. when permeabilization was required, cells were incubated in presence of 0.1% triton x-100 for 15 min at room temperature. a monospecific antiserum against p54 was raised in rabbit and used to visualize p54 (diluted 1:300). to detect tubulin a monoclonal antibody against β-tubulin (sigma) was used (diluted 1:200). secondary antibodies used were an anti rabbit igg antibody conjugated to alexa 488 fluor (molecular probes) and anti mouse igg antibody conjugated to alexa fluor 647 (molecular probes). specificity of labeling and absence of signal crossover were determined by examination of single labeled control samples. p54-egfp was directly observed by detection of fluorescence at λ = 509 nm. nuclei as well as dna in virus factories were detected by staining with hoechst 33342. finally, cells were mounted onto slides using fluorsave reagent (calbiochem). conventional microscopy was carried out in a leica photomicroscope with a digital camera, and digitized images were obtained with qwin software (leica). confocal microscopy was carried out in a leica confocal microscope tcs sp2-aobs equipped with a 63 or 100× objectives. vero cells were plated at 60% confluence onto 35 mm dishes (willco wells) and infected with 1 or 20 pfu of b54gfp-2 per cell, depending on the experiment. for the majority of experiments, synchronization of virus infection was achieved by performing the adsorption at 4°c for 90 min and following removal of unbound virus. at indicated time points, cells were imaged by confocal microscopy carried out in a leica confocal microscope tcs sp2-aobs. in either case, cells were maintained on a heated 35 mm stage with the temperature set at 37°c and fresh dmem supplemented with 2.5% fetal bovine serum was perfused onto the dish at a rate of 0.1 ml/min throughout the experiment. the acquisition of images was every 5 min in long experiments (4 to 8 h length) and every 2-3 s in shorter experiments (from 1 to 4 hpi). nevertheless, in long experiments only indicated postinfection times images are shown in figures. simultaneous acquisition of egfp fluorescence emission and transmitted light was performed. microtubules depolymerization was achieved with 10 μm nocodazole (sigma) 2 h before the infection without removing it during the acquisition of images. detection of mitochondria in live cells was carried out with mitotracker red cmxros (molecular probes) 100 nm for 30 min after virus infection. golgi complex was detected in live cells with 5 μm bodipy-trc 5 -ceramide complexed to bsa (molecular probes) for 30 min before infection, and emission detected at λ = 617. manipulation and subsequent analysis of acquired images were carried out with leica confocal software v.2.0. african swine fever virus protein p54 interacts with the microtubular motor complex through direct binding to light-chain dynein assembly of african swine fever virus: role of polyprotein pp220 african swine fever virus is enveloped by a two-membraned collapsed cisterna derived from the endoplasmic reticulum african swine fever virus protease, a new viral member of the sumo-1-specific protease family repression of african swine fever virus polyprotein pp220-encoding gene leads to the assembly of icosahedral core-less particles electron microscopic observation of african swine fever virus development in vero cells characterization of african swine fever virion proteins j5r and j13l: immunolocalization in virus particles and assembly sites design and construction of african swine fever virus chimeras incorporating foreign viral epitopes purification and properties of african swine fever virus vaccinia virus cores are transported on microtubules association of african swine fever virus with the cytoskeleton the major structural protein of african swine fever virus, p73, is packaged into large structures, indicative of viral capsid or matrix precursors, on the endoplasmic reticulum african swine fever virus interaction with microtubules the preparation of orthopoxvirus dna two-dimensional analysis of african swine fever virus proteins and proteins induced in infected cells tracking fluorescence-labeled rabies virus: enhanced green fluorescent protein-tagged phosphoprotein p supports virus gene expression and formation of infectious particles inducible gene expression from african swine fever virus recombinants: analysis of the major capsid protein p72 movements of vaccinia virus intracellular enveloped virions with gfp tagged to the f13l envelope protein microtubule-independent motility and nuclear targeting of adenoviruses with fluorescently labeled genomes improvement of african swine fever virus neutralization assay using recombinant viruses expressing chromogenic marker genes aggresomes resemble sites specialized for virus assembly the african swine fever virus dynein-binding protein p54 induces infected cell apoptosis switching on and off the cell death cascade: african swine fever virus apoptosis regulation vaccinia virus utilizes microtubules for movement to the cell surface the dynein microtubule motor cytoarchitecture and physical properties of cytoplasm: volume, viscosity, diffusion, intracellular surface area green fluorescent protein-tagged adeno-associated virus particles allow the study of cytosolic and nuclear trafficking visualization of the intracellular behavior of hiv in living cells construction and characterization of a fluorescently labeled infectious human immunodeficiency virus type 1 derivative mitochondrial distribution and function in herpes simplex virus-infected cells virus factories: associations of cell organelles for viral replication and morphogenesis ultrastructural study of african swine fever virus replication in cultures of swine bone marrow cells apoptosis: a mechanism of cell killing and lymphoid organ impairment during acute african swine fever virus infection interaction of the rabies virus p protein with the lc8 dynein light chain genetic manipulation of african swine fever virus: construction of recombinant viruses expressing the beta-galactosidase gene characterization and molecular basis of heterogeneity of the african swine fever virus envelope protein p54 the structural protein p54 is essential for african swine fever virus viability african swine fever virus structural protein p54 is essential for the recruitment of envelope precursors to assembly sites migration of mitochondria to viral assembly sites in african swine fever virus-infected cells african swine fever virus is wrapped by the endoplasmic reticulum human cytomegalovirus labeled with green fluorescent protein for live analysis of intracellular particle movements break ins and break outs: viral interactions with the cytoskeleton of mammalian cells microtubule-mediated transport of incoming herpes simplex virus 1 capsids to the nucleus vimentin rearrangement during african swine fever virus infection involves retrograde transport along microtubules and phosphorylation of vimentin by calcium calmodulin kinase ii microtubule-dependent plus-and minus end-directed motilities are competing processes for nuclear targeting of adenovirus visualization and characterization of the intracellular movement of vaccinia virus intracellular mature virions visualization of intracellular movement of vaccinia virus virions containing a green fluorescent protein-b5r membrane protein chimera this study was supported by grants from the spanish comisión interministerial de ciencia y tecnología projects agl2002-00668, agl2004-07857-c03-03, bio2004-00690 and bio2005-0651. supplementary data associated with this article can be found in the online version at doi:10.1016/j.virol.2006.01.021. key: cord-329625-hx2rsi91 authors: you, jae-hwan; howell, gareth; pattnaik, asit k.; osorio, fernando a.; hiscox, julian a. title: a model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (prrsv) nucleocapsid protein based on live cell imaging date: 2008-08-15 journal: virology doi: 10.1016/j.virol.2008.04.037 sha: doc_id: 329625 cord_uid: hx2rsi91 porcine reproductive and respiratory syndrome virus (prrsv), an arterivirus, in common with many other positive strand rna viruses, encodes a nucleocapsid (n) protein which can localise not only to the cytoplasm but also to the nucleolus in virus-infected cells and cells over-expressing n protein. the dynamic trafficking of positive strand rna virus nucleocapsid proteins and prrsv n protein in particular between the cytoplasm and nucleolus is unknown. in this study live imaging of permissive and non-permissive cell lines, in conjunction with photo-bleaching (frap and flip), was used to investigate the trafficking of fluorescent labeled (egfp) prrsv-n protein. the data indicated that egfp-prrsv-n protein was not permanently sequestered to the nucleolus and had equivalent mobility to cellular nucleolar proteins. further the nuclear import of n protein appeared to occur faster than nuclear export, which may account for the observed relative distribution of n protein between the cytoplasm and the nucleolus. a model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (prrsv) nucleocapsid protein based on live cell imaging introduction porcine reproductive and respiratory syndrome virus (prrsv) is a spherical, enveloped virus, which causes highly contagious, severe disease in the natural host, the swine, with a spectrum of disease states ranging from respiratory failure in neonates to abortions in sows and persistence (wills et al., 2003) . prrsv is endemic to most swine producing countries and imposes a heavy economic burden due to the high mortality associated with the disease. prrsv is a positive strand rna virus belonging to the arteriviridae family, order nidovirales. although the primary site of replication is the cytoplasm, the viral encoded nucleocapsid (n) protein (an rna binding protein) localises predominately in the cytoplasm and nucleolus during virus infection and when over-expressed in cell culture (rowland et al., 1999) . this property is common with related viruses including the equine arterivirus (eav) n protein (tijms et al., 2002) and the coronavirus n proteins wurm et al., 2001) with the possible exception of the severe acute respiratory syndrome coronavirus (sars-cov) n protein (li et al., 2005; rowland et al., 2005; timani et al., 2005; you et al., 2005; you et al., 2007) . localisation and trafficking of the avian coronavirus infectious bronchitis virus (ibv) n protein to the nucleolus (cawood et al., 2007) correlates with the stage in the cell cycle (g2) when virus protein synthesis and progeny virus production is most efficient harrison et al., 2007; li et al., 2007) . the nucleolus is a dynamic sub-nuclear compartment found principally during interphase in eukaryotic cells (andersen et al., 2005; boisvert et al., 2007; hernandez-verdun, 2006; matthews and olson, 2006) . solution of the nucleolar proteome (andersen et al., 2002) has indicated that the nucleolus has a number of functions ranging from ribosome subunit biogenesis to non-classical functions such as the stress response (mayer and grummt, 2005; rubbi and milner, 2003) and regulation of cell growth (hernandez-verdun and roussel, 2003) . virology 378 (2008) there are over 700 proteins associated with the nucleolar proteome (leung et al., 2006) and these include nucleolin, fibrillarin and b23.1. localisation of positive strand rna virus capsid proteins and replication proteins (and in one case partial rna synthesis) in the nucleus and/or nucleolus appears to be an emerging feature of positive strand rna viruses. the reason why positive strand rna virus capsid proteins localise to the nucleolus is unknown, and several hypotheses have been proposed including; as part of a cellular defense mechanism to sequester viral proteins away from sites of virus replication and assembly, to recruit nucleolar proteins to facilitate virus replication, to usurp cellular processes by disrupting the nucleolar proteome or because the viral protein contains motifs which mimic nucleolar localisation signals (nolss) (hiscox, 2002 (hiscox, , 2003 (hiscox, , 2007 uchil et al., 2006; weidman et al., 2003) . however, the majority of rna synthesis in positive strand rna viruses (ahlquist, 2006; de haan and reggiori, 2008; taylor and kirkegaard, 2008) including arteriviruses (posthuma et al., 2008; van hemert et al., in press ) is thought to occur on cytoplasmic membrane bound replication complexes. assembly also occurs in the cytoplasm; therefore capsid proteins are required in this cellular compartment to facilitate these processes. this would infer the need for appropriate trafficking signals to ensure import and export from the nucleus and several such motifs have been identified. mutational analysis and abrogation of nuclear/nucleolar localisation of viral proteins for the prrsv n protein (lee et al., 2006; pei et al., 2008) and the positive strand rna plant rna virus groundnut rosette virus orf3 (kim et al., 2007a,b) have elegantly demonstrated the importance of nucleolar localisation in the biology of these viruses. for prrsv n protein, the motifs which direct nucleolar localisation are composed of a nuclear localisation signal (nls) acting in concert with a nols (lee et al., 2006; . whereas for ibv n protein an eight amino acid motif was shown to be necessary and sufficient to act as an nols (reed et al., 2006) . for nuclear export of the arterivirus and coronavirus n proteins, nuclear export signals (ness) have either been identified or inferred from molecular genetic and/or bioinformatic analysis, structural modeling or use of specific metabolic inhibitors of nuclear export tijms et al., 2002; you et al., 2007) . the dynamic trafficking of positive strand rna virus capsid proteins between the nucleolus and the cytoplasm or the mobility in the nucleolus is not clearly understood. previous studies which have been mainly based on preparations of fixed cells could not distinguish whether there were for example two populations of capsid protein; one which sequestered to the cytoplasm and one which sequestered to the nucleolus or whether protein trafficked between the two compartments. in order to distinguish between these possibilities and derive a model for prrsv n protein trafficking, this study used live cell confocal microscopy to investigate the trafficking of fluorescent labeled n protein in porcine and non-porcine cell lines which were permissive and non-permissive for prrsv. to investigate whether the localisation of n protein within live cells was equivalent to that previously observed in fixed cells and for subsequent trafficking analysis, the prrsv n gene was pcr cloned downstream of the enhanced green fluorescent protein (egfp) in the expression vector pegfpc2. this created plasmid pegfp-prrsv-n, which when transfected into cells led to the expression of prrsv n protein fused to egfp. although several cell lines were used in this study, primarily, the continuous porcine monocytic cell line, 3d4/31, was used in the majority of experiments. although not permissive for prrsv infection, these cells were derived from porcine alveolar macrophages (which are permissive) following expressing of the sv40 large t antigen (weingartl et al., 2002) . to investigate the localisation of egfp-prrsv-n protein, 3d4/31 cells were co-transfected with pegfp-prrsv-n and a vector which directed the expression of the nucleolar protein b23.1 fused to dsred (dsred-b23.1) (reed et al., 2006; you et al., 2005) to act as a marker for the nucleolus (fig. 1a) . two different localisation patterns of egfp-prrsv-n protein were observed: in 3d4/31 cells which expressed lower amounts of egfp-prrsv-n protein, localisation was predominately nucleolar with some nuclear signal (fig. 1a, panel a) . in 3d4/31 cells which expressed higher amounts of protein localisation was predominately either cytoplasmic and nucleolar (fig. 1a , panel c) or predominately nucleolar with some cytoplasmic and nuclear signal (fig. 1a, panel d) . this difference in expression was evident in that the fluorescent signal from egfp-prrsv-n protein in fig. 1a , panel a was captured in the linear range. however, using the same setting led to over-saturated pixels in cells expressing higher amounts of egfp-prrsv-n protein e.g. fig. 1a , panel b (red indicates that the image range is not linear). gain adjustment to ensure pixels were captured in the linear range restored image clarity (fig. 1a , panel c). nucleolar localisation of egfp-prrsv-n protein was confirmed due to colocalisation with the nucleolar marker protein dsred-b23.1 (yellow signal). previously, n protein has been shown to interact with the nucleolar protein fibrillarin . there are several different mechanisms which may account for the concentration dependent localisation pattern of n protein. n protein could diffuse and/or be actively transported into the nucleus through the nuclear pore complex (npc), diffuse through the nucleoplasm and be permanently sequestered in the nucleolus. as the nucleolar concentration of n protein becomes saturating so excess protein could either diffuse or be actively transported through the npc to the cytoplasm. alternatively, n protein could be continuously exchanged between the nucleolus and the nucleus and the cytoplasm, with localisation being driven by possible differential relative trafficking rates between nuclear import and export. to investigate and discriminate between these possibilities dynamic live cell imaging using fluorescence loss in photobleaching (flip) and fluorescence recovery after photo-bleaching (frap) was used to study movement of egfp-prrsv-n protein. relative trafficking within the nucleolus and trafficking from the cytoplasm to the nucleolus was investigated using flip to continuously photo-bleach a defined portion of the nucleolus in 3d4/31 cells expressing egfp-prrsv-n protein. this was compared to the trafficking of three nucleolar marker proteins (reed et al., 2006; you et al., 2005) ; egfp-nucleolin, egfp-b23.1 and efp-fibrillarin (fig. 1b) . loss of fluorescence is then a result of the egfp-tagged fusion protein trafficking into and out of the photo-bleached area with time. the data indicated that by 180s, loss of fluorescence from cells expressing egfp-nucleolin and egfp-fibrillarin was greater than egfp-b23.1. in cells expressing lower amounts of egfp-prrsv-n protein, fluorescence was completely photo-bleached by 120s and in higher expressing cells egfp-prrsv-n protein fluorescence was completely photo-bleached in the nucleus and reduced in the cytoplasm by 180s (fig. 1b) . this indicated that prrsv n protein was potentially as mobile as the cellular nucleolar proteins examined. to compare the relative nucleolar mobility between egfp-prrsv-n protein and egfp-nucleolin, egfp-b23.1 and efp-fibrillarin in 3d4/ 31 cells, frap was used to photo-bleach a defined portion of the nucleolus ( fig. 2a ) and the time taken to refill the photo-bleached area was measured (fig. 2b ) and t 1/2 values were compared (fig. 2c ). the data indicated that egfp-prrsv-n protein had the lowest t 1/2 value and therefore had a slightly faster mobility than the three cellular nucleolar marker proteins used in this study. cellular nucleolar proteins can have different mobility rates and nucleolar localisation patterns depending on their functions (andersen et al., 2005) and can show a continuous rapid turn over between the nucleus and the nucleolusyet the overall structure of the nucleolus is maintained (misteli, 2001) . the diffusion coefficient (d) value of egfp-prrsv-n protein and egfp-nucleolin, egfp-b23.1 and efp-fibrillarin was calculated from the recovery curves (phair and misteli, 2000) . this value is the rate at which a protein repopulates a photo-bleached area. this confirmed that egfp-prrsv-n protein had an equivalent (or slightly higher) mobility (d = 0.839 μm 2 /s) than egfp-nucleolin (d = 0.156μm 2 /s), egfp-fibrillarin (d = 0.278μm 2 /s) or egfp-b23.1 (d = 0.166μm 2 /s). in a previous study the half time of egfp-fibrillarin in bhk cells was reported to be approximately 3s with a d value of 0.53μm 2 /s (phair and misteli, 2000) , which is in similar agreement to this study. phair and misteli (2000) noted that the d values of egfp-fibrillarin and other fluorescent tagged nuclear/nucleolar proteins were 100 times lower than that reported for free solutes in the nucleus and suggested that this was due to interactions with other nuclear components. therefore, similar to the cellular nucleolar proteins the mobility of egfp-prrsv-n protein indicated that n protein probably localised to the nucleolus by diffusing through the nucleoplasm until it located an appropriate binding site, possibly a nucleolar protein or ribosomal rna, and thus appeared to accumulate in this compartment. prrsv n protein has been shown to interact with fibrillarin . the precise mechanism of trafficking of n protein within a cell and to the nucleolus is unknown, although n protein has been shown to interact with and pull down fibrillarin and thus may potentially localise to the nucleolus via this interaction . for example, the nucleolin binding activity of hepatitis delta antigen is associated with nucleolar localisation (ning and shih, 2004) . prrsv n protein has also been shown to interact with importin-α and importin-β and is sensitive to leptomycin b (lmb), an inhibitor of crm1-dependent export . several different nuclear-cytoplasmic trafficking pathways are utilized by viruses (whittaker et al., 2000) and have been described for a number of different virus nucleocapsids and nucleocapsid proteins. for example, in vesicular stomatitis virus nucleocapsids are trafficked via a microtubule mediated process (das et al., 2006) , as are the hantaan virus nucleocapsid protein (ramanathan et al., 2007) and the west nile virus capsid protein (chu and ng, 2002) . to investigate whether the trafficking of n protein between the cytoplasm and the nucleus/nucleolus was associated with microtubules or energy dependent, the mobility of egfp-prrsv-n protein was compared in 3d4/31 cells incubated at 37°c in the presence and absence of nocodazole (60ng/ml nocodazole (sigma) for 16h), a microtubule inhibitor, or incubated at 10°c. in preparations of fixed cells, in the absence of nocodazole (fig. 3a ) microtubules were visualized (observed as filaments) using an anti-tubulin antibody. as before egfp-prrsv-n protein localised predominately to the nucleolus. in preparations of fixed cells in the presence of nocodazole (fig. 3b ) microtubule polymerization was inhibited (diffuse staining of tubulin), although similar to untreated cells, egfp-prrsv-n protein remained predominately localised in the nucleolus. the activity of nocodazole was also confirmed by analyzing the cell cycle stage of treated cells (using flow cytometry as described previously harrison et al., 2007) ), which demonstrated enrichment in the g2/m stage (fig. 3b ) compared to untreated cells (fig. 3a) , and is a result of the inhibition of spindle formation in metaphase. to investigate the trafficking of egfp-prrsv-n protein in live cells, a nucleolus in 3d4/31 cells expressing egfp-prrsv-n protein was continuously photo-bleached (flip) in the presence of nocodazole at 37°c (fig. 3c ) and in the absence of nocodazole in cells that had been pre-cooled to 10°c (fig. 3d) . in both cases, photo-bleaching resulted in the loss of fluorescent signal in both the cytoplasm (where appropriate) and nucleus and the non photo-bleached nucleolus, indicating that cytoplasmic to nuclear trafficking occurred, and was not inhibited by either the presence of nocodazole or cooling to 10°c. this suggested that the trafficking of n protein from the cytoplasm to the nucleus was not microtubule or energy dependent. to investigate trafficking within the nucleolus a defined portion of the nucleolus was photobleached (frap) in cells incubated at either 37°c, 10°c or 37°c in the presence of nocodazole (fig. 4a ) and the time taken to refill the photobleached area was measured (fig. 4b) . the data indicated that egfp-prrsv-n protein at 37°c had a t 1/2 value of 0.74 ± 0.07 which was similar to the t 1/2 value (0.75 ± 0.18) at 4°c and at 37°c in the presence of nocodazole (t 1/2 value of 1.37 ± 0.55). the mobility of egfp-prrsv-n protein between the nucleolus and the cytoplasm and vice versa was compared to egfp using frap and flip on the cytoplasm or the nucleus/nucleolus in 3d4/31 cells. egfp is approximately 27kda and contains no known nls, nols or nes motifs and therefore the relative localisation of egfp between the nucleus and the cytoplasm would reflect the relative trafficking of the protein across the npc, most likely due to diffusion and concentration gradients, such that equilibrium would be reached depending on the relative balance between the rate of nuclear import versus nuclear export and the relative volume of the cytoplasm versus the nucleus. prrsv n protein is approximately 15kda (and with the addition of egfp approximately 42kda) and although the fusion protein has the potential to freely diffuse through the npc (which has a size exclusion limit for passive transport of approximately 50kda (macara, 2001; terry et al., 2007) ), clearly egfp-prrsv-n protein has a discrete localisation pattern being cytoplasmic and nucleolar (with some nuclear signal) (rowland et al., 1999) . without the presence of trafficking signals n protein would be predicted to display similar trafficking and localisation to egfp. the trafficking of egfp compared to egfp-prrsv-n protein between the nucleus and the cytoplasm and vice versa was analyzed by frap (fig. 5 ) and the relative level of fluorescence between the nucleus and the cytoplasm was measured using imagej software (nih) as described for the 3d4/31 cell line. this takes into account the reduction in fluorescence during photo-bleaching due to ongoing trafficking into and out of the bleach area. in egfp-expressing 3d4/31 cells the pre-bleach level of egfp in the nucleus to that of the cytoplasm was 0.56 ± 0.05 to 0.44 ± 0.03, respectively. to examine trafficking into the nucleus, this area was photo-bleached and subsequent recovery of fluorescence in the nucleus (due to import of egfp from the cytoplasm) indicated that after 180s the relative level of egfp between the nucleus and the cytoplasm was 0.60± 0.04 compared to 0.41 ± 0.05, respectively, which was not significantly different from the pre-bleach levels (fig. 5a) . analysis by 180s was then used as a base line for comparison of trafficking of egfp and egfp-prrsv-n protein between the nucleus and the cytoplasm in frap analysis. in 3d4/31 cells expressing egfp-pprsv-n protein with nucleolar/nuclear and cytoplasmic distribution, the prebleach level of egfp-prrsv-n protein between the total nuclear content and the cytoplasm was 0.78 ± 0.07 and 0.22 ± 0.03, respectively. 180s after photo-bleaching, the nuclear to cytoplasm level was 0.81 ± 0.08 to 0.24 ± 0.04, respectively (fig. 5b) , which was not significantly different from the pre-bleach level. to examine trafficking of egfp into the cytoplasm, this area was photo-bleached in 3d4/31 cells and subsequent recovery of fluorescence in the cytoplasm (due to import of non photo-bleached egfp from the nucleus) was compared to levels before photo-bleaching (nucleus to cytoplasm level of 0.58 ± 0.04 to 0.41 ± 0.05, respectively) to levels 180s after photo-bleach when the nuclear to cytoplasm ratio was 0.72 ± 0.05 compared to 0.28± 0.04, respectively (fig. 5c ). this indicated that egfp had not reached pre-bleach levels, which was different when considering cytoplasm to nucleus trafficking. to examine trafficking of egfp-prrsv-n protein into the cytoplasm, this area was photo-bleached and subsequent recovery of fluorescence in the cytoplasm (due to import of egfp-prrsv-n protein from the nucleus) was compared before photo-bleach (nucleus to cytoplasm relative level of 0.71 ± 0.06 to 0.29 ± 0.03, respectively) to 180s after photo-bleach, when the nuclear to cytoplasm ratio was 0.92 ± 0.08 compared to 0.08 ± 0.05, respectively (fig. 5d ). (note that this experiment was performed on cells in which egfp-prrsv-n protein localised also in the cytoplasm). this data indicated that the ratio of egfp-prrsv-n protein had not reached pre-bleach levels, which was different when considering cytoplasm to nucleus trafficking. this indicated that the export of egfp-prrsv-n protein from the nucleus was potentially slower than import and given the difference in relative ratios, slower than the export of egfp from the nucleus to the cytoplasm. the relative difference between nuclear import and nuclear export observed with egfp-prrsv-n protein was investigated using flip in which either a defined area of the nucleus (not the nucleolus) was photo-bleached for either egfp (fig. 6a ) or egfp-prrsv-n protein (fig. 6b ) expressing 3d4/31 cells or a defined area of the cytoplasm was photo-bleached for either egfp (fig. 6c ) or egfp-prrsv-n protein (fig. 6d ) expressing 3d4/31 cells. again, comparison of the data from the two proteins indicated that more fluorescent signal of egfp-prrsv-n protein was lost during import than export, suggesting that the former process operates faster than the latter. reflecting the frap analysis, the relative trafficking of egfp-prrsv-n protein between the nucleus and the cytoplasm was in favor of nuclear import. the observed live cell trafficking patterns of egfp-prrsv-n protein may have been specific to 3d4/31 cells. therefore, to investigate this possibility, the localisation and dynamic trafficking of this protein was compared to egfp in marc-145 cells (an african green monkey kidney cell line permissive for prrsv infection (kim et al., 1993) ) and hela cells (derived from a human cervical cell line, and non-permissive for infection). the data indicated that in both marc-145 and hela cell types egfp-prrsv-n protein localised predominately to the nucleus/ nucleolus compared to egfp which localised throughout the cell (figs. 7a and 8a, respectively). very few cells (less than 5%) exhibited more than 10% of the total fluorescence from egfp-prrsv-n protein in the cytoplasm. note that for egfp, the transmission phase image is presented to highlight the differentiation between the cytoplasm, nucleus and nucleolus. (such images were used in subsequent analysis for placement of photo-bleaching in the egfp fluorescent cell, where distinction between the nucleus/nucleolus/cytoplasm was not possible). the sub-cellular localisation patterns for both fluorescent proteins were similar to that observed in 3d4/31 cells. investigation of the trafficking of egfp-prrsv-n protein compared to egfp between the cytoplasm and the nucleus and vice versa by continuous photo-bleaching (flip) a defined portion of the nucleus or the cytoplasm in both marc-145 cells (fig. 7) and hela cells (fig. 8) , indicated that photo-bleaching in the nucleus in cells expressing egfp resulted in apparent simultaneous loss of fluorescence from the nucleus and the cytoplasm (figs. 7b and 8b), indicating rapid trafficking of egfp between the two compartments. in comparison, little fluorescence (approximately~5% of total fluorescence) of egfp-prrsv-n protein was observed in the cytoplasm in both marc-145 and hela cells and photobleaching in the nucleus resulted in loss of fluorescence from the nucleoplasm and then the nucleolus (figs. 7c and 8c upper panels, respectively) . in approximately 10% of cells expressing egfp-prrsv-n protein fluorescence was observed in the cytoplasm, but only if imaging was adjusted so that the fluorescent signal in the nucleus/nucleolus was above the linear range (for example fig. 8c, lower panels) . in this case photo-bleaching in the nucleus resulted in loss of fluorescence from the cytoplasm which could be accounted by faster trafficking into the nucleus of non-photo-bleached protein which was then photo-bleached than the export of photo-bleached protein into the cytoplasm. photobleaching in the cytoplasm of either marc-145 cells or hela cells expressing egfp resulted in loss of signal from both the nucleus and the cytoplasm (figs. 7d and 8d, respectively) . in comparison, photobleaching in the cytoplasm of either marc-145 cells or hela cells expressing egfp-prrsv-n protein resulted in less loss of fluorescence when compared to egfp (figs. 7e and 8e) . the photo-bleaching analysis for egfp-prrsv-n protein also indicated that when the nucleus was photo-bleached loss of fluorescence in the cytoplasm was due to the trafficking of non photo-bleached protein into the nucleus rather than the rapid export of photo-bleached protein from the nucleus into the cytoplasm. thus the photo-bleaching analysis of egfp and egfp-prrsv-n protein in marc-145 and hela cells was similar to that found in 3d4/ 31 cells. thus we propose a model of n protein trafficking in which the nuclear import of n protein is faster than the nuclear export and n protein is apparently localised in greater amounts in the nucleolus (fig. 9) . the deficiency of the model is that the analysis was conducted in the absence of other viral proteins or rna (i.e. infected cells) which may associate with n protein and alter its relative trafficking rates. this may be certainly true for the cytoplasm, where n protein will complex with viral rna and possibly components of the replicase complex. although there is currently no published data that other prrsv macromolecules localise in the nucleus/nucleolus, the eav non-structural protein 1 (nsp1) has also been observed in the nucleus (tijms et al., 2002) , and therefore prrsv nsp1 may have similar properties. prrsv n protein does not accumulate or is sequestered in the nucleus/nucleolus per se and in common with other cellular nuclear/nucleolar proteins is continuously being exchanged between the nucleolus and the nucleoplasm and indeed is as mobile as the cellular nucleolar proteins examined. the implication of this study is that all de novo synthesized n protein (when over-expressed) traffics to the nucleolus. together with molecular genetic analysis showing that virus replication is reduced when n protein nucleolar localisation is abolished (lee et al., 2006; pei et al., 2008) , suggests that the nucleolar trafficking of n protein is playing a crucial role in virus biology. what this role is remains to be elucidated. cell culture 3d4/31 cells were grown at 37°c with 5% co 2 in dmem and rpmi 1640 (50:50). marc-145 and hela cells were maintained in dmem supplemented with 10% foetal bovine serum and grown at 37°c with 5% co 2 . 30mm glass bottom tissue culture dishes were seeded with 2 × 10 5 cells 24h prior to transfection. the plasmid pegfp-prrsv-n, which expressed prrsv n protein fused c-terminal to egfp, was constructed by amplifying the n gene using the amplicon fl12 (truong et al., 2004) (genbank accession number ay545985) as template. primers were designed to the 5′ and 3′ end of the n gene and contained appropriate restriction enzyme sites; 5′ hindiii and 3′ ecori respectively. the amplification product was ligated into the topo vector pcr2.1 and then cloned into hindiii and ecori restricted pegfp-c2 (clontech). the insert was sequenced in both directions to confirm authenticity and western blot analysis to confirm expression. plasmids which led to the expression of the nucleolar marker proteins egfp-nucleolin, egfp-b23.1, dsred-b23.1 and efp-fibrillarin have been described previously (reed et al., 2006; you et al., 2005) . transfected cells were imaged at 37°c in co 2 -independent media (gibco) supplemented with 5% fbs on an inverted lsm510 meta confocal microscope (carl zeiss) using a 63× objective and a 4 factor zoom. five images were captured before a short period of photobleaching using conditions that had negligible effects on total cell fluorescence. photo-bleaching was performed on an area of 12 by 12pixels (approx 20.16μm 2 ) with a 25mw argon laser at 100% power, bleaching took approximately 1.2s. fluorescence was analyzed with lsm510 software and raw data was imported into microsoft excel for analysis and generation of graphs. raw data were averaged before normalizing. normalization of the data was performed in such a way as to ensure that the recovery profiles were set to zero at the initial time point post bleaching, and set to 1 at the final time point; as previously described (marcelli et al., 2006; stenoien et al., 2002) . time to half life recovery (t 1/2 ) was performed on the mean raw data, determined by the equation (i e − i o ) / 2, where i e was the intensity at the final time point, and i o the intensity at the time point immediately following photo-bleaching. diffusion coefficient (d) values were calculated from the half life recovery (t 1/2 ) using the following diffusion equation d =(w 2 / 4 t 1/2 ) × 0.88 where w is the width of the bleach area, approx 1.68μm, and a constant factor of 0.88 was used for a gaussian beam profile (axelrod et al., 1976) . data from 5 experiments were used per construct and standard deviation was calculated in the usual manner using microsoft excel. transfected cells were imaged in glass base dishes as outlined above. imaging and photo-bleaching was performed with the same laser settings as detailed in the frap microscopy. in each flip experiment cells were imaged 5 times followed by a period of photobleaching for a total time of 3min. photo-bleaching was performed on a defined area (12 by 12pixels (approx 20.16μm 2 )) within the cell for 50 iterations (mean bleach time 2.1s). parallels among positive-strand rna viruses, reverse-transcribing viruses and double-stranded rna viruses nucleolar proteome dynamics directed proteomic analysis of the human nucleolus mobility measurement by analysis of fluorescence photobleaching recovery kinetics the multifunctional nucleolus cell cycle dependent localisation of the coronavirus nucleocapsid protein trafficking mechanism of west nile (sarafend) virus structural proteins visualization of intracellular transport of vesicular stomatitis virus nucleocapsids in living cells are nidoviruses hijacking the autophagy machinery? cell cycle perturbations induced by infection with the coronavirus infectious bronchitis virus and their effect on virus 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respiratory syndrome virus generated from an infectious cdna clone retains the in vivo virulence and transmissibility properties of the parental virus nuclear localization of flavivirus rna synthesis in infected cells the in vitro rna synthesizing activity of the isolated arterivirus replication/transcription complex is dependent on a host factor the interaction of cytoplasmic rna viruses with the nucleus continuous porcine cell lines developed from alveolar macrophages: partial characterization and virus susceptibility viral entry into the nucleus duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus localisation to the nucleolus is a common feature of coronavirus nucleoproteins and the protein may disrupt host cell division colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar rnaassociated protein fibrillarin subcellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein trafficking motifs in the sars-coronavirus nucleocapsid protein this research was supported by a national pork board grant to jah. drs amanda stuart and t. david k. brown at the university of cambridge are thanked for providing the materials and reagents. key: cord-335482-nx7odchj authors: makino, shinji; taguchi, fumihiro; fujiwara, kosaku title: defective interfering particles of mouse hepatitis virus date: 1984-02-29 journal: virology doi: 10.1016/0042-6822(84)90420-3 sha: doc_id: 335482 cord_uid: nx7odchj abstract after six to eight serial undiluted passages of mouse hepatitis virus (jhm strain) in dbt cell culture, a decrease in the yield of infectious virus occurred, and with further passages fluctuating yields of infectious virus were observed. the serially passaged virus interfered with the multiplication of the standard jhm virus, but not with vesicular stomatitis virus. after sucrose equilibrium centrifugation of high passage virus, a single peak contained both infectious virus and interfering activity. this virus population resembled the original jhm virus in its structural proteins, but it contained an increased proportion of a protein with a molecular weight of 65 × 103. genomic rna from standard jhm virus contained a single species of rna with a molecular weight of 5.4 × 106. after five undiluted passages, however, the virion population contained two rna species with molecular weights of 5.4 × 106 and 5.2 × 106. rnase t1 resistant oligonucleotide finger-printing of these rnas showed that the lower molecular weight rna had lost several oligonucleotide spots that were present in the genomic rna of the standard jhm virus. after several serial diluted passages of passage 10 virus, a single virus population was obtained which again had only standard virus rna with a molecular weight of 5.4 × 106 and lacked interfering activity. these results indicated that defective interfering particles were generated by serial undiluted passages of jhm virus. defective interfering (di) particles have been described in most virus groups (holland et al, 1980; huang and baltimore, 1977) . characteristically di particles (a) lack part of the viral genome, (b) possess a normal complement of structural proteins, (c) replicate only with the help of standard virus, (d) interfere with replication of the homologous virus. many studies have focused on the role of di virus genomes in virus evolution and their involvement in persistent infection (friedman and ramseur, 1979; holland and villarreal, 1975; holland et al, 1980; jacobson and pfau, 1980; rima and martin, 1976) . the coronavirus group has a positive single-stranded rna genome with a molecular weight (mw) of 5.4-6.9 x 10" (siddell et al., 1982) . so far, there have been no reports on the existence of coronavirus i author to whom requests for reprints should he addressed. di particles. the jhm strain of mouse hepatitis virus (mhv), a coronavirus (bailey et al, 1949; cheever et az., 1949; robb and bond, 1979; siddell et al, 1982) , has now been examined for the generation of di particles. we have observed marked reduction of infectivity in the course od serial undiluted passages of jhm virus in dbt cell culture. the progeny viruses in-' terfered with the replication of the original jhm virus but not with vesicular stomatitis virus. this interference was mediated by particles of jhm virus which possessed the properties of di particles cited above. materials and methods dbt cells (hirano et ah, 1974; kumanishi, 1967) were used for propagation of viruses and infectivity assays. they were grown in eagle's minimal essential medium (mem, nissui, tokyo) containing 10% calf serum and 10% tryptose phosphate makino, taguchi, and fujiwara broth (difco) (gm). the concentration of calf serum was reduced to 5% in the maintenance medium (mm) used for virus harvesting. to avoid contamination of the standard virus stock with di particles, plaque purified mhv (jhm strain) (hirano et al, 1981; makino et al, 1983) was propagated on dbt cells at a multiplicity of infection (m.o.i.) of 0.0002 and at 15 hr postinfection (p.i.) culture fluid was harvested and clarified by low speed centrifugation. this virus was then propagated twice on dbt cells at an m.o.i. of 0.01. at 16 hr p.i. the culture fluid was harvested, clarified, and then stored at -70". the infectivity of this standard jhm virus was 2.0 x lo6 plaque-forming units (pfu)/o.z ml. vesicular stomatitis virus (vsv), strain new jersey, was propagated on dbt cells and stored at -70". infectivities of jhm virus and vsv were determined by the plaque assay system described previously (hirano et al, 1974; makino et a& 1982) . dbt cell monolayers (2 x lo6 cells) in 35-mm plastic dishes (corning, new york) were washed once with mem and inoculated with 0.2 ml of the standard jhm virus at an m.o.i. of 1.0. after adsorption for 60 min at 37", unabsorbed virus was removed and the cells were washed once with mem and cultured with 2 ml of mm/dish at 37'. the culture fluids were harvested at 14 hr p.i. and clarified, and the supernatants were stored at -70". two hundred microliters of undiluted samples of passage 1 was used to infect a fresh dbt culture, and each successive passage was carried out in the same way. preparation of the standard jhm virus for interference assay jhm virus was propagated on dbt cells after infection at an m.o.i. of 1.0 and was harvested at 14 hr p.i. the culture fluid was clarified by centrifugation at 8000 rpm for 30 min. one liter of the supernatant was subjected to salt precipitation with ammonium sulfate (50% saturation) followed by centrifugation at 8000 rpm for 30 min. the pellet was resuspended in nte buffer (containing 0.1 mnacl, 0.01 mtris-hcl, ph 7.2, and 0.001 m ethylenediaminetetraacetic acid (edta)) and the suspension was centrifuged at 3000 rpm for 10 min. the supernatant was then placed on a discontinuous sucrose gradient consisting of 60, 30, and 20% (w/v) sucrose (3:2:2 in volume, respectively) and centrifuged at 27,000 rpm for 3 hr at 4o in a beckman sw 28.1 rotor. a virus band at the interface between 60 and 30% sucrose was collected and diluted sixfold with gm. this diluted virus stock contained 2.0 x 10' pfu10.2 ml and was stored at -70". this partially purified jhm virus was used as the standard jhm virus for interference assay. interference assay dbt cell monolayers (3 x 105 cells) prepared in 24-well multiwell plates (coster, cambridge) were washed once with mem, and inoculated with the mixture of 50 ~1 of the standard jhm virus or vsv and 100 ~1 of test virus samples. a mixture of 50 ~1 of the standard jhm virus or vsv and 100 ~1 of mm was inoculated as a control. after 60 min at 37', unabsorbed viruses were removed and the cells were washed once with mem and incubated at 37' for 13 hr with 1 ml of mm/well. dbt cells grown in 60-or 35-mm plastic dishes were inoculated with 0.4 and 0.2 ml of virus samples, respectively. after adsorption the inoculum was removed and the cells were labeled with [3h]uridine or ri-ijleucine according to methods described previously (makino et d, 1983) . for the preparation of =p-labeled virus rna, cultures were incubated for 2 hr before virus inoculation with mem in which the concentration of phosphate was reduced to 11 10. after virus adsorption, the inoculum was replaced by phosphate-free mem containing 10% dialyzed calf serum and 200 &i of bp (japan radioisotope association, interfering particles of mhv 11 tokyo) per milliliter. radiolabeled virus was collected after 14 hr of infection and purified from the medium of the infected culture as described previously (makino et al, 1983) . the discontinuous sds-polyacrylamide system of laemmli (1970) was used as described previously (makino et al, 1983) . after electrophoresis, gels were further processed for fluorography using sodium salicynate (chamberlain, 1979) . pellets of purified viruses were suspended in nte buffer containing 0.5% sds and 100 pg/ml of proteinase k (boehringer-mannheim, west germany). the suspension was incubated at 50" for 5 min, at 25" for 25 min, and then rna was extracted with phenol-chloroform-isoamyl alcohol (50:5&l). the final aqueous phase was made 0.25 m in sodium chloride and the rna was precipitated at -20" after the addition of 2.5 vol of ethanol. after centrifugation and one washing with 75% ethanol, rna was dried and dissolved in a small volume of redistilled water. agarose gel electrophoresis 1. analytical electrophoresis. following denaturation of the rna with glyoxal treatment, agarose gel electrophoresis was performed as previously described (mcmaster and carmichael, 1977) . after electrophoresis the gels were washed with water and dried under vacuum, and then exposed to a kodak xs-1 film with an intensifying screen. 2. preparative electrophoresis. radiolabeled rna was dissolved in 10 mm sodium phosphate, ph 7.0, containing 1 mmedta, 0.1% sds, 0.1% bromophenol blue, and 0.1% xylene cyan01 ff and mixed with equal volume of glycerol. the solution was heated for 5 min at 56", rapidly cooled on ice, and placed in a slot of a horizontal 1% agarose in electrophoresis buffer (40 mm tris, 20 mm sodium acetate, 33 mm acetic acid, 2 mm edta, ph 7.4) supplemented with 6 m deionized urea. electrophoresis was performed at 4". rna bands were located by autoradiograph of the wet gel, excised, and kept at -20". the gel slices were melted for 10 min at 65" and 20 pg of yeast trna (type x; sigma) was added to each sample. then the rna was eluted from gel slices by the methods of langridge et al. (1980) . the purified 32p-labeled rna was exhaustively digested with rnase t1 (sankyo, tokyo) and analyzed by two-dimensional polyacrylamide gel electrophoresis by a modification of the procedure of de wachter and fiers (1972). the first dimension was performed on 7.2% polyacrylamide-0.09% bisacrylamide gel slabs at ph 3.3 containing 6 m urea. electrophoresis was carried out at 1000 v for 4 to 5 hr. the second dimension was performed on 22% polyacrylamide-1.54% bisacrylamide gel slabs at ph 8.2 and 650 v for 16 to 19 hr. after electrophoresis, the gel was wrapped with saran wrap and exposed to kodak xar5 or xs-1 film with an intensifying screen at 4' for an appropriate length of time. the culture fluid from 25 undiluted passages was assayed for infectivity at each passage ( fig. 1) . from passages 1 to 5, a constant level of infectious viruses was produced. during passages 6 to 8, however, the yield of infectious viruses was significantly reduced. at passage 9, infectivity of the culture supernatant increased, but it was again reduced from passages 10 to 12. these cyclic increases and decreases in infectivity were observed until passage 25. severe syncytial formation was observed in all 25 passages. to determine if the reduction in the yield of infectious virus was due to the presence of di particles, a'n interference analysis was performed with several culture fluid samples at different passage levels. as shown in table 1 , the multiplication of the standard jhm virus was specifically inhibited by passaged virus stock samples at passage levels 10, 15, and 20. a slight inhibition was observed with a sample at passage 5. however, the growth of vw was not interfered with by passages 10 and 20 virus samples. to test whether this interference was mediated by di particles or another agent, such as interferon, passage 1 and 10 viruses were first metabolically labeled with [3hjuridine and centrifuged to equilibrium. infected culture fluid (400 ml) from passages 1 and 10 was then precipitated with ammonium sulfate, as described under materials and methods, and this unlabeled material was mixed with the partially purified labeled virus from the corresponding passage level. these mixtures were then applied to a 20 to 60% continuous sucrose gradient. as shown in fig. 2 , a single radioactive peak, corresponding to the infectivity peak, appeared in both virus preparations at a buoyant density of 1.196 g/cm3. the activity of interference with standard jhm virus was detected in the virus preparation of passage 10 but not in that of passage 1. this interference activity coincided with the peak of radioactivity and infectivity. the results indicated that the agent causing interference could not be separated from standard infectious virions by this sucrose equilibrium centrifugation. "dbt cells (3 x 106 cells) in 24-well plates were infected with 50 pl of standard jhm or vsv and 100 pl of test samples. after 60 min at 3'7" inoculum was removed and eelis were washed once with mem. the cells were cultured with 1 ml of mm for 13 hr at 37" and medium was harvested, centrifuged, and assayed for infectivity. b jhm virus titer. c vsv titer. virus was labeled for 13 hr p.i. with mimidine (100 &i/ml) and partially purified by a discontinuous sucrose gradient (60,30, and 20% sucrose). then the partially purified virus was mixed with unlabeled virus of the same passage level, prepared by precipitation with ammonium sulfate, and then this mixture was applied to a 20 to 60% continuous sucrose gradient and centrifuged at 27,000 rpm for 18 hr at 4" by beckman sw 23.1 rotor. then the gradient was fractionated into 25 x 0.35 ml portions. portions of each fraction were analyzed for radioactivity (0) and pfu (0). the interference activity (a) was measured by the use of 1:6 dilution of the sample with gm and selected fractions were also examined for density (x). interfering particles of mhv 13 fraction virion rnas labeled with bp were exof passages 1 and 10 were metabolically tracted from purified virus samples at paslabeled with [3h]leucine, purified, and subsage levels of 1, 3, 4, 5, 10, 15, and 20. the jected to sds-page analysis (fig. 3) . vi-rnas were denatured with glyoxal and ruses of passages 1 and 10, both showed subjected to agarose gel electrophoresis. four major polypeptides with mw of 26ok, virus rnas from passages 1, 3, and 4 105k, 6ok, and 23k. a 21k polypeptide was showed a single band with a mw of 5.4 also sometimes found, but in variable x 106, which was identical to the mw of amounts. the only obvious difference be-mhv genome rna described previously tween the structural polypeptides of viby others (fig. 4) (lai et al, 1981 ; lai and ruses from passages 1 and 10 was a 65k stohlman, 1978). on the other hand, virion protein, which was present in much larger rnas from passages 5, 10, 15, and 20 amounts in passage 10 virus. this protein showed two bands, one identical to that was also present in passage 1 virus but to from passages 1 to 4 and a second having obtain clear identification it was necessary a lower mw of 5.2 x 106. to confirm that to apply a greater amount of radioactivity this lower mw rna species was a defective to the gel (lane c). rna and not due to conformational fig. 2 were pooled, diluted, and pelleted at 45,000 rpm for 60 min at 4' in a beckman sw 50.1 rotor. the pelleted viruses were treated with sample buffer (0.06 mtris-hcl, ph 6.8,2% sds, 25% glycerol, 5% 2-mercaptoethanol, 0.1% bromophenol blue), and boiled for 2 min. electrophoresis was performed on a 7.5 to 15% polyacrylamide gel. the ratio of radioactively labeled virus applied was a:b:c = 1:1:2.5, lane a, passage 1 virus; lane b, passage 10 virus; lane c, passage 1 virus. changes of the genomic rna, the rnas with mw of 5.4 x lo6 (genomic rna) of passages 1 and 10 viruses and the lower mw rna species of passage 10 viruses were compared by oligonucleotide fingerprinting (fig. 5) . poly(a) tracts are seen as streaks in the lower left-hand corners of every fingerprint indicating that all rna species were polyadenylated. the oligonucleotide fingerprinting pattern of genomic rna of passage 1 virus is almost identical to that reported by others. the numbering of oligonucleotides in this paper is therefore as described previously (lai et al, 1981; stohlman et cz& 1982) . however, three oligonucleotides (numbered as 2a, 2b, and 42a) appear not to be present in the previously reported fingerprints. oligonucleotide fingerprinting of genomic rna of passage 10 was essentially identical to that of passage 1 virus. however, an oli-gonucleotide (42b) (indicated by an arrow) appeared in the genomic rna of passage 10 and an oligonucleotide, 42a, had disappeared (indicated as an open circle). the oligonucleotide fingerprinting of the lower mw rna species of passage 10 virus showed the loss of nine oligonucleotides including 42a and 42b (indicated as open circles) and the appearance of three additional oligonucleotides (indicated by arrows). these studies clearly indicated that the lower mw rna species had undergone deletions. thus, these data strongly suggested that di particles were present in culture fluids after passage 5. for further confirmation of the presence of di particles, high dilution serial passages of virus stock containing di particles were conducted. dbt cells were first infected with passage 10 virus samples at an m.o.i. of 0.0002, and the virus yield was subsequently inoculated onto dbt cells at an m.o.i. of 0.1. this virus stock after passages with dilution (spd) was shown to possess an infectivity of 1.5 x lo6 pfuio.2 ml and did not interfere with the multiplication of the standard jhm virus (data not shown). viral rna extracted from the spd contained a single rna with a mw of 5.4 x lo6 (fig. 6 ) being identical to that of the virus passage 1. these data confirmed that the smaller size rna was di rna and that di particles were eliminated by serial passages with dilution. discussion di particles are deletion mutants which cannot replicate by themselves but interfere specifically with replication of the homologous virus, which is itself required for the generation and replication of the di particles. after serial passages without dilution, di particles were generated in our virus stock of jhm virus. these particles were characterized by (1) interference with the multiplication of homologous jhm virus but not with that of vsv, (2) being inseparable from infectious jhm virions by sucrose equilibrium centrifugation, (3) having structural proteins almost identical to the original jhm virus, (4) having a smaller size rna (mw: 5.2 x 106) that had lost several rnase t1 resistant oligonucleotides, (5) being excluded readily from the virus stock by serial passages with dilution. we and others have also searched for the presence of di particles in mhv per-sistent infection in vitro (hirano et al, 1981; robb and bond, 1979; stohlman and weiner, 1978) and the generation of di particles by serial passages without dilution has been attempted by several laboratories (leibowitz et d, 1981; robb and bond, 1979; fig. 6 (1978) failed to detect di particles using murine neuroblastoma cells and dbt cells by serial undiluted passages of viruses which were released from the culture persistently infected with jhm virus. the reason for failure to detect the generation of di particles of jhm virus in these experiments is unclear, but minor genetic differences might play some roles, because although the oligonucleotide fingerprinting pattern of our standard jhm virus was very similar to that reported by others (lai and stohlman, 1981) , some differences existed (see fig. 5 ). during undiluted passages di rna was first detected at passage 5, and the virus yield was gradually reduced thereafter until passage 8, indicating that di particles, generated at passages 4 to 5, accumulated in the culture and played a crucial role in the reduction of the infectivity. after reduction of the yield of infectious virus, fluctuating patterns of infectivity were observed. such a phenomenon has been described in other viruses during passages without dilution (holland et al, 1980; huang and baltimore, 1977) , indicating that the di particles can replicate only with the aid of helper standard infectious virus and that the replication of the helper standard virus was inhibited by di particles. the fluctuating pattern of infectivity has also been observed in the case of persistent infections with many viruses in tissue culture, in which di particles have been detected. the di particles of jhm virus were not separable from infectious jhm particles by sucrose centrifugation. this was presumably due to the relatively small deletions present in the rna of di particles and there being no major difference between the structural proteins of these particles and standard jhm virions. however, bruton and kennedy (1976) reported that semliki forest virus di particles could be separated from the standard virus using cscl but not sucrose centrifugation. so the purification and separation of di particles of jhm virus might be possible using these techniques. in our studies, four major proteins (26ok, 105k, 6ok, and 23k) and minor 21k and 65k proteins were identified at passage 1 of jhm virus. as already described (makino et al, 1983 ) the 260k protein in our studies is the counterpart of gp170 described by others (siddell et &, 1981; siddell et al, 1982) . the proteins of passage 10 virus, rich in di particles, contained no significant difference from the structural proteins of passage 1 jhm virus, except for an increased amount of the 65k protein. at the moment this observation cannot be explained. the mapping of t1 resistant oligonucleotides on the genome of jhm virus has been reported by stohlman et cd (1982) , and therefore it should be possible to localize the deletion site(s) in the rna of the di particles reported here. comparing the oligonucleotide fingerprinting of di rna with standard virus rna, it seems that the deletion(s) were located within a region 2 to 7 kilobases (kb) from the 3' end of the genome. it is not possible to conclude, however, whether there is only a single, or multiple deletion ,sites. more detailed mapping of the oligonucleotides in this region of the genome would presumably define the precise location of the deletion site(s). the mechanisms of interference and generation of mhv di particles are not understood. studies on the detail gene structure of mhv di rna described in this paper are now in progress and may help to elucidate these questions. proc. nat. acad sci. plenum defective fection of tissue culture cells by rna viruses. i'tfed interfering rna viruses and the host-cell response fication of defective interfering t particles of ve-plenum coronavirus jhm: intracellular protein 449 comprehensive vi-the structure and replication of coronaviruses esis and resistance to defective interfering particles stability sarcoma virus, schmidt-ruppin strain. 1. induction of neurotropic mouse hepatitis virus (jhm strain) of brain tumors in adult mice with rous chicken during chronic infection of neuroblastoma cells. sarcoma cells weiduring the assembly of the head of bacteriophage pa~on, morphology variants of the jhm neurotropic strain references bailey, 0. t., pappenheimer, a. m., cheever, f. s.. and daniels, j. b. (1949) . a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin. ii. pathology. j. exp. med so, [195] [196] [197] [198] [199] [200] [201] [202] [203] [204] [205] [206] [207] [208] [209] [210] [211] [212] c. j., and kennedy, s. i. t. (19'76 immunol 27, 445-4&l. (1981) . persistent infection with mouse hepatitis mcmaster, g. k., and carmicheal, g. g. (1977) . virus jhm strain in dbt cell culture. in "the bio-analysis of single-and double-stranded nucleic acchemistry and biology of coronaviruses" (v. ter ids on polyacrylamide and agarose gels by using key: cord-346514-vyo8l14p authors: chen, i-hsuan; cheng, jai-hong; huang, ying-wen; lin, na-sheng; hsu, yau-heiu; tsai, ching-hsiu title: characterization of the polyadenylation activity in a replicase complex from bamboo mosaic virus-infected nicotiana benthamiana plants date: 2013-06-13 journal: virology doi: 10.1016/j.virol.2013.05.032 sha: doc_id: 346514 cord_uid: vyo8l14p bamboo mosaic virus (bamv) has a positive-sense single-stranded rna genome with a 5′ cap and a 3′ poly(a) tail. to characterize polyadenylation activity in the bamv replicase complex, we performed the in vitro polyadenylation with various bamv templates. we conducted a polyadenylation activity assay for bamv rna by using a partially purified bamv replicase complex. the results showed that approximately 200 adenylates at the 3′ end of the rna were generated on the endogenous rna templates. specific fractions derived from uninfected nicotiana benthamiana plants enhanced the polyadenylation activity, implying that host factors are involved in polyadenylation. furthermore, polyadenylation can be detected in newly synthesized plus-strand rna in vitro when using the exogenous bamv minus-strand minigenome. for polyadenylation on the exogenous plus-strand minigenome, the 3′ end requires at least 4a to reach 22% polyadenylation activity. the results indicate that the bamv replicase complex recognizes the 3′ end of bamv for polyadenylation. bamboo mosaic virus (bamv), which is a member of the potexvirus group (lin et al., 1992 (lin et al., , 1994 , has a single-stranded positive-sense rna genome with a 5′m 7 gpppg structure (lin et al., 1994) and a 3 poly (a) stretch of approximately 150 to 300 adenylates (chen et al., 2005) . the 3′ untranslated region (utr) of the bamv rna folds into a series of stem-loops, including a tertiary pseudoknot structure . these stem-loops in the 3′ utr, including the binding site for rnadependent rna polymerase (rdrp), are involved in the accumulation, minus-strand rna initiation, polyadenylation, and long-distance movement of viral rna (chen et al., 2002 (chen et al., , 2003 (chen et al., , 2005 huang et al., 2001; tsai et al., 1999) . polyadenylation modification is a signal for the accelerated degradation of rna in prokaryotes (sarkar, 1997) , chloroplasts (kudla et al., 1996; lisitsky et al., 1996) and mitochondria (chang and tong, 2012) . in contrast, poly(a) tails play an important role in mrna translation and turnover (proudfoot, 2011; sachs and wahle, 1993; weill et al., 2012) and the movement of mrna from the nucleus to the cytoplasm in eukaryotes (huang and carmichael, 1996; millevoi and vagner, 2010) . for positive-sense rna viruses, poly(a) tails have been implicated in rna stability, translation (bergamini et al., 2000) , and replication (guilford et al., 1991; tsai et al., 1999) . a positive correlation between the length of the poly (a)-tail and virus infectivity has been reported for several viruses including bamv , white clover mosaic virus (guilford et al., 1991) , poliovirus (sarnow, 1989) and cowpea mosaic virus (eggen et al., 1989) . furthermore, the 3′ poly(a) sequences of several positive-sense rna viruses, such as bovine coronavirus (spagnolo and hogue, 2000) and bamv (lin et al., 2007) , interact with the host proteins required for viral rna replication. the mechanism and elements involved in polyadenylating plus-strand bamv rna have to be elucidated. in eukaryotes, the polyadenylation of mrna occurs in the nucleus. however, large numbers of positive-sense rna viruses with a poly(a)-tailed genome complete their life cycle in the cytoplasm. although cytoplasmic polyadenylation has been reported previously in eukaryotes, the polyadenylation activities are mainly involved in oogenesis (the early development of many animal species) and regulating the diverse forms of translational activation in the cytoplasm (radford et al., 2008; villalba et al., 2011) . sequence analysis of the 5′ terminus of the bamv minus-strand rna synthesized both in vitro and in vivo contained predominantly 7 to 10 uridylates (from 1 to 15 u residues were reported), indicating that the minus-strand rna synthesis is initiated at approximately the 7th to 10th adenylate downstream from the 3′ utr of the genomic rna (cheng et al., 2002) . accordingly, only 7 to 10 adenylates at the 3′ end of the plus-strand rna are initially synthesized from the minus-strand rna template during viral replication. the 3′ utr of bamv rna also contains a polyadenylation signal (aauaaa) commonly found in eukaryotic mrna. the polyadenylation signal in the 3′ utr of bamv was shown to be involved in the synthesis of minus-strand viral rna and regulating the length of the poly(a) tail (chen et al., 2005) . in this study, we established a polyadenylation activity assay using a partially purified bamv replicase complex isolated from virus-infected plants. polyadenylation was detected on plus-strand rnas synthesized from endogenous and exogenous rna templates in this assay. furthermore, the effects of host plant extracts on polyadenylation and the 3′ sequence of the polyadenylated rna products were analyzed. models of the mechanism for bamv rna polyadenylation are proposed and discussed in this paper. we have demonstrated that a crude membrane-bound replicase complex isolated from bamv-infected n. benthamiana plants could generate genomic and subgenomic rnas from endogenous bamv rna templates (cheng et al., 2001; lin et al., 2005) . in this study, we test whether the same extract contains the polyadenylation activity. for a polyadenylation activity assay, rna products synthesized from endogenous rna were labeled with radioactive [α-32 p] atp during rna replication and subsequently analyzed based on digestion with the rnase a/t1 (specific on the unpaired pyrimidines and guanine, respectively) digestion (fig. 1a) . the poly(a) rna fragments with a higher radio-labeling intensity were generated by np40-treated replicase complex rather than by untreated replicase complex (fig. 1b, lanes 2 and 3) . the sizes of the poly(a) fragments ranged from approximately 40 to 200 nts, which is consistent with the reported 3′ poly(a) length of bamv genomic rna (chen et al., 2005) . all subsequent polyadenylation activity assays were conducted using np40 solubilization. to confirm that the radioactively labeled fragments (the poly (a) fragments) generated by rnase a/t1 digestion were polyadenylates, the replication products were labeled with either [α-32 p] atp or [α-32 p] utp. the smeared radioactive rna fragments were detected in the presence of [α-32 p] atp (fig. 1c , lane 2), but not [α-32 p] utp (fig. 1c, lane 4) . furthermore, the radioactive rna fragments generated by rnase a/t1 digestion were hybridized with oligo-dt 39 and subjected to rnase t2 (cleavage at the unpaired nucleotides) digestion ( fig. 2a) . the results indicate that the treatment converted the smeared radioactively labeled rna fragments (fig. 2, lane 2) into a 39-base ladder (up to 195 bases) (fig. 2, lanes 3-5) . therefore, the rna smears detected on the gel were likely polyadenylates generated in vitro by the replicase complex. overall, these results indicate that the partially purified bamv replicase complex is capable of producing polyadenylated rnas from endogenous rna templates. host factors are involved in the polyadenylation activity of the bamv replicase complex the partially purified bamv replicase complex isolated from bamv-infected n. benthamiana leaves can be separated into 10 fractions (bamv-infected fraction; bf1 to 10) in a 20% to 60% sucrose gradient (cheng et al., 2001; lin et al., 2005) . the highest endogenous rna polyadenylation activity was detected in bf3 ( the radioactive rna products were digested with (lanes 2 and 3) or without (lane 1) 6 mg/ml rnase a and 2000 u/ml rnase t1 in dmso buffer. (c) rna products were labeled with [α-32 p] atp (lanes 1 and 2) or [α-32 p] utp (lanes 3 and 4) in the in vitro rna replication assay. the radioactive labeled products were treated with rnase a and t1 (lanes 2 and 4), analyzed on an 10% denaturing gel, and visualized by phosporimaging analysis. the sizes of 32 p-labeled rna markers (m) are indicated on the right. the addition of bamv 3′ utr containing the aauaaa sequence suppresses the polyadenylation activity of the bamv replicase complex the aauaaa sequence has been implicated in regulating polyadenylation efficiency in bamv (chen et al., 2005) . to further demonstrate that the aauaaa motif in the 3′ utr of bamv rna is involved in the regulation of polyadenylation, we tested the inhibitory effects of different bamv 3′ utr-related rna molecules on polyadenyaltion. rna r138/10a contained the bamv 3′ utr with 10 adenylates at the 3′ end, whereas r138/10a/δbulge and r138/10a/δil were r138/10a with a bulge and an internal loop deletion, respectively, ( fig. 4) . the effects of the 3 rna molecules on polyadenylation activity were evaluated in the in vitro replication assay. the results showed that the signal intensity of polyadenylation was largely unaffected by the addition of rna in which the conserved aauaaa sequence was deleted (r138/10a/δil) (fig. 4c , lane 2), but reduced and shortened in size by the rna containing the aauaaa motif (r138/10a and r138/10a/ δbulge) (fig. 4c, lanes 3 and 4) . it is possible that the exogenous aauaaa motif affected polyadenylation by sequestering one or more of the polyadenylation factors in the replicase complex or by interfering directly with the enzyme complex (chen et al., 2005) . to test whether the replicase preparation could polyadenylate exogenous plus-strand rna templates, 5′ end-labeled radioactive bamv plus-strand rna substrate containing the entire 3′-utr and 7, 15, or 40 adenylates (r138/7a, r138/15a, or r138/40a, respectively), were subjected to the polyadenylation activity assay. the results indicated that the polyadenylation signal was not detected on any of the templates in this assay (data not shown) because of inefficient polyadenylation or because the substrates were inadequate for polyadenylation. to overcome these two possible problems, we used a minigenome (a hybrid bamv satellite/bamv minigenome) (huang et al., 2009 ) as template for rdrp assay and then performed 3′ rapid amplification of cdna ends (3′ race) to analyze the 3′ terminal sequence after the reaction. bamv minigenomes were demonstrated to be suitable templates for an in vitro replication assays (huang et al., 2009) ; therefore, the exogenous plus-strand or minus-strand bamv minigenome was added to the reaction to help determine whether the poly(a) tail could be added to the 3′ end of preformed or newly transcribed plus-strand rna, respectively. the bamv minigenome is constructed to comprise the coding region of bamv satellite rna and the utrs of bamv. the template rnas of the plus-strand and minus-strand minigenomes, bsm13 and bsm13(−), respectively, were prepared (fig. 5a ). the 3′ end sequences of the bsm13 templates and the plus-strand rna products synthesized from the bsm13(−) templates were examined using 3′ race analysis after the the radioactive rna products from the in vitro rna replication assay were incubated in the absence (lane 1) or presence of rnase a and t1 (lanes 2 to 5) in dmso buffer. after the rnase treatment, the products were hybridized with oligo-dt 39 and digested with increasing amounts (20, 40, and 60 u) of rnase t2 (lanes 3, 4, and 5). the products were separated on an 10% denaturing gel and visualized by phosphorimaging analysis. the sizes of 32 p-labeled rna markers (m) are indicated on the right. containing bamv replicase complex (bf3 to 5) treated with (lanes 2, 4, and 6) or without (lanes 1, 3, and 5) np40. (b) the soluble fraction hs30 (supernatants from 30,000 â g ultra-centrifugation) from healthy n. benthamiana plants was centrifuged through a 20-60% sucrose gradient to obtain fractions hf1 to 10 and solubilized by np40. bf3 (lane 13) and bf4-5 (lane 2) from bamv-infected plants, and bf4-5 supplemented with hs30 (lane 1) or hf1 to 10 (lanes 3 to 12) were subjected to polyadenylation activity (top panel) and rna replication (bottom panel) assays. for polyadenylation assay, the rna replication products were labeled with [α-32 p] atp and digested with rnase a and t1 (top panel). the replicative form (rf) of bamv genomic rnas (arrow) labeled with [α-32 p] utp was expected to be 6.4 kb (bottom panel). the radioactive labeled products were separated on a 1% agarose gel and visualized by phosphorimaging. in vitro replication (fig. 5b) . the dna sequence of 12 clones derived from the bsm13 template showed that all clones contained a 20adenylate sequence downstream of the 3′ utr (fig. 5c ). the 20 adenylates were possibly derived from the race primer dt 20 , which can prime to the bsm13 rna template harboring the -a 13 gaauu sequence (gaauu is derived from ecori when preparing the run-off transcription) (fig. 5a) . the results indicate that either no or considerably few adenylates were added to the 3′ end of bsm13 rna templates. in contrast, the newly synthesized plus-strand rnas derived from bsm13(−) included 20 to 74 consecutive adenylates (fig. 5c ). because the 5′-terminal sequence of bsm13(−) is gggu 13 -(ggg is derived from the t7 promoter; fig. 5a ), the 3′-end sequence of the newly synthesized plus-strand rna copied from the template should contain -a 13 ccc. the results indicate that adenylates were added to the 3′ end of newly synthesized rnas. although the clones containing the polyadenylates ranging from 20 to 31 (10 clones) did not result from polyadenylation, the remaining clones with consecutive polyadenylates ranging from 35 to 74 (7 clones) did. because the template provides 13 us for copying and the primer dt 20 for reverse transcription, only rnas with active polyadenylation can have more than 33 adenylates in the clones. in 2 clones (containing an -a 15/19 c 3 a 20 sequence), the poly (a) track is interrupted by a ccc sequence, which is derived from the template ggg. the results indicate that polyadenylation might occur before reaching the ggg sequence (only 13 uridylates in the template; fig. 5a ). however, the polyadenylation enzyme complex could return to the template and copy the g 3 sequence during polyadenylation. this could be due to the enzyme copying slippage, although the cloning artifact could not be disregarded. overall, the results indicate that polyadenylation began before the replication enzyme complex reached the ggg sequence and that bamv rna replication was accompanied by an exonuclease activity in which the c 3 sequence was removed before the poly (a) tail was added. polyadenylation on the preformed genomic rna requires at least 4 adenylates at the very 3′ end in the mentioned results, we could not detect adenylates longer than 20 in all the clones derived from bsm13 after the reaction (fig. 5c) . because bsm13 is a suitable template for minus-strand rna synthesis, the in vitro reaction could be more efficient in rna replication than in polyadenylation. to exclude minus-strand rna synthesis from the reaction, we included only atp and gtp in the rdrp assay. to test the requirements of the 3′ end structure for polyadenylation, we used the rna transcripts derived from pcr products to obtain the precise sequence instead of using run-off transcripts, which contain a nonviral sequence (the ecori site). bsm13/noa, −/4a, −/7a, and −/13a templates were prepared and incubated with the bamv replicase complex, and atp and gtp were included in the reaction. the reaction products were examined using a head-to-tail ligation and rt-pcr analysis (wu and brian, 2010) instead of 3′ race in which the primer (dt 20 ) might cover the authentic 3′-end sequence. the sequence results indicate that no adenylate can be added to the template without a preexisting adenylate (table 1 , bsm13/noa). long preexisting adenylates at the very 3′ end of the template (with 4, 7, and 13 as) cause efficient for polyadenylation (22%, 46%, and 68%, respectively). the results indicate that the partially purified bamv replicase complex is capable of polyadenylating the preformed exogenous rna templates. the rna template required at least 4 adenylates to obtain a minimum of 22% efficiency for polyadenylation. the bamv is a positive-sense rna viruses with a poly(a)-tailed genome. the results (figs. 1 and 2 ) of this study confirm that bamv positive-sense rna is polyadenylated. compared with the untreated bamv replicase complex preparation, the np40-treated preparation resulted in enhanced poly(a) signal intensity in the products of the in vitro rna replication assay (fig. 1b) . this was probably due to particular conformational changes or subunit rearrangements in the enzyme complex induced by the detergent np40. numerous studies have not connected the bamv infection cycle with the nucleus. therefore, it is unlikely that the addition of the poly(a) tail to the bamv genome during its replication cycle is processed by the nuclear polyadenylation machinery of the host cell. plants encode enzymes, such as nucleotidyltransferase and polynucleotide phosphorylase, for the rna polyadenylation involved in the poly(a)-stimulated rna degradation pathway in mitochondria and chloroplasts (lange et al., 2009; zimmer et al., 2009) . accordingly, the possibility that the polyadenylation of viral rna occurs through this type of host-encoded cytoplasmic proteins cannot be disregarded. poly(a)-tailing of the newly synthesized plus-strand rna from the minus-strand bamv minigenome was clearly detected (fig. 5) , indicating that polyadenylation coupled with the plus-strand rna synthesis is efficient and that the aauaaa motif is accessible to the replicase complex during plus-strand rna synthesis. in contrast, incubating exogenous plus-strand rnas with the bamv replicase complex does not lead to the polyadenylation of bsm13/noa rna (table 1) , but leads to a minimal activity of approximately 22% for bsm13/4a rna. a presence of a large number of preexisting adenylates at the 3′ end of the templates (bsm13/7a and bsm13/13a) resulted in the efficient addition of adenylates (46% and 68%, respectively; table 1 ). it is possible that the 3′ utr of bamv is arranged in a way that the polyadenylation signal aauaaa is hidden and not initially accessible to the enzyme complex. when the 3′ end extended with more adenylates such as 7a and 13a (copying from the templates during replication), the pseudoknot structure formed (fig. 4b ) and the aauaaa motif was exposed for the switch from replication to polyadenylation. when the competitor rnas were added to the replicase preparation, rdrp activity persisted, but polyadenylation activity decreased if the competitor rna contained the aauaaa sequence (fig. 4c) . these results suggest that the 2 enzymatic activities (rdrp activity and polyadenylation) could be separated during bamv replication. the plus-strand rna copies from the minusstrand rna template with rdrp activity until the end of the template that the plus-strand rna forming the pseudoknot structure and exposing the aauaaa sequence. the host factor (s) could recognize the sequence and switch to polyadenylation activity. therefore, the competitor rna containing the aauaaa sequence could eliminate the host factor(s) in replicase preparation through competition. in eukaryotes, the polyadenylation of mrna precursors may be a two-phase process. approximately 9 adenylates are added to the 3′ end of the rna containing a conserved aauaaa sequence during the first phase. subsequently, independent of the aauaaa motif and the associated factors, the polyadenylation complex extends efficiently from the short oligo(a) to generate a full-length poly(a) tail (approximately 200 adenylates) (sheets and wickens, 1989) . in general, the polyadenylation process of bamv plus-strand rna is similar to that of eukaryotic mrna. these similarities include the requirements of the aauaaa motif (chen et al., 2005) , the synthesis of a short oligo(a) (derived from the minusstrand template), and the synthesis of a poly(a) tail of a similar length. bamv minus-strand rna synthesis was shown to initiate between the seventh to tenth adenylate abutting the 3′ utr on plus-strand rna (cheng et al., 2002) , leading to the presence of a few uridylates at the 5′ end of the minus-strand rna. these uridylates in minus-strand rna could serve as the template for the first few adenylates of the poly(a) tail on plus-strand rna. these adenylates could be used as the primer for the synthesis of up to 300 adenylates (chen et al., 2005) . moreover, mutation of the aauaaa sequence in the bamv system led to poly(a) tails of a shorter length (chen et al., 2005) , indicating that the polyadenylation complex of the bamv replication machinery is associated with the aauaaa motif during the tailing process. adding particular subcellular fractions (hs30 and hf1) from uninfected plants increased the polyadenylation activity but not adenylating range bsm13/no a 0 (0%) a 9 (100%) 0 (0%) 0 a bsm13/4 a 1 (6%) 13 (72%) 4 (22%) 6-18 as bsm13/7 a 0 (0%) 7 (54%) 6 (46%) 9-13 as bsm13/13 a 9 (29%) 1 (3%) 21 (68%) 14-35 as a data are numbers of clones, with percentages in parentheses. the rdrp activity (fig. 3b ). host factors are possibly responsible for regulating the switch between template-dependent rna synthesis and the polyadenylation mode. in another possible mode of polyadenylation, the enzyme complex simply shuttles back and forth on the oligo(u) region of the template to generate the poly (a) tail, which is similar to the poly(a) tail generation process of influenza virus mrna synthesis (poon et al., 1999) . terminal adenyltransferase activity has been demonstrated in the nsp4 (rdrp) of sindbis virus (tomar et al., 2006) and the 3d pol of poliovirus (neufeld et al., 1994) . in contrast, an internal poly (a) tract in the 3′ utr of dulcamara mottle virus (tzanetakis et al., 2009) , chikungunya alphavirus (khan et al., 2002) , and hibiscus latent singapore virus (srinivasan et al., 2005) has been proposed to lead to viral polymerase slippage during rna replication. the replicase complex isolated from bamv-infected plants contains both replication activity and polyadenylation activity. these activities could be regulated with different host factor (s) and replication can switch to polyadenylation during genomic rna synthesis. pbsm13 was constructed previously to allow in vitro synthesis of bsm13 rna, a chimeric bamv minigenome rna containing the open reading frame from bamv satellite rna flanked by the 5′ and 3′ utrs of bamv genomic rna (huang et al., 2009) . for in vitro synthesis of bsm13(−), a minus-strand bamv minigenome rna, pbsm13(−) was constructed from pbsm13 by pcr using the bamhi-bamv5′+1 (5′gaattcgaaaaccactccaaacgaaa3′) and t7-13t bamv3′end (5′ggtacctaatacgactcactatagggttttt-ttttttttggaaaa3′) primers. the pcr product was cloned into a smai-linearized puc119 vector and verified by dna sequencing analysis. templates for in vitro transcription were generated by pcr instead of restriction enzyme linearized-plasmids to get the expected 3′ ends. pbsm13 was used as the template in the pcr. the forward primer t7-bamv5′+1 (5′gctctagataatacgactcac-tatagaaaaccactccaaacgaa3′) and reverse primers bamv 3′ end noa (5′ggaaaaaactgtaga aaccaaaag3′), bamv 3′end 4a (5′ttttggaaaa aactgtagaaacc3′), bamv 3′end 7 a (5′ttt-ttttggaaaaaactgtagaaa3′), and bamv 3′end 13a (5′tttttt-tttttttggaaaa3′) were generated bsm13/noa, −/4a, −/7a, and −/13a. the pcr products were gel purified and used for in vitro transcription by t7 rna polymerase (promega, madison, wi, usa). after transcription, the mixture was treated with rq1 rnase-free dnase (promega, madison, wi, usa) at 37 1c for 30 min. transcripts were extracted with phenol/chloroform and precipitated with ammonium acetate and ethanol. rna concentration was determined by spectrophotometry and agarose gel electrophoresis. in vitro bamv rna replication assay bamv replicase complex was purified from bamv-infected n. benthamiana leaves as described previously (cheng et al., 2001; lin et al., 2005) . in brief, the healthy and bamv infected leaves were kept in −80 1c. the frozen leaves were homogenized in rdrp complex extraction buffer (50 mm tris-hcl ph 7.6, 15 mm mgcl 2 , 120 mm kcl, 0.1% β-mercaptoethanol, 20% glycerol, 1 μm pepstatin a, 0.1 mm phenylmethenylsulphonyl fluoride). the homogenate was filtrated through miracloth (calbiochem) and centrifuged at 500 â g for 10 min to remove the cell debris. the clarified supernatant was applied to a 30,000 â g ultracentrifugation for 35 min, and the pellet (p30) was re-suspended in solubilization buffer (50 mm tris-hcl ph 8.2, 10 mm mgcl 2 , 1 mm dithiothreitol, 1 μm pepstatin a, 1 μm leupeptin). the 50-ml bamv rna replication assay containing 20 ml of 1.5% np40solubilized replicase fractions (cheng et al., 2001) , 4.8 mg/ml bentonite, 10 mm dithiothreitol, 10 mm mgcl 2 , 2 mm atp, 0.066 mm [α-32 p]-atp (3000 ci/mmol, dupont-nen, boston, ma ), and 2 mm utp, ctp, and gtp was carried out at 30 1c for 1 h. for labeling with [α-32 p]-utp, the concentrations of utp and atp were changed to 2 mm and 2 mm, respectively. the rna products were extracted with phenol/chloroform and precipitated with ethanol. for assays using exogenous templates, np40-treated bamv replicase complex was treated with micrococcal nuclease (usb corporation, cleveland, oh, usa) and the reaction stopped by adding 30 mm egta before the addition of the rna template (1 mg). for the competitor rnas, the cdna fragments containing the 3′ utr of bamv and its derivatives were pcr amplified with the primer set, t7-bamv+6228 (5′gcgaattctaatacgactcactatag-ggcgttgcatgatcg3′) and bamv 3′end 10a (5′ttttttttttgg-aaaaaactgtagaaa3′), and the plasmid templates, pbamv40a, −/δil and −/δbulge (chen et al., 2005) . these fragments were cloned into puc18 vector with the smai site. the resultant clones, pba138/10a, pba138/10a/δil and pba138/10a/δbulge, were linearized with bamhi. the rnas, r138/10a, −/δil and −/δbulge, were transcribed from these linearized plasmids and were gel-purified. in the competition rdrp assay, 200 ng of the competitor was added into the reaction. the radioactively labeled rna products generated in bamv rna replication assay were digested with rnase a (6 mg/ml) and rnase t1 (2000 u/ml) in dmso buffer (30.8% dmso and 13.2 mm tris-hcl ph 7.8) at 37 1c for 20 min. the products were extracted with phenol/chloroform, precipitated with ethanol, and dissolved in 1x urea sample buffer (4.5 m urea, 15 mm sodium-citrate ph 5.0, 0.5 mm edta, 0.01% xylene cyanol ff, and 0.01% bromophenol blue). the radioactively labeled rnas were denatured in boiling water for 2 min, separated on a 10% denaturing gel and visualized by a bas-1500 phosphorimaging analyzer (fujifilm, tokyo, japan). to demonstrate the presence of poly (a) tails, the rnase a/t1treated rna products were incubated with oligo-(dt) 39 at 70 1c for 10 min. the mixture was cooled down to 37 1c and treated subsequently with 20, 40, and 60 u of rnase t2 in dmso buffer at 37 1c for 20 min. the rna products were extracted with phenol/ chloroform, precipitated with ethanol, separated on a 10% denaturing polyacrylamide gel, and visualized by phosphorimaging. the products from polyadenylation assay were converted into cdna by using primer oligodt 20 /gc-clamp (5′gccccgggat-cctttttttttttttttttttt3′) and superscript iii reverse transcriptase (invitrogen, carlsbad, ca, usa). the cdnas were amplified by pcr for 35 cycles using forward primer basat+607 (5′gctgacg-cgtggctccctgaccgtg3′) corresponding to nt 607-631 of bamv satellite rna, reverse primer oligodt 20 /gc-clamp, and gotaq s flexi dna polymerase (promega). the pcr products were cloned into the pgem t-easy vector (promega). head-to-tail ligation of viral rna products and sequence analysis of the 3′ end rna rnas extracted from in vitro exogenous rna replication assay were dephosphorylated with shrimp alkaline phosphatase (promega, madison, wi, usa) and followed by phosphorylation with t4 polynucleotide kinase (new england biolabs, beverly, ma, usa). before head-to-tail ligation, the rnas were denatured at 95 1c for 5 min and then quick-chilled on ice. the denatured rnas were added to 1 ml of 10x ligase buffer and 1 u of t4 rna ligase 1 (neb) in a total of 10 ml reaction, and the mixture was incubated at 16 1c for 16 h. after phenol/chloroform extraction, the ligated rnas were converted into cdna with improm-ii™ reverse transcriptase (promega) and ba-77 (5′gggcgattgtagggga3′) priming at nt 62-77 in plus strand of bamv. the cdnas were further amplified by pcr for 35 cycles using forward primer basat+607, reverse primer ba-21 (5′gtttcgtttggagtggtt3′) corresponding to nt 4-21 of bamv, and gotaqs flexi dna polymerase (promega). the pcr products were sequenced after cloning into the pgem t-easy vector (promega). picornavirus ireses and the poly (a) tail jointly promote cap-independent translation in a mammalian cell-free system mitochondrial poly(a) polymerase and polyadenylation the aauaaa motif of bamboo mosaic virus rna is involved in minus-strand rna synthesis and plusstrand rna polyadenylation functional analysis of the cloverleaflike structure in the 3′ untranslated region of bamboo mosaic potexvirus rna revealed dual roles in viral rna replication and long distance movement structural and functional analysis of the 3′ untranslated region of bamboo mosaic potexvirus genomic rna the partial purified rnadependent rna polymerases from bamboo mosaic potexvirus and potato virus x infected plants containing the template-dependent activities the synthesis of minus-strand rna of bamboo mosaic potexvirus initiates from multiple sites within the poly (a) tail improvements of the infectivity of in vitro transcripts from cloned cowpea mosaic virus cdna: impact of terminal nucleotide sequences influence of the poly(a) tail and putative polyadenylation signal on the infectivity of white clover mosaic potexvirus sequences at the 3′ untranslated region of bamboo mosaic potexvirus rna interact with the viral rna-dependent rna polymerase role of polyadenylation in nucleocytoplasmic transport of mrna structural and functional analyses of the 3′ untranslated region of bamboo mosaic virus satellite rna complete nucleotide sequence of chikungunya virus and evidence for an internal polyadenylation site polyadenylation accelerates degradation of chloroplast mrna polyadenylation-assisted rna degradation processes in plants chloroplast phosphoglycerate kinase, a gluconeogenetic enzyme, is required for efficient accumulation of bamboo mosaic virus characterization of the infectivity of bamboo mosaic virus with its correlation to the in vitro replicase activities in nicotiana benthamiana nucleotide sequence of the genomic rna of bamboo mosaic potexvirus genome properties of bamboo mosaic virus addition of destabilizing poly (a)-rich sequences to endonuclease cleavage sites during the degradation of chloroplast mrna molecular mechanisms of eukaryotic pre-mrna 3′ end processing regulation identification of terminal adenylyl transferase activity of the poliovirus polymerase 3dpol direct evidence that the poly(a) tail of influenza a virus mrna is synthesized by reiterative copying of a u track in the virion rna template ending the message: poly(a) signals then and now translational control by cytoplasmic polyadenylation in xenopus oocytes poly(a) tail metabolism and function in eucaryotes polyadenylation of mrna in prokaryotes role of 3′-end sequences in infectivity of poliovirus transcripts made in vitro two phases in the addition of a poly(a) tail host protein interactions with the 3′ end of bovine coronavirus rna and the requirement of the poly(a) tail for coronavirus defective genome replication determination of complete nucleotide sequence of hibiscus latent singapore virus: evidence for the presence of an internal poly(a) tract catalytic core of alphavirus nonstructural protein nsp4 possesses terminal adenylyltransferase activity sufficient length of a poly(a) tail for the formation of a potential pseudoknot is required for efficient replication of bamboo mosaic potexvirus rna a tymovirus with an atypical 3′-utr illuminates the possibilities for 3′-utr evolution cytoplasmic polyadenylation and translational control translational control by changes in poly(a) tail length: recycling mrnas subgenomic messenger rna amplification in coronaviruses polyadenylation in arabidopsis and chlamydomonas organelles: the input of nucleotidyltransferases, poly(a) polymerases and polynucleotide phosphorylase this work was supported by grants from national science council (nsc 96-2752-b-005-012-pae and 97-2752-b-005-004-pae. key: cord-342901-ca2xxkb2 authors: lloyd, richard e. title: nuclear proteins hijacked by mammalian cytoplasmic plus strand rna viruses date: 2015-05-31 journal: virology doi: 10.1016/j.virol.2015.03.001 sha: doc_id: 342901 cord_uid: ca2xxkb2 abstract plus strand rna viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular rna-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building rna replication complexes. in the course of infectious cycles many nuclear-cytoplasmic shuttling proteins of mostly nuclear distribution are detained in the cytoplasm by viruses and re-purposed for their own gain. many mammalian viruses hijack a common group of the same factors. this review summarizes recent gains in our knowledge of how cytoplasmic rna viruses use these co-opted host nuclear factors in new functional roles supporting virus translation and virus rna replication and common themes employed between different virus groups. viral spread and ultimately pathogenesis require efficient replication in key host cells that aid spread of the virus within hosts and throughout host populations. rna viruses are typically small, encoding as little at three genes, and thus must rely on many host factors interacting with viral rnas to assist with essential replication functions, and control many interaction points within host cells to promote replication. this often results in redirecting host metabolism on several levels to support the infection and at the same time suppress innate host defense systems that are triggered. comparing plus and minus stranded rna viruses, there are stark differences at the time of uncoating of genomic viral rna in the cytoplasm. the plus strand rna virus genome that is released is naked, however the minus strand rna virus genome is completely enclosed in a functional nucleocapsid with rna replicase poised ready to produce transcript mrnas. thus, the plus strand virus rna can, and does, interact with many host rna binding proteins (rbps), whereas there is a little opportunity for minus strand virus genomic rna to interact directly with host rbps. most rna-binding proteins are nuclear shuttling proteins and many more nuclear rbps have been reported to play roles in replication of plus strand rna viruses than minus strand rna viruses. accordingly, this review focuses heavily on plus stranded rna viruses, particularly mammalian viruses. rna viruses interact with a multitude of host factors during the course of infection. several screening approaches have been employed to identify which of the 15-20,000 proteins that may be expressed in a given cell are host factors required for rna virus replication. these include genetic screens in yeast that implicated 130 proteins that could affect plant virus replication (tomato bushy stunt virus) (jiang et al., 2006) and about 100 genes that affect brome mosaic virus (kushner et al., 2003; panavas et al., 2005) . rnai knockdown studies in mammalian cells with hepatitis c virus (hcv), dengue virus (denv) and west nile virus (wnv) have identified several hundred other genes that affect virus replication. however, many or most of these may function quite indirectly, affecting pathways that produce metabolites or products the virus needs, movement or trafficking of constituents that are directly required, factors that control divalent cation fluxes and atpase pumps, the stress or innate immune activation levels that counteract general cellular biosynthetic potential, or include general off-target effects from the silencing step. it is likely that the spectrum of factors that directly interact in meaningful ways with virus rna and proteins will be larger than that known today, but also smaller than the first lists that have emerged from such screenings (box 1). recently the novel approach of thiouracil cross-linking mass spectroscopy (tux-ms) was used to more precisely identify host proteins bound to poliovirus rna during replication. this procedure identified all proteins known to interact with enterovirus rna, plus 66 additional factors previously unidentified (lenarcic et al., 2013) . eight of the new proteins were chosen and validated as playing roles in replication, indicating this new method is powerful and should be applied to other virus systems. however, standard molecular biology and biochemical approaches will still be required to tease out the functions and impact of each of these factors on virus replication. proteins that interact with viral rna do not present interesting targets for antiviral development unless it is determined that they play critical roles in virus replication. plus strand rna viruses must translate incoming viral genomic rna as the first biosynthetic step in replication cycles, thus, control of translation becomes the first battleground with the host that involves co-opted nuclear factors. it makes sense for the virus to utilize the host factors it commonly encounters at sites of replication. thus, translation regulation involves virus co-opting of cellular translation factors. these are mostly cytoplasmic resident proteins since translation is a cytoplasmic process. however, translation does not occur on transcripts that are naked and devoid of rna-binding proteins, rather, cellular transcripts are continually bound to a host of rna binding proteins from the instant they emerge from rna polymerase during their synthesis. in mammalian cells, rna binding proteins control most aspects of rna biology and the rna cycle; from splicing, transport out of the nucleus, cellular function, transcript-specific translation control, and cytoplasmic localization and mrna half-life. mammalian cells encode hundreds of rbps ( $ 860), most with several splice variants (castelló et al., 2012) . the cytoplasmic milieu encountered by plus strand rna virus genomes as they are released from capsids is poised to greet the interloper as any other mrna, with a ready store of rna binding proteins ready to interact and impart functions. no wonder viruses have evolved to interact with rbp in diverse ways to promote replication. recent research indicates that interactions of viruses with rbp can both benefit and inhibit virus replication, depending on stages of the replicative cycle. rbp interaction with complexes may afford molecular switching events that promote rna replication over translation as one example. the complete virus "interactome" will contain proteins that provide a diverse range of functions during virus replication; including protein and rna chaperone functions, transport functions, helicases, factors and enzymes that regulate vesicular traffic, membrane functions and lipid metabolism. notably, many of these are cytoplasmic proteins, thus beyond the purview of this review. most of the nuclear rbps are constantly shuttling between the nucleus and cytoplasm, thus, the definition of nuclear factor in this context refers solely to shuttling proteins that are more concentrated in the nucleus under normal physiological conditions. nuclear pore disruption: how cytoplasmic viruses get their stuff an infecting virus may encounter sufficient nuclear shuttling proteins to initiate translation or the first rounds of rna replication. however, rapidly expanding rna virus replication requires a growing storehouse of supplies and will quickly expend the limited cytoplasmic supply of many nuclear factors. it was noticed early during studies of poliovirus-infected cells that many nuclear resident proteins showed improper cytoplasmic localization, including ptb, la autoantigen, sam68, nucleolin and others (back et al., 2002; mcbride et al., 1996; meerovitch et al., 1993; waggoner and sarnow, 1998) . in fact, many viruses disrupt trafficking of proteins and mrnps at the nuclear pore as a mechanism to restock the cytoplasmic storehouse with needed supplies. this dysregulation is usually required for efficient viral replication and can involve increased export of outbound cargo from the nucleus and/or blockage of inbound cargo from the cytoplasm. detailed descriptions of the mechanisms involved are beyond the scope of this article, but recent reviews cover advances in virus disruption of nuclear pores (le sage and mouland, 2013; yarbrough et al., 2014) . the range of viruses known to disrupt nuclear-cytoplasmic traffic is diverse and includes viruses that replicate in the cytoplasm (enterovirus, cardiovirus, coronavirus, rhabdovirus) and viruses that replicate in the nucleus (influenza, hiv-1, herpes simplex virus 1, adenovirus). the mechanisms that disrupt nuclear transport are also diverse. just two examples are enteroviruses that employ viral 2a proteinase to cleave at least three nuclear pore proteins (nup 62, nup 98, and nup 153) (park et al., 2008 (park et al., , 2010 watters and palmenberg, 2011) and cardiovirus that employs l protein to induce a phosphorylation cascade that results in hyperphosphorylation of nup 98, nup62, nup153 and nup 214 as well as dysregulation of the pore complex (bacotdavis and palmenberg, 2013; basta et al., 2014; porter and palmenberg, 2009; ricour et al., 2009; watters and palmenberg, 2011) . in addition, l protein directly binds and inhibits the active cellular transport protein ran gtpase (bacotdavis and palmenberg, 2013) . notably some of the nup targets are the same for both picornavirus genera, an example of convergent evolution providing two different approaches to accomplish similar functional goals. picornaviruses such as poliovirus (pv) and encephalomyocarditis virus (emcv) do not contain m7gtp cap structures on genomic rna to recruit ribosomes, instead they use an internal cap-independent mode of translation that requires a large folded rna structure to recruit ribosomes called an internal ribosome entry site (ires). ires elements have complex rna folds and are only active when bound with specific proteins called ires transactivating factors (itafs) that are thought to provide rna chaperone functions (table 1) . itafs are not canonical translation initiation factors that function in cap-dependent translation but are proteins known to play primary roles in other aspects of rna biology in the cell. itafs are all of cellular, not viral origin, which makes sense for plus strand viruses because the ires must function before any viral proteins can be synthesized. the ires plus the required itafs and canonical translation factors that make up a functional unit are referred to as iresomes because they function together as a complex. the functional relationships between itafs, canonical translation factors and ribosome recruitment are still unclear but significant strides have been made in understanding them in three virus systems. although the first itafs were discovered in the context of viral ires translation, many of the same itafs are thought to play similar roles promoting cap-independent translation for cellular ires elements. for example, ptb is an itaf for bip, bag1, apaf-1, unr, p53 iress; hnrnp a1 is an itaf for cyclin d1 and c-myc iress; and hnrnp c1/ c2 is an itaf for xiap and c-myc iress (reviewed in ). there are distinct classes of virus iress that are referred to by a somewhat inconsistent and evolving nomenclature. picornavirus ires elements can be classified into at least five types based on structure, limited sequence homology and phylogeny. type 1 iress are large $ 450 nucleotide segments that are encoded by poliovirus and other enteroviruses; type 2 iress are similarly large and occur in cardioviruses (emcv), aphthoviruses (fmdv) and parechoviruses; and type 3 ires occurs only in hepatitis a virus (hav). type 4 iress (sometimes called class 2 or type 3) are smaller $ 330 nucleotide segments and occur in non-picornaviruses (hepatitis c virus and pestiviruses) but are also found among the newer picornavirus families teschovirus, sapelovirus, senecavirus, tremovirus. type 5 iress are encoded in the newly defined picornavirus genera kobuvirus, salivirus and paraturdivirus (sweeney et al., 2012) . one of the first host proteins ever shown to interact with picornavirus rna by uv-crosslinking was polypyrimidine tract binding protein 1 (ptb1, shortened to ptb) . ptb is a shuttling, but mostly nuclear resident protein that associates with pre-mrnas and plays roles in pre-mrna processing, alternative splicing, mrna metabolism and transport (kafasla et al., 2010; sawicka et al., 2008) . ptb binds polypyrimidine tracts in pre-mrna introns to repress exon inclusion but can actually bind quite varied rna structures. thus, ptb can also stabilize certain mrnas against degradation by binding to the 3 0 untranslated regions. in virus replication schemes ptb plays roles that support and stimulate cap-independent translation driven by picornavirus and hcv rna. ptb exists as one of three alternatively spliced isoforms (ptb1, ptb2, ptb4) and contains four-rna binding domains (rbds) of the rnp1/rnp2 class (kafasla et al., 2010; sawicka et al., 2008) . though ptb was the first, a wide spectrum of factors, largely nuclear rbps, have also been proposed as poliovirus itafs, including lupus autoantigen (la) (meerovitch et al., 1993 (meerovitch et al., , 1989 , poly(rc)binding proteins (pcbps) (blyn et al., 1996; gamarnik and andino, 1997) , upstream of n-ras (unr) (anderson et al., 2007; boussadia et al., 2003; hunt et al., 1999) , srp20 (bedard et al., 2007) and glycyl-trna synthetase (gars) (andreev et al., 2012) (table 1 ). in order to make sense of the relative impact of this range of factors on basic translation, pestova and hellen have used in vitro translation initiation experiments reconstituted with a complete set of purified factors to define the minimal set of factors absolutely required for ires translation. in these reconstituted translation systems, the hcv class of ires minimally requires no itafs and only translation factor eif3 to bind ribosomes (hellen, 2009; pestova et al., 1998) . for both picornaviruses emcv and pv, several canonical translation factors and only one itaf is minimally required. emcv requires only ptb as an itaf and poliovirus requires only pcbp2 as an itaf to initiate translation (pestova et al., 1996; sweeney et al., 2014) . however, minimal requirements in vitro likely do not reflect the conditions of severe competition among mrnas for ribosomes in cells, suggesting the other factors proposed as itafs may play less fundamental, but still critical roles during infection and can contribute to pathogenesis. in addition, certain itafs play crucial roles in regulating the conversion of translation-competent genomic rnas into replicationcompetent rnas. ptb was known as a nuclear splicing factor when its role as an itaf of poliovirus and emcv was discovered; a classic hijacked nuclear protein pressed into a new role required for the virus. however, ptb is now known to also support cap-independent translation of cellular ires-containing mrnas that were discovered after the viral iress. these include apaf-1, bag-1 and mutant forms of c-myc transcripts associated with more aggressive tumor growth sawicka et al., 2008) . thus ptb can interact with a wide range of rnas to promote cap-independent translation and may be considered a pro-translation, general itaf. continuous investigations of ptb have revealed biochemical details about how it supports iresome function in virus translation. the rna binding function of ptb is modular and split between four rna-binding domains (rbds) that are distributed along an overall extended and flexible structure. this extended flexible nature of ptb is important for ires function. the ptb rbds recognize short pyrimidine-rich sequences but have distinct rna structural preferences. the two n-terminal rbds (rbd1 and rbd2) recognize short pyrimidine tracts contained in loops, while the rbds3-4 preferentially bind to larger flexible rna sequences. these features give ptb the ability to bind to a variety of rna structures and enables ptb to function as a versatile adapter protein that facilitates formation of many rna-protein regulatory complexes (clerte and hall, 2009) . recently, emcv and poliovirus ires structures have been used most extensively as models to study the itaf functions of ptb. mapping studies indicated that two ptb moieties bind the emcv ires, one upstream in a non-essential region of the ires and another in the ires core. in the core, the orientation of a single ptb binding to ires was determined by binding of ptb mutants and hydroxyl radical probing. rbd1 and rbd2 bind near the 3 0 end of the core ires sequence, and rbd3 binds near the 5 0 end. binding of ptb to multiple regions on the ires simultaneously enables stabilization and constraint of the ires tertiary structure and is consistent with multiple rbd-rna interactions proposed in the splicing functions of ptb (oberstrass et al., 2005; kafasla et al., 2009) . this illuminates how an itaf mechanistically provides an rna-chaperone function. ptb is thought to promote rna looping also, partly because rbds 3 and 4 bind rna in anti-parallel directions (oberstrass et al., 2005; lamichhane et al., 2010) . further work established which of the multiple rbd-ires interactions are critical for itaf function. using ptb with point mutations in various rbds, it was shown that both poliovirus and emcv ires rnas required simultaneous interaction with three of the four ptb rbds, however the list of most critical rbds differed strand shown in red) breathes allowing cloverleaf and anti-cloverleaf to form, stabilized by binding pcbp and hnrnp c respectively. polymerase replicase complex also builds on 5 0 cl and initiates replication on negative strand template that is properly positioned for precise-end initiation. hnrnp c can also interact with sl on the 5 0 end of the negative strand that also requires breathing to form. in this scenario pabp may be able to rebind to poly(a) tail and hnrnp oligomerization and pabp interaction with pcbp may facilitate genome circularization in double-stranded rf intermediate. between the viruses (kafasla et al., 2011) . this indicates that the modular and elongated nature of ptb can adapt to disparate rna structures using different sets of rbds to provide similar chaperone function. besides providing an rna chaperone function, ptb also provides a second critical function as it helps recruit or position the canonical translation initiation factor eif4gi on the ires (fig. 1a ). in the case of pv, both ptb and eif4g bind the same rna stem loop structure (stem loop v) comprising much of the ires core. the orientation of ptb binding is such that rbds 3 and 4 bind the base of stem loop v, whereas rbds 1 and 2 bind the stem loop itself in the same region proposed to bind the central domain of eif4gi. the two proteins do not compete for binding, rather ptb is thought to stimulate ires activity by ensuring that eif4gi binds in the correct orientation and position to function properly for ribosome entry at a nearby aug (kafasla et al., 2010) . other picornaviruses also utilize ptb as an itaf. coxsackievirus b3 is another enterovirus closely related to poliovirus with a type 1 ires, thus there is no surprise that ptb interacts with cvb3 rna and promotes translation in a similar fashion as an itaf. however, ptb also interacts with the cvb3 3 0 utr and this interaction may stimulate ires translation through long-range looping or genome circularization bridged by ptb (verma et al., 2010) . foot-andmouth disease virus ires is a type 2 ires structure distantly related to emcv, sharing little sequence homology but some secondary structural similarity. however, fmdv requires both ptb and itaf 45 (also known as ebp1) to minimally support translation, whereas emcv requires only ptb. the fmdv ires also binds three of four ptb rbds, where rbds 3 and 4 bind distally located regions of the ires and can function as a minimal ires when supplied as a truncated form of ptb (song et al., 2005) . changes in ires structure were analyzed to compare effects of itaf binding on rna conformation shifts in emcv and fmdv iress. despite the different itaf requirements, in both cases when itafs interacted with their cognate iress, similar conformation changes occurred where two domains were brought into closer compacted proximity (yu et al., 2011a) . thus, ptb provides the same structural role building the functional iresome, whether alone or in conjunction with other itafs. poly(rc)-binding protein (pcbp) is the only obligate itaf for enteroviruses using type 1 iress (sweeney et al., 2014) . unlike ptb, that binds widely separated sites on the emcv ires, pcbp binds to a restricted area of stem loop iv of the pv ires (fig. 1a) . this binding region of sliv is distant from the ires core in the two dimensional secondary structure (blyn et al., 1996) . but pcbp also binds stem loop b of the cloverleaf structure (cl) at the 5 0 terminus of enterovirus rna (fig. 1) andino, 2000, 1997) . there are four pcbp isoforms, however pcbp1 and pcbp2 are expressed in more abundance and both bind pv ires. pcbp1/2 isoforms can form dimers or heterodimers and each can interact with pv ires rna, but pcbp2 plays a more dominant role. pcbp binds rna through three kh domains and interacts 6-fold more strongly with the ires than cloverleaf rnas in isolation (gamarnik and andino, 2000) . pcbp2 is the isoform required for both translation initiation and also for rna replication. kh1 is the primary domain that interacts with pv ires domain iv (silvera et al., 1999; walter et al., 2002) . for closely related cvb3, individual kh1 and kh3 domains of pcbp bind to ires stem loop iv, kh1 interacts with subdomain iv/c rna, whereas kh3 interacts with subdomain iv/b (zell et al., 2008a) . another host factor, srp20, interacts with pcbp2 through its kh3 domain and was found to be critical for itaf function of pcbp2 in cells. in vitro translation in hela cell extracts depleted of srp20 are deficient in supporting poliovirus translation initiation and srp20 sirna knockdown in hela cells restricted poliovirus translation. (bedard et al., 2007) . like other itafs, srp20 is strongly relocalized from the nucleus to cytoplasm during poliovirus infection. srp20 may bind pcbp2 rather than viral rna directly since deletion of its rrm-rna interaction motif does not alter its localization during infection, however, this truncated form strongly repressed virus translation, likely via a dominant negative process (fitzgerald and semler, 2011) . many other factors have been shown to promote virus translation and have considered itafs or itaf ancillary factors, but compared to pcbp and ptb much less is known about their function at the biochemical level in promoting ires translation. the binding sites of some of these other itafs are poorly characterized. la enhances and corrects aberrant translation of pv in reticulocyte lysates that lack other itafs (meerovitch et al., 1993) and 40s ribosome subunit binding to pv ires is inhibited by a dominant negative la protein (costa-mattioli et al., 2004) . nucleolin interacts with poliovirus ires and enhances ires-dependent translation (izumi et al., 2001) . unr has also been reported to interact with ires elements (pv and rhinovirus) acting as an rna chaperone and also binds pabp that is bound to poly(a) tails in cellular mrnas (anderson et al., 2007; chang et al., 2004; hunt et al., 1999) . presumably this interaction may also occur with viral polysomes and can contribute to genome looping or circularization that makes continuous translation more efficient. that unr is important in vivo was demonstrated by gene knockout of both unr alleles in mouse cells that reduced translation of pv and human rhinovirus (hrv) ires's by 90% and could be rescued by introduction of unr expression plasmid (boussadia et al., 2003) . rna affinity pulldown of cellular proteins using ev71 5 0 utr bait revealed ptb, unr, pcbp1/2, hnrnp a1 and 10 other proteins. hnrnp a1 interacts with stem-loops ii and vi of the ev71 5 0 utr, and is proposed to be another enterovirus itaf. the roles of this interaction in replication are unclear since knockdown of hnrnp a1 had no effect on viral replication. in contrast, knockdown of both hnrnps a1 and a2 reduced viral rna synthesis and virus output, suggesting that hnrnp a2 can substitute for hnrnp a1 (lin et al., 2009) , however the relative importance of hnrnp a1 versus pcbp2 or ptb in ev71 replication was not evaluated. gars is perhaps the most unusual proposed itaf; a trna synthetase house-keeping enzyme that binds the ires core in the apical portion of domain v that mimics the anticodon-stem-loop of trna gly (andreev et al., 2012) . so what are we to make of all the proposed itafs for pv and other closely related enteroviruses? recently, in vitro reconstitution of translation with purified factors has been accomplished on the pv ires and other type 1 iress and those experiments indicate that pcbp2 is the only mandatory itaf required to accomplish 48s ribosome assembly and initiation. ptb showed minor stimulation of translation, and other proposed itafs (la, unr, srp20, gars) produced no measureable effect on 43s or 48s translation complex formation in vitro. pcbp2 may also promote recruitment of 43s complexes by direct interaction with eif3 (sweeney et al., 2014) . the weak stimulation of pcbp2-dependent 48s complex formation by ptb in vitro is likely due to its imposed reorientation of eif4g binding to the ires (kafasla et al., 2010) . but many itafs promote translation in cells by providing functions that promote efficiency, beyond the minimal requirements for ires translation initiation. intracellular conditions encountered by viruses include intense competition among mrnas for ribosomes in cells, forcing viruses to utilize multiple mechanisms to seize translation control. thus, the other itafs that aid ires translation efficiency in vivo play less fundamental, but still critical roles during infection and contribute to pathogenesis. future work will discern new molecular details how this happens. comparatively little is known about factor requirements for the type 3 ires of hav. unlike most of its picornavirus cousins, this virus replicates very slowly, it does not cause host translation shutoff and is relatively non-cytopathic in tissue culture cells. the ires is unique in that it requires intact eif4g1, whereas the pv ires functions better with only the cleaved central domain of eif4gi (borman and kean, 1997; hambidge and sarnow, 1992) . both ptb and pcbp have been implicated as itafs for hav (yi et al., 2000; zhang et al., 2007) . hav 3cpro inhibits hav ires-dependent translation and cleaves ptb. this finding suggests ptb cleavage regulates the switch from viral translation to rna replication and strengthens a role of ptb as an itaf for this ires (kanda et al., 2010) . the smaller type 4 ires elements, ( $ 300 nt) hepatitis c virus (hcv) and pestiviruses such as bovine viral diarrheal virus (bvdv) and classical swine fever virus (csfv) have been extensively studied. the hcv ires has simpler factor requirements than type 1 or 2 iress. it requires only one canonical initiation factor, eif2, and no itafs to form 48s translation initiation complexes in vitro with reconstituted systems, it interacts directly with ribosomes and ribosomal 18s rna (malygin et al., 2013; pestova et al., 1998) and can recruit translation factor eif3 (ji et al., 2004; siridechadilok et al., 2005) . despite its simpler minimal requirements in vitro, like poliovirus, several factors have been described that enhance hcv translation in lysate systems and cells, including la protein. ptb also weakly interacts with the ires, but appears to attenuate translation instead of stimulating it and its putative role as a functional itaf involved in ribosome recruitment has been challenged (brocard et al., 2007; domitrovich et al., 2005; ito and lai, 1999) , several lines of evidence indicate that la is an itaf. la enhances translation in the context of the competitive translation environment during infection in cells with replicons or virus. sirna depletion of la inhibits hcv translation and a dominant negative form of la inhibits translation (costa-mattioli et al., 2004) in vivo. further, the fact that la protein can be exploited as a therapeutic target for hcv suggests a significant role in hcv replication. a cell permeable peptide corresponding to the n-terminal 18 amino acids of la inhibits hcv ires-mediated translation and also inhibits hcv replication in cells (fontanes et al., 2009 ). lapeptide-mediated inhibition of hcv ires-translation blocked interaction of la and other itafs (ptb and pcbp2) with the ires, but could be rescued with exogenous ptb and pcbp2. this implied that la peptide sequesters ptb and pcbp2 as its mode of action (fontanes et al., 2009) . other knockdown experiments implicated several host factors in hcv translation or hcv virus replication in cells, including la, ptb, pcbp2 and proteasome alpha-subunit 7 (psma7) (kruger et al., 2001; shirasaki et al., 2010) . all four factors are induced in huh-7.5 cells during hcv replication, suggesting the virus has coopted factors that are naturally up-regulated during infection. shrna knockdown of la repressed production of hcv core protein 70%, further supporting a role as an itaf in vivo. la has been reported to be a telomerase component, thus telomerase activity and expression of other telomerase subunits increased coordinately with la induction during chronic hcv infection (shirasaki et al., 2010) . the linkage of hcv-induced la expression to increased telomerase activity in chronically-infected liver may be important in development of hepatocellular carcinoma. evidence from in vitro translation experiments in somewhat artificial rabbit reticulocyte lysates suggest that ptb is not a true hcv itaf that facilitates 40s ribosome subunit binding to the hcv ires. rather, ptb may stimulate translation indirectly through bringing other factors bound to the x region of the 3 0 utr into proximity with the ires (brocard et al., 2007) . ptb is proposed to loop hcv rna by binding the 3 0 utr x region and 5 0 utr ires region simultaneously. another factor, nsap1 (also called hnrnp q) stimulates hcv ires translation by binding an a-rich region downstream of the initiator aug and facilitating formation of 48s ribosome initiation complex by also binding the 40s ribosome proteins. this is the first reported interaction of an itaf directly with a ribosome, thus nsap1 provides more of a translation-factor role than other canonical itafs that play "chaperone-like" roles (kim et al., 2004; park et al., 2011) . ptb also supports translation initiation of aichivirus (av) that contains a novel type of picornavirus ires that differs structurally from type 1 and 2 iress. in reconstitution experiments this ires requires interaction with both eif4g and ptb. unlike other iress, the av ires has a strong requirement for a novel nuclear factor dhx29, that is also a ribosome-associated helicase. dhx29 releases the av ires initiation codon from a strong stable hairpin to aid anticodon base pairing by the ribosome (yu et al., 2011b) (table 1) . various itafs are either essential for virus translation or play less critical roles that enhance efficiency of translation. high translation efficiency is critical for most viruses to produce sufficient proteases or other factors to control activation of innate immune pathways that would block further replication. therefore, itafs have long been considered host range factors that determine tissue and host tropism and pathogenesis. the attenuated sabin vaccine strains of poliovirus lost neurovirulence partly through nucleotide changes in the ires that alter ptb binding . restricted ires function will result from a lack of the proper itaf in certain cells. for instance, a neuronal variant of ptb (nptb) is required for the ) with sl-i through iv, the 3 0 db region containing duplicate dumbbell structures (db1 and db2) (light blue) and the 3 0 cs/sl region (red) containing the conserved 3 0 sl. all structures from sl-1 through db-2 participate in pseudoknots (not depicted). to facilitate translation poly(a)-binding protein binds a-rich sequences in the db region allowing protein-bridge looping to translation factor eif4g associated with the 5 0 cap structure. yb-1 binds the 3 0 sl and represses translation and replication. ptb and la bind 3 0 utr may also facilitate translation. (b) long range interaction between complimentary sequences in 5 0 and 3 0 regions (5 0 cs, 3 0 cs; 5 0 uar, 3 0 uar) facilitate genomic looping associated with rna replication. this looping remodels rna into new conformations that may promote binding of additional host rbps not associated with translating denv rna such as eef1, and the dsrna binding proteins nf90, nf45 and rha that bind the 3 0 sl. in hcv, nf90 binds both 5 0 and 3 0 utr of hcv rna and rha is involved in bridging 5 0 and 3 0 utrs (not depicted). a similar arrangement may exist in flaviviruses where nf90 and rha may also bind 5 0 utr stem loops and stabilize the looped structure to promote negative strand rna replication. the ns5 rdrp binds to the 5 0 sl1 which due to looping helps position the polymerase near the 3 0 terminus . ns5 may also be recruited by la, which binds the 5 0 utr. neurovirulence of the gdvii strain of tmev (pilipenko et al., 2001) . ptb1 expression patterns have a powerful tropic effect on a chimeric poliovirus containing the hrv2 ires element (called pv1(ripo)). this virus has a growth defect in mouse cells because it cannot interact with endogenous murine ptb (jahan et al., 2013 (jahan et al., , 2011 . this is proposed to interfere with the thermodynamics of folding of the iresome into a functional structure. the virus also has a severe neuro-attenuation defect in humans but retains highly specific virulence against glioblastoma cells and hela cells that express high levels of ptb (gromeier et al., 2000) . modern proteomic approaches are now being applied to identify the full complement of host factors that interact with viral rnas, and have identified over 30 proteins that interact with fmdv ires directly or indirectly (pacheco et al., 2008) . of these gemin5 was found to bind to the ires at a domain 5 hairpin flanked by a/u/c-rich sequences via its c-terminal domain. gemin5 is the rna-binding component of the survival of motor neuron (smn) complex that assembles sm proteins onto spliceosomal snrnas. gemin5 binding did not enhance fmdv translation, but rather inhibited it (table 1 ) (box 1). rna structure analysis using 2 0 hydroxyl acylation analyzed by primer extension (shape) revealed gemin5 induced conformation changes that out-competed shape changes induced by ptb. thus gemin5 may competitively inhibit ptb itaf binding (piñeiro et al., 2013) . gemin5 also interacts with the hcv ires and may provide similar functions (piñeiro et al., 2013) . gemin5 is cleaved in fmdv infection by l protease (piñeiro et al., 2012) similar to gemin3 cleavage by poliovirus that blocks assembly of the smn complex (almstead and sarnow, 2007) . gemin5 cleavage alleviates translational repression of the ires. the similarity of gemin 5 and gemin 3 cleavages suggests that inactivation of the smn complex may be required by a wide range of picornaviruses. rna decay regulator auf1 (which has 4 isoforms) is cleaved by poliovirus and rhinovirus 3cd proteinase. auf1 is a factor that binds au-rich elements in mrna 3 0 utrs and promotes rapid mrna decay and turnover. auf1 follows the familiar pattern of strong relocalization from nucleus to cytoplasm during poliovirus infection . surprisingly though, auf1 does not bind pv 3 0 utr but rather binds the ires at stem loop iv and negatively regulates rhinovirus and poliovirus infections via translation inhibition. however, discordant findings with closely related cvb3 indicated that auf1 was bound to the 3 0 utr (wong et al., 2013) . 3cpro cleavage inactivates rna binding function of auf1 and relieves translation restriction (cathcart et al., 2013) . thus, both auf1 and gemin5 act as a "hijacked" restriction factor in translation and must be remedied by inactivation by a viral proteinase. however, auf1 may not be a restriction factor for all picornaviruses. emcv infection induces strong cytoplasmic relocalization of auf1, but was not cleaved by emcv 3cpro, even at late times after infection (cathcart and semler, 2014) . caliciviruses such as feline calicivirus and human norovirus share a non-structural protein coding region that is distantly related to picornaviruses, yet translate by an unusual capindependent mechanism that does not involve an ires. rather, translation of norovirus involves interaction of initiation factor eif3 with the viral vpg protein covalently linked to the 5 0 end (daughenbaugh et al., 2003 (daughenbaugh et al., , 2006 . ptb interacts with both 5 0 and 3 0 ends of feline calicivirus genomic rna, and also subgenomic rna. fcv infection induces nuclear to cytoplasmic relocalization of ptb that is coincident with the switch from early translation to late rna replication. ptb inhibited fcv translation in vitro, thus ptb is proposed to be a negative regulator that may aid the switch from translation to rna replication . flavivirus rna is unique in containing a 5 0 cap structure, but no poly(a) tail to facilitate typical pabp binding that promotes 5 0 -3 0 looping. thus, flaviviruses use cap-dependent translation machinery but it is unknown how viral translation is promoted over cellular translation. dengue virus promotes both cap-dependent translation and a form of non-canonical translation that is not ires dependent and does not require a functional m7g cap structure (edgil et al., 2006) . surprisingly, denv can bind pabp in an a-rich region upstream of the 3 0 sl, resulting in promotion of translation (polacek et al., 2009a (polacek et al., , 2009b likely by binding cap-binding translation factor eif4g and promoting 5 0 -3 0 interactions similar to host mrna (fig. 2) . denv rna is also known to bind several host factors that could play roles in translation or rna replication, including ptb, la, y box-binding protein1 (yb-1), translation elongation factor eef-1a and p100/tudor-sn (table 1 ) (de nova-ocampo et al., 2002; garcía-montalvo et al., 2004; lei et al., 2011; paranjape and harris, 2007; polacek et al., 2009a polacek et al., , 2009b yocupicio-monroy et al., 2007; yocupicio-monroy et al., 2003) . yb-1 binds the 3 0 sl of denv and represses translation, possibly in a role that regulates the switch from translation to rna replication discussed below (paranjape and harris, 2007) , and binding of eef-1a to the 3 0 sl has no effect on translation (davis et al., 2007) (fig. 2) . la binds to both 5 0 and 3 0 utrs (garcíamontalvo et al., 2004) and could play a role of stabilizing looped rna structures via protein bridging and indirectly support translation through ribosome recycling. ptb has been proposed to play positive roles in virus replication but the mechanism(s) remains elusive. ptb relocalizes to the cytoplasm with variable completeness in infections in different cell types. ptb cytoplasmic localization is weak in huh7 cells, however ptb binds the 3 0 stem-loop region of denv rna in these infections (fig. 2) and could play roles in rna looping to promote translation and rna replication. nuclear to cytoplasmic relocalization of ptb was associated with increased denv translation and replication and sirna knockdown of ptb inhibited replication and overexpression increased replication (agis-juárez et al., 2009). however, in huh7 cells knockdown studies indicated that ptb did not have an effect on denv rna translation, but promoted negative strand rna synthesis and interacted with viral protein ns4a (jiang et al., 2009 ). thus, additional work is required to pin down the specific mechanisms of ptb-specific stimulation of denv replication. in theory, nuclear factors may not be necessary for support of viral rna synthesis. each virus synthesizes its own complete rnadependent rna replicase, an enzyme function that is uniquely not present in host cells. further, in the cytoplasm where virus replication occurs, viruses duplicate functions such as mrna capping and polyadenylation that are carried out in the nucleus for host transcripts. thus, one may not expect many dependent interactions between virus rna replicases and host factors to have evolved to support viral rna replication. for negative strand viruses that contain rna covered in nucleocapsid proteins, this may be more expected. however, plus strand rna viruses make extensive use of host rbps for important roles in the replicative process, not in rna polymerase enzymatic activity per se, but in promotion of its temporal and spatial regulation. a key feature for host factors is helping to organize complex 5 0 -3 0 genome interactions in alternate configurations that sequentially promote translation, then rna synthesis. all plus strand rna viruses are faced with the problem that the same genomic template is used for both 5 0 -3 0 transit by ribosomes and 3 0 -5 0 transit by the viral rna polymerase. since translation and rna replication machinery proceed in opposite directions there must be a regulated switch from translation to rna replication that clears the template of all elongating ribosomes before negative strand rna synthesis can take place. for plus strand rna viruses the details of this regulation are emerging. a common theme is that sufficient translation/production of key virus proteins is required before modifications are triggered in host factor structure/protein interactions that shift host factor roles from promoting translation to promoting rna synthesis. accumulating evidence indicates that both translation and negative strand rna replication occur on templates circularized via complex rna-rna, protein-rna and protein-protein interactions. further, the switch in template use involves complex shifts in these interactions allowing host factors to change roles simultaneously. pcbp2 helps mediate a switch from poliovirus translation to rna replication due to changes in its rna binding properties. pcbp binds both the ires and 5 0 cloverleaf (cl) stem b, and its binding the cloverleaf stimulates translation early during infection (gamarnik and andino, 1998) . the pcbp complexed on the 5 0 cloverleaf promotes translation in conjunction with the 3 0 poly (a) tail in a circularization model based on protein-protein interactions. the c-terminal kh3 domain of pcbp is required to stimulate translation and can be tethered directly to pv rna and still promote translation. in the current model the 5 0 cl-pcbp complex interacts with the 3 0 poly(a)-pabp complex to form a 5 0 -3 0 circular structure that enhances translation by facilitating ribosome reloading as ribosomes recycle from the stop codon ( fig. 1b) (ogram et al., 2010) . this is consistent with the finding that pcbp2 and 5 0 cloverleaf function during de novo assembly of polysomes (kempf and barton, 2008) . thus, pcbp strongly promotes translation by interaction with the 5 0 cl in the phase before viral proteins accumulate. but once virus proteins accumulate from sustained translation, pcbp bound to the 5 0 cl also promotes binding of viral polymerase precursor 3cd to the adjacent cloverleaf stem loop d (sld). the recruited 3cd promotes translation inhibition (gamarnik and andino, 1998) . further, cleavage of pcbp1 and 2 mediated by 3cpro occurs during this phase of the replication cycle (fig. 1b) . cleavage occurs between the kh2 and kh3 domains, resulting in truncated pcbp2 lacking the kh3 domain that cannot support translation but binds the cl and supports rna replication (perera et al., 2007) . in fact, several itafs for pv are cleaved by 3cpro in midphase of the replication cycle. in addition to pcbp, ptb is cleaved by 3cpro in a reaction that was shown to inhibit pv translation (back et al., 2002) and la is similarly cleaved (shiroki et al., 1999) . even though cleavage of pcbp2 and ptb inhibits poliovirus translation and contributes to a switch in rna template usage, for related rhinoviruses things may be different. pcbp2 and ptb are differentially cleaved during infection with three hrv serotypes in hela cells but are not cleaved in human lung epithelial cells that sustain productive infections. this suggests that some itaf cleavages may not be required by hrv to mediate a switch in template usage (chase and semler, 2014) . like picornaviruses, hcv must clear genomic rna templates of ribosomes to allow use by rna replicase complexes. several insights have emerged about how hcv regulates the switch and mechanisms involve both host factors and accumulation of nascent hcv proteins. early studies showed hcv core protein binds to the ires and linked translation production of hcv core protein to translation repression in a direct feedback loop (zhang et al., 2002) . expression of hcv ns3 protease also has the effect of blocking ires translation and increasing replicon replication. ns3 protease and la protein compete for binding the ires in the same sliv region, thus ns3 was proposed to directly inhibit itaf binding to the ires, thereby reducing translation activity (ray and das, 2011) . additional work indicated that la protein binds to a gcac sequence near the initiator aug in sliv of the ires, but this enhances rna replication, not translation, by promoting linkage between 5 0 and 3 0 utrs through la (kumar et al., 2013) . since both la and ns3 have similar dissociation constants for binding hcv ires rna, ns3 may not actually evict all the ires-bound la, but competitively reach a binding equilibrium that promotes replication by recruiting ns5b to a replication complex involving la, ns3 and both ends of the viral rna (kumar et al., 2013) . another class of nuclear factors called nf/nfar proteins nf90/nfar (nuclear factor associated with dsrna, also called ilf3) bind to both ends of the genome of hcv and the pestivirus bvdv (isken et al., 2007 (isken et al., , 2003 (isken et al., , 2004 . nf90/nfar is a part of group of interacting nuclear factors containing dsrna-binding motifs and multiple isoforms that include nf45 and rna helicase a (rha). nf90/nfar is associated with hcv translation inhibition and increased rna replication (isken et al., 2007 (isken et al., , 2004 (isken et al., , 2003 . another group used sirna approaches to show that efficient hcv translation was dependent upon several members of the cellular 5 0 -3 0 deadenylation-dependent mrna decay pathway, rck/p54, lsm1, and patl1. the requirement of these factors for efficient translation was linked to the 5 0 and 3 0 -utrs. the 3 0 utr also interacted with lsm1-7 complexes. all of these factors are core components of p-bodies where non-translating silenced mrnas are stored before undergoing decay. this raises the possibility that the decay factors could play roles in the switch to replication, but the proposed role from experiments for hcv interaction with reconstituted lsm1-7 complexes was support of translation (scheller et al., 2009) . it is possible that co-opting p-body components downregulates functions of p-bodies that repress hcv translation. hcv infection results in significant reductions in the number of p-bodies in cells both in vitro and also in hepatocytes from patients infected with hcv (pérez-vilaró et al., 2014 . rna synthesis of viral negative strands initiates replication of genomic templates from the 3 0 -terminus. thus, it was a surprise when rna polymerase complexes for poliovirus were found to bind near the 5 0 terminus of the plus strand genome template on the cloverleaf (andino et al., 1993) . this suggested that the template is circularized rather than linear to allow close proximity of the replicase complex and 3 0 end of the template where the polymerase must initiate. cellular mrnas and viral genomes are thought to translate optimally on rnas circularized by interactions between translation factors eif4g, eif4b and pabp that promote ribosome recycling (kuyumcu-martinez et al., 2004) . thus, rna replication may first begin on templates transitioning from a circular configuration. indeed, this paradigm seems to be conserved among many plus strand rna viruses. in viruses, the long-range interactions that circularize templates are mediated by either protein-protein interactions or rna-rna interactions or both. for pv, circularization is also promoted by a complex of pcbp bound to the cloverleaf that interacts with pabp bound to poly (a) tail forming a protein-protein bridge (herold and andino, 2001 ). an analogous arrangement occurs in coronavirus mouse hepatitis virus (mhv) where interaction between 5 0 and 3 0 ends is promoted by another protein bridge involving ptb bound to 5 0 utr and hnrnp a1 bound to the 3 0 utr (huang and lai, 2001) . for dengue virus cyclization occurs via long range rna-rna interactions of complementary sequences in the corresponding 5 0 and 3 0 utrs (filomatori et al., 2011; lodeiro et al., 2009; polacek et al., 2009a polacek et al., , 2009b sztuba-solinska et al., 2013; villordo et al., 2010) (fig. 2) . in hcv an rna sequence in the ns5b gene region interacts with the 3 0 utr via a kissing-loop to facilitate placement of the polymerase at the exact 3 0 end of the genome (friebe et al., 2005) . the latter two examples above emphasize rna-rna interactions without factors, but it is possible that host factors could promote or stabilize these long-range interactions. for instance, hcv interactions may be aided by hnrnp a1, which binds ns5b and both ends of the genome (kim et al., 2007) . finally, the host rbp nf90/nfar circularize the hcv genome by binding both ends of the genome. these interactions may occur sequentially after kissing loop and hnrnp a1 interactions form as nf90 binding blocks translation and stimulates rna replication (isken et al., 2007 (isken et al., , 2004 (isken et al., , 2003 . rha can also play a role in the complex with nf90/nfar-1 as a bridging factor between the 5 0 and 3 utrs of hcv that promotes genome circularization (isken et al., 2007) . several of these factors also bind to denv2 3 0 utr (nf90, nf45, rha) and nf90 is critical for replication (gomila et al., 2011) . together, these findings suggest that conformations of circularized genomic rna can differ between translation and replication. accordingly, switching between translation and replication can be facilitated by virus recruitment of host factors that provide conformational remodeling of rna structures. for the coronavirus mouse hepatitis virus (mhv), crosstalk between the viral utrs relies on interactions between ptb and hnrnp a1, which bind the 5 0 utr and 3 0 utr, respectively. mutations that block interaction of these factors with mhv rna reduce replication and transcription (huang and lai, 2001) . mhv replication was not restricted in cells that do not express hnrnp a1, since the viral rna can also bind several other hnrnp proteins in replacement roles. these were identified by rna affinity with negative strand utr to be hnrnp a2/b1, hnrnp a/b and hnrnp a3, all which are closely related to hnrnp a1 (shi et al., 2003) . for enteroviruses there is a clear dependence on co-opted pcbp2 to promote negative strand rna synthesis built on the platform of the extended 5 0 cloverleaf. only intact pcbp functions in translation but either intact or cleaved (by 3cpro) pcbp2 can bind the 5 0 cloverleaf (cl) on stem-loop b(slb) (perera et al., 2007) and promote genome circularization and recruitment of 3cd to the cloverleaf on the adjacent loop, positioning near the 3 0 poly (a) template due to the protein-bridge circularization (herold and andino, 2001) . initial reports indicated the pcbp binding region was cl slb, however this was later shown to be insufficient, that an additional c-rich spacer region located just downstream of the cl is also required for pcbp binding. mutation of six c residues in this region to a abolished replication in hela cells but had no effect on translation. it was found that pcbp did not bind to 88 nt cl alone and but did bind 142 nt extended cl including this region (toyoda et al., 2007) . point mutations in this region strongly affect pv neurovirulent phenotypes (de jesus et al., 2005) . similarly, pcbp2 was shown to bind the analogous c-rich spacer region in cvb3 rna (fig. 1c) . pcbp2 did not bind cvb3 cl alone but 3cpro binding to sld allowed recruitment of pcbp2 to the minimal cl (zell et al., 2008b) . also, pcbp kh domains 1 and 3 interact with the extended cloverleaf rna of cvb3 (zell et al., 2008a) . taken together these reports make it clear that pv co-opts pcbp to provide multiple functions for virus replication, including: promoting genome circularization, as an itaf, switching from translation to rna synthesis, and properly assembling the replicase on the 5 0 cl. the cl is a required partner in several of these steps and can be seen as a general promoter for rna synthesis that provides a platform for binding host factors and polymerase (vogt and andino, 2010) . nucleolin is a shuttling rna helicase that is largely concentrated in the nucleolus and binds pre-rrnas. nucleolin was initially reported to bind poliovirus 3 0 utr and enhance replication and may play roles in negative strand rna replication (waggoner and sarnow, 1998) . subsequently another report indicated nucleolin interacts with poliovirus ires and enhances ires-dependent translation (izumi et al., 2001) . whether interactions at two sites on opposite ends of the viral genome require one nucleolin moiety or looped circularized rna has not been determined and more precise functional roles of nucleolin have not been identified. fmdv interacts with rna helicase a (rha) and alters its distribution in the cell from mostly nuclear to cytoplasmic. rha is involved in diverse cellular functions as it enhances gene expression by interacting with cbp/p300 and rna polymerase ii and responds to ifn alpha signaling to increase its activity (aratani et al., 2001; fuchsová et al., 2002) . during fmdv infection rha can bind the s domain of the virus 5 0 utr and co-precipitates with fmdv 2c and 3a proteins that function in the replicase complex. though the precise function of rha remains undetermined, knockdown of rha reduces virus titers, indicating that rha plays roles in supporting replication (lawrence and rieder, 2009 ). fmdv-induced movement of rha out of the nucleus is associated with demethylation or arginine residues at the c terminus and did not require activity of fmdv leader protein. rather nuclear egress involves demethylation activity provided by jumonji c-domain containing protein 6 (jmjc) that is stimulated by fmdv (lawrence et al., 2014) . flavivirus 3 0 utrs are complex structures that contain cisacting elements required for translation, cyclization and replication. accordingly, this regulatory rna region binds several host factors that may play roles in rna synthesis, including la, ptb, yb-1, eef1a, nf90, rha, and nf45 (fig. 2) . flavivirus rna replication is dependent on many of these co-opted host factors, however not all are thought of as nuclear factors. translation elongation factor 1a (eef1a), which is abundant in both the cytoplasm and nucleus, binds to three sites within the 3 0 utr of west nile virus and other flaviviruses. this interaction supports negative strand rna synthesis but not translation (blackwell and brinton, 1997; davis et al., 2007) . eef1a immunoprecipitates with the ns5 replicase complex proteins of wnv and colocalizes with replication complexes, further supporting a role for this factor in minus strand rna synthesis (davis et al., 2007) . flavivirus rna shifts to an alternate conformation based on complex long-range binding interactions that form a new panhandle structure. this panhandle structure positions 5 0 and 3 0 termini close together and promotes rna replication (fig. 2) . binding of a complex of double-strand binding factors nf90, rha and nf45 to the 3 0 sl likely occurs in concert with the formation of the panhandle and promotes rna replication (gomila et al., 2011) . the possibility exists that these factors may also bind the 5 0 utr and stabilize the long-range looped structure similar to their role with hcv rna (isken et al., 2007) . further, la binds to both 5 0 and 3 0 utrs and could play a role stabilizing this structure as well as recruiting ns5 and ns3 (garcía-montalvo et al., 2004) . the ns5 rdrp binds to the 5 0 sl1 which due to looping helps position the polymerase near the 3 0 terminus . similar to picornaviruses and hcv, ptb also binds the 5 0 utr of dengue virus (denv) and japanese encephalitis virus (jev) (anwar et al., 2009; kim and jeong, 2006) . although the 5 0 utr binding site suggested a possible role of ptb in flavivirus translation, ptb knockdown inhibits denv negative strand rna replication and does not affect virus translation or viral entry. ptb also interacts with ns4a protein, a component of the replicase, further suggesting that ptb plays a role in viral rna replication (anwar et al., 2009; jiang et al., 2009) . surprisingly, ptb knockdown did not inhibit jev replication so its requirement is not conserved. ptb also binds 3 0 utrs of both viruses. though the 5 0 utr of both viruses are conserved, the differential requirement for ptb may stem from the fact that the 3 0 utrs are divergent. in addition, ptb translocates to the cytoplasm in denv-infected cells and is found colocalized with calnexin endoplasmic reticulum marker, ns1 and ns3 (agis-juárez et al., 2009) . further work will be required to elucidate precise functional roles of ptb in flavivirus replication. for hcv, as discussed above, the switch from translation to rna replication is stimulated by binding of core and ns3 to the ires. however, some translation may still occur and be coupled to rna replication at the same subcellular membrane compartment termed a replicasome. pulse chase and other analyses in infected cells supported this conclusion since restriction of rna synthesis with an ns5b polymerase inhibitor reduced translation even when levels of hcv rna were unaltered (liu et al., 2012) . as with any rna virus, the overlapping, ongoing replication and translation processes make separation of the roles of host factors very difficult to tease apart. even though ptb may not be a bona fide itaf for hcv, it interacts with the poly(u/c) tract in the hcv 3 0 utr and may promote replication. during infection in huh7 cells a phosphorylated form of ptb not found in uninfected cells associates with the membrane-bound hcv replication complex (chang and luo, 2006) . synaptotagmin-binding cytoplasmic rna-interacting protein (syn-crip)(nsap1)(hnrnp q) is another member of the heterogenous nuclear ribonucleoprotein (hnrnp) family that plays roles in mrna maturation and also binds hcv rna and enhances ires-dependent translation. syncrip/hnrnp q also plays a role in rna replication as it associates with the replication complex in membrane fractions and colocalizes with nascent virus rna. immunodepletion or sirna knockdown of nsap1 decreases rna replication, indicating dual functions of nsap1 in the hcv replicative cycle . finally, the rna chaperone nucleolin has also been proposed to play a role in hcv replication, as it binds the virus ns5b rna polymerase via its glycine-arginine-rich domain. transient expression of ns5b recruits or sequesters nucleolin from the nucleus to cytoplasm and may modulate the oligomerization of ns5b that is required for rnrp activity (hirano et al., 2003; kusakawa et al., 2007) . after sindbis virus (sinv) infection numerous nuclear proteins shift to the cytoplasm including hnrnp k, hnrnp a1, and hur. hnrnp a1 interacts with the viral 5 0 utr, and with the genomic (g) and subgenomic (sg) rna promoters. knockdown of hnrnp a1 resulted in markedly decreased synthesis of g and sg rna both in infected cells and in vitro (gui et al., 2010; lin et al., 2009) . a recent report also indicates that sinv infection results in cytoplasmic relocation of ptb and tia1, but no specific function of these factors in replication is known (sanz et al., 2015) . several reports indicate that sinv nonstructural proteins interact with stress granule nucleating proteins g3bp1 and g3bp2, including nsp4 polymerase (cristea et al., 2010) , nsp3 and nsp2 (cristea et al., 2006) (gorchakov et al., 2008) . the interaction of g3bp1 with nsp3 has been shown to have the proviral effect of inhibiting stress granule formation (panas et al., 2012) . tia1 and ptb also enter the cytoplasm during sinv infection but not during ectopic expression of nsp proteins, indicating replicative processes may be required (sanz et al., 2015) . recently the combined approach of isotope labeling of purified semliki forest virus replicase/ modified lysosome complexes with proteomics analysis identified 78 host proteins associated with the functional replicase. several familiar proteins colocalized with replicase including pcbp1, hnrnp m, hnrnp c, and hnrnp k. silencing experiments indicated that hnrnp m and c are antiviral for sfv, chikungunya and sinv. differential silencing results with hnrnp k indicated opposite roles of this rbp in chikv and sinv versus sfv. this suggests that interactions of host factors with replicase complexes are not always proviral and that the roles of hnrnps may be interchangeable or more nuanced in various cells/ virus combinations (varjak et al., 2013) . nuclear factors modulate astrovirus and norovirus rna replication ptb also binds the 3 0 utr of astrovirus. seven host proteins from caco2 cell lysates could be cross-linked to 3 0 -utr rna probes in vitro and mobility shift assays indicated that two complexes of host factors form on 3 0 utr probes and that ptb is part of one of the complexes. sirna knockdown of ptb reduced virus replication, suggesting that ptb is required for replication (espinosa-hernández et al., 2014) . it is unclear if negative strand or plus strand rna replication is affected by ptb. human norovirus was shown to bind la and ptb to the 3 0 utr that contains a small stem-loop structure (gutiérrez-escolano et al., 2003) , however, since human norovirus cannot be cultivated in vitro, most recent work has focused on murine norovirus that replicates in macrophage cell lines. murine norovirus rna contains three stable stem loop structures and a single stranded polypyrimidine (py) tract within the 3 0 terminal stem loop. both ptb and pcbp bind the py tract, but this is not essential since viruses lacking this region are viable in cells and in mice. however, py-deleted virus suffer a fitness cost and is less virulent in mice . rna affinity chromatography-mass spectroscopy identified over 50 host factors that bound to discreet structures in murine norovirus rna. many were common to other rna viruses discussed above, including ptb, la, ddx3, nucleolin, hnrnpk, pcbp1/2, eef1a and hnrnp a species. sirna knockdown of ptb, la and ddx3 in raw264.7 cells resulted in deficient virus replication (vashist et al., 2012) suggesting these play roles in replication, but much more work will be required to elucidate how these factors function in norovirus infections. a long range 5 0 -3 0 rna-rna looping interaction was described in murine norovirus rna that is stabilized by binding pcbp2 and hnrnp a1. mutations in the rna-rna complimentary binding motif reduced binding of both host factors to rna and also inhibited rna replication; and pcbp2 and hnrnp a1 colocalized to virus replication complexes in cells. all these results indicate the looping interaction stabilized by the host factors plays an important roles in replication (lópez-manríquez et al., 2013) . feline calicivirus also binds nucleolin on the 3 0 utr together in a complex with viral protein ns6/7 and infection of feline kidney cells results in nuclear-cytoplasmic relocalization of nucleolin to sites enriched for ns6/7 (cancio-lonches et al., 2011) . finally, ptb plays a negative role in calicivirus translation and therefore may promote the switch from translation to replication . several host proteins interact with the 3 0 end of the transmissible gastroenteritis coronavirus (tgev) genome. genomic rna ends were used as bait in rna affinity pulldowns to identify host factors. the only protein to bind the 5 0 end was ptb, whereas nine other proteins bound the 3 0 terminus rna, including several hnrnps, pabp and two amino-acyl trna synthetases. knockdown of pabp, hnrnp q and glutamyl-prolyl-trna synthetase inhibited replication of a replicon, indicating that they may play functional roles in replication (galán et al., 2009) . since hnrnps oligomerize, they may participate in rna-protein complexes by bringing transcription-regulating sequences (trs) trs-l and trs-b into close proximity to facilitate coronavirus discontinuous transcription (sola et al., 2011b) . further investigation of the role of ptb in tgev replication indicated that ptb affects virus rna accumulation and relocalizes viral rnas to novel cytoplasmic domains different from replication-transcription sites. interestingly, sirna knockdown of ptb in two cell lines increased virus mrna levels and virus titer, suggesting that ptb interaction inhibits virus replication, the opposite of most other rna viruses. ptb relocalized from the nucleus to discreet structures that contained tia1 and tiar and may be reminiscent of stress granules. these foci also contained viral genomic and subgenomic rna and ptb could play a role in sequestration of some virus rna into novel foci involved in posttranscriptional regulation of virus gene expression (sola et al., 2011a) . there is not substantial evidence that the tia1-foci are bonafide stress granules but may represent replication complexes. nuclear factors regulate enterovirus plus strand rna synthesis compared to minus strand synthesis, less is known about the synthesis of plus strands from the minus strand anti-genomic template in rna virus systems such as poliovirus. the template for plus strand synthesis is different, being mostly double-stranded rna instead of single stranded rna, and this may have profound influence on the host factors that interact with virus rna. this double stranded structure is called replicative form (rf) in picornaviruses. hnrnp c supports virus positive strand replication by binding to the anti-cloverleaf that forms on the 3 0 end of the minus strand of rf (fig. 1d ). since the rf template is completely double stranded, the anti-cloverleaf can only form by dsrna breathing allowing strand separation at the terminus. the strand separation is aided progressively by host factors that recognize the cognate plus strand stem-loop b and anti-stem-loop b, which are pcbp2 and hnrnp c1/c2, respectively and prevent renewed basepairing that would zip the rf back to completely double-stranded structure. in this situation there is opportunity for the factors pcbp and hnrnp c to interact to support positive strand rna synthesis, yet this has not been reported. hnrnp c also is relocalized from the nucleus to the cytoplasm during infection and like pcbp2, can interact with pv 3cd polymerase precursor. a current model proposes that hnrnp c maintains the 3 0 end of the template in a single strand form via its chaperone function and then recruits the polymerase (brunner et al., 2005) . using the hela in vitro replication system, depletion of hnrnp c inhibited production of plus strand rna product, and replenishment with full length but not truncated hnrnp c restored rna synthesis (brunner et al., 2005) . hnrnp c also interacts with the 5 0 end of the negative strand rna on a stem loop structure that can only form through breathing of the full length double strand rf replication intermediate (ertel et al., 2010) . although rf is typically depicted in cartoons as a linear structure, this finding raises the possibility that oligomerization of hnrnp c could maintain a loop with both ends of the double-stranded template in close proximity to promote rna strand synthesis (fig. 1d ). hnrnp c functions in pre-mrna processing as a tetramer that forms via a coil-coil interactions involving its oligomerization domain (whitson et al., 2005) . an engineered tandem cloverleaf approach was used to separate functions required for pv minus strand rna synthesis from plus strand rna synthesis. this approach revealed several requirements for plus strand synthesis that were surprising. first, the short stem a is essential for plus strand synthesis but not minus strand synthesis, suggesting the stem-loop structure is functional on the anti-cloverleaf. second, the complete plus strand version of the cl is required, including binding sites for pcbp and 3cd polymerase precursor. a trans-initiation model was proposed where replicase builds on the plus strand cl to prime plus strand synthesis the same way it does for negative strand rna synthesis (vogt and andino, 2010) . however, hnrnp c binding to the anti-cl will also help position the 3 0 end of the negative strand to allow precise rna replication initiation by the replicase. after synthesis of pv plus strand transcripts an rna processing step removes vpg at the 5 0 end of a portion of transcripts. all pv rna associated with polysomes lacks vpg, having been removed by a host unlinkase enzyme, whereas all encapsidated rna retains vpg. cellular 5 0 tyrosyl-dna phosphodiesterase-2 (tdp2) is a hijacked cellular dna repair enzyme that performs the vpg unlinkase function for enteroviruses. pv infection relocalizes the bulk of tdp2 from the nucleus to cytoplasm. a hypothesis predicts that vpg is a capsid packaging signal that must be removed so that a pool of nascent transcripts can continue to engage ribosomes and produce additional virus proteins (virgen-slane et al., 2012) . the effect of preventing vpg unlinking by using click chemistry to form an uncleavable bond for tdp2 was tested and found to not affect translation or replication efficiency, indicating that vpg does not inhibit initial steps in either process (langereis et al., 2013) . in flaviviruses, plus strand rna synthesis is initiated by a promoter-protein complex that builds on a conserved stem-loop structure on the 3 0 terminal 96 nt of minus strand rna. this structure is specifically bound by four host proteins, one of which is la autoantigen (yocupicio-monroy et al., 2003) and another is tiar. tiar and its closely related paralog tia1 are nuclear proteins that shuttle into the cytoplasm during environmental stress and help nucleate formation of rna stress granules. tia1 also binds wnv ( à ) rna and both proteins bind wnv rna through their conserved rrm2 motifs (li et al., 2002) . wnv replication was repressed in tiar knockout cells and mutations of the uaauu tiar recognition motif in the 3 0 sl affected replication but not translation. several mutant viral rnas that only weakly bound tiar rapidly reverted to wild type phenotypes in vivo, suggested that tiar interaction was not required for low level minus strand replication but allowed efficient high level plus strand rna synthesis from the minus strand template (emara et al., 2008) . the ability of wnv and denv to hijack tiar and tia1 from the nucleus and place it into new roles in rna synthesis provides another advantage; hijacking also sequesters these proteins from their role in forming stress granules (sg) (emara and brinton, 2007) . many viruses induce sgs by interrupting cellular pathways and homeostasis, particularly protein synthesis, and by triggering activation of pkr. these virus-derived perturbations drive sg formation through phosphorylation of eif2 à or other mechanisms (kedersha et al., 2013 (kedersha et al., , 1999 reineke and lloyd, 2013) . accumulating evidence suggests that sgs may provide platforms to activate innate immune functions and they are seen as antiviral (lloyd, 2013) . indeed, many viruses have evolved mechanisms to suppress sg formation, and co-opting key sg nucleating factors such as tiar and g3bp1 is one mechanism employed by flaviviruses and hcv (ariumi et al., 2011; emara and brinton, 2007; ruggieri et al., 2012) . further experiments with chimeras of wnv that had different rna replication efficiencies and different abilities to induce or control sgs indicated that early viral rna synthesis cannot exceed the ability to protect product dsrna with virusinduced membranes. virus-induced membranes block access of pkr to the dsrna intermediates. viruses that replicate too quickly overwhelm the limited virus-induced membranes that protect dsrna, allowing pkr activation and sgs formation that inhibits replication (courtney et al., 2012) . several plus strand viruses have multiple open reading frames and use subgenomic plus strand transcripts (sgrna) to express structural proteins. the synthesis of subgenomic plus strand rna is regulated differently than genomic rna, however is still controlled by transcription elements that function in cis or trans. this provides the opportunity for subgenomic transcription elements to assemble alternate replicase complexes containing different host factors than those modulating genomic rna replication. coronaviruses promote sgrna transcription using interactions between 5 0 terminal leader sequences and intergenic (ig) sequences just upstream of each orf. one host factor, hnrnp a1 binds to both leader and ig sequences in mhv negative strand template rna (li et al., 1997) . overexpression of hnrnp a1 promotes mhv rna replication while a dominant negative hnrnp a1 reduces replication (shi et al., 2000) and the interaction of hnrnp a1 with the leader and ig sequences is critical for in vitro sgrna transcription . hnrnp a1 could function by recruiting virus n nucleocapsid protein (wang and zhang, 1999) . ptb is another prominent nuclear factor that may regulate sgrna transcription as it binds the plus strand leader sequence and also the 5 0 utr in minus strand rna in reactions that promote sgrna replication specifically li et al., 1999) . the alphavirus sindbis virus induces a shift of hnrnp k from the nucleus to cytoplasm where it colocalizes with viral nonstructural protein 2 and co-immunoprecipitates with subgenomic but not genomic rna, nsp1 and nsp2. sirna knockdown of hnrnp k reduced gene expression from a subgenomic promoter. these findings indicate that hnrnp k has a role, details yet to be determined, in sgrna synthesis (burnham et al., 2007) . viruses must maintain the integrity of their viral rnas and suppress rna decay pathways. alphavirus and coronavirus genomes are capped and poly-adenylated, retaining two modifications that stabilize mrnas against rna decay. however, binding of certain host factors to virus rna can also protect against rna decay machinery. hur is associated with prolonged mrna stability in cells and binds to u-rich elements (ures) in alphavirus 3 0 utrs (garneau et al., 2008; sokoloski et al., 2010) . as with many other factors, hur is selectively relocalized to the cytoplasm during sindbis virus infection. two other alphaviruses, ross river virus and chikungunya virus, lack the conserved 3 0 ures but still tightly bind hur via alternative binding elements (dickson et al., 2012) . knockdown of hur increased decay of sindbis virus rnas and reduced virus replication yields in both human and mosquito cells, indicating that hur binding to sinv rna blocks components of the cellular rna decay pathway (sokoloski et al., 2010) . other viruses lack either 5 0 m7gtp caps or poly(a) tails and may specifically co-opt or target host factors to protect against decay. many of these are resident cytoplasmic factors but some are nuclear shuttling proteins. the pv 5 0 cl binds pcbp to support rna replication as discussed above, but this also stabilizes pv plus strand rna in hela lysates. a mutant pcbp protein that cannot bind the cl was associated with increased decay of pv rna in the hela in vitro replication system and it was proposed that pcbp binding blocks xrn1 exonuclease activity. this was supported by showing that capped pv rnas bypassed the requirement for pcbp2 in stability assays (murray et al., 2001) . poliovirus also achieves greater rna stability through cleavage and proteolytic degradation of rna decay factors xrn1, dcp1a and pan3 by poliovirus proteinases and virus-activated proteasomal decay (dougherty et al., 2011) . further, 3cpro cleavage of the rna destabilizing factor auf1 likely contributes to poliovirus rna stability wong et al., 2013) . flaviviruses generate short non-coding rnas (sfrna) from the 3 0 utr due to the ability of this structure to stall and sequester xrn1. denv2 sfrna accumulates during infection and acts a sponge and binds host factors g3bp1, g3bp2 and caprin1. sequestration of g3bp was linked to dysregulation of translation of several mrnas of specific interferon stimulated genes, thus interfering in innate immunity . importantly sfrna also sequesters xrn1, and results in dysregulation of host mrna stability and accumulation of uncapped mrna decay intermediates in cells (moon et al., 2012) . the stem-loops and u-rich tract contained in the 3 0 utr of hcv bind la protein and inhibit decay of hcv rna in hela extracts (spångberg et al., 2001) . similar to alphaviruses, the hcv 3 0 ure can bind hur and knockdown of hur expression in cells resulted in reduced hcv translation and rna replication using replicon models (korf et al., 2005; spångberg et al., 2000) . the stimulatory effects of hur on ires translation were confirmed using dicistronic vectors in rabbit reticulocyte lysates, xenopus laevis oocytes and hela cell extracts but may not involve direct interaction of hur with the ires (rivas-aravena et al., 2009) . though hur is associated with rna stability in many systems, its importance for hcv rna remains unclear. proteomics analysis indicates more than 70 host proteins are enriched from cells with hcv 3 0 utr probes and several of these are associated with regulation of rna stability in cells, including yb-1, nf90, hnrnp c and hnrnp d . the discussion herein mostly focused on nuclear shuttling rbps and their roles in regulating mammalian plus strand rna virus translation and rna replication. however, cytoplasmic viruses exploit many other types of cytoplasmic factors involved in translation regulation, membrane formation and remodeling, and virus assembly and egress that were not discussed. as research on rna virus replication has continued, several common themes have emerged. first, the array of host proteins that plays significant roles in replication will continue to grow as the newly expanded list of interacting factors identified from proteomics approaches undergo analysis. second, although many host proteins are hijacked by viruses, several ubiquitous host proteins, such as ptb, pcbp and hnrnp a1 are used by several classes of viruses, often in different ways. third, the production of key virus proteins shifts the function of some host factors from support of translation to rna replication. fourth, the rna chaperone functions described for host factors in supporting iresome activity often provide similar or analogous chaperone functions supporting rna structures and long range interactions in rna synthesis. fifth, the new concept of host factors being sequestered or sponged away from normal host roles may be common, and may be a variant form of hijacking. sixth, some interactions of host factors with virus rna inhibit replication or translation and must be opposed by viruses. many nuclear host factors discussed are highly conserved, which can enable cross-species infection in the case of alphavirus and flaviviruses, but also restrict virus tissue tropism and control pathogenesis in mammals where gene expression patterns are so variable. also, the complete lists of factors used by any one virus are unique evidence that viruses evolve many different ways to take over cell functions. however, many of the diverse host factors that are hijacked perform related functions for the virus in replication. our understanding of co-opted factors, though expanding rapidly, is far from complete. we certainly do not have a comprehensive list of factors utilized by any single virus. future expansion of proteomics research and new approaches such as tux-ms will fill in the catalog, but substantial traditional biochemical wet bench science will be required to tease apart functions and impact of newly discovered factors, many of which may be novel to science as a whole. adaptation of in vitro replication systems used for poliovirus to study other virus families will help dissect the roles of factors in viral replication complexes, particularly where the same factor may play roles in sequential replicative processes. finally, the increased use of super-resolution fluorescence microscopy and cryo-electron microscopy of whole cells will also be important to finely localize factors and virus-induced structures in cells. polypyrimidine tract-binding protein is relocated to the cytoplasm and is required during dengue virus infection in vero cells inhibition of u snrnp assembly by a virus-encoded proteinase internal initiation of translation from the human rhinovirus-2 internal ribosome entry site requires the binding of unr to two distinct sites on the 5 0 untranslated region poliovirus rna synthesis utilizes an rnp complex formed around the 5 0 -end of viral rna glycyl-trna synthetase specifically binds to the poliovirus ires to activate translation initiation the polypyrimidine tract-binding protein is required for efficient dengue virus propagation and associates with the viral replication machinery dual roles of rna helicase a in crebdependent transcription hepatitis c virus hijacks p-body and stress granule components around lipid droplets translation of polioviral mrna is inhibited by cleavage of polypyrimidine tract-binding proteins executed by polioviral 3c(pro) encephalomyocarditis virus leader protein hinge domain is responsible for interactions with ran gtpase functional analysis of rna structures present at the 3 0 extremity of the murine norovirus genome: the variable polypyrimidine tract plays a role in viral virulence encephalomyocarditis virus leader is phosphorylated by ck2 and syk as a requirement for subsequent phosphorylation of cellular nucleoporins a nucleo-cytoplasmic sr protein functions in viral ires-mediated translation initiation g3bp1, g3bp2 and caprin1 are required for translation of interferon stimulated mrnas and are targeted by a dengue virus non-coding rna flaviviral rnas: weapons and targets in the war between virus and host translation elongation factor-1 alpha interacts with the 3 0 stem-loop region of west nile virus genomic rna poly(rc) binding protein 2 binds to stem-loop iv of the poliovirus rna 5 0 noncoding region: identification by automated liquid chromatography-tandem mass spectrometry intact eukaryotic initiation factor 4g is required for hepatitis a virus internal initiation of translation unr is required in vivo for efficient initiation of translation from the internal ribosome entry sites of both rhinovirus and poliovirus evidence that ptb does not stimulate hcv ires-driven translation functional interaction of heterogeneous nuclear ribonucleoprotein c with poliovirus rna synthesis initiation complexes heterogeneous nuclear ribonuclear protein k interacts with sindbis virus nonstructural proteins and viral subgenomic mrna nucleolin interacts with the feline calicivirus 3 0 untranslated region and the protease-polymerase ns6 and ns7 proteins, playing a role in virus replication insights into rna biology from an atlas of mammalian mrna-binding proteins cellular mrna decay protein auf1 negatively regulates enterovirus and human rhinovirus infections differential restriction patterns of mrna decay factor auf1 during picornavirus infections the polypyrimidine tract-binding protein (ptb) is required for efficient replication of hepatitis c virus (hcv) rna unr, a new partner of poly (a)-binding protein, plays a key role in translationally coupled mrna turnover mediated by the c-fos major coding-region determinant differential cleavage of ires trans-acting factors (itafs) in cells infected by human rhinovirus the domains of polypyrimidine tract binding protein have distinct rna structural preferences upregulated c-myc expression in multiple myeloma by internal ribosome entry results from increased interactions with and expression of ptb-1 and yb-1 la autoantigen is necessary for optimal function of the poliovirus and hepatitis c virus internal ribosome entry site in vivo and in vitro west nile virus infections suppress early viral rna synthesis and avoid inducing the cell stress granule response tracking and elucidating alphavirus-host protein interactions host factors associated with the sindbis virus rna-dependent rna polymerase: role for g3bp1 and g3bp2 in virus replication the genomelinked protein vpg of the norwalk virus binds eif3, suggesting its role in translation initiation complex recruitment 22 vpg of murine norovirus binds translation initiation factors in infected cells interaction between the cellular protein eef1a and the 3 0 -terminal stem-loop of west nile virus genomic rna facilitates viral minus-strand rna synthesis mutation of a single conserved nucleotide between the cloverleaf and internal ribosome entry site attenuates poliovirus neurovirulence translation elongation factor-1alpha, la, and ptb interact with the 3 0 untranslated region of dengue 4 virus rna dephosphorylation of hur protein during alphavirus infection is associated with hur relocalization to the cytoplasm role of la autoantigen and polypyrimidine tract-binding protein in hcv replication poliovirus-mediated disruption of cytoplasmic processing bodies dengue virus utilizes a novel strategy for translation initiation when cap-dependent translation is inhibited interaction of tia-1/tiar with west nile and dengue virus products in infected cells interferes with stress granule formation and processing body assembly mutation of mapped tia-1/ tiar binding sites in the 3 0 terminal stem-loop of west nile virus minus-strand rna in an infectious clone negatively affects genomic rna amplification mechanistic consequences of hnrnp c binding to both rna termini of poliovirus negative-strand rna intermediates ptb binds to the 3 0 untranslated region of the human astrovirus type 8: a possible role in viral replication rna sequences and structures required for the recruitment and activity of the dengue virus polymerase re-localization of cellular protein srp20 during poliovirus infection: bridging a viral ires to the host cell translation apparatus a cell-permeable peptide inhibits hepatitis c virus replication by sequestering ires transacting factors kissing-loop interaction in the 3 0 end of the hepatitis c virus genome essential for rna replication nuclear dna helicase ii is recruited to ifn-alpha-activated transcription sites at pml nuclear bodies host cell proteins interacting with the 3 0 end of tgev coronavirus genome influence virus replication two functional complexes formed by kh domain containing proteins with the 5 0 noncoding region of poliovirus rna switch from translation to rna replication in a positive-stranded rna virus interactions of viral protein 3cd and poly(rc) binding protein with the 5 0 untranslated region of the poliovirus genome la protein binds to ns5 and ns3 and to the 5 0 and 3 0 ends of dengue 4 virus rna the 3 0 untranslated region of sindbis virus represses deadenylation of viral transcripts in mosquito and mammalian cells nf90 binds the dengue virus rna 3 0 terminus and is a positive regulator of dengue virus replication different types of nsp3-containing protein complexes in sindbis virus-infected cells intergeneric poliovirus recombinants for the treatment of malignant glioma hnrnp a1 interacts with the genomic and subgenomic rna promoters of sindbis virus and is required for the synthesis of g and sg rna la, ptb, and pab proteins bind to the 3( 0 ) untranslated region of norwalk virus genomic rna translational enhancement of the poliovirus 5 0 noncoding region mediated by virus-encoded polypeptide 2a identification of cellular factors associated with the 3 0 -nontranslated region of the hepatitis c virus genome a cytoplasmic 57-kda protein that is required for translation of picornavirus rna by internal ribosomal entry is identical to the nuclear pyrimidine tract-binding protein ires-induced conformational changes in the ribosome and the mechanism of translation initiation by internal ribosomal entry poliovirus rna replication requires genome circularization through a protein-protein bridge direct interaction between nucleolin and hepatitis c virus ns5b polypyrimidine tract-binding protein binds to the complementary strand of the mouse hepatitis virus 3 0 untranslated region, thereby altering rna conformation heterogeneous nuclear ribonucleoprotein a1 binds to the 3 0 -untranslated region and mediates potential 5 0 -3 0 -end cross talks of mouse hepatitis virus rna unr, a cellular cytoplasmic rnabinding protein with five cold-shock domains, is required for internal initiation of translation of human rhinovirus rna nuclear factors are involved in hepatitis c virus rna replication members of the nf90/nfar protein group are involved in the life cycle of a positive-strand rna virus complex signals in the genomic 3 0 nontranslated region of bovine viral diarrhea virus coordinate translation and replication of the viral rna an internal polypyrimidine-tract-binding protein-binding site in the hepatitis c virus rna attenuates translation, which is relieved by the 3 0 -untranslated sequence nucleolin stimulates viral internal ribosome entry site-mediated translation a host-specific, temperature-sensitive translation defect determines the attenuation phenotype of a human rhinovirus/poliovirus chimera, pv1(ripo) polypyrimidine tract binding protein-1 (ptb1) is a determinant of the tissue and host tropism of a human rhinovirus/ poliovirus chimera pv1(ripo) coordinated assembly of human translation initiation complexes by the hepatitis c virus internal ribosome entry site rna polypyrimidine tract-binding protein influences negative strand rna synthesis of dengue virus identification of essential host factors affecting tombusvirus rna replication based on the yeast tet promoters hughes collection activation of picornaviral iress by ptb shows differential dependence on each ptb rna-binding domain polypyrimidine tract binding protein stabilizes the encephalomyocarditis virus ires structure via binding multiple sites in a unique orientation polypyrimidine tractbinding protein stimulates the poliovirus ires by modulating eif4g binding hepatitis a virus (hav) proteinase 3c inhibits hav ires-dependent translation and cleaves the polypyrimidine tract-binding protein polypyrimidine tract binding protein functions as a negative regulator of feline calicivirus translation stress granules and cell signaling: more than just a passing phase? rna-binding proteins tia-1 and tiar link the phosphorylation of eif-2 alpha to the assembly of mammalian stress granules poly(rc) binding proteins and the 5 0 cloverleaf of uncapped poliovirus mrna function during de novo assembly of polysomes an rna-binding protein, hnrnp a1, and a scaffold protein, septin 6, facilitate hepatitis c virus replication a cellular rna-binding protein enhances internal ribosomal entry site-dependent translation through an interaction downstream of the hepatitis c virus polyprotein initiation codon polypyrimidine tract-binding protein interacts with the 3 0 stem-loop region of japanese encephalitis virus negative-strand rna the role of ires trans-acting factors in regulating translation initiation inhibition of hepatitis c virus translation and subgenomic replication by sirnas directed against highly conserved hcv sequence and cellular hcv cofactors involvement of proteasome alpha-subunit psma7 in hepatitis c virus internal ribosome entry site-mediated translation human la protein interaction with gcac near the initiator aug enhances hepatitis c virus rna replication by promoting linkage between 5 0 and 3 0 untranslated regions functional interaction of hepatitis c virus ns5b with nucleolin gar domain systematic, genome-wide identification of host genes affecting replication of a positive-strand rna virus cleavage of poly(a)-binding protein by poliovirus 3c protease inhibits host cell translation: a novel mechanism for host translation shutoff rna looping by ptb: evidence using fret and nmr spectroscopy for a role in splicing repression modification of picornavirus genomic rna using "click" chemistry shows that unlinking of the vpg peptide is dispensable for translation and replication of the incoming viral rna redistribution of demethylated rna helicase a during foot-and-mouth disease virus infection: role of jumonji cdomain containing protein 6 in rha demethylation identification of rna helicase a as a new host factor in the replication cycle of foot-and-mouth disease virus functional interaction between cellular p100 and the dengue virus 3 0 utr viral subversion of the nuclear pore complex thiouracil cross-linking mass spectrometry: a cell-based method to identify host factors involved in viral amplification polypyrimidine tract-binding protein binds to the leader rna of mouse hepatitis virus and serves as a regulator of viral transcription heterogeneous nuclear ribonucleoprotein a1 binds to the transcription-regulatory region of mouse hepatitis virus rna cell proteins tia-1 and tiar interact with the 3 0 stem-loop of the west nile virus complementary minus-strand rna and facilitate virus replication hnrnp a1 interacts with the 5 0 untranslated regions of enterovirus 71 and sindbis virus rna and is required for viral replication syncrip (synaptotagmin-binding, cytoplasmic rna-interacting protein) is a host factor involved in hepatitis c virus rna replication hepatitis c virus translation preferentially depends on active rna replication regulation of stress granules and p-bodies during rna virus infection structural and functional studies of the promoter element for dengue virus rna replication norovirus genome circularization and efficient replication are facilitated by binding of pcbp2 and hnrnp a1 hcv ires interacts with the 18s rrna to activate the 40s ribosome for subsequent steps of translation initiation human protein sam68 relocalization and interaction with poliovirus rna polymerase in infected cells a cellular protein that binds to the 5 0 -noncoding region of poliovirus rna: implications for internal translation initiation la autoantigen enhances and corrects aberrant translation of poliovirus rna in reticulocyte lysate a noncoding rna produced by arthropod-borne flaviviruses inhibits the cellular exoribonuclease xrn1 and alters host mrna stability poly(rc) binding proteins mediate poliovirus mrna stability structure of ptb bound to rna: specific binding and implications for splicing regulation the 5 0 cl-pcbp rnp complex, 3 0 poly(a) tail and 2a(pro) are required for optimal translation of poliovirus rna riboproteomic analysis of polypeptides interacting with the internal ribosome-entry site element of foot-and-mouth disease viral rna sequestration of g3bp coupled with efficient translation inhibits stress granules in semliki forest virus infection yeast genome-wide screen reveals dissimilar sets of host genes affecting replication of rna viruses y box-binding protein-1 binds to the dengue virus 3 0 -untranslated region and mediates antiviral effects differential targeting of nuclear pore complex proteins in poliovirus-infected cells specific cleavage of the nuclear pore complex protein nup62 by a viral protease translation-competent 48s complex formation on hcv ires requires the rna-binding protein nsap1 cellular protein modification by poliovirus: the two faces of poly(rc)-binding protein canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry a prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis c and classical swine fever virus rnas hepatitis c virus infection inhibits p-body granule formation in human livers hepatitis c virus infection alters p-body composition but is independent of p-body granules cell-specific proteins regulate viral rna translation and virus-induced disease gemin5 promotes ires interaction and translation control through its c-terminal region gemin5 proteolysis reveals a novel motif to identify l protease targets conformational changes in the solution structure of the dengue virus 5 0 end in the presence and absence of the 3 0 untranslated region poly(a)-binding protein binds to the nonpolyadenylated 3 0 untranslated region of dengue virus and modulates translation efficiency leader-induced phosphorylation of nucleoporins correlates with nuclear trafficking inhibition by cardioviruses interplay between ns3 protease and human la protein regulates translation-replication switch of hepatitis c virus diversion of stress granules and p-bodies during viral infection inhibition of mrna export and dimerization of interferon regulatory factor 3 by theiler's virus leader protein the elav-like protein hur exerts translational control of viral internal ribosome entry sites picornavirus modification of a host mrna decay protein dynamic oscillation of translation and stress granule formation mark the cellular response to virus infection inhibition of host protein synthesis by sindbis virus: correlation with viral rna replication and release of nuclear proteins to the cytoplasm polypyrimidine-tractbinding protein: a multifunctional rna-binding protein translation and replication of hepatitis c virus genomic rna depends on ancient cellular proteins that control mrna fates heterogeneous nuclear ribonucleoprotein a1 regulates rna synthesis of a cytoplasmic virus la protein required for internal ribosome entry sitedirected translation is a potential therapeutic target for hepatitis c virus replication intracellular redistribution of truncated la protein produced by poliovirus 3cpro-mediated cleavage the n-terminal k homology domain of the poly(rc)-binding protein is a major determinant for binding to the poliovirus 5 0 -untranslated region and acts as an inhibitor of viral translation structural roles for human translation factor eif3 in initiation of protein synthesis sindbis virus usurps the cellular hur protein to stabilize its transcripts and promote productive infections in mammalian and mosquito cells the polypyrimidine tract-binding protein affects coronavirus rna accumulation levels and relocalizes viral rnas to novel cytoplasmic domains different from replication-transcription sites rna-rna and rna-protein interactions in coronavirus replication and transcription evidence for an rna chaperone function of polypyrimidine tract-binding protein in picornavirus translation hur, a protein implicated in oncogene and growth factor mrna decay, binds to the 3 0 ends of hepatitis c virus rna of both polarities binding of the la autoantigen to the hepatitis c virus 3 0 untranslated region protects the rna from rapid degradation in vitro the mechanism of translation initiation on type 1 picornavirus iress a distinct class of internal ribosomal entry site in members of the kobuvirus and proposed salivirus and paraturdivirus genera of the picornaviridae structural complexity of dengue virus untranslated regions: cis-acting rna motifs and pseudoknot interactions modulating functionality of the viral genome replication of poliovirus requires binding of the poly(rc) binding protein to the cloverleaf as well as to the adjacent c-rich spacer sequence between the cloverleaf and the internal ribosomal entry site magnetic fractionation and proteomic dissection of cellular organelles occupied by the late replication complexes of semliki forest virus identification of rnaprotein interaction networks involved in the norovirus life cycle polypyrimidine tract-binding protein interacts with coxsackievirus b3 rna and influences its translation a balance between circular and linear forms of the dengue virus genome is crucial for viral replication an rna virus hijacks an incognito function of a dna repair enzyme an rna element at the 5 0 -end of the poliovirus genome functions as a general promoter for rna synthesis viral ribonucleoprotein complex formation and nucleolar-cytoplasmic relocalization of nucleolin in poliovirus-infected cells distinct poly(rc) binding protein kh domain determinants for poliovirus translation initiation and viral rna replication the nucleocapsid protein of coronavirus mouse hepatitis virus interacts with the cellular heterogeneous nuclear ribonucleoprotein a1 in vitro and in vivo differential processing of nuclear pore complex proteins by rhinovirus 2a proteases from different species and serotypes solution structure of the symmetric coiled coil tetramer formed by the oligomerization domain of hnrnp c: implications for biological function genetics of poliovirus cytoplasmic redistribution and cleavage of auf1 during coxsackievirus infection enhance the stability of its viral genome viral subversion of nucleocytoplasmic trafficking functional significance of the interaction of hepatitis a virus rna with glyceraldehyde 3-phosphate dehydrogenase (gapdh): opposing effects of gapdh and polypyrimidine tract binding protein on internal ribosome entry site function mosquito la protein binds to the 3 0 untranslated region of the positive and negative polarity dengue virus rnas and relocates to the cytoplasm of infected cells cellular proteins from human monocytes bind to dengue 4 virus minus-strand 3 0 untranslated region rna common conformational changes induced in type 2 picornavirus iress by cognate transacting factors the mechanism of translation initiation on aichivirus rna mediated by a novel type of picornavirus ires interaction of poly(rc)-binding protein 2 domains kh1 and kh3 with coxsackievirus rna poly(rc)-binding protein 2 interacts with the oligo(rc) tract of coxsackievirus b3 rna interaction and cleavage of poly(c)-binding protein 2 by hepatitis a virus protease autogenous translational inhibition of core protein: implication for switch from translation to rna replication in hepatitis c virus formation of a ribonucleoprotein complex of mouse hepatitis virus involving heterogeneous nuclear ribonucleoprotein a1 and transcription-regulatory elements of viral rna research in the author's laboratory is supported by nih public health service grant ai50237, by nci, national institutes of health cancer center support grant p30ca1251230; and by national institutes of health grants hd007495, dk56338, and ca125123 (to the integrated microscopy core at baylor college of medicine). key: cord-344515-e0g911le authors: voss, kelsey; amaya, moushimi; mueller, claudius; roberts, brian; kehn-hall, kylene; bailey, charles; petricoin, emanuel; narayanan, aarthi title: inhibition of host extracellular signal-regulated kinase (erk) activation decreases new world alphavirus multiplication in infected cells date: 2014-11-30 journal: virology doi: 10.1016/j.virol.2014.09.005 sha: doc_id: 344515 cord_uid: e0g911le abstract new world alphaviruses belonging to the family togaviridae are classified as emerging infectious agents and category b select agents. our study is focused on the role of the host extracellular signal-regulated kinase (erk) in the infectious process of new world alphaviruses. infection of human cells by venezuelan equine encephalitis virus (veev) results in the activation of the erk-signaling cascade. inhibition of erk1/2 by the small molecule inhibitor ag-126 results in inhibition of viral multiplication. ag-126-mediated inhibition of veev was due to potential effects on early and late stages of the infectious process. while expression of viral proteins was down-regulated in ag-126 treated cells, we did not observe any influence of ag-126 on the nuclear distribution of capsid. finally, ag-126 exerted a broad-spectrum inhibitory effect on new world alphavirus multiplication, thus indicating that the host kinase, erk, is a broad-spectrum candidate for development of novel therapeutics against new world alphaviruses. the new world alphaviruses, venezuelan equine encephalitis virus (veev), eastern equine encephalitis virus (eeev), and western equine encephalitis virus (weev), can infect humans and potentially cause encephalitic disease (weaver and reisen, 2010) . in 2013, latin america recorded multiple confirmed cases of new world alphavirus infections with a total of 19 patients hospitalized for encephalitis. among them, 3 patients died, 1 of whom had confirmed vee (carrera et al., 2013) . owing to their propensity to cause naturally-occurring disease, these alphaviruses have been classified as emerging infectious agents. the new world alphaviruses have the potential to be extremely infectious by the aerosol route and have therefore been explored previously as potential bioweapons (zacks and paessler, 2010) . for this reason, veev and eeev are classified by the centers for disease control (cdc) as category b select agents. there are currently no fda-approved therapeutic candidates or vaccines for the protection of humans from new world alphavirus infections. the attenuated tc-83 strain of veev is used to vaccinate select at-risk personnel; however, tc-83 has concerns regarding safety and is considered to be a reactogenic vaccine (barrett and stanberry, 2009) . around 40% of all vaccinees have developed disease with some symptoms typical to that of natural vee infection (volkova et al., 2008) . therefore, there are ongoing efforts to establish a more effective and safer vaccine, some of which include vaccine candidates derived from the v3526 attenuated strain of veev martin et al., 2010; paessler and weaver, 2009; sharma et al., 2011) . fda-approved antiviral therapies for rna viruses, such as ribavirin, have been ineffective against veev and weev (canonico et al., 1984) , further highlighting the importance of new therapeutic approaches as medical countermeasures against new world alphaviruses. viruses rely on their host cell for the establishment of a productive infectious cycle. viruses are obligate pathogens that are known to modulate and utilize many host events, including host signal-transduction mechanisms. a deeper understanding of the dynamics of the interactions between the host and the pathogen can help in the identification of novel targets for therapeutics. for example, many alphaviruses have developed the ability to interfere with the induction of the host cell antiviral response (burke et al., 2009; garmashova et al., 2007) . animal models have also revealed changes in gene expression in veev-infected mouse brains (sharma et al., 2008) . we have demonstrated that host kinases, such as contents lists available at sciencedirect journal homepage: www.elsevier.com/locate/yviro glycogen synthase kinase-3β (gsk-3β) and the inhibitor of nuclear factor kappa-b kinase subunit beta (ikk-β), were modified upon veev infection amaya et al., 2014) . targeting these kinases with small molecule inhibitors resulted in a decrease of viral replication. such observations underscore the potential of hostbased candidates as therapeutic targets for development of antivirals against new world alphaviruses. the advantages conferred by hostbased therapeutics include a decreased potential for the development of resistant strains and an increased probability of broadspectrum applicability to treat many viral indications. the concerns around host-based therapeutics include a low threshold for toxicity and hence, a requirement of a stringent analysis of inhibitor induced toxicity profiles in the host. the host mapk, extracellular signal-regulated kinase (erk), responds to stress events including infection by directing multiple downstream events like inflammation and cell death (hong et al., 2009; hu et al., 2004; xing et al., 2010) . erk exists as erk1 and erk2 (hereafter referred to collectively as erk1/2), both of which have a central position in the mapk cascade, downstream in ras-raf-mek-erk signal transduction (roskoski, 2012a (roskoski, , 2012b . briefly, raf kinases act by phosphorylating and therefore activating mek1 and mek2 (collectively referred to as mek1/2). mek1/2 have dual specificity for erk1/2, phosphorylating first at tyrosine and then threonine sites in the activation segments of erk1/2, causing subsequent activation (roskoski, 2012a (roskoski, , 2012b . activated erk1/2 act as protein-serine/ threonine kinases, phosphorylating more than 150 cytosolic and nuclear substrates (yoon and seger, 2006; shaul and seger, 2007) . erk1/2 form an activated dimer and translocate to the nucleus, where they phosphorylate transcription factors regulating gene transcription (chuderland and seger, 2005; parra et al., 2005) . multiple viruses are known to activate the ras-raf-mek-erk signaling cascade in the host cell, and in many cases, this activation has been correlated with viral replication (pleschka, 2008) . specifically, publications have implicated the erk signaling pathway in the regulation of viral replication and gene expression for coxsackievirus b3 (luo et al., 2002) , human cytomegalovirus (boldogh et al., 1990; johnson et al., 2001) , junin virus (rodríguez et al., 2014) , human immunodeficiency type 1 (furler and uittenbogaart, 2010; jacqué et al., 1998) , coronavirus (cai et al., 2007) , and influenza virus . of particular relevance to our studies, it was reported that borna disease virus, an rna virus with high neurotropism, also manipulates the raf/ mek/erk signaling cascade in vitro and appears to be essential for viral spread (hans et al., 2001; planz et al., 2001) . here we demonstrate that veev infection of human astrocytoma cells results in the phosphorylation of multiple target proteins in the erk signaling cascade. using a small molecule inhibitor of erk, ag-126, we demonstrate that erk1/2 phosphorylation plays an important role in veev multiplication in infected cells. we provide data which suggest that early and late events in the viral infectious cycle are susceptible to erk1/2 inhibitors. we extend our studies to the virulent strains of veev, weev and eeev, and demonstrate that erk1/2 signaling is a broad-spectrum requirement for new world alphaviruses for the establishment of a productive infectious cycle. our previous studies have indicated that host kinases including ikk-β and gsk-3β were modulated in veev-infected cells and that inhibition of these kinases with small molecule inhibitors resulted in decreased viral multiplication (amaya et al., 2014; kehn-hall et al., 2012) . we reported in our ikk-β study that multiple components, which collectively constitute the nfkb response (ikk-β, iκbα, p65), were phosphorylated as part of a cascade activation process (amaya et al., 2014) . based on our previous lines of evidence, we hypothesized that veev infection will activate other host phospho-signaling cascades in infected cells and inhibition of specific events in those cascades will have an effect on veev multiplication. we utilized a reverse phase protein microarray (rppa) approach to identify changes in phospho-signaling cascades in veev-infected cells. we have successfully used this approach in previous studies to identify host events that are altered in virus-infected cells (popova et al., 2010a (popova et al., , 2010b wulfkuhle et al., 2006) . the rppa analysis was carried out as described previously (popova et al., 2010a (popova et al., , 2010b . briefly, u87mg cells were either mock infected or infected with tc-83, the attenuated strain of veev. infections with tc-83 as indicated throughout the manuscript involved exposure of the cells to the virus for one hour unless otherwise indicated. we are referring to as the time frame during which infection was allowed to happen after which the viral overlay was removed. the antibodies used for the analysis included a diverse range of signaling cascades such as growth factor signaling, akt signaling, nfkb signaling, and erk signaling. fold differences in phosphorylation of the candidate proteins seen in infected cells were calculated in comparison with those in the uninfected cells. we observed that multiple signaling molecules, which are part of the ras-raf-mek-erk signaling cascade, were phosphorylated in veev-infected cells. the schematic of the erk signaling cascade is shown in fig. 1a . the rppa analysis revealed that levels of phosphorylated erk1/2 were 2-fold higher in infected cells when compared with uninfected cells (fig. 1b) . the data also indicated that the phosphorylation of the erk1/2 upstream activator mek1/2 was also increased (fig. 1b) . furthermore, many downstream targets of erk1/2 including elk-1 (cruzalegui et al., 1999) , p65 (carter and hunninghake, 2000) , and stat-1 (zhang et al., 2004) were activated in infected cells (fig. 1b) . cumulatively, preliminary rppa data indicated that the erk signaling cascade was activated in veev-infected cells. we performed independent validation experiments with a focus on the phosphorylation status of erk1/2 in tc-83 infected u87mg cells. we carried out infections of u87mg cells and collected total protein lysates at multiple time points between 5 min after the removal of initial viral inoculum (referred to hence forth as the infection period) and 6 h after the infection period. the lysates were subsequently fractionated by sds-page and analyzed by western blot using antibodies against phosphorylated erk1/2 (thr202/tyr204), unphosphorylated (total) erk1/2, and βactin. the results showed an increased phosphorylation of erk1/2 in the infected cells after the infection period (fig. 1c , compare lanes 1 and 7). levels of p-erk1/2 remained consistently higher in the infected samples than the mock samples for the entire time course which distinguished the phospho-profile of erk1/2 in infected cells from the mock-infected control cells (fig. 1c) . we performed additional validation experiments of the rppa data by subjecting our protein lysates to western blot using antibodies against p-p90rsk and β-actin. the activation profile of p90rsk paralleled that of erk1/2 and agreed with the data obtained by the rppa process (fig. 1d ). to quantify levels of p-erk1/2 in the tc-83 infected samples, three independent experiments were performed and the fold changes over the mock samples in each experiment averaged. levels of p-erk1 and p-erk2 remained at around a minimum of a 2-fold increase over the mock throughout the time course (fig. 1e ). our analysis of total erk1/2 levels did not indicate any significant differences between mock and infected samples, thus indicating the phosphorylation event to be independent of total protein levels. cumulatively, the data included in fig. 1 suggest that infection of u87mgs with the attenuated tc-83 strain of veev results in an activation of the erk signaling cascade and phosphorylation of erk1/2 at early time points after infection. following validation of erk phosphorylation in veev-infected cells, we wanted to determine if this phosphorylation event was essential for viral replication. to define the role of erk1/2 in tc-83 infection, an inhibitor of erk1/2 phosphorylation, tyrphostin ag-126, was employed. tyrphostin ag-126 (α-cyano-[3-hydroxy-4nitro]cinnamonitrile), has been reported as a cell-permeable inhibitor of lipopolysaccharide (lps)-induced synthesis of tumor necrosis factor-alpha (tnf-α) and nitric oxide (nos) in murine peritoneal macrophages (novogrodsky et al., 1994) . it also blocks lps-induced tyrosine phosphorylation of erk and its substrates and reduces the expression of inos and cox-2 in the lungs of rats treated with carrageenan (chatterjee et al., 2003) . before we evaluated the antiviral efficacy of ag-126, we determined the toxicity profile of ag-126. u87mg cells were seeded in 96-well plates and 24 h later, increasing concentrations of ag-126 were added to wells in triplicate. dmso was added as a negative solvent control. the cells were maintained in the presence of ag-126 for 24 h after which viable cells were quantified by measuring luminescence in a cell-titer-glo assay. the assay results showed that the inhibitor was non-toxic to u87mg cells when compared to the dmso treated cells at the concentration range tested ( fig. 2a) . for all our subsequent experiments, we have used ag-126 at a concentration of 10 mm unless otherwise indicated. as a next step, we wanted to test whether inhibiting erk1/2 phosphorylation with ag-126 would have an effect on tc-83 replication. u87mg cells were plated in a 96-well format and were pretreated for 2 h with ag-126. the media with the inhibitor was removed and cells were infected with tc-83 for 1 h at 37 1c. the viral overlay was removed at the 1 h time frame and media with ag-126 was added back to the cells. the cells were then incubated for up to 24 h at 37 1c after which the supernatants were collected to quantify infectious viral titers using plaque assays. controls included infected cells treated with dmso. as the concentration of ag-126 treatment increased, the amount of viral inhibition increased, demonstrating a dose-dependent response (fig. 2b ). to determine whether the inhibitory effects of ag-126 were dependent on the multiplicity of infection (moi), u87mg cells were pretreated for 2 h with ag-126 or dmso alone and infected with tc-83 at three different mois (0.1, 0.5, 1). the cells were incubated for 24 h as described previously and the supernatants were collected for quantification of infectious viral titers. our data indicated that even at higher viral titers, ag-126 continued to be inhibitory to viral multiplication (fig. 2c) . to assess the effect of ag-126 on expression of viral proteins, whole cell lysates were obtained at 8 and 24 h post-infection, and subjected to western blot analysis using antibodies to veev capsid and envelope glycoproteins. at 8 h post-infection (8 hpi), veev capsid protein can be detected in the treated cells; however, the amount of capsid protein detected at this time point in the untreated cells was higher in the untreated cells (fig. 2d , compare lanes 1 and 2). the difference between the amounts of capsid protein produced in ag-126 treated versus untreated cells was more prominent at the 24 h time point (compare lanes 3 and 4). while we were unable to detect the veev e2 glycoprotein (gp) at 8 hpi, we could detect fairly robust expression of glycoprotein in tc-83 infected, untreated cells when compared to treated cells at the 24-hour time point (fig. 2d, lanes 3 and 4) . we quantified the differences of treated and untreated cells at the 24-hour time point in two independent experiments and found that ag-126 treatment modestly reduced capsid expression, and had a greater effect on gp expression (fig. 2d ). this indicated that decrease in viral structural protein levels by ag-126 treatment may be partially responsible for decreased viral titers. we also conducted western blot analysis on infected and ag-126 treated and infected cell lysates to ensure that ag-126 treatment did indeed inhibit phosphorylation of erk1/2. using antibodies against phosphorylated erk1/2 (thr202/ tyr204), unphosphorylated (total) erk1/2, and β-actin, we were able to document a decrease in the amount of phosphorylated erks at all time points tested in the presence of ag-126 ( fig. 2e) . we performed a similar inhibition efficacy study in vero cells to determine if the inhibition we observed in u87mg cells was a cell type dependent phenomenon. to that end, we first evaluated the cytotoxicity of ag-126 in vero cells and determined that up to 50 mm concentrations, ag-126 did not evoke any cytotoxic responses (fig. 3a) . we then determined if ag-126 could inhibit tc-83 multiplication in vero cells by pretreating cells with ag-126 for 2 h and then infecting with tc-83. infectious titers were measured by plaque assays at 24 h post the infection period. the result shown in fig. 3b indicates that ag-126 could inhibit tc-83 even in a permissive cell line such as vero cells, albeit, the extent of inhibition being lesser than what we observed in u87mg cells. cumulatively, our data suggested that at nontoxic concentrations, ag-126 inhibited erk1/2 phosphorylation, decreased viral protein expression and negatively influenced tc-83 multiplication in infected cells. inhibition of erk1/2 phosphorylation with ag-126 inhibits nuclear accumulation of erk, but does not interfere with nuclear accumulation of veev capsid protein phosphorylated erk1/2 has both nuclear and cytosolic targets, but typically is known to dimerize and translocate to the nucleus where it acts on multiple transcription factors (roskoski, 2012a) . inhibition of erk1/2 phosphorylation by small molecule inhibitors affected nuclear translocation of erk1/2 (roskoski, 2012a) . therefore, to confirm that ag-126 treatment indeed resulted in decreased phosphorylation of erk1/2 in u87mg cells, we investigated the intracellular p-erk1/2 localization during veev infection in the presence or absence of ag-126 at different time points using confocal microscopy as described previously (amaya et al., 2014) . briefly, u87mg cells were seeded in 8-well chambered slides and infected with tc-83 virus. at 6 and 24 h after the infection period, the cells were fixed and probed with antibodies to erk1/2 and veev capsid. at 6 h, nuclear enrichment of p-erk1/2 was observed in 71% of cells that were infected with tc-83 (fig. 4a ). we defined nuclear enrichment by a visual assessment, in which p-erk1/2 was localized to the nucleus. in contrast, 38% of mock cells showed enrichment, whereas the ag-126 treated cells had 22% enrichment. therefore, treatment with ag-126 resulted in a decrease in nuclear p-erk1/2, thus attesting to an inhibition of erk phosphorylation. this observation added support to the data presented in fig. 2e . at 24 h, 30% of tc-83 infected cells demonstrated a nuclear enrichment of p-erk1/2, but only 16% of the mock-infected cells showed nuclear erk1/2 (fig. 4b ). capsid staining was detected in 90% of cells exposed to tc-83 virus, but not treated with ag-126. in comparison, capsid staining was reduced by 68% in the infected, ag-126 treated cells. while overall capsid staining was reduced in ag-126 treated cells, we did not observe any difference in the distribution of capsid between the nucleus and cytoplasm of infected cells, regardless of ag-126 treatment thus indicating that erk1/2-mediated signaling events did not play a role in the nuclear-cytoplasmic transport of veev capsid protein in infected cells. cumulatively, our microscopy studies, while attesting to the inhibitory action of ag-126 in negatively influencing the nuclear accumulation of erk1/2, did not support the potential of erkmediated phosphorylation to control nuclear-cytoplasmic transport of veev capsid protein. our study thus far, has analyzed inhibition of tc-83 multiplication by ag-126 as a static phenomenon, at 24 h after the infection period. it would be more informative to determine if the overall replication kinetics of the virus was impacted by ag-126 treatment. to this end, u87mg cells were pre-treated with ag-126 for 2 h or dmso alone as a control. cells were infected with tc-83, supernatants were collected at multiple time points (3, 6, 16, 24 and 30 h post-infection), and plaque assays were performed. the results, as shown in fig. 5a , demonstrate an overall decrease in viral replication kinetics that could be clearly observed as early as 6 h post the infection period and was not exacerbated over subsequent time points. we performed time-of-addition experiments in an attempt to identify specific time frames in the infectious cycle when the inhibition by ag-126 could be detected. cells were treated with either ag-126 or dmso alone, at the indicated times (fig. 5b ). all supernatants were collected at 24 h and quantified for infectious viral titers. we observed that when cells were pretreated with ag-126 two hours before infection (2 h pre), robust inhibition was apparent. while ag-126 continued to be inhibitory when added 2 hpi (2 h post), the extent of inhibition was not as robust as the 2-hour pretreatment. when the drug was added at 4 hpi, the inhibitory potential decreased (fig. 5c) . we also observed a modest, but statistically significant reduction in titers when drug was added at 8 h post the infection period, which could potentially implicate ag-126 in late events as well. however, it is unclear whether the apparent inhibition seen at the 8 h time point was due to late events in the first round of viral multiplication, or the early events of the second round of multiplication. cumulatively, our experimentation with the addition of ag-126 at fig. 4 . ag-126 treatment decreases nuclear pools of p-erk1/2 without influencing capsid localization in the nucleus. u87mg cells were seeded in a 8-chambered slide and infected with tc-83 (moi:1). for every variable, at least two independent wells were analyzed. cells were fixed at 6 (a) and 24 (b) hpi and stained with antibodies against veev capsid (red) and p-erk1/2 (green). the slides were also stained with dapi to stain the nucleus (blue). images were collected using a nikon eclipse te2000-u and acquired with a 60x objective. images are representative of multiple fields that were imaged for each variable. three independent experiments were conducted. multiple time points after the infection period revealed that ag-126 may affect viral multiplication at early and late time frames of the infectious process. the fact that the pretreatment strategy was the most efficacious in our hands could also imply that the longer the drug is available in the system, the greater the extent of inhibition observed. at this time, we wanted to determine if ag-126 was directly virucidal in nature, which may also contribute to the observed inhibition with a concentration towards the early time points. to address this possibility, we performed a modified plaque reduction neutralization assay. we incubated tc-83 directly with ag-126 for 1 h at room temperature. after the incubation, the infectivity of the virus was quantified by plaque assay. our data indicated that when ag-126 was added to u87mg cells in the presence of virus, there was inhibitory action as expected; this acted as our positive control for the inhibitory potential of ag-126 (fig. 5d) . however, when we incubated the virus directly with ag-126 as a part of the same experiment, in the absence of u87mg cells and then quantified the infectivity of the virus by plaque assay, there was no difference between virus exposed to the drug and virus not exposed to the drug, thus indicating that ag-126 did not exert any direct virucidal activity. to gain more insight into the antiviral effects of ag-126 on tc-83 multiplication, we performed a slight variation of the time of addition study in which the drug was made available only for limited blocks of time. this experimental organization involved adding the drug at different time points followed by removing the drug and replacing with fresh media (fig. 5e ). u87mg cells were treated with ag-126 at the indicated times and all supernatants were collected at 24 h and quantified for infectious viral titers (fig. 5e ). we observed that when cells were pre treated for 2 h (pre), there was a robust inhibition of tc-83 (fig. 5f ). however, the inhibition was even more robust when the drug was added directly after the one hour infection period for one hour (0-1 hpi). later time points were modestly efficacious. paralleling our earlier observation, addition of the drug at the 6-8 h window resulted in a statistically significant decrease in viral multiplication. this may be due to the influence of ag-126 on late stages of viral multiplication or reflective of the early events during the second round of viral multiplication. we attempted to determine whether inhibition of viral multiplication by ag-126 may also be a result of aberrant transport and/ or localization of structural proteins such as the glycoproteins to the endoplasmic reticulum (er) and the golgi apparatus (snyder and mukhopadhyay, 2012) . to that end, we performed confocal microscopy experiments using antibodies to the er and the golgi apparatus in the context of tc-83 infection, with and without ag-126 to assess glycoprotein distribution in the er and golgi. we carried out this experiment using veev glycoprotein antiserum and analyzed the distribution of the glycoprotein in the er and golgi at 1, 2, 4 and 6 h post the infection period. in our hands, we were able to detect glycoprotein in the er at the 1 h time point and the er signal steadily decreased during the time course of analysis ( fig. 6a and b) . we were not able to observe any distinct colocalization of glycoprotein with the golgi apparatus within the time frames included in our study (fig. 6a ). in the presence of ag-126, we noticed that the glycoprotein signal in the er was reduced at the 1 h time point and the difference in the er signal between the treated and untreated cells increased by the 2 and 4 h time points suggesting that ag-126 may have an effect on the er localization of veev glycoprotein. we hypothesized that if inhibition of erk1/2 activation resulted in down-regulation of veev multiplication, activation/ priming of erk1/2 could contribute to an up-regulation of veev multiplication. we employed a mapk activator of erk, ceramide fig. 6 . inhibition of erk1/2 phosphorylation with ag-126 results in less veev glycoprotein in the endoplasmic reticulum. u87mg cells were seeded in a 8-chambered slide and infected with tc-83 (moi:1). for every variable, at least three independent wells were analyzed. (a) cells were fixed at 1, 2, 4, and 6 hpi and stained with antibodies against veev glycoprotein (red) and an endoplasmic reticulum (er) marker (green). the slides were also stained with dapi to stain the nucleus (blue). slides were also fixed and stained with a golgi marker (green) at the same time points. images were collected using a nikon eclipse te2000-u and acquired at 60 â objective. images are representative of multiple fields that were imaged for each variable. two independent experiments were conducted. (b) cells from triplicate wells in two experiments were counted at random for co-localization of veev glycoprotein and the er in the presence and absence of ag-126. c6 (raines et al., 1993) , to prime the cells and activate erk1/2 phosphorylation prior to infection. as a first step, we assessed the toxicity of the compound in u87mg cells and found no loss of viability at 1 μm concentration of the compound (fig. 7a ). treatments greater than or equal to 5 μm, however, had a noticeable level of toxicity. therefore, we decided to proceed with 0.1 μm treatments for analysis of viral replication kinetics when erk was activated prior to infection. to validate that ceramide c6 treatment was activating erk1/2 under our experimental conditions, we collected whole cell protein lysates at multiple time points in the presence or absence of ceramide c6 and performed western blot analysis. the data indicated that ceramide c6 treatment increased the amount of phosphorylated erk1/2 at 0.1 μm and 1 μm treatments. addition of 1 μm ceramide c6 resulted in a earlier activation of erk signaling (fig. 7b, compare lanes 1 and 6) , than 0.1 μm treatment confirming that ceramide c6 does indeed activate erk1/2 signaling in this cell type. to determine if ceramide c6-based activation of erk prior to infection contributed to an increase in viral multiplication, u87mg cells were treated for 2 h with either dmso alone or 0.1 μm ceramide c6 and then infected with tc-83 for 1 h. supernatants were collected at 6, 8, and 16 hpi and quantified by plaque assay. our data indicated that virus titers were statistically significantly higher with ceramide c6 treatments at the 6 h analysis point (fig. 7c) . by 16 h, the ceramide c6-treated samples had more than a 2-log increase over the dmso samples. we then assessed the effect of ceramide c6 treatment on the expression of viral proteins. we saw a modest increase of veev capsid and glycoprotein (gp) when cells were treated with 0.1 μm ceramide c6 before infection with tc-83 (fig. 7d ). two independent experiments were conducted and protein levels normalized to β-actin quantified from both experiments. the average is shown by the bars in fig. 7d . we next wanted to determine if activation of erk1/2 had an effect on the amount of viral rna produced in the treated cells. to assess the levels of intracellular viral rna, we performed quantitative rt-pcr (qrt-pcr) with veev specific primers. at 16 hpi, where we saw the greatest difference of infectious viral titers, we isolated intracellular rna and subjected it to qrt-pcr analysis. the genomic copies did not show any noticeable differences in the viral rna accumulation in the cells dependent on ceramide c6 treatment (fig. 7e) . the supernatants that corresponded to the 16 h time point that was analyzed by the pcr method was subjected to plaque assay to quantify infectious virus in those same supernatant samples. interestingly, the plaque assays revealed an increase in the infectious viral titers in the presence of ceramide c6, although viral rna levels were similar to the dmso control (fig. 7e) . we assessed whether ag-126 treatment resulted in a reduction of cytopathic effects (cpe) in context of tc-83 infection. our earlier experiments using host-based inhibitors of tc-83 multiplication have alluded to the possibility of increased survival of infected cells when the kinetics of viral multiplication are decreased (e) qrt-pcr results of veev genomic copies at 16 hpi. to the right, the supernatants from the same exact wells examined in the qrt-pcr for intracellular genomic copies were subjected to plaque assay to quantify infectious virus. (amaya et al., 2014) . to test whether ag-126 treatment reduced cpe in tc-83 infected u87mg cells, we imaged infected, untreated cells and infected, ag-126-treated cells 24 hpi (fig. 8a) . the infected, ag-126-treated cells (panels d, h and l) appeared to be healthier than the infected, untreated cells (panels c, g and k) and phenotypically resembled the mock infected, untreated cells (a, e and i). ag-126 treatment by itself did not change the morphology of the cells (b, f and j) when compared to the mock infected, untreated cells. next we adopted a quantitative cell survival assay to verify increased cell viability when treated with ag-126. in the survival assay, we measured the fraction of live cells by cell-titer-glo at 24 and 48 hpi in the presence and absence of ag-126 ( fig. 8b and c) . at 24 hpi, infected cells treated with dmso alone dropped to 75% of the luminescence seen in the uninfected cells. however, ag-126-treated cells showed 11% more luminescence when compared to dmso alone. at 48 hpi, treated cells had 32% more survival than the dmso alone control. collectively, the data supports the idea that in conjunction with exerting antiviral activity, inhibition of erk phosphorylation during early stages of infection by inhibitors, such as ag-126, can contribute to increasing survival of infected cells. we extended our analysis of ag-126 dependent inhibition of erk1/2 phosphorylation to the virulent strains of new world alphaviruses. in this experiment, we utilized the trinidad donkey (trd) strain of veev, the ga97 strain of eeev, and the california 1930 strain of weev. to this end, u87mg cells were pretreated with dmso or ag-126 and then infected with each of the virulent strains. all infections were performed for 1-hour and conditioned media were replaced and the supernatants collected 24 h later. plaque assays were performed to quantify infectious viral particles (fig. 9) . addition of ag-126 treatment reduced viral titers of veev trd from 8.67 â 10 8 pfu/ml to 8.17 â 10 4 pfu/ml, a 4-log decrease. in the case of eeev, treatment resulted in an overall 1-log drop, from 1.48 â 10 10 pfu/ml to 9.17 â 10 8 pfu/ml. for weev, ag-126 treatment contributed to a 2-log decrease in viral titer from 2.83 â 10 5 pfu/ml to 5.33 â 10 3 pfu/ml. the results supported the hypothesis that erk1/2 was likely to be a broad-spectrum target for the development of antivirals against new world alphaviruses. finally, we wanted to know if other inhibitors of the ras-raf-mek-erk signaling cascade would similarly affect veev multiplication. to this end, we employed various mek1/2 inhibitors to characterize their effect on tc-83 multiplication in our cells. we chose to evaluate pd184352, gsk1120212, azd6244, and u0126. among these, gsk1120212 is already fda approved for cancer therapy, and would therefore be a strong candidate to repurpose as an antiviral therapeutic for alphaviruses. the toxicity profile of each compound was evaluated at 50 and 20 μm concentrations in u87mg cells by cell-titer-glo assay as described previously. pd184352 showed a significant decrease in cell viability to 74% at 50 μm treatment, but was relatively nontoxic at 20 μm (fig. 10a ). all other compounds were non-toxic at both concentrations. u87mg cells were then pre treated with 50 μm of each mek inhibitor, except for pd184352 which was tested at 20 μm, and then infected with tc-83 virus. supernatants were collected 24 h later and subjected to plaque assay. the results showed that gsk1120212 was able to decrease veev multiplication by up to 2 logs while ad184352 was also effective, but to a lesser extent. u0126 and azd6244 were not effective at inhibiting tc-83 multiplication in this system (fig. 10b) . cumulatively, our data indicated that inhibition of erk1/2 signaling is an attractive option to inhibit alphavirus multiplication in human cells. innate immune responses that are established early following exposure to infectious agents play an essential role in pathogen control and protection of the host. such innate immune mechanisms are shaped by host phospho-signaling responses that undergo fairly elaborate modulation upon cellular insult, which includes exposure to infectious agents. virus infection is well documented to activate many host signaling responses that often shift the balance between host survival and cell death. many viruses have been known to hijack some of these intrinsic host responses to enable establishment of a productive viral infection. often such hijacking mechanisms involve additional host responses that are also modified, cumulatively resulting in a suppressed host environment where the virus can multiply without interference by the innate host protective mechanisms such as interferon responses. in fact, new world alphaviruses including veev and weev have been known to suppress the host interferon response by a mechanism that involves the viral capsid protein (atasheva et al., 2010; peltier et al., 2013) . as part of the pathogenesis, new world alphaviruses have also been known to trigger apoptosis of the infected cells, mediated by host signaling pathways (griffin and hardwick, 1997) . our previous studies using gsk-3β and ikk-β inhibitors have established that inhibition of host phospho-signaling events exerts an inhibitory effect on viral multiplication in infected human cells (amaya et al., 2014; kehn-hall et al., 2012; narayanan et al., 2012) . in the current study, we have provided evidence for the activation of erk1/2 phosphorylation at early time points following exposure to the tc-83 strain of veev (fig. 1) . our observation that phosphorylated erk1/2 increased in tc-83 infected cells while there were no significant changes observed in the levels of total erk1/2 protein indicated that the increase in phosphorylated forms was independent of de novo protein synthesis (fig. 1c) . small molecule inhibitors of erk1/2 phosphorylation, such as ag-126, when used in the nontoxic range, have the potential to inhibit viral multiplication (fig. 2) . we have observed that the inhibition is mediated through interruption of potentially early and late events in the infectious process. our data suggests that the compound is not directly virucidal. we hypothesize that ag-126 is likely to interfere with a host process that is essential for the internalized virus to go through the subsequent steps of capsid shedding and release of viral nucleic acid into the cytoplasm of the cell. additional steps in the inhibitory process may involve abberations in packaging and viral egress. ongoing research to address these possibilities will include analysis of host events that are essential for the intracellular transport of the virus, which may involve cytoskeletal dynamics. the hypothesis does not negate the possibility of direct erkmediated modification of viral proteins. for example, viral nonstructural proteins (nsps) may be phosphorylated by host kinases that influence their function. one such alphavirus protein that is known to be heavily phosphorylated is nonstructural protein 3 (nsp3) (li et al., 1990; peränen et al., 1988; vihinen and saarinen, 2000) . nsp3 is a critical regulator of negative strand synthesis and affects virulence. however, the exact role of nsp3 in the viral infectious cycle continues to remain unknown. our earlier studies have indicated that nsp3 may be phosphorylated by ikk-β. we will be characterizing the phosphorylation status of nsp3 in the presence of inhibitors, such as ag-126, because nsp3 contains other potential phosphorylation sites. these interesting aspects are currently under investigation in our laboratory. in addition, it appears that localization of e2 glycoprotein to the er of infected cells may be affected by ag-126 (fig. 6) . it is therefore interesting to speculate that ag-126 may have an effect on viral particle assembly and packaging. our studies suggest that inhibition of erk phosphorylation may also contribute to increase in survival of infected cells and decrease in cpe following alphavirus infection (fig. 8) . viruses such as varicella-zoster virus (vzv) have been documented to control apoptosis of infected cells by modulating multiple aspects of the apoptotic signaling responses, often culminating in differential phosphorylation of key effector molecules like bad, bax, bim, etc. (liu and cohen, 2014) . interestingly, it was demonstrated that the vzv protein orf12 triggered the phosphorylation of erk in order to inhibit apoptosis (liu et al., 2012) . it would be informative to quantify the status of various apoptotic events in veev-infected cells in the presence and absence of effective inhibitors like ag-126 to determine mechanisms that mediate cell death of infected cells. our observation that mek and erk are phosphorylated in veev-infected cells and erk inhibitors effectively interfere with viral multiplication open the doors for fda-approved mek and erk inhibitors including trametinib, selumetinib, and cobemetinib, for repurposing as antivirals with a novel indication as new world alphavirus therapeutics. repurposing such mek and erk inhibitors is additionally attractive because of our evidence that inhibition of erk has a broad-spectrum effect on the multiplication of all three new world alphaviruses (fig. 9) . in our hands, at least one other fda approved inhibitor of the mek-erk cascade was efficacious at inhibiting veev multiplication even though ag-126 had a better inhibitory potential (fig. 10) . the rppa analysis identified many signaling nodes in the ras-raf-mek-erk signaling cascade as being phosphorylated in tc-83 infected cells. we have focused the current study only on erk and its phosphorylation status. this study opens new avenues of exploration on the consequences of the phosphorylation of additional targets in this cascade on alphavirus replication. for example, p90rsk phosphorylation has been demonstrated to be intimately associated with replication of sars-coronavirus (mizutani et al., 2006) . it would be interesting to determine whether inhibitors of these multiple erk phospho-signaling nodes will exert a synergistic and more potent inhibition of the virus when used in a combinatorial capacity. human astrocytoma cell (u87mg cells) and african green monkey kidney epithelial cells (vero cells) were maintained in dmem supplemented with 10% fetal bovine serum (fbs), 1% penicillin/streptomycin, and 1% l-glutamine at 37 1c and 5% co 2 . cells were seeded in a 96-well plate in order to attain confluence within 24 h. the media was removed prior to infection and saved and is referred to as conditioned media. the cells were infected for 1 h at 37 1c to allow for viral adsorption, and then the viral inoculum was removed. cells were washed once with pbs, and the conditioned media replaced. then the cells were incubated at 37 1c, 5% co 2 overnight, and the supernatant collected 24 hpi and stored at à 80 1c until analyzed. small molecules included , ceramide c6 (santa cruz biotechnology, catalogue no. sc-3527), u0126 (cell signaling technology, catalogue no. 9903), pd184352 (selleckchem, catalogue no. s1020), gsk1120212 (selleckchem, catalogue no. s2673), and azd6244 (selleckchem, catalogue no. s1008). compounds were dissolved in dimethyl sulfoxide (dmso). u87mg cells were seeded in a 96-well plate at a density of 10,000 cells per well. the next day the cells were pretreated with the compound for 2 h. drug concentrations were maintained in a manner that did not exceed 0.1% dmso final concentration per well. the conditioned media containing the compound were removed and viral infections proceeded at multiplicity of infection (moi) of 0.1 for 1 h at 37 1c. the viral inocula were then removed and replaced with the conditioned media with compound. the cells were incubated for 24 h at 37 1c, 5% co 2 , and the supernatant was collected and stored at à 80 1c until analyzed. cell viability was measured using a cell-titer-glo luminescent cell viability kit (promega, catalog no. g7570) according to the manufacturer's instructions. briefly, u87mg cells were seeded in 96-well white wall plates at 10,000 cells per well and incubated for 24 h at 37 1c, 5% co 2 . cells were then treated with ag-126 or dmso (control) and incubated for 24 h at 37 1c, 5% co 2 . to determine cell viability, cell-titer-glo reagent was added to the cells in a ratio of 1:1. the plate was shaken for 2 min at room temperature and then incubated for 10 min at room temperature. luminescence was detected using the dtx 880 multimode detector (beckman coulter). the methods for rppa have been described previously (popova et al., 2010a (popova et al., , 2010b . u87mg cells were plated in a 6-well format and infected with tc-83 (moi:1) for one hour at 37 1c, 5% co 2 . after 6 h, the cells were washed with 1 â pbs, lysed and boiled for 10 min. the lysis buffer composition is as follows: 1:1 mixture of t-per reagent (pierce, il) and 2 â tris-glycine sds sample buffer (novex, invitrogen, ca) in the presence of 2.5% β -mercaptoethanol, and protease and phosphatase inhibitors (1 â halt cocktail, pierce). lysed samples were stored at à 80 1c until they were ready for the rppa study. to conduct the rppa study, approximately 30-50 nl of each sample were printed onto nitrocellulose slides (whatman, ma) using a high-resolution 2470 arrayer (aushon biosystems, billerica, ma). randomly chosed slides were stained with cyproruby stain to quantify total protein which will be used in signal normalization (described below). the printed slides were stained using a dako autostainer with specific antibodies using a biotin-linked peroxidase-catalyzed signal amplification (dako, csa kit). the slides were stained with a secondary biotinylated goat anti-rabbit igg antibody (vector labs, burlingame, ca) and imaged (umax powerlook iii scanner, umax, dallas, tx). the images were analyzed with software alphaase fc (alpha innotech). for every antibody used, the average pixel intensity value for negative control (staining with second antibody only) was subtracted from the average pixel intensity value and normalized by the corresponding average value of the total protein intensity. the signal intensity for every antibody spot was statistically evaluated using graphpad prism ver5 software (graphpad software, ca). the antibodies used in rppa array were against the following phosphorylated proteins and purchased from cell signaling: (catalog numbers of antibodies indicated in parenthesis): b-raf c-raf erk (9101), mek1/2 (9121), p65 (3031), p90rsk (9341), rsk3 (9348), stat1 (9134), u87mg cells were seeded at a density of 1.0 â 10 5 cells per well in a 12-well plate. cells were mock-treated, treated with ag-126, or treated with ag-126 for 2 h and then infected for 1 h. each condition was performed in triplicate wells. random sections of each well were imaged at 24 hpi and 48 hpi using a zeiss axiovert-25 microscope with pixera's viewfinder 3.0 software. vero cells were seeded at a density of 1.5 â 10 5 cells per well in a 12-well plate. viral supernatants, diluted in dmem, were used to infect vero cells in technical duplicate. the plates were incubated at 37 1c and 5% co 2 for 1 h with occasional rocking. a 1 ml overlay comprised of 2xe-mem and 0.6% agarose (1:1) was added to each of the wells. once solidified the plates were incubated for an additional 48 h at 37 1c and 5% co 2 . to complete the assay, an addition of a 10% formaldehyde solution was added to the surface of the agarose plugs followed by 1 h incubation at room temperature. the plates were then rinsed with dih 2 o and the agarose plugs removed. a 1% crystal violet solution containing 20% ethanol was added to each of the wells and incubated at room temperature with rocking for 30 min. the plates were rinsed with dih 2 o, and visible plaques counted to determine viral titers as plaque forming units per ml (pfu/ml). tc-83 viral stock at 1.6 â 10 7 pfu/ml was incubated at room temperature for 1 h either in the presence or absence of 10 μm ag-126 with gentle mixing. samples were then directly subjected to plaque assay. in order to obtain whole cell lysates, the media was removed from u87mg cells and the cells were washed once with pbs. next the cells were lysed with lysis buffer that consisted of a 1:1 mixture of t-per reagent (pierce, catalogue no. 78510), 2 â trisglycine sds sample buffer (invitrogen, catalogue no. lc2676), 2.5% β-mercaptoethanol, and protease and phosphatase inhibitor cocktail (1 â halt mixture, pierce). cells with lysis buffer were incubated at room temperature for one minute, and then collected. cell lysates were boiled for 10 min and stored at à 80 1c until analyzed. u87mg cells were pre-treated with 1 μm ceramide c6 or 10 μm ag126 for 2 h and then infected with tc-83 at an moi: 0.1. as a control cells were treated with dmso. at 16 h post infection cells were lysed using the magmax™-96 total rna isolation kit (life technologies, am1830) as per the manufacturer's instructions. intracellular viral rna was quantitated using q-rt-pcr with primers and probe for nucleotides 7931-8005 of veev tc-83 (kehn-hall et al., 2012) . the q-rt-pcr cycling conditions were as follows: 1 cycle at 50 1c for 30 min, 1 cycle at 95 1c for 2 min and 40 cycles at 95 1c for 15 s and 61 1c for 1 min using the stepone plus real time pcr system. the primer and probe pairs used were originally described by julander; forward primer (tctgacaagacgttcccaatca) and reverse primer (gaataactt ccctccgaccaca) and taqman probe (5 0 6-carboxyfluorescein-tgttggaagggaagataaacggctacgc-6-carboxy-n,n,n 0 ,n 0 -tetramethylrhodamine-3 0 ) (julander et al., 2008) . the q-rt-pcr assays were performed using biorad itaq universal probes onestep 2 â mix (biorad, 172-5140). the absolute quantification was calculated based on the threshold cycle (ct) relative to the standard curve. whole cell lysates were separated on a 4-20% tris-glycine gel at 100 v and transferred to a polyvinyl difluoride (pvdf) membrane using the iblot gel transfer system (invitrogen). the membranes were blocked in 1% dry milk in pbs-tween 20 or 2% bsa in pbs-tween 20 at room temperature. primary antibodies to veev capsid (bei resources, nr 9403), veev glycoprotein (bei resources, nr 9404), p-erk 1/2 (thr 202/tyr 204) (cell signaling technology, catalogue no. 4370), erk 1/2 antibody (cell signaling technology, catalogue no. 9102), p-p90rsk (ser380) (cell signaling technology, catalogue no. 11,989) and hrp-conjugated actin (abcam, catalogue no. ab49900) were used according to manufacturer's instructions. the blots were incubated with primary antibody overnight at 4 1c. following 2 washes with pbs-tween 20 the blots were then incubated with respective secondary hrp-coupled antibody for 2 h. after 3 washes with pbs-tween 20 and 1 wash with pbs, the membranes were visualized by chemiluminescence using supersignal west femto maximum sensitivity substrate kit (thermoscientific) on a bio-rad molecular imager chemidoc xrs system (bio-rad). immunofluorescence u87mg cells were seeded at a density of 20,000 cells/well in an 8-well chambered slide. the cells were either, mock-treated, treated with 10 μm ag-126 or infected with tc-83 at an moi of 5. cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.5% triton x-100 in pbs for 15 min. slides were washed with pbs and blocked at room temperature for 10 min with 3% bsa. the slides were incubated with primary antibody for 1 h in the dark at 37 1c, and then washed three times with pbs and incubated with respective secondary antibody alexa fluor antibodies (invitrogen) for 1 h in the dark at 37 1c. primary antibody markers for the endoplasmic reticulum and golgi included pdi (c81h6) and rcas1 (d2b6n), respectively (cell signaling technology, catalogue no. 8653s). slides were washed three times with pbs and incubated with dapi for 10 min in the dark at room temperature. following an additional pbs wash, the slides were mounted with fluoromount g (southernbiotech, catalogue no. 0100-01) and stored in the dark at 4 1c overnight. the cells were imaged using nikon eclipse te2000-u. images were taken at 60 â objective. graphing and data analyses were performed using the r statistical language. we performed three independent experiments using separately harvested viral supernatants each time. each experiment included three biological samples per group. we recorded the raw plaque counts for each of the plaque assays and determined the pfu/ml. the figures present scatter plots overlaid on box and whisker plots. the box and whisker plots have their conventional meanings. we use our graphical representations as well as performing wilcoxon rank sum tests to assess differences among treatment groups. p-values less than 0.01 were considered statistically significant. significant results are indicated on the figures in the following fashion: p-value r0.0001 (nnn), p-value r0.001 (nn), p-value r0.01 (n). the role of ikkβ in venezuelan equine encephalitis virus infection interplay of acute and persistent infections caused by venezuelan equine encephalitis virus encoding mutated capsid protein preface activation of proto-oncogenes: an immediate early event in human cytomegalovirus infection characteristics of alpha/beta interferon induction after infection of murine fibroblasts with wild-type and mutant alphaviruses suppression of coronavirus replication by inhibition of the mek signaling pathway in-vivo activity of antivirals against exotic rna viral infections eastern equine encephalitis in latin america a constitutive active mek -4 erk pathway negatively regulates nf-kappa b-dependent gene expression by modulating tata-binding protein phosphorylation the tyrosine kinase inhibitor tyrphostin ag126 reduces renal ischemia/reperfusion injury in the rat protein-protein interactions in the regulation of the extracellular signal-regulated kinase erk activation induces phosphorylation of elk-1 at multiple s/t-p motifs to high stoichiometry a multisystem approach for development and evaluation of inactivated vaccines for venezuelan equine encephalitis virus (veev) signaling through the p38 and erk pathways: a common link between hiv replication and the immune response the old world and new world alphaviruses use different virus-specific proteins for induction of transcriptional shutoff regulators of apoptosis on the road to persistent alphavirus infection borna disease virus persistent infection activates mitogen-activated protein kinase and blocks neuronal differentiation of pc12 cells noncatalytic function of erk1/2 can promote raf/mek/erk-mediated growth arrest signaling critical role of endogenous akt/iaps and mek1/erk pathways in counteracting endoplasmic reticulum stressinduced cell death modulation of hiv-1 infectivity by mapk, a virion-associated kinase the role of mkk1/2 kinase activity in human cytomegalovirus infection c3h/hen mouse model for the evaluation of antiviral agents for the treatment of venezuelan equine encephalitis virus infection modulation of gsk-3β activity in venezuelan equine encephalitis virus infection phosphorylation of sindbis virus nsp3 in vivo and in vitro inhibition of bim enhances replication of varicella-zoster virus and delays plaque formation in virus-infected cells varicella-zoster virus orf12 protein triggers phosphorylation of erk1/2 and inhibits apoptosis coxsackievirus b3 replication is reduced by inhibition of the extracellular signal-regulated kinase (erk) signaling pathway evaluation of formalin inactivated v3526 virus with adjuvant as a next generation vaccine candidate for venezuelan equine encephalitis virus regulation of p90rsk phosphorylation by sars-cov infection in vero e6 cells curcumin inhibits rift valley fever virus replication in human cells prevention of lipopolysaccharide-induced lethal toxicity by tyrosine kinase inhibitors vaccines for venezuelan equine encephalitis features of the catalytic domains and c termini of the mapk signal-integrating kinases mnk1 and mnk2 determine their differing activities and regulatory properties neurotropic arboviruses induce interferon regulatory factor 3-mediated neuronal responses that are cytoprotective, interferon independent, and inhibited by western equine encephalitis virus capsid semliki forest virusspecific non-structural protein nsp3 is a phosphoprotein mek-specific inhibitor u0126 blocks spread of borna disease virus in cultured cells rna viruses and the mitogenic raf/mek/erk signal transduction cascade influenza virus propagation is impaired by inhibition of the raf/mek/erk signalling cascade reverse-phase phosphoproteome analysis of signaling pathways induced by rift valley fever virus in human small airway epithelial cells reverse-phase phosphoproteome analysis of signaling pathways induced by rift valley fever virus in human small airway epithelial cells sphingomyelinase and ceramide activate mitogen-activated protein kinase in myeloid hl-60 cells raf/ mek/erk pathway activation is required for junin virus replication erk1/2 map kinases: structure, function, and regulation mek1/2 dual-specificity protein kinases: structure and regulation venezuelan equine encephalitis virus infection causes modulation of inflammatory and immune response genes in mouse brain safety and protective efficacy of ina-inactivated venezuelan equine encephalitis virus: implication in vaccine development the mek/erk cascade: from signaling specificity to diverse functions the alphavirus e3 glycoprotein functions in a clade-specific manner phosphorylation site analysis of semliki forest virus nonstructural protein 3 iresdependent replication of venezuelan equine encephalitis virus makes it highly attenuated and incapable of replicating in mosquito cells present and future arboviral threats technology insight: pharmacoproteomics for cancer-promises of patient-tailored medicine using protein microarrays roles of the erk mapk in the regulation of proinflammatory and apoptotic responses in chicken macrophages infected with h9n2 avian influenza virus the extracellular signal-regulated kinase: multiple substrates regulate diverse cellular functions encephalitic alphaviruses evidence of stat1 phosphorylation modulated by mapks, mek1 and msk1 veev ptc83 was a kind gift from ilya frolov of the university of alabama at birmingham. the following reagents were obtained through the nih biodefense and emerging infections research resources repository, niaid, nih: (i) venezuelan equine encephalitis virus, tc83 (subtype iab), nr63, (ii) venezuelan equine encephalitis virus trinidad donkey (subtype ia/b), nr-332, (iii) polyclonal anti-venezuelan equine encephalitis virus, tc83 (subtype ia/b) capsid protein (antiserum, goat), nr-9403, (iv) polyclonal anti-venezuelan equine encephalitis virus, tc-83 (subtype ia/b) glycoprotein (antiserum, goat), nr-9404. we are thankful for the following reagent, which was obtained from dr. jonathan jacobs from mriglobal: eeev ga97. the following reagent was obtained from atcc: weev (california 1930 strain), vr-70. we would like to thank jyoti shankar from the j craig venter institute for providing statistical programming mentorship to kv. key: cord-334133-61om170g authors: hollier, mark j.; dimmock, nigel j. title: the c-terminal tail of the gp41 transmembrane envelope glycoprotein of hiv-1 clades a, b, c, and d may exist in two conformations: an analysis of sequence, structure, and function date: 2005-07-05 journal: virology doi: 10.1016/j.virol.2005.04.015 sha: doc_id: 334133 cord_uid: 61om170g in addition to the major ectodomain, the gp41 transmembrane glycoprotein of hiv-1 is now known to have a minor ectodomain that is part of the long c-terminal tail. both ectodomains are highly antigenic, carry neutralizing and non-neutralizing epitopes, and are involved in virus-mediated fusion activity. however, data have so far been biologically based, and derived solely from t cell line-adapted (tcla), b clade viruses. here we have carried out sequence and theoretically based structural analyses of 357 gp41 c-terminal sequences of mainly primary isolates of hiv-1 clades a, b, c, and d. data show that all these viruses have the potential to form a tail loop structure (the minor ectodomain) supported by three, β-sheet, membrane-spanning domains (msds). this means that the first (n-terminal) tyrosine-based sorting signal of the gp41 tail is situated outside the cell membrane and is non-functional, and that gp41 that reaches the cell surface may be recycled back into the cytoplasm through the activity of the second tyrosine-sorting signal. however, we suggest that only a minority of cell-associated gp41 molecules – those destined for incorporation into virions – has 3 msds and the minor ectodomain. most intracellular gp41 has the conventional single msd, no minor ectodomain, a functional first tyrosine-based sorting signal, and in line with current thinking is degraded intracellularly. the gp41 structural diversity suggested here can be viewed as an evolutionary strategy to minimize hiv-1 envelope glycoprotein expression on the cell surface, and hence possible cytotoxicity and immune attack on the infected cell. viruses of the lentivirus genus of the retroviridae have a single virus-encoded, envelope glycoprotein (env). this is a type i membrane protein with the n-terminus on the outside of the cell. env mrna is synthesized in the nucleus, and exported with the help of the viral rev protein to the cytoplasm where it is translated as a gp160 precursor. gp160 is then translocated through the rough endoplasmic reticulum (er) (dettenhofer and yu, 2001) , and folded with the assistance of the chaperone proteins, calreticulin, and calnexin (otteken et al., 1996) . glycans are added in the golgi, and subsequently trimmed (dash et al., 1994; fenouillet and jones, 1995) . gp160 oligomerizes in the er, forming a non-covalently linked trimer (earl et al., 1991; willey et al., 1988) . most gp160 is degraded, but about 5-15% is cleaved into gp120 (distal) and gp41 (membrane anchor) components. these remain associated non-covalently and are targeted to the cell surface (see below). gp120-gp41 trimers are incorporated into progeny virions. gp120 can be shed from the surface of cells and virions (willey et al., 1988 (willey et al., , 1991 . enzymatic cleavage of gp160 to gp120-gp41 is essential for env function. uncleaved gp160 cannot induce syncytium formation, and virions with uncleaved gp160 are not infectious. gp160 is rarely incorporated into virions, probably because so little reaches the cell surface (duensing et al., 1995; pal et al., 1991; pfeiffer et al., 1997; willey et al., 1988) . hiv-1 gp160 is cleaved between residues 518 r and 519 a by the subtilisin/kexin-like ca 2+ -dependent convertases such as furin, pace4, pc5/6-b, and pc1 (morikawa et al., 1993; moulard et al., 1994; vollenweider et al., 1996) . gp160 is probably cleaved by more than one cellular protease. gp160 cleavage occurs in the trans-golgi network (tgn) or after it exits the tgn (bultmann et al., 2000; pal et al., 1991; pfeiffer et al., 1997; stein and engelman, 1990) . brefeldin a, a1fn, monensin, and tunicamycin inhibit gp160 cleavage even when gp160 is allowed to accumulate in the tgn, suggesting that cleavage occurs in the late compartment of the golgi or after gp160 has exited from the golgi (dewar et al., 1989; kantanen et al., 1995; pal et al., 1991 pal et al., , 1988 . cleavage may occur in an acidic compartment, as it was inhibited by nh 4 cl and partially inhibited by chloroquine (courageot et al., 1999; willey et al., 1988) . however, these inhibitors may prevent gp160 from reaching the correct intracellular site for cleavage. methionine methyl ester failed to inhibit gp160 cleavage, indicating that cleavage may occur in a non-lysosomal compartment (willey et al., 1988) . only a small proportion of gp160 (5-40%) is cleaved into gp120-gp41, and this depends on the host cell (bird et al., 1990; hallenberger et al., 1993; jose et al., 1997; kimura et al., 1996; kozarsky et al., 1989; moulard et al., 1999; pfeiffer et al., 1997; willey et al., 1988 willey et al., , 1991 . for example, 10 -20% of gp160 is cleaved in peripheral blood lymphocytes (willey et al., 1988) . uncleaved, trimeric gp160 is degraded in the er and lysosomes. however, the proportion degraded in each location is not known. according to willey et al., most gp160 leaves the er and reaches the golgi, and the minority that remains in the er is degraded there (willey et al., 1988) . however, others find that most gp160 remains in the er and is degraded (bultmann et al., 2000; courageot et al., 1999; hallenberger et al., 1993; jabbar and nayak, 1990; pfeiffer et al., 1997) , or is rapidly sent to lysosomes and degraded (jabbar and nayak, 1990; pfeiffer et al., 1997) . whichever the location, degradation is relatively rapid, and only 10 -20% of gp160 remains after 8 h (willey et al., 1988) . when prevented from leaving the er, gp160 accumulates there and is degraded (courageot et al., 1999; pal et al., 1991; willey et al., 1991) . gp160 degradation has also been observed in proteosomes (bultmann et al., 2000) . the consensus view is that most gp160 is degraded in the er, and only a minority reaches the golgi. of the latter, most (85 -95%) is degraded in lysosomes, and the remainder is targeted as gp120-gp41 to the cell surface (willey et al., 1988) . gp120 is relatively stable, and 30 -50% can be detected as secreted or intracellular protein 24 h later (bird et al., 1990; willey et al., 1988 willey et al., , 1991 . the amount of gp160 secreted from the cell is less than 10% of the gp120 secreted (willey et al., 1988) , although the amount varies with the type of host cell (moulard et al., 1999) . tyrosine-dependent sorting signals (yxxb, where x represents any amino acid residue and b represents a bulky hydrophobic residue), and possibly di-leucine-sorting signals are found in the c-terminal tail of gp41, and have been implicated in transport of gp160 and gp120-gp41 (see results and discussion section and berlioz-torrent et al., 1999; boge et al., 1998; bu et al., 2004; deschambeault et al., 1999; egan et al., 1996; lodge et al., 1997; ohno et al., 1997; owens et al., 1991; rowell et al., 1995; west et al., 2002; wyss et al., 2001) . gp120 is the distal part of the envelope glycoprotein trimer, and recognizes the primary cd4 receptor, and coreceptors on the target cell. gp41 anchors the envelope glycoprotein in the cell or virion membrane, and mediates the virion fusion entry process that is activated by receptor binding. the n-terminal peptide of gp41 is inserted into the membrane of the target cell and leads to fusion of the virion and cell membranes, entry of the virus genome and associated proteins into the cell, and infection. gp41 comprises an ectodomain, a single membrane-spanning domain (msd), and a long c-terminal tail that in the past has been viewed as being entirely contained inside the cell or virion (gallaher, 1987; gallaher et al., 1989; gonzalez-scarano et al., 1987; levy, 1998; white, 1990) . however, there is abundant evidence that hiv-1 virions can be neutralized by antibodies directed to an epitope in the cterminal tail (buratti et al., 1998; chanh et al., 1986; cleveland et al., 2000a cleveland et al., ,b, 2003 dalgleish et al., 1988; durrani et al., 1998; ho et al., 1987; mcinerney et al., 1999; mclain et al., 1995 mclain et al., , 1996 mclain et al., , 2001 newton et al., 1995; reading et al., 2003) . since antibodies do not cross lipid bilayers, this means that part of the tail is exposed on the virion surface. we have called this exposed region of the gp41 tail the minor ectodomain, to distinguish it from the better known, and larger, major ectodomain. non-neutralizing antibodies specific for the minor ectodomain have also been shown to bind virions (cleveland et al., 2003; mclain et al., 2001) , providing further evidence of the externalization of the minor ectodomain. binding of antibody is abrogated by pre-treatment of virions with protease, again supporting the external location of this part of the tail (cleveland et al., 2003) . more recently, we demonstrated for the first time that neutralizing and non-neutralizing antibodies to the minor ectodomain bind to infected cells and that the neutralizing antibodies inhibit fusion of infected and non-infected cells (cheung et al., 2005; heap et al., 2005) . thus, part of the gp41 c-terminal tail is exposed on the surface of both infected cells and virions. until now, evidence for the exposure of part of the gp41 c-terminal tail has been based on the study of hiv-1 b clade, t cell line-adapted (tcla) viruses, and the generality of the existence of the minor ectodomain, and its structural basis, have not been explored. to remedy this, we have here analyzed 357 gp41 c-terminal tail database sequences from clades a, b, c, and d. this analysis shows that all these could potentially have a minor ectodomain of approximately 40 residues, supported by three msds, and an internal tail of approximately 100 residues. we shall further suggest that this 3-msd form of gp41 coexists with the accepted 1-msd form of gp41, and that the 3-msd form represents a minor population of intracellular gp41 that is destined for incorporation into virions. in contrast, 1-msd gp41 is the majority form of intracellular gp41, most of which is degraded. regions of conservation in the gp41 sequence 690 -793 of hiv-1 clades a to d are summarized in fig. 1 , with residues numbered according to ratner et al. (1985) . the only very highly conserved region throughout all clades is 713 nrvrqgysplsfq 725 , which contains the most n-terminal (first) tyrosine-dependent sorting signal ( 719 yspl 722 ). as expected, the region comprising the accepted msd, 690 kifimivggliglrivfavlsiv 712 , is highly conserved and forms part of a larger highly conserved sequence 690 kifimivggliglrivfavlsivnrvrq-gysplsfq 725 , which includes the region surrounding the first tyrosine-dependent sorting signal. however, there are conservative substitutions in individual clade consensus sequences of 700 i-v (clade b), 705 v-i (clade c), 711 i-l (clade d), and 712 v-i (clade a). the region 767 rslclfsyhrlr 779 containing the second potential tyrosine-dependent sorting signal ( 775 yhrl 778 ) is highly conserved. the highly conserved 790 ellg 793 contains a potential di-leucine signal, but the only di-leucine signal known to be functional in gp41 is 862 ll 863 (wyss et al., 2001) . the kennedy sequence ( 731 prgpdrpgrieeeg-geqdrdrs 752 ) in clade b viruses contains the neutralizing epitope core sequence 746 erdrd 750 , and two non-neutralizing epitopes ( 734 pdrpeg 739 and 740 ieee 743 ) (chanh et al., 1986; dalgleish et al., 1988; evans et al., 1989; ho et al., 1987; kennedy et al., 1986; niedrig et al., 1992; vella et al., 1993) . overall the kennedy sequence is only poorly to moderately conserved. specifically 731 prgpdrpgri 740 and 747 qdrdrs 752 are poorly to moderately conserved, but 741 eeegge 746 is moderately to highly conserved. 741 eeegge 746 is notable in having 67% acidic residues. 746 erdrd 750 is present only in clade b and the recombinant crf03 _ ab clade. clades a and c have 746 eqdrd 750 and clade d has 746 eqgrg 750 . the exchange of 747 r (basic, ionizable, hydrophilic, with a long side chain) for q (polar, hydrophilic, amidic, with a shorter side chain) might create a different epitope, and 746 erdrd 750 -specific neutralizing antibody may not recognize 746 eqdrd 750 . the gp41 tail reading frame overlaps the second exons of tat (+1 relative to env) and rev (+2). both of the latter start fig. 1 . summary of the conservation of gp41 c-terminal amino acid residues 690 -793 of hiv-1 clades a to d, using a 7-residue moving window. at the same position, equivalent to the codon for residue 725 of env. tat and rev stop at the equivalent of the codons for residues 740 and 816 of env, respectively. this is the only part of the env sequence where all three orfs are used, yet counter-intuitively, conservation of 725 -740 of env ranges from poor to high. it may be that the tat and/or rev sequences are conserved at the expense of env. all clades have a very highly conserved 745 g (100%), 758 g (99.9%), and 764 w (99.4%). the conservation of 745 g and 758 g may be a consequence of the reading frame shared with rev. the last two bases of a rev codon are the first two bases of the env codon. thus, if rev requires a tryptophan in its second exon, the overlapping codon of env has to be glycine, since tryptophan is only encoded by ugg, while the codon for glycine is ggx. the reason why 764 w is very highly conserved is not clear, but any change in its codon results in a different amino acid residue or a stop codon (uga or uag). regions of conservation for clades a to d consensus sequences of gp41 residues 690 -793, and an overall consensus sequence are summarized in fig. 2 . the consensus sequences of hiv-1 clades a to d derived above were analyzed individually (see materials and methods), but as the predicted structures were virtually identical, only clade a data are presented. according to kyte and doolittle (1982) , four regions (690 -699, 702-711, 758 -763, and 783-787) have hydropathy values of >1.6, indicating that they are potential msds (fig. 3a ). there are two single point peaks of 1.6 at residues 771 and 781, but the values of surrounding residues are too low for these to form an msd. the kennedy region (731 -752) is highly hydrophilic, with values of mainly à1, suggesting that it is exposed to solvent. essentially the same conclusions were reached (data not shown) using other hydropathy prediction algorithms (eisenberg et al., 1984; hopp and woods, 1981; sweet and eisenberg, 1983) . five regions (690 -694, 704 -711, 740 -747, 758 -766, and 779 -793) have a-helix potentials of >1.03 (fig. 3b) , and four regions (690 -719, 753-761, 770 -777, and 779-788) have h-sheet values of >1.05 (fig. 3c ). the most likely conformation is that with the highest predicted value (chou and fasman, 1978) . thus, 690 -719 (1.34) is likely to be hsheet. in this region there is a dip to 1.05 at residue 702, suggesting that 690-719 may comprise two discrete regions (690 -701 and 703-719). this is consistent with the two hydropathic regions predicted above. there are two regions with different conformations adjacent to each other at 753-766, with 753 -761 showing higher h-sheet potential and 761 -766 showing greater a-helix potential. 779-788 could fig. 2 . consensus sequences for gp41 c-terminal amino acid residues 690 -793 of hiv-1 clades a to d combined (top line), and for clades a to d individually. the potential tyrosine-dependent sorting signals are in red. a potential di-leucine signal is pink. residues common to the majority of sequences are in black, while residues that vary are in green. where two residues are equally represented between the consensus sequences of the four clades, the overall consensus sequence is based on the larger number of sequences analyzed. the proposed first, second, and third msds are overlined. underlined is the antigenically active kennedy sequence. be either a-helix or h-sheet. the shows no potential for h-sheet formation with values <0.95. however, 740 -747 of the kennedy region that in clade b tcla viruses contains the highly immunogenic and anti-genic epitope 740 ieee 743 (cleveland et al., 2000a ) may form an a-helix (>1.03). there are potential h-turns at 702, 721, 731, 733, 748, 768, and 778 (fig. 3d) . the h-turn at 702 is consistent with the hydropathy and h-sheet predictions that this links two discrete msds. residue 721 p is present in most of the first tyrosine-dependent sorting signals ( 719 yspl 722 ) and is discussed later. the kennedy region contains potential hturns at 731, 733, and 748, and with the prediction of only one short region of a-helix and no h-sheet, the region is probably unstructured. the potential h-turn at 749 r might be important for maintaining the complex conformational neutralizing epitope 746 erdrd 750 found in clade b virions (buratti et al., 1998; cheung et al., 2005; cleveland et al., 2000a cleveland et al., ,b, 2003 heap et al., 2005; mclain et al., 2001; reading et al., 2003; vella et al., 1993) . h-turns at 768 and 778 would occur after the putative third msd (see below). there are four highly non-polar regions (694 -699, 708 -711, 720-725, and 759-762) (fig. 3e) . regions 694 -699 and 708 -711 coincide with the msd 1 and 2 predicted above. the unequal polarity of these two domains adds to the suggestion that they are separate entities. residues 720 -725 contain the first tyrosine-dependent sorting signal, but the significance of its lack of polarity is not clear. residues 759 -762 coincide with the possible location of the third msd predicted above (754 -763). region 780 -793 is moderately polar and unlikely to form an msd. the kennedy region is the most polar region in the analyzed sequence, consistent with it being exposed to aqueous solvent and with its antigenic properties. regions 694 -712 (containing the predicted msd 1 and 2 at 691-700 and 703 -712) and 755 -763 are likely to be inaccessible to aqueous solvent (fig. 3f) . the dip at the center of 694 -712 (residue 703) supports the prediction that this region comprises two msds. region 755-763 coincides with the predicted third msd (754 -763). the kennedy region is accessible to solvent. taken together, these data suggest that the gp41 of hiv-1 clades a, b, c, and d all have the potential to have three msds in a h-sheet conformation. msd 1 and 2 are connected by a short h-turn ( 701 gl 702 ), and msd 2 and 3 support the highly antigenic kennedy sequence on the outside of the cell or virion. further the msds all have significant parallel and anti-parallel h-strand potential (lifson and sander, 1979 ; data not shown). the 1-and 3-msd forms of gp41 are shown schematically in figs. 4a and b. the significance of the new positioning of the potential tyrosine-sorting signals is discussed below. while msds are typically a-helical and approximately 20 residues in length (sabatini et al., 1982; singer, 1990) , h-sheets as short as 7 residues form msds as part of transmembrane h-barrel proteins of bacteria, choroplasts, and mitochondria (schultz, 2003) . short h-turns connect sequential msds and do not normally occur within msds (jahnig, 1990) , but the h-turn may intrude into the membrane, as suggested for herpes simplex virus glycoprotein b (pellett et al., 1985) . an arginine residue is unlikely to occur in the middle of an msd (singer, 1990) , and r703 of the 3-msd form of gp41 is predicted to be on the membrane surface, where the polar head groups of membrane lipids can neutralize its positive charge. however, we shall suggest below that the 3-msd gp41 is a minority form that is selectively incorporated into the plasma membrane and virions, and that most intracellular gp41 exists in the 1-msd conformation. finally preliminary sequence and structural analysis suggests that other primate lentiviruses (hiv-2, siv) have the potential to form a 3-msd structure (unpublished data). gp41 molecules of hiv-1 clades a to d all have the potential to form three short msds, which most likely have a h-sheet conformation. the position of the third msd places the first potential tyrosine-dependent sorting signal outside the membrane, where it is non-functional. all viruses have a hydrophilic, unstructured region of 41 residues supported by msds 2 and 3 that probably equates to the antigenically and biologically active minor ectodomain of hiv-1 clade b, tcla viruses. the definition and values of the peaks obtained in the above analysis make the data highly significant. the position and number of residues in the msds and other regions of interest are summarized in table 1 and 1999; egan et al., 1996; ohno et al., 1997; rowell et al., 1995; west et al., 2002) and basolateral sorting (deschambeault et al., 1999; lodge et al., 1997; owens et al., 1991) . the signal sequence and upstream residues are highly to very highly conserved in clades a to d: 716 r = 100%, 717 q = 96.4%, 718 g = 99.95%, 719 y = 99.7%, 720 s = 99.5%, 721 p = 99.8%, 722 l = 96.9%. the glycine immediately before a tyrosine signal signifies that it can function in the tgn to target the protein to lysosomes. also for signal functionality there is a strict requirement that the tyrosine residue is the 7th -11th residue from the membrane (rohrer et al., 1996) . gp41 is also involved in basolateral localization of envelope protein in the plasma membrane of polarized cells (owens et al., 1991) , a property that is lost when the tyrosine of the first signal is substituted (deschambeault et al., 1999; lodge et al., 1997) . in the 1-msd model of gp41, gallaher et al. (1989) have proposed that the msd ends at residue 712 v, and thus 719 y will be the 7th residue from the membrane, and . only a monomer is represented. there may be interactions between msd 1, 2, and 3, between the minor ectodomain and the gp41 major ectodomain, between the minor ectodomain and elements of gp120, or with the other gp41 monomers that form the trimer (arrows). the nine msds of the trimer could also interact with each other. the antigenically active kennedy sequence (731 -752) containing neutralizing and non-neutralizing epitopes is shown as the outer face of the minor ectodomain. table 1 predicted location of residues in the 3-msd conformation of the gp41 of hiv-1 clades a to d in the virion or cell membrane within the required distance for optimum function. much depends on knowing precisely which residue ends the msd. often a charged residue that acts as a stopper defines the end, and it is possible that the gp41 msd could end at 714 r. if so, 719 y would be 5 residues from the membrane, and this might compromise the endocytosis, basolateral sorting, and lysosomal targeting functions of the signal. the minimum distance of the tyrosine-signal from the membrane is required for both direct lysosomal targeting and endocytosis (collawn et al., 1990; pytowski et al., 1995; trowbridge and collawn, 1992; trowbridge et al., 1993) . however, only lysosomal targeting signals have a strict maximum distance from the membrane (collawn, 1990; rohrer et al., 1996; trowbridge and collawn, 1992; trowbridge et al., 1993) . the fact that the first tyrosine signal functions in endocytosis (as referenced above) argues that it is indeed at the required distance from the membrane, but at the time of writing there are no experimental data to show if the signal is active in directing gp41 to lysosomes. the 3-msd model, as stated above, puts the first tyrosine signal outside the membrane where it is non-functional for any transport function (fig. 5) . adaptor protein (ap) complexes interact with tyrosinesorting signals. ap-1 and ap-3 complexes are mainly found in the tgn and function in lysosomal targeting, while ap-2 is predominantly localized to the plasma membrane and functions in endocytosis. they have distinct preferences for specific residues or combinations of residues of the tyrosine signals, although there is overlap, particularly with ap-1 and ap-3 complexes (table 2) (boll et al., 1996; ohno et al., 1996 ohno et al., , 1998 . table 2 shows that the consensus sequence of the first tyrosine-sorting signal of the gp41 tail of clades a to d most closely matches the preferences of ap-1 and ap-3 complexes. the arginine residue at position y à 3 of the signal is 100% conserved, and there is almost complete conservation of the glycine at y à 1 (99.95%), the tyrosine itself (99.7%), and the proline at y + 2 (99.8%). the leucine residue at y + 3 is 96.9% conserved, but if leucine, isoleucine, and valine at y + 3 (all with similar properties and tolerated at this position of the tyrosine signal) are summed, conservation reaches 98.4%. thus, the first tyrosine-sorting signal ( 719 yspl 722 ) could interact with the ap-1 and ap-3 complexes in the tgn and target gp41 to the lysosomes. however, as stated above, this signal functions at the plasma membrane (berlioz-torrent et al., 1999; boge et al., 1998; deschambeault et al., 1999; egan et al., 1996; ohno et al., 1997; rowell et al., 1995; west et al., 2002) , and is not known to be active in the tgn. it may be that the amount of gp41 synthesized saturates the lysosomal targeting system in the tgn, allowing gp41 to reach the cell surface and the first tyrosine-sorting signal to function as an endocytosis signal. the possibility that the signal targets gp41 to the lysosomes is consistent with the observation that the majority of gp160 that reaches the golgi is degraded in the lysosomes (willey et al., 1988) . ap-2 complexes have the broadest specificity range and associate with the same signals as ap-1 and ap-3 complexes (ohno et al., 1998) . the endocytosis function of the first tyrosine-sorting signal may enable plasma membrane gp41 to be re-directed to the lysosomes if it escapes that route initially (ohno et al., 1998) . the second tyrosine-dependent sorting signal ( 775 yhrl 778 ) has no preceding glycine, and is non-functional in the context of the 1-msd model of gp41 (boge et al., 1998; rowell et al., 1995) . it is situated 63 and 12 residues from the membrane in the 1-msd and 3-msd models, respectively, the latter being close to the optimal 7-11 residue distance. the residues of the second signal are variably conserved: 772 l = 94.5%, 773 f = 96.8%, 774 s = 86.0%, 775 y = 99.7%, 776 h = 89.3%, 777 r = 86.5%, and 778 l = 99.8%. the f at position y à 2, r at y + 2, and l at y + 3 suggest that the signal interacts with the ap-2 complex ( table 2 ). the lack of an r at y à 3, glycine at y à 1, and proline at y + 2, and the fact that it is not within the favored distance from the membrane (rohrer et al., 1996) , indicate that this signal is not optimal for interacting with ap-1 or ap-3 complexes. ap-3 complexes disfavor serine at position y à 1 and makes this interaction less likely (ohno et al., , 1998 . as yet there is no evidence that this signal is functional in gp41 (boge et al., 1998; rowell et al., 1995) . a peptide containing the signal interacted table 2 comparison of the preferences of ap complexes for cellular tyrosine-dependent sorting signals a with the consensus sequence for the first and second potential tyrosine-dependent sorting signals of the gp41 of hiv-1 clades a to d position (relative to y) ap-1 preferences ap-2 preferences ap-3 preferences hiv-1 tyrosine-dependent sorting signal sequences first second np, no preference known at this position; x, any amino acid residue; b, a bulky hydrophobic residue. a boll et al. (1996) , heilker et al. (1999 ), hö ning et al. (1996 , ohno et al. (1995 ohno et al. ( , 1996 ohno et al. ( , 1998 , ooi et al. (1997) , owen and evans (1998) , simpson et al. (1997) , stephens and banting (1998) , stepp et al. (1997) . strongly with the medium subunit of ap-2 (boge et al., 1998; ohno et al., 1997) , but the presence of a major upstream sequence in the 1-msd model could alter its environment and its possible interaction with the ap-2 complex. all hiv-1 gp41 tail sequences have a potential n-terminal gyxxf-sorting signal of the type that would be expected to interact strongly with ap-1 and ap-3 complexes. thus, with the critical 7-residue spacing from the membrane, the signal is likely to be functionally important in targeting tgn gp41 to lysosomes. if the signal was required only for endocytosis, it is unlikely that the g at position y à 1, r at y à 3, and the 7-residue spacing from the membrane would be so highly conserved. however, this would not preclude it from functioning as an endocytosis signal at the cell surface. the second yxxf sequence is not so well conserved, is 63 residues from the membrane in the 1-msd model, and is unlikely to interact with ap-1 and ap-3 complexes, or to be involved in lysosomal targeting from the tgn. however, the y is almost completely conserved and the bulky hydrophobic residue is highly conserved. in the 3-msd structure of gp41, only the second signal is on the cytoplasmic side of the membrane, where it could be functional. in the infected cell there may be populations of gp41 with one msd and three msds until recently all primate lentivirus envelope protein anchor components were viewed as having a single msd (691 -712 in hiv-1; fig. 4a ) (gallaher, 1987 (gallaher, , 1989 gonzalez-scarano et al., 1987; levy, 1998; white, 1990) . however, published work and the discussion above suggest that the gp41 tail crosses the membranes of hiv-1 virions and infected cells three times (cheung et al., 2005; cleveland et al., 2003; mclain et al., 2001) . to resolve this apparent conflict, we shall argue here that the 1-and 3-msd forms of gp41 coexist in the infected cell, with the 1-msd version being the major form. however, we shall propose that only the minor 3-msd form is incorporated into virions. these proposals are consistent with, and rationalize the observed degradation of the majority (85 -95%) of the1-msd form of cellular gp160 (bultmann et al., 2000; courageot et al., 1999; jabbar and nayak, 1990; pfeiffer et al., 1997; willey et al., 1988) , the apparently contradictory evidence of a functioning first tyrosine-sorting signal (berlioz-torrent et al., 1999; boge et al., 1998; deschambeault et al., 1999; egan et al., 1996; ohno et al., 1997; rowell et al., 1995; west et al., 2002) and basolateral-sorting signal (deschambeault et al., 1999; lodge et al., 1997; owens et al., 1991) , with the immunogenic and antigenic properties of the kennedy region (chanh et al., 1986; dalgleish et al., 1988; evans et al., 1989; ho et al., 1987; kennedy et al., 1986; niedrig et al., 1992) . the c-terminal tail of the 1-msd form of hiv-1 gp41 starts at residue 713 (fig. 4a) . as already stated, the majority of this type of gp41 (85 -95%) is degraded intracellularly. the y residue of the first tyrosine-dependent sorting signal ( 718 gyspl 722 ) is situated precisely 7 residues from the membrane and conforms closely to the requirements for cellular gyxxf lysosomal targeting signals (see above). in the 1-msd form of gp41, the second tyrosinedependent sorting signal is apparently not needed, and we suggest may not be functional. the 3-msd form of hiv-1 gp41 has msds at 691-700, 703-712, and 754 -763, and its 718 gyspl 722 sequence is outside the virion. this model is supported by antigenic and other data showing that residues of the c-terminal tail are exposed on the surface of the virion (buratti et al., 1998; chanh et al., 1986; cleveland et al., 2000a cleveland et al., ,b,2003 dalgleish et al., 1988; durrani et al., 1998; ho et al., 1987; mcinerney et al., 1999; mclain et al., 1995 mclain et al., , 1996 mclain et al., , 2001 newton et al., 1995; reading et al., 2003) and the infected cell (cheung et al., 2005; heap et al., 2005) . furthermore, we propose that this form of gp41 represents the 5 -15% of the tgn gp160 that is directed to the cell surface. its gyxxf signal is outside the cell membrane and cannot function in lysosomal targeting or endocytosis, but the second potential tyrosine-dependent sorting signal ( 775 yhrl 778 ), which has none of the requirements for lysosomal targeting (see above), is 12 residues from the membrane, and well situated to function in endocytosis and recycling of cell membrane-inserted gp41. because of its location this gp41, in association with gp120, is the major gp41 form in the cell membrane, and the major gp41form incorporated into virions. co-existence of two forms of gp41 raises questions, which will require further work to answer. the mechanism by which two forms of gp41 arise is not known, although translocational pausing may be involved in formation of the multiple msds (dettenhofer and yu, 2001) . the 1-and 3-msd forms are proposed to be a-helix and h-sheet, respectively, and it is noted above that structure predications allow for either conformation. conformation may be determined by the length of unbroken msd sequence, as a long sequence of lower value a-helix, can take precedence over a higher value, shorter h-sheet region (chou and fasman, 1978) . the conventional 1-msd model still has the problem of 703 r being centrally located in the membrane with no counter charge. however, a recent suggestion that the 703 r equivalent in siv is situated in a position where it can react with the polar lipid head groups may provide a solution (west et al., 2001) . degradation of the 1-msd gp41 and recycling of the 3-msd form would both act to reduce the surface expression of gp120-gp41. this may be important, as high intracellular concentrations of gp41 can be cytotoxic (arroyo et al., 1995; chernomordik et al., 1994; comardelle et al., 1997; gawrisch et al., 1993; miller et al., 1993; zhang et al., 1996) , and provoke immune responses against the infected cell. it may be that such post-translational control measures, utilizing tyrosine-dependent sorting signals and the host cell's degradation pathways, have evolved as envelope expression is not easily controlled at a genetic level due to the overlap of the env, tat, and rev orfs. the suggestion above that a membrane-inserted viral protein can have different numbers of msds is not unique. for example, the gl envelope protein of equine arteritis virus is proposed to have 1 or 3 msds (snijder and meulenberg, 1998) , the m protein of transmissible gastroenteritis coronavirus and equine arteritis virus, and the s antigen of hepatitis b virus are proposed to have three or four msds (prange and streeck, 1995; risco et al., 1995; snijder and meulenberg, 1998) , and the herpes simplex virus glycoprotein b (pellett et al., 1985) , and the epstein -barr virus 58 kda latent protein hennessy et al., 1984) both have multiple msds. based on an analysis of their sequence and structure, we propose that the gp41 transmembrane region and c-terminal tail of all hiv-1 clades a to d can exist in two conformations, with either 1 msd (the conventional structure) or with 3 msds. we suggest that these are, respectively, the majority and minority forms of intracellular env. in the 3-msd form, msd 1 and msd 2 are separated by a highly conserved beta turn, while the msd 2 and msd 3 support an unstructured hydrophilic loop/minor ectodomain of 41 residues that in clade b strains is highly antibody-reactive and involved in fusion. all viruses have two potential tyrosine-dependent sorting signals within the region analyzed. in the 1-msd model it is likely that only the n-terminal signal is functional, and that this interacts with ap-1 and ap-3 to direct env from the tgn to degradation in the lysosomes. in the 3-msd version, the nterminal signal is situated outside the membrane and nonfunctional, thus allowing this form of gp41 to reach the cell membrane. thus, it seems that the 3-msd form is the majority species on the cell surface and hence in virions. we propose that the second signal is functional in the 3-msd gp41, and controls recycling of cell surface env. the 1-and 3-msd strategy can be seen as an evolutionary adaptation that allows hiv-1-infected cells to evade the immune system or to avoid gp41-induced cytotoxicity. hiv-1 gp41 sequences from infectious viruses, molecular clones, and pcr products from blood samples from infected individuals were obtained from http://hiv-web.lanl.gov/. we analyzed residues 690-793 of hiv-1 (numbering according to ratner et al., 1985) of the following number of sequences: clade a, n = 25; clade b, n = 245; clade c, n = 61; clade d, n = 26. the sequence analyzed comprises the msd and approximately two thirds of the c-terminal tail. conserved regions of sequence were aligned, with spaces as necessary to maintain alignment. the residue occupying each position was recorded as a percentage, and consensus sequences constructed for each clade. sequence conservation is defined here as poor (<80%), moderate (80 -89.9%), high (90 -96.4%), and very high (96.5 -100%). for structure predications we used the consensus sequences derived in this report for the gp41 msd and cterminal region of hiv-1 clades a to d. hydropathy values assigned to the amino acids are based on water vapor transfer free energies and the interior -exterior distribution of amino acid side chains (kyte and doolittle, 1982) . this system predicts that a region with a value >1.6 is likely to be an msd, and that a region with a value >1.09 is likely to be sequestered inside the protein. a-helices and h-sheets were predicted according to chou and fasman (1978) , where p a is the helix conformational parameter and p h is the h-sheet conformational parameter. any segment of !6 residues with ( p a ) !1.03 and ( p a ) > ( p h ) is predicted to be aàhelical, and any segment of three residues or longer in a native protein with ( p h ) !1.05 and ( p h ) > ( p a ) is predicted to be h-sheet. a segment containing overlapping aand hresidues is resolved through conformational boundary analysis so that ( p a ) > ( p h ) is a-helical, and ( p h ) > ( p a ) is h-sheet. a h-turn occurs where a polypeptide folds back on itself by nearly 180-and typically requires four consecutive residues (chou and fasman, 1978) . however, a h-turn of two residues occurs between two msds of herpes simplex virus glycoprotein b (pellett et al., 1985) . the lower cut-off value for h-turns of 0.75 was used here (chou and fasman, 1978) . polarity was determined according to zimmerman et al. (1968) . accessibility of a region of a protein to solvent was predicated using the percentage buried residues index (janin, 1979) . low values indicate a region that is likely to be accessible to solvent and hence surface exposed, and high values indicate regions not accessible to the solvent. predications used a moving window of 7 residues. membrane permeabilization by different regions of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41 interactions of the cytoplasmic domains of human and simian retroviral transmembrane proteins with components of the clathrin adapter complexes modulate intracellular and cell surface expression of envelope glycoprotein expression of human immunodeficiency virus type 1 (hiv-1) envelope gene products transcribed from a heterologous promoter a membraneproximal signal mediates internalization of the hiv-1 envelope glycoprotein via interaction with the ap-2 clathrin adaptor sequence requirements for the recognition of tyrosine-based endocytic signals by clathrin ap-2 complexes enhancement of immunogenicity of an hiv env dna vaccine by mutation of the tyr-based endocytosis motif in the cytoplasmic tail ubiquitination of the human immunodeficiency virus type 1 env protein the neutralizing antibody response against a conserved region of hiv-1 gp41 (amino acid residues 731 -752) is uniquely directed against a conformational epitope induction of anti-hiv neutralizing antibodies by synthetic peptides an amphipathic peptide from the c-terminal region of the human immunodeficiency virus envelope glycoprotein causes pore formation in membranes part of the c-terminal tail of the envelope gp41 transmembrane protein of human immunodeficiency virus type 1 (hiv-1) is exposed on the cell surface and is involved in virus-mediated cell-cell fusion prediction of the secondary structure of proteins from their amino acid sequence immunogenic and antigenic dominance of a non-neutralizing epitope over a highly conserved neutralizing epitope in the gp41 transmembrane envelope glycoprotein of hiv-1: its deletion leads to a strong neutralizing antibody response properties of a neutralizing antibody that recognises a conformational form of epitope erdrd in the c-terminal tail of human immunodeficiency virus type 1 a region of the c-terminal tail of the gp41 envelope glycoprotein of human immunodeficiency virus type 1 contains a neutralizing epitope: evidence for its exposure on the surface of the virion transferrin receptor internalization sequence yxrf implicates a tight turn as the structural recognition motif for endocytosis a synthetic peptide corresponding to the carboxyterminus of human immunodeficiency virus type 1 transmembrane protein induces alterations in the ionic permeability of xenopus laevis oocytes intracellular degradation of the hiv-1 envelope glycoprotein. evidence for, and some characteristics of, an endoplasmic reticulum pathway neutralization of diverse strains of hiv-1 by monoclonal antibodies raised against a gp41 synthetic peptide deletion of a single n-linked site from the transmembrane envelope protein of human immunodeficiency virus type 1 stops cleavage and transport of gp160 preventing env-mediated fusion polarized human immunodeficiency virus budded in lymphocytes involves a tyrosine-based signal and favors cellto-cell viral transmission characterization of the biosynthesis of human immunodeficiency virus type 1 env from infected t-cells and the effects of glucose trimming of env on virion infectivity biosynthesis and processing of human immunodeficiency virus type 1 envelope glycoproteins: effects of monensin on glycosylation and transport processing of the envelope protein gp160 in immunotoxin-resistant cell lines chronically infected with human immunodeficiency virus type 1 intranasal immunization with a plant virus expressing a peptide from hiv-1 gp41 stimulates better mucosal and systemic hiv-1-specific iga and igg than oral immunization folding, interaction with grp78-bip, assembly, and transport of the human immunodeficiency virus type 1 envelope protein human immunodeficiency virus type 1 envelope protein endocytosis mediated by a highly conserved intrinsic internalization signal in the cytoplasmic domain of gp41 is suppressed in the presence of the pr55gag precursor analysis of membrane and surface protein sequences with the hydrophobic moment plot an engineered poliovirus chimaera elicits broadly reactive hiv-1 neutralizing antibodies nucleotide sequence of an mrna transcribed in latent growth-transforming virus infection indicates that it may encode a membrane protein the glycosylation of human immunodeficiency virus type 1 transmembrane protein (gp41) is important for the efficient transport of the envelope precursor gp160 detection of fusion peptide sequence in the transmembrane peptide of human immunodeficiency virus a general model for the transmembrane proteins of hiv and other retroviruses interaction of peptide fragment 828 -848 of the envelope glycoprotein of human immunodeficiency virus type 1 with lipid bilayers sequence similarities between human immunodeficiency virus gp41 and paramyxovirus fusion proteins secretion of a truncated form of the human immunodeficiency virus type 1 envelope glycoprotein an antibody specific for the c-terminal tail of gp41 of hiv-1 mediates post-attachment neutralization probably through inhibiting virus-cell fusion recognition of sorting signals by clathrin adapters a membrane protein encoded by epstein -barr virus in latent growthtransforming infection human immunodeficiency virus virus-neutralizing antibodies recognise several conserved domains on the envelope glycoprotein the tyrosinebased lysosomal targeting signal in lamp-1 mediates sorting into golgiderived clathrin-coated vesicles prediction of protein antigenic determinants from amino acid sequences intracellular interaction of human immunodeficiency virus type (arv-2) envelope glycoprotein gp160 with cd4 blocks the movement and maturation of cd4 to the plasma membrane structure predictions of membrane proteins are not that bad surface and inside volumes in globular proteins megalomycin inhibits hiv-1 replication and interferes with gp160 processing endoproteolytic cleavage of hiv-1 gp160 envelope precursor occurs after exit from the trans-golgi network (tgn) antiserum to a synthetic peptide recognizes the htlv-iii envelope glycoprotein uncleaved env gp160 of human immunodeficiency virus type 1 is degraded within the golgi apparatus but not lysosomes in cos-1 cells glycosylation and processing of the human immunodeficiency virus type 1 envelope glycoprotein a simple method for displaying the hydropathic character of a protein hiv and the pathogenesis of aids antiparallel and parallel beta-strands differ in amino acid residue preferences the membraneproximal intracytoplasmic tyrosine residue of hiv-1 envelope glycoprotein is critical for basolateral targeting of viral budding in mdck cells analysis of the ability of five adjuvants to enhance immune responses to a chimeric plant virus displaying a hiv-1 peptide human immunodeficiency virus type 1 neutralizing antibodies raised to a gp41 peptide expressed on the surface of a plant virus stimulation of neutralizing antibodies to human immunodeficiency virus type 1 in three strains of mice immunized with a 22-mer amino acid peptide expressed on the surface of a plant virus different effects of a single amino acid substitution on three epitopes in the gp41 c-terminal loop of a neutralizing antibody escape mutant of human immunodeficiency virus type 1 alterations in cell membrane permeability by the lentiviral peptide (llp-1) of hiv-1 transmembrane protein legitimate and illegitimate cleavage of human immunodeficiency virus glycoproteins by furin kex2p: a model for cellular endopeptidase processing human immunodeficiency virus type 1 envelope glycoprotein precursor processing and routage of hiv glycoproteins by furin to the cell surface expression and immunogenicity of an 18-residue epitope of hiv-1 gp41 inserted in the flagellar protein of a salmonella live vaccine murine monoclonal antibodies directed against the transmembrane protein gp41 of human immunodeficiency virus type 1 enhance its infectivity interaction of tyrosine-based sorting signals with clathrin-associated proteins structural determinants of interaction of tyrosine-based sorting signals with the adaptor medium chains interaction of endocytic signals from the hiv-1 envelope glycoprotein complex with members of the adaptor medium chain family the medium subunits of adaptor complexes recognise distinct but overlapping sets of tyrosine-based sorting signals altered expression of a novel adaptin leads to a defective pigment granule biogenesis in the drosophila eye color mutant garnet folding, assembly, and intracellular trafficking of the human immunodeficiency virus type 1 envelope glycoprotein analyzed with monoclonal antibodies recognizing maturational intermediates a structural explanation for the recognition of tyrosine-based endocytotic signals human immunodeficiency virus envelope protein determines the site of virus release in polarized epithelial cells processing and secretion of envelope glycoprotein of human immunodeficiency virus type 1 in the presence of trimming glucosidase inhibitor deoxynojirimycin brefeldin a inhibits the processing and secretion of envelope glycoprotein of human immunodeficiency virus type 1 anatomy of the herpes simplex virus 1 strain f glycoprotein b gene: primary sequence and predicted protein structure of the wild type and of monoclonal antibody-resistant mutants transfer of endoplasmic reticulum and golgi retention signals to human immunodeficiency virus type 1 gp160 inhibits intracellular transport and proteolytic processing of viral glycoprotein but does not influence the cellular site of virus particle budding novel transmembrane topology of the hepatitis b envelope proteins an internalization motif is created in the first cytoplasmic domain of the transferrin receptor by substitution of a tyrosine at the first position of a predicted turn complete nucleotide sequence of the aids virus, htlv-iii a novel monoclonal antibody specific for the c-terminal tail of the gp41 envelope transmembrane protein of human immunodeficiency virus type 1 that preferentially neutralizes virus after it has attached to the target cell and inhibits the production of infectious progeny membrane protein molecules of transmissible gastroenteritis coronavirus also expose the carboxy-terminal region on the external surface of the virion the targeting signal of lamp1 to lysosomes is dependent on the spacing of its cytoplasmic tail tyrosine sorting motif relative to the membrane endocytosis of endogenously synthesized hiv-1 envelope protein: mechanism and role in processing for association with class ii mhc mechanisms for the incorporation of proteins in membranes and organelles transmembrane h-barrel proteins characterization of the adaptor-related protein complex, ap-3 the structure and insertion of integral proteins in membrane the molecular biology of arteriviruses intracellular processing of the gp160 hiv-1 envelope precursor. endoproteolytic cleavage occurs in a cis or medial compartment of the golgi complex specificity of interaction between adaptor-complex medium chains and the tyrosine-based sorting signals of tgn38 and igp120 the yeast adaptor protein complex, ap-3, is essential for the efficient delivery of alkaline phosphatase by the alternate pathway to the vacuole correlation of sequence hydrophobicities measures similarity in three dimensional protein structure structural requirements for high efficiency endocytosis of the human transferrin receptor signal-dependent membrane protein trafficking in the endocytic pathway characterization and primary structure of a human immunodeficiency virus type 1 (hiv-1) neutralization domain as presented by a poliovirus type 1/hiv-1 chimera comparative cellular processing of the human immunodeficiency virus (hiv-1) envelope glycoprotein gp160 by the mammalian subtilisin/kexin-like convertases mutations within the putative membrane-spanning domain of the simian immunodeficiency virus transmembrane glycoprotein define the minimal requirements for fusion, incorporation and infectivity mutation of the dominant endocytosis motif in human immunodeficiency virus type 1 gp41 can complement matrix mutations without increasing env incorporation viral and cellular membrane fusion reactions biosynthesis, cleavage, and degradation of the human immunodeficiency virus type 1 envelope glycoprotein gp160 mutations with the human immunodeficiency virus type 1 gp160 glycoprotein alter its intracellular transport and processing the highly conserved c-terminal dileucine motif in the cytosolic domain of the human immunodeficiency virus type 1 envelope glycoprotein is critical for its association with the ap-1 clathrin adaptor amphipathic domains in the c terminus of the transmembrane protein (gp41) permeabilize hiv-1 virions: a molecular mechanism underlying natural endogenous reverse transcription the characterization of amino acids in proteins by statistical methods we thank richard compans for reading the manuscript and his advice and are pleased to acknowledge the financial support of avert, uk. key: cord-353467-wbtzvm4i authors: lambert, carsten; thomé, nicole; kluck, christoph j.; prange, reinhild title: functional incorporation of green fluorescent protein into hepatitis b virus envelope particles date: 2004-12-05 journal: virology doi: 10.1016/j.virol.2004.09.031 sha: doc_id: 353467 cord_uid: wbtzvm4i the envelope of hepatitis b virus (hbv), containing the l, m, and s proteins, is essential for virus entry and maturation. for direct visualization of hbv, we determined whether envelope assembly could accommodate the green fluorescent protein (gfp). while the c-terminal addition of gfp to s trans-dominant negatively inhibited empty envelope particle secretion, the n-terminal gfp fusion to s (gfp.s) was co-integrated into the envelope, giving rise to fluorescent particles. microscopy and topogenesis analyses demonstrated that the proper intracellular distribution and folding of gfp.s, required for particle export were rescued by interprotein interactions with wild-type s. thereby, a dual location of gfp, inside and outside the envelope, was observed. gfp.s was also efficiently packaged into the viral envelope, and these gfp-tagged virions retained the capacity for attachment to hbv receptor-positive cells in vitro. together, gfp-tagged virions should be suitable to monitor hbv uptake and egress in live hepatocytes. hepatitis b virus (hbv), a human hepadnavirus, is an enveloped dna virus that infects hepatocytes and causes acute and chronic liver disease. despite considerable understanding of the details of hepadnaviral genome replication, fundamental insights into the early and late steps of an hbv infection are still lacking. infection initiates by virus attachment to the hepatocyte and is determined by the hbv envelope. among the three related large l, middle m, and small s envelope proteins, l has been shown to play the key role in receptor recognition and cell attachment (le seyec et al., 1999; neurath et al., 1986) . the route of subsequent hbv uptake is a yet poorly identified process and may proceed by direct fusion of the viral envelope with the plasma membrane, receptor-mediated endocytosis, or alternate mechanisms (for review, see cooper et al., 2003) . in favor of a fusion mechanism at the cell surface is the previous demonstration of a ph-independent internalization of hbv in primary human hepatocytes (hagelstein et al., 1997) . by contrast, infectivity studies performed with the related duck hbv in duck liver cells have demonstrated that viral entry depends on endocytosis (breiner et al., 1998) . in the late stages of an hbv infection, progeny virions are formed by budding of the pre-assembled cytosolic nucleocapsids, enclosing the partially double-stranded dna genome 3.2 kb in length and the viral polymerase through intracellular membranes accommodating the viral envelope proteins (for review, see nassal, 1996) . during this process, an excess of envelope proteins is not incorporated into virion envelopes but self-assembles into secreted subviral lipoprotein particles referred to as hepatitis b surface antigen (hbsag) particles or empty envelopes. these particles have been shown to mature by budding into intralumenal cisternae of post-endoplasmic reticulum (er)-pre-medial-golgi compartments and to exit the cell by the constitutive secretory pathway (huovila et al., 1992) . in contrast, the intracellular budding site of viral particles has not been defined to date. the three l, m, and s envelope proteins contribute differently to subviral and viral particle formation. assembly and secretion of subviral particles are solely driven by the s protein and are initiated by co-translational integration of s molecules into the er membrane (simon et al., 1988) . the current models for the transmembrane structure of s assume a lumenal disposition (i.e., external in the mature particles) of both the n-and c-termini and four membranespanning segments (berting et al., 1995; stirk et al., 1992) . following dimerization, about 100 transmembrane s monomers then self-assemble with host-derived lipids into spherical empty envelopes, 20 nm in diameter, which are secreted from the infected liver and transfected cell lines with high efficiency. this process is also sustained by the m protein that shares the sequence of s but differs in its nterminal pres2 extension. by contrast, the l protein along with its n-terminal pres1 plus pres2 domains blocks subviral particle production but is the key player in virion formation (bruss and ganem, 1991; chisari et al., 1986) . the pivotal role of the l protein in the viral life cycle is related to its dual transmembrane topology (bruss et al., 1994; ostapchuk et al., 1994; , as its internal (cytosolic) pres domain is needed for envelopment of cytosolic nucleocapsids , while the same external (lumenal) pres domain mediates receptor binding during host cell attachment (le seyec et al., 1999) . in the present study, we addressed whether the hbv envelope could be tagged with the green fluorescent protein (gfp) without impairing its functionality. we reasoned that such gfp-tagged particles should be a promising tool to study the early and late stages of the hbv life cycle in live cells. for tagging of the hbv envelope with gfp, the s protein was chosen as the target because this protein is an essential and abundant constituent of the virion envelope. two chimeras were constructed by fusing either a fluorescenceenhanced gfp in frame to the n-terminus of s (gfp.s) or the yellow-shifted yfp variant to the c-terminus of s (s.yfp) (fig. 1a) . the fusion constructs were transiently expressed in cos-7 cells and analyzed by gfp-specific western blotting. the unfused s protein with a c-terminal ha-tag (sha) was included as reference. as shown in fig. 1b , lysates of cells contained sha in its characteristic doublet of a non-glycosylated (p24) and single-glycosylated (gp27) form as a consequence of partial modification at asn-146. n-linked glycosylation was confirmed by treatment with peptide/n-glycosidase f (pngase f), which converted the gp27 form of s to its faster migrating p24 form. the gfp.s and s.yfp fusion proteins were stably expressed as 51-kda polypeptides in accordance with the molecular masses calculated for their non-glycosylated forms (p51) (fig. 1b) . as evidenced by enzymatic deglycosylation, both fusion proteins appeared in glycosylated forms (gp54) in addition, indicating that they were fig. 1 . the n-terminal gfp.s fusion is co-secreted with wild-type s. (a) schematic representation of the hbv s protein and the gfp.s and s.yfp fusion constructs. numbers below the domains refer to the corresponding amino acid positions of s (hatched bar) and gfp/yfp (white bars). for gfp.s, the two domains are linked by the amino acid sequence sglrsraqasn and terminated by the extra sequence wdppdldn, while s and yfp are interspersed by the sequence dppvat. (b) synthesis, glycosylation, and secretion of sha and the fusion constructs. lysates (c) from transfected cos-7 cells were divided into two portions and either left mock treated or digested with pngase f, as indicated. cellular supernatants (m) were analyzed without enzymatic de-glycosylation. proteins were processed by sds-page and ha-specific (lanes 1-3) or gfp-specific (lanes 4-9) immunoblotting. nonglycosylated (p) and glycosylated (gp) forms are indicated on the left with the numbers referring to the molecular masses of the polypeptides. (c) secretion of gfp.s is rescued by coexpressed wild-type s molecules. supernatants of cells synthesizing gfp.s in the absence (à) or presence (+) of s were assayed by gfp-specific western blotting. competent for co-translational integration of the s region into the er lumen. however, unlike wild-type sha, each chimera failed to be secreted from transfected cells into the supernatants (fig. 1b) . although these results implicated some misfolding of gfp.s and s.yfp, we nonetheless assessed whether their block to secretion could be abrogated by co-expression of wild-type s. cos-7 cells were co-transfected with each fusion construct, and the wild-type vector at a ratio of 1:1 and cellular supernatants were assayed by gfp-specific immunoblotting. the s.yfp fusion turned out to act in a trans-dominant-negative manner and even inhibited export of co-expressed wt s chains (data not shown). by contrast, gfp.s was secreted in the presence of s (fig. 1c) , and thus was chosen for further analysis. for quantitative evaluation of the secretion efficacy of gfp.s, lysates and supernatants of gpf.s + s co-transfected cells were analyzed with a gfp-specific elisa. this determination revealed that 68 f 5.2% of the intracellular gfp.s was released into the supernatant when co-expressed with wt s. in order to get insights how s rescued the secretion of gfp.s, we comparatively examined the intracellular distribution of gfp.s in the presence and absence of sha. when expressed alone, gfp.s surprisingly yielded an intense micropunctate fluorescence (fig. 2a) . these bdotsq partially overlapped with er structures, as shown by co-staining of the cells with antibodies to the er-resident protein disulfide isomerase (pdi) (figs. 2b and c) . although the nature of these structures remained to be determined, they might present aggregates of malfolded gfp.s destined for degradation. importantly, however, on co-expression of gfp.s with sha, the chimera now exhibited a widely dispersed membranous staining pattern together with a significant colocalization with the er marker pdi (figs. 2d-f ). under the same conditions, a nearly 100% co-localization of gfp.s with sha was observed when cells were stained with haspecific antibodies . from these data we concluded that the correct distribution of gfp.s, required for export, was warranted by interaction with co-expressed s chains. to determine whether the co-secreted gfp.s and sha proteins resembled authentic subviral hbv envelope particles, the culture supernatant of transfected cells was fractionated by isopycnic cscl gradient centrifugation and fractions were analyzed by an s-specific elisa. as shown in fig. 3a , the co-secreted proteins banded at a buoyant density of about 1.22 g/ml, typical for wild-type s lipoprotein particles (heermann and gerlich, 1991) . peak fractions were then subjected to specific western blot analysis that confirmed the presence of both the gfp.s fusion and the sha protein (fig. 3a ). in addition, when fig. 2 . gfp.s co-localizes with wild-type s at the er. shown is the intracellular distribution of gfp.s expressed either alone (squares a-c) or together with sha (squares d-i) in cos-7 cells. cells were fixed, permeabilized, and examined by fluorescence microscopy. (a, d, and g) gfp fluorescence (green); (b and e) immunostaining with a mouse antibody to pdi followed by alexafluor 494-conjugated goat anti-mouse igg (red); (h) immunostaining with a mouse anti-ha antibody followed by alexafluor 494-conjugated goat anti-mouse igg (red) to visualize sha. squares c, f, and i are the corresponding merged images so that overlapping red and green signals appear yellow. gfp.s + sha and wild-type sha particles were analyzed by sucrose gradient velocity centrifugation, their sedimentation profiles were nearly coincident with a buoyant density of about 1.130 g/ml in sucrose (fig. 3b) . together, these data demonstrated that gfp.s was successfully incorporated into subviral particles that closely resembled s spheres with respect to density and size. because the fluorescence of gfp depends on its native three-dimensional structure that might be altered within the chimeric context, we next examined whether purified gfp.s + sha particles were fluorescent. as imaged by fluorescence microscopy, a strong vesicular gfp staining was observed, indicating a native display of gfp within the particle (fig. 3c ). the intensity of fluorescence and hence the apparent size of the particles appeared slightly variable and brighter than expected, perhaps because of some particle aggregation or variation in the amount of gfp.s incorporation. the production of fluorescent subviral particles indicated that the individual constituents of the gfp.s chimeric protein were properly folded, at least when co-expressed with wild-type s chains. nonetheless, for further studies outlined below, it was important to know its precise topology, that is, the orientation of the gfp domain relative to the membrane. according to current models for the transmembrane structure of s, its n-terminus and hence the gfp fusion site are located to the lumenal side of intracellular membranes that is topologically equivalent to the virion surface ( fig. 4a ) (berting et al., 1995; stirk et al., 1992) . to analyze the topological features of gfp.s, expressed in the absence or presence of s molecules, we used proteinase k protection experiments of microsomes prepared from transfected cos-7 cells. both gfp.s and s proteins were synthesized in ha-tagged form to enable their simultaneous detection by immunoblotting. consistent with previous works , proteinase k did not cleave the p24/gp27 forms of sha unless the protecting microsomal membrane was disrupted by detergent ( fig. 4b , lanes 4-6). by contrast, gfp.sha synthesized in the absence of sha chains was accessible to cleavage with the protease in intact microsomes (fig. 4b , lanes 1-3). thereby, two ha-reactive fragments were generated that closely resembled the full-length p24 and gp27 forms of sha. therefore, cleavage of gfp.sha must have occurred at least at a very distal site within the nterminal fused gfp domain. in support of a fully cytoplasmic location of the gfp domain, we were unable to detect gfp-reactive proteolysis products by western blotting (data not shown). when expressed together with sha, the topology of gfp.sha surprisingly changed in such that a fraction of polypeptides was now protected from cleavage with proteinase k by the microsomal membrane (fig. 4b, lanes 4-6) . these results indicated that the presence of extra sha chains caused a partial topological change of the n-terminal gfp domain from cytosolic to lumenal, likely as a consequence of gfp.sha/sha interprotein subunit interaction. given the strict correlation between partial n-tail translocation and secretion competence and vice versa, we finally investigated the topological properties of gfp.sha complemented in trans with a secretion-defective s mutant protein. to this aim, we took advantage of sdtm1ha that lacks the first transmembrane domain and is blocked in the assembly and secretion of subviral particles (prange et al., 1992) . consistent with our previous results (prange et al., 1992) , the deletion mutant predominantly appeared in a glycosylated 24-kda form (gp24; fig. 4b, lane 7) . importantly, sdtm1ha failed to impart a partial lumenal orientation of gfp.sha (fig. 4b , lanes 7-9). together, these data indicated that the topological change of gfp.sha in the presence of extra s molecules was likely established during the formation and extracellular release of the gfp.sha/sha mosaic particles. if this notion was correct, we would expect the gfp tag located outside and inside of the secreted particle. for a formal proof of a surface display of the gfp domain, we tried to immunoprecipitate the particles under non-denaturing conditions using two monoclonal a-gfp antibodies directed against different epitopes. however, neither antibody brought down the particles in the absence of detergent (data not shown; and see below). although these results might argue against an exterior location of gfp, it seemed equally possible that the epitopes were not accessible in the bulky beta barrel of gfp on the intact particle. incorporation of gfp.s into envelopes allows viral particle formation next we examined whether gfp.s would also be incorporated into the viral envelope. for virus production, human hepatoma huh-7 cells were transiently transfected with a cloned over-length copy of the hbv genome. on cotransfection with the gfp.s expression plasmid at a 1:1 ratio, virus secretion into the cellular supernatant was reduced about 3-fold (data not shown), whereas no inhibitory effect of gfp.s on the virus secretion profile was observed if a 4-fold excess of the hbv encoding vector was used for co-transfection. because transfected huh-7 cells released viral and subviral particles into the culture medium, virions were separated from empty envelope particles by isopycnic cscl gradient centrifugation. a resolution of these particle types was verified by assaying of fractions for the presence of hbv dna by the endogenous polymerase reaction. as shown in fig. 5 (a and b) , dna-containing virions banded at a density near 1.24 g/ml around fractions 18-22, with the strongest signal in fraction 20, while empty envelopes were found at their typical density of 1.22 g/ml. importantly, when probed with a gfp-specific elisa, the virus-containing fractions were clearly reactive, thus giving first hints to an incorporation of the gfp-tagged s protein during virus assembly. as a final proof, supernatants of hbv + gfp.s co-transfected huh-7 cells were immunoprecipitated with a gfp-specific antibody prior to detection of the viral genome by dot-blot analysis. for reasons mentioned above, such a precipitation, however, required the presence of detergent (0.5% np-40) that decreased the limits of detection, likely because some nucleocapsids might leak from the disordered envelope network. nonetheless, despite these restraints, we could specifically detect the viral dna in a-gfp-precipitated supernatants (fig. 5c) . to investigate whether these fluorescent hbv virions supported attachment to hepatocytes, cell binding assays were performed. although most permanent cell lines are not permissive to hbv infection, the human hepatoblastoma hepg2 cell line is able to bind the virion (lu et al., 1996) . for binding, we applied the same assay conditions as validated previously for in vitro infection of primary human hepatocytes (gripon et al., 1993) . accordingly, recombinant hbv + s.gfp particles secreted from co-transfected huh-7 cells (see above) were precipitated with polyethylene glycol (peg) and reacted with hepg2 cells grown on glass bottom fig. 4 . gfp.s forms a mixed topology when co-expressed with wild-type s. (a) model of the transmembrane topology of s at the er membrane and the virion envelope. the predicted four membrane spanning segments of s project its n-and c-terminus into the er lumen that is topologically equivalent to the virus outside. the n-glycosylation site at asn-146 is indicated by w. (b.) proteinase k protection assay of gfp.s. cos-7 cells were transfected with ha-tagged gfp.s either alone (lanes 1-3) or together with ha-tagged s (lanes 4-6) or the secretion-defective sdtm1 mutant (lanes 7-9). two days after transfection, microsomes were prepared and either left untreated or digested with proteinase k (prot. k) in the absence (à) or presence (+) of np-40, as denoted above each lane. samples were analyzed by ha-specific immunoblotting. the gfp.s-and s-specific p and gp forms are indicated on the left; the corresponding forms of sdtm1ha are denoted on the right. dishes. after incubating cell cultures overnight, followed by several washes, a strong gfp fluorescence almost evenly distributed on the plasma membrane of the hepatocytes was observed (figs. 6a and b) . to test the specificity of adsorption, we analyzed the binding properties of gfp.s + s subviral particles that lacked the l envelope protein and thus the receptor binding site. as expected, these particles failed to attach to hepg2 cells thereby yielding only some diffuse background staining (figs. 6c and d) . however, because infected liver cells and hbv-producing cell lines are known to also produce subviral particles containing the l protein (nassal, 1996) , we could not totally exclude the possibility that the gfp fluorescence, shown in figs. 6a and b, might be also generated by binding of this particle type. in recent years, the adaptation of the aequorea victoria gfp for visualization of protein expression and protein localization in living organisms has provided a powerful new tool. here we applied this approach to hepatitis b virus and obtained fluorescent subviral and viral particles by incorporation of the viral s envelope protein, tagged with gfp, in trans, thereby preserving all the functions necessary for the viral life cycle. because hbv empty envelope particles, built from the s protein, provide a safe antigen delivery system, their use as a carrier for the presentation of various antigens is a long established practice. to date, chimeric particles have been produced in mammalian cells with inserts in the range of 10-80 amino acids (delpeyroux et al., 1986; michel et al., 1988; . for example, the n-terminal ectodomain of the s protein has been shown to tolerate 84 foreign residues while its c-terminus accommodated 42 residues without effects on particle assembly and secretion (michel et al., 1988; . in agreement and extension, the current study demonstrated that the nterminus of s is more permissive for insertions than the c-terminus and is even amenable for addition of the complete gfp protein. hence, proteins of at least up to 238 amino acids can be displayed on hbv envelope containing virion particles were further subjected to an envelope-specific immunoprecipitation and radioactive labeling of the viral genome by the endogenous viral polymerase. the migration of the hbv dna genome as visualized by agarose electrophoresis and phosphorimaging is indicated. (c) pooled extracellular viral particle fractions from hbv + gfp.s cotransfected cells were immunoprecipitated with either an envelope-specific (a-hbsag) or a gfp-specific (a-gfp) antibody prior to extraction of the viral genome and dna dot blot analysis. for control of nonspecific immunoprecipitation, native hbv particles (control) were reacted with the a-gfp mab (third panel from the left). in addition, the control material was probed with a-hbsag, as shown in the right panel. fig. 6 . gfp-tagged hbv binds to hepg2 cells. in squares a and b are shown the adsorption of gfp-tagged hbv particles (hbv + gfp.s) to live hepg2 cells, grown on glass bottom dishes, as detected by gfp autofluorescence (magnification, â1600). under the same assay conditions, gfp-tagged subviral particles (s + gfp.s) did not bind to cells (squares c and d). particles, raising intriguing perspectives for their future use as a carrier system. particle formation of the gfp.s chimeric protein, however, depended on the presence of the wild-type s protein, indicating that the fusion construct itself failed to fold into a functional conformation required for assembly. nonetheless, as judged by the autofluorescence of gfp.s in cells, at least the gfp-tag must have folded properly. thereby, gfp.s surprisingly appeared in sequestered speckles that overlapped with the er compartment. the formation of similar structures, termed concentric membranous er bodies, has been recently observed upon expression of a misfolded mutant of the cystic fibrosis transmembrane conductance regulator that accumulates in these bodies prior to erassociated degradation (okiyoneda et al., 2004) . accordingly, the s moiety of gfp.s might not attain its native transmembrane structure thereby tending to aggregate into discrete speckles. on co-expression with wild-type s, the intracellular distribution of gfp.s completely changed in such that it now yielded a typical membranous er staining pattern and a high degree of co-localization with s. this result indicates that the proper folding and localization of gfp.s were warranted by interprotein interactions with wildtype s chains. as a consequence thereof, highly fluorescent mosaic particles were formed and secreted, which closely resembled authentic spherical s lipoprotein particles according to density and size. furthermore, the gfp.s fusion protein could be also natively incorporated into the virion envelope. gfp, by virtue of its properties, has been successfully used to tag various viruses like, for example, herpes simplex virus type i, vesicular stomatitis virus, vaccinia virus, hiv, and mouse hepatitis coronavirus (bosch et al., 2004; dalton and rose, 2001; desai and person, 1998; stauber et al., 1999; ward and moss, 2001) . however, unlike these viruses, hbv is a very small particle with a diameter of 42 nm and a dense packed structure. even so, it can apparently accommodate multiple copies of gfp, indicating that there is substantial space available between the viral envelope and the nucleocapsid as well as sufficient flexibility allowing the incorporation of proteins in trans. although we were unable to unequivocally define the location of the gfp-tag within the secreted hbv envelope, our topological analysis of gfp.s suggested an interior (i.e., cytosolic side of microsomes) and exterior (i.e., lumenal side of microsomes) display of gfp. the formation of such a mixed topology is rare among membrane proteins but is a prominent feature of the large l envelope protein of hbv (bruss et al., 1994; ostapchuk et al., 1994; . in that case, it is established post-translationally at the er membrane and is uncoupled from envelope assembly (lambert and prange, 2001) . by contrast, the two differently orientated isoforms of gfp.s were only generated upon coexpression with secretion-competent wild-type s molecules, thus hinting to a link between envelope subunit interactions and topogenesis of gfp.s. while the underlying mechanism remains to be determined, it is tempting to speculate that the dual location of the gfp domain might be beneficial for the functionality of gfp-tagged hbv. by splitting the tag to both sides of the virion envelope, the local density of gfp might be lowered thereby limiting the risk of sterical hindrance of the envelope/nucleocapsid interactions at the interior and the envelope/receptor binding at the exterior. by using hepg2 cells, an established hepatocyte-derived cell line that is permissive for hbv attachment but nonpermissive to a productive infection (lu et al., 1996) , a specific binding of gfp-tagged viral particles was observed. hence, these particles must imitate the natural virus and viral attachment appeared to proceed through the authentic pathway. however, whether these particles even retained their infectivity remains to be addressed. in vitro hbv infection studies had been restricted to primary human hepatocytes that are not readily available. more recently, however, successful experimental infections of tree shrews were carried out that might present a new animal model (köck et al., 2001) . in addition, a human hepatoma cell line has been established that supports hbv infection (gripon et al., 2002) . therefore, the gfp-tagged hbv envelope might be useful for the investigation of the early events of virus penetration such as monitoring entry and uncoating in living infected cells and tracing the fate of the envelope structure. with gfp as a marker, it should also be possible to approach sites of viral assembly and to follow the intracellular trafficking and egress of (sub)viral particles during the late stages of an hbv infection. the mammalian expression vectors carrying the hbv s gene with or without a c-terminally tagged influenza virus hemagglutinin (ha) epitope under the control of the human metallothionein iia promoter had been described (pni2.-sha and pni2.s, respectively) (hartmann-stühler and . for construction of the gfp.s chimera, plasmid pegfp-c1 (bd biosciences clontech) was cut within the multiple cloning site by using ecori and smai. to introduce an ecori restriction site downstream of the start codon of the hbv s gene, site-directed mutagenesis with a recombinant m13mp19 bacteriophage carrying a 2.3kb bglii-bglii fragment (nucleotide [nts] 2839 to 1986, as referred of the hbv genome, subtype ayw) with the antisense oligonucleotide 5v-gaatcctgaattcatgttctc-3v (the ecori site is italic) was performed. the newly created ecori site, cutting between codon positions 4 and 5 (nts 170) of the s gene, together with the cognate filled-in acci site (nts 827) was then used to generate an s encoding fragment for in-frame insertion into pegfp-c1, giving rise to plasmid pegfp.s. in parallel, the ha-tagged version of the s gene was similarly cloned, giving rise to gfp.sha. a reciprocal chimera, containing the yellow-shifted yfp colinearly fused to the c-terminus of s (s.yfp), was created using plasmid peyfp-n1 (bd biosciences clontech), which was opened with xhoi and bamhi. the ha-tagged s gene was derived from pni2.sha by cleavage with xhoi (nts 127) and psii (cutting between the ha-tag and the translational stop codon) and inserted into peyfp-n1 after filling-in of its bamhi site. plasmid pni2.sdtm1ha carries an in-frame deletion of tm1 that was achieved by removal of a styi (nts 180)-xbai (nts 249) fragment from pni2.s. to produce viral envelope proteins, transient transfection of cos-7 cells by electroporation was used. unless otherwise indicated, 5 â 10 6 cells were transfected with 12 ag of plasmid dna, while in the case of co-transfections, 12 ag of each dna was used. three days posttransfection, cellular supernatants were harvested and clarified by low-speed centrifugation. cells were washed twice in tris-buffered saline (tbs, 50 mm tris-cl [ph 7.5], 150 mm nacl) and lysed with 1 ml of tbs-0.5% nonidet p-40 (np-40), supplemented with a protease inhibitor cocktail (roche), for 30 min on ice. after centrifugation for 5 min at 13 000 â g and 4 8c, proteins of lysates and cell supernatants were precipitated with 10% trichloroacetic acid (tca), washed twice with 5% tca, and once with acetone. tca precipitates were resolved by sds-page and western blotted to nitrocellulose membranes. enzymatic n-deglycosylation of proteins with pngase f (new england biolabs) was done according to the instructions of the supplier. immunoblots were incubated with a mouse monoclonal antibody (mab) against the ha epitope (babco), diluted 1:2000 in blotting buffer (pbs with 5% skim milk) or a mouse mab specific for gfp (jl-8; bd biosciences clontech) applied in 1:4000 dilution. peroxidase-labeled secondary antibodies (dianova) were diluted as instructed by the manufacturer, and the blots were developed with enhanced chemiluminescence detection reagents (amersham biosciences). in parallel, cell lysates and supernatants were analyzed by elisas. hbsag was measured using the auszyme ii diagnostic kit (abbott laboratories). for simultaneous detection of gfp and hbsag, a sandwich elisa with gfp-specific antibodies in the solid phase and s-specific antibodies in the detection phase was employed. briefly, the a-gfp-mab jl-8 was coated to microtiter plates (1:500 dilution in tbs) for 3 h at room temperature. nonspecific binding sites were blocked with tbs-1% bovine serum albumin for 18 h at 4 8c, followed by three washes with tbs-0.1% tween 20. samples were reacted for 2 h at 37 8c in the presence of 0.5% np-40, washed out, and incubated with peroxidase-labeled hbsag-specific mabs (auszyme ii) for 1 h at 37 8c. transfected cos-7 cells on glass cover-slips were fixed and permeabilized with ice-cold methanol containing 2 mm egta for 15 min at à20 8c. for staining of internal antigens, cells were incubated with the ha-specific mab (1:200 dilution in pbs) or a mouse mab specific for protein disulfide isomerase (spa-891, stressgen biotechnologies) (1:2000 dilution in pbs) prior to incubation with an alexafluor 594-conjugated goat anti-mouse immunoglobulin g (2 ag/ml in pbs; molecular probes). spontaneous gfp fluorescence and immunostaining were visualized with a fluorescence microscope (axiovert 200m, zeiss), and images obtained with a zeiss axiocam digital camera were processed using the zeiss axiovision software. to assess for fluorescent particles, cellular supernatants were concentrated 50-fold by ultracentrifugation (see below). a 10-al aliquot was spotted on glass bottom dishes (bd biosciences), briefly air dried, and imaged with a â100 objective. three days after transfection of cos-7 cells, microsomes were prepared essentially as described . briefly, cells were disrupted by dounce homogenization, and microsomes were recovered by ultracentrifugation prior to proteolysis with proteinase k (100 ag/ml) in the presence or absence of 0.5% np-40. after incubation on ice for 60 min, proteinase k was inactivated by the addition of 10 al/ml phenylmethylsulfonyl fluoride. each sample was then adjusted to 0.5% np-40 and solubilized for 20 min on ice. cleared samples were precipitated with 10% tca and subjected to western blot analysis. cesium chloride and sucrose gradient purification of particles subviral particles of cellular supernatants were pelleted through a 1-ml cushion of 20% sucrose in tne (10 mm tris-cl [ph 7.5], 150 mm nacl, 10 mm edta) using a sw 40 rotor (beckman) at 37 000 rpm for 4 h at 4 8c. the resuspended pellets were separated by isopycnic gradient centrifugation through 10-50% (wt/vol) cesium chloride in tne. after centrifugation at 35 000 rpm and 10 8c (sw 40 rotor), fractions were collected from the top and screened for chimeric particles by elisa, immunoblotting, and fluorescence analysis. for velocity sedimentation, pelleted particles were layered on top of a 12-ml linear sucrose gradient (10 to 50% [wt/wt] sucrose in tne buffer) with a 68% sucrose cushion, centrifuged at 35 000 rpm and 10 8c for 16 h (sw 40 rotor), and processed as above. for replication of hbv in the huh-7 liver cell line, plasmid phbv was used that carries a 1.1 mer of the hbv dna genome (radziwill et al., 1990) . this plasmid was cotransfected with pegfp.s by the calcium phosphate precipitation technique. four days after transfection, extracellular virions were separated from subviral empty envelopes and non-enveloped core particles by isopycnic cscl gradient centrifugation. to this aim, 3.8 g of cscl was dissolved in 10 ml cleared supernatant and 28 ml of this solution was spun for 20 h at 45 000 rpm at 10 8c in a beckman 50vti rotor. fractions were collected from the bottom, supplemented with the protease inhibitor cocktail, and were screened for hbsag and gfp by immunoassays. following dialysis of fractions against 10 mm tris-cl [ph 7.5], 150 mm nacl at 4 8c, virions were isolated by an envelope-specific immunoprecipitation, as described previously (löffler-mary et al., 2000) . for detection of gfp.s within the virion envelope, particles were immunoprecipitated with the a-gfp mab jl-8 that had been coated to a 10% (wt/vol) suspension of protein g-sepharose (10-al antibody was bound to 100 al sepharose solution in an overnight reaction at 4 8c). immunoprecipitation was performed in the presence of 0.5% np-40 and precipitates were then washed two times with ten buffer (50 mm tris-hcl [ph 7.5], 1 mm edta, 75 mm nh 4 cl). detection of the encapsidated viral progeny dna by radioactive labeling of the partially double-stranded genome with 10 aci [a 32 p]datp (amersham biosciences) by the endogenous polymerase, isolation, and separation of the dna by agarose gel electrophoresis have been described (löffler-mary et al., 2000) . the radioactive signal was detected by phosphorimager scanning. for detection of hbv dna on dot-blots, an ecorilinearized unit-length hbv genome was labeled with digoxygenin-dutp by random priming as instructed by the manufacturer (roche). after extraction of the dna genomes from the immunoprecipitated samples as described, they were denatured by boiling in 0.4 m naoh-10 mm edta and filtered with a dot-blot manifold (schleicher and schqll) onto nylon membranes. the membrane was baked at 120 8c for 30 min and hybridized with the labeled probe according to the roche dig dna labeling and detection kit. for binding analysis, human hepatoma hepg2 cells were cultivated in glass bottom dishes in dulbecco's modified eagle's medium with 10% fetal calf serum (fcs). hbv and subviral particles secreted from huh-7 (co)-transfected cells were precipitated from 10-ml culture supernatant in the presence of 6% polyethylene glycol 8000 (peg; sigma). the pellet was resuspended in 200-al phosphate-buffered saline (pbs) containing 25% fcs. hepg2 cells were incubated with 50 al of this concentrate, diluted in 1 ml culture medium with 4% peg, for 20 h at 37 8c. cells were washed three times with pbs and analyzed for gfp fluorescence by microscopy. computer-aided studies on the spatial structure of the small hepatitis b surface protein coronavirus spike glycoprotein, extended at the carboxy terminus with green fluorescent protein, is assembly competent carboxypeptidase d (gp180), a golgi-resident protein, functions in the attachment and entry of avian hepatitis b viruses the role of envelope proteins in hepatitis b virus assembly post-translational alterations in transmembrane topology of the hepatitis b virus large envelope protein functions of the large hepatitis b virus surface protein in viral particle morphogenesis expression of hepatitis b virus large envelope polypeptide inhibits hepatitis b surface antigen secretion in transgenic mice the earliest steps in hepatitis b virus infection vesicular stomatitis virus glycoprotein containing the entire green fluorescent protein on its cytoplasmic domain is incorporated efficiently into virus particles a poliovirus neutralization epitope expressed on hybrid hepatitis b surface antigen particles incorporation of the green fluorescent protein into the herpes simplex virus type 1 capsid reproducible high level infection of cultured adult human hepatocytes by hepatitis b virus: effect of polyethylene glycol on adsorption and penetration infection of a human hepatoma cell line by hepatitis b virus phindependent uptake of hepatitis b virus in primary human hepatocytes hepatitis b virus large envelope protein interacts with g2-adaptin, a clathrin adaptor-related protein surface proteins of hepatitis b viruses hepatitis b surface antigen assembles in a post-er, pre-golgi compartment efficient infection of primary tupaia hepatocytes with purified human and woolly monkey hepatitis b virus dual topology of the hepatitis b virus large envelope protein: determinants influencing post-translational pre-s translocation infection process of the hepatitis b virus depends on the presence of a defined sequence in the pre-s1 domain hepatitis b virus assembly is sensitive to changes in the cytosolic s loop of the envelope proteins protease-induced infectivity of hepatitis b virus for human hepatoblastoma cell line induction of anti-human immunodeficiency virus (hiv) neutralizing antibodies in rabbits immunized with recombinant hiv-hepatitis b surface antigen particles hepatitis b virus morphogenesis. cmti identification and chemical synthesis of a host cell receptor binding site on hepatitis b virus df508 cftr pool in the endoplasmic reticulum is increased by calnexin overexpression a dramatic shift in the transmembrane topology of a viral envelope glycoprotein accompanies hepatitis b viral morphogenesis novel transmembrane topology of the hepatitis b virus envelope proteins deletions in the hepatitis b virus small envelope protein: effect on assembly and secretion of surface antigen particles properties of modified hepatitis b virus surface antigen particles carrying pres epitopes mutational analysis of the hepatitis b virus p gene product: domain structure and rnase h activity secreted hepatitis b surface antigen polypeptides are derived from a transmembrane precursor direct visualization of hiv-1 entry: mechanisms and role of cell surface receptors a topological model for hepatitis b surface antigen visualization of intracellular movement of vaccinia virus virions containing a green fluorescent protein-b5r membrane protein chimera characterization of early hepatitis b virus surface protein oligomers we acknowledge the perfect technical assistance of tatjana dfring and thank rolf e. streeck, martin sapp, and hans-christoph selinka for helpful discussion. this work was supported by grants to r. p. from the deutsche forschungsgemeinschaft (sfb 490-a1, pr305/1-1). key: cord-329794-msxrdhb3 authors: lu, aili; zhang, huanqing; zhang, xiaoyan; wang, hongxia; hu, qikuan; shen, li; schaffhausen, brian s.; hou, weimin; li, linsong title: attenuation of sars coronavirus by a short hairpin rna expression plasmid targeting rna-dependent rna polymerase date: 2004-06-20 journal: virology doi: 10.1016/j.virol.2004.03.031 sha: doc_id: 329794 cord_uid: msxrdhb3 abstract severe acute respiratory syndrome (sars) is a highly contagious and sometimes a lethal disease, which spread over five continents in 2002–2003. laboratory analysis showed that the etiologic agent for sars is a new type of coronavirus. currently, there is no specific treatment for this disease. rna interference (rnai) is a recently discovered antiviral mechanism in plant and animal cells that induces a specific degradation of double-stranded rna. here, we provide evidences that rnai targeting at coronavirus rna-dependent rna polymerase (rdrp) using short hairpin rna (shrna) expression plasmids can specifically inhibit expression of extraneous coronavirus rdrp in 293 and hela cells. moreover, this construct significantly reduced the plaque formation of sars coronaviruses in vero-e6 cells. the data may suggest a new approach for treatment of sars patients. severe acute respiratory syndrome (sars) is a highly contagious disease. an outbreak in 2002-2003 affected more than 8000 patients in over 30 countries across five continents. in some areas, the mortality reached as high as 10% of the infected patients (who). viruses isolated from sars patients have been confirmed to be a new type of coronavirus that is responsible for the outbreak (enserink and normile, 2003; guan et al., 2003; lai, 2003; yan et al., 2003) . coronaviruses are large enveloped, positive-stranded rna viruses that cause diseases in human and animals (chiu, 2003; martina et al., 2003) . they have the largest genomes in all rna viruses and replicate by a unique mechanism, which results in high frequency of recombination. similar to influenza virus, coronaviruses are highly mutable and therefore the infection is extremely difficult to treat (knipe and howley, 2001) . rna interference (rnai) is a native process in which the introduction of a short double-stranded rna into cells results in posttranscriptional silencing of the targeted genes. this gene silencing was found to be a genespecific mechanism for regulation of complex biological functions. rnai has been successfully used in c. elegans, fungi, drosophila, and mammalian cells to study gene functions (elbashir et al., 2001a (elbashir et al., , 2001b fire et al., 1998; kadotani et al., 2003) . recently, several reports have demonstrated the use of rnai for attenuation of viral infection and replication in animal cells, suggesting that rnai might become a therapeutic approach in the future for treating viral diseases (gitlin et al., 2002; jacque et al., 2002; jia and sun, 2003; kapadia et al., $ supplementary data associated with this article can be found, in the online version, at doi:10. 1016 /j.virol.2004 .03.031. 2003 novina et al., 2002; qin et al., 2003; randall et al., 2003) . the goal of this study is to test the inhibitory effect of rnai on the expression of rna-dependent rna polymerase (rdrp) of sars coronavirus, and the potential of rnai to attenuate the plaque formation of sars coronavirus in infected animal cells. here, we provide the evidence that rnai targeting at coronavirus rdrp using short hairpin rna (shrna) expression plasmids can specifically inhibit expression of extraneous coronavirus rdrp in 293 and hela cells. moreover, this construct significantly reduced the plaque formation of sars coronaviruses in vero-e6 cells. the results may suggest a new approach for treatment of sars. design of specific shrna expression plasmids targeting at coronavirus rdrp coronavirus isolated from sars patients has a large genomic rna (approximately 30 kb). when the virus replicates, errors are generated with a high frequency, making the virus particularly difficult to target. we therefore chose the target genes in the viral genome that are conserved in different strains of coronavirus. one of the candidates is rdrp (genebank ay 268070). this gene has 645 bp and codes a 215-amino-acid peptide. blast analysis shows that there is almost no mismatch in this region of coronaviruses. this sequence has no homology with any known human genes. among 25 candidate fragments, five panels of dna sequences (table 1) were synthesized and inserted into psilence1.0-u6. with the u6 pol iii promoter, the plasmids express a 55-nt rna stem-loop. after transfecton, about 40-90% of rdrp gene expression was inhibited, depending on the sequence of the shrna inserts, based on rt-pcr analysis in both hela and 293 cells transfected with rdrp (data not shown). therefore, we selected the most potent plasmid inserted with sequence a (plasmid-a, see table 1 ) for subsequent experiments. a mutated version of the inserted sequence-a with 6-nt mismatch was also synthesized as a specific negative control (plasmid-a*, table 1 ). shrna reduced the expression of sars rdrp mrna in 293 and hela cells as shown in fig. 1a , based on the rt-pcr analysis, the expression of extraneous rdrp gene was observed peaking at 48 h after the transfection. when 293 cells were cotransfected with pires-2-egfp/rdrp plasmid along with shrna expression plasmid-a, the rdrp mrna was reduced to 25% of that of the control cells measured at 24 h after the transfection, and the rdrp mrna can no longer be detected 48 h after the transfection (fig. 1b, lane 4) . no inhibitory effect was observed in the cells co-transfected with pires-2-egfp/rdrp along with plasmid-a* (fig. 1b , lane 5). the inhibitive effect on rdrp by plasmid-a was also observed in hela cells (data not shown). shrna reduced the production of sars rdrp proteins to confirm the effect of shrna on rdrp expression, we performed western blot analysis. because anti-rdrp antibody is not commercially available, antibody against ha tag was used to detect the production of rdrp proteins, in which an ha epitope tag had been introduced at the nterminus. the results showed that the production of rdrp proteins was reduced by more than 90% in cells co-transfected with plasmid-a (fig. 2b, lane 4) . the specificity of the rnai was again demonstrated in that no inhibitory effect on rdrp protein expression was observed in the cells co-transfected with plasmid-a* (fig. 2 , lane 5). the reduction of rdrp protein in the target cells was also analyzed by immunofluorescence. again, rdrp production was significantly reduced by plasmid-a (fig. 3d ), but not by plasmid-a* (fig. 3e ). in addition, we noticed an interesting phenomenon: rdrp proteins appeared to be located in the cytoplasm, but somehow accumulated in the nucleus of the cells 48 h after the transfection (fig. 3) . the significance of this finding is as yet unknown. to test whether shrna expression plasmid could indeed attenuate sars coronavirus infection, the plaque formation was analyzed in vero-e6 cells. plaques are routinely counted at 48 h after coronavirus infection. no difference table 1 the sequences of shrna inserts for psilence 1.0 u6 sequence targeting in the number of plaques was observed between control cells and cells transfected with either psilence 1.0-u6 or plasmid-a*. however, the plaque number was significantly reduced in the cells transfected with shrna expression plasmid-a, indicating that this shrna expression plasmid could effectively inhibit plaque formation of sars coronavirus in vero-e6 cells (fig. 4) . the etiologic agent for sars has been proven to be coronavirus. because rdrp is the key enzyme for coronavirus replication and its sequences are very conserved in different isolates of coronaviruses, it might be a good target to inhibit the replication of coronavirus in sars patients. to test this possibility, in the present study, hela cells and 293 cells were transfected with sars coronavirus rdrp, and the effect of rnai on inhibition of rdrp of coronavirus was tested. our data showed that the shrna expression plasmid for rdrp, plasmid-a, reduced the expression of rdrp in both mrna and protein levels by more than 90% in both 293 and hela cells. inhibition of rdrp by the shrna expression plasmid-a in the rdrp-transfected cells may not necessary to be warranted in cells infected with sars coronavirus. we therefore tested the inhibition of coronavirus infection by the shrna expression plasmid-a in vero-e6 cells infected with the virus. the data clearly demonstrated that the plaque formation of sars coronavirus was significantly inhibited by this plasmid-a, suggesting the shrna expression plasmids might indeed inhibit the replication of sars coronavirus. numbers of studies have shown that rnai is an important mechanism for host defense in plant as well as in mammalian cells (baulcombe, 2002; ge et al., 2003 ; randall xia et al., 2002) . it has been reported that short interfering rna (sirna) worked as antiviral tools in cell culture for hiv, hcv, and herpesvirus (jia and sun, 2003; kapadia et al., 2003; novina et al., 2002; qin et al., 2003) . there have also been reports in regard to treat diseases by sirna in mice (mccaffrey et al., 2002; song et al., 2003) . delivery of rnai in those animal studies all depends on a high-pressure injection via mouse-tail vein, which is not practical for humans. delivery method is therefore a limiting factor for application of rnai in treating human diseases. combined the information together with our study, we therefore proposed that inhibition of rdrp by the shrna expression plasmid, plasmid-a, might be an anti-coronavirus agent for treating of sras patients. in fact, dr. he et al. most recently reported that sirna targeting at rdrp could knock down sars viral genes in frhk-4 cells . our data not only confirmed their findings, but also provide the new rnai tool, the shrna expression plasmids. considering that the shrna expression plasmids can be easily incorporated into viral vectors for in vivo delivery, this study may suggest a new method to treat sars patients in the future. vero-e6, 293, and hela cells were routinely cultured in dulbecco's modified eagle's medium supplemented with 10% of fetal bovine serum. the rdrp gene of coronavirus was synthesized by geneart company (geneart gmbh, rosenberg, germany) based on the rdrp sequence (genebank accession number ay 268070). the synthetic rdrp gene was cloned into kpni/saci sites of pcrscript amp (stratagene). the rdrp gene was pcr amplified using primers: forward: 5vgcggaattcatgcaggacgctgtagcttc and reverse: 5vgcgggatcctcat aggttat tttcagtgtc. the pcr product was digested with ecori/bamhi and cloned into mammalian expression vector pires-2-egfp or pcmv-ha that introduced an ha epitope tag at the nterminus of the proteins. all the inserted sequences were verified by dna sequencing. the shrna inserts (table 1) were designed by webbased tools (www.ambion.com/techlib/misc/sirna _ finder. html). we designed 25 different pairs of shrna inserts based on the following criteria: (1) the 19 nucleotides are immediately downstream of an aa di-nucleotide in the coding region of the target gene. (2) the sequence should not contain a repeats of more than four as or ts. a total five pairs of shrna inserts were synthesized and inserted into linearized psilence1.0-u6 (ambion) according the manufacturer's manual. hela and 293 cells were seeded in a 6 well plate at 3 â 10 5 /well the day before transfection. one microgram of rdrp expression plasmids, pires-2-egfp/ rdrp or pcmv-ha-rdrp, was co-transfected along with 1 ag of shrna expression plasmid-a (the best of shrnas tested) in lipofectamine 2000 (gibcobrl), respectively. the plasmids without inserts or the mutant shrna expression construct, plasmid-a*, was used as negative controls. total rna from each well of cells was isolated using rnaeasy kit (qiagen). rt-pcr was carried out with m-mlv kit (gibcobrl) at 42 jc for 1 h. the reverse transcriptase was then inactivated by heating at 70 jc for 15 min. pcr was performed with 2 al of cdna and specific primers: rdrp: forward: 5vgcggaattcatg caggacgctgtagcttc3v and reverse: 5vgcgggatc ctcataggttattttcagtgtc 3v; g3pdh: forward: 5v gaaggtgaaggtcggagt and reverse: 5v gaaga tgtg atgg gatttc. pcr was carried out at 95 jc 2 min, then 95 jc 30 s, 55 jc 1 min, 72 jc 1 min for 40 cycles. five microliters of pcr product were run on a 1% agarose gel. cells were harvested at the indicated time after transfection, washed with cold pbs, and total proteins were extracted in the extraction buffer (150 mm nacl, 50 mm tris, ph 7.5, 1% glycerol, and 1% np-40). protein extracts were separated by 10% sds-page and transferred to nitrocellulose. anti-ha antibodies (roche), antia-tubulin (santa cruz biotechnology) antibodies, and hrp-conjugated second antibodies (chemicon) were used for western blotting, respectively. bands were visualized by ecl reagent and recorded on image station 440cf (kodak). cells grown on the glass coverslip were fixed by immersion in 3.7% formaldehyde solution for 30 min at 37 jc. coverslips were treated in ice-cold methanol/ acetone (3:7 v/v) for 15 min at 4 jc. air-dried coverslips were incubated at 37 jc in a humidified atmosphere box, blocked with 1% bsa in pbs, and then incubated with primary antibody (1:100 in pbs 1% bsa) for 1 h. after three washes with pbs, the coverslips were incubated with fluorochrome-conjugated second antibody (1:100 in pbs 1% bsa) for 30 min, washed with pbs and mounted with 50% glycerol. photographs were taken on an olympus immunofluorescent microscope (model bx51tr) and apogee instruments microscopy fluorescence system (model kx85). sars coronavirus (sars cov-p9) was kindly provided by the chinese national institute for viral disease control and prevention. the viruses were replicated in vero-e6 cells at 35 jc. the cell culture supernatant was harvested 48 h after virus infection and stored at à80 jc for future use. to test the anti-sars virus potential of the shrna expression plasmids, 1 â 10 5 vero-e6 cells were seeded in a 24well plate and transfected with 0.5 ag shrna expression plasmid-a, its mutant plasmid-a* and another negative control, psilence1.0-u6, respectively. twenty-four hours after transfection, the sars cov-p9 viruses in the cell culture supernatant mentioned above were diluted at 1/ 100 000 and added to each well. after infection for 45 min at 35 jc, 1 ml modified eagle's medium containing 0.8% methylcellulose was added to each well, and the plaques were visualized by staining with crystal violet 48 h after infection. statistical analysis was based on t test and performed on microsoft excel. all graphs represent the mean f sd. rna silencing taiwanese scientists find genetic link to sars duplexes of 21-nucleotide rnas mediate rna interference in cultured mammalian cells functional anatomy of sirnas for mediating efficient rnai in drosophila melanogaster embryo lysate infectious diseases. search for sars origins stalls potent and specific genetic interference by double-stranded rna in caenorhabditis elegans rna interference of influenza virus production by directly targeting mrna for degradation and indirectly inhibiting all viral rna transcription short interfering rna confers intracellular antiviral immunity in human cells isolation and characterization of viruses related to the sars coronavirus from animals in southern china inhibition of sars-associated coronavirus infection and replication by rna interference modulation of hiv-1rep-lication by rna interference inhibition of gamma herpesvirus replication by rna interference rna silencing in the phytopathogenic fungus magnaporthe oryzae interference of hepatitis c virus rna replication by short interfering rnas sars virus: the beginning of the unraveling of a new coronavirus virology: sars virus infection of cats and ferrets rna interference in adult mice sirna-directed inhibition of hiv-1 infection inhibiting hiv-1 infection in human t cells by lentiviral-mediated delivery of small interfering rna against ccr5 clearance of replicating hepatitis c virus replicon rnas in cell culture by small interfering rnas rna interference targeting fas protects mice from fulminant hepatitis cumulative number of reported cases of severe acute respiratory syndrome sirna mediated gene silencing in vitro and in vivo assessment of putative protein targets derived from the sars genome this work is supported by grants from the chinese national 973 project (2001cb510100), 863 project (2001aa216171), 211 project (2002project ( -2005, and the grants from beijing ministry of science and technology (2002 -489) and china medical board (cmb). we thank dr. junqi he and dr. biao kan for their help.